NEURAL - BIQGHEMWAL CONTROL OF MASCUUNE
SEXUAL BEHAWOR EN THE LABORATORY RAT
(RAUUS NORVEQCUS)

Dissertatien for the Degree of Ph. D.
MSCHIGAN STATE UNIVERSITY
LARRY WAYNE CHRISTENSEN

1973

   
 
  
   

   
 
  

L I L“)? A R Y
Bd§c¥ gan State
Urzrvcrsity

'HES'H

LABORATORY RAT (RATTUS NORVEtBICUS)
presented by
Larry Wayne Christensen
has been accepted towards fulfillment

of the requirements for

Ph . D . degree in Zoology

Major professor

 

 

 

ABSTRACT
NEURAL—BIOCHEMICAL CONTROL OF

MASCULINB SEXUAL BEHAVIOR IN THE
LABORATORY RAT (RATTUS NORVEGICUS)

By
Larry Wayne Christensen

Four experiments were carried out to investigate possible neuro—
biochemical events associated with the stimulation of masculine sexual
behavior by testosterone. Castrated adult male rats were used in all
experiments to investigate 3 problems; 1) Is testosterone necessarily
converted to an estrogen before stimulating the resumption of mating
behavior in long term castrated rats? 2) Is 3'5' cyclic adenosine mono-
phosphate involved as an intracellular mediator in the action of testos-
terone on the brain in stimulating sexual behavior? and 3) What influence
do drugs designed to alter the adrenergic or cholinergic milieu of the
nervous system, have on copulatory performance, when they are applied
directly to the preoptic area (POA) of the brain?

In Experiment I adult male rats that had been screened for copula-
tory behavior and subsequently castrated, where bilaterally implanted
with stainless steel cannulae in the brain after meeting a criterion
for the loss of sexual behaivor. The cannulae were placed 80 the tips
rested in one of two areas, the preoptic area (POA) or the posterior
hypothalamic area (PHA). Steroid hormones (ie. testosterone, estradiol
or cholesterol) where then applied to either the FDA or the PHA in an
attempt to reinstate c0pulatory behavior. The application of testosterone
or estradiol to the FDA significantly increased sexual responses compared to
scores exhibited by animals receiving the control steroid, cholesterol,

or animals receiving either testosterone or estradiol in the PHA. Estradiol

Larry W. Christensen
was more effective than testosterone in stimulating the mean number of
mounts and intromissions when applied to the FDA.

Experiment II tested the hypothesis that the aromatization of testos-
terone to estradiol is necessary for the stimulation of masculine sexual
behavior. This hypothesis was tested by administering directly to the
POA, in combination with systemically administered testosterone, a compound
(metapirone) that is known to inhibit the conversion of testosterone to
estradiol. When animals were treated with systemic testosterone + intra-
cerebral metapirone (infused directly into the FDA) a significant increase
in mounting was not observed, as was the case when animals received sys—
temic testosterone + an intracerebral control solution or systemic estra-
diol + intracerebral metapirone.

In Experiment IV long term castrated male rats that showed no ejac—
ulatory response on 3 consecutive weekly tests, were bilaterally implanted
with cannulae resting in the POA. Solutions of CAMP, S'AMP(the inactive
metabolite of CAMP) or Dibutyryl CAMP (Db—CAMP) were then administered
in an attempt to mimic the effects of testosterone on sexual behavior.
None of the subjects showed any sexual responses during the 13 days of
nucleotide treatment. However, when subsequently combined with systemic
testosterone treatment S'AMP but not CAMP or Db—CAMP significantly eli—
vated the mean number of mounts and intromissions exhibited.

In Experiment IV drugs were administered via cannulae, directly to
the FDA of sexually active male rats in an attempt to determine the influ—
ence of adrenergic and cholinergic neural systems on the control of sex-
ual behavior. Applications of norepinephrine (an adrenergic neurotrans—
mitter) retarded the temporal pattern of COpulation, the animals taking

longer to initiate copulation and likewise longer between events in the

Larry W. Christensen

pattern once it was begun. Alpha-methyl-tyrosine (an inhibitor of
norepinephrine synthesis) significantly decreased the number of ejacula-
tions exhibited during a test for sexual behavior given A hrs after the
initial application to the POA. lpha-methyl-tyrosine affected no other
component of sexual beahvior when compared to the sham test, when no

drug was given. When carbachol (a Cholinomimetic) was applied to the

POA of copulating rats, all components of the copulatory pattern were
eliminated. Application of a compound to the FDA that blocks the synthe—
sis of achetylcholine (hemicholinium—B) decreased the mean number of
mounts, intromissions and ejaculations in a test given u hrs after the
initial application. However, it had no effect on any component of
copulation in a test given 15 minutes after the initial exposure.

The decrease in copulation at u hrs was due to a prolonged period of sex~
ual quiescence exhibited in a significant percentage of the hemicholinium-3
treated animals, after achieving 1 or 2 ejaculatory responses.

The data presented in this dissertation suggest: 1) the aromatiza—
tion of testosterone to estradiol is necessary for the stimulation of
masculine sexual behavior; 2) CAMP is involved in regulating the dis-
play of masculine copulatory responses, perhaps through an inhibitory mechan-
ism; and 3) adrenergic neural systems are involved in temporally pattern-

ing copulation, where as cholinergic systems regulate the occurrence of

the pattern.

NEURAL-BIOCHEMICAL CONTROL OF
MASCULINE SEXUAL BEHAVIOR IN THE

LABORATORY RAT (RATTUS NORVEGICUS)

By

Larry Wayne Christensen

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Zoology

1973

g 555537

"In scientific research there is no such thing as bad results,
just unexpected ones."

R. Buckminster Fuller

ACKNOWLEDGMENTS

My special appreciation and thanks is extended to Dr. Lyn Clemens.
His genius and approach to science provided a guidance far beyound any
formal instruction. The assistance from each of the members on my guid-
ance committee is also greatly appreciated; Dr. Jack Johnson, Dr. John
King and Dr. Harold Hafs. Each provided a valuable and personal exper-
tise. I would like to thank all the students in Animal Behavior and
especially those graduate students in the Hormone and Behavior Laboratory,
Linda Coniglio, John Dwyer, Ray Humphrys and Archie Vomachka. A special
thank you also must be given to, Ray Humphrys for his help in Experiment
IV, Mary Vallender for her always friendly assistance, Jan Harper for her
assistance in the typing, and Dr. Jean McManus for her generous help
in the histology of brains. I want lastly but most importantly to extend
an appreciative thanks to my lady, Glenda Rogers. She provided assist—
ance with the actual manuscript, as well as giving graciously of her
love and understanding. This research was supported, in part, by USPHS
training grant: GM 01751-01 from the National Institute of General Med-

ical Sciences, and a USPHS Research Grant: HD 06760-01 to Dr. L. G. Clemens.

ii

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES.

LIST OF ABBREVIATIONS.

 

INTRODUCTION
BACKGROUND

Hormonal Concomitants of Female Sexual Behavior
Female Sexual Responses
Masculine Sexual Pattern. .
Hormonal Control of Masculine Mating Behavior .
Neuro— hormonal Control of Adult Male Sexual Behavior.

Neural control

Hormonal control .

Aromatization process.

Biochemical mechanisms involved in sexual behavior
Parmacological Control of Masculine Sexual Behavior
Objectives of the Present Study .

EXPERIMENT I. DETERMINE IF ESTRADIOL IS AS EFFECTIVE AS
TESTOSTERONE FOR INDUCING THE RESUMPTION OF MASCULINE
SEXUAL BEHAVIOR.

Part A. Determine a Site of Testosterone Action
Methods.
Results. . . . . . .
Part B. Determine if Estradiol is as Effective as.
Testosterone When Implanted in the Same Area
Methods.
Results.

EXPERIMENT II. THE BLOCKING OF TESTOSTERONE METABOLISM TO AN

ESTROGEN BY METAPIRONE AND ITS EFFECT ON THE INDUCTION OF

MASCULINE SEXUAL BEHAVIOR.

iii

Page

.vi, vii

. viii

. 10
ll
11
12

. 1M

. 16
18
2O

22

22
22
23

27
28

38

Methods.
Results. . . .

EXPERIMENT III.

SEXUAL BEHAVIOR.

Methods.
Results.

EXPERIMENT IV.

Methods. . . . .

Results.
Part A:
Part B:

DISCUSSION. . . . . .

1) Neural Hormonal Specificity . . .

2) Intracellular Events Associated With Testosterone
Reactivation.

3) Pharmacological Control of Male Sexual Behavior

CONCLUSION.

APPENDIX.

I. Subject Selection For all Experiments.
II. Cannulae Implantation Procedure

III. Histology.

LIST OF REFERENCES.

DETERMINE IF CAMP CAN MIMIC THE EFFECTS OF .
TESTOSTERONE IN INDUCING THE RESUMPTION OF MASCULINE

EFFECTS OF POA ADRENERGIC OR CHOLINERGIC.
MANIPULATION ON MASCULINE SEXUAL BEHAVIOR.

Adrenergic manipulation
Cholinergic manipulation.

Page

38
39

 

SO

SO
51

55
55
59

59
64

72

73
75

79

83

85

85

86
9O

LIST OF TABLES

Page

Table l. The effects of intracerebrally implanted testos- . . . . . . 26
terone or cholesterol on the mean mount and intromission
.frequency as well as percentage of animals ejaculating.

Table 2. The effects of implanted estradiol or cholesterol . . . . . . 31
on the mean mount and intromission frequency as well as
the percentage of animals ejaculating.

Table 3. The effects of implanted steroids on the mean number. . . . . 37
of penile spines and the mean length of penile papillae.

Table A. The means t SE of mount and intromission scores for
three groups of animals receiving testosterone or estra—
diol in combination with intracerebrally infused metapirone
or sucrose.

. . . . 44

Table 5. Percentage of castrated male rats exhibiting mount, . . . . . ”5
intromission or ejaculatory responses after receiving sys—
temic testosterone or estradiol plus intracerebrally infu-
sed metapirone or sucrose.

Table 6. The effects of intracerebrally infused S'AMP, CAMP or . . . . 5”
Db-CAMP by themselves or with 8 days of testosterone, on
mean mount and intromission frequency as well as percentage
of animals ejaculating.

Table 7. The effects of andrenergic manipulation of the POA on . . . . 63
parameters of masculine sexual behavior.

Table 8. The effects of Cholinergic manipulation of the POA on . . . . 67
the parameters of masculine sexual behavior.

LIST OF FIGURES

Figure l. A shcematic representation of a working.
hypothesis for possible biochemical mechanisms
involved in testosterone activation of the ner-
vous system. -

Figure 2. A hypothesized pathway for the conversion.
of androgens to estrogens.

Figure 3. The effect of intracerebral application of . .
of testosterone or cholesterol on the mean mount
and intromission frequency as well as percentage
of animals mounting.

Figure A. The effect of intracerebral application of
estradiol or Cholesterol on the mean mount fre-
quency, intromission frequency and percentage of
animals ejaculating.

Figure 5. Locations of maximum POA cannulae penitra-
tion in Experiment I, represented on cross sec—
tional maps.

Figure 6. Locations of cannulae penitration in Exper_.
iment I, represented in a longitudinal map.

Figure 7. Locations of cannulae implanted in the PHA . .
during Experiment I, represented in cross section-
al maps.

Figure 8. Locations of cannulae in the PHA of Exper- . .
iment I, represented in a longitudinal map.

Figure 9. The effects of testosterone + metapirone,.
testosterone + sucrose or estradiol + metapirone
treatment on the mean mount and intromission fre-
quency.

Figure 10. Percentage of animals mounting, intromite
ting and ejaculating after being treated with
testosterone + metapirone, testosterone + sucrose
or estradiol + metapirone.

Figure ll. Locations of maximum cannulae penitration . .
in Experiment II, represented in cross sectional

maps.

Figure 12. Locations of cannulae in Experiment II, .
represented in a longitudinal map.

vi

 

LIST or FIGURES (CONT.)

Figure 13. Effects of intracerebrally applied nucleo—
tides in combination with systemic theophylline
or testosterone, on the mean mount and intromis—
sion frequency and percentage of animals ejac—
ulating.

Figure 1H. Locations of cannulae in Experiment III. . .
implanted in the POA, represented on cross
sectional maps.

Figure 15. The locations of cannulae implants in
Experiment III, represented in a longitudinal
map.

Figure 16. Effects of intracerebrally administered.
drugs designed to affect the adrenergic milieu
of the POA on parameters of masculine sexual
behavior.

Figure 17. The effect of intracerebrally adminis—
tered drugs that affect the cholinergic milieu
of the POA, on parameters of male sexual behav-
ior.

Figure 18. The locations of maximum cannulae peni—. .
tration in Experiment IV, represented in cross
sectional maps.

Figure 19. The locations of cannulae placements in.

Experiment IV, represented in a longitudinal
map.

vii

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. S8

. 62

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.70

.71

LIST OF ABBREVIATIONS

Ach. . . . . . . . . . . . . . Acetylcholine

a-MT . . . . . . . . . . . . . Alpha methyl tyrosine

S'AMP. . . . . . . . . . . . . 5' Adenosine monophosphate

CAMP . . . . . . . . . . . . . 3'5' Cyclic adenosine monophosphate

Db-CAMP. . . . . . . . . . . . Dibutyryl 3'5' cyclic adenosine monophosphate

DHT. . . . . . . . . . . . . . Dihydrotestosterone

DNMR . . . . . . . . . . . . . Duncan's new multiple range test
HC-3 . . . . . . . . . . . . . Hemicholinium

ICT. . . . . . . . . . . . . . Intracerebral treatment

IF . . . . . . . . . . . . . . Intromission frequency

IL . . . . . . . . . . . . . . Intromission latency

IM . . . . . . . . . . . . . . Intramuscular

MF . . . . . . . . . . . . . . Mount frequency

MFB. . . . . . . . . . . . . . Median forebrain bundle

MIII . . . . . . . . . . . . . Mean inter intromission interval
ML . . . . . . . . . . . . . . Mount latency

NE . . . . . . . . . . . . . . Norepinephrine

PEI. . . . . . . . . . . . . . Post ejaculatory interval

PHA. . . . . . . . . . . . . . Posterior hypothalamic area

POA. . . . . . . . . . . . . . Preoptic area

TP . . . . . . . . . . . . . . Testosterone propionate

viii

INTRODUCTION

The importance of testicular hormones, especially testosterone, for
the deveIOpment and display of masculine copulatory behavior in mammalian
species is well documented (see Young 1961 for review). The development
and maturation of sexually dimorphic morphology and behavior depend upon
the presence or absence of gonadal hormones during two critical periods
in the animal's life, once around the time of sexual anatomical and
behavioral differentiation and again during adulthood (postpuberal).

