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ADOLESCENT ANABOLIC STEROID EXPOSURE: EFFECTS
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ADOLESCENT ANABOLIC STEROID EXPOSURE:
EFFECTS ON SOCIAL BEHAVIORS AND NEURAL PLASTICITY
By

Kaliris Y. Salas—Ramirez

A DISSERTATION

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

Department of Neuroscience

2007

ADOLESCENT ANABOLIC STEROID EXPOSURE:
EFFECTS ON SOCIAL BEHAVIORS AND NEURAL PLASTICITY

By
Kaliris Y. Salas-Ramirez
Anabolic-androgenic steroids (AAS) are synthetic derivatives of
testosterone used by over half a million adolescents in the US for their tissue-
building potency and performance-enhancing effects. Recent studies show AAS
use is increasing among teenagers. AAS have behavioral effects such as
heightened aggression and changes in sexual libido in humans. The maturation
of adult behavior during adolescence involves complex interactions among
hormones, experience, and the developing brain. Our research using Syrian
hamsters demonstrates that the absence of gonadal hormones during
adolescence irreversibly impairs male reproductive and social communication
behaviors in adulthood, indicating that adolescence is a particularly sensitive
period for steroid-dependent remodeling of neural circuits underlying complex
social behaviors. The overall goal of my work was to determine how AAS
interact with the adolescent brain to inﬂuence social behaviors during
adolescence and in adulthood. Results from my studies show that a cocktail of
AAS containing testosterone cypionate, nandrolone decanoate, and boldenone
undecylenate increase aggressive and reproductive behaviors in male Syrian
hamsters treated for two weeks and tested during adolescence. Nevertheless,
the same cocktail has different effects on adult males treated for the same length
of time. Both adolescent and adult males suffered from long term effects due to

AAS exposure, but only for particular aspects of sexual and agonistic behaviors.

Finally, adolescent AAS exposure affects amygdaloid neural plasticity. These
data indicate that the adolescent brain responds differently to exogenous
hormones than the adult brain, suggesting that the still developing adolescent

brain is vulnerable to perturbations in steroid milieu.

Dedication:

I dedicate this body of work to my parents, Dr. Salvador Salas-Quintana and Dr.
Doris Ramirez, for being my role models, inspiration and motivation to obtain the
highest degree I could in science. To my sister, Ing. Karilin Salas and her
children, Kayra Yimar and Rambn Esteban, who have been my pride and joy
since the day they were born. And ﬁnally, to my husband Dr. Ken Hoyte, for
always reminding me to challenge myself intellectually and that one day, we are

going to change the world. Thank you, for being my support network.

iv

Acknowledgements

It’s been 6.5 years since I arrived at MSU from Puerto Rico to complete a
PhD in Neuroscience in the College of Natural Sciences. There were days when
I thought this process would never end. There were days when I wanted to leave,
but the majority of the days l was grateful to have the opportunity to do what I
love day in and day out. I deﬁnitely could not do it without the help and support
of a lot of people.

F irstoand foremost, to my parents, Drs. Salvador Salas-Quintana and
Doris Ramirez-Soto, I thank you for being my lifetime mentors. For listening to
me every single time I called and asked for a plane ticket home, for not letting me
give up on all of my dreams (because I ALWAYS wanted to be like my Marni and
Papi and get a PhD) and for showing me that today we may have to sacriﬁce
some things so that later we can reap the benefits. Not one day in my life did I
ever feel like I went without, the complete opposite, I always feel lucky and
blessed because I get to be your daughter and get learn from you about life, love
and family. Thank you so much.

My sister, Ing. Karilin Salas, and her family Ramén, Kayra and Monchito,
you are my pride and joy. The love and laughter that you give me every time I
hear your voices and see you are heaven sent... I hope one day Kayra and
Monchito will admire and respect their Titi as much as I love and admire you,

Kary. You are such a hard worker, and have been through so much, but you

V

always prevail. Thank you, for always taking my opinion into account, even when
you think I am speaking nonsense, and for loving me no matter what!

Clearly, I could not have done a thing without my academic family...

I would like to thank my advisor and mentor, Dr. Cheryl Sisk, for all of her
guidance and dedication towards my education. I am a Neuroscientist today
because you pushed me to strive for excellence. You set the bar high. Through
your words and instruction l have learned that it’s never going to be easy, nothing
in science is done quickly, and that as long as quality is the priority, it’s a job well
done. You always challenged me to think above and beyond. I have learned a
lot from you, and grown so much, in the last 6.5 yrs, more than you will ever
know, and more that I could ever thank you for. Thank you for giving me the
liberty and ﬂexibility to do experiments I designed. I appreciate you for allowing
me to attend professional conferences that provided me with additional
opportunities for funding and promoting my research. I know that sometimes that
came at the expense of spending more time in the lab, but that was never an
excuse not to go. You deﬁnitely made sure that I moved forward, even when I
didn’t think I would make it; you supported me, believed in me and pushed me,
so there would never be any regrets. Thank you for being a role model and an
inspiration.

To my committee members:

Dr. Antonio Nunez. .. you will forever be my academic father. You wiped
tears off my face many times, and always encouraged me to keep going. Thank

you for always asking if everything was ok and for reassuring me that I had your

vi

complete trust and conﬁdence. Having you in my academic life made a
signiﬁcant difference in my training... always. for the better. Thank you so very
much.

Dr. David Kreulen... out of all the members of my committee you were the
farthest from behavioral neuroscience, but you challenged me to think further
about my data! I feel like you were one of the few people here at MSU that really
wanted to know me, not only for my science and ideas, but also for the person I
am. You made sure I always had the directions to move forward. I haven’t met a
lot of people here that wanted to genuinely understand where l was coming from,
but you always asked the right questions and made me feel at home (even
though I am so far from it!). That alone made it an honor to not only have you as
part of my committee, but as a mentor.

Dr. S. Marc Breedlove... thank you for showing me that you can be a
“regular person” with a large family, a large lab, and a crazy schedule, that can
also run a successful research program and stay on top of our science. You
have taught me that even when you were are the one that makes a discovery,
mistakes are made along the way, humor is important and humility is key to
success. If lever needed comfort during our meetings, I would just look at you
and you would say a joke or explain your interpretation of my experiments, and it
refocused me on the task at hand. Thank you for appreciating me for me, and
always giving me a pat on the back... that l was all I needed to get motivated and
not forget my purpose.

To the members of the Sisk Lab, old and new:

vii

Dr. Kalynn Schulz, you are my colleague, my mentor, my partner, my best
friend, my conﬁdant, my sister... thank you, for helping me design experiments,
for challenging my thinking and for always helping me put things in perspective. I
never imagined that our bond would surpass being lab mates... but I am thankful
it did. Most of all, thank you for having Alden, and letting me be a part of his life,
he always puts a smile on my face and that is priceless. God bless you both!

Dr. Eman Ahmed, you have always looked out for me. I can honestly say
you are an incredible mentor, I admire you and I respect you not only for your
science, but for the person you are. You always told me that I could do it, and no
matter what obstacles came my way, you were right by my side rooting me on!
You always reminded that family and work go hand in hand, and that as long as
we always believe in who we are, sky is the limit. Thank you for believing in me,
even when I didn’t believe in myself.

Dr. Julia Zehr and Dr. Heather Molenda-Figueira, thank you for always
providing me with valuable advice so that I could become a better student, a
better scientist and better prepared for a post doctoral position. I appreciate all
the time you spent reading my papers and providing me with professional advice.
Julia, thank you, for always reminding me that I should be prepared for anything
and everything in life and science. Heather, thank you for always being “real”
and showing me that you can to take care of yourself, and look good, while being

a professional woman in science.

viii

Margaret Bell, thank you for making my last 1.5 yrs so fun! I love
collaborating with you... You are going to be an incredible teacher and mentor
one day, please remember the little people when you become famous.

Jane Venier, Lisa Rogers and Pamela Montalto... the three best
laboratory technicians ever! I hope one day I can have professionals in my lab
that work as hard and are as focused as you are. We ran T assays, tested for
behavior, injected a lot of animals and performed a lot of perfusions... basically,
there’s no way I could have ﬁnished my dissertation work without you!!! I know
there were times you preferred to be doing something else that wasn’t cutting, or
mounting or injecting... but I thank you for your technical assistant and for always
coming though for me!

