EFFECTS OF ESTROGEN ON THE FUNCTIONAL OUTPUT OF THE
BASAL GANGLIA





By





JEFFREY NEAL JOYCE


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






UNIVERSITY OF FLORIDA















ACKNOWLEDGEMENTS


In the completion of this dissertation and attainment of this

graduate degree, I was never truly alone. It would be difficult to express

all my true appreciation for everyone's help. I would like to thank

specifically those individuals that helped me learn how to think, as well

as write, as a scientist. Thanks go to Dr. William Luttge and Dr. Adrian

Dunn especially for their patience and helpful criticism over the last

few years. I would like to further thank my primary mentors for all their

help in forming me into a capable scientist: Dr. Merle Meyer, Dr. Neil

Rowland and Dr. James Simpkins.

In addition, tremendous support was given by my friends and my

colleagues Ron Smith, Leslie Chambers, Cathy Gonzales, Linda Bellush and

Janis Carlton. I also wish to express tremendous gratitude to Dr. Neil

Rowland, Dr. Merle Meyer and the Center for Neurobiological Sciences for

providing the necessary employment while pursuing my doctoral degree.

The research included in this dissertation was supported by a

Biomedical Research Grant to Dr. Carol Van Hartesveldt. I am pleased to

acknowledge my gratitude to Elsevier Biomedical Press, publisher of

European Journal of Pharmacology, for granting me permission to use the

following article in my dissertation:

J.N. Joyce, R.L. Smith and C. Van Hartesveldt. Estradiol suppresses
then enhances intracaudate dopamine-induced contralateral
deviation. European Journal of Pharmacology, Vol. 81, 1982,
pp. 117-122.











Finally, my dream to be a scientist and academician was carefully

nurtured and supported by a few very important people in my life.

Thanks go to Dr. Carol Van Hartesveldt for seeing me through the last

six years towards the realization of that dream. I send my love to my

closest friend and very special lady, Roberta Franks, whose love and

caring have been a joy and blessing for me. My love and thanks go most

of all to my parents, James and Maxine Joyce, who have always made a

difference. I send this to them as a late Father's Day and Mother's Day

present.













TABLE OF CONTENTS


CHAPTER PAGE

ACKNOWLEDGEMENTS ............................................ ii

ABSTRACT .................................................... vi

I GENERAL INTRODUCTION ........................................ 1

II EFFECTS OF ESTROGEN ON BRAIN DOPAMINE NEUROTRANSMITTER
SYSTEMS ..................................................... 5

Introduction ............................................. 5

Effects of Estrogen on the Tuberoinfundibular
Dopamine System .......................................... 8

Effects of Estrogen on the Mesostriatal Dopamine
Transmitter System............... ..... .................... 18

III EFFECTS OF ESTROGEN ON BEHAVIORS MEDIATED BY THE
MESOSTRIATAL DOPAMINE SYSTEM ............................... 32

Introduction .................................. ........ 32

Assessment of Central and Peripheral Actions
of Estrogen.............................................. 34

Functional Effect of Estrogen on Striatal
DA-Mediated Behaviors.................................... 38

Experiment 1. Estradiol Suppresses Then Enhances
Intrastriatal Dopamine-Induced Contralateral
Deviation................................................ 40

Experiment 2. Behaviors Induced by Intrastriatal
Dopamine Vary Independently Across the Estrous
Cycle...... ....... ................ .. ...... .............. 59

Experiment 3. Behaviors Induced by Intrastriatal
Dopamine are Suppressed Differentially by
Estradiol Benzoate....................................... 82

Experiment 4. Intrastriatal Implant of Estradiol
Suppresses Apomorphine-Induced Postural Deviation......... 121

IV GENERAL DISCUSSION.......................................... 147

Experimental Studies..................................... 147










PAGE

Eztrapyramidal Disorders and Estrogen ................. 154

REFERENCES................................................ 161

BIOGRAPHICAL SKETCH ........................................ 188















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



EFFECTS OF ESTROGEN ON THE FUNCTIONAL OUTPUT OF THE
BASAL GANGLIA

By

Jeffrey Neal Joyce

August, 1983

Chairman: Carol Van Hartesveldt
Major Department: Psychology


The present experiments were conducted to analyze the effects of

estrogen on basal ganglia function in rats. Treatment with different

doses of estrogen produced different effects on intrastriatal dopamine-

induced postural deviation. Pharmacological doses of estrogen produced

an initial suppression, followed by an enhancement of postural deviation

in response to intrastriatal dopamine. In the second experiment, it was

found that the magnitude of the intrastriatal dopamine-mediated behaviors,

postural deviation and rotation, varied with changes in serum levels of

estrogen across the estrous cycle of the rat. Both were suppressed when

plasma titers of estrogen should be high (proestrus) and enhanced when the

levels should be low (estrus, diestrus days 1 and 2). However, when moni-

tored across the day of proestrus the postural deviation and rotation varied

independently in magnitude. Moreover, neither behavior was enhanced when

serum levels of estrogen should be low. Consistent with those findings,

systemic administration of a small dose of estradiol benzoate to









ovariectomized rats produced suppression, without later enhancement of

intrastriatal dopamine-mediated postural deviation and rotation.

The behaviors induced by dopaminergic stimulation of the striatum

were differentially modulated by estrogen. Postural deviation was

suppressed within 1/2 hour of one treatment with estradiol benzoate.

Rotation was also suppressed, but the latency was longer and required

that two treatments with estradiol benzoate be made. In contrast, loco-

motor activity mediated by ventral striatal dopamine was not suppressed

with estradiol benzoate treatment, and may require estrogen for a normal

response to dopaminergic stimulation.

Direct application of estrogen to the dorsal striatum suppressed

postural deviation in response to apomorphine. The sites for this estrogen

effect occur in the same region in which application of dopamine produces

postural deviation. Estrogen did not produce a nonspecific suppression

of the striatum. Increasing doses of apomorphine reduced, and at the

highest dose completely reversed, the suppressive effects of estrogen. The

specificity of estrogen's effect was also suggested by comparison of the

effects of estrogen with 17a-estradiol; the implantation of 17a-estradiol

into the striatum did not suppress apomorphine-induced behavioral activity.

Additionally, the estrogen receptor antagonist CI-628 blocked estrogen's

suppression of striatal dopamine-mediated postural deviation.















CHAPTER I

GENERAL INTRODUCTION


The proposal that estrogen might regulate the functional output

of the basal ganglia was based initially on observations in human patients

suffering from disorders of DA systems in the basal ganglia (Bedard

et al., 1979; Villeneuve et al., 1978; Donaldson et al., 1978; Zegart

and Schwartz, 1968; Bickerstaff, 1975; Nausieda et al., 1979a). The

response of those patients to drugs that act on DA systems of the basal

ganglia was affected by their sex and hormonal condition (e.g., receiving

estrogen). The proposal was strengthened by research in the early 1970's

that indicated that gonadal hormones had biochemical effects on DA

systems of the basal ganglia in animals (Jori and Cecchetti, 1973;

Holzbauer and Youdin, 1973; Greengrass and Tonge, 1974). More recent

behavioral and biochemical studies in animals have extended those findings

emphasizing the diverse consequences of gonadal hormone treatment on

the DA systems of the basal ganglia. Unfortunately, no consensus has

been reached regarding the precise nature of the actions of estrogen on

the DA systems of the basal ganglia; support for suppressive and

enhancing effects of estrogen has been documented in both human patients

(e.g., Bedard et al., 1979; Nausieda et al., 1979a) and laboratory animals

(e.g., Becker et al., 1982; Bedard et al., 1983; Gordon, 1980). Nor

has it been established where the estrogens act to alter the DA systems

of the basal ganglia. Cell bodies of the DA fibers terminating in the

basal ganglia and DA-sensitive neurons in the basal ganglia do not









concentrate [3H]estradiol intracellularly (Heritage et al., 1980; McEwen,

1979; Stumpf and Sar, 1976). Therefore, there has been a tacit assump-

tion that estrogen is exerting its effects on the basal ganglia through

actions elsewhere. However, the 3H-steroid autoradiographic method of

locating estrogen receptors is not an infallible guide in deciding where

in the brain to look for the actions of estrogen (McEwen, 1980; McEwen

et al., 1982). Not all brain areas where steroid receptors can be

demonstrated by biochemical means contain estrogen-concentrating cells

which can be visualized by autoradiography (McEwen et al., 1982). Con-

sequently, it is not clear whether estrogen interacts with DA in the

striatum to mediate behavior.

In order to determine the nature of the dopamine-estrogen interaction

in modulating the behavioral output of the striatum, several experiments

were designed. In order to evaluate the hypothesis that estrogen can

directly modify the actions of DA in the striatum, DA will be applied intra-

cerebrally while plasma levels of estrogen are modified. First,

unilateral injections of DA into the anterior-dorsal striatum result in a

contralateral asymmetry of the animal's ongoing behaviors, which can be

quantified by measuring the amount of time the animal spends producing

behaviors to the side contralateral to the intrastriatal injection (Joyce

et al., 1981). The behavioral response produced by the intrastriatal

injection of DA, termed postural deviation, is a highly localized response

(Joyce et al., 1981) which makes it useful for examining the effects of

estrogen. Since the estrogen treatment-test interval is considered to be

an important variable (Gordon, 1980), the effects of a single treatment

with EB on the amount of contralateral postural deviation elicited by

unilateral intrastriatal injection of DA will be tested at 2 and 6 days









after hormone treatment in male rats. These two time points were chosen

because the literature indicated that treatment with large doses of EB

or EV induced an initial suppression of DA agonist effects lasting 24 to

48 hours (Gordon, 1980; Gordon et al., 1980) and a clear enhancement by

6 days post treatment (Hruska and Silbergeld, 1980a; 1980b).

In previous studies, nonphysiological doses of estrogen were used,

and it is of concern that only pharmacological responses to estrogen were

measured. In order to test the effects of natural fluctuations of plasma

levels of estrogen on dopamine-mediated functional output of the basal

ganglia, the effects of intrastriatal dopamine on behavior will be measured

throughout the estrous cycle of the female rat. The day of proestrus

will be examined in greater detail because it shows the greatest changes

in plasma levels of estrogen. Both dopamine and amphetamine will be

injected intrastriatally, and both postural deviation and rotation will

be measured.

In a further test of the effects of physiological doses of estrogen

on intrastriatal dopamine-mediated behaviors, female rats will be

ovariectomized and administered a small dose of estrogen. Both dopamine

and amphetamine-induced postural deviation and rotation will be measured

at several time points after the treatment with estrogen. The specificity

of the estrogen effects on behavior will be tested by administering an

anti-estrogen.

Finally, the technique of intracerebral application of estrogen

has proved to be valuable in determining where in the brain estrogen acts

to modify sexual behavior (e.g., Lisk, 1962; Davidson, 1972) and

gonadotropin release (e.g., Ramirez and McCann, 1964). In this experiment,

the intracerebral cannulation method will be utilized to investigate









characteristics of estrogen action in the striatum. The behavioral

measure, postural deviation, will be used to assess the effects of

estrogen in the striatum. Postural deviation can be induced by altering

the balance of dopaminergic activity between the two striata. If

estrogen can directly alter the responsiveness of DA-sensitive neurons

in the striatum, then unilateral application of estrogen to the striatum

will result in postural deviation. In order to increase the sensitivity

of this method, a DA agonist, APO, will be given which acts post-

synaptically on DA-sensitive neurons in the striatum when administered

systemically. Estrogen's alteration in the responsiveness of DA-

sensitive neurons in the striatum will be monitored by measuring (1) the

direction of the postural deviation, and (2) the potency of the effect.

To determine the potency of estrogen's effects different doses of APO were

tested. Examination of the regional specificity of estrogen's actions

will also be initiated in order to determine whether estrogen was acting

directly in the striatum. Since intrastriatal DA-induced postural

deviation is elicited from a circumscribed region of the anterior dorsal

striatum (Joyce et al., 1981), it will be determined if the effects of

estrogen are also limited in this region. The specificity of estrogen's

effects will be detailed by comparing the effects of E2 with cholesterol

and 17cL-E2.














CHAPTER II

EFFECTS OF ESTROGEN ON BRAIN DOPAMINE
NEUROTRANSMITTER SYSTEMS


Introduction

Recent research suggests that dopamine (DA) containing fibers

originating in the midbrain and terminating in the basal ganglia, and/or

DA-sensitive cells in the basal ganglia, are subject to regulation by

estrogen. It is unclear how this regulation takes place because this

region does not contain cells that concentrate estrogen. Hypothalamic

neural systems thought to play a major role in the mediation of repro-

ductive behavior and gonadotropin secretion are also modulated by estrogen,

and the mechanisms by which estrogen acts to mediate these functions

have been extensively studied. The guiding hypothesis, developed over

the last 20 years, has been that estrogen acts in the brain by genomic

mechanisms, involving intracellular steroid receptors (see Luttge, 1983;

McEwen, 1979; 1980).

Four areas of research catalyzed the development of this hypothesis

with respect to estrogen-dependent behaviors. First crystalline hormone

was implanted into particular brain regions to identify sites of action

of systemic hormone treatment (Davidson, 1972; Davis et al., 1979; Lisk,

1962; Luttge, 1976; see also McEwen, 1979; McEwen et al., 1979). Second,

3H-steroids were used to delineate biochemically the temporal and regional

profiles of hormone uptake and retention in the brain, followed by the

characterization of the cytosol and cell nuclear receptors (Eisenfeld









and Axelrod, 1965; Kahwanago et al., 1970; McEwen and Pfaff, 1970;

McEwen et al., 1975). Subsequent work, utilizing 3H-steroid auto-

radiography, mapped the brain for cell groupings containing high levels

of steroid receptors (Stumpf and Sar, 1976; Pfaff and Keiner, 1973;

McEwen, 1979). Third, to provide evidence that estrogen receptor-

mediated genomic events could underlie reproductive behavior and

gonadotropin secretion, antiestrogens, and biosynthetic inhibitors of

RNA and protein synthesis, have been used to determine the time course

of estrogen-dependent changes in genomic expression (McEwen et al.,

1975; Quadagno and Ho, 1975; Rainbow et al., 1980; Roy and Wade, 1977;

Terkel et al., 1973; Whalen et al., 1974; Morin et al., 1976). Fourth,

estrogen induction of gene products in neural tissue has been examined,

providing a rationale for the mechanism by which estrogen could mediate

reproductive behavior and gonadotropin secretion (see below; Luttge,

1983; McEwen et al., 1981, 1982).

Since the hypothalamus-preoptic area contains high concentrations

of estrogen receptors ([3H]estradiol binding sites, McEwen, 1979; Pfaff

and Keiner, 1973; Stumpf and Sar, 1976), it has been the focus of much

of the research on estrogen's actions upon neural tissue. An integrated

picture, at least with respect to certain aspects of reproductive

behavior and control of gonadotropin secretion, has begun to emerge that

is consistent with the hypothesis that estrogen acts through receptor-

mediated genomic mechanisms to alter neural events (see McEwen et al.,

1979, 1982). For example, estrogen is known to alter catecholamine

release from hypothalamic tissue (Becker and Ramirez, 1980; Paul et al.,

1979); modulate the number of noradrenergic (Vacas and Cardineli, 1980;

Wilkinson et al., 1979) and serotonergic receptors in the hypothalamus









(Biegon et al., 1980; Biegon and McEwen, 1982; Biegon et al., 1982);

and increase the accumulation of cyclic AMP in hypothalamic tissue

(Gunaga and Menon, 1973; 1974; Paul and Skolnick, 1977; Weissman and

Johnson, 1976). However, even in the hypothalamus there are neural

effects of estrogen, such as the modification of neuronal firing (Kelly

et al., 1976; 1977a; 1977b; 1978; 1980; Poulain and Carette, 1980),

which are so rapid as to preclude genomic mediation (see also; McEwen

et al., 1981; 1982). This revelation has become increasingly important

as evidence has accumulated which suggests that extrahypothalamic regions

are also sensitive to the effects of estrogen, even regions which appear

to contain no intracellular receptors for estrogen.

