EFFECTS OF ESTROGEN ON INDIVIDUAL APOMORPHINE-INDUCED BEHAVIORAL RESPONSES

By

RONALD L. SMITH

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

UNIVERSITY OF FLORIDA

2004 ACKNOWLEDGMENTS

I would like to thank Dr. Rowland and the members of my committee for their

guidance in the last stages of my work. I would also like to think Dr. Carol Van

Hartesveldt for her help over the years. In particular, I would like to thank Dr. Meyer, who has been a reliable source or encouragement and support from the very beginning.

To my family, I would like to thank my wife, Barbara who is a sustaining force in my life and who inspires me to be better everyday. Finally, to my mother, who has given so much and deserves so much more, my gratitude can not match your sacrifice. This

work is dedicated to my mother and to my grandmother, who started it all. Thanks for the experience and thank God for all of you.

ii TABLE OF CONTENTS

gage

ACKNOWLEDGEMENTS H

LIST OF FIGURES v

LIST OF TABLES v

LIST OF ABBREVL\TIONS vii

ABSTRACT ix

CHAPTER

1 LITERATURE REVIEW 1

Dopamine-Agonist Induced Behaviors 1 Effects of Estrogen on DA-Related Behaviors 7 Estrogen and Striatal DA Neurochemistry and Physiology 14 Aim 25

2 GENERAL METHODS 27

Subjects 27 Apparatus 27 Treatments Groups 28 Surgical Procedures 28 Drug and Hormone Preparation and Administration 30 Testing Procedure 31 Behavioral Measures 32 Histology 33 Statistical Analysis 34 Experiment Outline 34

3 EFFECTS OF APOMORPHINE DOSE ON BEHAVIORS 36

Introduction 36 Methods 37 Results 38 Discussion 43

4 EFFECTS OF OVARIECTOMY ON BEHAVIORS 62

Introduction 62

iii 1

Methods 63 Results 64 Discussion 72

5 EFFECTS OF ESTRADIOL TREATMENTS ON BEHAVIORS 84

Introduction 84 Methods 85 Results 86 Discussion 89

6 EFFECTS OF ESTRADIOL IMPLANTS ON BEHAVIORS 1 09

Introduction 109

Methods 1 1 Results 113 Discussion 115

7 GENERAL DISCUSSION 120

BIBLIOGRAPHY 125

BIOGRAPHICAL SKETCH 143

iv LIST OF TABLES

Table page

2- 1 . Behavioral Descriptions used to identify behaviors for measurement 35

3- 1 . Effects of apomorphine dose on behavior response totals 49

3-2. Effects of apomorphine dose on behavior response onset 50

4- 1 . Mean body weights after ovariectomy and intact rats 75

4-2. Behavioral response totals after ovariectomy 76

4-3. Behavioral response latencies after ovariectomy 83

5- 1 . The effects of 1 7-p E2 treatment on individual behaviors 93

5-2. The effects of 1 7-P E2 treatment on response latencies for behaviors 94

5-3. The effects of EB treatment on individual behaviors 95

6- 1 . Response Totals for behaviors after intracerebral implants 118

7- 1 . Comparison of behavior classifications among different studies 124

V LIST OF FIGURES

Figure page

2- 1 . Illustration of method of behavioral recording 33

3- 1 . Effects of apomorphine dose on biting behavior 51

3-2. Effects of apomorphine dose on continuous biting 52

3-3. Effects of apomorphine dose on discontinuous biting 53

3-4. Effects of apomorphine dose on sniffing behavior 54

3-5. Effects of apomorphine dose on continuous sniffing 55

3-6. Effects of apomorphine dose on discontinuous sniffing 56

3-7. Effects of apomorphine dose on line crossings 57

3-8. Effects of apomorphine dose on ambulation 58

3-9. Effects of apomorphine dose on rearing 59

3-10. Effects of apomorphine dose on grooming 60

3- 11. Effects of apomorphine dose on behavioral inactivity 61

4- 1 . Biting behavior totals after ovariectomy 77

4-2. Biting behavior displayed over time after ovariectomy 78

4-3. Continuous biting displayed over time after ovariectomy 79

4-4. Discontinuous biting displayed over time after ovariectomy 80

4-5. Line crossing totals after ovariectomy 81

4-6. Line crossings displayed over time after ovariectomy 82

5- 1 . Sniffing Behavior totals after treatment with 1 7-p E2 96

5-2. Biting Behavior totals after treatment with 1 7-p E2 97

5-3. Time-course for Biting 24 hours after 1 7-|3 E2 treatment 98

5-4. Continuous Biting totals after treatment with 1 7-P E2 99

vi 5-5. Time-course for continuous biting after 17-P E2 treatment 100

5-6. Time-course for discontinuous biting after 1 7-P E2 treatment 101

5 7. Biting Behavior totals after treatment with EB 102

5-8. Time-course pattern for biting after EB treatment. Panel E 103

5-9. Time-course pattern for biting after EB treatment. Panel F 104

5-10. Time-course pattern for biting after EB treatment. Panel G 105

5-11. Rearing behavior totals after treatment with 1 7-P E2 1 06

5-12. Sniffing behavior totals after treatment with EB 107

5- 13. Response totals for line crossings after treatment with EB 1 08

6- 1 . Location of cannula tip for intracerebral estradiol implantation 119

vii LIST OF ABBREVIATIONS

Ach Acetylcholine AD Dorsal Anterior Striatum ANOVA Analysis of Variance Apo Apomorphine C Centigrade cm centimeter CPU Caudate Putamen DA Dopamine DL Dorsal Lateral Striatum

DOPAC 3, 4-dihydroxyphenylacetic acid E2 Estradiol EB Estradiol Benzoate ga gage GAD Glutamic Acid Decarboxylase GP Globus Pallidus h hour HVA Homovanilic Acid kg Kilogram LA Locomotor Activity

Jig microgram mg milligram N-K Neuman Keuls N. Acc Nucleus Accumbens sc subcutaneous TH Tyrosine Hydroxylase VS Ventral Striatum 60HDA 6 hydroxy dopamine

1 7-a E2 1 7 alpha estradiol

1 7-P E2 1 7 beta estradiol

viii Abstract of Dissertation Presented to the Graduate School of the University' of Florida as Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

EFFECTS OF ESTROGEN ON INDIVIDUAL APOMORPHINE-INDUCED BEHAVIORAL RESPONSES

By

Ronald Leon Smith

May, 2004

Chair: Neil Rowland Cochair: Merle Meyer Major Department: Psychology

Clinical findings have suggested that ovarian hormones can influence the course of neurological disorders such as Parkinson's disease, Huntington's Disease as well as some forms of schizophrenia. Subsequent experimental investigations have shown a close association between the ovarian hormone, estrogen and dopaminergic activity in the

striatum of the rat. Additionally, estrogen has been shown to influence dopamine (DA)- related behaviors. However, findings in the literature have produced contradictory conclusions concerning the effects of estrogen on various DA-related behaviors, including apomorphine-induced behaviors. The aim of our study was to investigate the

specific effects of several estrogen treatment conditions on individual apomorphine-

induced behaviors.

Apomorphine, at doses from 0.15 mg/kg through 0.90 mg/kg (sc.), was shown to

induce individual behaviors, each displaying distinct dose and time-course response

ix patterns. The effects of ovariectomy on behavioral responses were examined and found to decrease locomotor activity, while increasing oral behavior. Estrogen treatments were found to differentially affect individual behaviors produced by a reference dose of 0.45 mg/kg of apomorphine. The effects of estrogen treatments were both dose and time dependent. Apomorphine-induced behaviors were influenced as early as 3 hours after administration of 50 ^g/kg of 17-P E2. Daily injections of estradiol benzoate were also found to influence apomorphine-induced behaviors after 3 and 7 days of treatment, but not after 20 days of treatment. The behavioral response to estrogen implants placed in different parts of the basal ganglia of the rat indicated that estrogen may be acting on

specific regions in the striatum to influence these behaviors.

These studies enhance our understanding of interactions between hormones and the brain which could potentially enhance our understanding of the possible influences of these hormones on various neurological disorders.

X CHAPTER 1 LITERATURE REVIEW

Previous studies have shown that estrogen can affect several clinical conditions

involving neurological and psychiatric conditions (Smith et al., 1978; Bedard et al., 1979;

Villeneuve et al., 1980). For instance, estrogen treatment improved some symptoms

associated with tardive dyskinesia (Villeneuve et al., 1980). However, subsequent

experimental investigations have presented contradictory findings as to the mechanisms

and site of estrogen actions in the brain. The aim of our study was to investigate various

elements of the interaction between estrogen and the brain by examining the effects of

estrogen on apomorphine-induced changes in specific behaviors. Estrogenic effects on

apomorphine-induced changes in behaviors were examined in three steps: by examining

the effects of the removal of the intrinsic source of estrogen; by examining the effects of

administration of exogenous estrogen; by examining the behavioral effects of

intracerebral implants. Apomorphine produces a behavioral response consisting of

several recognizable response patterns. The present study attempted to measure specific

behavioral responses in order to determine if estrogen produced differential effects on

distinct behaviors.

Dopamine-Agonist Induced Behaviors

Basic Description

Dopamine-related behaviors represent a diverse group of behaviors, displayed in response to the administration of dopamine (DA) agonists, whose form is generally characterized as being displayed in a repetitive and highly stereotypical manner. These

1 behaviors, often referred to as stereotyped behaviors, are displayed in response to a

variety of DA agonists, including amphetamine and apomorphine.

DA-related behaviors have been used both as a behavioral model for various basal

ganglia-related syndromes, and as a behavioral assay for basal ganglia fionction (Fog,

1969; Ellinwood and Balster, 1974; Randrup and Munkvad, 1974; Randall, 1985;

Dourish, 1987). The use of stereotyped behaviors as a behavioral assay of striatal DA

activity is based primarily on pharmacological and neuroanatomical data associating

changes in behaviors with changes in striatal DA activity and the condition of the

striatum in general.

Despite the strong evidence suggesting the involvement of the striatum in specific

DA agonist-induced behaviors, the full constellation of behaviors typically associated with the stereotypy syndrome, including sniffmg, locomotion, rearing, along with oral behaviors, are not completely reproduced by intracerebral injections of DA agonists.

Instead, it seems that intracerebral injections of DA agonists into various regions of the forebrain can produce different and specific behavioral effects. In addition, the sequence in which behaviors are displayed after intracerebral injections of DA or DA agonists may differ from the behavioral sequence usually observed after systemic injections of DA agonists. Intracerebral injections of DA agonists into the nucleus accumbens (N. Acc) or olfactory tubercle produce an initial display of sniffing and increased locomotion (Costall and Naylor, 1975a; Makanjuola and Ashcroft, 1982).

However, intrastriatal injections of DA or amphetamine in some portions of the striatum produce biting behavior first, followed by bouts of sniffing and locomotion sometime later (Fog and Pakkenberg, 1971). Fog & Pakkenberg, (1971) also performed intrastriatal 3

injections of tritiated DA to determine the spread of drug within the brain. The authors

observed that radioactive DA was retained within the caudate for at least 25 minutes after

injections. Biting and licking occurred for the first 20 minutes, after which sniffing

predominated, suggesting that biting is mediated primarily by the striatum, but also that

surrounding areas might mediate other stereotyped behaviors.

Neuroanatomical Substrate for DA-Related Behaviors

The main common characteristics shared by these behaviors is their essential

reliance on central DA systems (Melzacka et al, 1978; Melzacka et al., 1979; Smith et

al., 1979; Campbell et al., 1980; Watanabe et al., 1981; Lloyd et al., 1982; Watanabe et

al., 1982; Smith et al., 1985; Bianchi et al., 1986). The impression that DA agonist-

induced behaviors are dependent on the striatum was established relatively early.

Apomorphine-induced biting was associated with the basal ganglia even before detailed

descriptions of brain DA systems were available (Fog, 1972). Subsequently, it was

determined that almost all of the DA in the mammalian brain is found in the extrapyramidal system, unlike noradrenalin, which is widely distributed throughout the brain. These studies pointed to an intimate involvement of the basal ganglia in DA agonist-induced behaviors.

Neuroanatomy of the mesencephalic DA system

The relationship between brain DA systems and stereotyped behaviors is characterized, in part, by the neuroanatomical organization of DA systems. DA pathways

compose seven projection systems, of which only the mesencephalic efferent system is considered essential to the development and display of DA agonist-induced stereotyped behaviors (Schultz, 1982; Albanese et al., 1986). Originally the mesencephalic efferent

DA system was thought to contain two anatomically distinct DA pathways, the mesolimbic DA pathway and the mesostriatal DA pathway (Anden et al., 1966a; Anden

et al., 1966b; Ungerstedt, 1971). The mesostriatal DA pathway system is composed of

DA-containing cell bodies in the substantia nigra which project primarily to the dorsal portion of the caudate nucleus and globus pallidus, while mesolimbic DA cells in the ventral tegmental area project primarily to the N. Acc, olfactory tubercle, cerebral cortex and other regions of the limbic forebrain including the central nucleus of the amygdala

(Anden et al., 1966a; Anden et al., 1966b; Ungerstedt, 1971).

A number of reciprocal pathways have been described indicating there is

significant overlap of the terminal fields of midbrain DA neurons. The substantia nigra

and ventral tegmental area are continuous and their designation as separate nuclei is often

overemphasized. While DA cells in the substantia nigra primarily project to the caudate nucleus, they also project to the N. Acc, central nucleus of the amygdala and cerebral cortex (Lindvall and Bjorklund, 1978; Moore and Bloom, 1978). Similarly, DA neurons

in the ventral tegmentum primarily project to the N. Acc, but also project to the ventral caudate, globus pallidus, cerebral cortex and other parts of the limbic system (Lindvall

and Bjorklund, 1978; Moore and Bloom, 1978). Therefore, there is considerable overlap of both the terminal fields and cell-rich regions of the mesostriatal and mesolimbic DA pathways. Consequently separation of the mesolimbic and mesostriatal DA pathways based primarily on the midbrain projection pathways may not be valid.

Involvement of the CPU in DA-related behaviors. Placement of localized lesions in cerebral tissue or intracerebral injections of DA agonists have been the primary tools used to investigate the behavioral significance of the striatum and other DA-rich

forebrain regions in the mediation of DA agonist-induced stereotyped behaviors. Initial 5

studies showed that intracerebral application of DA agonists could produce biting behavior (Ernst and Smelik., 1966). Bilateral implants of crystalline apomorphine in the

dorsal striatum induced continuous biting. However, the increase in biting after

crystalline implants only occurred after a delay of 30-40 minutes, possibly indicating that the behavioral effects resulted from diffusion of the drug to other portions of the brain.

Nevertheless, biting and licking behaviors have also been produced by bilateral

intrastriatal injections of DA or DA agonists, occurring in a much shorter time frame

(Fog and Pakkenberg, 1971; Jackson et al., 1975). Additionally, 60HDA lesions in the

caudate nucleus decrease DA agonist-induced biting, indicating the involvement of the striatum in this behavior (Divac, 1972; Costall and Naylor, 1973c; Costall and Naylor,

1975b; Kelly et al., 1975; Costall et al., 1977a).

Extrastriatal involvement in DA-related behaviors

Olfactory Tubercle. The involvement of extrastriatal areas in DA agonist- induced behavioral response has been suggested by numerous studies (Glick, 1970;

Costall and Naylor, 1973a; Costall and Naylor, 1974; Braestrup, 1977; Redgrave et al.,

1980). Whereas the striatum has been implicated as essential for the expression of DA agonist-induced biting and licking (Fog and Pakkenberg, 1971; Asher and Aghajanian,

1974), DA agonist-induced sniffing and locomotor activity have been suggested to be primarily under the influence of the olfactory tubercle and N. Acc (Pijnenburg and

Rossum, 1973; Asher and Aghajanian, 1974; Kelly et al., 1975).

Nucleus Accumbens. In support of the general consensus, there is considerable evidence suggesting that the N. Acc is the principal forebrain region controlling locomotor activity. Intra-accumbens injections of DA or DA agonists can stimulate locomotor activity (Pijnenburg and Rossum, 1973; Carr and White, 1987). Furthermore, 6

local injections of DA antagonists into the N. Acc inhibit amphetamine-induced

locomotor activity (Jackson et al., 1975; Pijnenburg et al., 1975; Makanjuola and

Ashcroft, 1982; Morgenstem and Fink, 1985; Towell et al., 1987; van den Boss et al.,

1988). Additionally, destruction of dopaminergic input to the N. Acc by 60HDA lesions

abolishes spontaneous as well as amphetamine-induced locomotion (Kelly et al., 1975;

Kelly and Iversen, 1976; Costall et al., 1977a; Iversen and Koob, 1977; Joyce et al.,

1983; Joyce and Iversen, 1984). Finally, 60HDA lesions in the N. Acc can increase

apomorphine-induced locomotor activity, probably as a result of denervation

supersensitivity of DA receptors in the N. Acc, indicating that DA receptors in the N. Acc

mediate DA agonist-induced locomotor activity (Asher and Aghajanian, 1974).

However, there have been reports that 60HDA and kainic acid lesions in the N.

Acc fail to decrease amphetamine-induced locomotion (Fink and Smith, 1979; Fink and

Smith, 1980; Carey, 1982; Kafetzopoulos, 1986). Fink & Smith (1980) reported that

60HDA lesions in the N. Acc alone failed to reduce amphetamine-induced locomotor

activity. Locomotor activity was reduced only when areas surrounding the N. Acc were

included in the lesion. Similarly, Carey (1982) observed that electrolytic lesions in the N.

Acc failed to influence amphetamine-induced locomotor activity unless the septum was

also damaged. Moreover, many of the studies reporting a reduction in locomotion after

60HDA lesions of the N. Acc, also reported lesions in the olfactory tubercle, and in some cases, portions of the caudate nucleus (Creese and Iversen, 1974; Kelly et al., 1975; Kelly and Iversen, 1976; Costall et al., 1977b; Joyce et al., 1983). It appears that contrary to earlier studies that suggested that the caudate nucleus was not involved in DA agonist- induced locomotor activity, more recent studies suggest otherwise. Intrastriatal injections of amphetamine or 60HDA lesions specifically in the caudate nucleus stimulated and

reduced locomotor activity respectively (Fink and Smith, 1979; Fink and Smith, 1980;

Can- and White, 1987).

Estrogen Effects on DA-Related Behaviors

Estrogen and DA-Related Behaviors - Antagonist Effects

There is now substantial evidence suggesting that estrogen can influence midbrain

DA systems. Animal studies suggest that indeed estrogen treatment can antagonize

several DA agonist-induced behaviors including apomorphine-induced circling (Bedard

et al., 1978), locomotion (Bedard et al., 1980) and other DA-related behaviors (Gordon,

1980; Gordon et al., 1980a; Gordon et al., 1980b; Fung et al., 1986). Estrogen acts as an

antagonist when it blocks or decreases a response otherwise stimulated by the

administration of a DA agonist.

In addition to antagonizing apomorphine-induced behaviors in a manner similar to

neuroleptics, estrogen treatment can augment the effects of neuroleptics on DA-related

behaviors. Estradiol (E2) treatment increases haloperidol catalepsy. The E2 treatment

effects are additive with the effects of haloperidol administration. Several studies have

reported that estradiol treatment has been reported to augment neuroleptic-induced

catalepsy (Chiodo et al., 1979; Di Paolo et al., 1981; De Ryck et al., 1982; Johnson and

Stevens, 1983a; Johnson and Stevens, 1983b; Nicoletti et al., 1983; Palermo-Neto and

Dorce, 1990). Further, estrogen treatment reduced the rebound increase in behavioral ratings often found after withdrawal from haloperidol treatment (Gordon et al., 1980a;

Fields and Gordon, 1982).

If estrogen is acting as a DA antagonist, it should acutely antagonize DA-induced behaviors and potentiate neuroleptic-induced responses. It appears to do so. Second, 8

over time as the estradiol plasma levels decline, estrogen should provide less antagonistic

effects on DA-related behaviors and provide less augmentation of neuroleptic-induced

responses such as catalepsy. There is evidence for this second effect as well. Behavioral

ratings are higher six days after a single injection of estradiol valerate (Hruska et al.,

1980). Estradiol fails to produce cataleptic responses two or six days after injection,

when E2 levels are likely declining (De Ryck et al., 1982; Johnson and Stevens, 1983b).

There is disagreement as to whether estrogen treatment alone can produce

catalepsy. Estrogen treatment has been reported to increase duration of the bar grid

response (Johnson and Stevens, 1983a), but others have reported that estrogen had no

effect on similar catalepsy indicators (De Ryck et al., 1982). Nevertheless, the effects of

estradiol on neuroleptic-induced catalepsy are consistent with the antidopaminergic

effects of E2 treatment on apomorphine-induced behaviors.

Estrogen and DA-Related Behaviors - Agonist Effects

Despite numerous reports suggesting an antagonistic effect of estrogen on DA

agonist-induced behavioral responses, there are also reports that estrogen treatment

increases some DA agonist-induced behaviors (Hruska and Silbergeld, 1980a; Hruska

and Silbergeld, 1980b; Chiodo et al., 1981; Becker and Beer, 1986). Disagreements

among studies have been attributed to a number of factors including differences in

treatment procedures, the type of behavior measured and the animal subject used for

study. Inconsistencies in fmdings concerning the effects of estrogen treatment on DA

agonist-induced behaviors and striatal DA neurochemistry are further compounded by the undetermined location and mechanism by which estrogen acts to influence DA-related 9

behaviors and striatal neurochemistry. These variables will be discussed in the next sections.