I have trained and mentored many undergraduates. Without their help the
nights that we had to run experiment, it would have been impossible. Sara
Hunter, Jason Thomas, Nada Zohdy, Liana Nichols, Anibal Rivera-Rivera, Janice
Chesney, Rachel Christensen, Adam Stowie, Meriem Benjelloum, Keaghan
Cronin and Josh Olsewisc, all played a signiﬁcant role in the completion of these
experiments.

The APA Fellowship Program that sent me to SPINES in Woods Hole for
a summer and provided me with lifelong mentors like, Dr. Joe Martinez, Jr and
Dr. Anne Etgen; along with lifelong friends: Dr. Dario Dieguez, Dr. Nelson Cruz-
Bermudes, Dr. Mayra Padilla, Dr. Annelyn Torres-Reverén, and Dr. Unja Hayes.
Since the summer of 2002 you have been in my life teaching me that no one’s

research experience is worth comparing to your own and that the PhD

ix

professional journey is unique to everyone; however, we all have the same goals,
which is to use our science and training to make a difference, it gives me great
honor and pleasure to have you in my life.

Ms. Della Sutton-Hoyte, my mother-in-law... Ms. Della, you are the
deﬁnition of a woman with heart and conviction. Thank you for blessing me with
your words of encouragement and unconditional love.

Last, but certainly one of the most signiﬁcant, my husband, Dr. Ken Hoyte.
The day we got married was one of the most incredible days of my life. You
complete my personal and professional goals. You have passion for what you
do, and for all the differences you want to make in academia and in some one’s
life. You, have a purpose in this world, and are not scared of fulﬁlling your
vocation. Although we later had to learn about the deﬁnition of a marriage, and
have had tough times, there’s no one in this Universe that I would ever want to
share my life with if it’s not you. You have seen me at my highest and my
lowest... and you have not feared any situation, even when you didn’t know what
to do. The sunshine you bring into my life, even when we were at our darkest
point, would shine through and helped me overcome so many obstacles. Thank
you for being my biggest fan, my number one supporter, for making sure I had at
least 3 meals a day, and for putting up with all the late night phone calls. We
have a healthy competition, that demonstrates that the only people we want to
become better for is each other... and you are the only person that I want to
share my accomplishments with. Thank you for being my hero, a role model and

my partner in changing the world! 100 more yrsll Go TEAM HOYTE!!

TABLE OF CONTENTS

LIST OF TABLES ......................................................................... xiii
LIST OF FIGURES ....................................................................... xiv
KEY TO SYMBOLS AND ABBREVIATIONS ...................................... xviii

CHAPTER 1: ANABOLIC-ANDROGENIC STEROID EXPOSURE: POTENTIAL
EFFECTS ON THE ADOLESCENT BRAIN AND SOCIAL BEHAVIORS 1

AAS and Adolescent Development .......................................... 4
Pharmacology of Anabolic-Androgenic Steroids ......................... 10
Characterization of Social Behaviors in the Male Syrian Hamster ..... 11
The Amygdala: Social Behaviors and Neural Plasticity ................. 12
AAS and the Amygdala .......................................................... 18
Preliminary Experiments ......................................................... 21

CHAPTER 2: ANABOLIC-ANDROGENIC STEROID EXPOSURE
DIFFERENTIALLY AFFECT SOCIAL BEHAVIORS IN ADOLESCENT AND

ADULT MALE SYRIAN HAMSTERS ................................................. 27
Introduction ......................................................................... 27
Material and Methods ............................................................ 30
Results ............................................................................... 37
Discussion ........................................................................... 41
Figures ................................................................................ 48

CHAPTER 3: ADOLESCENT ANABOLIC-ANDROGENIC STEROID EXPOSURE
ENHANCES AGGRESSIVE BEHAVIORS REDUCES THE POTENTIAL TO
REACH SEXUAL SATIETY AFTER DISCONTINUATION OF TREATMENT.. 55

Introduction ............................................................................ 55
Material and Methods ............................................................... 58
Results .................................................................................. 65
Discussion ............................................................................. 69

Figures ............................................................................... 76

CHAPTER 4: ADOLESCENT ANABOLIC-ANDROGENIC STEROID AFFECTS
MEDIAL AMYGDALA VOLUME AND CELL ADDITION IN THE MALE SYRIAN

HAMSTER ..................................................................................... 83
Introduction ........................................................................... 83
Material and Methods .............................................................. 87
Results ................................................................................. 92

xi

Discussion ........................................................................... 94
Figures ............................................................................... 98

CHAPTER 5: IMPLICATIONS AND CONTRIBUTIONS TO UNDERSTANDING
THE DEVELOPMENT OF SOCIAL BEHAVIORS AND THE EFFECTS OF
ANABOLIC-ANDROGENIC STEROIDS ON THE ADOLESCENT BRAIN 106

Chapter 2 Summary ............................................................... 108
Chapter 3 Summary ............................................................... 109
Chapter 4 Summary .............................................................. 112

Normal Development of Social Behaviors in Male Syrian Hamsters... 1 14
Implications and Contributions: Understanding Human Anabolic-
Androgenic Steroid Use ....................................................................... 117

BIBLIOGRAPHY ............................................................................ 121

xii

LIST OF TABLES

Table 2.1 AAS- Treated adolescent males were less likely to display submissive
behaviors than vehicle controls and adult males ...................................... 52

Table 2.2 Peripheral measures in vehicle- and AAS-treated adolescent and adult
males .............................................................................................. 54

Table 3.1 Peripheral measures in males exposed to AAS during adolescence or
adulthood ........................................................................................ 82

Table 5.1 Short-tenn vs. Long-term Effects: Summary of the Effects of AAS on
Social Behaviors Compared to Adult-Typical Social Behaviors ................... 111

xiii

LIST OF FIGURES

Figure 1.1 Effects of 17d-methyltestosterone on attack latency and aggression in
adolescent male Syrian hamsters ......................................................... 22

Figure 1.2 Attack number, bites and attack duration were signiﬁcantly increased
with an AAS cocktail containing TC, ND, and BU (B) compared to a cocktail
containing TC, ND and 17a-MT and vehicle- controls ............................... 26

Figure 2.1 AAS affect male sexual behaviors differently in adolescent and adult
hamsters. Two-way ANOVA revealed age by treatment interactions in which,
relative to age-matched vehicle-treated males [adolescent (n=8); adults (n=9)],
AAS-treated adolescents (n=7) displayed more (A) mounts [Age = p < n.s., TMT
= p < 0.05, Age* TMT = p < 0.001], (B) intromissions [Age, TMT = p < n.s., Age *
TMT = p < 0.01]; and (C) ejaculations [Age, TMT = p < n.s., Age * TMT = p <
0.001], while these behaviors were decreased in AAS-treated adults (n=7).
Asterisk indicates signiﬁcant difference (p<0.05) between AAS- and vehicle
treatment within an age as determined by post hoc analysis. [Vehicle-treated
(white bars); AAS-treated gray bars).] ..................................................... 48

Figure 2.2 AAS increase aggression in adolescents but not in adults. Two-way
ANOVA revealed age by treatment [adolescent vehicle-treated (n=13), AAS-
treated (n=14); adult vehicle-treated (n=11), AAS-treated adults (n=13)]
interactions on a global aggression score [(ﬂank marks + attacks + bites) - (tail-
up walking)] [Agez p-value < 0.001; TMT: p—value < 0.0001; Age*TMT: p-value <
0.001]. Asterisk indicates signiﬁcant difference (p<0.05) between AAS- and
vehicle treatment within an age as determined by post hoc analysis ............... 49

Figure 2.3 AAS affect male aggressive behaviors differently in adolescent and
adult hamsters. Two-way ANOVA revealed age by treatment interactions on (A)
attacks [Age = p < 0.001; TMT = p < 0.001; Age*TMT = p < 0.001], (B) bites

[T MT = p < 0.001; Age*TMT = p < 0.05] and (C) attack durations (min) [T MT = p
< 0.01; Age*TMT = p < 0.01]. Asterisk indicates signiﬁcant difference (p<0.05)
between MS and vehicle treatment within an age as determined by post hoc
analysis. .......................................................................................... 50