The proposal that estrogen might regulate the functional output

of the basal ganglia was based initially on observations in human patients

suffering from disorders of DA systems in the basal ganglia (Bgdard

et al., 1979; Villeneuve et al., 1978; Donaldson et al., 1978; Zegart

and Schwartz, 1968; Bickerstaff, 1975; Nausieda et al., 1979a). The

response of those patients to drugs that act on DA systems of the basal

ganglia was affected by their sex and hormonal condition (e.g.,receiving

estrogen). The proposal was strengthened by research in the early 1970's

that indicated that gonadal hormones had biochemical effects on DA

systems of the basal ganglia in animals (Jori and Cecchetti, 1973;

Holzbauer and Youdin, 1973; Greengrass and Tonge, 1974). More recent

behavioral and biochemical studies in animals have extended those findings

emphasizing the diverse consequences of gonadal hormone treatment on

the DA systems of the basal ganglia. Unfortunately, no consensus has

been reached regarding the precise nature of the actions of estrogen on

the DA systems of the basal ganglia; support for suppressive and







8

enhancing effects of estrogen has been documented in both human patients

(e.g., Bedard et al., 1979; Nausieda et al., 1979a) and laboratory animals

(e.g., Becker et al., 1982; Bedard et al., 1983; Gordon, 1980). Nor

has it been established where the estrogens act to alter the DA systems

of the basal ganglia. Cell bodies of the DA fibers terminating in the

basal ganglia and DA-sensitive neurons in the basal ganglia do not

concentrate [3H]estradiol intracellularly (Heritage et al., 1980; McEwen,

1979; Stumpf and Sar, 1976). Therefore, there has been a tacit assump-

tion that estrogen is exerting its effects on the basal ganglia through

actions elsewhere. However, the 3H-steroid autoradiographic method of

locating estrogen receptors is not an infallible guide in deciding where

in the brain to look for the actions of estrogen (McEwen, 1980; McEwen

et al., 1982). Not all brain areas where steroid receptors can be

demonstrated by biochemical means contain estrogen-concentrating cells

which can be visualized by autoradiography (McEwen et al., 1982). Con-

sequently, several questions concerning the role of estrogen in the

mediation of DA systems in the basal ganglia remain unresolved. Before

introducing the experiments designed to resolve some of these issues,

evidence will be presented that estrogen can modulate brain DA systems.

In this paper the term estrogen will refer to estradiol (E2), unless

otherwise specified, because it is the principal ovarian estrogen and

has been the most systematically studied (Luttge, 1983).

Effects of Estrogen on the Tuberoinfundibular Dopamine System

Evidence for estrogen-induced alteration of the release of prolactin,

from the pituitary gland, through actions on the hypothalamic tubero-

infundibular DA (TIDA) neurotransmitter system has had a significant

impact on much of the research directed at understanding how estrogen









acts on extrahypothalamic DA systems. DA-containing neurons of the

arcuate nucleus and adjacent periventricular zone project a dense

terminal system to the external layer of the median eminence, in close

apposition to the hypophyseal portal capillaries (Moore and Johnston,

1982; Sawyer and Clifton, 1980). There is considerable support for the

hypothesis that DA is released from the TIDA into the hypophyseal portal

blood system, and is carried to the anterior pituitary gland, where it

acts to inhibit tonically the release of prolactin (PRL) (Boyd and

Reichlin, 1978; MacLeod, 1976; Neill, 1980). This system appears to

operate as a classic short feedback loop. Conditions which result in

elevated plasma levels of PRL lead to an enhancement in activity of the

TIDA and a resulting suppression of PRL release (Anderson et al., 1981;

Perkins and Westfall, 1978; Annunziato and Moore, 1978; Fuxe et al.,

1977; Gudelsky et al., 1976). During the estrous cycle of the rat,

fluctuations in the concentration of PRL in plasma are correlated with,

and apparently stimulated by, similar fluctuations in the concentration

of estrogen (Boyd and Reichlin, 1978; MacLeod, 1976; Neill, 1980). These

two lines of evidence have prompted investigators to speculate that the

estrogen-induced surge in the release of PRL that occurs on the after-

noon of proestrus (Butcher et al., 1974; Smith et al., 1975; Demarest

et al., 1981a; Anjika et al., 1972; Neill et al., 1971) might be mediated

by actions of estrogen on the TIDA (Crowley et al., 1978a; Ben-Jonathan

et al., 1977; Demarest et al., 1981a; Simpkins et al., 1979), and of

estrogen on DA's actions upon PRL containing cells of the anterior

pituitary gland (Heiman and Ben-Jonathan, 1982a; 1982b; Labrie et al.,

1979; 1980).









Estrogen's Suppression and Enhancement of the TIDA

It has been assumed that during proestrus the increased concen-

tration of estrogen antagonizes the tonic inhibitory actions of the

TIDA, which results in an increase in the secretion of PRL. This is

then followed by a decline in the concentration of estrogen, a

resurgence in the inhibitory effects of the TIDA, and a decrease in the

secretion of PRL. This proposed scheme is based primarily on the

evidence that, during the estrous cycle in the rat, TIDA activity is

correlated inversely with the concentration of estrogen in serum (Ben-

Jonathan et al., 1977; Crowley et al., 1978a; Cramer et al., 1979;

Demarest et al., 1981a; Fuxe et al., 1977; Simpkins et al., 1979). The

concentration of estrogen in serum of rats increases dramatically on

the morning of proestrus, with a peak surge at noon, then a rapid de-

cline during the afternoon of proestrus (Butcher et al., 1974; Smith

et al., 1975). Direct measurement of the concentration of DA in the

hypophyseal portal blood of cyclic rats indicates that concentrations

are lowest at noon of proestrus and highest on the morning of estrus;

that is correlated inversely with the concentration of estrogen in

systemic blood (Ben-Jonathan et al., 1977; Cramer et al., 1979). Con-

sistent with the direct measure of DA release from TIDA is the evidence

that the activity of tyrosine hydroxylase (TH) in the median eminence

is low on the morning of proestrus and increases significantly between

then and the morning of estrus (Crowley et al., 1978a; Demarest et al.,

1981a). Measures of the turnover of DA in the median eminence indicate

low activity of the TIDA on proestrus, with a rapid rise in activity by

the morning of estrus (Demarest et al., 1981a; Fuxe et al., 1977). This

would indicate that during the estrous cycle when estrogen levels are









high, there is a suppression of the TIDA; and when they are lower,

there is a rebound in the activity of the TIDA.

Consistent with the hypothesis that estrogen suppresses the

activity of the TIDA, ovariectomy or castration produces an increase

in the activity of the TIDA (Honma and Wuttke, 1980; Kizer, 1978), which

can be reversed with a single treatment of 50 pg estradiol benzoate (EB)

(Crowley, 1982; Cardinali and Gomez, 1977). However, if measures of DA

turnover are taken at 12 or more hours after an acute treatment with

EB (Crowley, 1982; Fuxe et al., 1977b; L6fstr6m et al., 1977; Mansky

et al., 1982; Simpkins et al., 1979), or with chronic EB treatment

(Beattie et al., 1972; Demarest and Moore, 1980; Endr6czi and Szab6,

1973; Tobias et al., 1981; Dupont et al., 1981; Eikenburg et al., 1977;

Wiesel et al., 1978), there is an increase in activity of the TIDA. It

has been postulated that the increase in TIDA activity that occurs at

long intervals after an acute injection of EB, or with chronic admin-

istration of EB, is due to a compensatory feedback loop mediated by PRL

(Demarest and Moore, 1980; Fuxe et al., 1977b; Gudelsky et al., 1976), an

hypothesis supported by several lines of evidence. First, the increase

in activity of the TIDA, induced by EB, is correlated with increases in

the concentration of PRL in serum (Crowley, 1982; Fuxe et al., 1977b;

Mansky et al., 1982). Secondly, hypophysectomy, which removes the source

of PRL, abolishes both estrogen- and DA antagonist-induced increases in

turnover of the TIDA(Gudelsky et al., 1976; Demarest and Moore, 1980;

Eikenburg et al., 1977; Perkins et al., 1979). Third, TIDA activity is

accelerated by systemic or cerebroventricular injection of PRL

(Annunziato and Moore, 1978; Fuxe et al., 1977; Gudelsky et al., 1976),

or by chronically increasing serum levels of PRL (Hohn and Wuttke, 1978;









Krieger and Wuttke, 1980; Morgan and Herbert, 1980; Perkins et al.,

1979). Fourth, it is now evident that PRL could act directly on the

TIDA neurons to alter their activity (Gudelsky et al., 1978; Poulain

and Carette, 1976; Yamada, 1975; Walsh et al., 1978). Recent studies

show that PRL, like other anterior pituitary gland hormones, is able

to reach the brain via retrograde portal blood flow (Bergland and Page,

1978). The results of these studies support the hypothesis that a

prolactin-mediated feedback loop could be involved in the estrogen-

induced enhancement of activity in the TIDA. The enhancement would,

of course, follow the estrogen-induced suppression of the TIDA that is

involved in the elevated plasma levels of PRL, and be consistent with

the proposal that estrogen controls the fluctuations in the TIDA across

the estrous cycle.

Effects of Estrogen in the Pituitary

While evidence presented in the preceding paragraphs does indicate

that estrogen can alter the secretion of PRL by modulating the activity

of the TIDA, it is also evident that there are effects of estrogen on

the pituitary gland. Estrogens have long been known to directly cause

hypertrophy of the anterior pituitary gland and an increase in the

synthesis of PRL (Ajika at al., 1972; Kanemaster and Sawyer, 1963; Neill

et al., 1971; Nicol and Meites, 1962; Ramirez and McCann, 1964). Con-

sistent with the presumed genomic mechanism of action by estrogen

(McEwen et al., 1979; McEwen, 1980), it requires many hours for these

effects of estrogen to become apparent (MacLeod et al., 1969; Neill et al.,

1971). It is also clear that this is not the sole means by which

estrogen causes an increase in synthesis and secretion of PRL; more

rapid effects are now well established that involve antagonism of the









PRL-suppressing effects of DA. DA receptors located on the cells of

the anterior pituitary gland, thought to mediate the inhibitory actions

of DA on the PRL secreting cells (Labrie et al., 1979; 1980; Cronin,

1982), fluctuate in number during the estrous cycle (Heiman and Ben-

Jonathan, 1982a), and can be altered with systemic administration of EB

(Heiman and Ben-Jonathan, 1982b). As determined by [3H]spiroperidol

binding, elevated serum levels of estrogen result in a decrease in the

density of DA receptors, and a consequent decrease in the ability of DA

to inhibit the secretion of PRL (Heiman and Ben-Jonathan, 1982b). There

appears to be no change in the affinity of these binding sites. The

estrogen-induced decrease in [3H]spiroperidol binding to anterior

pituitary gland is probably not due to direct competition of estrogen

with DA receptors, since estrogen does not have any significant affinity

for pituitary gland DA receptors (Paden et al., 1982; Schaeffer and Hsueh,

1979). Moreover, estrogen's antagonism of the DA-inhibitory effect on

PRL secretion in vivo and in vitro is noncompetitive (Ferland et al., 1979;

Gudelsky et al., 1981; Nansel et al., 1981; Labrie et al., 1979; 1980).

While admittedly a still highly speculative idea, there is in-

creasing evidence that steroids may have membrane receptors which can

mediate some of their cellular effects (Pietras and Szego, 1977; Szego,

1978). It is possible that some of the short-term effects of estrogen

may be mediated by a membrane receptor (Zyzek et al., 1981), and could

include changes in the membrane properties of the PRL- secreting cells

that lead to a reduction in the effectiveness of DA (Dufy et al., 1979b).

Several lines of evidence indicate this possibility and suggest that

more than one receptor-mediated event could underlie the suppression of

the effect of DA. Pretreatment with estrogen for one hour markedly









suppresses the responsiveness of anterior pituitary gland cells to the

inhibitory actions of DA agonists in vivo (Ferland et al., 1979;

Gudelsky et al., 1981; Nansel et al., 1981) and in vitro (Labrie et al.,

1979; 1980). It has also been shown, with anterior pituitary gland cells

in culture, that incubation with DA consistently results in inhibition

of electrical activity on the lactotropes; and this effect can be

antagonized by very low concentrations of estrogen (0.1 nM) within

milliseconds of estrogen application (Dufy et al., 1979b). Extracellular

iontophoresis of estrogen onto anterior pituitary gland cells (Dufy et al.,

1979a) or neurons in the anterior hypothalamus (Kelly et al., 1976; 1977a;

1977b; 1978; Poulain and Carette, 1981) produces immediate changes in

electrical activity of these neurons. The results of these studies

indicate that E2 is acting at a specific membrane receptor since

(1) the effect of E2 is immediate; (2) the effect is not due to non-

specific actions of the injection procedure itself, e.g., changes in

current flow or pH; and (3) the selectivity for E2 was shown, since the

inactive isomer of E2, 17 c-E2, is ineffective in producing changes.

Recently, Kelly and associates (1980) have been able to record intra-

cellularly from neurons of hypothalamic slices. They have shown that

E2, which is biologically much more active than estrone (El) in other

tissues, is also much more effective than El in altering the electrical

activity of the hypothalamic neurons. Therefore, estrogen may inhibit

the TIDA by altering the activity of neurons that synapse on TIDA neurons,

or perhaps by modulating the electrical activity of the TIDA neurons

directly.

Estrogenic Effects of the Catechol Estrogens

A major route of estrogen metabolism in brain and pituitary gland

tissue leads to the formation of 2- and 4-hydroxylated estrogens (Ball









and Knuppen, 1980; Paul et al., 1980). Several observations have led

to the proposal that the formation in situ of these "catechol"

estrogens could provide a direct biochemical link between estrogens,

inductions of PRL secretion and suppression TIDA function (Fishman, 1981).

First, 2-OH-E2 administered subcutaneously can induce the secretion of

PRL in ovariectomized rats (Adashi et al., 1980; Shin et al., 1981;

Yanai and Nagasawa, 1979; Rodriguez-Sierra and Blake, 1982). Conversion

of estrogen (E2) to either 2-OH-E2 or 4-OH-E2 reduces its affinity for

the estrogen receptor (Duax et al., 1983; Davies et al., 1975; Ball and

Knuppen, 1980; Merriam et al., 1980; Fishman, 1981) and, therefore, its

potency in eliciting estrogen receptor-mediated effects (Ball and

Knuppen, 1980; Fishman, 1981; Luttge and Jasper, 1977; Naish and Ball,

1981).

Secondly, by virtue of their catechol structure, 2- and 4-OH-E2 are

capable of directly interacting with the hypothalamic TIDA system

(Duax et al., 1983), being potent inhibitors of TH activity (Foreman

and Porter, 1980; Lloyd and Weiz, 1978; Lloyd and Ebersole, 1980). It is

hypothesized that under conditions where high concentrations of the

catechol estrogens could be maintained, it is possible that a significant

reduction in DA synthesis would occur (Ball and Knuppen, 1980; Hiemke

and Ghraf, 1982), and be a means by which estrogen could suppress TIDA

function. Consistent with this hypothesis, there are several lines of

direct evidence that it is the catechol derivatives of E2, and not E2

itself, that suppress the activity of the TIDA. For example, ovariec-

tomized rats treated with 4-OH-E2 (100 lg), but not with an equimolar

dose of ethenylestradiol (EE2, 100 pg), showed a reduction in activity

of the TIDA (Hiemke and Ghraf, 1982). Furthermore, although 17 a-E2









has very low affinity for the intracellular estrogen receptor the 17

a isomers of 2- and 4-OH-E2 are effective inhibitors of hypothalamic TH

activity (Hersey et al., 1982).

Third, it is extremely unlikely that 2- and 4-OH-E2 act in the

pituitary gland to antagonize the effects of TIDA, since the catechol

estrogens have low affinity for the pituitary gland DA receptor (Paden

et al., 1982; Schaeffer and Hsuech, 1979). Additionally, the latency

between the administration of 2-OH-E2 and the induction of PRL secretion

is longer than with E2 (Adashi et al., 1980; Shin et al., 1981; Yanai

and Nagasawa, 1979), and the latency with 2-OH-E2 is not affected by E2

pretreatment (Shin et al., 1981). This suggests it is not DA receptor

blockade that is the site of action of 2-OH-E2 induction of PRL secretion.

This is further substantiated by the finding that superfusion of the

pituitary gland in vitro with 2-OH-E2 inhibits, not enhances, PRL release

and fails to antagonize DA-induced suppression of PRL secretion (Linton

et al., 1981).