Effects of Endogenous Estrogens on DA-Related Behaviors

Consistent with evidence showing that various exogenous estrogen treatments can influence DA agonist-induced behaviors are reports that endogenous sources of estrogen can also influence these behaviors. This is interesting since traditionally, ovarian hormones, and estrogens specifically, have been closely associated with reproductive functions. Estrogens are a class of steroidal hormones, of which estradiol 17-P (17-P E2) is the most biologically active form. The primary source of endogenous estrogens is the

ovary, although, the adrenal cortex produces measurable quantities as well, hi the rat, the ovarian cycle is completed every 4-5 days. During the day of proestrus, plasma estradiol levels display a dramatic but short-lived increase, followed by a more gradual decline

over successive days. The significance of the estrus cycle in the rat is that dramatic

changes in behavior correspond with changes in estrogen secretion. The implication is that the changes in estrogen secretion have a significant impact on the animal's behavior, presumably by acting on the brain.

If estrogen can affect the behavioral response to DA agonists, then DA agonist- induced behaviors should be influenced by the natural variations of endogenous estrogens. There have been reported that apomorphine-induced behaviors vary across the estrous cycle, and that behavioral ratings were lower on proestrus, when plasma estradiol levels are highest, than on other days (Miller, 1983). Miller (1983) suggested that changes in plasma estradiol secretions across the estrous cycle might be responsible for the changes in behavioral responding. Additionally, both amphetamine- and electrical 10

stimulation-induced rotations are greater on estrus than on other days of the estrous cycle

(Robinson et al., 1982; Becker and Beer, 1986; Becker et al., 1987). DA agonist-induced postural deviation and rotation are displayed more on the day of estrus than proestrus

(Joyce and Van Hartesveldt, 1984b). These studies indicate that endogenous estrogens can influence some DA agonist-induced behaviors. In contrast, several other reports failed to observe any changes in apomorphine-induced behaviors during the estrous cycle

of the female rat (Steiner et al., 1980; Hruska et al., 1982a; Kazandjian et al., 1987).

Additional studies reported that neither apomorphine-induced locomotion nor

haloperidol-induced catalepsy varied across the estrous cycle (Kazandjian et al., 1987;

Kazandjian et al., 1988). Differences in apomorphine dose and method of behavioral measure, as well as the time during estrus cycle when tested, may account for disagreements among studies. Furthermore, differences in plasma estradiol levels over the 4-day estrous cycle may not be significant enough to produce recognizable changes in some behaviors. Finally, the method of behavioral measure may not have been sensitive

enough to detect subtle changes in behavior.(Beatty et al., 1972; Savageau and Beatty,

1981; Miller, 1983; Johnson and Stevens, 1984)

Effects of Ovariectomy

If endogenous estrogens can influence the behavioral response to DA agonists, then the removal of the major source of endogenous estrogens, by ovariectomy, should affect the behavioral response to DA agonists as well. Indeed several reports have suggested that bilateral ovariectomy results in an increase in stereotypy ratings of apomorphine-induced behaviors in the rat (Lai and Sourkes, 1972; Gordon, 1980; Gordon et al., 1980b; Verimer, 1981; Hruska et al., 1982a). On the other hand, there were conflicting reports that ovariectomy failed to affect DA agonist-induced behaviors in the 11

rat (Beatty et al., 1972; Savageau and Beatty, 1981; Miller, 1983; Johnson and Stevens,

1984).

One possible explanation for the differences among studies includes the time after

ovariectomy, and the dose of drug given. Gordon and associates have suggested that

behavioral ratings are not enhanced until three months after ovariectomy (Gordon, 1980;

Gordon et al., 1980b). Indeed, behavioral testing shortly after ovariectomy might

explain, in part, the failure to observe any change in behaviors (Beatty et al., 1982;

Miller, 1983). Nevertheless, others reported that tests performed more than 3 months

after ovariectomy still failed to result in any change in behavioral responding (Savageau

and Beatty, 1981; Johnson and Stevens, 1984). Therefore, it appears that factors in

addition to the interval between the time of ovariectomy and behavioral testing may

determine the behavioral response to DA agonist after ovariectomy.

Effects of Exogenous Estrogen

A more direct approach to investigating the influence of estrogen on DA agonist-

induced behaviors involves the administration of estrogen to ovariectomized females.

The systemic administration of estrogen has been shown to influence a variety of DA-

related behaviors, including DA agonist-induced circling behavior and postural deviation

(Bedard et al., 1980; Hruska and Silbergeld, 1980b; Joyce et al., 1984; Joyce and Van

Hartesveldt, 1984b; Becker and Beer, 1986). Despite the evidence suggesting that estrogen treatment can influence DA agonist-induced behaviors, there is dispute as to whether estrogen treatment inhibits or enhances DA agonist induced behaviors (Gordon,

1980; Gordon et al., 1980b; Gordon and Diamond, 1981; Hruska et al., 1982a; Johnson and Stevens, 1984). 12

The possible causes of the disagreements among studies have centered around

three likely factors. (1) The time between termination of estrogen treatment and

behavioral testing, also called the treatment-test interval; (2) The length of time estrogen

is administered, or the treatment interval; (3) the dose of estrogen administered, or the

dose-interval; (4) A fourth source of variability, which has not been carefully examined,

may involve the type of behavior measured. These four factors may be important enough

to determine the direction and extent to which estrogen affects DA agonist-induced

behaviors.

Effects of treatment-test intervals. Gordon (1980) was first to provide

convincing evidence that the treatment-test interval might determine the effects of

estrogen on stereotyped behaviors. In general, stereotypy rating scores are lower in

animals tested within 24 hours after the last of several estrogen treatments, while

stereotypy rating scores are increased in animals tested 48 hours or more after cessation

of estrogen treatment (Lai and Sourkes, 1972; Gordon, 1980; Hruska et al., 1980; Hruska

and Silbergeld, 1980b; Pittman and Fibiger, 1983). Gordon (1980) suggested that the

apparent biphasic affects of estrogen treatment on stereotypy ratings of DA agonist-

induced behaviors maybe attributed to an initial inhibition of stereotyped behaviors during elevated estradiol plasma levels within 24 hours of injection. Behavioral inhibition is followed by a rebound increase in behavioral rating scores 48 hours or more after treatment cessation, during the period in which plasma estradiol levels are declining.

Although the treatment-test interval may be important in determining the effects of estrogen treatment on DA-related behaviors, it alone does not completely explain the differences in findings among studies. For example, at least two studies suggest that the 13

treatment-test interval does not determine the affect of estrogen treatment on DA-related

behaviors. Johnson & Stevens (1984) failed to observe a decrease in DA-related

behaviors 24 hours after estrogen treatment. In contrast Pitman & Fibiger (1983)

observed a decrease in behavioral ratings 24 hours after cessation of estrogen treatment,

and stereotypy rating scores remained suppressed at least 72 hours after treatment.

EfTects of estrogen dose. The effects of the treatment-test interval on DA-related

behaviors maybe determined, in part, by the dose of estrogen. Studies have reported that

stereotypy ratings are lower after treatment with doses of estradiol ranging from 10 ^ig/kg

to 100 \ig/kg (Gordon, 1980; Gordon et al., 1980b; Clopton and Gordon, 1985).

Stereotypy ratings are decreased by as little as 10 |ig/kg, but the latent increase in

stereotypy ratings after withdrawal from estrogen treatment is only produced by doses of

30 ^g/kg or more (Gordon, 1980; Clopton and Gordon, 1985). Additionally, the latent

increase in stereotypy rating scores of DA agonist-induced behaviors is dose dependent.

Accordingly, stereotypy ratings were greater after withdrawal from 100 ng/kg of estradiol than after withdrawal from 50 }xg/kg of estradiol.

Effects of estrogen treatment duration. If the effects of estrogen treatment on

DA-related behaviors are dependent on the length of treatment, then the increase in stereotypy ratings observed after treatment would be a function of the time exposed to estrogen, rather than the treatment-test interval. There seems to be agreement among several studies concerning the effects estrogen administration has on DA agonist-induced behaviors measured within 24 hours after estrogen treatment. The inhibitory effects of estrogen are found after both singular and multiple injections of the hormone if behavioral measurements are taken within 24 hours of the last estrogen treatment. A 14

single injection of a very high dose of estradiol benzoate (2.0 & 10 mg/kg) resulted in

lower stereotypy ratings of amphetamine-induced behaviors 90 minutes after injection

(Naik et al., 1978). Furthermore, a single injection of 10 ^ig/kg of estradiol benzoate 2

hours before testing prevented the increase in stereotypy ratings normally observed after

withdrawal from chronic neuroleptic treatment (Gordon et al., 1980a).

Studies utilizing longer treatment intervals, in which estrogen was administered

for 3 - 10 days also reported a decrease in the behavioral response to apomorphine

(Bedard et al., 1980; Gordon, 1980; Hruska et al., 1982a; Miller, 1983; Fung et al., 1986).

In contrast, stereotyped ratings have been reported to be unaffected by estrogen

treatments lasting longer than 10 days (Menniti and Baum, 1981). These studies suggest

that behavioral tolerance may develop in response to prolonged estrogen treatment. Thus

while the length of treatment may influence the effects of estrogen on DA agonist-

induced behaviors, the body of evidence presented here suggest that there is an upper

limit of time after which estrogen treatments are ineffective.

Estrogen and Striatal DA Neurochemistry and Physiology

Biochemical and physiological studies provide fiirther evidence that estrogen is affecting DA-related behaviors by acting on striatal neurochemistry. Estrogen treatment has been shown to influence biochemical measures of striatal DA activity in a manner consistent with some behavioral changes. Estrogen treatment can increase striatal DA receptors and decrease DA concentrations in the striatum (Cheronis et al., 1979; Dupont et al., 1981; Di Paolo et al., 1982a; Di Paolo et al., 1982b; Fields and Gordon, 1982).

Estrogen treatment has been reported to influence electrical activity of nigral and striatal cells (Amauld et al., 1981; Chiodo and Caggiula, 1983; Tansey et al., 1983). The 15

conclusion from these studies is that estrogen can influence striatal DA activity, possibly

resulting in a related change in behavior.

Estrogen treatment has been shown to affect different aspects of DA activity. The

significance of the effects of estrogen on DA-ergic activity must be maintained within the

context of the chain of events from DA synthesis to the terminal points of DA receptor

activity and DA metabolism.

Estrogen Effects on DA Receptors

Estrogen-induced increases in DA receptors

The effects of E2 on striatal DA receptors are consistent with a DA antagonistic

effect on DA-ergic transmission. DA receptors are reported to increase after E2 treatment

by numerous studies. With a fairiy high dose of 125 ^g/kg of estradiol valerate DA

receptors first increase 4 days after E2 treatment (Hruska, 1986). At least 24 hours after

the last of 3 daily injections is required to observe an increase. Measurement of DA

receptors less than 24 hours after the last E2 injection found no differences in DA

receptors among groups (Hruska, 1986; Fernandez-Ruiz et al., 1989). Therefore the

effects of E2 on DA receptors are conditional. A minimum of 4 days after E2 treatment is needed before an increase in striatal DA receptors are found (Hruska, 1986). Estradiol treatment in intact females is ineffective, producing no noticeable changes in striatal DA receptors (Hruska et al., 1982a). The effects differ across brain regions also. Estradiol treatment increased DA receptors in the caudate putamen (CPU), but variable results have been reported for other regions.

The increase in DA receptors after E2 treatment is not a fimction of declining E2 plasma levels since the increase in DA receptors is observed during treatment when 16

plasma levels are still elevated (Hruska, 1986). Although a minimal dose of E2 is needed

to produce an increase in DA receptors, even large doses of E2 are ineffective if the

measurements are made earlier than 96 hours after beginning treatment. It appears that

E2 treatment can maintain elevated DA receptors only for a limited time. A decline in

DA receptor number was observed 10 days after the onset of treatment even though

plasma E2 levels were still elevated (Hruska, 1986). However, others have detected

increased DA receptors 24 and 48 hours after 21 and 28 day treatments, suggesting a

much larger DA receptor elevation (Di Paolo et al., 1982c; Ferretti et al., 1988; Ferretti et

al., 1989). These studies suggest DA receptor up-regulation in the presence of high circulating E2 levels. Gordon and associates, on the other hand, point to receptor down regulation by estrogen, when estrogen levels are high. They offer two lines of evidence for this. First, the authors report that within 24 hours of E2 treatment striatal DA receptor affinity is decreased (Fields and Gordon, 1982; Gordon and Perry, 1983). Second, that E2 treatment can eliminate the increase in DA receptor number normally seen after long- term neuroleptic treatment.

Estrogen-induced decreases in DA receptors

Several studies have reported decreased striatal receptor number after E2 treatment (Fields and Gordon, 1982; Gordon and Perry, 1983; Miller, 1983; Fernandez-

Ruiz et al., 1989; Tonnaer et al., 1989). Decreased receptor number or affinity is usually observed within 24 hours of the end of estrogen treatment. This would support behavioral observations associated with lower behavioral ratings 24 hours after E2 treatment. Within 24 hours of E2 treatment several studies reported decreases in striatal

DA receptors (Fields and Gordon, 1982; Gordon and Perry, 1983; Miller, 1983; 17

Fernandez-Ruiz et al., 1989; Tonnaer et al., 1989). Measurements taken less than 10 hours after the last injection of E2 did not detect changes in striatal DA receptors

(Fernandez-Ruiz et al., 1989). The duration of E2 treatment is also critical. At least 3

days of E2 treatment is necessary to produce a reliable difference in receptor activity.

Estradiol treatments shorter than 3 days were ineffective (Hruska, 1986). A very low dose of 8 i^g/kg of E2 for 4 days was reported to decrease striatal DA receptor density

(Fields and Gordon, 1982). Others using higher doses of E2 observed decreases in DA

receptor affinity, but always within 24 hours of the treatment end (Ramos et al., 1987;

Fernandez-Ruiz et al., 1989; Tonnaer et al., 1989). Therefore, studies have reported that both low and high E2 treatment doses can reduce DA receptor activity. An important variable in determining the effects of E2 on DA receptors is the treatment-test interval, which refers to the time between the end of E2 treatment and the time of measurement.

Testing between 10 and 24 hours after the last of several daily E2 injections produced a decrease in receptor activity.

Testing occurring after only 24 hours or 4 hours after 3 days of treatment were ineffective, suggesting that a critical time frame is present (Hruska, 1986; Fernandez-

Ruiz et al., 1989). A single injection of a large dose of E2 valerate (E2V) can produce elevated striatal DA receptor numbers 6 days later, but not 10 days later (Hruska, 1986).

The effectiveness of E2 on DA receptors and related behaviors is diminished after long-

term E2 treatment (Gordon et al., 1980b; Gordon and Diamond, 1981 ; Menniti and Baum,

1981; Fung et al., 1986; Hruska, 1986). 18

Estrogen treatment effects on DA receptor subtypes

Evidence that estrogen differentially affects DA receptor subtypes will be

approached by discussing the effects on specific receptor type, and whether the

recognized variables such as treatment dose and duration affect the receptor subtypes the

similarly or differently. Estrogen treatments have been reported to lead to an increase in

the density of striatal Di DA receptors (Hruska and Nowak, 1988; Levesque and Di-

Paolo, 1989; Levesque and DiPaolo, 1991) and D2 DA receptors (Di Paolo et al., 1979;

Hruska and Silbergeld, 1980a; Di Paolo et al., 1982a; Gordon and Perry, 1983; Hruska,

1986; Levesque and DiPaolo, 1991). Levesque & DiPaolo (1991) observed that chronic

E2 treatment increased both striatal Di and D2 DA receptor densities, but no change in

receptor affinity was observed.

Hruska et al. (1982) interpreted the absence of an effect on DA receptors and

suggested there is no evidence that E2 exerts either a rapid or prolonged inhibition of the

DA system in the striatum (Hruska et al., 1982a). Instead, the authors interpreted the

changes in receptors as an indication of increased DA transmission. Estrogen has been

reported to increase DA receptors in the striatum, N. Acc and olfactory tubercle (Bedard

et al., 1979). DA receptors were not affected by estrogen dose. With doses ranging from

10 to 500 ^g no further increases in DA receptor number (Bmax) were observed above

that shown in response to the 14 ^g estradiol dose.

Anatomical regional effects of estrogen treatment on DA receptors

Estradiol treatment was found to increases DA receptors in the N. Acc (Menniti

and Baum, 1981). The authors refer to earlier work in which chronic E2 caused an increase in DA concentration and HVA and DOPAC in the N. Acc and septal region, but 19

not the CPU of castrated male rats (Menniti and Baum, 1981). The fact that E2 increased

apomorphine-induced locomotor activity in animals with 60HDA lesions suggests that

E2 effects are postsynaptic.

Summary effects of estrogen treatments on striatal DA receptors

Estrogen treatment effects on striatal DA receptors appear to act according to

region- as well as time-dependent factors. Estrogen effects on DA receptors vary in

different regions of the striatum (Amt, 1985; Savaster et al., 1987). Moreover, the

duration of treatment can result in either a decrease in the affinity of the receptor or an

increase in the receptor density. Finally, there is evidence that the increase in DA

receptor density after estrogen treatment including reports of possible acute estrogen

effects on DA receptors is not mediated by protein synthesis which would be necessary to

produce more receptors. Apparently, estrogen decreases the degradation of DA receptors

without affecting receptor synthesis. Such an effect of estrogen would be consistent with

the reports of acute effects of estrogen on DA receptors, which would occur to quickly to

increase protein synthesis. But a change in receptor degradation might occur in a very

short time. The end result would be an accumulation or increase in receptor number.

Estrogen Effects on Striatal DA Content

It seems that E2 effects on striatal DA concentration are significant only under certain conditions. Numerous studies failed to observe an effect of estrogen treatment on striatal DA content (Gordon et al., 1979; Crowley, 1982). There appear to be, fi-om the literature, two variables responsible for a change in DA content after estrogen treatment, and they are the treatment dose and duration.

Estradiol treatment along with septal lesions result in decreased DA content

(Gordon et al., 1977; Gordon et al., 1979). Striatal DA content is decreased by treatment 20

with high E2 doses (Jori and Dolfini, 1976), or striatal DA content is decreased by E2

treatment over extended time (Jori and Dolfini, 1976; Dupont et al., 1981; Di Paolo et al.,

1982a; Di Paolo et al., 1982b). The treatment duration is important, because acute

measures after both low and high E2 dose treatment were ineffective (Gordon et al., 1979;

Crowley, 1982). The high E2 dose administered by the same treatment schedule produced a decrease in striatal DA content. Finally the results, suggest collectively that what ever the conditions are, E2 treatment is either ineffective or produces a decrease in

DA content, there were no reports of increased content.

No change in DA content is observed within 3 hours of E2 treatment, over a dose range of 0.03 to 50 ^g, or after E2 treatment lasting three days (Eikenburg et al., 1977;

Gordon et al., 1977; Gordon et al., 1979; Euvard et al., 1980; Crowley, 1982; Piccardi et al., 1983). However, long-term E2 treatment causes a decline in DA content (Dupont et

al., 1981; Di Paolo et al., 1982a; Di Paolo et al., 1982b).

Steroid contraceptive drugs can cause a decrease in striatal DA concentration after chronic treatment lasting 30 days (Jori and Dolfini, 1976). Treatment with a higher dose of E2B (50 mg) caused a decrease in DA concentration 72 hrs later, but not 3 hrs later

(Crowley et al., 1982). Interestingly, a second injection of E2 or progesterone reversed the effects of a single injection of E2.

Striatal DA concentrations are unaffected by long-term estrogen treatment

(Eikenberg et al., 1977; Alderson & Baum, 1981), but DA concentration was decreased in the nucleus accumbens (N. Acc). While chronic treatment with low E2 dose can reduce DA content in both the striatum and N. Acc, striatal DA content is unchanged 30 21

minutes after 30 ^ig of E2 (DuPont et al., 1981; Di Paolo et al., 1985). Finally chronic

steroid treatment lasting 4 or 30 days decreased DA concentration (Jori & Dolfmi, 1976).

There are time-related factors when considering the effects of estrogen treatment

on DA content. Estradiol treatment was ineffective on striatal DA content when

measures were taken within 3 hours of a single E2 injection or 24 hours after the last E2

injection. Prolonged estradiol treatment (14 days) followed by testing within 24 hours of

the last injection produces a decrease in DA content.

Estrogen Treatment Effects on DA Turnover

Measurements of DA turnover are obtained by more than one approach. The

inhibition of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of

DA, allows determination of the decline in DA content, and so provides one estimate of

DA turnover. A second approach is to measure the concentration of DA metabolites as

an indication of DA turnover.