Figure 2.4 AAS increase ﬂank marking in both adolescent and adult males. Two-
way ANOVA revealed a signiﬁcant effect of treatment (p< 0.01). Asterisk
indicates signiﬁcant difference (p<0.05) between AAS- and vehicle treatment
within an age as determined by post hoc analysis .................................... 51

Figure 2.5 Adults engage in more offensive posturing than adolescents during a

male-male social interaction. Two-way ANOVA revealed a signiﬁcant main effect
of age (p<0.05) ................................................................................. 51

xiv

Figure 2.6 Plasma luteinizing hormone (LH) and testosterone (T) concentrations
immediately after each behavior test. (A)In males tested with a receptive female
for sexual behaviors (A), there were no effects of age or treatment on LH (1A)
concentrations. AAS decreased T concentrations (2A) in both AAS-treated
adolescent and adult males (main effect of treatment, p < 0.05). (B) In males
tested with another male for aggressive behaviors, there were no effects of age
or treatment on either LH (2A) or T (2B) concentrations. Asterisk indicates
signiﬁcant difference (p<0.05) between AAS- and vehicle treatment within an age
as determined by post hoc analysis ...................................................... 53

Figure 3.1 Adult sexual behavior remains consistent from P 56 to P106. A one-
way ANOVA revealed no signiﬁcant differences in the number of mounts and
intromissions displayed by vehicle-treated males tested on P56, P70, P72 and
P106 ............................................................................................. 76

Figure 3.2 AAS exposure reduces the potential to reach satiety independently of
age of AAS exposure and time of post-treatment behavioral test. A 3-way
ANOVA was used to determine that AAS exposure during adolescence and
adulthood signiﬁcantly (p < 0.05) (A) decreases the number of long intromissions
and (b) increases long intromission latency ............................................. 77

Figure 3.3 Developmental changes were found in the expression of agonistic
behaviors. A one-way ANOVA revealed that the older the vehicle-treated males
were when tested, the fewer (A) attacks (*, p < .002) and the more (B) tail-up
walking (*, p < 0.005) they displayed ...................................................... 78

Figure 3.4 A main effect of treatment was found on (A) attacks and (B) bites (p <
0.05). A three-way ANOVA also revealed a main effect of age of exposure on
the expression of both behaviors (p < 0.0001). No signiﬁcant interactions were
found. The insets are ANOVAs comparing age of exposure and treatment. A 2-
way ANOVA comparing age of exposure and treatment determined that
adolescent AAS exposure signiﬁcantly increased (A) attacks (*, p < 0.05) and (B)
bites (*, p < 0.05) .............................................................................. 79

Figure 3.5 AAS exposure during adolescence signiﬁcantly increased ﬂank
marking behaviors and overall aggression. (A) A 3-way ANOVA showed that
ﬂank marking behavior was increased after AAS exposure during adolescence
and adulthood (p < 0.02). A main effect of age of AAS-exposure was observed
(p < 0.002). The inset is a 2-way ANOVA revealed that adolescent AAS
exposure signiﬁcantly increases ﬂank marking behavior when compared to
vehicle- and adult-treated males (*, p < 0.05). (B) A 3-way ANOVA showed that
AAS treatment increases aggression in males treated during adolescence and
adulthood (p < 0.02). A main effect of age was also observed (p < 0.001). The
inset is a 2-way ANOVA determined males exposed to AAS treatment during

XV

adolescence had signiﬁcantly higher composite aggression scores than vehicle-
and adult-treated adult (*, p < 0.005) ...................................................... 80

Figure 3.6 Males exposed to AAS during adolescence did not express tail-up
walking, escape dashes or stretch-attend. A three-way ANOVA determined a
main effect of age of exposure for (A) number of tail-up walking (p < 0.001), (b)
escape dashes (p < 0.02) and (c) stretch attend (p < 0.0005) ..................... 81

Figure 4.1 Photomicrographs of Nissl-stained tissue at 4X of the amygdaloid
nuclei. Neurolucida tracings outline (A) central amygdala (CeA), anterodorsal
medial amygdala (MeAD), anteroventral medial amygdala (MeAV); (B) CeA,
posterodorsal medial amygdala (MePD), posteroventral medial amygdala
(MePV)] .......................................................................................... 98

Figure 4.2 Photomicrographs at 40X of BrdU-labeled cells (arrows) in the
amygdala (scale bar = 500 pm). In general, a signiﬁcantly higher number of
BrdU-labeled cells was identiﬁed in males perfused 4-hrs after the last BrdU
injection (A) than in males perfused 4 wk after the last BrdU injection (B).
Approximately one third of the cells born during adolescence survive to
adulthood ....................................................................................... 99

Figure 4. 3 AAS exposure during adolescence does not affect cell proliferation or
cell survival In the CeA. (A) A 2-way ANOVA revealed a main effect of age on
the number of BrdU- labeled celzls/mm2 (*p < 0. 0001). No effects of treatment
were found In BrdU- ir cells/mm2 or volume (mm3 ) (B) ............................... 100

Figure 4. 4 AAS exposure during adolescence does not affect cell proliferation or
cell survival In the MeAZD. (A) A 2-way ANOVA revealed a main effect of age on
BrdU- labeled cells/mm: (*p< 0. 001). No effects of treatment were found on

BrdU- labeled cells/mm2 or volume (mm3 ) (B) .......................................... 101

Figure 4.5 AAS exposure during adolescence does not affect cell proliferation or
cell survival in the MeAV, however AAS exposure signiﬁcantly increases MeAV
volume even 4 wks after treatment. (A) A 2-way ANOVA revealed a main effect
of age on BrdU- ir cells/mm2 (* p<0. 0001). (B) AAS-treatment increased MeAV
volume (mm3 ) In males perfused immediately after treatment and 4 wks after
treatment ( p < 0. 05) ...................................................................... 102

Figure 4. 6 AAS exposure during adolescence decreases the number of cells
added to the MePD. (A) A signiﬁcant interaction between age and AAS-
treatment was found on BrdU- -ir cells/mm2 (* p <0. 05). AAS treated males had
signiﬁcantly less cells added to the MePD than vehicle-treated animals when
perfused 4 hr after treatment but not when perfused 4 wk after treatment. (B) No
effects of age or treatment were found on MePD volume (mm3 ) .................. 103

xvi

Figure 4. 7 AAS exposure during adolescence does not affect cell proliferation or
cell survival In the MePV. (A) A 2-way ANOVA revealed a main effect of age on
BrdU- ir cells/mm2 (p < 0. 03001). No signiﬁcant of treatment was found on BrdU- ir
cells/mm2 or volume (mm3 ) (B) ............................................................. 104

Figure 4.8 AAS exposure during adolescence decreases plasma testosterone
levels and testes weight immediately after treatment. (A) A 2-way ANOVA
revealed a significant main effect of treatment (*p < 0.01) and age (p < 0.01) on
plasma testosterone levels. (B) A 2-way ANOVA revealed an interaction between
age and treatment (p < 0.05) on testes weight .......................................... 105

Figure 5.1 Depiction of the development of male social behavior from
adolescence to adulthood ................................................................... 116

Figure 5.2 Depiction of development of male social behaviors after adolescent
and adult AAS exposure ..................................................................... 120

xvii

KEY TO AND ABBREVIATIONS SYMBOLS

Anabolic-Androgenic Steroids ..................................................... AAS
Hypothalamic-Pituitary-Gonadal Axis ........................................... HPG axis
Drug Enforcement Agency ......................................................... DEA
Testosterone ........................................................................... T
Testosterone Propionate ........................................................... TP
Testosterone Cypionate ............................................................. TC
Nandrolone Decanoate .............................................................. ND
Boldenone Undecylenate ........................................................... BU
17a-methyltestosterone ............................................................. 17a—MT
Dihydrotestosterone ................................................................. DHT
Estrogen ................................................................................. E
Estradiol Benzoate ................................................................... EB
Progesterone ........................................................................... P
Luteinizing Hormone ................................................................. LH
Follicle Stimulating Hormone ...................................................... FSH
Gonadotropin Releasing Hormone ............................................... GnRH
Androgen Receptor .................................................................. AR
Estrogen Receptor ................................................................... ER
Gonadectomy ......................................................................... gdx
Overiectomy .......................................................................... OVX
Postnatal day ......................................................................... P#

xviii

Medial Amygdala ..................................................................... Me