These findings would suggest that the most likely site of action of

the catechol estrogens, for inducing PRL secretion, is suppression of

TIDA activity through inhibition of TH (Ball and Knuppen, 1980), but

there are some inconsistencies in the literature. There are no reports

that 2-OH-E2 alter the activity of the TIDA (Heimke and Ghraf, 1982), and

two groups have failed to find any evidence that the 2-hydroxylated

catechol estrogens have any effects on serum PRL levels in women (Franks

et al., 1981; Merriam et al., 1981). It may be that the 4-hydroxlated

"catechol" estrogens, which are more potent than 2-hydroxylated estrogens

on a number of comparative tests for their estrogenic effects (Ball and

Knuppen, 1980; Franks et al., 1980; Merriam et al., 1980), might mediate









some of estrogen's effects on the TIDA (Ball and Knuppen, 1980;

Hiemke and Ghraf, 1982; Kirchoff et al., 1981).

Summary. Considerable progress has been made in the past 10 to 15

years on determining the interaction of estrogen with hypothalamic

neurotransmitter systems, and much of the attention has been focused on

estrogen's modulation of the TIDA. In part, this emphasis has been due

to the relatively good evidence that the TIDA is the principle system

inhibiting PRL secretion (Boyd and Reichlin, 1978; MacLeod, 1976; Neill,

1980), and thus is logically a site for estrogen's active modulation of

PRL secretion. There is sufficient evidence to propose that estrogen

can suppress the activity and/or actions of the TIDA, as a part of the

mechanism by which estrogen enhances PRL secretion. The suppression

of the TIDA system appears to occur at several levels or mechanisms.

Estrogen can inhibit the activity of TH (directly or indirectly) in

TIDA neurons, inhibit release of DA, and could alter electrical

activity of the TIDA neurons either trans-synaptically or directly.

Following the suppression of the TIDA system by estrogen, the result-

ing increase in secretion PRL leads to an enhancement in the activity

of the TIDA.

Estrogen also has important "postsynaptic" effects on the DA-sensitive

PRL-secreting cells of the anterior pituitary gland. These effects can

be broken down by their latency to produce (1) increased synthesis of

PRL, which requires 24 hours from an injection of estrogen; (2) an

alteration of DA receptor number, with a latency of many hours; (3) non-

competitive antagonism of DA on PRL-secreting cells, with a latency of an

hour or more; and (4) an almost immediate change in the electrical response

of the anterior pituitary gland cells to DA, possibly through estrogen's









actions at a membrane receptor. Additionally, systemic administration

of DA produces rapid and extensive changes in lactotrope ultrastructure

(Reifel et al., 1983; Gudelsky et al., 1980). These changes are thought

to underlie some aspects of DA inhibition of PRL release, and are re-

duced by pretreatment with estrogen (Gudelsky et al., 1981).

A hormone, by definition, is a substance released into the blood-

stream, to reach and act at distant targets. In that context, the TIDA

system is a hypothalamic hormone system (Meites and Sonntag, 1981).

Since interactions between hormones is a well established concept, it

was not unreasonable for researchers to investigate interactions between

estrogen and the TIDA system. Extension of these concepts to extra-

hypothalamic DA systems has been considerably slower. This is indicated,

for example, by the use of extrahypothalamic DA systems as control tissue

in studies that have investigated the effects of estrogen on the activity

of hypothalamic DA systems (e.g., Demarest and Moore, 1980; Eikenburg

et al., 1977; Crowley et al., 1978). This is probably due, in part, to

the considerable differences in the anatomy and physiological regulation

of the TIDA and extrahypothalamic DA systems (Moore and Johnson, 1982;

Moore and Wuerthele, 1979). Recent research does suggest, however, that

the fact that estrogen alters the activity of the TIDA, and the means by

which it does, are relevant to extrahypothalamic DA systems.

Effects of Estrogen on the Mesostriatal
Dopamine Transmitter System

The DA-containing cell bodies of the midbrain substantiala nigra and

ventral tegmental area) give rise to long ascending axons that terminate

in circumscribed regions of the forebrain (Lindvall and Bjorklund, 1978;

Moore and Bloom, 1978). These DA projection systems are referred to as

the mesostriatal (nigrostriatal), mesolimbic and mesocortical systems.









The mesostriatal DA system projects predominantly to the dorsal caudate-

putamen (dorsal striatum) in the rat (Fallon and Moore, 1978; Fallon

et al., 1978). The mesolimbic system terminates in the region referred

to as the ventral striatum (Fallon et al., 1978; Heimer, 1978), which

in the rat includes the ventral caudate-putamen, nucleus accumbens and

olfactory tubercle. The mesocortical system terminates in the frontal

cortex and cingulum (Moore and Bloom, 1978). It is now common to refer to

those regions in which the DA system terminate by the name of the pro-

jection system, i.e., mesostriatal (dorsal striatum), mesolimbic (ventral

striatum) and mesocortical (frontal cortex) regions.

Effects of Estrogen on Presynaptic Components of the Mesostriatal DA System

Although it is well established that estrogen can modulate the

activity of the TIDA, it has become apparent only recently that estrogen

can alter the activity of the mesostriatal DA system. This was first

suggested by the reports of sex differences in striatal DA content and

turnover in rats. Gordon and Shellenberger (1974) found that DA levels

were higher in whole striata of female than male rats. However, two

other groups report lower striatal DA levels in female rats (Crowley

et al., 1978b; Greengrass and Tonge, 1974), and amphetamine (AMPHET)-

induced efflux of DA from striatum in vivo and in vitro is greater in

female than male rats (Becker and Ramirez, 1981b; Robinson et al., 1980).

Further support for the hypothesis that estrogen modulates the meso-

striatal DA system are the reports of variations in striatal DA content

and turnover across the estrous cycle of rodents. Striatal DA levels

are highest, and DA turnover lowest, on the day of proestrus (high plasma

estrogen levels), with a significant increase in turnover by 12-24 hours

after the proestrus surge in estrogen (Crowley et al., 1978a; Jori et al.,









1976; Jori and Cecchetti, 1973; Holzbauer and Youdim, 1973). Additionally,

amphetamine-induced efflux of DA from striatal tissue in vitro is low-

est on proestrus and highest by estrus (Becker and Ramirez, 1981b).

These studies, taken together, would suggest that increasing serum levels

of estrogen result in a suppression of activity of the mesostriatal

DA system.

Attempts to determine whether estrogen treatment itself alters the

activity of the mesostriatal DA system have resulted in contradictory

reports. If measurement occurs within 6 to 18 hours of acute or chronic

treatment with EB, E2 in saline or synthetic estrogens (mestranol, EE2),

there is a decrease in TH activity coupled with an increase in DA con-

tent of striatal tissue (Crowley, 1982; DiPaolo et al., 1982b; Dupont

et al., 1981; Greengrass and Tonge, 1974; Gordon et al., 1977; Lzfstrim

et al., 1977). If, however, the latency from treatment is less than

3 hours (Crowley, 1982; Wiesel et al., 1978) or more than 24 hours

(Becker and Ramirez, 1981b; Crowley, 1982; Crowley et al., 1978c; Gordon

et al., 1977) then alterations in the activity of the mesostriatal DA

system are not observed.

Although the discrepancies between those reports could be due to

several variables, three methodological issues are of particular relevance.

First, the interval between hormone treatment and measurement of meso-

striatal DA activity is probably critical. The activity of the meso-

striatal DA system can be modified rapidly through several feedback

mechanisms (see Moore and Wuerthele, 1979), and any alterations in

synthesis or release of DA induced by low doses of estrogen would exist

for only a short time after termination of treatment. Additionally, by

12 hours after estrogen administration high serum levels of PRL are









typically observed, and might cause an acceleration in striatal DA

turnover (Perkins and Westfall, 1978; Chen and Ramirez, 1982; Perkins

et al., 1979). Secondly, the sex of the animal used in the assay of

estrogen's effects on the mesostriatal DA system may be important.

Several groups have utilized male rats in the examination of the actions

of estrogen and androgenic steroids on mesostriatal DA activity (Becker

and Ramirez, 1981a; 1981b; Demarest and Moore, 1980; Eikenburg et al.,

1977; Gordon et al., 1979; Alderson and Baum, 1981; Vermes et al., 1979).

In contrast to the results found when using female rats and mice (Crowley,

1982; DiPaolo et al., 1982a; 1982b; Dupont et al., 1981; Greengrass and

Tonge, 1974; Gordon et al., 1977; L6fstr6& et al., 1977), it has not been

found that steroid hormones induce changes in DA turnover or AMPHET-

induced efflux of DA from striatal tissue in male rats. This may indicate

that there are sex differences in the ability of gonadal steroids to

modulate the mesostriatal DA system. Third, another methodological problem

refers to the use of the entire striatum in the biochemical studies of

estrogen's effects, even though the striatum contains the fiber terminals

from more than one DA system. This is relevant since these systems may be

modulated differently by estrogen. Acute intravenous injections of estrogen

can immediately alter the activity of dopaminergic cells located in the

midbrain substantiala nigra), but the activity of one cell type is increased

whereas the other is decreased (Chiodo and Caggiula, 1980). Furthermore,

when these same cell types are examined 48 hours after EB administration

for their suppression to low doses of apomorphine, the cells show either

an enhanced or depressed response, respectively, depending on the cell type

(Chiodo and Caggiula, 1980). It would be useful to determine if these

separate cell types project to different parts of the striatum and thus

differentially alter the output of the striatum.









In summary, future investigation of estrogen's effects on the

mesostriatal DA system should take into account the treatment-test

interval, the sex of the research animal, and the possible differential

effects of estrogen on separate populations of DA fibers terminating in

the striatum.

Effects of Estrogen on Postsynaptic Components of the Mesostriatal DA
System

In addition to modulation of the mesostriatal DA neurons, estrogen

modulates, directly or indirectly, DA-sensitive events postsynaptic to

the DA terminals. When measured shortly after acute estrogen treatment,

estrogen suppresses the effects of DA agonists on striatal neurons, as

indicated by a number of independent lines of research. First, the

administration of DA agonists stimulates the accumulation of cAMP in

striatal neurons, through activation of a DA receptor (Greengard, 1976;

for additional references see Joyce, 1983), and this effect of DA agonists

is antagonized by estrogen treatment (Tang and Cotzias, 1977). Second,

the DA agonist apomorphine acts through a DA receptor to inhibit the activ-

ity of acetylcholine (ACh) neurons, as indicated by the increased

accumulation of ACh in these neurons, and estrogen modulates this apomorphine

effect. Shortly after the administration of estrogen, the apomorphine

(APO)-induced accumulation of ACh in striatal cholinergic neurons is

antagonized (Euvrard et al., 1979; 1980). The fact that estrogen does

not appear to alter the synthesis of ACh in these neurons (Euvrard at al.,

1980; Daabees et al., 1981) further suggests a direct effect of estrogen

on antagonism of DA agonist responses. Estrogen's antagonism of the APO

effect can even occur in DA-denervated striatal tissue, thus suggesting

that estrogen's actions can be independent of any modulation of the pre-

synaptic DA fibers (Euvrard et al., 1979). Third, the inhibitory actions









of DA on striatal neurons can be recorded extracellularly, and an intra-

venous injection of estrogen reverses the effects of DA applied ionto-

phoretically onto the striatal neurons (Arnauld et al., 1981). Fourth, at

least one output from the striatum, the DA-sensitive striatonigral GABA

neural system (Dray, 1981), has also been shown to be responsive to

estrogen. Activity in the striatonigral GABA system is measured by changes

in GAD activity in the substantial nigra (Dray, 1981), and shortly after an

acute treatment with estrogen there is a measurable decline in GAD

activity of the substantial nigra (Gordon et al., 1977; 1979; Perry et al.,

1981a; Nicoletti et al., 1982; Tyler et al., 1979). While this response

to estrogen is thought to be due to antagonism of dopaminergic events in

the striatum, it is also possible that estrogen can directly inhibit

GAD activity in neural tissue (Wallis and Luttge, 1980).

On the basis of two pieces of evidence, one group has proposed

that PRL mediates estrogen's dopaminergic antagonism (Euvrard et al., 1980).

Using APO-induced accumulation of striatal ACh as the measure of DA agonist

activity, they reported that estrogen suppression of the apomorphine

response is eliminated by hypophysectomy. Secondly, these same authors

reported that hyperprolactinemia for 10 days, produced by pituitary

transplants under the kidney capsule, produces a partial antagonism of the

APO effect (Euvrard et al., 1980). In contrast, two other lines of evidence

suggest that PRL does not mediate estrogen's antidopaminergic effect. First,

it has been shown that implantation of estrogen directly into the striatum

on one side of the brain produces DA antagonism, as indicated by a specific

decrease in activity of the DA-sensitive striatonigral GABA system of

that brain (McGinnis et al., 1980b). Secondly, it has been shown that

the estrogen-induced reversal of the effect of iontophoretically applied









DA on striatal neurons (Arnauld et al., 1981) occurs well before estrogen

could increase plasma PRL levels (DeLian et al., 1977; Horowski and Durow,

1981). Moreover, suppression of striatal DA activity during the estrous

cycle (Becker and Ramirez, 1981b; Crowley et al., 1978a; Jori et al.,

1976; Jori and Cecchetti, 1973; Holzbauer and Youdim, 1973) would not be

correlated with elevated plasma levels of PRL (Smith et al., 1975;

Butcher et al., 1974). However, in the DA iontophoresis study by Arnauld

and coworkers (1981), hypophysectomy did eliminate both estrogen's

alteration of striatal cellular activity, and estrogen's antidopaminergic

effect. Thus, although a pituitary cofactor may be necessary for estrogen

to have antidopaminergic effects, these datado not suggest that PRL

mediates estrogen's antidopaminergic effects in the striatum.

Investigators have been interested in determining if estrogen's

suppression of DA agonist responses in the striatum might involve

alterations in the affinity or number of DA receptors on striatal neurons.

It is known that intact female rats have fewer [3H]spiroperidol binding

sites in the striatum as compared to ovariectomized females and intact

males (Hruska et al., 1982a; Fuxe et al., 1979). Although this finding

suggests that estrogen decreases (down-regulates) the number of striatal

DA receptors, there is still no convincing evidence that estrogen, or

even the catechol estrogens, can directly interact with striatal DA

receptors, except at extremely high (micromolar) concentrations (Paden

et al., 1982; Schaeffer and Hsueh, 1979). Nonetheless, there are pre-

liminary reports indicating that shortly after estrogen treatment there

is a decrease in the amount of striatal binding of [3H]DA (Inaba and

Kamata, 1979; Perry et al., 1981a). More complete reports, however, have

shown that the binding density of [3H]spiroperiodol is unchanged at 24 hours









after an acute treatment with EB (Fields and Gordon, 1982; Hruska

et al., 1980a).

These discrepancies may be explainable on the basis of estrogen-

induced changes in the affinity of striatal DA receptors. Fields and

associates (Fields et al., 1982) reported recently that within four hours

after estrogen treatment there is a decrease in the affinity of striatal

DA receptors for [3H]spiroperidol and [3H]2-amino-6,7 dihydrotertralin

(ADTN), and that this alteration in affinity is lost by 48 hours after

estrogen treatment (see also Fields and Gordon, 1982). Although this

effect of estrogen needs further investigation, it might explain the

results of the earlier, brief reports on estrogen-induced changes in

binding of [3H]DA (Inaba and Kamata, 1979; Perry et al., 1981a). Thus,

it appears unlikely that estrogenic modulation of the number of striatal

DA receptors is a mechanism for the antidopaminergic actions of estrogen.

With respect to the estrogen-induced antagonism, it may be more profitable

to investigate estrogen modulation of DA receptor-linked mechanisms,

such as adenylate cyclase (Tang and Cotzias, 1977; Kumakura et al., 1979).