Increased DA turnover has been reported to occur as early as 30 minutes after a

very small E2 dose (Di Paolo et al., 1985). Estradiol treatment increased DA turnover, as

evidenced by homovanilic acid (HVA) and dihydroxyphenyl acetic acid (DOPAC) levels

(Bedard et al., 1983; Bedard et al., 1984; Bedard et al., 1985; Di Paolo et al., 1985). Also

E2 treatment reversed the apomorphine-induced decline m DOPAC levels (Piccardi et al.,

1983). In vitro measures of striatal DA release are increased after estradiol treatment

(Becker and Ramirez, 1981; Becker and Beer, 1986). DA turnover was also increased when tested 72 hours after E2 treatment (Crowley, 1982).

single A injection of E2 produced an increase in HVA 24 hours later in monkeys

(Bedard et al, 1 984). However, other studies have failed to observe an effect of estrogen 22

treatment on striatal DA turnover (Eikenburg et al., 1977; Bedard et al., 1979). DA turnover is unchanged in most cases when tested 24 hours after the end of E2 treatment

(Eikenburg et al., 1977; Crowley et al., 1978; Bedard et al., 1979; Euvard et al., 1979;

Euvard et al., 1980; Dupont et al., 1981; Crowley, 1982; Di Paolo et al., 1982a).

Estrogen Treatment Effects on DA Synthesis

Various studies have reported that E2 treatment can either increase or decrease

DA synthesis. Estrogen treatment has been reported to cause an increase in DA synthesis

(Algeri et al., 1976), and a decrease in DA content (Dupont et al., 1981 ; Crowley, 1982).

Gordon & Perry (1983) observed that DA synthesis was decreased by E2 treatment.

DA synthesis, as determined by TH activity, was decreased up to 24 hours after cessation of E2 treatment (Gordon et al., 1979; Foreman and Porter, 1980; Gordon and

Perry, 1983). When tested 72 hours after termination of E2 treatment, DA synthesis was increased (Gordon and Perry, 1983).

Human contraceptives have been shown to increase DA synthesis (Algeri et al.,

1976). Gordon and colleagues utilizing different methodology reported that EB caused a decrease in the Km of the pterine cofactor for striatal TH, presumably resulting in an increase in TH activity and DA synthesis within 24 hours after cessation of EB treatment

(Gordon and Perry, 1983).

Estrogen Effects in the Striatum - Synaptic Elements

Estrogen treatment effects can occur independently of presynaptic terminals and are therefore not due completely to alteration in presynaptic activity. Unilateral 60HDA lesions of the nigrostriatal fibers do not abolish E2 effects on apomorphine-induced

increases in striatal acetylcholine (ACh) levels (Bedard et al., 1979) or the increase in striatal DA receptors when compared to the intact side (Bedard et al., 1979; Hruska & 23

Silbergeld, 1980). The minimal effect of E2 treatment on DA turnover, suggests E2

effects are primarily postsynaptic (Euvard et al., 1980)

Estrogen Treatment Effects on Unit Activity

Chiodo et al (1980) reported that E2 can alter the firing rate of substantia nigra neurons and that there are probably two distinct populations of nigrostriatal DA neurons which differ in their response to E2 (Chiodo and Caggiula, 1980). He suggests that perhaps various testing environments therefore differentially affect or influence the way these neurons respond to drug, both in the presence or absence of hormone.

Estrogen Effects on ACh

DA stimulation results in an increase in striatal ACh content. Estrogen treatment

had no effect on basal striatal ACh levels (Bedard et al., 1979). However, E2 treatment reversed the apomorphine-induced stimulation of striatal ACh in a dose-dependent

manner (Bedard et al., 1979; Euvard et al., 1979; Euvard et al., 1980; Hamon et al.,

1983). Also estrogen treatment (moxestrol) potentiated the haloperidol-induced decrease in striatal ACh levels. In contrast, whole brain levels of ACh were not affected by long-

term estradiol treatment (Daabees et al., 1981).

Effects of Antiestrogens on Estrogenic Effects

Several studies examined the specificity of estrogenic effects by trying to reverse the estrogen effects. This is done in two ways. First, ovariectomized female were examined to determine if the removal of the major source of endogenous estrogen would produce an effect other than what is found when estrogen is present. Second, antiestrogens were used to determine if competitive inhibition of the estrogen receptor.

Three antiestrogens, Mer25, CI628 and tamoxifen, are commonly used as competitive inhibitors. Ferretti et al. (1988) observed that the antiestrogen, tamoxifen blocked the 24

estrogen-induced increase in striatal DA receptors (Ferretti et al., 1988). Similarly,

tamoxifen blocked the estrogen-induced increase in muscimol binding in the striatum,

three hours after E2 injection (Maggi and Perez, 1986). Joyce et al. (1984) observed that

the antiestrogen C1628 blocked the suppressive effects of EB on postural deviation and

circling behavior (Joyce et al., 1984). In contrast, tamoxifen failed to affect the reduction

in nigral Glutamic Acid Decarboxylase (GAD) activity observed after estrogen treatment

(Nicoletti et al., 1985). These studies suggest that the changes observed after estrogen

treatment were due to specific activity at the estrogen receptor and not resulting fi-om a

non-specific effect.

Intracerebral Effects of

The view that estrogen may act directly on striatal tissue is based primarily on

observations of changes in behaviors known to be intimately involved with the striatum

and striatal neurochemistry after estrogen treatment. Furthermore, estrogen implantation

within the striatum produces specific changes in behavior and striatal neurochemistry

(Colvin and Sawyer, 1969; Joyce and Van Hartesveldt, 1984a; Van Hartesveldt et al.,

1989; Van Hartesveldt et al., 1990). Estrogen is thought to act on intracellular receptors

operating through genomic mechanisms (Jensen et al., 1968; Gustafsson, 1999).

Autoradiographic studies have been very successful at mapping estrogen-sensitive cells

by identifying cells that concentrate tritiated steroids (McEwen et al., 1975; Akesson et al., 1988; Loy et al., 1988; Pelletier et al., 1988). However, there is no autoradiographic evidence that estrogen significantly accumulates in cells in the striatum.

Indirect Actions of £2 Treatment

Inconsistent evidence of estrogen receptors in the striatum suggests that estrogen may indirectly influence changes in DA-related behaviors and striatal neurochemistry. 25

There are a niunber of extrastriatal areas closely associated with DA activity, and are

significantly affected by estrogen, that could mediate the changes in DA-related

behaviors and striatal neurochemistry. Neuroactive substances may mediate estrogen

effects on striatal DA activity. Indeed, both prolactin, an estrogen-induced pituitary

hormone, and the estrogen metabolites catecholestrogens, have been reported to influence

DA agonist-induced behaviors and striatal neurochemistry in a manner consistent with

reported estrogen effects (Hruska and Pitman, 1982; Hruska et al., 1982b; Clopton and

Gordon, 1985; Hruska, 1987). Therefore, it has yet to be established convincingly

whether estrogen influences DA agonist-induced behaviors by acting directly in the

striatum, or indirectly through some intermediary factor, or at some site distant to the

striatum.

Aim

Behavioral Ratings

In general, DA agonist-induced changes in behaviors are evaluated by means of

behavioral rating scales. However, behavioral rating scales have been criticized as being

an inadequate measure of stereotyped behaviors (Fray et al., 1980; Lewis et al., 1985;

Szechtman and Eilam, 1988). Recent evidence suggests that some assumptions

concerning the use and interpretation of rating scale measures of DA agonist-induced

stereotyped behaviors are unfounded and possibly erroneous (Nickolson, 1981; Geyer et

al., 1986). For instance, behavioral classifications used in the formation of rating scales

use subjective behavioral descriptions, such as "exploratory behavior". These terms may

be interpreted to mean different response patterns and don't provide an objective

reference for the behavior. The behaviors used to form the behavior classifications are grouped differently among studies, limiting the usefulness of the scale among studies. 26

Consequently, contradictions concerning the effects of estrogen on DA agonist-induced changes in behaviors may be based, in part, on problems related with the way changes in

behaviors are measures. One alternative to behavioral rating scales is to measure the individual behavioral responses.

Effects of Estrogen Treatment on Individual Behaviors

At least two studies have reported that estrogen treatments influenced DA agonist-induced locomotor activity differently fi-om other DA-related behaviors (Naik et al., 1978; Menniti and Baum, 1981). Similarly, Chiodo et al., (1981) observed that repeated injections of 100 ^g/kg of estradiol benzoate influenced the number of animals displaying specific behaviors. However, the individual behaviors were not measured separately. Therefore, it is unclear how estrogen actually affected the display of theses behaviors. Based on previous studies, estrogen treatments might differentially affect DA agonist-induced behaviors. In our study, the effects of estrogen treatments on individual apomorphine-induced behavioral responses were examined. CHAPTER 2 GENERAL METHODS

The primary goal of our study was to investigate the behavioral effects of

estrogen on DA agonist-induced behavior. The assessment of individual response

patterns was used to evaluate the effects of estrogen on specific apomorphine-induced

behavioral responses. Different hormone conditions were used to characterize the

relationship between estrogen and apomorphine-induced behavioral responses. Because

there is evidence that the differential effect of estrogen may result from actions at various

areas within the brain, we further examined the effects of intracerebral estradiol

implantation into various regions of the basal ganglia on DA agonist-induced behaviors.

Subjects

Adult female Long-Evans hooded rats between 3 and 4 months of age were used

in all experiments. Animals were individually housed with free access to food and water

for at least two weeks before behavioral testing. The animals were maintained on a 12:12

hour light: dark cycle throughout the experiment, with lights on at 08:00 hours. All

testing was conducted between 09:00 hours and 17:00 hours.

Apparatus

Observation Chamber

Two identical observation chambers were constructed of Plexiglas walls with a wire mesh floor (Appendix A-1). The dimensions of the chambers were 39 cm x 34.5 cm x 60 cm. The wire mesh was 1.2 cml Three pellets of Purina rat chow and a small block of wood cm x (9.0 18 cm X 1.9 cm) were placed in the chamber. The chamber walls were

27 28

white except for the front panel, which was clear. The clear side of the chamber was

positioned against a one-way mirror, allowing the observer to watch from another room.

The chambers were illuminated by a 52 watt light bulb positioned approximately 10 cm

above the top of the chamber. The chamber temperature was maintained at

approximately 22-23 degrees Celsius with the chamber light on. A noise generator was

placed in the room along side the observation chambers

Three lines, one longitudinal and two bisecting the longitudinal lines at

equidistant points, were painted on the floor and used to measure locomotion in the

chamber. The lines formed six squares (20 cm x 17 cm) on the floor.

Intracerebral Cannulae

Intracerebral cannulae were made from 27 ga stainless steel tubing of differing

lengths depending on the site of implantation. The tubing was cleansed for implantation

by boiling in distilled water.

Treatments Groups

(1) In studies utilizing intact females, rats were monitored by vaginal smears for at least ten days to determine their cyclicity. Only females displaying regular 4-day estrous cycles were included in behavioral testing.

(2) In studies utilizing short-term ovariectomized females, females were bilaterally ovariectomized and placed in single cages for two weeks before behavioral testing.

(3) In studies utilizing long-term ovariectomized females, females were bilaterally ovariectomized and returned to group housing for 16 weeks. Two weeks before behavioral testing the animals were placed in individual cages.

Surgical Procedures

Two surgical procedures were employed in the course of our study. Bilateral ovariectomies were performed on all female subjects. Intracerebral cannulations were additionally performed in experiment 4. These studies were performed in 1987 and 29

reflect standard procedures of that era. We are aware that more stringent requirements

for animal welfare and rodent surgery have become required, including sterile technique

and use of postoperative analgesics.

Ovariectomy

Bilateral ovariectomies were performed by placing the animals in a desiccator

containing ether until the subject became unresponsive to foot pinch. Once anesthetized,

a midline abdominal incision was made after which both ovaries were located and

removed. The incision was then sutured and the animal returned to its home cage.

Tissue removal was very conservative, such that very little peri-ovarian fat or uterine

horn was removed. This may be important since it has been shown that the amount of

uterine tissue removed during ovariectomy can influence the animal's response to

subsequent hormone replacement therapy (Ahdieh and Wade, 1982). Sham

ovariectomies were performed in the same manner as complete ovariectomies, except

ovaries were located, displaced and left intact in the abdominal cavity. The incision was

then sutured and the animal returned to its home cage.

Cannula Placement

Animals were implanted bilaterally with 21 ga stainless steel guide caimulae

under sodium pentobarbital (50 mg/kg) anesthesia. A 27 ga stylet was inserted into each

cannula to prevent prolonged exposure to air. After bilateral cannula implantation, the

animals were given a one-week recovery period before behavioral testing.

Coordinates for intracerebral implantation were taken from Pellegrino and

Cushman (1978). Anterior-posterior coordinates were taken from bregma and vertical measurements from the dural surface of the brain. All guide cannulae were implanted to a depth of 2.0 mm below the surface of the brain, so as to prevent damage at the site if 30

hormone implantation. Hormone implants were directed at various brain nuclei having the following coordinates;

1. Dorsal-anterior caudate: anterior, 2.6; lateral, + 3.0; ventral, -4.0

2. Ventral caudate: anterior, 2.6; lateral, ± 3.5; ventral, -7.0

3. N. Acc: anterior, 3.2, lateral, ±2.0 ventral, -7.5

4. Globus pallidus: anterior, 1.2, lateral, ±2.8 ventral, -6.4

Drug and Hormone Preparation and Administration

Three systemic and one intracerebral treatments were administered over the

course of these studies. Animals were given apomorphine in all four experiments to elicit

the behavioral response for study. In experiment 3, subjects were given one of two systemic hormone preparations. In experiment 4, subjects were given intracerebral estradiol implants, which will be discussed in more detail later.

Apomorphine hydrochloride (Sigma Chemical Co., St. Louis, MO.) was dissolved in a 0.9% saline solution and shielded from direct light until injection. The apomorphine solution was freshly prepared before each behavioral test, and was injected into the subject within 15 minutes after preparation. Apomorphine injections were given subcutaneously in the right rear flank of the animals. Saline served as the vehicle control and was injected in the same manner as apomorphine.

Estradiol benzoate (Steraloids, Inc., New Port, R.I.) was dissolved in peanut oil heated to a temperature of approximately 60° C. All systemic injections of estradiol

benzoate were given subcutaneously in the left rear flank of the animals. Peanut oil served as the vehicle control and was injected in the same manner as estradiol benzoate.

Estradiol 17-p (Steraloids, Inc., New Port, R.I.) and estradiol 17-a were dissolved in peanut oil heated to temperature of approximately 60° C. The hormone solutions were allowed to return to room temperature before use. All subsequent injections of estradiol 31

17-|3 (I7-P-E2) and estradiol 17-a (IT-a-Ei) were injected subcutaneously in the left rear

flank of the subjects. Estradiol 17-a in peanut oil served as the control solution and was

injected in the same manner as estradiol 17- p.

Estradiol 17-p or estradiol 17-a was placed in mtracerebral implants for

placement in various positions in the brain. The implants were loaded with crystalline

17-a or 1 7-p (Sigma) by placing the hormone on wax paper and tapping the open end of

the implant tubing 20 times into the hormone powder. The tubing was then examined

under a dissecting microscope and the portion of the tubing cleaned of any extra hormone

by clean surgical gauze.

At the time of hormone implantation, the 27 ga stylet was removed and the 27 ga

implant placed into the 21 ga guide cannulae. The hormone implant remained in place

for 48 hours and through the behavioral test session. In order to maintain the implant in

place, paraffin was melted and a few drops placed over the implants. After removal, the

implants were examined for the presence of hormone.

Testing Procedure

Animals were habituated to the testing chamber 30 min before injection with

apomorphine or saline. Animals were then taken out of the chamber and injected with

either saline or apomorphine, and their behavior was observed and recorded starting 4

minutes later. Two animals were tested simultaneously in identical chambers and

observed continuously in alternating one-minute intervals. An electrical metronome produced an audible signal every 5 sec. and a second signal at one-minute intervals.

During the one minute observation interval, behavioral recordings were made every 5 seconds. That is, the animal was observed for the presence of one of several behaviors 32

during each 5 sec recording interval for a total of 12 such intervals equaling one minute.

At the end of the one-minute observation interval, there was a one-minute time-out

period, during which the animal's behavior was not observed (Figure 2-1). During the

time-out period, the observer recorded the behavior of the animal in the second chamber

for one minute. Behavioral observations of the two animals continued in this alternating

fashion until the end of the 1-hour test session. Only one behavior was recorded for each

5 sec recording interval. Therefore, for any one minute observation interval, a particular

behavior could be recorded a maximum of 12 times or a minimum of 0 times. The only

exception was the number of lines crossed, which was recorded in the presence of other

behaviors.

Behavioral observations were terminated 60 minutes after injection. The latency

of a behavior was determined as the time between injection and the first occurrence of the

behavior.

Behavioral Measures

Behavioral measurement of stereotyped behaviors consisted of the observation and recording of seven behavioral classes (Table 2-1). The behaviors measured were rearing, sniffing, licking, biting, grooming, immobility and locomotor activity (LA). In addition to measuring these behavioral classes, qualitative aspects of sniffing, biting and

LA were also examined. To this end, continuous and discontinuous sniffing and biting were distinguished in the measure. Measurements of locomotor activity were obtained by two methods; the number of lines crossed, and direct observations of ambulatory behavior. 33

4 minute Interval

-2 min 2 min

Observation Interval Observation Interval

1 min 1 min 1 min 1 min

Recording time-out Recording time-out

\

\

\

/ 1 minute recording interval

S sec recording intervals

Figure 2-1. Illustration of method of behavioral recording.

In this figure the relationship between the 1 minute recording interval and two minute observation intervals are shown. Each observation interval was composed of a 1 -minute period in which the animal's behavior was recorded, followed by a one-minute time-out period in which behavioral recordings were not made. The alternating pattern between recording and time-out

periods constituted the entire 60-minute test session. The 1 -minute recording

interval is composed of 1 2, five second, recording intervals.

Histology

At the end of the experiment the animals were deeply anesthetized with a high dose of sodium pentobarbital (100 mg/kg), then were perfused with 0.9% saline followed by a buffered 10% formalin solution. The brains were removed and placed in formalin solution. Twenty-four to 48 hours before they were sectioned the brains were placed into a 20% sucrose formalin solution. TTie brains were frozen and sectioned coronally at approximately 30 micron thickness. The sections were mounted and stained with cresyl violet. The positions of the hormone implants were then determined. Subjects with cannulae implants outside of the defined areas were excluded from data analysis. 34

Statistical Analysis

For statistical analysis, the test session was partitioned into 14 consecutive 4-

minute time intervals. All of the behaviors occurring within the 4-minute time intervals

were summed and the totals used for statistical analysis. For descriptive purposes, each 4

minute period is referred to by the fourth minute of the interval. Therefore, the 8 minute

time interval refers to events occurring fi-om 4 minutes after apomorphine injection

through 8 minutes after injection. The 12 minute time interval refers to events occurring

from 8 minutes after injection through 12 minutes after injection, and so on.

Behavioral data were analyzed by two-way mixed design analysis of variance

(ANOVA) with repeated measures on time as the within factor. Behavioral latencies

were analyzed by one-way ANOVA. The Newman-Keuls multiple range test was used

for all post-hoc analysis with significance level at p < 0.05.

Experiment Outline

Our study is separated into four sections. The first section establishes the

parameters of the instrument of measurement, and in particular whether these

measurements can determine individual response patterns to small changes in

apomorphine dose. The second section examined the role of endogenous estrogen on

apomorphine-induced behavioral responses. Ovarian hormone levels in female rats

change rapidly over a short period of time (4 days on average), and considerable variability in hormone levels could possibly interfere with an accurate assessment of the effects of endogenous estrogen on apomorphine-induced behaviors. Therefore, the effects of removing the primary source of endogenous estrogen on apomorphine-induced response patterns were examined. The effects of both short-term and long-term ovariectomy were examined. The third section examines the effects of exogenous 35

estrogen on apomorphine-induced behavioral responses. The effects of estrogen dose and

length of treatment were examined. The fourth section examined the effect of

intracerebral estradiol implants on apomorphine-induced behavioral responses.

Table 2-1 . Behavioral Descriptions used to identify behaviors for measurement. Behavior Behavior Description

Rearing Behavior Rr. Animal stands on hind legs with forelimbs in the air or against the wall, at least once during the 5 sec recording interval.

Sniffing Behavior Sn. Rhythmic movement of the head and snout along the cage floor or walls, accompanied by rapid back and forth movement of the vibrissae.

Continuous Sniffing C.S. Sniffing behavior which is expressed without interruption by another behavior during the 5 sec recording interval

Discontinuous Sniffing D.S. Sniffing behavior that is interrupted by the display of another behavior during the 5 sec recording interval.

Biting Behavior Bt. The wire mesh floor was gripped between the teeth.

Continuous Biting C.B. Biting behavior which is expressed without interruption by another behavior during the 5 sec recording interval

Discontinuous Biting D.B. Biting behavior that is interrupted by the display of another behavior during the 5 sec recording interval.

Grooming Behavior Gr. Animal licks, scratches or bites any part of body during the 5 second recording interval.

Behavioral Inactivity B.I. Animal was inactive and displays no other behavior.

Locomotion L.C. Animal passed over floor line with all four paws.

Ambulation Amb. Locomotor movements of at least two limbs resulting in body displacement.

The criteria above were used to identify and record different behavioral responses during testing. See text for more details. Two measures were included to detect different qualitative expressions of sniffing and biting behavior.

Continuous behavior. Continuous behavior was defmed as behavior displayed without interruption by another behavior, during a 5 sec recording interval.