When rats (Gerall and Ward 1966), guinea pigs (Phoenix, Goy, Gerall and
Young 1959), hamsters (Eaton 1970) or mice (Edwards and Burge 1971) are
exposed to testosterone during sexual differentiation and again post-
puberally (in the form of testicular secretion in males and injected
testosterone propionate in females) they develop the sexual morphology
and sexual behavior patterns characteristic of normal males. On the
other hand, lack of testosterone or certain of its metabolites, during
either period, prevents the activation of masculine behavior and morphol-
ogy.

The mechanisms by which testosterone influences the display of mas-
culine sexual behavior in the adult animal are for the most part unknown.
However, evidence suggests that in the male rat, the preoptic area (POA)
of the brain is one site of testosterone action which is involved in the
regulation of male copulatory behavior. Autoradiographic studies using
castrated male rats have shown a high concentration of radioactivity in
this neural area an hour after the administration of tritiated testos-

terone (Pfaff 1968, Resko, Goy and Phoenix 1967, Sar and Stumpf 1972).

I...)

2
Likewise, direct application of crystalline testosterone propionate to
the POA of long term castrated rats resulted in an increase of sexual
behavior (Davidson 1966, Lisk 1967, Johnston and Davidson 1972).
The present study was designed to provide information on possible
neurobiochemical mechanisms involved in the reactivation of masculine

sexual behavior in the castrated male rat (Rattus norvegicus). Inves-

 

tigation has centered around a working hypothesis which conceptualizes
the biochemical events occurring in the POA during testosterone stimula—
tion. In this hypothesis testosterone (H—androsten-l7sol-3—one) reaches
a nerve cell via the blood and is bound to the cell by a protein recepe
tor at the plasma membrane (see Figure l). The testosterone-receptor
complex is taken into the cell where it is metabolized to an estrogenic
form, probably estradiol (l,3,5(lO)—estratrien-3,17B—diol). The estradiol
now bound to the same or a different receptor protein enters the nucleus
where it has anabolic effects, the products of which may be directly
involved in preparing the cell for activity. Another intracellular path-
way important in this working hypothesis involves the synthesis of cyc-
lic 3'5'—adenosine monophosphate (CAMP). Cyclic AMP stimulates nuclear
controlled anabolic pathways, the products of which may either be invol-
ved directly in the activation of the nerve cell or may be important
indirectly in processes such as the conversion of testosterone to estra-
diol or in carbohydrate metabolism.

One obvious class of anabolic products needed by the nervous system
for successful operation is neurotransmitter material. In the present
working hypothesis it is suggested that testosterone restores the ability
of the nerve cells to function by increasing the synthesis and avail-

ability of neurotransmitters.

T...

NERVE CELL

 

  
  
 

 

'9‘Hydroxylase
R ._. T-R r ‘ A E-R
\ \\ //
\\\
\ /

adenyl cyclase

 

Neurotransmitter .—
(N'U
Synthesis

 

fl

 

N'T

Figure l. A working hypothesis of possible biochemical mechanisms involved

in testosterone activation of the nervous system. In this working
hypothesis testosterone (T) bound to an intracellular receptor (R)
is converted to an estrogen (E). The estrogen then has effects on
anabolic processes the products of which may be important in stim-
ulating the nerve cell (ie. neurotransmitters). CycliCAMP is also
involved in this hypothesis either directly or indirectly.

u
The experiments reported here concern three aspects of this

working hypothesis outlined above. Experiments I and II test the
hypothesis that conversion of testosterone to an estrogen in the POA
of castrated male rats is necessary for the resumption of mating
behavior. Experiment III tests the hypothesis that CAMP acts as an
intracellular mediator of testosterone in activating the resumption
of masculine sexual behavior. Experiment IV tests the hypothesis that
within the POA the synthesis or presence of two neurotransmitters,
norepinephrine and acetylcholine, are essential for the display of

masculine mating behavior.

BACKGROUND

Sexual behavior is essential for successful reproduction and species
survival in all vertebrate species. Through the use of various species
specific signals (visual, olfactory and auditory) the process of mate
selection insures that prospective participants are of the same Species
and are in mature reproductive condition. Other mechanisms involved in
the mechanics of copulation are also important in isolating undesirable
mates as well as activating specific physiological events necessary for
reproductive success. For example, hormonal changes in the female ring
dove in response to visual cues from the male integrate her behavior with
that of the male (Lott, Scholz and Lehrman 1967); intromissions in the
rat determine hormonal changes in the female necessary for implantation
success (Adler, Resko and Goy 1970) and also trigger ovulation in cats
and rabbits (Heape 1905). Thus, sexual behavior, from an evolutionary
standpoint, is important in that it serves as an isolating mechanism
valuable for the preservation of species identity. Likewise, the mecha-
nics of courtship and copulation are important in activating physio-
logical responses necessary for the propogation of offspring and hence
the species.

The study of sexual behavior in laboratory mammals (ie. rats, ham-
sters, guinea pigs and mice) has proven extremely productive, largely
due to their ease in domestication and ease in handling. In the following
sections a description of female as well as male sexual behavior in the

laboratory rat is presented to provide a wider reference base to which

the present research must be related.

6
Hormonal Concomitants of Female Sexual Behavior

Many mammals (eg. deer, wolves, prairie dogs and bear) engage in
mating behavior only during specific periods of the year (seasonal
breeders), whereas others are sexually active throughout the entire year
(eg. rabbit, hamster and man). Males of species sexually active through—
out the entire year will generally mate on any occasion with a receptive
female. However, females of nonseasonal species are only receptive during
a particular phase of the estrous cycle. The estrous cycle of the labora-
tory rat for instance, is usually four days with the period of behavioral
heat or sexual receptivity, lasting about 12 hours. The onset of heat
occurs just prior to ovulation, and is a result of two days exposure to
estrogen and a surge of progesterone on the night of proestrus (Bar-
raclough, Collu, Mussa and Martini 1971, Feder, Resko and Goy 1968).

Thus, sexual behavior in the intact female rat is dependent upon the
ovarian release of estrogen and progesterone. The removal of the ovary
abolishes sexual behavior (Ball 1936, Beach l9u2). However, sexual
receptivity may be induced in ovariectomized female rats by injections
of estrogen and progesterone (Beach 19u2).

Two doses of estradiol benzoate (4 ug) given 24 hours apart will
induce estrus when synergized with an injection of progesterone (500 pg),
given 24 hours after the second estrogen injection. One to four hours
after progesterone treatment the female will reSpond sexually to the
male.~ Estradiol benzoate will, by itself, induce receptivity if admin-
istered daily for a week or more (Davidson, Rodgers, Smith and Bloch
1968). However, high estrogen levels in the intact cycling female are
never maintained for long periods, hence, the surge of progesteroneis needed

(Powers 1970). The mechanisms involved in estrogen priming and proges-

(IT

()1

,-

terone synergism are unknown.

Female Sexual ReSponses

 

Sexual behavior in the female rat consists of two main components
1) hopping and darting and 2) presentation or lordosis. The hopping and
darting component of receptive behavior is believed to be involved in
stimulating the male to mount (Coniglio and Clemens 1972). Hopping
and darting consists of a short rapid run (1-3 feet), with the female
usually coming to an abrupt halt in a crouch. A sexually active male
will usually follow Closely during the run and mount the female when she
stops. During this quick run, short hops may be exhibited, especially
at the beginning or end.

Lordosis usually occurs at the end of a run in response to the
male's mount. The lordotic response consists of a concave arching of
the back; the head and rump being raised with respect to the back. The
complete response, with extreme curvature of the back, will only occur
when the female is mounted by a male. However, slight lordotic responses
may be exhibited during investigative sniffing or licking of the perineum
by the male.

Lordosis in the rat is not limited to the female. Estrogen primed
males will also display the lordosis posture when mounted (Beach 1938,
19u5), although the amount of estradiol needed to prime the male is much

greater than in the case of the female (Davidson 1969).

Masculine Sexual Pattern

 

In general, three separate components of male sexual behavior can

be recognized in the rat: 1) nuzzling and chasing the female 2) mount

8
bouts and 3) intermount bout grooming. The nuzzling and Chasing consists
of anogenital licking and rooting of the female. This activity usually
elicits a short run by the receptive female. When the female ends her
run, the sexually vigorous male may engage in any one of three mount
bout activities: 1) mount (without intromission), 2) intromission
(mount with vaginal penetration) or 3) ejaculation. A mount bout is
defined as a sequence of mounts (one or more) with or without intro-
mission, uninterrupted by any behavior that is not oriented toward the
female or the male's own genitalia (Sachs and Barfield 1970). Each of
the three possible activities occurring in a mount bout are behaviorally
identifiable and easily distinguished by the experienced observer.

During a mount, the male, approaching from the rear of the female,
Clasps his forelegs around the female's laterolumbar region, in what is
called, a mount without palpation. While clasping the female, the male
may palpate her sides with rapid movements of his forelimbs and simul-
taneously move his pelvic region in rapid piston-like thrusts (mount
with palpation and thrusting). The male then slips off the female
rather weakly. This type of dismount invariably signifies there was no
vaginal penetration or "intromission” (Beach l9u4). In the intromission
pattern the palpation with thrusting does occur, but in addition, a final
and forceful thrust is observed during which time vaginal penetration
is achieved. Stone and Ferguson (19HO) estimated from film studies that
vaginal intromission lasts 1/3 to 2/3 of a second during which time 2—9
pelvic thrusts occur. Vaginal penetration is terminated after this short
period with a "forceful backward lunge which carries the male several
inches from the female" (Young 1961). If an ejaculation accompanies the

intromission, the backward lunge is absent. Instead, the male stays in

contact with the female and slowly raises his front paws laterally until
almost in an upright position. The ejaculate is eXpelled during this
response. In castrated males given TP replacement, sperm are absent but
the gross motor responses still occur. The penis is withdrawn following
ejaculation and the male lowers himself to all fours.

Ejaculation is usually preceded by several mounts (without intro-
mission) and 3 to 14 intromissions (Young 1961). The sequence of mounts
and intromissions preceding an ejaculation is termed an ejaculatory
series. Several ejaculatory series may be achieved by one male in a
single 20 minute test. Several time dependent measures are commonly
used in quantifying masculine sexual behavior. Mount and intromission
latencies is the time measured in seconds from the introduction of a
stimulus female into the testing area, until the occurrence of the first
mount and intromission, respectively. The ejaculation latency is defined
as the time from the first intromission in a series until the occurrence
of the ejaculatory pattern. Mean Inter—Intromission—Interval (MIII),
gives the mean time between intromissions and is calculated by dividing
the ejaculation latency by the number of intromissions preceding the
ejaculation. Following an ejaculation a period of sexual inactivity is
seen, lasting 5—10 minutes. This period is termed the Post Ejaculatory
Period (PEI) and is defined as the time from the ejaculatory response
until the next intromission. During the PEI the male is not influenced
by sexual stimuli (Larsson 1956). The length of the PEI increases with
each successive ejaculatory reSponse. After 3—10 ejaculatory series the
PEI may extend over 30 minutes (an arbitrary criterion frequently used
for defining sexual exhaustion or satiation) (Beach and Jordon 1956).

Beach and Jordon (1956) also reported Changes in behavior preceding

10

each successive ejaculation. The number of intromissions in an ejacula-
tory series decreased from an average of 10.6 intromissions in the first
ejaculatory series to 4.1 in the sixth. Ejaculation latency (here
measured in seconds as the interval from the first mount to ejaculation)
decreased from a mean of 450 to 130 seconds. The PEI increased from an
average after the first ejaculation of 324 to 818 seconds after the 6th
ejaculation. Sexual satiety was reached after 3—10 ejaculations, and

a recovery period of 24 hours or more usually followed, although effects

of sexual satiation could sometimes be noticed up to 7-8 days later in

some measures .

Hormonal Control of Masculine Mating Behavior

The development and display of masculine sexual behavior depends
upon the presence of testosterone during two critical periods in the
life of a rodent; once very early in life, near the time of sexual
differentiation, and again as an adult (postpuberal). In the absence
of testosterone exposure during either period, an animal will exhibit
very little adult sexual behaivor.

An hypothesis first expounded by Young and his co—workers (Phoenix,
Goy, Gerall and Young 1959) to eXplain the sexual differentiation of
male copulatory behavior, emphasized the necessity of testosterone
exposure during early development. When the developing organ systems
(genital and neural) of the young male animal are eXposed to testos—
terone normally secreted from its own testes during sexual differen-
tiation, these organ systems develop the capacity for male sexual behavior
to be exhibited subsequent to testosterone exposure in adulthood. On the

other hand, if testosterone is not present during sexual differentiation,

11

as is the case of genetic females, the animal will not exhibit masculine
sexual behavior as an adult, even if given massive doses of testosterone.

The necessity of testosterone for the development and display of
masculine sexual behavior in rodents has been well documented. When
male hamsters (Swanson 1970, Coniglio, Paup and Clemens 1973) and rats
(Beach and Holtz 1946) were castrated on the day of birth, they exhibi-
ted less male copulatory responses in adulthood than adult castrates
even though both groups were given large doses of testosterone pro—
pionate (TP). Conversely, when female rats (Gerall and Ward 1966),
guinea pigs (Phoenix, Goy, Gerall and Young 1959), hamsters (Eaton 1970)
or mice (Edwards and Burge 1971) were eXposed to testosterone propionate
during the early critical period (the first 5 days after birth for the
mouse, rat and hamster, and days 30-35 of gestation for the guinea pig)
they displayed an increased frequency of masculine copulatory behavior
when injected with TP postpuberally.