There is much better evidence that at long intervals after termination

of acute estrogen treatment (Hruska and Silbergeld, 1980a; 1980b; Hruska

et al., 1980a), or with chronic estrogen treatment (DiPaolo et al., 1979;

1981; Perry et al., 1981a), there is an increase in the number of DA

receptors in striatum, as indicated by an increase in the number of [3H]

spiroperidol binding sites. DiPaolo and associates (1982a) have observed

an increase in the binding of several dopaminergic ligands ([3H]spiroperidol,

[3H]spomorphine, [3H]haloperidol) with chronic estrogen treatment, even

after denervation of the mesostriatal DA system,suggesting estrogen has

direct postsynaptic effects. It has also been reported that GAD activity









in the substantial nigra is increased at long intervals after estrogen

treatment, suggesting that there is a rebound increase in activity of the

striatonigral GABA system following the initial estrogen-induced suppression

(McGinnis et al., 1980; Perry et al., 1981a).

Gordon and associates (Fields and Gordon, 1982; Gordon and Diamond,

1981) have further hypothesized that estrogen can prevent the up-

regulation of striatal DA receptors that normally occurs following

cessation of chronic haloperidol or estrogen treatment (DiPaolo et al.,

1981; Gordon and Diamond, 1981; Fields and Gordon, 1982). Estrogen

administration during the first four days following cessation of the

chronic administration of estrogen or haloperidol blocks the increase

in striatal [3H]spiroperidol binding observed with oil administration.

This effect was, however, not replicated (DiPaolo et al., 1981), and it

is inconsistent with the evidence that estrogen cannot decrease the

density of striatal DA binding sites when given acutely (Fields and

Gordon, 1982; Hruska et al., 1980a). Thus, although there is not strong

evidence that estrogen can antagonize DA agonist effects in the striatum

through a modulation of DA receptors, estrogen's apparent antagonism may

lead to compensatory changes in the striatum that includes an increase in

the density of DA receptors. Alternatively, treatment with estrogen may

trigger events that lead to the up-regulation of DA receptors, but be

separable from the initial antagonism.

Another area of research that has shown no signs of being clarified

concerns the issue of whether PRL mediates the increase in density of

striatal DA receptors observed both at 5-11 days (e.g., Hruska et al.,

1980a) after acute treatment with a large dose of estrogen and after

several days of chronic estrogen treatment (e.g., DiPaolo et al., 1982a).









Hruska and associates (Hruska et al., 1980a) have reported that hypo-

physectomy prevents the increase in 13H]spiroperidol binding to striatal

tissue that occurs after a single treatment with estradiol valerate to

intact male rats (Hruska et al., 1980a; Hruska and Pitman, 1982). Research

from that laboratory also indicates that constant infusion of ovine PRL

to male rats for 7 days increases striatal binding of [3H]spiroperidol

(Hruska et al., 1980b; 1982b; Pitman et al., 1981), a finding corroborated

by another laboratory (Levin et al., 1983). Consistent with those obser-

vations, it has been reported that hyperprolactinemia produced by

transplantation of anterior pituitary tissue under the kidney capsule

increases the activity of the DA-sensitive striatonigral GABA system

(Nicoletti et al., 1982).

DiPaolo et al. (1982c) have reported findings that only partly

support the proposal of Hruska (Hruska et al., 1980a; 1982b). DiPaolo

and associates (1982c) have found that chronic treatment of ovariectomized

female rats or intact male rats with E2 in saline or ovine-PRL results

in elevated numbers of [3H]spiroperidol binding sites in striatum.

However, the effects of E2 and PRL are independent and can occur in the

absence of a pituitary gland. Hypophysectomy, itself, results in a de-

crease in striatal [3H]spiroperidol binding, which is prevented with

simultaneous treatment with E2 and PRL.

To complicate matters further, Gordon and associates have found

diametrically opposite results to those of DiPaolo et al. (1982c) and

Hruska et al. (1980a; 1982b), reporting that hypophysectomy induces an

increase in striatal DA receptor density which can be counteracted by

simultaneous treatment with EB or PRL (Perry et al., 1980; Diamond and

Gordon, 1981). In their hands, hypophysectomized and ovariectomized









rats administered oil for 3 days show an increase in binding of

[3H]spiroperidol to striatum at 24 hours after the last administration

of oil, as compared to sham operated rats (Perry et al., 1980; 1981b).

Simultaneous treatment with EB attenuates the increase in striatal

[3H]spiroperidol binding of the hypophysectomized rats, but does not

decrease striatal [3H]spiroperidol binding in sham operated rats (Perry

et al., 1980; 1981b). Similar qualitative effects are seen with male

rats hypophysectomized for 3 days and simultaneously administered PRL

(Diamond and Gordon, 1981). The discrepancies between the work of Gordon

and associates (Perry et al., 1980; 1981b; Diamond and Gordon, 1981)

and the other laboratories (Hruska et al., 1980a; 1982b; DiPaolo et al.,

1982c) could be attributed to several experimental variables, but a

particularly important one may be the time from ovariectomy and hypo-

physectomy to measurement of DA receptor binding. In any case, it is

clear that a pituitary factor, possibly PRL, is important in the regulation

of striatal DA receptors. It is also reasonable to presume that estrogen

can modify the density of striatal DA receptors independently of a

pituitary factor, but the interaction of estrogen with pituitary hormones

in the striatum needs to be examined further.

Summary. The classical hypothesis has been that estrogen's actions

on the brain neurotransmitter systems are mediated by genomic mechanisms,

requiring nuclear receptors that bind estrogen (McEwen et al., 1979;

McEwen, 1980). Neurons in the neostriatum and the DA-containing cells of

the midbrain do not concentrate [3H]estradiol intracellularly (Heritage

et al., 1980; McEwen, 1979; Stumpf and Sar, 1976). Since striatal tissue

has often been used as a control for measurement of the effect of estrogen

on DA system (e.g., Eikenburg et al., 1977; Demarest and Moore, 1980;









Crowley et al., 1978), it would appear that a tacit assumption has been

that estrogen would not modify neuronal events in the striatum. Recent

research has shown otherwise, and suggested important parallels with

estrogenic modulation of the TIDA are clear. Acute treatment with estrogen

results in a suppression of both the presynaptic components of the

mesostriatal DA system, and DA-sensitive events in neurons postsynaptic

to the mesostriatal DA fibers. This parallels estrogen's modulation of the

TIDA, as does the apparent suppression of the mesostriatal DA system

during proestrus parallel that of the TIDA.

It appears that PRL is the major component of the feedback system

controlling the activity of the TIDA, and clearly, estrogen's antagonism

of the TIDA is modulated by PRL. The activity of the mesostriatal DA

system is modulated differently; a much more rapid modulation can be

effected by the presynaptic autoreceptors and the striatonigral feedback

system (Dray, 1981; Moore and Wuerthele, 1979). There is only limited

evidence that PRL administration alters the activity of the presynaptic

components of the mesostriatal DA system. Some investigators report that

hypophysectomy depresses striatal DA turnover (DiPaolo et al., 1982h;

Gudelsky and Moore, 1977; Wiesel et al., 1978); PRL administration

enhances release of DA from striatal nerve terminals (Perkins and Westfall,

1978; Chen and Ramirez, 1982); and there is one report that PRL increases

striatal DA turnover (Perkins et al., 1979). Yet most investigators find

no evidence that either intracerebroventricular or systemic injections of

PRL alter DA turnover in the dorsal striatum caudatee putamen) Annunziato

and Moore, 1978; Eikenburg et al., 1977; Fuxe et al., 1977a; 1977b; 1979;

Gudelsky et al., 1976), and DA turnover in ventral striatum (nucleus

accumbens) is enhanced (Fuxe et al., 1977a; 1977b; 1979; Wiesel et al.,









1978). The differences in these reports have been attributed both to

the treatment-test interval for PRL's effects, and the sex of the animal.

It has been suggested that the male is more sensitive to PRL's effects

(Chen and Ramirez, 1982). This would not, however, explain the differences

observed between PRL's effects on DA turnover in dorsal and ventral

striatum of the same rats.

Interestingly, the potential "postsynaptic" effects of PRL on the

TIDA system have not been studied. Yet, the postsynaptic effects of PRL

on DA sensitive striatal neurons, indicating an enhancing effect, have

been reported consistently (Hruska et al., 1980a; Pitman et al., 1981;

DiPaolo et al., 1982c; Wood et al., 1980; Nicoletti et al., 1982), but

the interactions between PRL and estrogen are far from being understood.

The demonstrated postsynaptic effects of estrogen and PRL may reflect the

enhanced ability of hormones to act at this point of control of the

mesostriatal DA system. It is very possible that the initial DA antagonism

by estrogen either results directly in, or initiates events leading to,

a delayed enhancement of postsynaptic components of the mesostriatal DA

system.

A cautionary note must be added to this discussion. It has been

pointed out in the preceding section that estrogen, either directly or

indirectly, may modulate more than one DA system terminating in the striatum.

Estrogen can modulate DA containing cells of the midbrain in different

directions (Chiodo and Caggiula, 1980); PRL may more potently modulate

DA systems terminating in the ventral but not dorsal striatum; and

estrogen treatment may not alter the density of DA receptors in the

ventral striatum while doing so in the dorsal striatum (Hruska and Pitman,

1982). This differential control of striatal DA systems may be very






31


important in understanding the effects of estrogen on the functional

output of the basal ganglia. Estrogen's potential regulation of other

transmitter systems in the striatum has only begun to be investigated

(Hong et al., 1982), but could be important for clarifying estrogen-DA

interactions in the striatum.














CHAPTER III

EFFECTS OF ESTROGEN ON BEHAVIORS MEDIATED BY
THE MESOSTRIATAL DA SYSTEM


Introduction

In the previous section, evidence was presented that estrogen

alters neuronal transmission in the basal ganglia, through modulation

of the mesostriatal DA system. Consequently, even though the striatum

and substantial nigra do not concentrate 3H] estradiol intracellularly

(Heritage et al., 1980; McEwen, 1979; Stumpf and Sar, 1976), estrogen

could affect DA-mediated functional output of the basal ganglia.

Evidence for such a proposition already exists in the literature on

extrapyramidal dysfunction in humans. For example, it has long been

known that the extrapyramidal symptoms associated with neuroleptic

useage are more frequent in females, particularly geriatric females

(Crane, 1968; Greyhan, 1957; Lehmann and Ban, 1974; Bell and Smith,

1978; Gratton, 1968; Ayd, 1961; Tepper and Haas, 1979; Simpson e al.,

1978). Since neuroleptic-induced extrapyramidal symptoms are thought to

be due to a disturbance of the mesostriatal DA system (Joyce, 1983;

Barbaccia and Trabucchi, 1979; Tarsy and Baldessarini, 1977), the

sex-linked exacerbation of these symptoms likely reflects a further

modification of the mesostriatal DA system. Estrogen, itself, may play

an important role in inducing these apparent sex differences since

(1) estrogens given to males or females increases the prevalence and

severity of parkinsonian symptoms induced by neuroleptic treatment

32









(Gratton, 1960; Villeneuve et al., 1978); (2) estrogens given to males

or females can decrease neuroleptic-induced tardive dyskinesia symptoms

(Bedard et al., 1979; Villeneuve et al., 1978, 1980); and (3) estrogen

administered to female parkinsonian patients can decrease the

dyskinetic episodes associated with L-dopa treatment (Bedard et al.,

1979; Villeneuve et al., 1978). Furthermore, in certain individuals,

estrogen treatment can reveal a latent hyperactivity of the extra-

pyramidal system. In asymptomatic patients with a previous history of

chorea and/or rheumatic fever, choreatic episodes have been known to

occur during pregnancy (Donaldson, 1978; Lewis et al., 1966; McDowell

et al., 1981; Zegart and Schwartz, 1968), or be induced by oral contra-

ceptives (Bickerstaff, 1975; Nausieda et al., 1979a). These reports

have led some investigators to suggest that estrogens may modify certain

extrapyramidal disorders by acting on the mesostriatal DA system

(Bedard et al., 1979; Nausieda et al., 1979a, 1979b).

Although many clinical studies suggest an interaction between

estrogen and the DA systems of the basal ganglia, two important issues

still remain to be addressed. First, it is unclear whether estrogen

modifies striatal DA-mediated behaviors by a direct (i.e., central) or

an indirect (i.e., peripheral) action on the central nervous system.

Second, it is also unclear what the acute, or initial, functional effect

of estrogen is on DA-mediated behaviors involving the basal ganglia.

The clinical reports are contradictory with respect to the presumed

actions of estrogen, indicating both a suppression and enhancement of the

mesostriatal DA system. In order to investigate these discrepancies,

as well as to examine the mechanism of action of estrogen on the meso-

striatal DA system, a variety of animal models have been developed.









For example, the stereotypy model and the rotational model of dopaminergic

activity have both been used to study the effects of estrogen on DA-

mediated behaviors. Both of these behavioral paradigms do, however, have

some inherent problems. The stereotypy model utilizes the systemic

administration of DA agonists to intact (nondamaged) animals, and then

a measurement of the resulting changes in their behaviors. In contrast,

the rotational model requires that a lesion be made in the animal so

that there is a loss of DA (i.e., as a result of denervation) from one

side of the brain. The developing imbalance in dopaminergic activity between

the two sides of the brain, particularly in the basal ganglia, is claimed

to be the cause of a circling behavior in response to the systemic admin-

istration of DA agonists. These pharmacological models have long been

used for studying the efficacy of various DA agonists in the central

nervous system (see Joyce, 1983).

Assessment of Central and Peripheral Actions of Estrogen

When rats are given high doses of amphetamine (AMPHET) or

apomorphine (APO) systemically, the animals will display a limited

repertoire of behaviors in a very repetitive (stereotypic) pattern for

the duration of the effect of the drug. It has been observed that the

particular type or class of stereotypic behavior displayed is dependent

on the dose of the DA agonist given (Costall and Naylor, 1973; 1974;

Costall et al., 1974; Creese and Iversen, 1974). Accordingly, most

authors assign each class of stereotypic behavior a number on an ordinal

scale. Effects of the hormone treatment are then evaluated by comparing

the mean ratings of the animals' behavior. The validity of this method

rests on the assumption that a difference in stereotypy rating indicates

a difference in dopaminergic stimulation. Second, research conducted









in the late 1960's indicated that dopaminergic manipulations of the

caudate nucleus could produce stereotypic behaviors (reviewed in Fog,

1972); consequently, alterations in DA agonist-induced stereotypy are

often presumed to occur in the caudate nucleus. Therefore, most authors

that examine the effects of estrogen treatment on APO- and AMPHET-

induced stereotypy assume that changes in the stereotypy rating reflect

changes in the "intensity" of dopaminergic stimulation in the caudate

nucleus (Beatty and Holzer, 1978; Chiodo et al., 1981; Gordon, 1980;

Hruska and Silbergeld, 1980a; Koller et al., 1980; Nausieda et al.,

1979b; Savageau and Beatty, 1981). It has been deduced from these

experiments that estrogen acts in the brain to directly modify the

response of DA agonists. However, the basic assumptions underlying these

paradigms have been criticized seriously. For example, in contrast to

the assumption that the caudate nucleus (dorsal striatum) is the sole

mediator of stereotypic behavior, it is now known that different classes

of stereotypic behaviors occur as a result of dopaminergic stimulation of

different regions of the forebrain (Costall et al., 1977; 1980; for review

see Joyce, 1983). Thus, a change in stereotypy ratings could be a

reflection of changes in the types of stereotypic behaviors displayed,

produced by an alteration in the distribution of dopaminergic agents in the

brain, or by changes in the activity of drugs in various regions of the

forebrain. Hence, an estrogen-induced alteration in stereotypy rating

does not necessarily mean that a modulation of dopaminergic stimulation

of the caudate nucleus has occurred. It is also possible that estrogen

might modify the behavioral response to dopaminergic drugs by altering

the baseline behavioral conditions of the animal (see Beatty, 1979, for

review), resulting in an altered threshold for behaviors produced by the









systemic administration of dopaminergic drugs. Neither of these effects

of estrogen would be considered to be a direct modulation of the

mesostriatal DA system; but animal models utilizing the systemic injection

of dopaminergic drugs cannot discriminate between central and peripheral

effects of estrogen.