Discontinuous behavior. Discontinuous behavior was defined as a behavior that is interrupted by another behavior during a 5 sec recording interval. CHAPTER 3 EFFECTS OF APOMORPHINE DOSES ON BEHAVIORS

Introduction

Previous studies have indicated that the nature of the relationship between

estrogen and DA-related behaviors is unclear. For instance, estrogen has been reported in different studies to either increase or decrease the apomorphine-induced changes in

behaviors (Gordon, 1980; Hruska and Silbergeld, 1980b; Hruska et al., 1982a). One possible cause for these differences is that endogenous estrogen or its removal may differentially affect individual DA agonist-induced behaviors. DA agonist-induced changes in behaviors often referred to as stereotyped behaviors, are commonly used as a

behavioral assessment of DA-ergic activity in the brain (Ernst, 1967; Angrist et al., 1974;

Randrup and Munkvad, 1974; Iversen and Creese, 1975; Robbins, 1977; Dantzer, 1986).

In most studies, behavioral rating scales were used to measure changes in behaviors after administration of DA agonists (Ernst, 1967; Asher and Aghajanian, 1974;

Costall et al., 1977b; Klawans et al., 1977). Recently, the use of rating scales have been

criticized as an inaccurate measure of specific changes in behaviors (Fray et al., 1980;

Rebec and Segal, 1980; Nickolson, 1981; Szechtman et al., 1982; Lewis et al., 1985).

Given the evidence that behavioral rating scales are unable to determine specific changes in behavioral responses, disagreements concerning the effects of estrogen on DA agonist- induced behaviors may be resolved by re-examining the effects of estrogen on individual apomorphine-induced behaviors. Behavioral checklists, which can be used to measure and record specific changes in multiple response patterns, is an alternative to behavioral

36 37

rating scales (Fray et al., 1980; Robertson and MacDonald, 1984; Lewis et al., 1985; Carr

and White, 1987).

In order to understand how estrogen might affect specific behaviors, a behavioral

checklist was used to determine the effects of different doses of apomorphine on specific

response patterns. Measurements were made to determine the pattern of behavioral

change under differing apomorphine doses. Although behavioral checklists provide

detailed information, they have been found to be inadequate in the measurement of

qualitative changes in DA agonist-induced behaviors, an aspect of behavior which rating

scale measures assess well (Fray et al., 1980; Lewis et al., 1985; Geyer et al., 1986).

Consequently, in our study, the behavioral checklist was designed to include measures of

qualitative aspects of behavior to determine the patterns of change for each behavioral

response. Information derived from the dose-response curve was used to evaluate

changes in specific apomorphine-induced behaviors in subsequent studies.

The principal advantage of measuring individual stereotyped behaviors is that it

provides more information concerning specific changes in behavior. Similarly, this

approach is most appropriate for the detection of subtle changes in behavior which might

go undetected by more global behavioral measures.

Methods

Subjects

Thirty-six aduh female Long-Evans rats were housed in single cages with free

access to food and water for at least two weeks before testing. Subjects were given bilateral ovariectomies and behaviorally tested two weeks later. Behavioral testing was performed as described in Chapter 2 ( see page 31). 38

Design

Six animals were randomly assigned to one of five apomorphine dose groups or

the saline control group. All injections were made subcutaneously on the dorsal portion

of the neck. At the time of testing the animals were injected subcutaneously (s.c.) with

either 0.9% saline (1.0 mg/kg) or one of the following five apomorphine doses: (1) 0.15

mg/kg, (2) 0.25 mg/kg, (3) 0.35 mg/kg, (4) 0.45 mg/kg, or (5) 0.90 mg/kg. Drug doses

are expressed in terms of the salt. The animals were used once and were habituated to the

test chamber 60 minutes before testing. After injection, animals were placed in the test

chamber and observed for one hour according to the procedures in the general methods

section.

Results

Table 3-1 and Table 3-2 show the means for the total number of observations and

latencies, respectively, for each behavioral response.

Biting Behavior

Apomorphine significantly increased biting behavior in a dose dependent manner,

F(5,30) = 205.08, p < 0.001. The 0.35 mg/kg, 0.45 mg/kg and 0.90 mg/kg apomorphine

dose groups displayed more biting behavior than all other groups (p < 0.05). The 0.90

mg/kg apomorphine dose group also displayed more biting than the 0.35 mg/kg and 0.45

mg/kg apomorphine dose groups (p < 0.05). The 0.45 mg/kg apomorphine dose group

displayed more biting than the 0.35 mg/kg apomorphine dose group (p < 0.05). The dose

response curve revealed a monotonic relationship (Figure 3-1).

There was a significant dose by time interaction, F(65,390) = 1 1.90, p < 0.001.

Analysis of dose effects at each time interval revealed that the 0.90 mg/kg apomorphine dose group displayed more biting than all other groups over the course of the session 39

beginning with the 8 minute through the 56 minute time intervals (p < 0.05). The 0.45

mg/kg apomorphine dose group displayed more biting than all other groups except the

0.90 mg/kg apomorphine dose group during the 20 minute through 40 minute time

intervals (p < 0.05).

Apomorphine decreased the latency to display biting in a dose-dependent manner,

= F (5, 30) 4.40, p < 0.01 . The latency to display biting behavior was significantly

shorter for the 0.35 mg/kg, 0.45 mg/kg and 0.90 mg/kg apomorphine dose groups than the saline and 0.15 mg/kg apomorphine dose groups (p < 0.05).

Continuous Biting

Increments in apomorphine dose increased continuous biting in a fashion very similar to the effects overall biting, = on F (5, 30) 75.57, p < 0.001 . Continuous biting displayed a bell-shaped time-response curve, the amplitude of which increased with increments in dose (Figure 3-2). Both the 0.45 mg/kg and 0.90 mg/kg apomorphine dose groups displayed more continuous biting than all other groups (p < 0.05). The 0.90 mg/kg apomorphine dose group also displayed more continuous biting than the 0.45 mg/kg apomorphine dose group (p < 0.05).

There was a significant dose-time interaction, F(65,390) = 3.03, p < 0.001.

Analysis of dose effects at each time interval revealed that the 0.90 mg/kg apomorphine dose group displayed more continuous biting than all other groups during the 8 minute through 56 minute time intervals (p < 0.05). During the 20 minute through 28 minute time intervals, the 0.45 mg/kg apomorphine dose group displayed more continuous biting than all other groups except the 0.90 mg/kg apomorphine dose group (p < 0.05). 40

Discontinuous Biting

Like continuous biting, discontinuous biting was also increased by apomorphine,

= F(55,30) 15.12, p < 0.001 . The dose response curve displayed a monotonic

relationship, in which the 0.90 mg/kg apomorphine dose group displayed more

discontinuous biting than all other groups (p < 0.05). In contrast, discontinuous biting

displayed a much flatter response curve, with little differences among dose groups in the

amplitude of the response curve (Figure 3-3). The major effect of apomorphine dose

increments was to prolong the display of discontinuous biting. Consequently, high

apomorphine dose groups displayed sustained levels of discontinuous biting longer than

lower apomorphine dose groups. Although discontinuous biting was also increased by

apomorphine, it occurred at a much lower rate than continuous biting. As a result, the

contribution of continuous biting and discontinuous biting to the overall biting response

differed across dose and biting became more continuous with increments in dose.

Sniffing Behavior

Sniffing behavior increased was by apomorphine F(5,30) 19.44, p < 0.01 . The

0.25 mg/kg, 0.35 mg/kg and 0.45 mg/kg apomorphine dose groups displayed more sniffing than all other groups (p < 0.05).

There was a significant dose by time interaction F(65,390) = 6.04, p < 0.001.

With increments in apomorphine dose, changes in the time-course response pattern for sniffing was transformed from a bell-shaped response pattern displayed by saline and apomorphine dose groups up to the 0.35 mg/kg dose levels, to a U-shaped response curve, most prominently displayed by the 0.45 mg/kg and 0.90 mg/kg dose groups

(Figure 3-4). During the 20 minute through 32 minute time intervals the 0.25 mg/kg and

0.35 mg/kg apomorphine dose groups both displayed more sniffing than all other groups 41

(p < 0.05). The mid-session depression in sniffing first observed with the 0.35 mg/kg

dose group increased in both size and duration with further increments in apomorphine

dose.

All animals displayed sniffing within the first 4 minutes of observation; therefore

apomorphine did not affect the onset of sniffing. However, the amount of sniffing

displayed at the onset of behavioral measurement was greatest for the 0.25 mg/kg and

0.35 mg/kg dose groups.

Continuous and Discontinuous Sniffing

The increase in sniffing behavior was primarily resulted from the dose-dependent

increase in continuous sniffing F(5,30) = 41.40, p < 0.001 (Figure 3-5). The 0.25 mg/kg

and 0.35 mg/kg apomorphine dose groups displayed more continuous sniffing than all

other groups (p < 0.05). The 0.45 mg/kg and 0.90 mg/kg apomorphine dose groups also

displayed more continuous sniffing than the saline groups (p < 0.05).

In contrast to the increase in continuous sniffing, discontinuous sniffing was

decreased with increments in apomorphine dose (Figure 3-6).

Overall latencies for sniffing were not affected, but the latencies for the specific

behavioral components, continuous sniffing and discontinuous sniffing were affected by

apomorphine dose in opposite directions (Table 3-2). Apomorphine advanced the onset

of continuous sniffing, but delayed the onset of discontinuous sniffing (p < 0.05). When

the two sniffing response patterns are combined to obtain the overall sniffing measures,

dose-related differences in the onset of sniffing are eliminated. A significant dose-time interaction was illustrated by the higher apomorphine dose groups (0.25 - 0.90 mg/kg), which displayed less discontinuous sniffing during the first 30 minutes of the test session.

The pattern of sniffing changed from primarily discontinuous sniffing for the saline and 42

0.15 mg/kg dose groups to essentially all continuous sniffing for the higher apomorphine

dose groups. Also note that even though the 0.45 mg/kg and 0.90 mg/kg dose groups

displayed a decline in sniffmg, the pattern of sniffing remained primarily continuous.

Locomotor Activity

Apomorphine had mixed effects on the two measures of locomotor activity, line

crossings and ambulatory behavior. All apomorphine dose groups, except the 0.45 mg/kg

dose group, executed fewer line crossings than the saline group (Figure 3-7). The effects

of apomorphine on line crossings were subtle. The dose of apomorphine did not affect

the onset of line crossings, nor did apomorphine dramatically affect the amplitude of the

response curve. Rather apomorphine produced a small suppression of line crossings

expressed over the course of the session. The cumulative effect was to decrease the

overall number of line crossings that occurred during the session. Line crossings were

suppressed by apomorphine, beginning with the threshold dose of 0.15 mg/kg (p < 0.05).

Only the 0.45 mg/kg dose failed to suppress line crossings.

In contrast to line crossings, measures of ambulatory behavior were increased by

apomorphine in a dose dependent manner (Figure 3-8). Ambulatory behavior, which also

served as a measure of locomotor activity, was greatest for the 0.45 mg/kg dose group(p

< 0.05). However, ambulation was decreased by the 0.90 mg/kg dose. The ambulation

time-course curves were specifically affected by apomorphine dose. The increase in

ambulation response totals was reflected by an increase in the amplitudes of the response

curve for ambulation, with no change in onset. This was true for doses up to the 0.45 mg/kg dose level. At the 0.45 mg/kg dose level, ambulation was displayed at the level of the previous dose for the first half of the session. During the second half, ambulation was increased above other dose levels, and the increase was sustained, even as ambulation for 43

other groups declined. Therefore, the increase in ambulation displayed by the 0.45 mg/kg

dose group resulted from activity during the second half of the session.

Rearing Behavior

Rearing behavior was decreased by apomorphine in a dose-related fashion. All

apomorphine dose groups displayed less rearing than the saline control group. The

latency to rear was also increased by apomorphine (p < 0.05). The 0.90 mg/kg

apomorphine dose group had the longest latency to rear. Therefore, animals given

apomorphine displayed less rearing and also began rearing behavior later than saline

injected animals (Figure 3-9).

Grooming and Behavioral Inactivity

Grooming behavior and behavioral inactivity are both considered terminal

responses to apomorphine, often seen as evidence of the end of the drug effects on

behavior (see Figure 3-10 and Figure 3-1 1). Both grooming and inactivity were

decreased by apomorphine. For the saline group, short bouts of inactivity and grooming

were dispersed throughout the observation period, and represented a substantial

proportion of the animal's behavioral record. Even in apomorphine treated animals short

bouts of grooming and inactivity were displayed. However, apomorphine generally

suppressed grooming and behavioral inactivity during the first 30 minutes of the test

session.

Discussion

The present study is the only report in which a comprehensive and fine-grained analysis of the effects of apomorphine on multiple behaviors has been presented.

Apomorphine was found to have different effects on specific behaviors. Although our 44

study involved the measurement of multiple behaviors the results were consistent with

single behavior studies.

Biting behavior

While biting increased with increments in apomorphine dose, the biting response

displayed in response to higher apomorphine dose was also qualitatively different from

the biting response displayed by the saline and low dose groups. Animals given saline or

low apomorphine doses gnawed almost exclusively on a wooden block placed in the test

chamber. The wood chewing behavior occurred at relatively low levels, but accounted

for the continuous biting scores attributed to the saline group. Animals given 0.35 mg/kg

of apomorphine or higher doses displayed a completely different type of oral behavior in

which animals would bite the wire-grating of the chamber floor. Both response patterns

have been designated as biting behavior in the literature (Chow & Beck, 1 984; Havemann

et al., 1986; Rastogi, Rastogi, Singhal, & Lapierre, 1982), but clearly represent

qualitatively distinct behavioral responses, which allowed subtle distinctions among the

biting response patterns produced by different apomorphine doses.

Competitive behavioral relationships - Sniffing and biting behaviors

The relationship between sniffing and biting is considered a very good indicator

of the level of DA-ergic stimulation (Costall et al., 1972; Sahakian et al., 1975; Iversen

and Koob, 1977). Changes from sniffing to biting are almost universally interpreted as

indicating an increase in behavioral intensity and DA-ergic stimulation (Joyce & Iversen,

1984; Ljungberg & Ungerstedt, 1977a; Morelli, Porceddu, & Chiara, 1980). The conclusion is based primarily on dose-response studies in which biting is reported to replace snilfmg behavior as the dose of drug is increased. Our results substantiate the 45

view that under conditions of increasing DA-ergic stimulation biting replaces sniffing

behavior. Indirect evidence that sniffing and biting compete with each other is presented

by their respective response patterns. Sniffing dominated as a behavioral response at

lower apomorphine doses. When biting was displayed, it was always at the expense of

sniffing as well as other behaviors.

The qualitative aspects of sniffing and biting proved to be another indicator by

which distinctions could be found among the different apomorphine doses. With

increments in apomorphine dose, sniffing and biting behaviors each became more

continuous. This finding is in agreement with results of previous studies that used

behavioral rating scale measures of apomorphine-induced behaviors (Costall et al., 1975;

Lewis et al., 1985). Furthermore, the inclusion of the continuous and discontinuous

sniffing classification was the primary distinction between the 0.25 mg/kg and 0.35

mg/kg dose groups. Both groups displayed equivalent levels of total sniffing, but the

0.25 mg/kg dose groups displayed significantly more continuous sniffing than the 0.35

mg/kg dose group. Total sniffmg measures alone, would not have distinguished between

the two dose groups.

Measurements of locomotor activity

Locomotion is not one-dimensional, but is expressed along several different dimensions, including, speed, distance and area (Ljungberg and Ungerstedt, 1978;

Nickolson, 1981; Havemann et al., 1986; Stable and Ungerstedt, 1986; Starr and Starr,

1 986). Complicating this issue of apomorphine effects on locomotor activity is the fact that different results are obtained with different measures of locomotor activity (Maj et al., 1972; Kelly et al., 1986; Stable and Ungerstedt, 1986). Previous reports suggesting 46

that locomotor activity was decreased or abolished by high doses of DA agonists have

disputed been by more recent studies (Maj et al., 1972; Nickolson, 1981 ; Kelly et al.,

1986; Stable and Ungerstedt, 1986; Starr and Starr, 1986). Evidence suggests that there

is indeed a reduction in forward locomotion after treatment with high doses of DA

agonists, particularly during periods in which continuous biting is displayed. However,

other forms of locomotor activity are maintained or even increased (Stable and

Ungerstedt, 1986). While rapid, forward locomotion displayed throughout the test

chamber is reduced after treatment with DA agonists, animals exhibit an increase in slow,

circular locomotion, usually displayed in a restricted portion of the test chamber

(Nickolson, 1981; Szechtman et al., 1985; Stable and Ungerstedt, 1986; Starr and Starr,

1986).

In our study, ambulation was more closely associated with line crossings and

sniffing in apomorphine dose groups up to the 0.45 mg/kg dose. A qualitative change in

ambulation was observed as biting increased, which involved a shift from forward

locomotion to a slower, more circular locomotor activity. The shift from forward

locomotion to slow, circular locomotion would explain why ambulation measures were

high even though line crossings remained low. The circular locomotor activity described

here may be related to drug-induced circling behavior described in other studies (Jerussi

& Click, 1976a; Jerussi & Click, 1976b; Pycock & Marsden, 1978; Szechtman, Omstein,

Hofstein, Teitelbaum, & Colan, 1980; Szechtman et al., 1982).

Rearing behavior

The apomorphine-induced decrease in rearing behavior resulted partly fi-om a

delay in the onset of this behavior during the test session. Apomorphine decreased rearing early in the test session, but rearing reappeared later in the session. This finding 47

is consistent with findings from earlier studies in which the onset of rearing was progressively delayed with increments in apomorphine dose (Molloy & Waddington,

1987). At low apomorphine doses, the response pattern for rearing behavior paralleled the response pattern for line crossings. In fact, the decrease in rearing behavior may have contributed to the decrease in line crossings. The locomotor response displayed by the saline group consisted of rapid locomotion, frequently punctuated by rearing behavior, usually displayed over the extent of the test chamber. When an animal terminated a rear, the animal returned to the chamber floor, not in the same position as before, but often extending the body while moving in some direction. The combined effect of rapid forward locomotion and frequent rears produced more line crossings than would be found with either behavior alone. However, with increments in apomorphine dose, rearing decreased dramatically, and line crossings also decline.

Similar response patterns for rearing grooming and inactivity

Rearing, grooming and inactivity all were affected by apomorphine in a similar fashion. In each of the three cases, the onset of the behaviors was delayed, while the duration of the response was decreased. Grooming and inactivity are usually displayed at the end of the behavioral response to drug. Since behavioral observations lasted for one hour, much of the grooming and behavioral inactivity exhibited by higher dose groups may have occurred after the end of the test session.

Reference dose

Based on the present results, the 0.45 mg/kg of apomorphine was chosen as the reference dose for subsequent studies involving the effects of hormonal manipulations on apomorphine-induced behaviors. The 0.45 mg/kg apomorphine dose level produced 48

measurable levels of the entire spectrum of the behaviors measured, which showed the relationships among several behaviors across the course of the test session. Lower doses

either produced very high doses of sniffing and very little biting behavior. Consequently lower apomorphine doses did not produce a baseline across behaviors allowing adequate comparisons among changes in behaviors over the session. In contrast, the 0.90 mg/kg apomorphine dose level produced biting almost to the exclusion of all other behaviors.

The behavioral responses of the 0.45 mg/kg dose group displayed were distinguishable from the responses of both the lower and higher apomorphine dose groups. This finding is important because in the following experiments some behaviors have been reported to both increase as well as decrease under some circumstances; therefore changes in both directions must be detectable by this measurement. 49

Table 3-1 . Effects of apomorphine dose on behavior response totals. Behavior Saline 0.10 0.25 0.35 0.45 0.90 Bt 3.8 2.7 2.8 35.7* 127.8* 272.0* (0.9) (1.1) (1.1) (11.0) (4.5) (13.9) CB 1.6 1.3 1.5 17.3 88.5* 216.5*

(0.5) (0.5) (0.8) (9.6) (12.5) (17.6) DB 2.2 1.3 1.3 18.3* 39.3* 55.7*

(0.6) (0.7) (0.4) (1.9) (9.6) (19.0) Sn 83.3 72.7 192.2* 194.3* 144.8* 55.5 (10.8) (13.1) (15.0) (12.3) (19.3) (10.8) CS 9.3 18.5 161.7* 178.7* 128.5* 47.3*

(2.4) (5.14) (14.4) (9.0) (20.4) (8.7) DS 76.8 54.2* 30.5* 10.2* 16.3* 8.2*

(9.6) (8.1) (5.1) (3.3) (2.8) (4.1) Amb 75.3 61.7* 133.2* 150.7* 171.8* 80.3 (10.2) (10.4) (12.4) (15.6) (21.4) (22.2) LC 46.7 23.3* 20.5* 24.7* 39.0 11.5* (13.3) (4.0) (2.7) (6.0) (10.4) (3.7) Rr 35.0 22.7 14.7* 4.0* 6.2* 5.2* (10.5) (4.3) (3.2) (1.4) (2.1) (2.6) Gr 25.7 21.8 25.7 12.2 12.2* 1.3* (5.6) (3.7) (8.3) (5.6) (2.4) (0.5) BI 157.7 191.0 29.3* 33.0* 21.8* 5.8*

(20.8) (16.4) (6.9) (3.4) (4.3) (3.8)

Response totals refer to the sum of observations of a behavior made over the course of the test session. Each value represents the mean score for this measure obtained from a 60 minute test session for 6 animals. The numbers in parentheses represent the standard error of the mean. The animals were given either saline or one of 5 doses of apomorphine (s.c), 4 min. before beginning behavioral testing.