Anterodorsal Medial Amygdala ................................................... MeAD
Anteroventral Medial Amygdala .................................................. MeAV
Posterodorsal Medial Amygdala ................................................. MePD
Posteroventral Medial Amygdala ................................................ MePV
Central Amygdala ................................................................... CeA
Medial Preoptic Area ............................................................... MPOA
Lateral Septum ...................................................................... LS
Bed Nucleus of the Stria Terminalis ........................................... BNST
Anterior Hypothalamus ........................................................... AH
Female Hamster Vaginal Secretions ......................................... FHVS
Dopamine ........................................................................... DA
Serotonin ............................................................................ 5-HT
v-aminobutyric acid ............................................................... GABA
Argenine Vasopressin ........................................................... AVP
Neural Stem Cells ................................................................. NSC
Bromodeoxyuridine ............................................................... BrdU
Lightzdark ........................................................................... L:D
Treatment ........................................................................... TMT
p-value .............................................................................. P < #
Analysis of Variance ............................................................ ANOVA
Coefﬁcient of Variation ......................................................... CV

xix

CHAPTER 1: ADOLESCENT ANDROGENIC ANABOLIC STEROID
EXPOSURE (AAS): POTENTIAL EFFECTS ON SOCIAL BEHAVIORS AND
NEURAL PLASTICITY

AAS are synthetic compounds structurally related to testosterone (T) that
promote muscle growth (anabolic) and have masculinizing (androgenic) effects.
They were originally used during World War II to treat soldiers suffering from
malnutrition due to their anabolic potency. Later, in the 1940’s high doses of
AAS were used to treat hypogonadal men that suffer from a reduced or absent
secretion of T. Clinically, high dose regimens are used to promote muscle
deposition after burns, surgery, radiation therapy and, more recently, to treat
HIV+ patients (Kuhn, 2002). AAS have also been used by psychiatrists to treat
mood disorders in order to improve mental alertness, mood elevation, and
concentration (Uzych, 1992). However, the advantages that anabolic steroids
introduce for treating physiological and psychological disorders may become

disadvantages when they are used inappropriately (Blue and Lombardo, 1999;

Corcoran and Longo, 1992).

Athletes and non-athletes (Bahrke and Yesalis, 2004), males and females,
of different ages use AAS for cosmetic and anabolic purposes in doses of 10-100
times higher than clinically prescribed. These high doses may cause acne,
cardiovascular disease, headaches, and liver disease. In males it may cause
baldness, prostate changes and a decreased libido. In females they cause
breast shrinkage, clitoral enlargement and menstrual irregularities (Yesalis et al.,

1990). In addition to these pathological changes in peripheral tissues, AAS may

create psychological dependency. Recently, a two stage model for AAS
dependency was described (Brower, 2002). The ﬁrst stage is deﬁned when AAS
are used solely for aesthetic purposes for their anabolic effects. The second
stage begins after users discontinue administration, but remain psychologically
dependent on the drug. Users can become depressed and suffer from
withdrawal, similar to what someone suffering from cocaine or heroin withdrawal
would, to the extent that suicide attempts may occur (Thiblin et al., 1999b;
Yesalis and Bahrke, 2000; Yesalis et al., 1997). Therefore, the user must re-
administer AAS in order to avoid these consequences. Some of the
psychological adverse effects that are increased by inappropriate. use of AAS
are: aggression, mania, emotional instability, irritability, hostility, and ultimately
violence, all describe under the “roid rage” phenomenon in humans. Within the
last decade the Drug Enforcement Agency (DEA) has classified androgenic-
anabolic steroids as illegal substances because of their potency and addictive

characteristics.

Within the adolescent community there are social pressures to be
attractive, which in turn, encourages the use of AAS among teenagers for
cosmetic purposes (Bahrke et al., 1998). Furthermore, some adolescents claim
to use AAS to become intoxicated and braver (Kindlundh et al., 1999). The onset
of AAS use has been documented to be as early as 10 years of age. Over
700,000 adolescents have used AAS (T erney and McLain, 1990), two thirds of

these do not use AAS for athletic purposes. In addition, among teenagers the

perception of risk of anabolic steroid use is decreasing over time (Monitoring the
Future Study 2004). Data from the Youth Surveillance Survey from Center of
Disease Control (1993) shows the prevalence of lifetime illegal steroid use
ranged from 3.2% to 7.1% across state surveys (median: 4.8%) and from 2.3% to
7.4% across local surveys (median: 3.1%). Surprisingly, a range of 4 - 12% of
high school males have admitted to using AAS (Yesalis and Bahrke, 2000).
Similar studies indicate that high school students have little understanding of the
adverse effects AAS can have when abused (Tanner et al., 1995), NIDA Steroid
Report, 2000; Monitoring the Future, 2004). As a result of steroid misuse human
adolescents become deﬁant, aggressive, and are described to have antisocial
personalities, along with increased or decreased sexual libido (Thiblin and
Petersson, 2005). They may also suffer from physiological adverse effects like

stunted growth and acne.

AAS have traditionally been taken in “cycles” which last 6 to 12 wks or
more. By staggering and combining these drugs users attempt to avoid
developing a tolerance to a particular AAS, this is called “stacking”. Other kinds
of drugs are used to prevent the adverse effects caused by the abuse of AAS,
like antiduiretics, antiestrogens and human chorionic gonadotrophins are used to
counteract the adverse effects cause by AAS abuse (Karila et al., 2004;
Martikainen et al., 1986). Nevertheless, studies in humans show that even when
a single compound is actively abused severe side effects on the body and

psyche still occur (Daly et al., 2001; Kouri et al., 1995; Pope et al., 2000).

AAS and Adolescent Development

A stage of development that is under appreciated as a time of neural
change is puberty (Romeo, 2003; Sisk et al., 2003). In the last decade, studies
on pubertal and adolescent development provide evidence that the adolescent
brain undergoes neural changes that result in the expression of adult-typical
behaviors. During this developmental period the emergence of sex-biased
psychopathologies such as depression and schizophrenia occurs, along with an
increase in risk-taking behaviors, such as sexual exploration, drug use and
aggression. Puberty and adolescence are sometimes used interchangeably,
however, there are distinct deﬁnitions for each: (1) puberty. the activation of the
hypothaIamic—pituitary-gonadal axis (HPG-axis) that results in gonadal maturation
and (2) adolescence: the maturation of adult social and cognitive behaviors (Sisk
and Foster, 2004). Thus, adolescence encompasses both the hormonal changes

of puberty and the neural changes associated with behavioral maturation.

Gonadal hormones are known to have both activational and organizational
effects on brain and behavior. Activational effects are deﬁned as the occurrence
of a given behavioral response only in the presence of a given hormone. In the
absence of hormone, the behavior is not expressed. The classical deﬁnition of
organizational effects is permanent and long-lasting effects of steroid hormones
on structural features of neural circuits that occur during a maximally sensitive

period of perinatal development and that program behavioral responses to

steroid hormones in adulthood. Therefore, since adolescence is characterized
by rapid neural development, steroid hormones may exert organizational or

activational effects, or both, during this time.

The HPG-axis is governed by a small population of cells in the
hypothalamus that secrete the gonadotropin releasing hormone (GnRH).
Activation of GnRH neurons at the onset of puberty causes increased released of
GnRH hormone pulses into the median eminence of the hypothalamus, where
GnRH enters the pituitary portal system. GnRH acts on gonadotropes in the
anterior pituitary, to induce pulsatile release of the gonadotropins, follicle
stimulating hormone (FSH) and luteinizing hormone (LH). In males, these
hormones act on target cells to stimulate the release of testosterone (T) from the
testes. As T levels rise, a neuroendocrine negative feedback loop is activated
and it shuts down GnRH neurosecretion and the release of T in male rodents.

An additional effect of T in the brain is to facilitate male social behaviors in
speciﬁc environmental contexts. Exogenous androgens, such as anabolic
androgenic steroids (AAS), can also result in the negative feedback inhibition of
the HPG-axis and facilitation of behavior (McGinnis, 2004). Because puberty is a
time of rapid hormonal and behavioral development, this dissertation focuses on
understanding the effects of pubertal exposure to AAS on social behaviors and

adolescent neural development.