It has also been assumed that estrogen's modulation of the

rotational response to DA agonists administered systemically reflects

a change in the intensity of dopaminergic stimulation in the caudate

nucleus (Bedard et al., 1978; Euvrard et al., 1980; Hruska and

Silbergeld, 1980b). Inherent in these experiments is the naive and false

assumption that an alteration in the rotational response is a conse-

quence of changes in a unidimensional mechanism (see Ungerstedt et al.,

1981). It has been presumed that the degree of rotation to DA agonists

is related directly to the magnitude of DA stimulation in the caudate

nucleus (Silbergeld and Calne, 1981). The evidence, however, clearly

indicates that the rotational response involves DA-sensitive regions out-

side the caudate nuclei (Kelly, 1977; Pycock and Marsden, 1978). It

has also been argued that the response measured, i.e., the number of turns,

is a reflection of activity in the nucleus accumbens (ventral striatum)

and not in the caudate nuclei. The direction of rotation or postural

deviation is thought to be under the control of the dorsal striatum,

reflecting an imbalance of dopaminergic activity between the two striata

(Kelly, 1977; Pycock and Marsden, 1978; Joyce et al., 1981). Yet, even

this model may be overly simplistic since postural deviation may not

reflect only striatal dopaminergic activity. For example, we have found

that contralateral postural deviation induced by intrastriatal administration

of DA is reversed to ipsilateral deviation with a systemic administration









of APO (Joyce et al., 1983). Since we could not obtain any evidence

that this effect was due to a change in the balance of dopaminergic

activity between the two striata, it is entirely possible that these

results were due to the activation of a DA-sensitive site outside the

striatum. Another rotational model, involving animals with a unilateral

lesion of the entopeduncular nucleus, may also have problems. Pazo and

associates (1982) have obtained evidence that the rotational response to

DA agonists, in animals with such a lesion, is not mediated by the

classical striato-pallidal efferent pathways. Consequently, estrogen-

induced alterations in rotational responses of these lesioned animals

(Bidard et al., 1978, 1981) may not occur through the normal efferent

output of the basal ganglia. Therefore, either rotational model, when

used to determine estrogen's effects on striatal DA-mediated behaviors, has

inherent problems.

There are several reports that estrogen administration changes

the duration and the rate of return to baseline of DA drug-induced behaviors,

and the authors have hypothesized that estrogen is acting centrally for

these effects (Hruska and Silbergeld, 1980b; Koller et al., 1980; Lal

and Sourkes, 1972; Nausieda et al., 1979b; Naik et al., 1978). Yet,

many of the dopaminergic drugs used in these studies are metabolized by

microsomal enzymes in the liver. Therefore, the intensity and duration

of their action may be altered by substances which either induce or

suppress microsomal enzymes. In rats, the pituitary hormones and most of

the steroid hormones influence the activity of liver microsomal enzymes

(Conney,1967; Kato, 1974; Selye, 1971). This may account for the

estrogen-induced differences in brain levels of [3H]AMPHET and [3H]

spiroperidol observed upon systemic administration of the drugs (Becker









et al., 1982; Chiodo, 1979; 1981; Groppetti and Costa, 1969; Meyer

and Lytle, 1978). Therefore, the hypothesis that estrogen's modulation

of DA-mediated behaviors occurs as a consequence of a modification of

the mesostriatal DA system is confounded by problems with the method-

ologies used to study the issue.

Functional Effect of Estrogen on Striatal DA-Mediated Behaviors

The second major issue addressed by the animal model literature

regards the functional effects on striatal DA-mediated behaviors. The

animal data, like that of the clinical literature, indicate that the

consequences of estrogen's effects include both suppression (Bedard et al.,

1978; Bedard et al., 1982; Naik et al., 1978) and enhancement (Becker

et al., 1982; Chiodo et al., 1981; DiPaolo t al., 1981; Hruska and

Silbergeld, 1980a; 1980b; Koller et al., 1980). Gordon (1980) has

hypothesized that an important variable to consider in evaluating the

effects of estrogen on brain DA systems is the time between estrogen

administration and the behavioral test. He reported that ovariectomized

rats showed a reversal in the effect of estrogen on the stereotypic

response to APO between 24 and 48 hours after the last estrogen adminis-

tration. At 24 hours after treatment with a large dose of EB, the rats

had lower stereotypy scores than oil treated control rats, but by 48 hours

or longer after EB treatment, they had higher scores. Gordon postulated

that the initial DA antagonism produced with estrogen leads to a

"withdrawal supersensitivity" to DA agonist effects (Gordon, 1980; Gordon

et al., 1980a; Fields and Gordon, 1982). The suggestion that the estrogen

treatment-test interval is a critical variable to consider in evaluating

the effects of this hormone is supported by a review of the literature.

A number of studies utilizing large doses of estrogen (EB, EV or E2 in









saline) and a treatment-test interval of 24 hours or less report that the

behavioral or pharmacological response to systemically administered DA

agonist drugs are antagonized (Bgdard et al., 1978; Euvrard et al., 1979;

1980; Gordon et al., 1980a; 1980b; Naik et al., 1978; Tang and Cotzias,

1977). On the other hand, studies employing a treatment-test interval

of 48 hours or greater report that the behavioral response to DA agonist

drugs is enhanced with a large dose of estrogen (Chiodo et al., 1981;

DiPaolo et al., 1981; Gordon et al., 1980a; 1980b; Hruska and Silbergeld,

1980a; 1980b; Koller et al., 1980).

Although these studies evidence a role for estrogen in the

suppression and enhancement of the behavioral responses of animals to

dopaminergic drugs, the studies suffer from methodological problems

associated with the use of systemic administration of drugs. In order

to get around these methodological problems, some authors have either

measured the brain levels of drugs given systemically, or made central

manipulations of the mesostriatal DA system. Becker and associates (1982)

measured whole brain and striatal levels of [3H]AMPHET produced by systemic

administration of the drug. They determined doses of AMPHET that produced

equivalent levels of the drug in both male and female rats, and then

they examined the rats for their rotational response to AMPHET. Females

were shown to have significantly more AMPHET-induced rotations than males,

which may reflect a greater amount of DA in the striata of females in

response to AMPHET (see Becker and Ramirez, 1981b; Robinson et al., 1980).

In addition, while there were no estrous cycle-dependent differences in

brain levels of AMPHET, the amount of rotations elicited by AMPHET did

fluctuate with the estrous cycle. These same authors have also reported

that contralateral rotation in response to electrical stimulation of the









mesostriatal DA system (SNC) is attenuated by ovariectomy (Robinson et al.,

1981) and varies in magnitude with different stages of the estrous cycle

(Robinson et al., 1982). Steiner and associates (1980; 1981) have also

reported that female rats show estrous cycle-dependent changes in magnitude

of intracranial (SNS) self-stimulation. Although these reports indicate

that fluctuations in plasma levels of hormones result in changes in the

striatal DA-mediated behaviors, the authors did not specifically alter

the plasma levels of estrogen. Chiodo et al. (1981) also measured

brain levels of [3H]AMPHET and [3H]APO after systemic administration of the

drugs in ovariectomized rats treated with 10 pg or 100 ig EB. Even

though there were no differences in brain levels of these drugs between

EB- and oil-treated animals, the EB-treated rats did have significantly

greater stereotypy scores in response to AMPHET or APO than the oil-

treated rats when assessed at 48 hours after hormone treatment. Chiodo

et al. (1981) did not test the response of the animals to AMPHET or APO

at any other time point after EB treatment, and may have missed different

effects of EB occurring at earlier time points. To insure that the

magnitudes of behaviors mediated by DA-sensitive neurons of the striatum

are measured concurrently with manipulations of serum levels of

estrogen, it is necessary to administer EB while directly manipulating

DA levels of the striatum. The following experiment was designed

specifically to provide this assessment.

Experiment 1: Estradiol Suppresses and Then Enhances Intrastriatal
Dopamine-Induced Contralateral Deviation

In order to evaluate the hypothesis that estrogen can directly

modify the actions of DA in the striatum, DA can be applied intra-

cerebrally while plasma levels of estrogen are modified. Such a method

allows for tests of behavioral responsiveness at different time points









after estrogen treatment. In collaboration with others in this

laboratory, I tested the behavioral effects of unilateral injection of

DA into the anterior-dorsal striatum of male rats at different time

points after a single subcutaneous (s.c.) injection of EB. Unilateral

injections of DA into the anterior-dorsal striatum result in a contra-

lateral asymmetry of the animal's ongoing behaviors, which can be

quantified by measuring the amount of time the animal spends producing

behaviors to the side contralateral to the intrastriatal injection

(Joyce et al., 1981). The behavioral response produced by the intra-

striatal injection of DA, termed postural deviation, is a highly localized

response (Joyce et al., 1981), which makes it useful for examining the

effects of estrogen. Since the estrogen treatment-test interval is con-

sidered to be an important variable (Gordon, 1980), the effects of a

single treatment with EB on the amount of contralateral postural deviation

elicited by unilateral intrastriatal injection of DA was tested at 2 and

6 days after hormone treatment. These two time points were chosen

because the literature indicated that treatment with large doses of EB

or EV induced an initial suppression of DA agonist effects lasting 24 to

48 hours (Gordon, 1980; Gordon et al., 1980), and a clear enhancement by

6 days post treatment (Hruska and Silbergeld, 1980a; 1980b).

Materials and Methods

Stereotaxic surgery

Male Long-Evans hooded rats weighing 250-300g at the time of surgery

were implanted bilaterally with permanent cannulae. Stereotaxic surgery

was carried out under sodium pentobarbital (W.T. Butler Co.) anesthesia.

Guide cannulae were constructed from 21 GA stainless steel tubing and

the injection cannulae were constructed using 27 GA tubing. Since the









injection cannulae terminated 3.0 mm below the guide cannulae, the

guide cannulae were located stereotaxically such that the injection

cannulae were aimed for the anterior dorsal striatum using the following

coordinates derived from Pellegrino et al. (1979): +2.0 to 3.0 mm with

respect to bregma; 2.0 to 4.0 mm lateral to bregma; and 3.5 to 5.0 mm

below the surface of the brain. Stainless steel stylets, made from

closed 27 GA tubing, kept the guide cannulae patent when the animals

were not being injected intracerebrally.

Behavioral observations and drug injection procedures

The intracerebral application of a drug was made by injecting the

drug solution through the 27 GA cannula which was connected by silastic

tubing to a Hamilton syringe mounted on a Sage syringe pump (Orion

Research). The injection was made at a constant rate of 0.5 pl/min,

and the injection cannula remained in place for an additional 30 sec

after the completion of the drug injection. After the drug administration,

the rat was placed into a circular, clear plexiglas observation chamber,

34 cm in diameter and 30.5 cm in height, and observed continuously for

postural deviation. The amount of time the rats deviated contralateral

and ipsilateral to the side of the intracerebral injection was recorded

continuously by the principal investigator using a two pole switch

connected in series to a time clock and a rack of cumulative counters.

The cumulative durations of contralateral and ipsilateral deviation were

recorded every 5 min for the entire 30 min observation period. Estradiol

benzoate in peanut oil or peanut oil alone was administered subcutaneously

into the neck through a 23 GA needle.

Drugs. DA (Sigma) was dissolved in a phosphate buffer to a final

pH of 7.4. The phosphate buffer solution was 7.0 mM sodium phosphate









monobasic/149 mM sodium phosphate dibasic solution. The concentration

of the DA solution was 25 ig/0.25 il. The drug control (VH) was a

solution of sucrose (Malinckrodt) dissolved in the phosphate buffer

adjusted to same osmolarity as the DA solution. Estradiol benzoate

Steraloids), at a concentration of 1 mg/ml was dissolved in peanut oil

by heating the oil to 60Q C.

Experimental procedure

Rats (n=30) were injected unilaterally in the anterior-dorsal

striatum first with the VH (0.25 pl) and four hours later with DA

(25 yg/0.25 pl). All animals were then placed immediately into the

observation chamber and observed for 30 min. Following removal from the

chamber, the animals were injected subcutaneously with either estradiol

benzoate (EB, 50 pg/100 g body wt) or the oil vehicle (peanut oil, OIL)

at 0.05 ml/100 g body wt. The behavioral effects of both VH and DA

injected into the anterior-dorsal striatum were again measured, as above,

at both 2 days and 6 days after the EB or OIL treatment.

Histology. After behavioral testing, rats were administered an

overdose of sodium pentobarbital and perfused intracardially with

0.9% saline followed by 10% buffered formalin. The brains were placed

in a 20% sucrose-l0% formalin mixture for at least 24 hr. They were

then frozen, sectioned at 30 im, stained with cresyl violet, and the

locations of the cannula tips verified (Figure 1).

Statistical analyses

In order to obtain an index of the dominant direction of deviation,

the time spent ipsilateral was subtracted from the time spent contra-

lateral to the side of the injection (difference score). The difference

scores for postural deviation were analyzed for each of the sequential














TABLE 2-1

Postural Deviation Score for Intrastriatal DA or VH at
Different Times Pre- or Post-Hormone Treatment

HORMONE TIME COURSE
Drug Pre-Treatment 2 Days 6 Days

OIL DA 781(41)ab 812(46)bd 782(52)b

VH -62(31) 45(40) 20(40)

EB DA 846(46)b 196(32)c 1446(93)b'e

VH 31(55) 35(31) -36(45)

a
mean score for total observation period, SD appears in brackets

significantly different from VH score for that condition, g <0.01
c
significantly different from pre-treatment EB, p <0.01 and 6 days EB

significantly different from EB treatment at 2 days, p <0.01

egn cantly different from OIL treatment at 6 days <0.01
significantly different from OIL treatment at 6 days, p <0.01



























Figure 1. Locations of cannula tips for unilateral injection of
25 yg DA or 0.25 ~i VH into the dorsal part of the anterior striatum
(diagrams derived from Pellegrino et al., 1979). Open circles
indicate rats treated with OIL (n=15); filled circles, rats treated
with EB (n=15). Courtesy of Eur. J. Pharmacol.





46








0





















O0O
0 0::0 \
.*
o00 o

* 'oo oo,^









5 min blocks of the 30 min observation period as well as for the

total 30 min observation period. In order to examine the time course

of effects of EB treatment as compared to the time course of effects

of OIL treatment, an analysis of covariance with sequence (6 levels

of 5 min blocks) as the quantitative covariable was run. Subjects were

nested within hormone treatment (2 levels, EB and OIL), crossed with

drug (2 levels, DA and VH) and time course of hormone treatment (3 levels;

0, 2, 6 days). In order to determine whether the hormone treatment

affected the sequence response for the drug (DA or VH), the slope of the

response to the drug across the six 5-min blocks of the 30 min observation

period was determined, and analyzed for differences in hormone effects

at 0 (pre-hormone), 2 and 6 days of hormone treatment.

Experimental Results

The location of the injection cannulae sites for all animals are

shown in Figure 1 (n=30), all sites are within the anterior dorsal

striatum. There was no difference in the distribution of sites between

EB-treated (n=15) and OIL-treated (n=15) rats. The pretreatment

response of the OIL and EB groups to the intrastriatal injection of DA

or the VH were not different (Table 2-1, Figure 2). Consistent with

previous findings (Joyce et al., 1981), DA injected into the anterior

dorsal striatum induced consistent contralateral deviation, whereas the

intrastriatal injection of VH produced no significant change in postural

deviation. For both the OIL and EB groups (Figure 2), the pretreatment

response to an intrastriatal injection of DA was significantly different

from the VH injections for 10-30 min after injection (p < 0.01). The

groups were not different in the slope of the response to the drug (DA

or VH) across the six sequential 5-min blocks of the 30 min observation

period.

























Figure 2. Pre-treatment postural deviation scores for OIL and
EB groups. The ordinate represents the average difference score
for postural deviation expressed in 0.01 min. Ipsilateral devi-
ation was subtracted from contralateral deviation for each animal
to obtain an absolute difference score. Positive scores represent
a predominantly contralateral deviation, and negative scores, an
ipsilateral deviation. The graph represents the mean time +
S.E.M. for each 5-min time block. For each hormone treatment group
the filled symbols indicate scores for dopamine (DA); open symbols,
for the vehicle (VH). The DA and VH scores for OIL group are indi-
cated by circles and those for the EB group by squares. Courtesy
of Eur. J. Pharmacol.