Response measures for behaviors were determined according to procedures presented in the methods section. Abbreviations for behaviors are listed here in parentheses: Biting behavior (Bt); continuous biting (CB); discontinuous biting (DB); sniffing behavior (Sn); continuous sniffing (CS); discontinuous sniffing (CS); ambulation (amb); line crossing (LC); rearing behavior (Rr); grooming behavior (Gr) and inactivity (BI). The * indicates the groups was significantly different from the saline control group. u U

50

Table 3-2 Effects of apomorphine dose on behavior response onset

n 1 n A OA Dendvioi Saline U. 1 u U.Zj U.4J \}.y\J

"iA A 1 1 "3 1 A 7 Q A rJi 16./ 30. Z4.U 1 1 .3 lU. / O.U A^ (8.4) (/.O) (2.U) (2.U) (0)

AA 7 Af\ 7 7A 7* 1 A 7* 0 74: 44. / 4U. / 4y.3 ZU. / lU. / O. / /'7 A\ ^^\ 7^ p.2J (0.2) (6.0) (5.1) (Z.O) (0.7)

10 7 7/1 A 1 1 "J 1 A 7 JJrS 1 0. / 3y.3 z4.U 1 1 .3 10./ O./*

/"I Q\ /"T A'k /A 7\ (y.o) (7.6) (1.9) (2.0) (0.7) 0 7 0 A 0 A 0 A 1X7 on o.U o. / O.U O.U O.U 16.Z

7\ /7 1 \ (0.7) (0) (0) (0) (7.1)

1 l T 1 C T O A O A O A Co 13.3 1 J.3 O.U 8.0 8.0 22.0

(2.46) (3.0) (0) (0) (0) (7.0)

0 7 1 /I A 0 A '3 * Do o.U O. / 14. 3z. 55.3* /n 7\ fA A\ A\ (0) (0.7) (4.6) (5.4) (5.8) (1.6) O A O A Amo o.U O.U O.U 8.7 8.0 9.3 (0\ I"/ (U. /) yy) (U.8) LC 12.7 14.0 12.0 14.7 14.0 24.0 (3.0) (3.0) (4.0) (5.2) (5.2) (9.6) Rr 8.7 14.0 19.3 42.0* 36.7* 55.3*

(0.7) (3.0) (4.2) (1.4) (4.5) (1.9) Gr 8.7 14.7 24.0* 32.0* 47.0* 56.7*

(0.7) (2.5) (5.3) (6.4) (1.3) (1.2) BI 12.0 8.0 32.7* 36.0* 41.3* 39.3*

(2.6) (0) (8.5) (6.1) (7.0) (9.5)

The response onset refers to the time interval during which the behavior was first observed. Each value represents the mean score for this measure obtained from a 60 minute test session for 6 animals. The numbers in parentheses represent the standard error of the mean. The animals were given either saline or one of 5 doses of apomorphine (s.c), 4 min. before beginning behavioral testing.

Response measures for behaviors were determined according to procedures presented in the methods section. Abbreviations for behaviors are listed here in parentheses: Biting behavior (Bt); continuous biting (CB); discontinuous biting (DB); sniffing behavior (Sn); continuous sniffing (CS); discontinuous sniffing (CS); ambulation (amb); line crossing (LC); rearing behavior (Rr); grooming behavior (Gr) and inactivity (BI). The * indicates the groups was significantly different from the saline control group. 51

25 n

8 12 16 20 24 28 32 36 40 44 48 52 56 60

Time (min)

Figure 3-1 . Effects of apomorphine dose on biting behavior. Each value represents the mean number of observations over a 4 min. period for 6 animals. The vertical bars represent the standard error of the mean. The animals were given either saline or one of 5 doses of apomorphine (s.c), 4 min. before begirming behavioral testing. Significant differences among group scores for each time interval are reported in the results section. 52

25 n

8 12 16 20 24 28 32 36 40 44 48 52 56 60 Time (min)

Figure 3-2. Effects of apomorphine dose on continuous biting. Each value represents the mean number of observations over a 4 min. period for 6 animals. The vertical bars represent the standard error of the mean. The animals were given either saline or one of 5 doses of apomorphine (s.c), 4 min. before beginning behavioral testing. Significant differences among group scores for each time interval are reported in the resixlts section. 53

Saline

-^0.15 mg/kg Apo

-*- 0.25 mg/kg Apo

0.35 mg/kg Apo

0.45 mg/kg Apo

-^0.90 mg/kg Apo

JD O

8 12 16 20 24 28 32 36 40 44 48 52 56 60 Time (min)

Figure 3-3. Effects of apomorphine dose on discontinuous biting. Each value represents the mean number of observations over a 4 min. period for 6 animals. The vertical bars represent the standard error of the mean. The animals were given either saline or one of 5 doses of apomorphine (s.c), 4 min. before beginning behavioral testing. Significant differences among group scores for each time interval are reported in the results section. 54

8 12 16 20 24 28 32 36 40 44 48 52 56 60 Time (min)

Figure 3-4. Effects of apomorphine dose on sniffing behavior. Each value represents the mean number of observations over a 4 min. period for 6 animals. See text for more details. 55

8 12 16 20 24 28 32 36 40 44 48 52 56 60

Time (min)

Figure 3-5. Effects of apomorphine dose on continuous sniffing. Each value represents the mean number of observations over a 4 min. period for 6 animals. See text for more details. 56

Saline 0.15 mg/kg Apo 0.25 mg/kg Apo 0.35 mg/kg Apo 0.45 mg/kg Apo 0.90 mg/kg Apo

8 12 16 20 24 28 32 36 40 44 48 52 56 60 Time (min)

Figure 3-6. Effects of apomorphine dose on discontinuous sniffing. Each value represents the mean number of observations over a 4 min. period for 6 animals. See text for more details. 57

- Saline 0.15 mg/kg Apo 0.25 mg/kg Apo 0.35 mg/kg Apo 0.45 mg/kg Apo 0.90 mg/kg Apo

12 16 20 24 28 32 36 40 44 48 52 56 60 Time (min)

Figure 3-7. Effects of apomorphine dose on line crossings. Each value represents the mean number of observations over a 4 min. period for 6 animals. See text for more details 58

Saline

0. 1 5 mg/kg Apo 0.25 mg/kg Apo 0.35 mg/kg Apo 0.45 mg/kg Apo 0.90 mg/kg Apo

12 16 20 24 28 32 36 40 44 48 52 56 60 Time (min)

Figure 3-8. Effects of apomorphine dose on ambulation. Each value represents the mean number of observations over a 4 min. period for 6 animals. See text for more details. 59

25 1

Saline -^0.15 mg/kg Apo 0.25 mg/kg Apo -* 0.35 mg/kg Apo 0.45 mg/kg Apo — 0.90 mg/kg Apo

12 16 20 24 28 32 36 40 44 48 52 56 60 Time (min)

Figure 3-9. Effects of apomorphine dose on rearing. Each value represents the mean number of observations over a 4 min. period for 6 animals. See text for more details 60

Saline

0.15 mg/kg Apo 0.25 mg/kg Apo 0.35 mg/kg Apo 0.45 mg/kg Apo 0.90 mg/kg Apo

8 12 16 20 24 28 32 36 40 44 48 52 56 60

Time (min)

Figure 3-10. Effects of apomorphine dose on grooming. Each value represents the mean number of observations over a 4 min. period for 6 animals. See text for more details 61

25 1

8 12 16 20 24 28 32 36 40 44 48 52 56 60

Time (min)

Figure 3-11. Effects of apomorphine dose on behavioral inactivity. Each value represents the mean number of observations over a 4 min. period for 6 animals. See text for more details CHAPTER 4 EFFECTS OF OVARIECTOMY ON BEHAVIORS

Introduction

The purpose of our study was to determine the effects of ovariectomy on

apomorphine-induced behaviors. If endogenous estrogen affects apomorphine-induced

stereotyped behaviors, then removal of the major source of endogenous estrogen by

bilateral ovariectomy should influence the behavioral response to apomorphine.

Contradictory findings in the literature make it difficult to draw definitive conclusions

the about effects of estrogen on apomorphine-induced behaviors. Previously, it has been

shown that the time after ovariectomy is an important factor in determining the response

to apomorphine-induced behaviors (Gordon et al., 1980b; Koller et al., 1980). Gordon

and associates have suggested that ovariectomy does not result in a significant

enhancement of stereotyped behaviors until three months after ovariectomy (Gordon,

1980; Gordon et al., 1980a; Gordon et al., 1980b). Others have failed to observe any

change in stereotyped behaviors three months after ovariectomy (Johnson and Stevens,

1984). Therefore, factors other than the length of time after ovariectomy may contribute

to the changes in DA agonist-induced stereotyped behaviors observed after long-term

ovariectomy.

Disagreements in these reports concerning the effects of ovariectomy on DA agonist-induced locomotion and other stereotyped behaviors suggest that ovariectomy may have different effects on specific apomorphine-induced stereotyped behaviors. In experiment 1, the behavioral responses to different doses of apomorphine were examined

62 63

and provided information on the pattern of behavioral change under different levels of

DA-ergic stimulation. The information from the dose-response study in experiment 1

will be used to evaluate the changes in apomorphine-induced behaviors after

ovariectomy.

In this experiment, two questions were addressed. First, how does ovariectomy

affect apomorphine-induced changes in behaviors? For instance, are the behaviors

differentially affected by ovariectomy? Second, are the effects of ovariectomy modified

by the length of time between ovariectomy and testing?

Methods

Subjects

Sixty-two adult female Long-Evans rats were grouped housed with free access to

food and water. The animals were exposed to a 12:12 hour light-dark cycle, with lights

on at 08:00 hours.

Design

Subjects were assigned to one of five groups:

(a) Fourteen intact four-month old females were monitored for regular estrous cycles by vaginal smears for at least two consecutive cycles (intact females). Only females exhibiting regular 4-day estrous cycles were used in this study. Intact females were tested for their behavioral response to apomorphine during either the estrus or diestrus phase of the estrous cycle.

(b) Eight four-month old females were sham ovariectomized two weeks before testing for their behavioral response to apomorphine (sham ovariectomized females). The females were monitored for regular estrous cycles prior to the sham ovariectomy. After sham ovariectomy, the animals were housed individually in cages until behavioral testing. Females exhibiting a regular 4-day estrous cycle were tested for their behavioral response to apomorphine on estrus or the first day of diestrus of the estrus cycle. 64

(c) Fourteen females were bilaterally ovariectomized at four months of age. After ovariectomy, the animals were housed individually in cages until behavioral testing two weeks later (short-term ovariectomized, 4 month old females).

(d) Eight females were bilaterally ovariectomized at 8 months of age and tested two weeks later for their behavioral response to apomorphine (short-term ovariectomized, 8 month old females). After ovariectomy, the animals were housed individually in cages until behavioral testing. This group served as a control for any effect that age or weight might have contributed to the eight month old, long-term ovariectomized females' behavioral response to apomorphine.

(e) Fourteen females were bilaterally ovariectomized at 4 months of age

and housed in group cages until the time of testing 4 months later. Two weeks before testing, the animals were removed and replaced in individual cages. These animals were approximately 8 months old at the time of testing (long-term ovariectomized females).

All animals were given s.c. injections of 0.45 mg/kg of apomorphine 4 minutes

before beginning behavioral observations.

Results

Tables 4-1 and 4-2 show the means for the total number of observations and

latencies, respectively, for each behavioral response.

Unlike experiment 1 in which each dose level produced specific and qualitative

changes in behavior, the effects of ovariectomy on apomorphine-induced behaviors were

more subtle. As the results indicate, there were no apparent differences in behavioral

responding between treatment conditions based on the type of behaviors displayed.

Treatment conditions did not produce the dramatic changes in the behavior profiles

across conditions that were observed with increments in apomorphine dose.

Biting Behavior

There was a significant effect of condition on biting, F(4,53) = 5.63, p < 0.001.

Figure 4-1 shows the long-term ovariectomized females displayed more biting than the 65

intact females, the sham-ovariectomized females and the short-term ovariectomized, 4

month old females (p < 0.05). The short-term ovariectomized, 8 month old females also

showed more biting than the sham-ovariectomized females (p < 0.05).

There was a significant interaction between time and treatment on total biting,

F(13,52) = 3.52, p < 0.001 (). The short-term ovariectomized, 4 month old females, short-

term ovariectomized, 8 month old females and long-term ovariectomized females all

displayed more biting than intact and sham ovariectomized females during the 24 minute

and 28 minute time period (p < 0.05). However, during the 32 minute time period, the

short-term ovariectomized, 8 month old females and long-term ovariectomized females

both displayed more biting than the sham ovariectomized females (p < 0.05). The long-

term ovariectomized females also showed more biting than the intact females and sham

ovariectomized, 4 month old females (p < 0.05).

During the 36 minute and 40 minute time periods the short-term ovariectomized,

8 month old females and long-term ovariectomized females displayed more biting than

all other groups (p < 0.05). During the 44 minute time period the short-term

ovariectomized, 8 month old females showed more biting than the sham ovariectomized

females only. The long-term ovariectomized females displayed more biting than all other

groups during the 44 minute through 56 minute time periods (p < 0.05). There was no

significant effect of condition on the latency to display biting.

Continuous Biting

The long-term ovariectomized females displayed more continuous biting than all other groups. Long-term ovariectomized females displayed more continuous biting than other groups during the last 20 minutes of the session than the other groups. 66

There was a significant effect of condition on continuous biting, F(4,53) = 7.08, p

0.001 figure 4-2 < . As shows, both 8 month old females displayed more continuous

biting than the sham ovariectomized females (p < 0.05).

The latency to display continuous biting was increased after ovariectomy F(4,53)

= 18.71, p < 0.001. The short-term ovariectomized, 8 month old females and long-term

ovariectomized females displayed longer latencies to display continuous biting than the

other groups (p < 0.05).

Discontinuous Biting

Discontinuous biting like continuous biting was increased after ovariectomy.

Both the short-term ovariectomized, 8 month old females and long-term ovariectomized

females displayed more discontinuous biting than the intact females and short-term

ovariectomized, 4 month old females. The short-term ovariectomized, 8 month old

females also displayed more discontinuous biting than the sham-ovariectomized females

and long-term ovariectomized females. The sham-ovariectomized females also showed more discontinuous biting than the intact females and short-term ovariectomized, 4 month old females.

There was a significant effect of ovariectomy on discontinuous biting, F(4,53) =

27.17, p < 0.001. As figure 4-3 shows, all groups displayed more discontinuous biting than the intact females and short-term ovariectomized, 4 month old females (p < 0.05).

The short-term ovariectomized, 8 month old females also displayed more discontinuous biting than the sham ovariectomized females and long-term ovariectomized females (p <

0.05). 67

There was a significant interaction between condition and time, F(13,52) = 5.92, p

< 0.001. During the 16 minute and 20 minute time periods the short-term

ovariectomized, 8-month-old females and long-term ovariectomized females displayed

more discontinuous biting than the intact females and short-term ovariectomized, 4

month old females (p < 0.05). The short-term ovariectomized, 8 month old females also

showed more discontinuous biting than the long-term ovariectomized females (p < 0.05).

The short-term ovariectomized, 8 month old females and long-term ovariectomized

females displayed more discontinuous biting than intact females during the 24 minute

time period (p < 0.05). The short-term ovariectomized, 4 month old females displayed

more discontinuous biting than all other groups during the 28-minute through 40 minute

time periods (p < 0.05).

The long-term ovariectomized females showed more discontinuous biting than the

short-term ovariectomized, 4 month old females during the 28 minute time period (p <

During 0.05). the 36 minute time period the long-term ovariectomized females displayed

more discontinuous biting than the intact females and short-term ovariectomized, 4

month old females (p < 0.05). During the 44-minute time period the short-term

ovariectomized, month old 8 females displayed more discontinuous biting than all other groups except the long-term ovariectomized females (p < 0.05). During the 52-minute through 56 minute time periods the long-term ovariectomized females showed more discontinuous biting than all other groups (p < 0.05).

Sniffing Behavior

There was no significant effect of condition on the display of sniffmg behavior.

There was a significant interaction between condition and time, F(13,52) = 3.33, p < 68

0.001. However, intact females and sham-ovariectomized females displayed more sniffing behavior during the mid-portion of the session, whereas both the short-term and long-term ovariectomized, 8 month old female groups displayed more sniffing during the

last 20 to 30 minutes of the session (p < 0.05). There was no difference in the onset of

sniffing behavior between groups.

Continuous Sniffing

There was no significant effect of ovariectomy on continuous sniffing behavior.

There was a significant interaction between the condition and time, F( 13,52) = 3.33, p <

0.001 . Intact females and sham-ovariectomized females showed more continuous

sniffing than all other groups during the midportion of the session. During the last 1 0 -

12 minutes of the session the short-term ovariectomized, 8 month old females and long-

term ovariectomized females displayed more continuous sniffing than all other groups.

Discontinuous Sniffing

Discontinuous sniffing decreased after ovariectomy, F(4,53) = 7.62, p < 0.001.

The long-term ovariectomized females displayed less discontinuous sniffing than the intact females (N-K, p < 0.05).

There was a significant effect of fime on discontinuous sniffing, F(l 3,689) =

47.22, 0.001 p < . Discontinuous sniffmg was primarily displayed during the last 20 minutes of the session, during which, the ovariectomized females group showed less discontinuous sniffing than the intact females group. There was no significant effect of condition on the latency to display discontinuous sniffing.

Locomotor Activity

The effects of ovariectomy on LA were consistent with an increase in DA-ergic tone. In experiment 1 , ambulation and line crossings were decreased with increments in 69

dose from the 0.45 mg/kg to the 0.90 mg/kg dose of apomorphine. Since ovariectomy

also decreased ambulation and line crossings, the effects of ovariectomy on LA are

consistent with an increase in DA-ergic stimulation. Although the direction of change in

ambulation and line crossings after ovariectomy were consistent, there were differences

in the sensitivity of total measures for line crossings and ambulation to the effects of

ovariectomy.

Only the long-term ovariectomized females displayed a decrease in ambulation.

Whereas, line crossings were lower in all of the ovariectomized females when compared

to intact females. The suggestion is that total measures for line crossings are more

sensitive to the effects of ovariectomy than ambulation.

Line Crossings

The number of lines crossed was decreased after ovariectomy, F(4,53) = 3.47, p <

0.05. The decrease in lines crossed was observed as early as 2 weeks after ovariectomy,

and was still depressed in animals ovariectomized for 16 weeks. There was no effect of

ovariectomy on the latency for line crossings.

As Figure 4-4 shows, ovariectomized females displayed fewer line crossing than the intact females and sham ovariectomized females (N-K, p < 0.05). There was a significant condition by time interaction, F(13,52) = 2.37, p < 0.001.

During the 8 minute time period the short-term ovariectomized 4 month old and 8 month old females displayed fewer line crossings than the intact females (N-K, p < 0.05).

During the 36 minute time period the short-term ovariectomized, 8 month old females and long-term ovariectomized females displayed fewer line crossing than the intact and sham ovariectomized females (N-K, p < 0.05). During the 40 minute through 44 minute 70

time periods the short-term ovariectomized, 8 month old females and long-term

ovariectomized females displayed fewer line crossings than the intact and sham

ovariectomized females (N-K, p < 0.05).

Rearing Behavior

Rearing behavior = was decreased by ovariectomy, F(4,53) 7.12, p < 0.001 .

Long-term ovariectomized females displayed less rearing behavior than sham-

ovariectomized females and short-term ovariectomized, 8 month old females (N-K, p <

0.05). All other groups displayed less rearing behavior than the sham-ovariectomized

female group (N-K, p < 0.05).

There was a significant interaction between condition and time on rearing

behavior, F(13,52) = 3.19, p < 0.001.

The short-term ovariectomized, 4 month old female group displayed more rearing than any other groups during the 8 minute through 36-minute time intervals (N-K, p <

0.05). During the 40-minute time interval the sham ovariectomized and short-term

ovariectomized, 4 month old female groups both displayed more rearing than all other groups. However, during the 44-minute time interval, intact females, sham ovariectomized and short-term ovariectomized, 4 month old female groups all displayed more rearing than the other groups (N-K, p < 0.05). The sham-ovariectomized females also displayed more rearing than the intact females during the 44-minute time interval

(N-K, p < 0.05).

The sham-ovariectomized and short-term ovariectomized, 4 month old female groups reared more than the long-term ovariectomized female group during the 48- minute, 52-minute and 60-minute time intervals (N-K, p < 0.05). 71

The short-term ovariectomized, 8 month old female group reared more than all

other groups during the 60-minute time interval (N-K, p < 0.05).

There was a significant effect of condition on the latency to rear, F(4,53) = 5.95, p

< 0.001. The latency to display rearing was greater for the long-term ovariectomized

female group than any other group

Rearing behavior was decreased after ovariectomy; however, only the long-term ovariectomized females were significantly different from the intact and sham- ovariectomized females. All treatment groups predominantly displayed rearing behavior in the last half of the session (N-K, p < 0.05). The latency to rear was increased only after long-term ovariectomy.

The decrease in rearing appears to be an effect of long-term ovariectomy, the decrease in immobility may be an age-related or weight-related effect, since both the short-term and long-term eight month old females displayed the decrease.

Rearing was only decreased by the long-term ovariectomized females, but both the eight month old, short-term ovariectomized and four month old, short -term ovariectomized females decreased inactivity. In both the effects on rearing and inactivity, the differences were among ovariectomized groups, and not between ovariectomized and intact females.