The use of AAS is increasing signiﬁcantly among adolescents. There are
several gaps in our current understanding that this work addresses: (1) a
comparison between the adolescent and adult exposure to AAS on brain and
behavior, (2) a comparison of short and long term effects of AAS on the
adolescent and adult brain and behavior, and (3) the effects of AAS on neural
changes in the brain during puberty. Work from our laboratory using the male
Syrian hamster has established that the absence of gonadal hormones during
adolescence impairs steroid-dependent social behaviors in adulthood,
suggesting that during adolescence, steroid hormones organize neural circuits to
program adult behavioral responses to hormone; however, how the
administration of supraphysiological levels of steroid hormones (which would

occur with AAS use) affect brain and behavior has not been well established.

Adolescence is the transition from childhood to adulthood and
encompasses neural, hormonal and behavioral maturation by both steroid
dependent and independent events. Gonadal steroid hormones play a critical
role during adolescent maturation in two important ways (Sisk et al., 2003). First,
they alter brain development and organization by inﬂuencing fundamental neural
processes such as neurogenesis, cell death, and synapse formation. Second,
they inﬂuence social experience both by activation of neural circuits underlying

social behaviors.

Recent studies in male animal models show that pubertal gonadal
hormones also organize the adolescent nervous system, resulting in an adult that
will more readily display male-typical behaviors (Sisk and Foster, 2004; Sisk et
al., 2003). Our laboratory has therefore proposed a two-stage model for
behavioral maturation of male social behaviors. During perinatal development
steroid hormones act on the central nervous system to sexually differentiate
neural circuits, which later are activated to result in the expression of adult
development. In addition to the perinatal period, puberty is a second time when
gonadal hormones further organize neural circuits to result in the full expression

of adult-typical behaviors (Romeo, 2003; Sisk et al., 2003).

Studies in our lab using the male Syrian hamster have repeatedly
supported the organizational hypothesis during puberty. Prepubertal males,
even when treated with adult physiological levels of T, do not express the full
repertoire of reproductive behaviors, suggesting that the prepubertal brain is not
prepared for the activation of these behaviors by T (Meek et al., 1997; Romeo et
al., 2001). In several studies, an experimental paradigm tested the hypothesis
that the presence of gonadal hormones during puberty organizes neural circuits
to allow the activation of reproductive behavior in adulthood. In this paradigm,
sexually na'ive males were gonadectomized (gdx) before or after puberty. Six
weeks after castration, in adulthood, a subcutaneous T pellet was implanted for
one week and animals were tested with a receptive female for reproductive

behaviors. Animals that were castrated before puberty expressed signiﬁcantly

lower mounts, intromissions and ejaculations (Schulz et al., 2004). Similar
results were observed in ﬂank marking behavior when using the same hormonal
manipulation and a resident intruder paradigm: animals that were gdx before
puberty displayed fewer ﬂank marks (Romeo et al., 2003; Sisk et al., 2003).
These results suggest that developmental events occurring during puberty affect
the responsiveness of the nervous system to the activational effects of gonadal

steroids in reproductive and agonistic behaviors.

Using a similar experimental paradigm, we found that the impairments in
reproductive behavior resulting from the absence of gonadal hormones during
puberty were long-lasting and not easily reversed. Neither prolonged T treatment
(17 days) nor sexual experience (3 exposures to a receptive female) abolishes
the behavioral differences between males undergoing adolescent development in
the presence versus absence of hormones. Taken together, we conclude that
steroid hormones in adolescence program the expression of social behaviors in
adulthood (Schulz et al., 2004). These results also suggest that adolescent
exposure to pharmacological levels of steroids, such as those resulting from AAS
use, may have more severe and permanent consequences than AAS exposure

in adulthood.

The adolescent brain undergoes profound change that results in both
synaptic reorganization and modiﬁcations in gross morphological features.

Imaging techniques show dramatic differences in grey matter and white matter

during this developmental period in humans. The brain’s grey matter volume
increases during early adolescence and then decreases during late adolescence
in both males and females. These changes are particularly robust in the frontal
cortex and cerebellum (Giedd et al., 1999). Interestingly, the average age at
peak gray matter volume occurs earlier in girls than in boys, and corresponds to
the earlier age at onset of puberty in girls, suggesting a potential role for gonadal
hormones in this process. Biphasic changes are also observed in striatal
dopamine receptors in adolescent male rats. There is a large increase of DZ
receptors in male rats between postnatal day (P) 25 and 40 (early puberty), and
then later there is a “pruning” process when the receptor density decreases
almost 55% by P80 (Andersen et al., 2002; Andersen et al., 2000). Interestingly,
this phenomenon is still observed in animals that are gonadectomized

prepubertally, indicating that this process is steroid-independent.

Pharmacology of Anabolic Andmgenic Steroids:

There are three different Classes (Figure 1.3) of AAS that can be
administered in a variety of ways, a transderrnal patch, skin cream, injectable or
an oral preparation. Like endogenous androgens, AAS are four-ringed structures
with nineteen carbon atoms. Modiﬁcations to these carbons were made in order
to prolong the metabolic half-life and the efﬁcacy of the synthetic AAS (Clark and
Henderson, 2003). Class I AAS, like testosterone cypionate (TC) and
testosterone propionate (TP), are injectable and are derived from an

esteriﬁcation of the 17B-hydroxyl group of testosterone. Class II AAS are also

injectable androgen esters; however, they are deﬁned as the 19-nor-testosterone
derivatives because of the substitution of a hydrogen bond for methyl group at
C19. This substitution extends the half-life of the compound that cannot be
attributed to esteriﬁcation alone. Among steroid users some popular class II AAS
are nandrolone decanoate (ND) and boldenone undecylenate (BU), which has
slow clearance from the body up to 12 wks (Ballard and Wood, 2005). Most
Class I and class II AAS are aromatizable and can have signiﬁcant effects in the
central nervous system (CNS) not only via the androgen receptor (AR) but also
by actions on the estrogen receptor. Class III AAS are mostly non-aromatizable
and cannot be converted to either dihydrotestosterone (DHT) or 178-estradiol
(E2) although other androgenic and estrogenic metabolites may be created by
the degradation of these compounds. They are known as the 17d-alkyl AAS
because they are alkylated in the C17 of the four-ring structure. Since the
alkylation slows down the metabolism by the liver, these are orally active. Some
class III AAS are 17a-methyltestosterone (17d-MT), oxymethalone and

stanozolol (ST) (Clark and Henderson, 2003).
Characterization of Social Behaviors in Male Syrian hamsters:

We use the male Syrian hamster to investigate the neural mechanisms
that are responsible for pubertal change in male social behaviors, and how these
are disrupted. In our lab we have characterized and deﬁned aggressive and

reproductive behaviors in male Syrian hamsters through development. We also

10

have a good understanding of their chemosensory processing neural pathway
(Wood, 1998; Wood and Coolen, 1997), along with the neural circuits that
mediate social behaviors in both pubertal and adult males. We have
demonstrated that behavioral responses to T change as a function of pubertal
development in this species (Meek et al., 1997) and the endocrinological proﬁle
has been established as the male progresses through puberty (Miller et al.,
1977). All these are important aspects for the use of this animal model and
understanding the effects of AAS on male social behaviors during adolescence

and adulthood.

The amygdala: Social behaviors 3nd Neural Plasticity

 

Pheromonal communication and hormonal signals are critical for the
expression of male social behaviors in male rodents. The neural circuits that
underlie male reproductive behaviors (which includes the bed nucleus of the stria
terrninalis (BNST) and the medial preoptic area (MPOA)) and inter-male
aggressive behaviors (which includes the anterior hypothalamus (AH), the BNST,
and the lateral septum (LS)) share efferent and afferent connections to and from
the medial amygdala (Me) (Ferris et al., 1987; KollaCk-Walker et al., 1999;
KolIack-Walker and Newman, 1995; Potegal et al., 1996; Romeo and Sisk, 2001)
The connections of the amygdala in rats, cats and monkeys can be divided into
three systems (1) sensory processing through connections with the olfactory
cortex, posterior thalamus, and sensory association cortex, (2) visceral function

in relation to the emotional stimuli through connections with the hypothalamus,

11

and (3) emotional behavior and mood through connections that compose the
forebrain circuit (Price, 2003). The Me is a complex structure that has been
subdivided based on cytoarchitectonic, synaptic and functional criteria, into four
main nuclei: anterodorsal (MeAD), anteroventral (MeAV), posterodorsal (MePD)
and posteroventral (MePV) (Alheid, 2003; Cooke and Simerly, 2005). The Me
subnuclei contribute to the interpretation of sensory information (Wood and
Coolen, 1997), for the regulation of intermale aggression and reproductive
behaviors, (KolIack-Walker and Newman, 1995) and to the interpretation of
emotionally loaded stimuli and memory modulation (Rasia-Filho et al., 2004) . In
addition, it is considered part of the “extended amygdala”, composed of parallel
rings of cells, extending through the medial and central amygdaloid nuclei, the
bed nucleus of the stria terminalis (BNST) and the substantia innominata, along
with structures in the basal forebrain, which are related by the shared
characteristics in cell morphology, reciprocal neuronal connections and
neurochemical/neurostransmitter identity (Alheid, 2003; Canteras et al., 1995;

Newman, 1999).