300 PRE OIL
DA
o---o VH
250- PRE EB

S--- DA
0---- VH


-50


5 10 15
MIN









Rats administered OIL and tested 2 days later did not differ in

their response to DA or VH as compared to pretreatment OIL scores

(Table 2-1, Figure 3). Rats administered EB and tested 2 days after EB

treatment did not differ in response to VH, but showed a significant

decrease in contralateral deviation induced by an intrastriatal injection

of DA (Table 2-1) as compared to pretreatment EB scores. This is indicated

by the fact that the response to DA was not significantly different from

VH for any 5 min block of the observation period (Figure 3). The reduction

in the response to DA at 2 days after EB treatment was due to a reduction

in the amount of contralateral deviation, and not due to an increase in

ipsilateral deviation. While the absolute amount of contralateral

deviation induced by DA was significantly different between EB and OIL

groups at 2 days post-hormone treatment (Figure 3), the slopes of the

response to the drug (DA or VH) across the six sequential 5-min blocks

of the 30 min observation period were not different by hormone treatment.

The OIL group was unchanged in its response to an intrastriatal

injection of DA or VH at 6 days after OIL treatment as compared to the

pretreatment OIL scores or 2 days post-OIL scores (Table 2-1, Figure 4).

Rats administered EB and tested 6 days after EB were not different in

response to an intrastriatal injection of VH, but had a significantly

greater response to DA (Table 2-1, Figure 4) as compared to pretreatment

EB scores or 2 days post-EB scores. This is indicated by the signifi-

cantly greater response to DA at 10-30 min of the sequence (p < 0.01,

Figure 4). The enhancement in the response to DA at 6 days after EB

treatment is due solely to an increase in the amount of contralateral

deviation, and not to a decrease in ipsilateral deviation. Six days

after injection of either EB or OIL the response to DA was greater at






































Figure 3. Scores for OIL and EB groups at 2 days after OIL
or EB treatment. All other details as in Figure 2. Courtesy
of Eur. J. Pharmacol.













300- 2 DAYS
*--* o
o---o
250 2 DAYS

o--a
200


POST OIL
DA
VH
POST EB
DA
VH


15 20 25 30


MIN




































Figure 4. Postural deviation scores for OIL and EB groups
at 6 days after OIL or EB treatment. All other details as
in Figure 2. Courtesy of Eur. J. Pharmacol.












6 DAYS POST OIL
p. DA
o----o VH
6 DAYS POST EBT
-- DA
0----0 VH


5 10 15 20 25 30
MIN









10-30 min in the EB group than in the OIL group, but the slopes of the

response to the drug (DA or VH) across the six sequential 5-min blocks

of the 30 min observation period were not affected by hormone treatment.

Discussion. The results of this study suggest that estrogen can

directly modify the actions of DA in the striatum; and,consistent with

the hypothesis of Gordon (1980), the time after hormone administration

is an important determinant of the behavioral response of animals to

DA agonists. After a single administration of a large dose of EB, the

male rats were initially depressed in their response to an intrastriatal

injection of DA (2 days post-EB), but were later supersensitive in their

response (6 days post-EB). A review of other animal research to date would

also indicate that the time after the last estrogen administration is an

important experimental variable. Administration of large doses of EB

(greater than 50 hg) to rats or mice reduces the stereotypy scores induced

by APO (Gordon et al., 1980; Fields and Gordon, 1982; Naik et al., 1978),

AMPHET (Gordon et al., 1980; Naik et al., 1978), 0-phenylethylamine

(Naik et al., 1978) and L-dopa (Tang and Cotzias, 1977), when tested within

24 hours of the last treatment of EB. Treatment with a smaller dose of

EB (10 pg) can also antagonize APO and AMPHET-induced stereotypy (Gordon

et al., 1980). Treatment with a 100 lg of moxestrol antagonizes the

rotational response produced by APO in rats with unilateral forebrain

depletions of DA (Euvrard et al., 1980). Similarly, treatment with 5 pg

EB twice daily suppresses the rotational response or activity response

produced by APO in rats with a unilateral lesion of the entopeduncular

nucleus, if testing is done within 24 hours of hormone treatment (Bedard

et al., 1980). In monkeys with a midbrain lesion involving the

substantial nigra, a lingual dyskinesia can be observed, which can be









markedly enhanced by systemic administration of APO (Bedard et al., 1982).

The administration of 500 jg EB subcutaneously will induce a suppression

of the APO effect at 24 hours after the EB treatment.

When the behavioral response to DA agonists is measured at 48 hours

or more after treatment with estrogen, the behavioral response is enhanced.

Female rats administered 10 Pg EB (Chiodo et al., 1981; Gordon et al.,

1980a; 1980b), 50 pg EB (Gordon, 1980) or 100 ig EB (Chiodo et al., 1981;

Gordon, 1980) and tested from 2 to 7 days post-hormone treatment show

enhanced stereotypy scores to APO and AMPHET. Male rats with lesion-

induced depletions of DA from one striatum show an enhanced rotational

response to AMPHET at 6 days after a single treatment with 125 hg estradiol

valerate (EV; Hruska and Silbergeld, 1980b). Male rats administered 125 Vg

EV and tested 6 days after treatment evidence an enhanced stereotypic

response to both APO and AMPHET (Hruska and Silbergeld, 1980a). Primates

with a midbrain lesion, exhibiting APO-enhanced lingual dyskinesia,show

an initial suppression with EB treatment, but this is reversed to an

amplification by two weeks post-EB treatment (Bedard et al., 1982).

Interestingly, two groups report that at 48 hours after a high dose

of EB (50 pg, 100 jg), female rats show an enhanced stereotypic response

to APO (Gordon, 1980; Chiodo et al., 1981), whereas the results of this

experiment evidence suppression of intrastriatal DA-induced deviation

at this same time point. This discrepancy could be due to methodological

differences, sex differences, dose of EB used or differences in the

behavioral measures utilized. First, the behavioral changes observed by

other groups, in response to systemic administration of dopaminergic

drugs, may be due to both central and peripheral effects of estrogen.

With the procedure used in this experiment, peripheral effects of estrogen,








such as altered pharmacokinetics of the dopaminergic drugs, can be

eliminated. Thus, in Gordon's experiment (Gordon, 1980) it is possible

that the peripheral effects of estrogen may be masking the central

effects of estrogen at 48 hours after EB administration. Second, the

sex of the animals used may also be an important variable, since male

rats were used in this experiment and female rats were used by Gordon

(1980) and Chiodo (Chiodo et al., 1981). In male rats, a single admin-

istration of estrogen does not result in an increase in striatal DA

receptors, measured with [3H]spiroperidol, until 5 days post-treatment

(Hruska et al., 1980a). Long-term ovariectomized (OVX) female rats are

more sensitive to the DA-suppressing effects of estrogen (Euvrard et al.,

1980), and may have different time-course to the enhancing effects of

estrogen. Third, with larger doses of estrogen, greater suppression of

various measures of striatal DA response are observed (Gordon, 1980;

Euvrard et al., 1980). The dose of EB used in this study is about 150 Pg,

which is approximately equal to that used by Hruska and Silbergeld

(1980a; 1980b), but greater than that utilized by Gordon (Gordon, 1980)

or Chiodo and associates (1981) with female rats. Finally, it is also

reasonable to suggest that the behavioral measure of striatal DA activity

used may be differentially sensitive to estrogen. The stereotypy,

rotational and intrastriatal DA-induced postural deviation measures

need not utilize the same neural systems, and could, therefore, be

differentially sensitive to estrogen. Bdard and associates (1982) report

that with monkeys given midbrain lesions, some APO-sensitive behaviors

are modulated by estrogen and some are not. It is also worth noting

that the suppression-enhancement switch that characterizes EB modulation

of APO-stimulated lingual dyskinesia has a time course different from

APO-stimulated behaviors measured in rats.









Previous animal studies dealing with the effects of estrogen on

striatal DA systems have made use of the systemic injection of DA

agonists, or have not directly modulated serum levels of estrogen, when

studying estrogen's modulatory effects. Nevertheless, these groups have

concluded that estrogen is acting in the brain to modulate the behavioral

output of the DA systems of the basal ganglia (Bedard et al., 1978; 1980;

Euvrard et al., 1980; Gordon, 1980; Hruska and Silbergeld, 1980a; 1980b;

Hruska et al., 1980a; Becker et al., 1982; Robinson et al., 1981). The

results of the present study support this hypothesis more convincingly

than previous studies, because DA was injected directly into the striatum,

while serum levels of estrogen were being altered. With this paradigm,

the time courses and slope of the response to the intrastriatal injection

of DA were unaltered by estrogen administration, which suggests that it is

an estrogen-induced alteration in the response of the cells postsynaptic

to the mesostriatal DA fibers that underlies the behavioral changes. I

cannot discount other possible estrogen-induced alterations in the

mesostriatal DA system. Although estrogen does not alter the uptake

of DA into striatal synaptosomes (Nixon et al., 1974; Wirz-Justice et al.,

1974), it may be involved in sex- and estrous cycle-dependent changes

in amphetamine-stimulated release of DA from striatal tissue (Becker

and Ramirez, 1981a; 1981b). An estrogen-induced alteration in DA release

has been postulated as a mechanism underlying sex- and estrous cycle-

dependent variation in the amount of rotation elicited in rats with

amphetamine (Becker et al., 1982) and electrical stimulation of the

mesostriatal DA system (Robinson et al., 1982). The results of this experi-

ment also support the hypothesis that the treatment-test interval is an

important variable to consider when evaluating estrogen's modulation









of the striatal DA system. This experiment suggests that the initial

effect of estrogen is to suppress the behavioral effects of striatal

dopaminergic activity.

Experiment 2: Behaviors Induced by Intrastriatal Dopamine Vary
Independently Across the Estrous Cycle

Introduction

Animal studies have been utilized to investigate the effects of

gonadal hormones on the functional output of the basal ganglia. The

intensity and duration of behaviors induced by the systemic administration

of dopaminergic drugs have been shown to be influenced by gender (Becker

et al., 1982; Robinson et al., 1980; 1981; 1982; Savageau and Beatty, 1981)

and hormones (Bedard et al., 1978; 1980; Chiodo et al., 1979; 1981; DiPaolo

et al., 1981; Euvrard et al., 1980; Gordon, 1980; Hruska and Silbergeld,

1980a; 1980b). In this and other laboratories animals given estrogen (EB)

show an initial suppression of the behavioral responses to dopamine (DA)

agonists, and only later an enhancement of the behavioral responses

(Experiment 1: Bedard et al., 1982; Gordon, 1980). However, in these

studies, nonphysiological doses of estrogen were used, and one must be

concerned that only pharmacological responses to estrogen were observed.

One way of studying the effects of physiological doses of estrogen is to

utilize the natural fluctuations of estrogen in the estrous cycle.

Researchers that have examined fluctuations of DA-mediated functional

output of the basal ganglia across the estrous cycle have obtained results

that are not clearly consistent with the findings reported in Experiment 1

and in other laboratories (Bedard et al., 1982; Gordon, 1980).

Steiner and associates (1980; 1981) report that intracranial self-

stimulation of the substantial nigra pars compact (SNC) shows regular









fluctuations across the estrous cycle of the rat with a peak on the

night between proestrus and estrus. Additionally, Steiner et al.(1980)

report that bromocriptine-augmented wheel running shows a peak response

on the same night, but apomorphine-induced stereotypic behavior shows no

variation across the estrous cycle in rats. In contrast, Robinson

and associates report that intranigral (SNC) electrical stimulation-induced

rotational behavior (Robinson et al., 1982) and amphetamine-induced

rotational behavior (Becker et al., 1982) in rats show a peak response on

the night of estrus and a suppression on the night of diestrus day 1.

Becker et al. (1982) also report that an additional measure of AMPHET-

induced rotations, extra quarter turns, show a different pattern of

variability across the estrous cycle. The fewest extra quarter turns occur

on proestrus, with all other days being equal. These reports suggest a

lack of any commonality in estrous cycle control of the various striatal

dopaminergic behaviors. Moreover, the observed changes in the behaviors

appear to bear little relationship to fluctuations in the plasma level of

estradiol. An important aspect of those experiments is that the behav-

ioral responses were obtained during the night (lights OFF) portion of

the estrous cycle, yet the most significant rise in concentration of

estradiol in the blood occurs on the morning (lights ON) of proestrus

(Smith et al., 1975; Butcher et al., 1974). In order to more specifically

test for the dopaminergic effects of physiologic concentrations of estradiol,

it would be useful to test the behavioral effects of dopaminergic agents

during the lights ON portion of proestrus.

Since the intrastriatal application of dopaminergic drugs is a

useful method to investigate the functional effects of estrogen on

striatal dopaminergic behaviors, I used this procedure to test the









effects of physiological changes in gonadal hormones on the functional

output of the basal ganglia. In order to examine the effects of changes

in levels of estradiol on intrastriatal DA-induced behaviors, I tested

the animals on the morning of proestrus and subsequent mornings of each

day of the estrous cycle (Experiment 2.1). Because I noted a dramatic

change between the days of proestrus and estrus, in the second part of

this experiment I tested for intrastriatal DA-induced behaviors at

several time points on the day of proestrus (Experiment 2.2).

Materials and Methods

Animals

Female Long-Evans hooded rats weighed 180-220 g at the beginning

of the experiment. They were housed individually and maintained on a

12:12 light:dark cycle (lights ON, 0800-2000). Vaginal smears were taken

twice daily (1000 hr, 1600 hr) and each day of the estrous cycle was

determined with reference to the day of estrus. The rats were smeared for

two weeks prior to surgery, and for two weeks prior to initiating behavioral

testing. The rats were monitored for regularity of 4 day estrous cycles.

Stereotaxic surgery

The rats were implanted bilaterally with permanent cannulae under

sodium pentobarbital (W.T. Butler Co.) anesthesia. Guide cannulae were

constructed from 21 GA stainless steel tubing and the injection cannulae

were constructed using 27 GA tubing. Since the injection cannulae

terminated 3.0 mm below the guide cannulae, the guide cannulae were located

stereotaxically such that the injection cannulae were aimed for the

anterior dorsal striatum using the following coordinates derived from

Pellegrino et al. (1979): +2.0 to 3.0 mm with respect to bregma; 2.0 to

4.0 mm lateral to bregma; and 3.5 to 5.0 mm below the surface of the brain.








Stainless steel stylets, made from closed 27 GA tubing, kept the guide

cannulae patent when the animals were not being injected intracerebrally.

Behavioral testing

The intracerebral application of a drug was made by injecting the

drug solution through the 27 GA cannula which was connected by poly-

ethylene tubing to a Hamilton syringe mounted on a Sage syringe pump (Orion

Research). The injection was made at a constant rate of 0.5 pl/min,

and the injection cannula remained in place for an additional 30 sec

after completion of the drug injection. After the drug administration

the rats were placed into a circular clear plexiglas observation chamber,

34 cm in diameter and 30.5 cm in height, and observed for 40 min.

The duration of postural deviation, duration of laterally directed groom-

ing and the number of 1/4 rotations that occurred both contralaterally

and ipsilaterally to the side of intrastriatal injection were recorded.

The amount of time the rats deviated and groomed contralateral and ipsi-

lateral to the side of the intrastriatal injection was recorded contin-

uously by the observer using a two pole switch connected in series to

a time clock and a rack of cumulative counters. The cumulative durations

of postural deviation, laterally directed grooming and number of 1/4

rotations were recorded every 5 min for 40 min.

Drugs. Amphetamine (AMPHET; Sigma) and dopamine (DA; Sigma) were

dissolved in the phosphate buffer solution to a final pH of 7.4. The

phosphate buffer solution was 140 mM sodium phosphate dibasic/7.0 mM

sodium phosphate monobasic solution. DA and AMPHET solutions were made

at a concentration of 25 pg/0.25 pl. The control drug (VH) was the

phosphate buffer adjusted to a pH of 7.4 with glacial acetic acid.

Histology. After behavioral testing, rats were administered an

overdose of sodium pentobarbital and perfused intracardially with 0.9%









saline followed by 10% formalin. The brains were placed in a 20%

sucrose-10% formalin mixture for at least 24 hr. The brains were

frozen, sectioned at 30 pm, stained with cresyl violet, and the locations

of the cannula tips verified. Cannula tip placements for Experiment 2.1

and Experiment 2.2 are shown in Figure 5.