Thus, exposure to testosterone during early development sensitizes
'the animal (be it genetic male or female) so that when exposed to testos-
txerone in adulthood the animal will display masculine mating patterns in
éippropriate stimulus conditions. Likewise, low levels of male mating

INESponses will be exhibited if testosterone is absent perinatally or

<fllxring testing postpuberally.

IEEELEEQ—hormonal Control of Adult Male Sexual Behavior
Ne uI‘al Control:

Results of several studies point to the preoptic area (POA) of the
1Drain as an important neural site for testosterone action in the adult

"Ha:L€3 rat. Fisher (1955), Davidson (1966), Lisk (1967) and Johnston and

l2
Davidson (1972) have reported increases in the sexual behavior of
castrated male rats when pellets of TP were implanted into the POA.
Autoradiographic techniques have also identified the POA as a neural
site which concentrated large amounts of radioactivity when tritiated
testosterone was given to castrated male rats (Pfaff 1968, Resko et a1
1967, Sar and Stumpf 1972). Lesion and stimulation experiments have
also implicated the POA in the control of masculine sexual behavior.
Lesions which interfere with the median forebrain bundle (MFB) disrupt
male sexual behavior in the adult rat (Hitt, Hendrics, Ginsburg and
Harris 1970, Rogers and Law 1967). Likewise, lesions which destroyed
the anterior portion of the POA, which is anatomically related to the
MFB, disrupted male sexual behavior (Singer 1968). Electrical stimulation
of the POA, on the other hand, accelerated masculine copulatory perfor-

mance (Caggiula and Szechtman 1972, Malsbury 1971).

Hormonal control:

Studies investigating the influence of testosterone on ventral
tprostate and seminal vesicles suggest that testosterone serves as a
"pre—hormone" for these tissues. That is to say, testosterone undergoes
iJatracellular metabolism before many of the physiological responses
éussociated with testosterone action are initiated. In these tissues,
‘teestosterone is converted to a reduced product, 5 -dihydrotestosterone
(IDFKF) (5 -androstan-l7 -ol-3—one) after being transported into the cell
(136n11ieu and Lasnitzki 1968, Bruchovsky and Wilson 1968). The intra—

Cellular DHT is then bound to the nucleus along with a protein receptor,
ESpecific for the DHT configuration. At the nucleus the receptor-DHT

<3<3TDE>1ex presumably initiates synthetic processes involving DNA-RNA

13

transcription, which eventually leads to the observed physiological
responses (Main—Waring and Mangon 1970).

Similar processes may be involved in androgen stimulation of the ner-
vous system. Testosterone bound at the plasma membrane to a protein recep-
to would be taken into the cell and metabolised to the effective configur—
ation. In fact, the conversion of testosterone to DHT was demonstrated
in slices of brain tissue (Jaffe 1969, Kniewald, Mussa and Martini 1971).
DHT was also effective in inhibiting gonadotropin release from the pitu—
itary of rats (Peder 1971). In the same study, Feder showed an accum-
ulation of radioactivity in the brain following administration of triti—
ated DHT. However, DHT failed to induce masculine sexual behavior, either
when given systemically (McDonald et a1 1970, Feder 1971, Whalen and Luttge
1971) or intrahypothalamically (Johnston and Davidson 1972) to castrated
male rats. Therefore, while DHT may be an important metabolite of testos—
terone in the stimulation of systemic tissues or regulation of gonadotro-
pin activity, it is less involved in the control of masculine sexual behav-
ior.

A number of investigators have suggested testosterone aromatization
to an estrogen as a possible step in the hormonal stimulation of male
copulatory responses (Young 1961, McDonald et al 1972, Beyer and Komisar-
ak 1971). Data from several behavioral experiments lend support to the
necessity of testosterone aromatization. Non—aromatizible androgens (DHT
and androsterone) failed to stimulate masculine sexual behavior in the
castrated male rat (McDonald et alil970, Whalen and Luttge 1971) or hamster
(Christensen, Coniglio, Paup and Clemens 1973). Furthermore, small quant-
ities of estradiol benzoate (10 ug/day) were more effective than oil in
inducing mounting and intromissions behavior in long-term castrated rats

(Pfaff 1970). Systemically injected estradiol benzoate was also more

14

effective than oil, but less effective than testosterone, in prolonging
the ejaculatory pattern in castrated male rats (Davidson 1969). Support
for this concept of hormone conversion also comes from work in which
androstenedione, a precursor to testosterone, was aromatized to estrone
in the diecephalon of adult male rats (Naftolin, Ryan and Petro 1972).
It thus appears as though.androgens, at least those which may be aroma-

tized to estrogens, or estrogens themselves are effective in stimulating

masculine sexual behavior.

Aromatization process:

The proposed biochemical pathway of gonadal steroid synthesis is illus-
trated in Figure 2. The conversion of either androstenedione or testos-
terone to estrone or estradiol respectively, involves the hydroxylation
of the C-19 carbon and aromatization of the A ring (removal of 1d and 2a
hydrogens) (Townsley and Brodie 1968, Brodie, Kripalani and Possanza 1969).
Although the aromatization process has not been completely delineated,
the hydroxylation and oxidation steps appear to be temporally separate
(Brodie et al., 1969). Presummably, the conversion process occurs intra—
cellularly at the level of the POA. In a study by Naftolin and co-workers
(1972) they reported a .17% conversion of androstenedione to estrone in
the diencephalon of adult male rats. At this conversion rate, one would
expect that if estradiol were the active metabolite, it would have to
be more potent than testosterone for inducing male behavior.

Thus a summary of the "aromatization hypothesis" suggests that testos-
terone undergoes C-19 hydroxylation and aromatization to an estrogen form,
presumably estradiol. Estradiol, therefore, would be the steroid affecting
intracellular anabolic processes involved in restoring masculine behavior.

One way of testing this hypothesis would be to block the conversion of

15

 

 

l
. l7-llydroxy.
progesterone

   

‘_———

   

.14- -\ndrostcnco

uo’ . ° 3J7.mnm no \\\
I
" Est'cne
Androstcronc
' I
‘ 0“ on

a.—
Testosterone

on
' |
[
5 I
:I

. 17,3-listradlol
Dihydrotestosterone

‘

Figure 2. A hypothesized pathway for the aromatization of androgen to
estrogen. In the aromatization process two mechanisms are involved:
1) the removal of two hydrogens from ring A and 2) the aromatization
of ring A.

16

testosterone to estradiol.
Biochemical mechanisms involved in sexual behavior:

Little information is available on how steroid hormones activate
nervous tissue in the restoration of masculine sexual behavior. However,
recent studies of testosterone action on seminal vesicles and ventral pros-
tate in rats (Singhal, Parulekas, Vijayvargiya and Robinson 1971) indicate
that the nucleotide 3'5'—cyclic adenosine monophosphate (CAMP) may function
as an intracellular mediator for testosterone. When testosterone was admin—
istered to castrated male rats, increases of CAMP were detected in the
seminal vesicles and ventral prostate gland. Likewise, when CAMP was injected
into castrated male rats it increased certain carbohydrate metabolizing
enzymes which are also increased by testosterone administration. Presumably,
a build-up in these enzymes increases the available stores of energy needed
by the cell for testosterone-induced functions. In the same study, effects
of submaximal doses of testosterone were potentiated by simultaneous inject—
ions of theophylline, an inhibitor of phosphodiesterase (the catabolic
enzyme for deactivating CAMP). Theophylline plus submaximal doses of
testosterone resulted in maximal dose responses (ie. increased enzyme levels).
Inhibition of phosphodiesterase by theophylline presumably prevented the
degredation of small amounts of CAMP activated by low levels of the steroid.
The subsequent build-up in CAMP resulted in intracellular responses normally
associated with maximal testosterone stimulation.

The possibility of CAMP being involved in the regulation of masculine
sexual behavior was demonstrated in recent experiments by Christensen and
Clemens (in press). Submaximal doses of testosterone propionate (TP) (Sug/
day) were ineffective in maintaining sexual behavior of castrated male rats.
However, when this same dosage was administered to castrated males in com—

bination with 10mg/day of theophylline, a significant maintenance of

17

intromission and ejaculatory patterns was achieved. Similarly, 25ug of TP
injected daily was ineffective in restoring sexual behavior of 6 long term
castrated male rats. However, this dose of TP, when injected in combin-
ation with 10 mg of theophylline daily, restored the ejaculatory response
in 60% of the castrated males. Intromission and mounting patterns were
restored in 80% of the animals receiving TP and theOphylline. Genital
stimulation (measured as the number of penile spines and the length of
penile papillae) was similar in the TP + saline and TP + theophylline
groups, suggesting potentiation of testosterone effects in the central
nervous system as a possible explanation of the observed behavioral dif-
ferences rather than effects of these compounds upon genital morphology.

Cyclic-AMP may be involved in the regulation of masculine sexual behav-
ior by affecting 1) all the intracellular biochemical events necessary
for nerve cell activation or 2) specific aspects of testosterone activa—
tion (eg. stimulating enzymes needed for intracellular steroid or carbo-
hydrate metabolism).

The brain has the highest capacity to make CAMP of any mammalian tis-
sue (Sutherland, Rall and Menon 1962). Various neurohumors have been shown
to increase the concentration of CAMP when applied to in vitro nerve prep-
arations: histamine (Kakiuchi and Rall 1968), dopamine (Kebabian and Green-
gard 1971), norepinephrine (Huang, Shimizu and Daly 1971) and serotonin
(Kakiuchi and Rall 1968). However, no definitive role of CAMP has emerged
in the regulation of behavior. In general, high doses of dibutyryl-CAMP
(Db-CAMP), a butyrized form of CAMP apparently resistant to metabolic inact—
ivation, when injected into some cerebral areas, may induce general motor
hyperactivity and convulsions (Gessa, Krishna, Forn Tagliamonte and Brodie
1970), hyperphagia, hyperthermia and prolonged estrous cycle (Breckenridge

and Lisk 1969).

18

Studies carried out in our laboratory (unpublished) and by Brecken—
ridge and Lisk (1969), suggest that CAMP is not involved in the activation
of female receptivity. Likewise, direct application of CAMP or Db-aCMP
to the anterior hypothalamus (a site for estrogen action) failed to stimulate
increases in female receptivity (Breckenridge and Lisk 1969). Some of
the possible roles for CAMP in testosterone stimulation may be: to increase
the available energy via enhanced carbohydrate metabolism, to increase
intracellular transport of synthesized products (ie. axoplasmic flow
of neurotransmitters), or to increase the C—19 hydroxilase needed for the

aromatization of testosterone.

Pharmacological Control of Masculine Sexual Behavior

Throughout the past decade, several neurotransmitters have been iso—
lated in the vesicular fraction of brain homogenates: acetylcholine (Ach),
serotonin, norepinephrine (NE), dopamine and histamine. Several studies
utilizing systemically administered substances have implicated some of
these transmitter substances in the control of masculine sexual behavior.
Dewsbury and Davis (1970) have suggested that high levels of biogenic
amines in the brain may retard COpulatory behavior in the male rat. Such
a hypothesis is consistent with the facilitory effects of monoamine depletors
such as reserpine (Soulairac and Soulairac 1961, Soulairac 1963, Dewsbury
and Davis 1970, and Dewsbury 1971a), tetrabenazine (Dewsbury1971b) and p-
Chlorophenylalanine (Shillito 1969, Sheard 1969, Tagliamonte, Tagiamonte,
Gessa and Brodie 1969, Gessa 1970 and Salis and Dewsbury 1971). However,
systemic administration of these drugs is known to affect the peripheral
as well as the central nervous system. Therefore, systemic administration
does not allow conclusions regarding behavioral effects in relation to

Changes in brain neurotransmitter levels. Likewise, general nonspecific

19

increases in neurohumoral agents throughout the entire brain offer little
insight into what areas of the brain'may be responsible for the observed
effects. Experiments.which alter the levels of neurotransmitters in loc—
alized regions of the brain are needed.

Fuxe (1965) has demonstrated through the use of histochemical fluo-
escence analysis that the POA has a high concentration of NE nerve fibers
and cell bodies. Since the POA has been shown to be involved in sexual
behavior, these data suggest that NE-containing nerve pathways may be
involved in controlling masculine sexual behavior. Another line of
evidence suggests that Ach-containing nerve pathways may be involved in
the regulation of sexual responses. In studies conducted in this labor-
atory (Christensen and Clemens unpublished observation) carbamylcholine
(carbachol), a sympathomimetic, when applied directly to the mesen—
cephalic reticular formation of estrogen—primed female rats, greatly
increased the number of lordoses. These results warranted investigation
as to whether Ach was also involved in the control of masculine sexual
behavior.

Following these lines of evidence, preliminary studies were carried
out to investigate adrenergic and Cholinergic control of masculine sexual
behavior (Humphrys, Christensen and Clemens 1972). These preliminary
studies utilized direct unilateral application of NE or carbachol to the
POA. The effects of these treatments on sexual behavior suggested that
cholinergic mechanisms may be involved in the inhibition of masculine

copulatory behavior, while the control by adrenergic systems was less Clear.

 

 

 

1.11

2O
ObJectives of the Present Study

The investigation reported in this dissertation was divided into three

parts: A, B and C.

Part A. Investigation of the hypothesis that testosterone is converted
to an estrogenic form in the activation of masculine sexual behavior.
Experiment I: The object of this experiment was to determine if esta—
diol is as effective as testosterone in inducing the resumption of mas-
culine sexual behavior in long—term castrated rats. Pellets of crystal-
line estradiol or testosterone were applied directly to an area of the
brain (POA) known to be responsive to testosterone implants. The data
from this experiment provided information on the relative strength of the
two steroids when applied directly to the brain. In addition, data on
the importance of steroid stimulation of penile morphology for the resump-
tion of copulatory behavior were collected.
Experiment II: The objective of this experiment was to determine
if direct application of metapirone, a compound known to inhibit the con-
version of testosterone to estradiol, to the POA of castrated male rats

would block the activation of masculine sexual behavior by testosterone.

Part B. An investigation into the possible involvement of CAMP in the
activation of masculine sexual behavior.