Grooming Behavior

There were no significant effects of ovariectomy on grooming behavior.

However, the latency to groom was increased by both short-term and long-term ovariectomized 8-month-old female groups. 72

There was a significant effect of condition on the latency to display grooming

- behavior F(4,53) 7.96, p < 0.001 . The latency to groom was greater for the short-term

ovariectomized, 8 month old females and long-term ovariectomized females than for all

other groups (N-K, p < 0.05).

Effects of Ovariectomy on Body Weight

A one-way ANOVA on body weight revealed a significant difference in body

weights between groups, F(4,53) = 38.30, p < 0.001 (Table 4). Both the long-term

ovariectomized females and short-term ovariectomized, 8 month old females had

significantly greater body weights than other groups (N-K, p < 0.05). To determine

whether group differences in body weight might contribute difference behavioral scores,

an analysis of covariance was performed on biting and line crossing, with weight as a

covariant. When behavioral scores were controlled for body weight, the effects of

ovariectomy on biting and line crossings were abolished.

To determine whether age differences among groups might contribute to

differences in behavioral scores an ANOVA on age was performed. There was a

significant effect of = age on biting behavior, F(2,55) 7.38, p < 0.01 . The

ovariectomized, 8 month old females displayed significant more biting than the 4 month

old intact and sham ovariectomized females (N-K, p < 0.05).

Ovariectomy had no effect on grooming, but decreased rearing and inactivity.

Discussion

Effects on Biting

In our study, biting was increased and locomotion decreased after ovariectomy.

While the effects on biting were modest, when combined with the decrease in line crossings, the changes in behavior are consistent with earlier studies which used rating 73

scale measures (Lai and Sourkes, 1972; Gordon, 1980; Gordon et al., 1980b; Verimer,

1981;Hruskaetal., 1982a).

Gordon and associates have suggested that stereotypy ratings are not enhanced

until three months after ovariectomy (Gordon, 1980; Gordon et al., 1980b). Indeed, behavioral testing shortly after ovariectomy might explain, in part, the failure to observe

any change in behaviors (Beatty et al., 1 982; Miller, 1 983). Several studies performed

behavioral tests more than 3 months after ovariectomy and still failed to observe any change in behaviors (Savageau and Beatty, 1981; Johnson and Stevens, 1984).

Nevertheless, Johnson «fe Stevens (1984) also observed a modest decrease in behavioral ratings for the ovariectomized females during the first 20 minutes of the test session compared to intact females (Savageau and Beatty, 1981; Johnson and Stevens, 1984). In both cases, according to their rating scale, the reduction in ratings involved a reduction in biting. In neither case was the effect great enough to produce an overall significant effect of condition. In our study, long-term ovariectomized females also displayed less biting during the early portion of the session, and more biting towards the end of the session.

However, because behaviors were measured separately, the effects of ovariectomy on biting were significant.

In contrast to biting, line crossings and rearing were significantly decreased after both short term and long-term ovariectomy. Both locomotor activity and rearing have been previously found to decline after ovariectomy (Savageau and Beatty, 1981; Johnson and Stevens, 1984), although the authors did not to quantify the changes in either behavior. Our study addressed this issue, however, there still are some problems with the manner in which biting behavior is affected by long-term ovariectomy. For instance, the 74

response patterns for biting after long-term ovariectomy did not indicate a greater

behavioral response, but rather an increase in the duration of responding. Furthermore,

the age and weight matched 8 month old short-term ovariectomized females showed a

biting response pattern similar to that of the long-tem ovariectomized females. The

similarity in response patterns between the two 8 month old female groups suggests that

the duration since ovariectomy may not completely explain the changes in behaviors.

It is well known that ovariectomy causes an increase in body weight, primarily in

the form of increased body fat (Wade, 1 972; Wade and Gray, 1 979). Therefore, it is

possible that an increase in body fat after ovariectomy could alter the distribution and

metabolism of apomorphine. For instance, it has been shown that the increase in body fat can greatly prolong the action of some drugs (Brodie et al., 1954). The increased body weight shared by both 8 month old groups might explain their similar response patterns.

Previous studies have reported an increase in stereotyped ratings after long-term ovariectomy, in which the authors interpreted the behavioral change as an indication of an increase in behavioral sensitivity to apomorphine (Gordon, 1980). The behavioral profile displayed by long-term ovariectomized females in the present study does not necessarily suggest an increase in behavioral sensitivity to apomorphine. In Experiment

1, increments in apomorphine doses increased the duration of biting behavior, but also increased the incidence of biting as illustrated by the increase in the peak of the response curve. However, the increase in total biting displayed after long-term ovariectomy only increased the duration of time in which biting was observed. Long-term ovariectomy had no affect on the peak amplitude or the onset of biting. In fact Verimer et al., (1981) 75

directly measured the duration of apomorphine-induced behaviors, and found that 6

weeks after ovariectomy the duration was increased.

In conclusion, the result suggests that ovariectomy in the short-term appears to

affect specific apomorphine-induced behaviors in a fashion consistent with an increase in

behavioral stimulation. But the effects of long-term ovariectomy on behaviors are more

problematic. While there were some effects of long-term ovariectomy that were

consistent with the changes in behavior after the short-term, but there was no indication

of greater behavioral stimulation. Instead some of the effects point to some other

influences such as peripheral changes in drug effects due possibly to ovariectomy-

induced increases in body fat and weight.

Group IF SO STA STB LTO Body Weight 287 218 276 *327 *335 (8) (9) (7) (11) (10) Number of Subjects 14 8 14 8 14

Table 4-1 Mean body weights after ovariectomy and intact rats. Body weights, in grams, are shown for the intact 4 month old females (IF), sham ovariectomized 4 month old females (SO), short-term ovariectomize'd, ' 4 month old females (STA), short-term ovariectomized, 8 month old females (STB) and long-term ovariectomized 8 month old females (LTO). The standard errors of the means are presented in parentheses below the mean body weights. The mean body weight of the short-term ovariectomized, 8 month old females and long-term ovariectomized females were significantly greater than the other groups (*). The weights listed above were obtained at the time of testing. 76

Table 4-2 Behavioral response totals after ovariectomy. IF SO STA STB LTO Rr b 15.4 6.6 9.2 b 10.0 b 3.7 b (3.2) (1.1) (1.4) (1.5) (1.1) Sn 172.7 196.7 139.0 175.1 132.7 (18.4) (22.8) (11.6) (15.2) (10.8) CS 151.9 165.9 124.5 157.4 122.5

(18.2) (22.7) (11.0) (14.7) (9.6) DS 19.4b 30.9 14.4 b 17.7b 9.4 a^b

(3.1) (4.4) (1.3) (2.6) (2.0)

Bt 110.1 79.5 129.0 147.0 b 177.4 a-b (16.4) (17.6) (9.3) (16.8) (14.4) CB 93.9 58.0 118.0b 96.9 123.8 b (17.5) (12.2) (10.3) (8.3) (20.1) DB 16.3 21.4 11.0 50.4 a,b 51.7

(2.9) (8.0) (1.9) (7.6) (8.2) Gr 9.1 10.6 11.1 0.5 5.9 (2.7) (2.1) (2.8) (0.4) (3.1) BI 19.9 11.7 30.7 0.1 ^b 6.1

(7.9) (6.7) (8.9) (.1) (2.7) Amb 78.8 96.2 61.1 68.4 52.8 (7.9) (8.1) (11.3) (12.4) (10.8) Lcm 35.6 35.6 16.8 20.1 a,b 18.6a,b

(5.2) (5.1) (2.8) (3.6) 1 (7.7)

Animals were placed into one of five groups: 4 month old intact females (IF, n=14);4 month old sham ovx females (SO, n=8), 4 month old short-term ovx females (STA, n=14); 8 month old short-term ovx females (STB, n=8); 8 month old long-term ovx females (LTO, 'n=14). Values represent the mean number of observations for each behavior. Numbers in parentheses are the standard error of the mean. The means the value is significantly different from intact females' behavioral "b" score; means the value is significantly different from sham ovx females All other details as in Table 3-1. 77

Figure 4-1 . Biting behavior totals after ovariectomy. Animals were placed into one of five groups: 4 month old intact females (n=14);4 month old sham ovx females (SO, n=8); 4 month old, short-term ovx females (STA, n=14); 8 month old short-term ovx females (STB, n=8); 8 month long-term ovx females. (LTO, n=14). Animals were given 0.45 mg/kg of apomorphine (s.c), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. The values represent the mean number of observations over a period of 4 min, vertical bars represent the standard error of the mean. Significant differences among group scores for each time interval are reported in the results section. 78

25 -I

> SI 20 - i3

Intact female 15 Sham ovx

I STA > 10 STB LTO s 5-

32 40 64 Time (mm)

Figure 4-2. Biting behavior displayed over time after ovariectomy. Animals were placed into one of five groups: 4 month old intact females (n=14);4 month old sham ovx females (SO, n=8); 4 month old, short-term ovx females (STA, n=14); 8 month old short-term ovx females (STB, n=8); 8 month long-term ovx females.(LTO, n=14). Animals were given 0.45 mg/kg of apomorphine (s.c), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. The values represent the mean number of observations over a period of 4 min, vertical bars represent the standard error of the mean. Significant differences among group scores for each time interval are reported in the results section. 79

Figure 4-3 . Continuous biting displayed over time after ovariectomy. Animals were placed into one of five groups: 4 month old intact females (n=14);4 month old sham ovx females (SO, n=8); 4 month old, short-term ovx females (STA, n=14); 8 month old short-term ovx females (STB, n=8); 8 month long-term ovx females.(LTO, n=14). Animals were given 0.45 mg/kg of apomorphine (s.c), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. The values represent the mean number of observations over a period of 4 min, vertical bars represent the standard error of the mean. Significant differences among group scores for each time interval are reported in the results section. 80

Figure 4-4. Discontinuous biting displayed over time after ovariectomy. Animals were placed into one of five groups: 4 month old intact females (n=14);4 month old sham ovx females (SO, n=8); 4 month old, short-term ovx females (STA, n=14); 8 month old short-term ovx females (STB, n=8); 8 month long-term ovx females.(LTO, n=14). Animals were given 0.45 mg/kg of apomorphine (s.c), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. The values represent the mean number of observations over a period of 4 min, vertical bars represent the standard error of the mean. Significant differences among group scores for each time interval are reported in the results section. 1

81

50

Figure 4-5. Line crossing totals after ovariectomy. Animals were placed into one of five groups: 4 month old intact females (n=14);4 month old sham ovx females (SO, n=8); 4 month old, short-term ovx females (STA, n=14); 8 month old short-term ovx females (STB, n=8); 8 month long-term ovx females.(LTO, n=14). Animals were given 0.45 mg/kg of apomorphine (s.c), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. The values represent the mean total number of lines crossed over a period of 60 min, vertical bars represent the standard error of the mean. Significant differences among group scores for each time interval are reported in the results section. 82

Figure 4-6. Line crossings displayed over time after ovariectomy. Animals were placed into one of five groups: 4 month old intact females (n=14);4 month old sham ovx females (SO, n=8); 4 month old, short-term ovx females (STA, n=14); 8 month old short-term ovx females (STB, n=8); 8 month long-term ovx females.(LTO, n=14). Animals were given 0.45 mg/kg of apomorphine (s.c), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. The values represent the mean number of lines crossed over a period of 4 min, vertical bars represent the standard error of the mean. Significant differences among group scores for each time interval are reported in the results section. .

83

Table 4 3 Behavioral response latencies after ovariectomy. IF SO STA STB LTO Rr 39.1 30.6 37.7 40.0 49 7

fl 9) (5 7) (2 1) (1 7) (2 3) Sn 4.0 4.0 4 0 4 0 4 0 CO) (0) (0) (0) (0) CS 14.1 4.0 4 0 4 0 4 0 (2.2) (0) (0) (0) (0) DS 29.3 24.2 34 3 31 5 76 4 (3 8) (4 9) (2 5) (5 7) 3'> Bt 6.2 6.2 5 6 5 7 7 9 fO 7) (0 9) (0 3) fO 5) (0 9'»

CB 6.4 13.0 6 1 10 5 111.11 1

(0 7) (2 5) (0 4) (1 3) DB 7.9 6.2 10 0 4 0 7 Q

(0.8) (0.9) (2.4) (0) (0 9) Gr 48.3 47.0 49.1 59.0 54.3

(1.9) (1.5) (1.8) (0.6) (1.4) BI 50.6 55.5 46.3 59.5 54.3 (2.2) (1.6) (2.3) (0.5) (2.9) Lcm 1.1 4.0 16.4 21.9 17.6 (2.2) (0) (4.7) (6.8) (5.3)

Values represent the mean latencies for each behavioral response.

Animals were placed into one of five groups: 4 month old intact females (IF, n=14);4 month old sham ovx females (SO, n-8), 4 month old short-term ovx females (STA, n=14); 8 month old short-term ovx females (STB, n=8); 8 month old long-term ovx females (LTO, n=14). Values represent the mean number of observations for each behavior. Numbers in parentheses are the standard error of the mean. The means the value is significantly different from intact females' behavioral score; "b" means the value is significantly different from sham ovx females. All other details as in Table 3-1 CHAPTER 5 EFFECTS OF ESTRADIOL TREATMENTS ON BEHAVIORS

Introduction

In experiment 2, the influence of endogenous estrogen on the behavioral response to

apomorphine was investigated by observing the effects of ovariectomy on apomorphine-

induced behaviors. The premise was that the effects of estrogen on DA agonist-induced

behaviors can be examined indirectly by removing the major source of estrogen. A more

direct approach to investigating the influence of estrogen on apomorphine-induced

stereotyped behaviors involves assessing the effects of the exogenous administration of

estrogen.

Previous studies investigating the effects of estrogen on apomorphine-induced

behaviors have reported contradictory results. While some studies conclude that estrogen

administration can inhibit or antagonize DA-related behaviors (Gordon et al., 1980b; Joyce

and Van Hartesveldt, 1984a), others have suggested that estrogen can facilitate the behavioral

response to DA agonists (Hruska and Silbergeld, 1980a; Hruska and Silbergeld, 1980b;

Chiodo et al., 1981; Hruska et al., 1982a; Johnson and Stevens, 1984). In those studies, the effects of estrogen on DA agonist-induced behaviors have been measured using a variety of estrogen treatment schedules and doses, either of which could be sources of considerable variation in the results. Consequently, it is unresolved as to whether the effects of estrogen on DA-related behaviors vary depending on the length of the treatment schedule or the dose of estrogen.

84 85

Another possible explanation for the differences in results among studies is that

estrogen treatment differentially affects specific DA-agonist-induced behaviors. As

mentioned previously, the use of behavioral rating scales may not provide an accurate or

complete assessment of individual behavioral change in response to estrogen treatment. In

experiment 2, specific behaviors were affected differentially by changes in hormone

condition. A similar relationship between the effects of estrogen administration and specific

apomorphine-induced behaviors may also exist.

In the present study, three issues were addressed.

(1) Does estrogen treatment differentially affect specific apomorphine- induced behavioral responses?

(2) Does the dose of estrogen influence the effects of estrogen on apomorphine-induced behavioral responses?

(3) Does the duration of the estrogen treatment schedule influence the effects estrogen has on apomorphine-induced behavioral responses?

Methods

Subjects. Sixty-eight individually housed 3 month old aduU female Long-Evans

hooded rats were placed in one of 7 treatment-interval groups. The treatment-interval refers

to the time between the first injection of oil or estradiol and the behavioral test with

apomorphine.

Treatment Schedule. Short-term treatment intervals: Animals were given injections

of either peanut oil (1 .0 ml/kg) or 50 ^g of 1 7-p E2 (1 ml/Kg) and tested for behavior with

0.45 mg/kg of apomorphine hydrochloride (s.c.) either 3 hours, 6 hours, 12 hours or 24 hours

later. Long-term treatment intervals: Animals were given one of the following treatment

schedules: (a) daily injections of either peanut oil (1.0 ml/kg), 25 ^g, or 100 ng of EB in peanut oil for 3 three days; (b) daily injections of either peanut oil (I.O ml/kg), or 25 ^g of 86

EB for 7 days, or; (c) daily injections of peanut oil (1 .0 ml/kg), or 25 ng of EB for 20 days.

Twenty-four hours after the last injection of either oil of EB, animals were tested with 0.45

mg/kg of apomorphine hydrochloride.

Hormone and drug Administration. Apomorphine hydrochloride was dissolved in

0.9% saline immediately before each behavioral test, and injected subcutaneously (s.c.) in the

right rear flank. EB and 17-p E2 were dissolved in peanut oil. EB, 17-p E2 and the peanut oil

vehicle were each injected s.c. in the left rear flank.

Statistics. Each treatment group was compared to the respective oil control group.

Behavioral scores were analyzed by three-factor ANOVA tests for treatment, treatment

schedule and with repeated measures on time. Further analyses of simple effects were

carried out by one-factor ANOVA tests and Neuman Keuls' test (P < 0.05). The F-values

and p-values for the ANOVAs have been tabled in appendix D.

Results

In this experiment both the short-term and long-term effects of estrogen treatment on

apomorphine-induced behaviors were examined. For each behavior the results for acute

effects of 50 ng/kg 17-P E2 treatment on apomorphine-induced behaviors will be presented

first, followed by the results for EB treatment on apomorphine-induced behaviors. The

effects of estrogen on behavioral observations for individual stereotyped behaviors have been

placed in Table 5.1 through Table 5.3.

Biting Behavior

Estradiol 17-p treatment effects

The treatment effects on biting behavior were significant, F(l,56) = 13.68, p < 0.001.

Total observations of biting behavior were decreased 6 hours and 24 hours after a single injection of 50 17-p E2 (Figure 5-1). 87

Figure 5-2 shows the time-course pattern for the 24 hour E2 treatment group, which

displayed less biting than the oil control group during the 12 minute 24 minute mtervals (P <

0.05). The 6 hour E2 treatment group displayed less biting than the oil control group during

the 16 minute, 24 minute and 28 minute intervals (P < 0.05). There was no effect of any E2

treatment on the latency to display biting.

Estradiol Benzoate treatment effects

Figure 5-3 shows that although biting was decreased after 3 days of 1 00 ng/kg of EB,

25 ^g/kg of EB decreased biting only after 7 days of treatment and not after 20 days of EB

treatment (P < 0.05). There were no effects of EB dose on the onset of biting behavior.

Continuous Biting

Estradiol 17- p treatment effects

Figure 5-4 continuous biting was decreased 3 hours, 6 hours and 12 hours after a

single injection of 50 ^g/kg of 1 7-p E2. The time-course patterns, as shown in figure 5-5,

indicates the 3 hour E2 treatment group displayed less continuous biting than the oil control group during the 12 minute through 28 minute time intervals (P < 0.05). The 6 hour E2 treatment group displayed less continuous biting than the oil group during the 12 minute through minute 40 time intervals (P < 0.05). The 12 hour E2 treatment group displayed less continuous biting than the oil group during the 12 minute through 28 minute time intervals (P

< 0.05). The latency to display continuous biting was increased after the 3 hour and 12 hour

E2 treatments.

Estradiol benzoate treatment effects

Similarly, continuous biting was decreased after either 3 days of 25 ^g/kg or 100

Mg/kg of EB, and after 7 days of 25 ^g/kg of EB (Table 5-3). 88

Discontinuous Biting

Estradiol 17- fi treatment effects

There was a significant main effect of treatment on DB, F(l,56) = 20.78, p < 0.0005.

In contrast to the effects on continuous biting, discontinuous biting was increased 3 hours, 6

hours and 12 hours after a single injection of 50 ^g/kg of 17-p E2 (p < 0.05). Figure 5-6

shows the time-course patterns for discontinuous biting.

Long-term EB treatment had no effect on discontinuous biting.

Sniffing Behavior

Estradiol 17- p treatment effects

There was a main effect of treatment on sniffing behavior, F(l,56) = 18.07, p <

0.0005. As Figure 5-7 shows, total measures of sniffing behavior were affected after the 12

and 24 hour estrogen treatment (p < 0.05). Sniffing behavior increased 12 and 24 hours after a single injection of 1 7-p E2. The increase in sniffing behavior after E2 treatment occurred primarily during the first half of the test session.

Estradiol benzoate treatment effects

Sniffing behavior was also increased after 3 days and 7 days of EB. Three days of

100 |ig/kg EB significantly increased sniffing behavior. Sniffing behavior was increased by

25 |ig/kg of EB only after 7 days of treatment.

Continuous Sniffing

There was a significant effect of treatment on CS, F(l,56) = 1 1.24, p < 0.005. Of the four short-term 17-p E2 treatment intervals, continuous sniffing was increased only after 24

hours of 1 E2 7-P (p < 0.05). Long-term EB treatment increased continuous sniffing after 7 days of 25 ^g/kg of EB. The increase in sniffing after EB occurred primarily during the last

15-20 minutes of the test session. 89

Rearing Behavior

Estradiol 17- p treatment effects

ITiere were no significant effects of 50 ^g/kg of 17-p E2 on rearing behavior after any

treatment interval. However, there was a significant treatment x time interaction. The 3 hour

and 6 hour estrogen treatment groups displayed more rearing than the oil group during the

last 20 minutes of the test session (P < 0.05).

Estradiol benzoate treatment effects

There was a significant effect of the p-ip on rearing behavior, F(2,42) = 4.68, p <

0.05. In contrast to acute effects of 17-P E2, rearing behavior was increased after 3 day EB

treatment (p < 0.05). The 100 ng/kg EB treatment group displayed more rearing than the 25

^g/kg EB and oil groups (Figure 5-8). The 7 day and 20 day EB treatments were ineffective.