Experiment 2.1

Procedure. All rats (n=10) were injected unilaterally into the

striatum with the VH, DA or AMPHET on separate days. Animals in this

study each received all drugs on each day of the estrous cycle in a

counterbalanced order, with a minimum of 48 hours between drug injections,

to allow within-animal comparisons. Animals were tested between 1000-1200

hr of the day of proestrus and between 1000-1500 hr on all other days of

the estrous cycle.

Data analysis. Only rats which had regular 4 day estrous cycles

during the entire experiment were used in the final analyses of the data.

In order to obtain an index of the dominant direction of postural deviation

(including lateralized grooming), the time spent ipsilateral was sub-

tracted from the time spent contralateral to the side of the intracerebral

injection (difference score). A dominant direction index was also

obtained for the number of 1/4 rotations by subtracting the number of

1/4 rotations ipsilateral from the number contralateral to the side of the

intracerebral injection. The difference scores were used as the drug

response (for each drug) to examine if there were differences due to

the day of the estrous cycle (HORMONE); and were analyzed for the total

40 min observation period (sum total) and across the eight 5-min blocks

of the observation period (TIME). For the sum total scores, an analysis

of variance was used to determine if the variables drug and day of the
























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estrous cycle (HORMONE) had significant overall effects. Tests for simple

main effects were then made using Scheffe's method for multiple compari-

sons (for equal sample size). An analysis of covariance was used to

determine if the variables drug and day of the estrous cycle (HORMONE)

had significant overall effects for the eight 5-min blocks of the obser-

vation period (TIME as the quantitative covariable). For each drug

response, differences due to the day of estrous cycle (HORMONE) were

tested for significance using least squares means estimation with a

quadratic function as the model. When testing for differences using

drug response across the eight 5-min blocks of the observation period, it

was assumed that the lines were parallel. Only the preplanned comparisons

for each day of the estrous cycle (HORMONE) by drug were tested.

Results. The difference score for postural deviation for each drug

by day of estrous cycle are shown in Figure 6. The data are presented

for the entire 40 min observation period by each 5-min block. The

response to the unilateral intrastriatal injection of the VH was not

significantly different from zero for any day of the estrous cycle. The

response to both DA and AMPHET was significantly different from the VH

for each day of the estrous cycle (p <0.01). Unilateral injections of

DA or AMPHET produced contralateral deviation, as indicated by the positive

difference scores across the eight 5-min blocks of the 40 min observation

period (Figure 6). The difference score for postural deviation for the

intrastriatal injection of DA for each day of the estrous cycle is shown

in Figure 7. The response to DA was least on the morning of proestrus

and greatest on the morning of estrus (p <0.01). Diestrus day 1 and

diestrus day 2 were not different from one another, but were different

from proestrus and estrus (p < 0.01). The difference score for postural


























Figure 6. Postural deviation scores for Experiment 2.1. The
ordinate represents the average difference score for postural
deviation expressed in 0.01 min. Ipsilateral deviation was
subtracted from contralateral deviation for each animal to obtain
an absolute difference score. Positive scores represent a pre-
dominantly contralateral deviation, and negative scores, an ipsi-
lateral deviation. The graph represents the mean time + S.D.
for each sequential 5-min block of the 40 min observation period;
the abscissa represents each 5-min time block. The response to
the intrastriatal injection of dopamine (DA, 25 Jg/0.25 111),
amphetamine (AMPHET, 25 ug/0.25 Il) and vehicle (VH, 0.25 il)
are shown for each day of the estrous cycle (PROESTRUS, ESTRUS,
DIESTRUS 1, DIESTRUS 2).













300 PROESTRUS ESTRUS


20 20
............ OA

2 AW2 / / {,
200 00



ioo \ N l











0 0 io 20 00 30 i 40 0 ,S 3 4

TIME TIME
300 300
DIESTRUS I DIESTRUS 2




200 200
E \

Z0 2 j i -








I. 0
< IS .- \










5 f IS 20 23 30 30 40 5 1 11 20 230 30 3 40


TIME


TIME































Figure 7. Postural deviation scores for intrastriatal
injection of DA at each day of the estrous cycle (Experiment
2.1); PROESTRUS, DIESTRUS 1, DIESTRUS 2, ESTRUS. All other
details as in Figure 6.













ESTROUS CYCLE DOPAMINE RESPONSE


o--o PROESTRUS
A.------A DIESTRUS I
A-----... DIESTRUS 2
oa---i--a ESTRUS


200 -


150 -


100 I


50 -


15 20 25


30 35 40


TIME


5 10


I I a a i g


|


I I































Figure 8. Postural deviation scores for intrastriatal
injection of AMPHET at each day of the estrous cycle
(Experiment 2.1); PROESTRUS, DIESTRUS 1, DIESTRUS 2,
ESTRUS. All other details as in Figure 6.















ESTROUS CYCLE AMPHETAMINE RESPONSE
----- PROESTRUS
300 A -------. DESTRUS I
A-.-.-.A DIESTRUS2
o-i-i-a ESTRUS

250 \,


200 -


150 1


too I-


50 I


I I I I
5 10 15 20

TIME


I I I I
25 30 35 40


~














TABLE 2-2

Intrastriatal DA- and AMPHET-Induced Deviation and
Rotation on Separate Days of the Estrous Cycle


PROESTRUS ESTRUS DIESTRUS 1 DIESTRUS 2

VH DEV 33.8(27)d 35(40) 10.8(28) 10.3(28)

ROT 1.3(0.7) .2(.2) -1.4(1.2) -1.1(2.2)

DA DEV 848.9(65)a'b 1496(121)a'b 1177(72)a 1189(77)a

ROT 18.6(4.4)c 69.1(14)a 65.5(11)a 94(19)a

AMPHET DEV 1017.8(34)a,b 1822(117) a,b 1508(51)a 1507(77)a

ROT 127.4(33)ac 331.8(51)a 222(69)a 198(52)a

different from VH for that hormone condition, p <.01
b
proestrus different from estrus for that drug, p < .01

pro different from all others, p < .01
d
same on Table 2-3









deviation for the intrastriatal injection of AMPHET for each day of the

estrous cycle is shown in Figure 8. The response to AMPHET was least

on the morning of proestrus and greatest on the morning of estrus

(p < 0.01). Diestrus day 1 and day 2 were not different from one another,

but were different from proestrus and estrus (p <0.01). Unilateral

intrastriatal injections of DA and AMPHET produced contralateral 1/4

rotations greater in number than that produced by the VH (Table 2.2,

p <0.01). Similar to the postural deviation response to DA and AMPHET,

the number of 1/4 rotations varied across the estrous cycle (Table 2.2).

The rotational response to DA was least on the morning of proestrus,

and increased significantly by the morning of estrus (p <0.01). The

rotational response to AMPHET was least on the morning of proestrus, and

had increased significantly by the morning of estrus (p < 0.01).

Experiment 2.2

The results of Experiment 2.1 indicate that behaviors induced by

intrastriatal injections of DA and AMPHET vary in magnitude across the

estrous cycle. Furthermore, consistent with the hypothesis that estrogen

suppresses the behavioral responses to intrastriatal dopaminergic

stimulation (Experiment 1) the responses were least on the morning of

proestrus. This is when serum estradiol levels are highest (Smith et al.,

1975; Butcher et al., 1974). Within 24 hours of the proestrus suppression,

there was a significant enhancement in the behavioral responses to intra-

striatal DA and AMPHET. This transformation potentially is related to

the level of estrogen, since the surge in serum concentration of estradiol

is over by the afternoon of proestrus (Butcher et al., 1974; Smith et al.,

1975). This inverse correlation between levels of estrogen and the

behavioral response to intrastriatal DA and AMPHET can be better examined









by testing at various times during the day of proestrus, since the most

significant changes in serum levels of estradiol occur then.

Procedure. Rats (n=5) were given unilateral intrastriatal injections

of DA and AMPHET at various times on the day of proestrus and the

morning of estrus. Injections of the drugs were made on separate days,

but all animals received each drug at 4, 7 and 11 hours after lights

ON and again at 4 hours after lights ON at estrus. The drugs were

administered in a counterbalanced order. All rats were monitored for

their estrous cycles for two weeks prior to the initiation of behavioral

testing.

Data analysis. Only rats with regular 4 day estrous cycles

throughout the entire experiment were used in the final analyses.

The difference scores for the behavioral responses postural deviation

and 1/4 rotations were analyzed for differences due to drug and time

of day (HORMONE) using the sum total for the 40 min observation period. An

analysis of variance was used to determine if the variables drug and

time of day on proestrus and estrus (HORMONE) had significant overall

effects. Tests for simple main effects were then made using Scheffe's

method for multiple comparisons (equal sample size).

Results. The behavioral responses deviation and rotations, induced

by the unilateral intrastriatal injections of DA and AMPHET, varied

across the day of proestrus. The postural deviation response to DA and

AMPHET was suppressed at 4 and 7 hours after lights ON at proestrus,

but was enhanced by 11 hours after lights ON (Table 2.3; p <0.01).

The rotational response to DA and AMPHET was suppressed at 4, 7 and 11

hours after lights ON at proestrus, but enhanced by the morning of estrus

(Table 2.3; p <0.01).















TABLE 2-3

Intrastriatal DA- and AMPHET-Induced Deviation and
Rotation at Various Times of Proestrus and Estrus

PROESTRUS ESTRUS
4 hrs 7 hrs 11 hrs 4 hrs


DA DEV 391(68)b'd 372(96)b 1075(162) 1092(159)

ROT 54(16) 40(14) 43(26) 153(37)

AMPHET DEV 753(135) b 669(153) b 1671(114)a 1572(150)

ROT 308(52) 278(72) 258(73) 681(88)c

aDA different from AMPHET, p <.01

different from Pro 11 hrs and Estrus 4 hrs, p < .01

Different from all other hormone conditions, p < .01

d
scores for postural deviation and number of 1/4 rotations, SD appears in brackets









Discussion

Consistent with the interpretation that high levels of estrogen

result in a suppression of intrastriatal DA-induced behaviors

(Experiment 1), I found in Experiment 2.1 that intrastriatal DA- and

AMPHET-induced behaviors were suppressed on the morning of proestrus.

Both of the behaviors measured, postural deviation and number of rotations,

were suppressed when plasma titers of estradiol should be high (Smith

et al., 1975; Butcher et al., 1974) and enhanced when the levels should

be low (estrus, diestrus days 1 and 2). This is in contrast to recent

reports on estrous cycle changes in striatal DA-induced behaviors,

indicating either no suppression on proestrus (Steiner et al., 1980) or

a suppression on diestrus (Becker et al., 1982; Robinson et al., 1982).

However, the results of this experiment are in agreement with biochemical

studies of DA activity in the striatum across the estrous cycle. When

animals are sacrificed during the lights ON portion of a standard

light:dark cycle, biochemical indices of mesostriatal DA neuronal activity

are lowest on proestrus, with a significant increase by estrus (Becker

and Ramirez, 1981b; Jori and Cecchetti, 1973; Jori et al., 1976; Crowley

et al., 1978a). Since previous authors have examined the estrous cycle vari-

ation in striatal DA-induced behaviors during the lights OFF portion of

the cycle, differences between studies might be due to the time of day

the behaviors were measured.

In Experiment 2.2, I examined changes in the intrastriatal DA-

induced behaviors across the day of proestrus and the morning of estrus.

I found that on proestrus, intrastriatal DA- and AMPHET-induced postural

deviation showed increases by 11 hours after lights ON, but rotation was

still suppressed. The rotational response, like that of postural









deviation, was enhanced by the morning of estrus. These data indicate

that the magnitude of one striatal DA-mediated behavior, postural

deviation, changes dramatically during the day of proestrus, while that

of another, rotation, does not. This suggests that striatal DA-

mediated behaviors can be modulated separately by gonadal hormones, an

observation made previously using monkeys (Bgdard et al., 1982). Those

authors reported that the DA-related behaviors tremor and lingual

dyskinesia, induced by a midbrain lesion involving the SNC, were

differentially affected by EB. Lingual dyskinesia was enhanced by

dopaminergic agonists, and this enhancement suppressed by the systemic

administration of EB. In contrast, tremor was suppressed by dopaminergic

agonists, and this suppression unaffected by estradiol benzoate. Thus,

conflicts between various research groups that have investigated alter-

ations in DA-mediated behaviors across the estrous cycle may be resolved

with reference to the behaviors studied. Rotational behaviors induced

by electrical stimulation of the SNC (Robinson et al., 1982) or systemic

AMPHET (Becker et al., 1982) need not be correlated with intrastriatal

DA-induced postural deviation.

However, in this study (Experiment 2.2) the rotational response

induced by the intrastriatal administration of DA and AMPHET is found

to be suppressed at the night of proestrus and enhanced by estrus. This

finding is inconsistent with the findings of other experimenters that

have utilized rotation as their behavioral measure of dopaminergic

activity of the striatum (Becker et al., 1982; Robinson et al., 1982).

Those authors reported that their rotational measure was enhanced on the

night of estrus, and suppressed on the night of diestrus day 1. However,

in the study by Becker et al. (1982) only full 360 degree rotations were









included in the data; when extra quarter turns are also included,

there is a different response across the estrous cycle. The addition

of the extra quarter turn data reveal that rotations were in fact

suppressed on proestrus, and augmented on estrus, a finding consistent

with the results of this study. The results of the present experiment

(Experiment 2.2) also may not be inconsistent with those of Robinson et al.

(1982). In that paper, electrical stimulation of the SNC at night pro-

duced more rotations on estrus than diestrus day 1 and day 2, with a

slight increase by proestrus. These data are inconsistent only with the

data in Experiment 2.2 for 11 hours after lights ON of proestrus.

However, since Robinson et al. (1982) took their measure later on proestrus

than I did for Experiment 2.2, then it is unlikely that the magnitude of

the rotational response would be the same. I have also noted that the

rotational response to intrastriatal DA or AMPHET is reduced by long-term

ovariectomy (more than 3 weeks), which again is consistent with the

findings of others (Robinson et al., 1980; 1981). The postural deviation

response to the intrastriatal administration of DA and AMPHET shows a time

course similar to intracranial reward (SNC stimulation) and bromocriptine-

augmented wheel running activity, which show an enhancement on the night

of proestrus (Steiner et al., 1980).

These data also suggest that rotation and deviation induced by

intrastriatal application of DA and AMPHET are not necessarily mediated

by the same neural system, since they showed separable variations in

intensity across the estrous cycle. It has been presumed that rotation

is mediated by the simultaneous activation of two DA systems, one terminat-

ing in the ventral striatum and the other in the dorsal striatum (Kelly,

1977; Pycock and Marsden, 1978; see for further references, Joyce, 1983).

The DA system terminating in the dorsal striatum is thought to mediate








the deviation component of rotation, whereas the DA system terminating

in the ventral striatum is thought to mediate the activity component of

rotation. Yet, in the present study (Experiment 2), injections of DA

or AMPHET into the dorsal striatum of female rats produced both

postural deviation and rotation (see also Wolfson and Brown, 1983).

This suggests that rotation and postural deviation could be mediated

by a common DA-sensitive system in the dorsal striatum, in accord with

the findings of others (e.g., Dunnett et al., 1981a). However, in the

present experiment, rotation and deviation did not covary in intensity

within the 40 min observation session. When plotted by the sequential

5-min blocks of the 40 min session, changes in the magnitude of rotation

and deviation did not covary (data not shown). Moreover, the two

responses did not covary across the day of proestrus with changes in

gonadal hormones. This may indicate that the responses are mediated by

separable neural systems within the dorsal striatum.

It is possible that the changes in magnitude of rotation are due to

fluctuation in general activity of the animal, but this is unlikely for

several reasons. First, the observations in Experiment 2.1 that the

responses to intrastriatal VH did not vary across the stages of the

estrous cycle suggests that no major changes in activity was occurring.

This conclusion is supported by reports that gonadal hormones do not alter

open field activity (Beatty, 1979; Robinson et al., 1982). Secondly,

any changes in active behaviors should be reflected in measures of postural

deviation as well as rotation, since the measure "postural deviation" is

a compilation of a number of separate behaviors. It has, however, been

reported that ambulatory activity, induced by the peripheral administration

of DA agonists, has a circadian variation (Holcslaw et al., 1975; Kuribara









and Tadokoro, 1982; Nakano et al., 1980). It is unlikely that this

circadian variation accounts for changes in the rotational response to

intrastriatal DA and AMPHET across the day of proestrus (Experiment 2.2).