EXperiment III: This experiment was designed to determine if direct
application of CAMP or Db-CAMP to the POA of long—term castrated rats,
would mimic the effects of testosterone in the resumption of copulatory
behavior. This experiment also provided information relavant to the question

of whether POA applied CAMP potentiates the behavioral effects of submaximal

doses of testosterone.

21

Part C: An investigation into the neuropharmacological control of male
COpulatory behavior.

Experiment IV: In this study various Chemicals designed to increase
or decrease the level of adrenergic and cholinergic activity, were applied
directly to the POA. The affect of these manipulations on the various

parameters of masculine sexual behavior were measured.

22

EXPERIMENT I. DETERMINE IF ESTRADIOL IS AS EFFECTIVE AS TESTOSTERONE FOR
INDUCING THE RESUMPTION OF MASCULINE SEXUAL BEHAVIOR.
Experiment I was divided into two parts (A88). In Part A an area
was located where testosterone implants would reactivate copulation in
castrated male rats. In Part B estradiol was implanted in the same area

to determine its effectiveness in stimulating masculine sexual behavior.

Part A. Determine a site of testosterone action.

Methods: Thirty male rats were used (see Appendix I, II and III for detailed
methods for all experiments). All animals were selected on the basis

of a screening test for sexual behavior and subsequently castrated and
allowed to rest for 3 weeks. At the end of 3 weeks each subject was tested
weekly for sexual behavior until they achieved a criterion of 3 consecu—
tive tests without an ejaculatory response. Upon meeting this criterion
all animals were bilaterally implanted with double-walled stainless steel
cannula (26 gauge inserts) which permitted intracerebral treatment with
crystalline hormones. Of the 30 males, 20 were implanted so the cannulae
tips rested on the POA. The remaining 10 animals were implanted so the
tips of the cannulae rested in the posterior hypothalamic area (PHA). The
methods of implantation are further detailed in Appendix.

Of the 20 males implanted in the POA, 10 received intracerebral treat-
ment (ICT) of testosterone and the other 10 received Cholesterol ICT. For
the 10 animals with cannulae in the PHA, ICT consisted of testosterone.
Each animal received one pellet of the respective steroid in each cannula

O,3,6,9 and 12 days after implantation. Thus each animal received a total

of 10 pellets (5 on each side). (See Appendix II-a,b). The weight of a

single pellet for each steroid used was 15 ug. All subjects were tested

23

for masculine sexual behavior 7, 10 and 13 days following the onset of
treatment.

Twenty four hours after the last test, all animals were perfused and
the brains and penises were prepared for histological examination. The
location of the implants was determined and the number of penile spines
and length of penile papillae were measured. Johnston and Davidson (1972)
and Christensen and Clemens (in press) have shown that the development of
these penile structures is a more sensitive index of Circulating testos-
terone than is seminal vesicle weight or masculine sexual behavior. See
Appendix III for histology.

Unless otherwise stated data from tests in this experiment and all
subsequent experiments were analyzed using an analysis of variance F test
for repeated measures (Winer 1962). The F statistic is given in the text
of the result section along with the cofiparative F value and the .05 level
of significance used. The degrees of freedom for the numerator and denom-
inator of the calculated F ratio are also included with the comparative F
statistic (ie. F.05,2,27)'

Results:

Masculine sexual behavior: As sumarized in Table 1 and Figure 3,
intracerebral applications of testosterone into the POA of long term cas—
trated rats were significantly more effective than treatments 6f testoster-
one in the PHA or Cholesterol in the POA for stimulating increases in mounts,
intromissions and ejaculations. An analysis of variance for repeated
measures (Winer 1962) indicated a significant difference among the three
treatment groups over the entire experiment (F=6.l2>F 05’2’”) and mean
intromission frequency (F=3.85>F 0532,27). In addition, a G statistic (a
variation of X2) indicated a significant difference among the treatment

groups in number of animals exhibiting the ejaculatory response (G=10.6O >

24

Figure 3. Represented for the three treatment groups are the mean number
of mounts and intromissions t SE as well as percentage of animals
ejaculating. Animals receiving intracerebral applicaitons of testos-
terone to the POA exhibited significantly more mounts and intromis—
sions as well as having a greater percentage of animals ejaculating
when compared to animals treated with testosterone in the PHA or ani—
mals receiving Cholesterol in the POA.

MEAN MOUNT FREQUENCY

INTRO. FREQUENCY

MEAN

R.)
U!

N6
8

O

% EJACULATI

 

 

Ui

    

 

 

 

 

 

 

 

 

 

 

 

T(POA)

.' . . gT( PHA)
. .................... . ::-.:-..-.:':.'..'.::.:::: a POA)

I 3

 

-2l -I4 '7

O 7 to
PRE AND POST IMPLANT TESTS

Figure 3

.J‘
new.

26

Table 1. Effects of intracerebrally implanted testosterone or cholesterol
on parameters of masculine sexual behavior.

 

Test Days Testosterone Cholesterol Testosterone
from (POA) (POA) (PHA)
Implantation 'R'i SE E': SE 3': SE
—21 0.6 1 0.43 4.8 t 3.62 3.1 t 2.57
~14 4.2 i 2.43 6.2 i 4.39 0.0
Mean
—7 1.8 i 1.06 0.0 0.0
Mount
7 9.9 t 4.09 0.0 0.0
Frequency
10 8.3 t 3.90 0.0 0.0
13 4.7 i 3.77 0.0 1.8 i 1.8
—21 0.0 1.3 i 1.3 0.8 t 0.8
—14 0.2 i 0 2 0.4 i 0.4 0.0
Mean
-7 0 0 0.0 0 0
Intromission
7 3 3 i 1.87 0 0 0 0
Frequency ,‘
10 2.7 f 1.75 0.0 0.0
13 3.0 i 2.03 0.0 0.8 t 0.8
-21 O 0 O
-14 0 0 0
Percent —7 0 0 0
Ejaculating 7 20 O 0
10 10 O O

13 33 0 0

(D

27

A priori comparisons on differences between groups in the mean number
of mounts indicated significantly more mounts were exhibited by the Testos-
terone (POA) group than by the Testosterone (PHA) group (F=10.04>F 05’1’”).
Application of testosterone to the POA was also more effective in stim—
ulating mounts than Cholesterol (POA) (p<.05 by Duncan's new multiple
range test [DNMR]). Testosterone, when placed in the PHA was no more
effective than Cholesterol in the POA in inducing mounting behavior (F<1).

Animals treated with testosterone in the POA were the only subjects
to increase their intromission frequency scores over their pretest level
(t=l.83>t.05’9 by a paired students t test). The Testosterone (POA) group
achieved more intromissions than Testosterone (PHA) group (an'23>F.05,1,27)
or the Cholesterol (POA) group (F=7.54>F 05,1927). There was no signifi—
cant difference between the Testosterone (PHA) group or Cholesterol (POA)
group (p>,05 DNMR),in the mean number of intromissions achieved.

Testosterone in the POA significantly increased the percentage of
animals ejaculating when compared to the testosterone in the PHA and

Cholesterol in the POA (G=5.45>X2 ).

05,2

Part B. Determine if estradiol is as effective as testosterone when implanted

in the same area.

 

Methods: Twenty sexually vigorous male rats were castrated and allowed

to rest for three weeks. At the end of 3 weeks, all subjects were tested
weekly for sexual behavior until the 3 test criterion for the loss of sexual
behavior was met. All animals were then implanted bilaterally with double-
walled stainless steel cannulae (21 gauge guide cannulae and 26 gauge inserts).
Ten males were implanted with cannulae located in the POA and the remaining

ten with cannulae in the PHAJ

28

Beginning on the day of implantation all animals received ICT of
estradiol (10ug per cannula) every 3 days. Thus each animal received
one 10 ug pellet of estradiol through each cannula 0,3,6,9 and 12 days
after implantation. Each animal received a total of 10 pellets. All sub—
jects were tested for masculine sexual behavior 7,10 and 13 days following
the onset of treatment.

At the conclusion of testing all animals were perfused and the brains
and penises were prepared for histological examination. The location of

the implants were determined and the number of penile spines and length

of penile papillae were measured.

Results:

Masculine sexual behavior: Intracerebral applications of estradiol
into the POA were effective in inducing mounting. intromitting and ejacu—
latory responses in long term castrated rats Figure 4 and Table 2. Estra-
diol when placed in the POA was significantly more effective than estradiol

in the PHA in restoring mount (F=8.19>F 05 1 27), intromission (F=12.37>
° 9 3

. 2_ 2 .
F 05’1’”) and ejaculatory responses (X - 9.51>X .05’1). Estrad1ol when

placed in the PHA was not significantly more effective than Cholesterol
in the POA for the induction of any of the sexual behavioral patterns (p<
.05 in mean mounts and intromissions as well as percent ejaculating).

In order to evaluate the effectiveness of estradiol and testosterone
as stimulatory agents of COpulatory behavior the data from Parts A and B
were analyzed together. It was found that the smaller amounts of estradiol
placed in the POA were more effective than the larger implants of testos-
terone in restoring mount, intromission and ejaculatory reSponses. Analy-
sis of variance utilizing all groups revealed that applying estradiol to

the POA was significantly more effective than testosterone in stimulating

\-

 

 

 

:pexantra
. r____ _ -. __-, l

P 9Q IIIM asaqL

—u__

m

29

Figure H. Represented for the three treatment groups are the mean number
of mounts and intromissions i SE as well as percentage of animals
ejaculating in tests for sexual behaivor. Estradiol when placed in
the POA was significantly more effective than estradiol placed in the
PHA or cholesterol in the POA, in reinstateing mount, intromission
and ejaculatory responses in long term castrated male rats.

MEAN MOUNT FREQUENCY

FREQUENCY

INTRO.

MEAN

% EJACULATING

70

60

50

40

30'

20

IO

 

    

   

 

 

 

 

 

 

 

1”
.. \ , ,
~§§~ ..---.I'
Q . -
IIOOOOOOOOO 2%: 0.0.0.0... .000 ....... I

   

 

  

lun132nnmuun I
3 ooooooooolooooooooooi ~ 0.000000... C (PO A)
- '21 - l A - 7 O 7 l O I 3

PRE AND POST |MPLANT TESTS

Figure u.

"m a“ {hutw‘ . "V - I

W‘ .-a

Table 2. Effects of implanted estradiol or cholesterol on masculine sexual
behavior.

 

Test Days Estradiol Estradiol Cholesterol
from (POA) (pHA) (POA)
Implantation ‘i t SE 2' t SE E' i SE
-21 4.9 t 2.77 0.3 i 0.2 4.8 i 3.62
-14 3.0 t 1.74 1.5 t 0.93 6.2 i 4.39
Mean
—7 1.6 t 1.01 0.0 , 0.0
Mount
7 22.0 i 6.66 6.7 t 4.40 0.0
Frequency
10 24.5 t 5.37 12.3 t 5.11 0.0
13 29.4 i 6.0 10.6 i 6.67 0.0
-21 9.1 i 0 1 0.0 1 3 i 1.3
-14 0.0 0.0 0.4 i 0.4
Mean
—7 0 2 t 0.2 0 0 0 0
Intromission
7 5 0 t 2.01 1 0 i 0.1 0.0
Frequency
10 9.7 t 3.10 0.9 i 0.64 0.0
13 10.1 t 2.34 2.5 i 1.36 0.0
—21 00 00 00
—14 00 00 00
PerCent -7 00 00 00
E57 a Culating 7 20 10 00
10 30 00 00

13 66 10 00

32

= 5.2> ° ° ' = .
mean number of mounts (F 1 F.OS,1,27) and intromiSSions (F 9 87>F 05’1927)
per test. Estradiol (POA) was also more effective than testosterone (POA)
in restoring the percent of tests during which ejaculation was achieved

2

although this difference Was not significant (X222.36<X )

.OS,1 ‘

Histological results: The location of the various steroid implants
for both Parts A and B is represented in Figures 5 and 6 (POA) and Figures
7 and 8 (PHA). A statistical analysis of the anterior, vertical and lateral
coordinates (according to Pellegreno's (1965) atlas) indicates no signif-
icant differences in the mean placement site between the 3 FDA treated
groups (1 way ANOVA p<.05 in all cases) or likewise between the 2 PHA implanted
groups. Through the use of regression analysis the coordinates of maximum
effectiveness were: estradiol; anterior 7.22, vertical 1.36, lateral 0.74
and testosterone; anterior 7.66, vertical 0.89, lateral 0.88. Comparison
of the percent of preoptic nucleus destroyed at the point of maximum pene-
tration using a plane planimeter, indicated there was no difference between
the Testosterone (POA), Estradiol (POA) or Cholesterol (POA) groups in the
percent of preoptic nucleus destroyed (F<1). Likewise, using regression
analysis a coefficient of correlation was calculated for all the POA implanted
groups using the percent of preoptic nucleus destroyed as the independent
variable and mean mount score for the three post implant tests as the depen-
dent variable.. The coefficients are: Testosterone (POA) r=-.u7, Estradiol
(POA) r=-.59 and cholesterol (POA) r=0.

A gross microscopic examination of the penises from all groups, revealed
the two testosterone implanted groups (POA and PHA) to be the only males
exhibiting significant penile development (Table 3). The two testosterone

treatment groups were not different from each other (p>.OS DNMR). Likewise,

no significant differences in penile spines or papillae were detected between

33

 

’7

0 =testosterone )

A =estradiol

0 =eholesterol \ a ‘
(3'13 I3=no sexual behavior fl ~H)1\ 611 u a. Z:}\ .. '31
O . . . \\ ‘~,__. ' .. .. .'
[:1 Qg=introm1551on observed .' yaw] '/\\\—,991J/"c ”-gp ‘ x

E @ejaculation observed W .

F ' . .
léngzREE 5. Locations of maximum FOA cannulae

Elbe represented in cross sectional maps

penitration in Experiment I
(Pellegrino et a1 1967).

 

34

:3

 

 

 

©m>ummno coflumazumhwug SE
vw>ummno newmmweouucwu®@©
xaco wcwucsoEnl t O
uoH>mswn Hmsxmm ocno d O

H0pmummaozun o.