Line Crossings

Estradiol 17-p treatment effects

There was a significant effect of repeated measures on LC F(l 3,546) = 2.74, p <

0.001. Figure 5-9 shows the response totals for line crossings after various EB treatments.

Line crossings were increased in the 3 day 100 ^g/kg and 7 day 25 ^g/kg EB treatment

groups compared to the oil controls (p < 0.1).

Discussion

Estradiol treatments had a selective and differential influence on individual behaviors.

Estrogen effects on DA-related behaviors and neurochemistry revolve around four sources of variability: The time (1) between termination of estrogen treatment and behavioral testing, also called the treatment-test interval; (2) The length of time estrogen is administered, or the 90

treatment duration; (3) the dose of estrogen administered, or the dose-interval; (4) A fourth

source of variability may involve the type of behavior measured.

In our study all testing was conducted within 24 or hours or less after the last E2

treatment. The current results suggest that not only does estrogen differentially affect

specific behavioral responses, but that the time-course of estrogen effects on specific

behaviors differs as well.

Any acute effects, occurring less than 24 hours, after a single injection of 17-P E2

suggests a non-traditional effect of estrogen on behaviors. Typically multiple estrogen

treatments over days are required to induce changes in behavior. In our study, a single

injection of 17-P E2 decreased biting behavior 6 and 24 hours after E2 treatment, but not after

3 and 12 hours after treatment. The irregular effects become clearer when biting components

are examined. Continuous biting decreased 3, 6 and 12 hours after E2 treatment, but

discontinuous biting increased during the same treatment intervals. Interestingly, there were no indications of an additive effect of E2 treatment over the course of 24 hours. For instance, the decrease in continuous biting was consistently lowered across time intervals. However, whether the decrease in total biting was significant was a reflection of the extent that component behaviors were affected. If the decrease in continuous biting was large enough and not cancelled by a corresponding increase in discontinuous biting, then total biting was significantly decreased. This is what occurred at 6 and 24 hours, while at other intervals changes in discontinuous biting cancelled the decline in continuous biting. This might explain why previous studies using more global behavior measures failed to observe acute effects of E2 treatments on apomorphine-induced behaviors (Fields & Gordon, 1982). 91

More behaviors were affected by 3 day and 7 day E2 treatment than after the acute

treatment intervals, which is more consistent with the traditional view of the time frame of

estrogenic effects. Estradiol treatment not only resulted in a decrease in biting behavior, but

rearing and sniffing were increased. Interestingly, most studies, based on behavioral ratings,

find that estrogen treatment antagonizes apomorphine -induced behaviors. In our study,

individual behavioral measures indicate that 3 and 7 day E2 treatments could both increase or

decrease specific behavioral responses There have also been previous reported that estrogen

decreased behavioral ratings of apomorphine -induced sniffing (Gordon, 1980; Gordon et al.,

1980b). However, Gordon and colleagues only rated behavior during a 20 minute period

after apomorphine administration. In our study the effects of E2 treatment involved only a

portion of the 60 minute test session, and it's possible that a shorter test session might not

detect the complete response. .

The 100 ng/kg treatment affected more behaviors than the 25 [ig/kg EB treatment

group, suggesting that the effects of estrogen treatment were additive either as a result of the

treatment duration or the dose. Since both doses of EB were given over 3 days, the higher

dose is the likely explanation for the greater behavioral effect. Gordon (1980) also reported

that apomorphine-induced behaviors were given lower rating scores after higher estrogen

treatment doses than lower. Additionally the 7 day EB treatment affected more behaviors

than the day EB treatment. 3 However, there was no indication that the effects of EB were

graded over time. In fact, the effect of 25 ^g EB treatment on sniffing and biting appeared to

be smaller at 7 days than days. 3 EB treatment had no effect on either behavior after 20 days.

Time-related effects of E2 treatment may derive from more than one mechanism.

Becker Beer (1986) suggested & that amphetamine-induced circling increased at 4 hours and 92

4 days after E2 treatment, while amphetamine-induced DA release increased only 4 hours

after E2, which the authors felt suggested more than one mechanism. The authors speculate

that changes in membrane polarity may explain some of the E2 treatment-induced changes in

behavior. The authors do not provide a clear time for these different mechanisms.

Biting behavior was very sensitive to estrogen treatments. The acute effects of 17-P

E2 treatment on biting were appeared to be inconsistent until the behavior components for

biting were examined. The effects of 17-P E2 consistently suppressed continuous biting over

the course of 24 hours, but the effects on discontinuous biting varied. The different patterns

for the components of biting illustrate how the qualitative features of a behavior may

determine how the observer interprets behavioral effects.

These results suggest that at least some of the effects of estrogen on apomorphine- induced behaviors occur faster than associated with the traditional view of the steroid receptor. In the next chapter, the possible central effects of estrogen on apomorphine- induced behaviors will be examined by applying E2 implants directly to various sites in the brain. 1

93

Table 5-1 The effects 1 . of 7-P E2 treatment on individual behaviors. OIL 17P-E2 3h 6h 12 h 24 h 3h 6h 12 h 24 h

Rr 12.3 1 1 7.1 17.5 20 0 16 4 *iI J. I 7 7 < o

(3.6) (1.5) (3.8) (4.3) (2.0) (3.8) (3.1) (4.2)

Sn 144.4 123.1 1 14.9 177 1 so 4 *1 T'i Q 126 7 4 1 OZ. / (10.5) (7.0) (13.3) (14.8) (8.0) (15.3) (10.6) (18.6)

CS 87.4 108.6 * 1 in Q 86 4 1 1 O.J ijV.y l-)o.6 (9.6) (7.0) (14.9) (15.5) (6.8) (17.2) (13.3) (8.5) DS 24.2 10.8 28 4 X) 0 JO.V J J.J JO.J z4.4 (7.1) (3.5) (4.6) (4.4) (2.7) (5.3) (3.9) (4.5) Bt 168.6 145.6 145.0 160.1 167.0 *81.9 128.6 *108 7 (8.0) (9.0) (8.4) (13.0) (8.3) (18.2) (10.3) (15.2) CB 127.3 125.3 88.4 89.4 *66.1 *27.4 *36.5 38.6 (12.5) (11.9) (15.5) (20.9) (11.6) (12.3) (10.8) (16.4) DB 40.1 21.2 52.7 70.7 * 100.9 54.0 *91.5 70.4 (14.0) (9.3) (8.2) (11.3) (9.1) (9.6) (2.8) (9.6) Amb 76.83 58.59 63.79 64.165 61.785 62.005 78.425 67.79 (11.28) (14.36) (7.89) (11.67) (9.09) (8.95) (9.25) (11.54) LC 23.8 13.6 38.7 30.3 19.7 24.1 38.5 23.9 (4.7) (3.5) (8.0) 1 (5.8) (4.0) (5.0) (8.2) (3.6)

The values represent the mean number of observations of each behavior after different lengths of time after a single injection of oil or 50 ^g/kg of 1 7-P E2. The numbers in parentheses represent the "*" standard error of the mean. The symbol signifies that the estrogen treatment group was significantly different from the respective oil control group. 94

Table 5-2. The effects of 1 7-p E2 treatment on response latencies for behaviors OIL 17- ?E2 3h 6h 12h 24 h 3h 6h 12h 24 h Rr 39.6 32.4 29.4 20.0 40.9 38.2 40.0 15.2

(1.9) (4.3) (3.8) (4.3) (1.6) (2.7) (1.8) (4.2) TS 4.0 4.0 4.0 4.5 4.4 4.0 4.0 4.0

(0) (0) (0) (0.5) (0.4) (0) (0) (0) CS 4.0 4.0 4.0 5.2 4.4 4.0 4.0 4.0

(0) (0) (0) (1.2) (0.4) (0) (0) (0) DS 33.8 39.1 35.5 38.0 36.0 32.9 31.0 38.0 (3.1) (3.3) (2.1) (1.5) (1.3) (1.5) (3.3) (1.8) IB 4.0 4.4 4.0 4.0 4.4 4.0 4.0 5.0

(0) (0) (0) (0) (0.4) (0) (0) (0.6) CB 4.0 6.2 4.0 6.0 7.1 20.0 10.0 18.5

(0) (0.7) (0) (1.1) (0.9) (7.7) (1.3) (6.8) DB 4.4 7.1 4.0 4.0 4.4 4.0 4.0 5.0 (0.4) (2.6) (0) (0) (0.4) (0) (0) (0.6) Lcm 8.4 16.0 7.0 22.0 31.6 20.4 9.5 4.5 (3.0) (7.0) (3.0) (6.8) (5.5) (6.8) (4.9) (0.5)

The values represent the mean number of observations of each behavior after different treatment durations of EE time after a single injection oil of or 50 ^ig/kg of 17-B E2 . The numbers in "*" parentheses represent the standard error of the mean. The symbol signifies that the estrogen treatment group was significantly different fi-om the respective oil control group. 95

Table 5-3. The effects of EB treatment on individual behaviors. OIL E]B 3 day 7 day 20 day 3 day (a) 3 day (b) 7 day 20 day Rr 5.6 2.9 10.2 5.6 *13.6 5.7 14.3

(1.1) (0.7) (1.8) (1.5) (1.5) (1.3) (1.8) TS 105.7 89.5 149.8 131.1 * 170.5 *118.0 121.2 (16.3) (7.4) (15.0) (10.6) (18.6) (11.0) (25.8) CS 92.2 77.4 129.8 110.6 142.0 *105.1 100.3 (14.8) (7.5) (16.3) (9.8) (19.4) (8.7) (23.2) DS 14.0 12.5 20.7 19.0 27.4 16.4 20.8

(3.7) (1.6) (3.4) (2.8) (5.3) (4.3) (4.1) TB 191.2 219.6 135.3 146.4 * 109.4 182.6 128.8 (19.4) (10.3) (20.5) (15.6) (13.3) (6.1) (19.0) CB 164.5 166.3 111.2 *87.7 *97.5 *105.7 114.7 (19.6) (20.7) (20.6) (10.7) (14.7) (13.8) (20.3) DB 26.5 57.7 24.2 57.5 22.7 76.9 14.2

(7.6) (12.7) (10.8) (13.6) (9.0) (11.9) (4.4) Gr 4.9 6.4 15.5 5.9 16.2 8.3 8.7 (1.6) (2.3) (4.4) (2.4) (9.0) (2.8) (3.8) BI 16.1 12.4 9.8 12.5 16.7 4.4 48.8 (8.8) (6.0) (8.5) (12.1) (6.4) (2.0) (19.9) Amb 79.7 50.4 94.8 102.1 *167.1 108.8 131.2 (15.8) (8.4) (16.0) (16.7) (10.9) (11.3) (19.8) LC 17.5 13.9 32.0 29.9 *37.1 *33.4 33.3 (6.0) (6.2) (8.1) (14.2) (5.6) (10.4) (17.1)

Animals were given either peanut oil or 25 ^ig/kg of EB (a) for 3 days, 7 days or 20 days. An additional group was given 100 ng/kg of EB (b) for 3 days. Twenty-four hours after the last treatment injection, animals were given 0.45 mg/kg of apomorphine (s.c.) and tested according to procedures as stated in the general methods section. The values represent the mean number of observations and standard error of the means respectively. 96

300 1

Oil ^ Estradiol

3 hour 6 hour 12 hour 24 hour

Treatment-test Interval

Figure 5-1 Sniffing . Behavior totals after treatment with 1 7-P E2. Sniffing behavior was observed 3 h, 6 h, 12 h or 24 h after a single injecfion of either peanut oil or 50 ng/kg of 17-p E2 in peanut oil. Animals were given 0.45 mg/kg of apomorphine (s.c), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. The values represent the mean number of observations over a 60 min time interval, vertical bars represent the standard error of the means. Significant differences between group scores at each time interval are reported in the results section. 97

250 1

Oil

0 Estradiol

3 hour 6 hour 12 hour 24 hour

Treatment-Test Interval

Figure 5-2. Biting Behavior totals after treatment with 1 7-P E2. Biting behavior was observed 3 h, 6 h, 12 h or 24 h after a single injection of either peanut oil or 50 ^g/kg of 1 7-P E2 in peanut oil. Animals were given 0.45 mg/kg of apomorphine (s.c.), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. The values represent the mean number of observations over a 60 min time interval, vertical bars represent the standard error of the means. Significant differences among group scores at each time interval are reported in the results section. 98

25 1

Time (min)

Figure 5-3. Time-course for Biting Behavior 24 hours after 17-p E2 treatment.

Biting behavior is observed 24 hours after a single injection of either peanut oil or 50 ^g/kg of 1 7-p E2 in peanut oil. Animals were given 0.45 mg/kg of apomorphine (s.c), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. Each value represents the mean number of observations over a 4 min. period. The vertical bars represent the standard error of the means. Significant differences among group scores at each time interval are reported in the results section. 99

160 -

3 Hour 6 Hour 12 Hour 24 Hour Treatment Intervals

Figure 5-4. Continuous Biting totals after treatment with 1 7-P E2. Continuous biting was observed 3 h, 6 h, 12 h or 24 h after a single injection of either peanut oil or 50 ng/kg of 1 7-P E2 in peanut oil. Animals were given 0.45 mg/kg of apomorphine (s.c), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. The values represent the mean number of observations over a 60 min time interval, vertical bars represent the standard error of the means. Significant differences among group scores at each time interval are reported in the results section. 100

3hroil 3hrEa

18 ?4 32 40 4« 56 64 Tim* (min) Tims (min)

20

12hroil 24hroU 12hrE2 15 24hrE2

10

• 16 24 32 40 41 58 64 I 18 24 32 40 41 56 64 Tima (min) Tims (min)

Figure 5-5. Time-course for continuous biting after 1 7-P E2 treatment. The effects of systemic injections 17-P E2 on continuous biting were observed 3 h (panel A), 6 h (panel B), 12 h (panel C) or 24 h (panel D)after a single injection of either peanut oil or 50 ^g/kg of 17-p E2 in peanut oil. Animals were given 0.45 mg/kg of apomorphine (s.c), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. The values represent the mean number of observations over a 60 minute time interval, vertical bars represent the standard error of the means. Significant differences among group scores at each time interval are reported in the results section. 101

25l

20

15 6hrE2

0 I If 24 32 40 41 H M Tfem^nta) Tkne inin)

23

20

24hrol 15 *- 24hrE2

10

8 16 24 32 40 48 56 64 Tlmt (min)

Figure 5-6. Time-course for discontinuous biting after 1 7-P E2 treatment. The effects of systemic injections of 17-p E2 on discontinuous biting were observed 3 h (panel A), 6 h (panel B), 12 h (panel C) or 24 h (panel D) after a single injection of either peanut oil or 50 ^ig/kg of 1 7-p E2 in peanut oil. Animals were given 0.45 mg/kg of apomorphine (s.c), 4 min before beginning behavioral observations according to procedures as stated in the general methods section. The values represent the mean number of observations over a 60 min time interval, vertical bars represent the standard error of the means. Significant differences among group scores at each time interval are reported in the results section. 103

Figure 5-7. Biting Behavior totals after treatment with estradiol benzoate. Animals were given either peanut oil or 25 ng/kg of EB for 3 days, 7 days or 20 days. An additional group was given 100 ng/kg of EB for three days. Twenty-four hours after the last treatment injection, animals were given

0.45 mg/kg of apomorphine (s.c.) and tested according to procedures as stated in the general methods section. The values and vertical bars represent the mean number of observations and standard error of the means respectively. 103

E

Time (min)

Figure 5-8. Time-course pattern for biting after estradiol benzoate treatment. Panel E. Animals were given either peanut oil or 25 ng/kg of EB for 3 days (panel E), 7 days (panel F) or 20 days (panel G). An additional group was given 100 ^ig/kg of EB for three days (shown in panel E). Twenty-four hours after the last

treatment injection, animals were given 0.45 mg/kg of apomorphine (s.c.) and tested according to procedures as stated in the general methods section. The values and vertical bars represent the mean number of observations and standard error of the means respectively. 1

104

F 25

' > 1 1 1 1 1 1 1 Ot 1 1 1 1 1 — 1 0 8 16 24 32 40 48 56 64

Time (min) Figure 5-9. Time-course pattern for biting after estradiol benzoate treatment. Panel F Animals were given either peanut oil or 25 |ig/kg of EB for 3 days (panel E), 7 days (panel F) or 20 days (panel G). An additional group was given 100 |ig/kg

of EB for three days (shown in panel E). Twenty-four hours after the last

treatment injection, animals were given 0.45 mg/kg of apomorphine (s.c.) and tested according to procedures as stated in the general methods section. The values and vertical bars represent the mean number of observations and standard error of the means respectively. 105

G

25-1 u o

Time (min)

Figure 5-10. Time-course pattern for biting after estradiol benzoate treatment. Panel G Animals were given either peanut oil or 25 ^g/kg of EB for 3 days (panel E), 7 days (panel F) or 20 days (panel G). An additional group was given 100 ^g/kg

of EB for three days (shown in panel E). Twenty-four hours after the last

treatment injection, animals were given 0.45 mg/kg of apomorphine (s.c.) and tested according to procedures as stated in the general methods section. The values and vertical bars represent the mean number of observations and standard error of the means respectively. 106

50-1

40 -

30 Oil EB25ug/kg EB 100 ug/kg E 20- T

10

3 day 7 day 20 day Treatment duration

Figure 5-11. Rearing behavior totals after treatment with 1 7-P E2. Animals were given either peanut oil or 25 ^g/kg of EB for 3 days, 7 days or 20 days. An additional group was given 100 ^ig/kg of EB for three days. Twenty-four hours after the last treatment injection, animals were given

0.45 mg/kg of apomorphine (s.c.) and tested according to procedures as stated in the general methods section. The values and vertical bars represent the mean number of observations and standard error of the means respectively. 107

O > (0 300

200- (0

X Oil to X EB25ug/kg c o X EB100ug/kg >(9 100

(0

c 2o 0 20 day c 3 day 7 day Treatment durations C

Figure 5-12. Sniffing behavior totals after treatment with estradiol benzoate. Animals were given either peanut oil or 25 [igfkg of EB for 3 days, 7 days or 20 days. An additional group was given 100 \ig/kg of EB for three days. Twenty-four hours after the last treatment injection, animals were given 0.45 mg/kg of apomorphine (s.c.) and tested according to procedures as stated in the general methods section. The values and vertical bars represent the mean number of observations and standard error of the means respectively. 108

50 n

40-

30- oil

EB25ug/kg

T 20- EB 100 ug/kg

10

3 day 7 day 20 day

Treatment durations

Figure 5-13. Response totals for line crossings after treatment with estradiol benzoate. Animals were given either peanut oil or 25 ^ig/kg of EB for 3 days, 7 days or 20 days. An additional group was given 100 ug/kg of EB for three days. Twenty-four hours after the last treatment injection, animals were given 0.45 mg/kg of apomorphine (s.c.) and tested according to procedures as stated in the general methods section. The values and vertical bars represent the mean number of observations and standard error of the means respectively. CHAPTER 6 EFFECTS OF ESTRADIOL IMPLANTS ON BEHAVIORS

Introduction

In experiment 3, various estrogen treatments were shown to differentially affect

individual stereotyped behaviors. Because of the close association between the striatum

and apomorphine-induced behaviors, E2 may influence apomorphine-induced behaviors

by acting directly on striatal tissue. Indeed, estrogen has been shown to influence the

activity of striatal and nigral neurons. Amauld et al. (1981), reported than the

intramuscular administration of EB significantly increased the number of spontaneously

active striatal neurons and their firing rate was also increased. Furthermore, striatal cell

activity was increased 2 - 5 days after subcutaneous E2 implants (Tansey et al., 1983).

Finally, E2 treatment has been reported to cause changes in striatal DA receptor

concentrations (Di Paolo et al, 1981b; Gordon & Diamond, 1981; Crowley et al., 1982;

Hruskaetal., 1982).

Although these studies imply that systemic estrogen treatment can directly

influence nigral and striatal neural activity, there is some evidence to suggest otherwise.

First, estrogen fails to display in vitro binding to striatal DA receptors, suggesting that

estrogen does not directly interact with DA receptors (Di Paolo et al., 1982; Hruska et al.,

1982). Second, estrogen receptors have not been identified in the striatum or in cell

bodies of DA fibers terminating in the striatum of adult animals arguing against the traditional estrogen mechanism (Stumpf and Sar, 1976; McEwen, 1982). Consequently, there is still the possibility that estrogen effects on DA agonist behaviors results from

109 110

action outside of the striatum, and it remains unresolved as to whether estrogens can

influence DA agonist-induced behaviors by acting directly on neural tissue.

One problem with systemic estrogen treatment is that the drug metabolism is

often affected, which could possibly alter the behavioral response to drugs (Kaul and

Conway, 1971; Masry and Mannering, 1974). Therefore, although systemic estrogen

treatment has been shown to influence DA-related behaviors and neurochemical events,

little information is available to determine whether those effects of estrogen are due to

direct or indirect actions of estradiol in the striatum. On the other hand, the direct

application of estradiol to striatal tissue would eliminate many of the alternative

explanations of how estrogen influences DA-related events. The use of intracerebral

estrogen implants has been used very successfully to elucidate the anatomical substrate

for estrogen's involvement in reproductive behaviors (McEwen, 1982). Moreover,

estrogen implants in the striatimi have been shown to influence some spontaneous and drug-related behaviors. In particular, unilateral implants of 1 7-P E2 placed in the dorsal

striatum caused ipsolateral postural deviation, after a systemic injection of apomorphine

(Joyce & Van Hartesveldt, 1984)

In the present study, two issues are addressed.