The results of several studies suggest that the subdivisions of the Me play
different roles in the mediation of male social behavior (Wood and Newman,
1995b). The MeA is primarily a relay for chemosensory information, while the
MeP relays mostly steroidal information (Meredith and Fewell, 2001). However,
their functions within circuits underlying social behaviors do overlap. The medial

amygdala may be involved in learning and memory for socially signiﬁcant cues

12

(KolIack-Walker & Newman, 1995). Both the MeA and MeP nuclei express
androgen and estrogen receptors (Wood & Newman, 1995), and both show a
signiﬁcant increase found in Fos-immunostaining, a marker of neuronal activity,
in response to pheromonal cues in estrous female vaginal secretions (in both
pubertal and adult males) (Romeo et al., 1998), and in response to the presence
of a stimulus male (Johansson-Steensland et al., 2002; KolIack-Walker and
Newman, 1995). Lesions in the medial amygdala, both the MeA and the MeP,
disrupt a male’s ability to engage in copulation (Lehman et al., 1980; Newman
and Bachevalier, 1997) . Reduced aggressive behavior is observed in lesioned
animals after experience with four aggressive interactions, suggesting that these

lesions interfere with social learning processes (Vochteloo and Koolhaas, 1987).

Gonadal hormones play a role in neural plasticity during puberty (Romeo
& Sisk, 2001). The MeA and MeP are steroid sensitive, and morphological
changes in these nuclei may reﬂect Changes in the expression of male social
behaviors. The removal of gonadal hormones in adult males results in a
decrease in somal size of individual cells and/or overall volume of the Me.
Interestingly, the MeA is signiﬁcantly larger in prepubertal male Syrian hamsters
when compared to adults; however the inverse in true about the MePD. This
may be explained by function of the amygdala and its sensitivity to gonadal
hormones. The MeA receives substantial projections from the accessory
olfactory bulb, and lesions to the MeA abolish chemoinvestigatory behavior,

suggesting the pubertal development may involve changes that allow appropriate

13

processing and integration of these cues (Gomez and Newman, 1992; Wood and
Newman, 1995b). However, the MeP contains more androgen (AR) and
estrogen receptors (ER) than the MeA (Cooke et al., 2003; Wood and Newman,
1995a), suggesting that it may retain the capacity for plasticity throughout life so
that the expression of male social behaviors is possible under speciﬁc steroidal
and/or environmental conditions (Romeo and Sisk, 2001).

When male rodents are gonadectomized and lack gonadal hormones the
MePD volume decreases. The MePD contains both androgen (AR) and estrogen
receptors (Cooke et al., 2003; Cooke et al., 2001) that may be regulating the
plasticity of this neural structure. When adult male rats are castrated and given
silastic capsules containing DHT and/or E2 or blanks, all groups were compared
to control shams. Steroid, alone or in combination, maintained ejaculatory
behavior and vocalizations patterns. Interestingly, when the volume measures
were taken of the amygdala, regional volume and cell soma size of the MePD
following castration was Iateralized (the left hemisphere neurons were larger than
the right). Both DHT and E2 each maintained neuronal soma size in both
hemispheres. This data suggests that steroid hormones that participate in the
maintenance of male sexual behavior mediated by both receptors in the MePD

contribute to the steroid—regulated structural plasticity in the brain region.

Estrogen increases the number of AR and the duration of the ligand
occupancy in brain regions involved in the control of mating, and androgens

downregulate the expression of ER (Brown et al., 1994) suggesting an interaction

14

between both steroids. Brains from adult male rats were used to assess whether
AR and ER receptors were co-localized in several structures in the brain that
mediate male reproductive behavior, one of which is the MeA, after being
exposed to several sexual interactions. Speciﬁcally, MeA contains both
receptors in male rodents (Greco et al., 1999). In the MePD of male Syrian
hamsters, 57.5% of neurons contain AR and 13% of neurons contain ER (Wood
& Newman, 1995). The differential distribution of AR and ER in the Me neurons
provide information for a potential synergistic actions of androgens and
estrogens on their receptors. T bound to AR is converted by aromatase to E2,
and within the same cells activation of ER facilitates androgenic actions by
increasing the duration of occupation of AR and the amount of AR. The
behavioral effects of androgens could be further enhanced by the binding of T to

AR in cells not containing ER.

Androgen—dependent plasticity in the MePD has also been by using Golgi-
staining in male Syrian hamsters. In castrated males, neurons in the posterior,
but not in the anterior, region of the Me undergo structural changes (Gomez and
Newman, 1992). This was indicated by a decrease in the mean highest dendritic
branch level and mean somal area when compared to intact hamsters. Animals
with implants of a testosterone (T), dihydrotestosterone (DHT) and/or 178-
estradiol (E2) were also compared to intacts and castrates; all of these were
subjected to a behavior test with a receptive female. Castration and treatment

with DHT resulted in a decrease in mean somal area, the mean highest dendritic

15

branch and the percentage of neurons with tertiary branch segments. DHT
treatment also resulted in a reduction of total dendritic length and spine density.
These results suggest that T or E is sufﬁcient to maintain normal morphology of
the neurons in the MePD. Recently, Zehr et al. (2005) demonstrated that that
MePD neurons go through dramatic changes and structural remodeling during
puberty such as changes in terminal spine densities, axons and dendritic
arborization. Prepubertal animals have a greater number of dendrites and higher
spine density than midpubertal and postpubertal animals. During puberty,
pruning of dendrites and spines, along with axonal changes occur with increasing

steroid levels.

Gonadal steroid hormones play an important role in the proliferation
(Orrnerod and Galea, 2001), survival (Leranth et al., 2000) and activation (lnsel,
1990) of neurons. Adult male voles were gdx and treated with TP, EB or DHT.
Animals also received injections of a proliferation marker, 5-bromo-
2’deoxyuridine (BrdU), which gets incorporated in the DNA of a mitotic cell during
the S phase of mitosis. Treatment with TP and EB exerted similar effects in
increasing the density of BrdU labeled cells in the Me (44% newly born neurons
as identiﬁed by a double labeled cells with TuJ1. TuJ1 is considered the earliest
marker for cells that have begun to differentiate into neurons), whereas DHT was
ineffective. Taken together, these data suggests that gonadal steroid hormones

inﬂuence the number of newly born cells in the amygdala (Fowler et al., 2002).

16

A study in adult male Syrian hamsters observed newly proliferated cells in
the medial amygdala in adulthood (Huang and Bittman, 2002). Animals were
exposed were exposed to an estrous female, an aggressive male or a cotton
swab containing FHVS. Tissue sections were stained for BrdU and c-Fos in the
olfactory bulb, MPOA, Me and BNST. The activated cells were seen in the
olfactory bulbs after the males were exposed to the estrous female. A
comparable pattern of activation was seen in the amygdaloid complex when
compared to the other nuclei quantiﬁed in this study. BrdU labeled cells were
observed 3 wks after the last BrdU injection in the amygdala. However, no
double labeled cells were seen in any of the hypothalamic nuclei. This data
supports previous studies seen in male voles suggesting a proliferation of newly

born cells in the Me, however, their function is still not deﬁned.