The circadian variation in the activity response to DA agonists,

administered systemically, is thought to be due to circadian variation

in the drug-metabolizing enzyme activities in the liver (Holcslaw et al.,

1975; Nakano et al., 1980). This could not account for the results of

Experiment 2.2, since DA and AMPHET were administered intracerebrally.

It might be argued that the rotational response to intrastriatal

DA and AMPHET is due to diffusion of the drugs to the ventral striatum,

where they induce increased locomotor activity, with a simultaneous

induction of postural deviation from the dorsal striatum. If, additionally,

the DA system terminating in the ventral striatum is sensitive to

gonadal hormones (Savageau and Beatty, 1981; Menniti and Baum, 1981),

then the changes in rotational activity could be due to an alteration of

the ventral striatal (mesolimbic) DA system. However, spread of DA

from the site of injection is probably minimal, and rotation appears not

to be dependent on spread of DA to ventral striatum following a dorsal

striatal injection (Brown and Wolfson, 1983; Wolfson and Brown, 1983).

In order to be sure that changes in activity are not contributing to

the changes in magnitude of the rotational response, experimental studies

of estrogenic effects on the DA system terminating in the ventral striatum

should be conducted.

In order to better test whether postural deviation and rotation are

indeed separately modulated by estrogen, it would be necessary to alter

the serum levels of estrogen directly, and measure the time for suppression

and enhancement of the response. It is also clear that changes in









magnitude of both behaviors, postural deviation and rotation, are not

correlated inversely with serum levels of estradiol on the day of

proestrus. Four hours after lights ON at proestrus, when serum levels

of estradiol should be high, the responses are suppressed; yet 3 hours

later when the serum levels of estradiol should be low, the responses were

not enhanced. This would suggest either that estrogen initiates events

leading to an increase in the responses, or other hormones are involved.

Experiment 3: Behaviors Induced by Intrastriatal Dopamine are
Suppressed Differentially by Estradiol Benzoate

Introduction

The results of Experiment 1 indicated that estrogen can modulate

the behavioral responses induced by the intrastriatal injection of DA,

producing first a suppression and later an enhancement of the response.

However, the study employed both male rats and a very large dose of EB, and

thus a pharmacological response of estrogen may have been measured. To

begin to determine if physiological levels of estrogen can alter striatal

dopaminergic behaviors, the responses to intrastriatal DA and AMPHET

were tested at various times of the estrous cycle, in Experiment 2. The

results from this experiment indicated that postural deviation and rotation

were both suppressed on the morning of proestrus and enhanced by estrus.

This inverse relationship between behavioral responses to striatal

dopaminergic stimulation and estrous cycle-related variations in the

concentration of estrogen in blood does not, however, appear to hold under

more careful scrutiny. For example, when tested at different times on

the day of proestrus, both responses were suppressed when serum levels of

estradiol should be high, but neither response was enhanced when the

serum levels estrogen should be low. It should be noted, however, that

serum levels of estrogen do not accurately reflect brain levels of estrogen









(Landau, 1977; McEwen et al., 1975; Blaustein et al., 1979; Eaton et al.,

1975); thus, the hypothesis of an inverse relationship between brain

levels of estrogen and the behavioral response to striatal DA may not

have been adequately tested in Experiment 2.2.

To test if exposure to endogenous estrogen is producing the

suppression of intrastriatal DA-mediated behaviors, and the withdrawal

of estrogen results in their enhancement, a direct alteration of serum

and brain estrogen levels, through peripheral injection of EB, should

produce down and up regulation of intrastriatal DA-induced behaviors

qualitatively similar to that observed during the estrous cycle.

Previously, experimenters have used high doses of EB in the range of

50 to 150 ig to suppress striatal DA-mediated behaviors (Naik et al.,

1978; Gordon et al., 1978), and the reversal of the suppression did not

occur for 24-48 hours post EB treatment (Gordon, 1980), however, since

low doses in the range of 1 to 3 pg EB, given suboutaneously, can induce

sexual receptivity (Eaton et al., 1975; McEwen et al., 1975; Davidson

et al., 1968), it would be instructive to see if a dose of EB in this

latter range can produce a significant modulation of striatal DA-mediated

behaviors.

The results of Experiment 2.2 indicated that the responses to intra-

striatal DA and AMPHET, postural deviation and rotation, did not covary

in magnitude across the day of proestrus. If estrogen is modulating

the behaviors postural deviation and rotation independently, then direct

alterations in serum levels of estradiol should lead to differential

changes in magnitude of the behavioral responses. The systemic injection

of EB should result in an independent variation in magnitude of the two

behaviors, over time, and not a covariation.









Finally, it is possible that the rotational response to intra-

striatal DA and AMPHET is due to spread of the drugs to the ventral

striatum. Moreover, there is some evidence that the DA system terminating

in the ventral striatum is sensitive to modulation by gonadal hormones

(Menniti and Baum, 1981; Savageau and Beatty, 1981). This could account

for the independent variation in magnitude of postural deviation and

rotation, to intrastriatal DA and AMPHET, observed across the day of

proestrus (Experiment 2.2). To test this possibility explicitly, intra-

cerebral application of dopaminergic drugs into the terminal regions of

the mesolimbic DA system can be made, while acutely altering serum

levels of estrogen. Experiments 3.1, 3.2 and 3.3 were designed to address

these issues.

Materials and Methods

Animals

Female Long-Evans hooded rats weighed 180-220 g at the beginning

of the experiment. They were housed individually and maintained on a

12:12 light:dark cycle (lights ON, 0800-2000). The rats were ovariec-

tomized bilaterally (OVX), under ether (Malinckrodt) anesthesia, 48 hours

before stereotaxic implantation of cannulae.

Stereotaxic surgery

The OVX rats were implanted bilaterally with permanent cannulae

under sodium pentobarbital (W.T. Butler Co.) anesthesia. Guide cannulae

were constructed from 21 GA stainless steel tubing and the injection

cannulae were constructed using 27 GA tubing. Since the injection cannulae

terminated 3.0 mm below the guide cannulae, rats in Experiments 3.1 and

3.2 had the guide cannulae stereotaxically implanted such that the injection

cannulae were located in the anterior dorsal striatum using the following









coordinates derived from Pellegrino et al. (1979): +2.0 to 3.0 mm with

respect to bregma; 2.0 to 4.0 mm lateral to bregma; 3.5 to 5.0 mm below

the surface of the brain. Rats in Experiment 3.3 had guide cannulae

stereotaxically implanted such that the injection cannulae were located

in the medial-ventral striatum using the following coordinates derived

from Pellegrino et al. (1979): +2.0 to 3.4 mm with respect to bregma;

1.0 to 2.0 mm lateral to bregma; 6.0 to 7.0 mm below the surface of the

brain. Stainless steel stylets, made from closed 27 GA tubing, kept

the guide cannulae patent when the rats were not being injected intra-

cerebrally.

Behavioral testing

The intracerebral application of a drug was made by injecting the

drug solution through the 27 GA cannula which was connected by poly-

ethylene tubing to a Hamilton syringe mounted on a Sage syringe pump

(Orion Research). The injection was made at a constant rate of 0.5 pl/min,

and the injection cannula remained in place for an additional 30 sec

after completion of the drug injection. For Experiments 3.1 and 3.2, after

the drug administration, the rats were placed into a circular clear

plexiglas observation chamber, 34 cm in diameter and 30.5 cm in height,

and observed for 40 min. The duration of postural deviation and the

number of 1/4 rotations that occurred both contralaterally and ipsilaterally

to the side of intrastriatal injection were recorded. A 90 degree movement

around the central axis of the rat was counted as a 1/4 turn. The amount

of time the rats deviated contralateral and ipsilateral to the side of

the intrastriatal injection was recorded continuously by the observer

using a two pole switch connected in series to a time clock and a rack

of cumulative counters. The cumulative durations of postural deviation








and number of 1/4 rotations were recorded every 5 min and 40 min.

For Experiment 3.3, the rats were administered intracerebral drugs

bilaterally, and then placed into a glass box (30 cm by 30 cm) that

rested on an electronic activity monitor (Stoelting 31400). The output

of the monitor was fed into a printout counter, and cumulative counts

for each 5-min block of the 60 min test were registered.

Drugs. Amphetamine (AMPHET; Sigma) and dopamine (DA; Sigma) were

dissolved in the phosphate buffer to a final pH of 7.4. The phosphate

buffer was a 7.0 mM sodium phosphate monobasic/140 mM sodium phosphate

dibasic solution. DA and AMPHET were made up at a concentration of

25 Vg/0.25 pl. Estradiol benzoate (Steraloids) at a concentration of

10 Ig/ml was dissolved in peanut oil by heating the oil to 600 C.

Histology. After behavioral testing, rats were administered an

overdose of sodium pentobarbital and perfused intracardially with 0.9%

saline followed by 10% formalin. The brains were placed in a 20% sucrose-

10% formalin mixture for at least 24 hours. The brains were then frozen,

sectioned at 30 nm, stained with cresyl violet, and the locations of

the cannula tips verified. Cannula tip placements for Experiment 3.1,

3.2 and 3.3 are shown in Figure 9.

Experiment 3.1

If, during the estrous cycle, estrogen is producing a suppression and

the withdrawal from estrogen an enhancement of intrastriatal DA behaviors,

then the direct alteration of brain estradiol levels should produce a

similar phenomenon. A peripheral injection of EB should produce down- and

up-regulation of intrastriatal DA-induced behaviors that show a time course

consistent with alterations in brain levels of estrogen. Administration of

1-3 ig EB subcutaneously produces a rapid increase in serum levels

of estrogen, and a decline to undetectable levels by 36 hours
























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after treatment (Cheng and Johnson, 1974). Changes in brain levels of

estrogen are delayed somewhat, and last for a longer period of time.

Using doses of EB in the range of 1-3 Ug, subcutaneous injections

produce significant increases in brain levels of estrogen by at least

3 hours after treatment, and are at undetectable levels by 24 (1 jg

EB, Landau, 1977) to 60 hours (3 pg EB, Eaton et al., 1975) after

treatment. Doses that are just slightly higher produce considerably

higher brain and serum levels, that remain at detectable levels up to

96 hours after subcutaneous injection of the EB (Eaton et al., 1975;

Cheng and Johnson, 1974).

Previously experimenters have used high doses of EB, 50 jig or

greater, to test for the time course of estrogen's modulation of striatal

DA-mediated behaviors. With high doses of EB, it has been reported that

suppression of striatal DA-mediated behaviors occurs as early as 2 hours

after EB administration (Naik et al., 1978; Gordon et al., 1978), and

the reversal from such a suppression does not occur for 24-48 hours post

EB treatment (Gordon, 1980). Because the short latency to, and long

duration of, the suppression seen with such large doses of EB could be

due to pharmacological effects of estrogen, more appropriate physiological

doses of EB need to be tested. Since, in Experiment 2.2, I did not

observe increases in magnitude of intrastriatal DA-induced postural

deviation with a decrease in serum levels of estrogen, withdrawal from

estrogen may not lead to the enhancement observed on the day of proestrus.

The enhancement of striatal DA-mediated behaviors observed with high doses

of estrogen (e.g., Experiment 1; Gordon, 1980) may also be due to

pharmacological effects of estrogen. Utilizing a smaller dose of EB, it

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of striatal DA-mediated behaviors; if so, the time course could then be

determined to see if it is correlated with a withdrawal from estrogen.

In this experiment, OVX rats were given different regimens of EB

treatment and then tested either for intrastriatal DA- or AMPHET-induced

deviation and rotation at 3, 24, 48 and 72 hours after the last EB

treatment. Animals were given 2 ig EB, s.c. in the neck. This treatment

paradigm should produce serum levels of E2 approximately equal to that

observed during proestrus, by 1 hour after treatment, and a return to

the pre-treatment baseline level by 36 hours post-treatment (Cheng and

Johnson, 1974). Such a dose is near the minimum amount needed to induce

sexual receptivity in OVX rats (Davidson et al., 1968).

Procedure. Rats (OVX) were divided into two groups that received

unilateral intrastriatal injections of either 25 Jg/0.25 pi DA or AMPHET

during each drug test. Rats were tested for intrastriatal DA- (n=6)

or AMPHET- (n=6) induced behaviors prior to each hormone treatment, in

order to obtain a PRE-HORMONE score. Each hormone regimen consisted of

two hormone treatments, separated by 96 hours (EB+EB, OIL+EB and OIL+OIL;

see Table 2-4). Rats were then injected intrastriatally with DA or AMPHET

and tested at either 3, 24, 48 and 72 hours (DA) or 3, 24 and 48 hours

(AMPHET) after the last hormone treatment. Rats (OVX) received each of

the three hormone regimens in a counterbalanced order. EB (2 ig) in the

oil vehicle (OIL) or OIL alone were given s.c. in the neck in a volume of

0.2 ml. No hormone was administered for 7 days after the last hormone

treatment of the previous regimen.

Data analyses. In order to obtain an index of the dominant direction

of postural deviation (including lateralized grooming), the time spent

ipsilateral was subtracted from the time spent contralateral to the side









of the intracerebral injection (difference score). A dominant direction

index was also obtained for the number of 1/4 rotations by subtracting the

number of 1/4 rotations ipsilateral from the number contralateral to

the side of the intracerebral injection. The difference scores for the

behavioral responses postural deviation and 1/4 rotations were analyzed

for differences due to intrastriatal injections of DA and AMPHET (DRUG),

and hormone regimen (HORMONE) using the sum total for the 40 min observation

period. An analysis of covariance was used to determine if the variables

DRUG (two levels) and HORMONE (3 levels) had significant overall effects,

with SEQUENCE (each drug test of HORMONE) as the quantitative covariable.

Because of the split-plot design, tests of HORMONE effects used the within

subject error term, and tests of between DRUG effects used subjects nested

within DRUG error term. In addition, in those HORMONE conditions in

which the SEQUENCE for the drug response to DA had one more value than

that for AMPHET, missing values were estimated according to the SAS

(Statistical Analysis System Institute) program. Tests for simple main

effects were then made using Scheffe's method for multiple comparisons

(equal sample size).

Results. Although the effects of the three separate hormone regimens

are qualitatively the same for both intrastriatal DA- and AMPHET-induced

postural deviation and rotation, the effects are not quantitatively the

same, and the data for each DRUG treatment will be presented separately.

For both drugs, DA and AMPHET, the effects of EB treatment were different

for the postural deviation and rotational responses.

When the rats were administered EB (regimens OIL+EB, EB+EB) they

showed a suppression of the contralateral postural deviation response to

intrastriatal DA at both 3 and 24 hours after the final EB treatment









(Figure 10-A, p <.01). By 72 hours after the last EB treatment the

postural deviation response had returned to PRE-HORMONE levels (Figure

10-A). The hormone regimen OIL+OIL produced no significant alteration

in the postural deviation response to intrastriatal DA at 3, 24 and 48

hours after the second OIL treatment, as compared to PRE-HORMONE scores.

In contrast to results observed with the postural deviation response,

the rotational response to intrastriatal DA was not altered by a single

treatment with EB (hormone regimen OIL+EB) at any time tested (Figure 10-B).

Two treatments with EB (hormone regimen EB+EB) did alter the rotational

response to intrastriatal DA, but the time course was not the same as that

observed with the postural deviation response measured at the same times.

Although there was no significant alteration in the rotational response

at 3 hours after the second EB treatment, there was a significant decrease

in the number of rotations at 24 hours after the second EB injection

(p <.01), as compared to the PRE-HORMONE drug response.

Intrastriatal DA-induced responses, contralateral postural deviation

and rotations did not show any carry-over effects for any hormone regimen.

The magnitude of the responses, measured prior to any hormone regimen

(Figure 10, PRE), did not differ significantly from the PRE-HORMONE

response measured at 5 days after each hormone regimen (3 replications,

data not shown).

The responses postural deviation and rotation produced by intra-

striatal AMPHET (Figure 11-A) showed a characteristic modulation to

EB treatment that was similar to that observed with DA (Figure 10-A).

Treatment with EB, hormone regimens OIL+EB and EB+EB, resulted in a

diminished contralateral postural deviation response to AMPHET at 3 and

24 hours after the last EB treatment (p < .01), and a return to PRE-HORMONE