D HOHumfiummu a
accumumOuwmuu o

 

 

The locations of POA implants in Experiment I are represented

Figure 6.

in this longitudinal map (Able—Fessard et a1 1067).

Ho

35

 

 

testosterone
,- A =estradiol

   

=no conulation
=mounting only
=intromission observed
=ejaeulation observed

II
\
fifléDIICJ
sa>>

The locations of maximum PHA cannulae penitration in Experiment

gure 7.
represented in these cross sectional maps (Pellegrino et al 1967).

I are

 

vu>uomno cowumHsommwu
ww>pmmbo scamm«50pucwu
maco mcwucsoen
cowumanmoo ocn

Howwmuummu
oCOHmumOumwuu

d

I

V~

 

 

 

 

The locations of PHA implants in Experiment I are represented

this longitudinal map (Able—Fessard et a1 1966).

in

37

Table 3. Effects of im 1 '
p anted ster01ds on the mean number f ° '
and the mean length of penile papillae. O Penlle Spines

Penile Spines Papillae Length

.—

 

x i SE i’ t SE
Testosterone (POA) 13.4 t 1.81 .62 t .02
Cholesterol (POA) 5.1 t 1.23 .50 i .02
Testosterone (PHA) 11.7 i 1.42 .59 t .01
Estradiol (POA) 4.3 i 1.28 .48 t .01
Estradiol (PHA) 4.5 i 1.00 .48 t .01

\I

38

the estradiol (POA), estradiol (PHA) or cholesterol (POA) groups (p>.05

DNMR) .

EXPERIMENT II. THE BLOCKING OF TESTOSTERONE METABOLISM TO AN ESTROGEN

BY METAPIRONE AND ITS EFFECT ON THE INDUCTION OF MASCULINE SEXUAL BEHAVIOR.

The results of the previous experiment suggest that estradiol when
applied to the preOptic area of castrated rats is more effective than tes-
tosterone in stimulating male copulatory behavior. Experiment II was sub-
sequently conducted to determine if the conversion of testosterone to
estradiol is a necessary step in the stimulation of masculine copulatory

behavior by testosterone.

Methods

Thirty adult intact male rats selected for their sexual vigor, were
castrated and bilaterally implanted in the POA with double-walled steel
cannulae (23 gauge guide cannulae and 30 gauge obdurator) as explained
in Appendix II. Ten males were then randomly assigned to each of the follow- I
ing treatment combinations: 1) 150ug of testosterone daily given intra-
muscularly (IM) + 150ug of sucrose ICT every 12 hr; 2) 150ug of testoster—
one (IM) daily + 150ug of metapirone ICT every 12 hr or 3) 50ug.of estradiol(IM)
daily + 150ug of metapirone ICT every 12 hr. Each group was administered
their respective steroid intramuscularly for 13 days. The dose of estradiol
was lowered from 50ug to 25 ug after 7 days of treatment, to prevent radical

weight loss in the estrogen treated group. Metapirone and sucrose were dis-

solved in 0.9% NaCl solution and adjusted to PM 7.4 with NaCO Metapirone

2.
and sucrose solutions were infused via the intracerebral cannulae in a

volume of .005 ul/cannula. (See Appendix II). Metapirone or sucrose appli-

cations began on the evening of implatation and steroid treatment began the

39

next morning. Males were tested for masculine sexual behavior on the
7th, 10th and 13th day of steroid administration. Histological verifica-
tion of implant sites was made as in Appendix III.

Results.

As seen in Figures 9 8 10 and Tables 4 8 5 intracerebral application
of metapirone in combination with systemic testosteorne, prevented the
increase in mounting activity exhibited by animals treated with sucrose +
testosterone or metapirone + estradiol. While the testosterone + sucrose
treated group showed more intromissions and ejaculations than the other
treatment groups these differences were not consistent.

An over—all analysis of variance for repeated measures revealed no
significant differences between the three treatment groups in mean mount fre-
frequency (F<1), even though a X2 statistic revealed the groups to be
different in the percent of tests during which mounting was exhibited
(X2=6.01>X2 05 2). An analysis over the entire treatment period indicated

0 9
a significant difference among the treatment groups in the mean number of
intromissions acheived (F=3.61>F ). A X2 analysis indicated a dif-

.05,2,27

ferenee among the groups in percent of tests during which an ejaculation

2 >.

. 2_
was achieved (X -21.18>X .05,2

The treatment of castrated males with systemic testosterone + intra-
hypothalamic infused sucrose or treatment with systemically injected estra-
diol + intrahypothalamic metapirone significantly increased the mean number
of mounts in the three postimplant tests, when compared to the three pre-

implant tests (t=2.78 and 1.84, respective1y>t by paired students t

.05,93
test). However, treatment with systemic testosterone + intrahypothalamically

infused metapirone failed to raise the mean number of mounts in the 3 post

tests (1.3<t 05 9). Likewise, the percent of tests during which mounting
. , .

40

Figure 9. The effects of testosterone + metapir ne (T+M), testosterone +
sucrose (T+S), or estradiol + metapirone (R +M) on the mean mount
and intromission frequency as a function of the pre and post implant
test days. Both T+S and E +M but not the T+M treated groups achieved
significantly more mounts in the three post tests compared to the
pre test scores. The T+S treated group also more intromissions than
the other treatment groups although the difference was not significant

in the case of the T+M treatment group.

MEAN MOU NT FREQUENCY

INTRO. FREQUENCY

MEAN

(”‘0

 

-2] -IA

ul

 

 

 

 

 

 

 

 

 

 

 

 

 

 

       

. c -.N. ,. .

PRE AND POST |MPLANT TESTS

figure 9.

42

Figure 10. Percentage of animals mounting, intromitting and ejaculating
during the 3 pre implant tests and 3 post implant tests are shown. The
following abbreviations a e used: T+M, testosterone + metapirone; T+S,
testosterone + sucrose; E +M, estradiol + metapirone. The T+S treat-
ed groups had significantly more animals mounting than the T+M treated
ggoup. The T+S treatment was significantly more effective than the
E +M treatment but not so the T+M treatment in inducing intomissions.
The percentage of animals ejaculating under T+S treatment was signif-
icantly greater than either of the other treatments.

% MOUNTING

% INTR OMITTING

% EJACULATING

100

50

40

3O

20

40

30

2O

 

 
  
  

43

 

 

-T&S

=-----------T&M

 

=EQ&M

 

-2l -14 -7 o 7 IO 13
PRE AND POST IMPLANT TESTS

Figure 10.

44

Table 4. The means t SE of mount and intromissions scores for three groups
of animals receiving testosterone or estradiol in combination with
intracerebrally infused metapirone or sucrose.

 

Testosterone Testosterone Estradiol
Test Days + + +
from Metapirone Sucrose Metapirone
Implantation R’ t SE E": SE 'f 1 SE
-21 0.4 i 0.4 3 S i 1.88 3.7 i 2.50
—14 5.0 i 3.29 0.1 i 0.1 0.0
Mean '
—7 3.6 t 2.83 0.0 1.1 i 1.1
Mount
7 3.6 t 2.83 0.9 i 0.8 5.5 i 2.85
Frequency
10 5.1 t 2.85 11.2 t 3.40 8.8 t 2.97
13 8.4 i 6.04 10.0 i 3.00 9.3 i 5.26
—21 0.0 0.0 0.2 t 0.2
—14 0.0 0.0 0.0
Mean
—7 0 1 i 0 1 0.0 0 2 i 0 2
Intromission
7 0 8 i 0.8 l 2 i 1.2 0.6 i 0.43
Frequency
10 2.2 i 1.99 5.4 i 2.46 0.3 i 0.3
13 2.0 i 1.24 4.66t 1.5 0.3 i 0.3

HS

Table 5, Percentage of castrated male rats exhibiting mount, intromission
or ejaculatory responses after receiving systemic testosterone or
estradiol plus intracerebrally infused metapirone or sucrose.

 

Test Days Testosterone Testosterone Estradiol
from + + +
Implantation Metapirone Sucrose Metapirone

~21 10 . 50 20

~14 30 10 00

Percent ~7 10 00 10
Mounting 7 10 20 30
10 30 70 60

13 33 80 77

~21 00 00 10

~14 00 00 00

Percent ~7 10 00 10
Imtromitting 7 10 10 20
10 20 40 10

13 . 33 50 20

~21 00 00 00

Percent ~14 00 00 00
Ejaculating ~7 00 00 00
7 10 10 00

10 10 30 00

13 10 40 00

46

was exhibited, was significantly greater in the testosterone + sucrose
and the estradiol + metapirone groups when compared to the testosterone +

metapirone treated animals (X2=5.10 and X2=5.50, respective1y>x2 05 1)
' 9

(Figure 10 Table 5).

Treatment with testosterone + sucrose was significantly more effective
in the induction of intromissions than was estradiol + metapirone (p<.05
DNMR). Testosterone + sucrose treatment tended to be more effective than
testosterone + metapirone although the difference did not reach the level
of significance 0.16 (F=2.7). Likewise, the percent of tests positive for
- intromissions was not significantly greater for the testosterone + sucrose
groups when compared to the estradiol + metapirone (X2=2.01<X2

.05,1
2

to the testosterone + metapirone group (X2=1.2<X 05 1). '
° 3

) or

Histological Results.

The location of the implantation sites for the three groups is shown
in Figures 11 and 12. An analysis of variance revealed no statistical dif-
ferences in the site of implantation between the three groups. The most
effective location for metapirone inhibition of mounting was: anterior
7.43, vertical 0.92 and lateral 0.68 (multiple quadratic regression). An
analysis of variance revealed that there was no difference between the three
groups in the percent of preoptic nucleus destroyed by the implantation
technique (F<1). The correlation coefficient between the percent of pre-
Optic nucleus destroyed and the mean mount score per test was calculated
for the three groups: Testosterone + Sucrose r=~.36, Testosterone + Meta—
pirone r=~.35 and Estradiol + Metapirone -.38.

It is interesting that the sites of metapirone application least effect-
ive in blocking the resumption of sexual behavior, were also the farthest

away from the most effective site for blockage. For instace, the three

47

Figure 11. The locations of maximum cannulae penitration in Experiment

II are represented in these cross sectional maps (Pellegrino et a1
1967).

[3C)t>c>

ao-o

 

0 =no copulation
.0 =mounting only
GD=intromission observed
Cj=ejaculation observed

48

O=testosterone + metapirone
D=testosterone + sucrose
A=estradiol + metapirone

7.)

 

Figure 11.

IO

49

   

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.132

 

 

Q71. U\._\ # ufliww C

.\.\.\
b

.\

     

The locations of maximum cannulae penitration in Experiment

II are represented in this longitudinal map (Able—Fessard 1966).

Figure 12.

50

animals that exhibited the most mounts and intromissions were out of the
POA completely. When an ANOVA was run on the three groups minus these
three exceptions plus one in the testosterone + saline group that was
implanted in the optic tract, the testosterone + metapirone treated group
showed significantly fewer mounts (F: 4.21>F 05,1,22), intromissions (F:

2 1

. . . 2-
11.44>F 05,1,22) and percent of tests showing ejaculations (X -7-78>X '05,?

when compared to the testosterone + sucrose group.

EXPERIMENT III. DETERMINE IF CAMP CAN MIMIC THE EFFECTS OF TESTOSTERONE
IN INDUCING THE RESUMPTION OF MASCULINE SEXUAL BEHAVIOR.
Methods.

Procedure for selection, castration and subsequent implantation of
bilateral cannulae into the POA (23 gauge guide and 30 gauge obdurator),
are descrided in Appendix. 0f 30 males, 10 each were randomly assigned
to 1 of 3 treatment groups: 19 25ug of CAMP administered intracerebrally
ICT every 24 hours + 5 mg of theOphylline (IM) every 12 hr; 2) 25ug of 5'
adenosine monophosphate (5'AMP) ICT every 24 hr + 5 mg of theophylline
(IM) every 12 hr; or 3) 25ug of Dibutryryl CAMP (Db—CAMP) ICT every 24 hr
+ 20% CQOH6 .9% NaCl solution (ethanoic saline) (IM) every 12 hours. The
CAMP, 5'AMP and Db-CAMP were administered in a volume of .005 ml 0.9 NaCl
solution per cannula. Theophylline or the control vehicle (ethanoic sal—
ine) was injected in a volume of 0.1 m1. All treatments began the day
after implantation and lasted 10 days. All animals were tested for mascu~
line sexual behavior on the 7th and 10th day of treatment.

Theophylline was given intramuscularly with CAMP to prevent CAMP from

being rapidly metabolized and hence inactivated. Db-CAMP is a diacylated

51

derivative of CAMP which is apparently resistant to phosphodiesterase deg-
radation (Posternak, Sutherland and Henion 1962, Menahan, Hepp and Weiland
1969, Drammand and Powell 1970). Five ' AMP is the inactive metabolite

of CAMP.

Following the behavioral test at day 10, all IM treatments (ie.
either theophylline or ethanoie saline) were terminated. On day 11 all
animals began 8 days of daily testosterone (150 ug/day) injection (IM)
in combination with their ICT (ie. CAMP, 5'AMP or Db—CAMP). Testosterone
was administered for 8 days, but the intracerebral treatment lasted only
7 days. On the 8th day of testosterone treatment all animals were tested
for masculine sexual behavior.

Results.

Masculine sexual behavior: As seen in Figure 13 and Table 6 none of the
nucleotides by themselves restored any component of masculine sexual
behavior. However, when testosterone was injected systemically along

with the nucleotide, the 5'AMP treated animals achieved significantly more
mounts intromissions and ejaculations. None of the animals receiving

CAMP or Db-CAMP exhibited any sexual behavior. Only 19 animals were
tested at the 8th day of steroid treatment (day 18 postimplant) due

to death or overt illness in 11 animals (4 each in CAMP + Testosterone

and 5'AMP + Testosterone and 3 in Db-CAMP + Testosterone). Four of the
six animals in the 5'AMP group achieved at least one mount in the day 18
test. The mean mount frequency for this group being significantly greater
than either CAMP + Testosterone or Db-CAMP + Testosterone (F=6.89>F ).

.05,1,16

The 5'AMP + Testosterone group also exhibited significantly more intromissions

than the other groups (F=5.59>F 05 1 16)’ but the percent of animals ejac-
' 3 3

ulating did not reach significance (X2z3.01<X2 ).