AAS and the Amvadgﬁ

The medial amygdala plays a role in the mediation of social behaviors that
are also affected by the use of AAS during adolescence and adulthood.
However, few studies have been performed to determine the effects of AAS on
androgen and estrogen receptors in the mammalian brain (Clark and Henderson,
2003; Lynch and Story, 2000). As stated previously, Class I and Class II AAS
can be aromatized to bind to AR and ER in the CNS. For changes to occur, AAS
must bind to AR and ER and affect cellular gene expression. Treatment of a
cocktail of AAS (2 mglkg testosterone cypionate, 2 mglkg nandrolone decanoate

and 1 mglkg boldenone undecylanate) to intact male rats for 2 wks increase the

17

expression of AR in the classical androgen target sites like the MePD, MPOA
and BNST (Menard and Harlan, 1993); all of these structures are involved in the
mediation of male social behaviors. High levels of AR-ir in non-classical
structures that usually express little AR (ie., caudate putamen, ventral tegmental
area, central gray, hippocampus, and locus coeruleus) as well as classical AR
dense structures were also observed. This provides evidence that AAS can
produce signiﬁcant changes in both “steroid-sensitive” and “steroid-insensitive”
brain regions, which in turn can affect the expression of behavior. These results

identify an up-regulation of AR in the male rat brain by AAS.

Another study compared the effects on AR-ir by the same AAS cocktail,
dihydrostestosterone (DHT), and E2 for 14 days in intact and castrated rats. The
male rats administered the cocktail and DHT fully upregulated AR-ir in the
ventromedial hypothalamus (VMH), MPOA and MeP. Estradiol caused partially
up-regulated AR-ir in the VMH and MeP, but not in the MPOA in castrated rats
when compared to intact males (Lynch and Story, 2000). Taken together, these
studies show that androgens can have profound effects on brain metabolism and
function via AR and ER. These measurements can be used to elucidate how

AAS can modify central nervous system mechanisms.

Studies examining neurochemical changes in the sexually maturing,
adolescent brain exposed to supraphysiological levels of AAS have determined

that AAS cause changes in the neurochemical communication pathways

18

associated social behaviors. It is well established that serotonin (5HT) and
arginine vasopressin (AVP) mediate aggression in male Syrian hamsters.
Reduced serotonin levels and increased vasopressin levels have been
associated with increased levels of aggressiveness. Adult male rats were treated
with nandrolone decanoate (ND) for 2 wks. Changes in 5HT receptors due to
AAS administration were visualized by using autoradiography. A decrease in
5HT2 R was observed in the medial amygdala (Kindlundh et al., 2003a).
Signiﬁcant changes are also seen in other hypothalamic regions. A cocktail of
AAS causes neuroanatomical changes after 30 days of consecutive
administration from 27-53 days of age in male Syrian hamsters. After
administration they were tested for aggressive behaviors. AAS treated males had
increased levels of offensive aggression. When examining serotonin ﬁbers (5-
HT) in the MeA, there was a signiﬁcant decrease in animals that were treated
with the AAS (DeLeon et al., 2002; Grimes and Melloni, 2002). Using the same
AAS regimen and autoradiography, a signiﬁcant increase was seen in the
expression of AVP V12, receptors in the cortical nucleus of the Me and other

regions that regulate aggressive behavior, like the anterior hypothalamus.

When same cocktail of AAS is used to look at GADss, the rate limiting
enzyme in the synthesis of v-aminobutyric acid (GABA), in adolescent brains of
aggressive male hamsters, a signiﬁcant increase was observed in the
immunoreactive puncta in the medial amygdala of these animals (Grimes et al.,

2003). Increased levels of GABA result in increased levels of aggression

19

(Potegal et al., 1982). Studies of neurohorrnonal receptors in the adolescent
male rodent brain show that a high dose of AAS during puberty can cause
changes in the GABA receptor subunits. No signiﬁcant changes can be
observed in adulthood. Taken together, these data demonstrate that the effects
of AAS exposure affect the adolescent developing brain and result in

modiﬁcations to male social behaviors.

Use of AAS can affect brain function leading to behavioral changes. A
study using rat neural stem cells (NSC) in culture, and the dentate gyrus in the
rat hippocampus was conducted to test the effects nandrolone decanoate has on
neurogenesis and cell proliferation. In vitro, NSC were stimulated by epidermal
growth factor, nandrolone reduced cell proliferation. Nandrolone also decreased
neurogenesis (measured by bromodeoxyuridine (Brd-U) labeling) in the dentate
gyrus of the rat hippocampus in both adult males and females after 5 days of

administration (Brannvall et al., 2005).

Overall, AAS can affect neural organization and circuitry, which in turn

results in changes in behavior.

Preﬁminafv Expen'meLts
In the ﬁrst preliminary experiment we examined the effects of pubertal
exposoure to 17-a-methyltestosterone (17o-MT), a class III non-aromatizable

AAS, on male aggressive behaviors. 17a-MT (0 mglkg, 1 mglkg, 3 mglkg, and 5

20

mglkg) was administered subcutaneously (sc) for three weeks beginning at
puberty onset (P21 - P41). One day after the last day of administration, mid-
pubertal (P42) male hamsters were then exposed to a weight and age matched
stimulus male in a neutral arena (with a 5 minute acclimation period before
testing); all tests were videotaped and quantiﬁed. Not all aggressive behaviors
were increased in AAS-treated males at all doses, but the results were
suggestive of an increase in aggression with the highest dose of 17d-MT. We
observed a signiﬁcant decrease in attack latency in a 5 mglkg dose (Fig. 4; p-
value = 0.008), and a trend towards an increase in ﬂank marking (p-value = 0.07)
and overall aggression score (Fig. 1.4; attacks + bites + ﬂank marks -
submissive behaviors; p-value = 0.07). In addition, when behavior of the
stimulus (untreated) males was compared with the experimental treated males,
the treated males expressed most, if not all, of the dominant behaviors within the
social interaction. It shows that a non-aromatizable AAS can affect some
aspects of aggressive behaviors when administered over three weeks during
puberty; nevertheless, a combination of aromatizable and non-aromatizable AAS
may be needed to activate the full repertoire of aggressive behaviors.
Physiological measures such as body weight from the beginning of
administration to the end, testes weight and T plasma levels where taken,

however there was no difference among any of the treatment groups.

21

 

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Attack Latency (min)
0|
Aggressron Score

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

o 5.6 0.0 1.0 3.0 5.0
Dose of 17d-MT Dqse if 119-! II

Figure 1.1 Effects of 17a-methyltestosterone on attack latency and aggression in
pubertal male Syrian hamsters.

 

 

The second experiment compared several doses of 17d-MT (0 mglkg, 2.5
mglkg, 5 mglkg, and 10 mglkg) with a cocktail of AAS. The cocktail included a
compound from all three classes of AAS: class I = testosterone cypionate (TC; 2
mglkg), class II = nandrolone decanoate (ND; 2 mglkg), and class III = 17a-MT
(5mglkg). Aggression tests used a neutral arena behavioral paradigm without an
acclimation period; therefore, neither animal had the advantage of investigating
their surroundings. Behaviorally we wanted to assess social dominance.
Stimulus males received saline injections to decrease handling differences
among animals. AAS administration was for a total of 4 weeks (P21-P49). After
matching the experimental males with age and weight matched partners, at 10
min behavior test was given and videotaped, without any acclimation period.
Aggressive behaviors were quantiﬁed; however there were no signiﬁcant
differences in any of the treatment groups. Physiological measures were also
taken including, seminal vesicle weight, testes weight, body weight and

testosterone (T) plasma levels. There was no signiﬁcant change in seminal

22

vesicle weight. However, the animals that were injected with the cocktail had
overall signiﬁcantly lower testes weight (p —value < .001) and body weight (p —
value <.01) when compared to all of the animals that received 17d-MT. Also,
their T plasma levels were signiﬁcantly lower (p — value <.02) than animals
treated with the vehicle. These data suggest that AAS effect reproductive target
tissues, and interferes in the normal function of the HPG axis by decreasing T
levels in pubertal males; this may result in deﬁcits of the expression of social

behaviors in adult males.