1 . Can estrogen affect apomorphine-induced behaviors by acting direcfly on neural tissues?

2. Are the effects of centrally administered estrogen on apomorphine- induced behaviors regionally specific?

In order to determine whether estrogen can affect apomorphine-induced behaviors by acting directly on neural tissues, the effects of 48 hour bilateral intracerebral implants containing either 17P-E2 or the biologically inactive stereoisomer 17a-E2 crystals on

apomorphine-induced behaviors. Also because there is evidence that the Ill

neuroanatomical substrate for individual dorsal-anterior striatum-related behaviors may be regionally specific, estradiol implants were placed in different regions of the basal

ganglia to determine if there is a differential sensitivity to the presence of estradiol

implants.

Methods

Subjects.

Adult, 3 month old female Long-Evans rats were housed in individual cages 2 weeks before the beginning of the experiment. One week before intracerebral

cannulation, animals were bilaterally ovariectomized.

Cannulae Implantation

Animals were implanted bilaterally with 21 ga stainless steel guide cannulae under pentobarbital anesthesia. A 27 ga stainless steel stylet was inserted into each cannula to prevent prolonged exposure to air. The coordinates for intracerebral implantation were taken from Pellegrino and Cushman (1978). Anterior-posterior coordinates were taken from bregma and vertical measurements from the dural surface of the brain. All guide cannulae were implanted at a depth of 2.0 mm below the surface of the brain, so as to prevent damage at the site if hormone implantation. Hormone implants were directed at various brain nuclei having the following coordinates;

1. Dorsal-anterior caudate: anterior, 2.6; lateral, ± 3.0; ventral, -4.0. 2. Ventral caudate: anterior, 2.6; lateral, ± 3.5; ventral, -7.0. 3. Nucleus accumbens: anterior, 3.2, lateral, ± 2.0; ventral, -7.5.

4. GP: anterior, 1 .2, lateral, ± 2.8; ventral, -6.4.

A 27 ga stainless steel stylet was inserted into each cannula to prevent prolonged exposure to air. After bilateral cannula implantation the animals were given a one week recovery period before behavioral testing. 112

Hormone Implant Preparation

Hormone implants were made from 27 ga stainless steel tubing of differing

lengths depending on the site of implantation. The tubing was cleaned of metal filaments

by boiling in distilled water. One end of the implant was closed preventing any air

passage. The implants were loaded with crystalline 1 7-a or 1 7-P E2 (Sigma) by placing

the hormone on wax paper and tapping the open end of the implant tubing 20 times into

the hormone powder. The tubing was then examined under a dissecting microscope and

the outer portion of the tubing cleaned of any extra hormone by clean surgical gauze. At

the time of hormone implantation, the 27 ga stylet was removed and the 27 ga implant

placed into the carmulae. After behavioral testing, the implant cannulae were removed

and examined for the presence of hormone.

Testing Procedure

Animals were behaviorally tested according to methods described in the General

Methods.

Histology

At the end of the experiment the animals were sacrificed by overdose of sodium pentobarbital anesthesia. The animals were perfused with 0.9% saline followed by a buffered 10% formalin solution. The brains were removed and placed in formalin solution. Twenty-four to 48 h before frozen sectioning, the brains were placed into a

20% sucrose-formalin solution. The brains were mounted and stained with cresyl violet.

The localization of the cannula tip placement was determined according to the rat brain atlas of Pellegrino et al., (Pellegrino and Cushman, 1979). 113

Statistical Analysis

The data for each stereotyped behavior were analyzed by a one-way analysis of

variance with treatment as the between subjects factor, and repeated measures on time.

Behavioral latencies were analyzed with one-way ANOVA. Animals were excluded

from statistical analysis if cannulae placements was not in one of the four intended

locations.

Results

The effects of 1 7-P E2 implants on response totals for individual behaviors have

been placed in Table 6-.

The hormone implant placements are shown in Figure 6-1. Initially, the cannulae

were directed toward 4 regions in the basal ganglia, including the anterior-dorsal portion

of the caudate-putamen (CPU). After histological examination of the implant

placements, the placements in the AD were found to be distributed into two distinct

regions; one group of implants was placed on the extreme dorsal region of the anterior portion of the CPU; another group was placed in a more lateral region of the dorsal CPU.

Because the placements were located in two distinct regions of the dorsal portion of the

CPU, they were grouped accordingly and statistically analyzed as separate placement groups.

Biting Behavior

There was a significant main effect of implant position on biting totals, F(4,70) =

8.99, p < 0.0005. The dorsal-anterior striatal implant group displayed less biting than groups with implants in the DA, VS, N. Acc and GP (p < 0.05). 114

There was a main effect of implant treatment on biting behavior totals, F(l ,70) =

6.10, p < 0.005. The 17 beta estradiol implant group displayed less biting than the 17

alpha estradiol implant group (N-K, p < 0.05).

Although the 1 7-p E2 implant group displayed less biting than the 1 7-a E2 implant

group (P < 0.05). There were no implant positions by treatment interactions.

Consequently, 1 7-P Ej did not a have a specific position effect on biting.

Sniffing Behavior

There was a significant implant position by treatment interaction, F(l,8) = 5.77, p

< 0.05. Sniffing behavior was significantly increased by the 17-P E2 implants in the AD

compared to the 17-a E2 implants (p < 0.05). 1 7-P E2 implants at other locations failed to

influence sniffing behavior.

Continuous Sniffing

There was no significant effect of 1 7-p E2 implants on continuous sniffing at any

brain implant location. However, there was significant treatment x time interactions after

17-p E2 implantations in the AD, CPU, N. Acc and GP.

There was a significant interaction between treatment and time after implantation

in the AD CPU, F(13,104) = 2.44, p<0.01. The 17-P E2 treatment group displayed more

CS than the 17-a E2 treatment group during the 28 minute and 32 minute time intervals

(P < 0.05).

There was a significant treatment x time interaction for continuous sniffing after implantation = for the N. Acc, F(13,91) 2.24, p < 0.05). The 17-P E2 treatment group displayed more continuous sniffing than the 17-a E2 treatment group during the 48 minute time intervals (N-K, p < 0.05). 115

Intracerebral implants in the GP produced a significant interaction between treatment and time, F(13,91) = 1.93, p < 0.05. The 17-P E2 treatment group displayed

more continuous sniffing than the 1 7-a E2 treatment group during the 8 minute through

12 minute time interval and 48 minute time interval (P < 0.05).

Line Crossings

Line crossings were significantly increased by 17-P E2 implants in the AD, F(l,8)

= 12.55, p < 0.01 . GP implants produced a significant treatment x time interaction,

= F(13,91) 2.60, p < 0.01 . The 1 7-p E2 treatment group displayed more line crossings

during the 44 minute time interval (P < 0.05).

Discussion

In our study, the central effects of estrogen on different behaviors were

investigated by placing 17p- E2 implants in specific regions of the CPU.

The diiration of tissue exposure to intracerebral implants is important because the

amount of tissue exposed is determined in part by the duration of time the implant is

present. The potential problem with using longer implant duration is that uncontrollable spread of the hormone may occur. For instance, although Yanase & Gorski (1976) reported that 17P-E2 implants in the striatum increase reproductive behavior 3 days after implantation, the females also displayed vaginal comification, indicating that hormone leakage into the general circulation may have occurred. Also, 27 ga implants in the hypothalamus or pituitary for 4 or 5 days produced radioactive estrogen diffusion as far as 2 mm away from the tip of cannula (Palka et al., 1966). There was also radioactivity in the uterus and plasma, indicating significant contamination. However, a 28 ga implant placed in the VMH for 2 - 3 days resulted in a diffusion of diluted E2 (0.3%) in an area of about 0.5 mm (Davis et al., 1982). Similarly 30 ga implants have been reported to 116

produce no more than 0.50 mm diffusion of estrogen after a much as 5 - 8 days. In these

two studies diluted estradiol was used, which might explain the low diffusion. Becker et

al., (1987) reported that 6 hours after bilateral implants of 30% 17P-E2 mixed with

tritiated 1 7P-E2 were positioned into the dorsal striatum, radioactivity was only found

less than 1 .5 cm away from implants.

Yet, in our study. Forty-eight hours after implantation of 17a-E2 or 17P-E2 in

various parts of the CPU, E2 implants in adjacent areas produced different effects

suggesting that the local effects of the implants were circumscribed. Implantation of 17P-

E2 into various portions of the CPU only influenced sniffing and line crossings, and to a

lesser degree biting. The AD and VS as well as GP were affected in different ways by

17P-E2 implants. However, clearly the AD portion of the striatum was affected most by this treatment since both sniffing and biting were influenced after 17P-E2 implants were placed in this region.

There was also some indication that line crossings are influenced by more than one region given that 17-p E2 implants in both the AD and VS, but not the N. Acc affected the number of line crossings. Given that the N. Acc is intimately associated with locomotion (Jackson et al., 1975; Costall et al., 1977a), the absence of an effect of the E2 implant in the N. Acc was surprising. However, the behavioral response to apomorphine is likely the result of actions at several sites in the brain, possibly even different sites in the striatum. Consequently, the effects of intracerebral hormone implants will not have the same magnitude of effects on behaviors that systemic treatments produce. For instance, although both E2 implants and systemic estrogen administration antagonized 117

apomorphine-induced postural deviation, it is unclear whether the two methods produced

comparable results (Joyce et al., 1984; Joyce and Van Hartesveldt, 1984a).

Indeed, the restoration of estrous behavior in ovariectomized females implanted with 17P-E2 does not result in the quality of behaviors displayed after systemic administration of hormone to ovariectomized females or in the intact female, yet fundamental aspects of the behavior were there (Barfield and Chen, 1977). In our study, the effects of E2 implants were modest compared to the effects of systemically administered E2. The results did not indicate if the placement of 1 7 p E2 implants or the treatment duration produced the optimal effects on apomorphine-induced behavioral responses.

However, our study achieved its objective. The results indicated that direct application of 17-P E2 to different regions of the CPU produced specific changes in the behavioral response to apomorphine. The effects of 17-P E2 implantation in the CPU were both behavior and region specific. 1 7 1

118

Table 6- 1 . Response Totals for behaviors after i ntracerebral implants. E2 17-a E2 17-B AD NJ A r*r* DL V 0 IN AVCL JUL t\\J V L> N Acc Or

1 1 1 1 'n Bt. I / J .*T 145 1 1 QK 0 0 n 70 Q OC T on lo/.o y\.l 185.3 It, 1.1 154.8 no 9'> r? 1^ /"1 7 ')^ (n./) (1 1.1) (22.7) (7.5) (19.8) C.B. 1 f,(\ n 1 IQ ^ 1110 U 1 .D lOVJ.U 1 1 j.y lUz.o4 144.6 135.6 86.0 ri6 (\1 \\ /"1 1 ^\ /^ A A\ yy^.y) (1J.6) (8.6) (22.1) (15.2) (24.4) 7Q f\ 8"? 1 HO Z" D.B. OJ. I jZ.j 00.0 65.6 68.6 42.0 52.1 69.2 (9. '^^ ^"1 5 1~\ {y.y) (lb.8) (12.9) (10.7) (10.9) (13.8 (13.1) 141 4 1 1 Q 1 m 1 Sn. 111.0 1 tl .H y 1 .J 112. '191.4 122.9 105.5 135.2 rii n 7 (Q 8^ ^"1 4 Q^ /")n 7 V* 1 -J) yv i.A) (12.7) (zU. / (zO.U) (8.6) (22.0) 105 7 1 T C.S. yJ .A. 01. /y.z 86.9 * 152.3 108.3 98.1 119.2 <^1 ^"1 \v 1 8 4^ 7 fi"\ /"I ^ n^ (13.0) (20.9) (20.5) (7.4) (19.9) D.S. 1 8 S 10.*^ zU.o zU.O 25.0 39.0 15.1 17.9 15.0 ^4 ('5 4^ p.i) (4.2) (3.0) (2.5) (3.8) (4.5) 11 1 14"? 9 117 1 1 no T Amb 1 1 /. 1 86.4 171.0 129.9 106.5 88.2 C9 9^ n? 7 r'1 7 7^ /'1 7 8^ (13.8) (10.1) (16.4) (20.3) (15.3) (19.9) L.C. 13.9 28.8 18.0 25.6 15.7 14.5 38.0 23 9 7^^ 7 1 1 (1.8) (4.2) (4.3) (6.3) (3.3) (2.3) (5.3) (4.5) (3.4) (3.4) Rr. 7.4 15.8 6.4 12.7 9.4 8.0 21.4 6.9 10.9 4.9 (1.2) (3.0) (1.3) (3.2) (2.1) (1.6) (3.5) (1.5) (2.2) (1.4) Gr. 6.1 5.6 3.6 1.6 9.4 11.7 6.4 2.3 5.6 5.0 (2.0) (2.0) (1.9) (0.7) (3.7) (2.2) (2.1) (0.9) (2.2) (2.3) B.I. 5.4 11.4 11.4 16.1 12.0 11.3 7.3 8.0 10.0 11.5 (3.1) (6.5) (6.2) (7.0) (6.3) (4.1) (4.4) (6.6) (4.9) (6.4)

Either 17-a E2 orl7-P E2 implants were placed in one of five sites in the CPU 48 hours before behavioral testing. The values represent the mean number of observations of each behavior. "*" The numbers in parentheses represent the standard error of the mean. The symbol signifies that the estrogen treatment group was significantly different from the respective oil control group. See text for more details. 119

Figure 6-1 Location of cannula tip for intracerebral estradiol implantation. The shaded areas indicate the group placement. Gray shaded area represents implant location in the dorsal anterior striatum (AD), yellow represents implants in the lateral portion of the dorsal-anterior striatum, green represents implant location in the ventral striatum (VS), blue represents the nucleus accumbens (N. Acc) and red represents the globus pallidus implant placements (GP). CHAPTER 7 GENERAL DISCUSSION

In the present study, the effects of estrogen on apomorphine-induced behavioral

responses were examined. The ultimate goal of our studies is to arrive at a unified,

comprehensive view of behavioral change in response to changes in condition, such as

dose or hormonal state. The method by which we arrive at this comprehensive view

determined, in part, what the ultimate conclusions were.

In experiment 1, we saw that increments in apomorphine dose produced

considerable changes in behaviors. We approached the measurement of change in these

behaviors by measuring the responses separately in contrast to other more global methods

of measurement. The measurements of changes in individual behaviors displayed in

response to apomorphine doses led to different conclusions than those obtain by more global methods. For example, measurements of individual behaviors indicated that most behaviors were expressed, at some level, across the fiill dose range and did not "drop out" at higher doses as indicated by rating scale measures (Costall and Naylor, 1973b; Carr and White, 1987). Another example is the conclusion, arrived at by rating scale measures, that the dominant behavior provides the best discrimination between doses.

However, our results indicated that any one of several individual behaviors could be used separately or in conjunction to distinguish between dose levels. Moreover, our approach provided a greater level of behavioral sensitivity between dose levels than is found with other more global measures.

120 121

Part of the increased behavioral sensitivity indicated by our results is due to the

behavioral definitions employed in our studies. Proper description of the behavioral

responses in objective, simple and clear terms provides a reliable basis for obtaining a

reproducible measurement outcome. For instance, Table 7.2 shows how behavioral

definitions across studies can show a reliable relationship between the response

description and the behavioral result. However, there were other methods considerations

in our studies, such as the frequency of behavior sampling and duration of the test session

all designed to increase information about each behavioral response and increase

sensitivity to behavioral change.

Using our approach, we found that ovariectomy does result in an increase in

apomorphine-induced biting, consistent with previous reports (Gordon, 1980; Gordon et

al., 1980b; Hruska et al., 1982a). However, we also found that the effects of long-term

ovariectomy are probably not related to direct effects of estrogen deprivation on striatal

activity. We observed that the increase in biting after long-term ovariectomy did not

follow the pattern of increased DA-ergic stimulation displayed in experiment 1 . Rather

the greater apomorphine-induced biting response were suggestive of systemic factors

such as weight and body fat gain associated with long-term ovariectomy (Marks, 1972;

Wade and Gray, 1979; Ahdieh and Wade, 1982).

In experiment 3, our comprehensive approach revealed that by including separate measures of the presence of qualitatively distinct forms of the behavior, subtle changes in behavior were detected that might have gone undetected otherwise. Separate measures of continuous biting and discontinuous biting revealed that acute E2 treatment actually affected biting even though the total biting score indicated other wise. Total biting was 122

unaffected by the 3 hour E2 treatment and not affected until 6 hours after treatment, but

continuous biting was decreased. The decrease in continuous biting 3 hours after E2

treatment was obscured by a corresponding increase in discontinuous biting. Although

the significance of acute estrogen effects causing a shift in the form of biting rather than

an out right decrease in all forms of biting is unclear at this time. But this type of

discriminative detailed information might prove helpful to subsequent studies examining

other aspects of estrogen effects on apomorphine-induced behaviors.

In conclusion, the approach to measuring apomorphine-induced behaviors

proposed by our studies suggest that to ultimately obtain a unified view of behavioral

expression and the influences on those behaviors, the specific changes in individual

behavioral responses must be considered first. Using this approach, the results suggest

that endogenous estrogen has differential influences on specific apomorphine-induced

behavioral responses. Endogenous estrogen can influence apomorphine-induced

behaviors by acting directly on striatal tissue. The effects of estrogen on striatal tissue

are regionally specific and correspondingly have differential effects on individual

behavioral responses. The exact mechanism by which estrogen acts on striatal tissue is

still unclear. Evidence has pointed to membrane receptors, in part, because of the rapid

time interval in which estrogen treatments influenced some apomorphine-induced

behaviors and the absence of traditional estrogen receptors in striatal tissue.

It is possible that long-term or chronic estrogen treatment effects may be mediated

by prolactin or catechol estrogens. However, the speed in which estrogen treatments can

influence apomorphine-induced behaviors precludes any likely involvement of prolactin or catechol estrogens as mediators of acute effects of estrogen (Hruska and Pitman, 1982; 123

Hruska et al., 1982b; Clopton and Gordon, 1985; Hruska, 1987). It remains for future studies to determine whether there are estrogen receptors located in striatal tissue and the extent to which it is involved in the hormonal influences on striatal activity including related behaviors. )

124

Table 7.2 Comparison of behavior classifications among different studies. Study Behavior Definition Results

Antoniou & Kafetzopoulos (1991) Amb. like like Frayetal., (1980) Amb. like like Molloy & Waddington (1987) Amb. like like

Frayetal., (1980) Bt. like like Havemann et al., (1986) Bt. like like Ljungberg & Ungerstedt, (1977a) Bt. diff like Ljungberg & Ungerstedt, (1977b) Bt. diff like Molleretal., (1987) Bt. like like Rastogi et al., (1982) Bt. diff like Redgrave etal., (1982) Bt. like like

Lewis etal., (1985) CB. like like Antoniou & Kafetzopoulos (1991) Gr. like diff Molloy & Waddington (1987) Gr. like like Szechtman et al., (1982) Gr. like like Antoniou & Kafetzopoulos (1991) LA(PI) diff diff Bemardi & Palermo-Neto (1983) LAfLC) like like Hitzmann & Curell (1982) LA(PI) diff like Ljungberg & Ungerstedt (1977b) LA(PI) diff like Ljungberg & Ungerstedt, (1977a) LA(PI) diff like Maj etal, (1972) LA(PI) diff diff Montanaro et al., (1983) LAfact diff diff Stable & Ungerstedt (1986) LA(PI) diff like Antoniou & Kafetzopoulos (1991) Rr. like like Bemardi & Palermo-Neto (1984) Rr. like like Frayetal., (1980) Rr. like like Hitzmann & Curell (1982) Rr. diff like Molloy & Waddington (1987) Rr. like like

Antoniou & Kafetzopoulos (1991) Sn. like like Chow & Beck (1984) Sn. like diff Frayetal., (1980) Sn. like like Havemann et al., (1986) Sn. like like Molleretal., (1987) Sn. like like Molloy & Waddington (1987) Sn. like like

Lewis etal., (1985) CS. like like Antoniou & Kafetzopoulos (1991) Sn-air like like

This table shows various aspects of different studies in comparison to our study. Including similarities or differences in the behavioral definition; type of behavior measured; data presentation and direction of behavioral change; and whether results agreed or disagreed. V

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BIOGRAPHICAL SKETCH

Ron received his bachelor's degree in psychology and masters degree in

physiological psychology from the University of Florida. He is currently employed at the

McKnight Brain Institute of the University of Florida. Ron is married to Barbara and has three kids, Jason, Leah and Rachel. I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fially adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

Rowland, Chair Professor of Psychology

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy/

Me Professor of Psychology

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

Marc BranciL Professor of Psychology

I certify that I have read this study and that m my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy .

a Peris ciate Professor of Pharmacodynamics

I certify that I have read this study and that in my opinion it conforms to acceptable

standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. I

Carol Van Hartesveldt Professor of Psychology

This dissertation was submitted to the Graduate Faculty of the Department of Psychology in the College of Liberal Arts and Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.

May, 2004 Dean, Graduate School