05,2

52

Figure 13. The effects of intracerebrally applied nucleotides on the mean
number of mounts and intromissions as well as the percentage of ani-
mals ejaculating during 10 days of intracerebrally applied (POA)
nucleotides + theOphylline or saline and then 1 week of intracerebral
nucleotide + testosterone. Testosterone when given with intracere-
brally applied 5'AMP significantly increased the mean number of mounts
and intromissions as well as increased the percentage of animals ejaC~
ulating when compared to CAMP or Db—CAMP in combination with,testosterone.

53

 

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Z

 

 

5 O 5 0

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.-—--

 

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CAMP

- {Db-CAMP

 

 

40

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02.53.430.43. R

 

a4
—
-
c
-
u.

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u

-

-14 -7 0 7 10 18
|MPLANT TESTS

PRE AND POST

-21

Figure 13.

50

Table 6. Effects of intracerebrally infused 5'AMP, CAMP or Db-CAMP by
themselves or with 8 days of testosterone (day 18), on male COpula-
tory patterns.

Test Days 5'AMP ’ CAMP Db-CAMP

 

from
Implantation X’ i SE 'i' t SE ‘R'i SE
-21 2.7 i 1.80 2.6 t 2.49 4.5 i 4.07
~14 0.5 i 0.27 1.0 t 0.90 0.1 i 0.1
Mean
~7 0.9 i 0.9 0.0 0.0
Mount
7 0.0 0.0 0.0
Frequency -
10 0.0 0.0 0.0
18(+Test.) 15.0 i 7.04 '0.0 0.0
~21 0.0 0.7 i 0.7 . 0.7 i 0.7
~14 0.0 0 0 0 0
Mean
—7 0 0 0.0 0 0
Intromission
10 0.0 0.0 0.0
Frequency
18(+Test.) 3.0 i 1.61 0.0 0.0
~21 00 00 00
~14 00 00 00
Percent —7 00 00 00
Ejaculating 7 00 ' 00 00
10 00 00 00

18(+Test.) 33 00 OO

55
Histological results: The location of the treatment application sites
are represented Figures 14 and 15. An analysis of variance reveals
there were no significant differences among the three groups in location
of implants (each dimension coordinate was analysized separately) or in
the percentage of the preOptiC nucleus destroyed by the implanted cannulae
(F<1, in all cases). A coefficient of correlation was calculated between

the percentage of the preOptiC nucleus destroyed and the mean mount score

for the 5‘AMP + Testosterone group, r=~.38.

EXPERIMENT IV. EFFECTS OF POA ANDRENERGIC OR CHOLINERGIC MANIPULATION ON
MASCULINE SEXUAL BEHAVIOR.
Methods.

Subjects were 18 male rats which had been given two 15 minute screen-
ing tests for sexual behavior. Animals which ejaculated on both tests
were castrated and given daily injections of testosterone propionate (TP)
(150ug) for the duration of the experiment. One week following castration.
all subjects were bilaterally implanted in the POA, with double-walled
stainless steel cannulae (21 gauge outer, 27 gauge inserts). All animals
were then assigned to one of two studies (9 animals/study). In Part A
all animals received each of the following treatments via cannula (a dif—
ferent treatment every week) 15 minutes before the start of a test for
masculine behavior: 1) Norepinephrine (NE), 2) e-methyl tyrosine (a-MT) and
3) the blank cannulae. The animals in Part B of the study received the
following drugs designed to manipulate the Cholinergic milieu of the POA:
1) Carbamylcholine (carbachol), a Cholinomimetic; 2) Hemicholinium-3 (HG-3)
(a synthesis inhibitor of acetylcholine) and 3) the blank cannulae. The

Chemical in both eXperiments were administered via the cannulae. A

56

Figure 14. The locations of maximum cannulae penitration in Experiment
III are represented in these cross sectional maps (Pellegrino 1967).

sooo

gov»
‘ae-o

o'1>

     

57

7)

5'AMP
CAMP
Db-CAMP

no COpulation
mounting only
intromission observed
ejaculation observed '“3~<'”

Figure 14.

 

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o I I n o I nt
The locations of maXimum cannulae penitration 1n Experime

III are represented in this longitudinal map (Albe—Fessard 1966).

Figure 15.

59
standard dose of 8 taps of the 27 gauge insert, into a thin layer of the
substance was used in both experiments (8—10ug). A new insert was used
for each chemical and remained in the guide cannulae throughout the testing
under that treatment. The experimental design for both studies (a latin
square design, Winer 1962) called for each animal to reveive all three
treatments during the course of the study. Animals were given an initial
15 minute test for sexual behavior starting 15 minutes after the chemical
was applied to the POA. A second 15 minute test was given 4 hours after
the initial treatment. The four hour interval from initial treatment
to the second test was Chosen in order to allow the synthesis inhibitors
sufficient time to act. A second administration of the treatment (half
the initial dose was given 15 minutes before the start of the 4 hour test
to control for the relative speed of action of the various compounds. Tests

for masculine sexual behavior were 15 minutes long, and the oecuranee of
mounts, intromissions and ejaculations was recorded on an Esterline-Angus
Event.Recorder.

Results.

Part A: Adrenergic manipulation:

NE when compared to the sham treatment reduced the number of intro—
missions and ejaculations in the first test and the number of mounts, intro-
missions and ejaculations in the second test. The applieaiton Cf a-MT had
no effect on sexual behavior in the first test but significantly decreased
the number of ejaculations in test 2. NE increased the latency to first
mount and first intromission as well as the time between intromissions..
That is to say it slowed down the COpulatory performance, resulting
in fewer mounts, intromissions and ejaculations being exhibited during the

test. Alpha-MT had no effect on the latency measures during either test.

60

The effects of intracerebral application of norepinephrine, or the
synthesis inhibitor, a-methyltyrosine on copulatory behavior are repre—
sented in Figure 16 and Table 7. In the ist test (Test I), given 15
minutes after treatment, neither NE or a-MT'significantly affected the
mean number of mounts achieved when compared to the sham treatment (blank
applicator) (F<1 in both treatments). However, NE significantly decreased
the mean number of intromissions (F=8.2>F ) and ejaculations (F=10.1

.05,1,6
>F 05 1 6) achieved during the 15 minutes of Test 1. Alpha-MT had no
° 5 ,

significant effect on intromission frequency (F=1'3<F.OS,1,6) or ejacula—
tory frequency (F<1). A further analysis of only the first ejaculatory
series in Test I (Table 7) revealed that neither NE or a-MT treatment had
effects on the mean number of mounts or intromissions. 0n the other hand,
NE treatment increased the mean mount latency (ML) (F=5.20>F 05,1,24) in
series 1, compared to the sham treatment. There was also a signifiCant
increase in the intromission latency of series 1 when the animals were
given NE (F=39.11>F 05,1,24)' There were however, no significant differ-
ences between the sham treatment and a-MT during the lat series in ML or
IL. NE but not a—MT, increased the mean inter-intromission—interval (MIII)
of series 1 (F=4.24[NE], F<1 [a—MT1). No significant differences were
detected (p<.05) between a—MT and sham treatments or between NE and sham
treatment in the percentage of animals exhibiting mounts, intromséions or
ejaculatory responses in series 1.

In Test 11, the 15 minute test given 4 hours after intracerebral treat—
ment, both a—MT as well as NE had significant effects on certain components
of masculine sexual behavior. NE 'but not a—MT significantly decreased
the mean number of mounts when compared to the sham treatment (F=3.98INE]

and F=0.86 Ia—MT]). Likewise, NE (F=9.5>F ) but not a-MT (F=3.15<

.05,1,2L1

E) 1

Figure 16. The effects of intracerebrally administered drugs on parameters
-of sexual behavior are represented. Two 15 min tests were given under
each treatment: Test I (15 min after the initial exposure) Test II
(given 4 hrs after the initial exposure). * = significantly differ-
ent from the sham treatment during that test.

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came .mm ”moconvmum cowmmHEouucw came .mH m%ocm:vouw ucaos cmme .mz

"pow: mum mCOHumH>munnm wCH30HH0w wee
mumumEMHmm co «om ecu mo cowumHschmE onHmamem mo muumwmm

.H0H>mnwn stxmm mcHasummE mo
.5 mabme

 

HIHHHZ

mm

mH

64

F 05 1 21+) significantly reduced the_mean number of intromissions. How-
“ , 3

ever, the application of either a—MT significantly decreased the mean

number of mounts when compared to the sham treatment (F=3.98 [NE] and

0.86 [a-MTJ). Likewise, NE (F=9.5>F ) but not a-MT (F=3.15<F

.05,1,24 05,

1,2“) significantly reduced the mean number of intromissions. However,
the application of either a—MT or NE significantly reduced the number of
ejaculations achieved in the 15 minute Test II (F=11.67 and 6'17>F.05,1,24
respectively). Neither a-MT or NE treatment significantly affected the

percentage of animals exhibiting mounts or intromissions. However, NE

but not a-MT treatment significantly reduced the percentage of animals

2 .
.05,2). As in

the case of Test I, neither NE or a-MT treatment had significant effects

exhibiting the ejaculatory response in Test II (X2=6.92>X

on the mean mount frequency of series 1 in Test II. Likewise, NE but not
a—MT treatment significantly increased the ML (F=8.13 INE]; F<1 [a—MT])
and IL (F=12.59 [NE]; F<1 Id-MTJ) in series 1. As in Test I, NE but not
a-MT significantly increased the MIII of series 1, Test II (F=12.93 [NE];
F=2.12 [a-MT]).

Part B: Cholinergic manipulation.

The effects of the carbachol, hemicholinium and sham treatments on
the number of mounts, intromissions and ejaculatory responses achieved
during Test I (given 15 minutes after the initial exposure) and Test II
(given 4 hours after the initial exposure) are represented in Figure 17
and Table 8. Application of carbachol abolished all masculine sexual
behavior patterns. A total of 3 mounts in Test I (and 1 mounts in Test II)
were achieved by the nine animals when treated with carbachol, compared
to the sham treatment (104 and 79 in Test I and Test II, respectively).

Carbachol effectively inhibited the display of any intromission or ejac~

65

Figure 17. The effects of intracerebrally administered drugs on the mean
number of mounts, intromissions and ejaculations during tests for
masculine sexual behavior as represented. * = significantly differ—
ent from the sham treatment during that test.

66

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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mpmumsmumm co «om mzu mo CoHumasawcmE onuoCHHono mo muomwmm .0 mHQmH

68
ulatory responses in Test I and II. Carbachol, however was not totally

behaviorally debilitating. A significant number of animals exhibited

2

fighting with the stimulus female (X2=8.8>X 05 2) when compared to the
.. ,

sham treatment.

The intracerebral administration of hemicholinium had no significant
effect on the oecuranee of mounts, intromissions or ejaculations in Test I
when compared to the sham treatment. However, in Test II when compared
to their sham treatment results, the animals under the influence of hemi~
Cholinium-3 (HC-3) achieved significantly fewer mounts (F=6.24), intromis-

sions per ejaculation (F=2l.01) and ejaculations (F=28.6) (all greater

than the comparative F 05 1 24). Likewise the percent of animals achieving
' 9 9

an ejaculation during Test II was significantly reduced with HC-3 treat-
ment.
An analysis of variance involving the first ejaculatory series of
both Test I and Test II reveals a significant increase in ML (F=50.8
ITest I and II, respectively]) when animals were given carbachol compared
to the sham treatment. Likewise, carbachol treatment significantly decreased
the mean MF and IF of series 1. No significant differences between HC-3
and sham treatment were detected in ML or IL during the lat series of Test
1. However, the HC-3 treatment, when compared to the sham test, signif—

icantly prolonged the ML (F=7.15>F ) and IL (F=8.2>F ) dur-

.05,1,18 .05,1,18

ing Test II. Similarly, the MF (F=5.08) and IF (F=6.40>F 054’2”) of the
animals under HC—3 (Test II) was significantly decreased compared to the
sham treatment. The increased ML and IL as well as the decreased MF and
IF observed during Test II were to a large extent, due to inactivity by

animals previously exhibiting a prolonged (over 20 minutes) post ejaculatory

interval (PEI). A significantly greater number of these prolonged PEI‘s

69
were exhibited during Test I by animals treated with HC-3 when compared
2

to the sham treatment (X2z8.87>X 05 2).
' 3

Histological results:

The location of the implants from animals in both Part A and B of
experiment IV, are exhibited in Figures 18 and 19. The location of the
majority of the implants were centralized around the preOptiC-anterior
hypothalamic continuum. Inspection of Figures 18 and 19 for the anterior—
posterior distribution of the implants reveals that they lie fairly well
restricted within a range from anterior 6.2 to 7.6. The vertical range
for both groups was from 1.1 to 3.3 and lateral from 0.8 to 1.6. The
anterior coordinate in this experiment tended to be somewhat more poster-
ior than in the other experiments. This experiment happened to be the
first of the four conducted, and corrections were made to move the implants
up to the coordinates in the other experiments. The mean location of the
implants for the adrenergic and Cholinergic groups were determined from

corrdinates utilizing Pellegrina et al (1965) Atlas of the Rat Brain:

 

Cholinergic; anterior 7.0, vertical 2.6, lateral 1.3; Adrenergic, anterior

6.9, vertical 2.8 and lateral 1.3.

7O

 

adrenergic experiment
Cholinergic experiment

()9
11

Figure 18. The locations of maximum cannulae penitration in Experiment
IV are represented in these cross sectional maps (Pellegrino 1967).

ucmEHHmme onHmCHHocu
HemEHuoaxw onnmcmuwm

 

 

. \

Dy
K

V

 

Zoom .c

 

(Able—Fessard 1966).

The locations of cannulae placements in Experiment IV are

repreSented in this longitudinal map

Figure 19.

72

DISCUSSION

The results.of this study extend the Current information on the neuro-
biochemical control of masculine sexual behavior in three areas: 1) the
neurohormonal specificity of behavioral activation, 2) the intracellular
events associated with testosterone restoration of mating behavior and 3)
the pharmacological control of male copulatory behavior.

Intracerebral application of testosterone or estradiol directly to
the POA restored copulatory behavior in long term castrated male rats.
Similar implants of testosterone or estradiol in the PHA were less effective
than their respective POA comparisons and no more effective than Cholesterol
(POA) controls, in reinstating copulation. The suggestion that testoster-
one is converted to estradiol before activating male sexual behavior, was
further supported by the finding that application of metapirone, an aroma-
tization inhibitor, to the POA of long term castrated males, blocked the
increase in sexual behavior normally associated with testosterone therapy.
This inhibition was not a result of the toxic effects of the drug, since
administration of estradiol in combination with metapirone resulted in an
increase in mounts over the pretreatment tests similar to that observed
with testosterone + sucrose treatment.

The infusion of solutions containing CAMP, 5'AMP or Db—CAMP directly
into the POA of long term castrates was ineffective in reinstating the cop-
ulatory behavior of any of the animals tested. When given in combination
with 150 ug of testosterone daily, only the animals treated with 5'AMP +
testosterone daily resumed significant amounts of mount and intromission
behavior.

Application of drugs to the POA designed to either increase or decrease

adrenergic or Cholinergic activity, all had similar effects on the gross

73
measures of sexual behavior, that is., all decreased some components of
sexual behavior. However, the mechanisms by which these Changes in behav-
ior were effected appear to be different. (In both. the Cholinergic and
adrenergic experiments, the effects of the mimetics were observed in both
Test I and II, suggesting an immediate action of the drugs. The inhibitors,
on the other hand, had no effects on COpulatory measures until the 4 hour
test (Test II), suggesting a longer action latency than the mimetics. Like~

wise, the various treatments affected different COpulatory measures (eg.

MF, IF, ML and IL).

1) Neural Hormonal Specificity

The findings of the present study support the concept that the POA
of male rats is intimately involved in the regulation of male COpulation
by testosterone. Testosterone implants in the POA facilitated male sexual
behavior but implants in the PHA did not. This suggests that the effects
of the hormone are Specific for the POA. Estradiol was also more effective
than Cholesterol in stimulating male mating behavior when placed in the
POA but not when placed in the PHA. Stimulation of penile morphology by
testosterone implants in both the POA and PHA suggests that significant
amounts of testosterone entered the systemic Cirulation. However, the ina—
bility of the PHA implants to effect increases in mating behavior suggests
that the levels of testosterone reaching areas of the brain other than the
implant site, via plasma Circulation or diffusion, were not sufficient to
influence behavior. Only when the POA was stimulated by high concentrations
of hormone was mating beahvior activated. Thes results indicate that testos-
terone and/or estradiol effect certain Changes in the POA to activate mas-

culine sexual behavior.

74

Two results in the present study support the concept that testosterone
is aromatized to an estrogen in the POA before stimulating masculine sex—
ual behavior. 1) Estradiol implanted in the POA was more effective than
POA implants of testosterone in stimulating male COpulation. 2) Inhibi-
tion of testosterone induced sexual reactivation by infused metapirone.
Clinical use of metapirone has shown that prolonged systemic adminsitration
of metapirone results in reduced adrenal production of cortisol through
its inhibition of adrenal 11 B—hydroxylation. As a result, a compensatory
increase in ACTH release follows and the secretion of 11-deoxycortisol is
accelerated (Goodman and Gillman 1970). However, the effective blockage
by metapirone seen in the present research, does not appear to be the result
of this toxic side effect, since when metapirone was given in combination
with estradiol the same increase in mounting was observed as with testos-
terone + sucrose treatment. The suggestion that testosterone is aromatized
before activating mating behavior also has support from studies reporting
the inability of the reduced androgen DHT, to stimulate male sexual behavior
when administered systemically (McDonald et a1 1970, Whalen and Luttge 1971)
or intrahypothalamically (Hohnston and Davidson 1972). Likewise, systemic
administration of estradiol has been shown to be stimulatory to male sexual
behavior (Pfaff 1970, Davidson 1966). The concept that the conversion of
testosterone to an estrogen occurs in the POA has also received support by
the finding that in vitro conversion of androstenedione to estrone occurs
in the diencephalon of male rats (Naftolin et a1 1972). Thus, for the stim—
ulation of masculine sexual behavior, it appears as though testosterone
serves as a prehormone, being carried to the target tissue (the brain)
where it is converted to the active configuration, presumably estradiol.
This model fits well the established prehormone role testosterone serves

in the stimulation of systemic tissues; testosterone being converted to DHT

75
instead of estradiol in the systemic targets.

2) Intracellular Events Associated With Testosterone Reactivation.

From the results of Experiments II and II information was gained on
two possible intracellular events associated with testosterone stimulation
of the POA: 1) Aromatization of testosterone to estradiol in the POA and
2) involvement of CAMP in neural—hormonal regulation of masculine sexual
behavior. The blockage of testosterone-induced sexual behavior, by admin-
istering an aromatization inhibitor (metapirone) adds support to the hypo—
thesis that testosterone is converted in the POA to an estrogen. If the
processes involved in the conversion of testosterone to DHT in the seminal
vesicle, prostate and epididymus are taken as a model for testosterone in
the brain, the converSion process would take place intracellularly. As in
the stimulation of androgen—sensitive accessory tissues, testosterone in
the POA wouls enter the target cell where it would bind with a cytosol
receptor. It would then be metabolized while attached to the receptor, to
the active configuration (estradiol) and transferred to the nuclear receptor,
which is at least of different molecular weight than the cytosol receptor,
although it may be related in part (Jensen, Numata, Brecher and DeSombre
1971). Although the cytosol and and nuclear receptors in the POA along
with.the corresponding structure of the hormones bound to them have not been
isolated as they have been in the ease of other tissues (Bruchousky and
Wilson 1968), the in vitro conversion of testosterone to estradiol in the
diencephalon of rat brains has been demonstrated (Naftolin et al 1972).

The known events in the aromatization process involve the removal of
C-19 carbon and the aromatization of the A ring. The removal of the C-19
carbon involves hydroxylation at this site and relies upon a C—19 hydroxyl-

ase dependent on NADP+. Thus, the production of the C~19.hydroxylase would

76

be one of the limiting steps in the stimulation of the nucleus by testos-
tePODE. If again, we conCider the working hypothesis developed for the
stimulation of genital tissues by testosterone via conversion to DHT, the r
activaty of the rate limiting enzyme in this system, Sa-reductase, is inver-
sly related to the concentration of testosterone (Kniewald, Massa and Mar-
tini 1971). Thus, testosterone seems to play a role in regulating the
enzymes involved in its conversion to DHT. This may also be the case in

the conversion process of testosterone to estradiol, although no experimental
data are available to confirm this assumption.

A number of studies have implicated the nucleotide 3'5'CAMP as a
rate determining factor in many steroidogenic processes, (ie. corticosterone
production in the adrenals, Robinson, Buther and Sutherland 1968 and gonadal
steroid synthesis in the testis and ovaries, Sandler and Hall 1966, Connell
and Eik-Nes 1968). Specifically, evidence has been presented that CAMP stim-
ulates steroidogenesis by increasing the conversion of cholesterol to
pregnenolone (Karaboyas and Koritz 1965). On the other hand, studies have
shown that in vitro administration of CAMP inhibits the NAD+ dependent 5-
ene-3B—hydroxysteroid dehydrogenase in adrenal cortex (Koritz, Yun and Fer-
guson 1968, McCune, Roberts and Young 1970) and ovarian tissues (Sulimovici
and Luneufeld). This last enzyme is involved in the conversion of testosterone
to androstenedione. Thus, CAMP may be involved in controlling steroidogenic
metabolism in a number of different tissues and systems.

The possibility of CAMP being involved in testosterone stimulation of
masculine sexual behavior was suggested in an earlier study by Christensen
and Clemens (in press). In results of that experiment theOphylline potent-
iated submaximal levels of TP in the stimulation of masculine behavior.

One conclusion drawn from these data was that theOpylline, in line with its

known effects of preventing the degradation of CAMP, stimulated sexual

77

behavior by preventing the metabolism of CAMP, synthesized during testos-
terone stimulation. The subsequent build-up in CAMP was thought to have
accentuated the TP. Experiment III in the present study ruled out the
possibility that CAMP was serving as the sole intracellular mediator for
all the action of testosterone, since intra-hypothalamic injection of sol-
utions containing CAMP or Db—CAMP failed to initiate copulation in long term
castrated males. An alternative consideration was that intracellular
CAMP was involved in the production of substrates (ie. receptor or C-19
hydroxylase) necessary for the incorporation of testosterone into the
cell and its eventual movement to the nucleus. However, the data generated
from the second half of experiment III imply that the role of CAMP in the
neuro-hormonal regulation of sexual behavior is somewhat more complicated.
Instead of potentiating low doses of testosterone as the previous study with
theOphylline suggested, infusion of CAMP or Db—CAMP inhibited the activation
of sexual behavior when compared to the 5'AMP treated group. Several hypo-
theses dealing with the apparent inconsistency between the previous study
(Christensen and Clemens in press) and the results of experiment III will
now be discused.

Firstly, in addition to the known effects of theophylline in preventing
CAMP degradation in many tissues (Greengard and Costa 1970) theophylline
has been shown to decrease the content of CAMP in in vitro brain slices
(Porn and Krishna 1973, Shimizu, Daily and Creveling 1969, Palmer, Sulser
and Robinson 1973). If this were, in fact, the effect of our in vivo admin-
istration of theOphylline, and its synergism with testosterone was due to
a decrease in intracellular CAMP, then these results would collaborate those
of experiment II in suggesting that low levels of CAMP potentiate testoster-
one. In fact, the data of experiment III suggest that high levels of CAMP

inhibit the action of testosterone. CycliCAMP has been shown to inhibit

78
several NAD+ dependent dehydrogenases, Specifically the 17B-hydroxylase

dehydrogenase involved in the in vitro conversion of testosterone to andro-
stenedione (Sulimovici and Lunenfeld 1972). The possibility therefore
exists that infusion of CAMP or Db-CAMP into the POA is preventing the
metabolism of testosterone to some effective compound (eg. estradiol), by
interfering with the NAD+ dependent process. Two such NAD+ dependent pro-
cesses which may be of importance are 1) the conversion of estradiol to
estrone and 2) the conversion of testosterone to androstenedione. In

human placental microsomes, androstenedione appears to be the immediate
precursor of estrogens rather than testosterone (Menimi and Engel 1967).

A second possible explanation for the variance in the observed
results in experiment II is that CAMP and Db-CAMP are duplicating the effects
of neurotransmitters. Specifically Rindi, Sciorelli, Poloni and Acanfora
(1972) have shown that when applied directly to the lateral hypothalamic
area of rats, Db—CAMP but not CAMP mimicked the effect of implanted carba-
chol, both of which significantly increased food and water intake. Although
the suggestion made by Rindi et al. that CAMP acts by releasing acetylcholine
at the synaptic Cleft, has not been directly proved, it is supported by the
finding that Db—CAMP increases miniature end-plate potentials (Goldberg and
Singer 1969). The similarity of Db—CAMP (experiment III) and carbachol
(experiment IV) in producing decrements in sexual behavior, suggests that
Db—CAMP may be having effects similar to carbachol. However, the fact that
Db—CAMP in addition to CAMP prevented the resumption of masculine sexual
behavior is at odds with the Rindi et a1 (1972) study where only Db—CAMP
and not CAMP duplicated the effects of carbachol. This general'hypothesis
also assumes there is no mediator role for CAMP in the stimulation of sexual
behavior by testosterone. The possibility exists that CAMP is involved in

this process, but with the procedure used here its mimicry of carbachol could

79
be masking this effect.

A third possible explanation of experiment III proposes that CAMP and
Db-CAMP are not inhibiting the reSponse to testosterone, but that 5'AMP
is potentiating the action of testosterone. Although no testosterone +
saline group was run in Experiment III, a comparison of 5‘AMP treated
group's mount scores with the Testosterone + Sucrose group of EXperiment
11 suggests that this may be the case. After 8 days of testosterone treat-
ment the mean number of mounts in the testosterone + 5'AMP group was 15.
Although there was no test on day 8 for the testosterone + sucrose
group, tests on days 7 and 10 produced mean mount frequencies of 9 and
10.1 respectively. Although this suggestion may be plaussible only one
specific role for intracellular 5'AMP has been identified in any mammalian
system (inhibition of precursor incorporation into nucleic acids in adrenal
steroidogenesis systems, Tsang and Johnston 1973). The possibility also
exists that any three of the presented explanation for the results are acting
in combination to produce the observed effects. Further experimentation

designed to investigate one or all of the alternatives is needed.

3) Pharmacological Control of Male Sexual Behavior

The data from Experiment IV provides information on the influence of
adrenergic and Cholinergic systems on the temporal sequence of masculine
sexual behavior. Application of either NE or carbachol to the POA of sex—
ually active rats decreased sexual behavior in a test given 15 minutes after
exposure to the substance. NE decreased the mean number of intromissions
and ejaculations in Test I and II, and likewise decreased the mean number

of mounts in Test II but not in Test I. The decreased number of mounts

and intromissions was not, however, due to the inability of the male to

80
COpulate, since there was no significant difference in the percent of animals

mounting or intromitting in either test. Likewise, there was no difference
in the number of mounts or intromissions achieved in the 1st series of
Test I or II when the animals were treated with NE compared to the sham treat-
ment. Instead, applieaiton of NE increased the time taken to initiate
the lst mount and intromission, and the time between successive intromissions.
It appears that high concentrations of NE in the POA retards the overall
temporal sequence of copulation.

When animals were treated with carbachol only a total of 4 mounts
was seen in both tests, with no intromissions or ejaculations being achieved.
The animals did exhibit interest in the female however, often following
her around the cage, but attacking her instead of attempting to mount.
Application of carbachol thus appears to interfere with the oecuranee of
COpulatory behavior.

The two systhesis inhibitors differed dramatically from the mimetics,
in that their effects on behavior were not apparent until Test II (4 hours
after the initial exposure), whereas the mimetics had effects on behavior
almost immediately (15 minutes after exposure). Neither e-MT or HC-3
influenced the mean number of mounts, intromissions or ejaculations in
Test I or the percent of animals exhibiting these responses. However,
HC-3 treatment resulted in significant decreases in the means of all these
measures and a decrease in the percentage of animals exhibiting mounting
or intromitting during Test II. This decrease in all components of sexual
behaivor under HC~3 was, to a large extent, a result of a prolonged PEI
(over 20 minutes) exhibited by~a significant number of the animals. This
prolonged PEI occurred either after 1 or 2 ejaculations. Since an average
of 0.8 ejaculations occurred during Test I, the PEI usually occurred at the

end of Test I or after the first ejaculation of Test II (mean number of

81

ejaculations in Test II:1.4 for the sham treatment). Only one animal resumed
COpulation after exhibiting this prolonged PEI and he copulated at a low
level. The prolonged PEI appeared very much like satiety, in that the

male moved about the cage after a standard period of inactivity, but showed
no interest in the female. This pattern of activity would seem to corres-
pond very well with the idea that the depletion of a substance, would term-
inate the display of copulatory responsess Since HC~3 is known to block

the synthesis of acetylcholine and the cholinomimetic (carbachol) both

have similar effects on the probability of cepulation, even though the
temporal and behavioral sequences leading to sexual quiescence are differ—
ent. One possibility for this paradox is that the application of carbachol
hyperpolarized the Cholinergic system involved in activating COpulation,

in effect, creating a Chemical lesion. With HC-3 treatment, the initiation
and maintenance of copulation is not affected until the acetylcholine stores
are depleted by one or two ejaculations, thus preventing the reinitiation
of copulation after a PEI.

Alpha—MT treatment significatnly decreased the mean number of ejac-
ulations exhibited in Test II without affecting any other measure of COpu-
latory behavior. If there were a decrease in the number of ejaculations
but no Change in IL, PEI or the time between intromissions (MIII), one
would expect there to be more intromissions under the a—MT treatment than
in the sham treatment. However, this was not the case. The variance between
individuals probably accounts for this discrepancy (eg. the difference
between individuals in Test II MF was significant p<.055).

These data on treatment with a—MT are at variance with results reported
by Malmnas (1973) who found a significant reduction in the percent of ani~

mals mounting and intromitting when given systemic injections of e—MT (lSOmg/kg).

82

However, this discrepancy was undoubtedly a dose response since a smaller
dose of systemic a—MT (75mg/kg) had no effect on sexual behavior in Malmnas'
study. Likewise, no detectable amount of e-MT was released from the cannulae
in the present study during the 4 hours of treatment.

Since a-MT inhibits the synthesis of both dopamine and NE (Spector,
Sjoerdsma and Undenfriend 1965), the behavioral effect of a-MT treatment
connot be delegated to either dopamine or NE systems at this time. Malmnas
(1973) however, was able to seperate the behavioral effect of a—MT treatment
by selectively blocking NE synthesis or dopamine postsynaptic receptors.

He determined that the behavioral deficits seen with e—MT were the result
of depleting neurotransmitter substances in dOpaminergiC systems.

In conslusion, it appears as though high concentrations of NE in the
POA significantly increased response latencies for masculine sexual responses.
The depletion of catecholaminergic fibers (via a-MT) decreases the occurrence
of ejaculation, probably through an effect on depaminergic systems. Chol-
inergic systems appear to be involved with whether a response will be shown
or not. High concentrations of a cholinomimetic prevent the display of
all sexual responses. The depletion of acetylcholine stores however,

results in the arresting of copulation.

83

CONCLUSION

The reinstatement of sexual behavior in long term castrated male rats
occurred in the present study when testosterone or estradiol was applied
directly to the POA. Estradiol was more effective than testosterone in
reactivating masculine sexual behavior, even though a smaller intracerebral
dose was used. These data confirm the concepts that 1) estradiol is an
effective stimulatory hormone for all components of masculine sexual
behavior and 2) the POA is a site for hormonal regulation of COpulatory
behavior. These data support the suggestion that testosterone may be
metabolized to an estrogenic form before acting on the neural substrate
to effect the behavioral Changes.

This latter hypothesis was tested directly in the present study. The
inability of testosterone to raise mounting scores above the preimplant levels
in animals that were given intracerebral metapirone, points directly to
the necessity of testosteorne aromatization for behavioral stimulation.

The ability of the aromatization inhibitor to block the reinstatement of
cepulation when applied to the POA also suggests that the aromatization
occurs in this neural area.

The present study also supports the hypothesis that CAMP is involved
in the hormonal regulation of masculine copulatory behavior. Although not
entirely Clear, the data imply that high levels of CAMP in the preOptiC
area inhibit the behavioral activation associated with testosterone
treatment. The mechanism of this blockage may involve the inhibition
of NAD+ dependent anabolism normally activated by testosterone stimulation.

The results of the present study further demonstrated that high

levels of NE in the preOptiC~anterior hypothalamic continuum. lengthened

84

the temporal sequence of copulation. 0n the other hand, carbachol and
hemicholinium treatment resulted in the arresting of sexual behavior.
Thus adrenergic systems appear to affect the temporal sequence of c0pula~

tion, where as the cholinergic regulate the activation of that sequence.

APPENDIX

85

APPENDIX: .METHODS

I. Subject Selection for all ExPeriments.

a) Subjects:

Adult male Long Evans rats, purchased from a commercial breeder
(Charles Rivers, Boston, Mass.) were used in all experiments. The
animals were 80-90 days of age at the beginning of testing, and were
maintained on food and water ad libitum, in a reversed light-dark cycle
of 14 hours light and 10 hours dark, with lights going off at 11:00 A.M.
Animals were housed 4—5 in a single 9x14x20 inch stainless steel cage.

Once implanted, however, they were transferred to individual 9x9x12 inch

stainless steel cages.

b) Testing procedure for masculine sexual behavior:

All tests for masculine sexual behavior were administered to.sub—
jects using a standard procedure. Individual male rats were placed in
an observation arena 3 minutes before commencement of the test to allow
the animal to adapt to the surroundings. At the start of the test, a
sexually receptive female was placed in the arena with the male.
Occurrence of sexual responses, mounts without intromissions (Mounts),
intromissions and ejaculations were recorded on an Esterline-Angus Event
Recorder. Unless otherwise noted, sexual behavior tests were continued
until one of the following criteria has been met: 1) occurrence of the
first intromission following the first ejaculation pattern; 2) 20

minutes has elapsed after the first intromission and no ejaculation

86
pattern has occurred; or 3) 20 minutes has elapsed after introduction

of the stimulus female and no intromission had occurred.

C) Screening for masculine sexual behavior:

Two weeks after arrival in the laboratory, all animals began a
series of 3, weekly tests for masculine sexual behavior. Males exhibi-
ting the ejaculatory response in two of the three screening tests were
retained for experimental testing. Animals meeting this selection cri-

terion were castrated within a week after completion of the third test.

Castration was done under ether anethesia, using a transscrotal approach.

Following castration, subjects in experiments I, II and III were main-
tained in the laboratory for one month without exogenous administration
of hormones. At the end of one month, these animals were given weekly
tests for sexual behavior until a criterion of 3 tests without an
ejaculation had been met by all animals. Once this criterion was
achieved, these males were assigned to one of the experimental proce- .
dures and subsequently implanted with stainless steel, double-walled
cannulae. In experiment IV, immediately after castration, all animals
were maintained on 150 ug TP daily for the entire experiment. All

males in experiment IV were also bilaterally implanted with double-

walled, stainless steel cannulae.
II. Cannulae Implantation Procedure.

a) Cannulae construction:

All animals were bilaterally implanted with double-walled stainless

steel cannulae. Each cannulae were made up of a larger outer guide

 

87

cannula (21 guage in experiments I and IV, 23 guage in experiments II and
III) permanently cemented in postition and an inner hollow removable
insert (26 and 27 guage in experiments I and IV, respectively and 30
guage in eXperiment II and II) which allowed for repeated chemical sti-
mulation. The outer guide cannulae were constructed in the laboratory
from 21 or 23 guage stainless steel hypodermic tubing. All guide can-
nulae used, were made equal in length so as to allow the use of a single
applicator insert for the administration of a chemical to all animals in
a particular treatment group.

Prior to the implantation, the two guide cannulae used in the
bilateral implantation, were fastened together with dental acrylic to
insure that the tubes remained parallel and the correct distance apart
during the implantation procedure. Each pair of connected guide can-
nulae were fitted with a pair of hollow inner cannulae or obdurators.
These remained in the guide cannulae throughout the eXperiment. The
obdurators were constructed in the laboratory from 26, 27 or 30 guage
stainless steel tubing. A small collar of outer guage tubing, about
4 mm in length, was crimped around the top of the obdurator tube to act
as a step, thereby preventing the obdurator from extending more than
1 mm beyond the end of the guide cannulae. A small lenght of poly-
ethalene intramedic tubing, 7—8 mm long, was fitted over both the collar
and t0p part of the guide cannulae, so as to make the step airtight.

The intramedic tubing comes off with the obdurator when the latter is
removed.

Chemicals were applied to specific neural loci by utilizing an
applicator made of two insert guage tubes. The applicator tubes were

soldered together so as to be the same distance apart as all the guide

 

88
cannulae used in a particular experiment. Small collars of outer guage
tubing were crimped around the applicator tubes to serve as stops,
which prevented the applicator from extending 5 mm beyond the tip of
all guide cannulae used in that treatment. The chemicals used were
applied to the brain in three different manners during the course of
this research. In experiment I, crystalline chemicals were loaded into
the lumen of the 26 guage applicator by tapping the ends of the tube into
a thin layer of the substance. For instance, 10 taps of the applicator
into testosterone loaded 15 ug of the hormone into each tube of the
applicator. Substances were ejected from this applicator directly to
the brain by passing a plunger through the lumen of the 26 guage appli-
cator. The plunger was fashioned out of 26 guage hypodermic needle
cleaning wire and was constructed so that the end did not extend more
than .25 mm beyond the tip of the applicator, thus expelling the
entire chemical pellet from the lumen of the applicator. In experiments
II and III the chemical treatments were applied directly to the neural
area by infusing a small amount of the chemical in solution through the
applicator. A Harvard Apparatus Infusion Pump hooked to an automatic
timer was used for the infusion of the solution. Two lines of intra-
medic tubing leading from two motor driven 2 cc surenges, were connected
to the 30 guage tubes of the applicator. The timer and pump were -
adjusted so that .005 ul of the solution would be expelled from the
applicator in 30 seconds. The use of chemical solutions allowed a
smaller guage of implant to be used, hence the 30 guage insert in
experiment II and II. In addition, it allowed the concentration and
PH of the treatment to be more rigidly controlled.

In experiment IV, 27 guage inserts were used as applicators. The

89 ,
lumen of the applicator was filled with powdered chemical as in experi—

ment I. However, the pellet of chemical was not pushed from the lumen
once the applicator was in place. Instead, the substance was allowed

to diffuse out of the lumen and into the surrounding tissue. Since

this experiment was done before II and III the solution, infusion method

was not used, although it is judged to be superior.

b) Chemical application:

In each application of a chemical to an individual animal, both
obdurators were removed from the unanethesized implanted animal and the
applicators inserted in their place. In experiment I with the appli—
cator in place, the plungers were pushed down, eXpelling the two pellets,
one into each hemisphere of the brain. The applicator was then removed
and the obdurator replaced until the next treatment administration. In
experiment II and III, with the applicator in place the infusion pump
was turned on, expelling .005 ul of solution/cannula into the brain.
The applicator was then removed and the obdurator replaced. In experi-
ment IV, the applicator, filled with chemical, was placed into the
guide cannulae and left there until the testing under that treatment

had finished. The obdurator was then replaced until the next treatment.

c) Implantation technique:

Implantation of the outer guide cannula was done under ether anes-
thesia with 108 mg of atrOpine sulfate being injected interperietally
20 minutes before the ether to prevent congestion during the 30 minute
operation. A KOpf Stereotaxic instrument was used for implantation.
The coordinates used for the placing of cannulae in the desired neural

area were taken from two stereotaxic atlases, Abel—Fessard, Stutinsky

90

and Libouban (1965) and Pellegrino and Cushman (1965). After histo—
logical verification of preliminary implants, minor adjustments necessary
to correct for strain differences were made to insure prOper cannulae
placement.

After securing the animal's head in the stereotaxic instrument,
the skin, muscle and fascia were cut and pushed aside to expose the
skull. At this point, four stainless steel screws were placed in the .
skull around the area of cannulae insertion. The screws served as an
anchor for the dental acrylic, which was used to fasten the implanted
cannulae to the skull. Each pair of guide cannulae was lowered with
the use of the stereotaxic instrument, through holes drilled equal dis-
tance on each side of the midline suture. The guide cannulae had
obdurators in them during implantation. After the cannulae were lowered
to the correct depth, dental acrylic was applied to and around the
scraws, cannulae and exposed skull, permanently securing the outer
guide cannulae in place. The remaining Open wound was closed and the

animal placed in an individual cage.

III. Histology

 

The neural location of cannulae implants was histologically veri-
fied after completion of each experiment. Following completion of an
experiment, all animals were perfused with saline and formalin solu—
tions, respectively. Animals were then decapitated and all skin
removed from the head. The brain was then extracted from the skull and
subsequently imbedded in a Paraplast block for sectioning. Brains were
(hit in a crossection plane, 30 um thick. Sections were mounted and

stained, using cresyl violet and methyl blue staining techniques.

91

Microsc0pic examination was then made to verify the site of implanta-
tion and also to determine the extent of any lesions made during the
experiment.

At the time of perfusion, the penises of some animals were also
removed. The penises were placed in Bowen's solution for one week,
subsequently imbedded in Paraplast and cut into crossections, 25 um
thick. The sections were placed on slides and stained with a hema-
toxylin and eosin procedure. After staining, microsc0pic examination
of the penises were made. The number of penile spines and length
of penile papillae were recorded from the dorsal half of the glands
penis. The number of penile spines from the dorsal half of the glands
were recorded from two different sections of the same penis. Similarly,
the length of papillae at the tap and both sides of the glands were
measured from two different sections. The data from these measurements
were used to determine if significant amounts of testosterone are

being released from the implant into the systemic circulation.

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