Taken together, these experiments suggest several things that affect the
expression of aggressive behaviors. First, social context is important when
testing for aggressive behaviors. In the ﬁrst experiment we used a neutral arena
paradigm with a ﬁve minute acclimation period for the treated male, which
resulted in a slight increase in the aggression score. However, there were no
behavioral effects of AAS in the second experiment, where there was no
acclimation period. Experiments in male rats treated with AAS used a tail-pinch
to provoke an aggressive response before a social encounter with another male
(McGinnis et al., 2002), suggesting that social context is important for the
expression of aggressive behavior. Second, a single compound may not be
sufﬁcient to increase aggressive behaviors relative to vehicle treatment. 17a-MT
is a Class III AAS that is not aromatized, suggesting that AAS that act on AR and
ER are more like to induce aggressive behaviors. Finally, a cocktail of class I and

II AAS affects the reproductive physiology of male hamsters, especially after a

23

long period of administration. These data suggest that AAS may induce a
negative feedback loop on the HPG axis, resulting in decreased testes weights

and decreased plasma levels.

A third experiment focused on comparing the cocktail used in the previous
experiment with one that has been documented in the literature to increase
aggressive behaviors in pubertal male Syrian hamsters and to upregulate AR in
classical and non-classical AR-dense structures in the brain (Lynch and Story,
2000; Menard and Harlan, 1993). Aggressive behaviors were assessed using a
resident-intruder paradigm, creating a context where the treated animal must
protect his “home” (Romeo et al., 2003) Animals arrived on P18 and were group
housed until P25. On P27 animals were divided in three groups in order to
compare two cocktails of AAS to a vehicle group. One cocktail (A) was
composed of all three classes: 2 mglkg TC, 2 mglkg ND and 5mg/kg 17a-MT.
The second (B) was made up of class I and class II AAS: 2 mglkg TC, 2 mglkg
ND and 1 mglkg boldenone undecylenate. After two weeks of daily injections
(P27-P41), one day after the last injection (P42) a behavior test was
administered with an age and weight matched gonad-intact male intruder.
Aggressive behaviors were signiﬁcantly increased (Figure 1.5; attacks, attack
duration, bites, and contact time; p - value <0.003) and defensive posturing was
signiﬁcantly decreased (p <0.06) by cocktail B. Cocktail A produced levels of
aggression intermediate to those seen groups treated with vehicle and cocktail B.

Physiological measures taken reﬂected that cocktail B also signiﬁcantly

24

decreased body weight in those two weeks (p - value < 0.03). Testes weight
was signiﬁcantly decreased by both cocktails (p — value < 0.01). There were no
signiﬁcant differences in T blood plasma levels. These results suggest that a
cocktail of aromatizable AAS has greater effects on the expression of aggression

during puberty that a cocktail with a non-aromatizable AAS and a single non-

aromatizable compound.

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

1 ﬂ
3
8
t
<
0 Vehicle
20 I
9'3 153
00 103
5:‘._I_| {
n‘ 3:3:i:
Vehicle
E 3
E
c 2.5
~5—3 2
x 1 if?
8 5 3:313.
< o I I
Vehicle

 

 

 

Figure 1.2 Attack number, bites and attack duration were significantly increased with an
AAS cocktail containing TC, ND, and BU (B) compared to a cocktail containing TC, ND
and 17a-MT (A) and vehicle.

25

These three experiments determined the optimal behavioral context for
assessing aggressive behaviors in intact male Syrian hamsters, along with
identifying that a cocktail of AAS results in an increase of aggressive behaviors
when compared with single compounds and vehicle controls. The following
experiments addressed (1) whether this effect is also seen in adult male
hamsters and in reproductive behaviors, (2) whether the AAS exposure have
long term effects on social behaviors in pubertal and adult animals and (3)

whether AAS affect neural plasticity speciﬁcally in the amygdala.

26

Chapter 2: ANABOLIC ANDROGENIC STEROIDS DIFFERENTIALLY
AFFECT SOCIAL BEHAVIORS IN ADOLESCENT AND ADULT MALE SYRIAN
HAMSTERS
Introduction

Adolescence is the developmental transition from childhood to adulthood
and it encompasses reproductive, neural, and behavioral maturation (Andersen,
2003; Casey et al., 2000; Lenroot et al., 2007; Sisk and Foster, 2004; Sisk and
Zehr, 2005; Spear, 2000; Yurgelun-Todd, 2007). The onset of puberty marks the
onset of adolescence, and gonadal steroid hormones, which become elevated
during puberty, inﬂuence subsequent adolescent brain development by both
organizing and activating neural circuits underlying social behaviors (Delville et
al., 1998; Primus and Kellogg, 1989; Primus and Kellogg, 1990; Romeo, 2005;
Schulz and Sisk, 2006a; Schulz and Sisk, 2006b; Sisk and Foster, 2004; Spear,
2004). lmportantly, the organizational effects of pubertal hormones program their
activational effects in adulthood. For example, studies in the Syrian hamster
show that the presence of testicular hormones during adolescence results in
adults that more readily display male-typical social behaviors relative to adults
that matured in the absence of testosterone (l') (Schulz et al., 2006; Schulz et al.,
2004; Schulz and Sisk, 2006a; Sisk et al., 2003). Thus, the adolescent brain is
exquisitely sensitive to endogenous testicular steroids, and the action of
testicular hormones during adolescence has both short-term and long-term

consequences on social behaviors.

27

Given the signiﬁcance of endogenous T in normal development of the
adolescent male brain, it is important to consider how anabolic androgenic
steroids (AAS) inﬂuence the adolescent brain and behavior. AAS are synthetic
compounds structurally related to T that promote muscle growth (anabolic) and
have masculinizing (androgenic) effects. AAS are clinically used to treat
malnutrition and mood disorders (Kopera, 1993; Perry et al., 2002); however, the
beneﬁts they have for treating physiological and psychological disorders may
become detriments when they are used inappropriately (Blue and Lombardo,
1999; Corcoran and Longo, 1992). Adolescents that use AAS are often
described by their peers, teachers, and psychologists as deﬁant, aggressive, and
antisocial. In addition, teenagers may display either increased or decreased
sexual libido after AAS misuse (Thiblin and Petersson, 2005). In the United
States, the onset of AAS use can be as early as 10 years of age, and use among
high school males has been as high as 12% in the last decade (Ahima and
Harlan, 1992; Bahrke and Yesalis, 2004; Bahrke et al., 1990; Bahrke et al., 1998;
Buckley et al., 1988; Terney and McLain, 1990). Although there is evidence that
severe consequences occur after misuse of AAS by adolescents, there has been
relatively little systematic investigation of how these exogenous compounds

affect the adolescent brain and behavior.

AAS affect the expression of social behaviors in most rodent models. The
behavioral responses to AAS depend on the chemical structure of the steroid

administered, whether steroid treatment consists of a single compound or as a

28

cocktail, and the age and duration of treatment. Intact adult male rats treated
with T and testosterone cypionate (TC) do not display altered reproductive
behaviors when compared to controls (Clark et al., 1997; Farrell and McGinnis,
2003). However, T administered to intact adolescent male rats increased sexual
behaviors in adulthood (Wesson and McGinnis, 2006). Also, long-terrn (12 wk)
treatment of intact male rats with oxymethalone, stanozolol, nandrolone or 170-
methyltestosterone during adolescence and continuing to adulthood, results in a
decrease in sexual behaviors (Clark et al., 1997; Farrell and McGinnis, 2003;
Feinberg et al., 1997). AAS also affect aggressive behaviors in rodents. Single
compounds such as T and testosterone propionate (T P) increase aggression in
intact adult male rats and mice (Albert et al., 1989; Clark and Barber, 1994;
Farrell and McGinnis, 2003; Lumia et al., 1994; Martinez-Sanchis et al., 1998).
However, adolescent exposure to single compounds such as nandrolone, 17a-
methyltestosterone, or stanozolol, has little or no effect on aggression in rats or
hamsters (Breuer et al., 2001; Farrell and McGinnis, 2003; McGinnis, 2004;
Wesson and McGinnis, 2006). Adolescent male Syrian hamsters exposed to a
cocktail of AAS for two or four weeks show an increase in aggressive behaviors
(DeLeon et al., 2002; Grimes and Melloni, 2002; Grimes et al., 2003; Melloni and
Ferris, 1996; Ricci et al., 2004; Salas-Ramirez and Sisk, 2005). It is clear that a
number of variables determine the overall effects of AAS on sexual and

aggressive behaviors, and age has not been systematically studied.

29

The use of anabolic steroids by adolescent males, and growing evidence
that the adolescent brain is particularly sensitive to hormones and experience call
for a better understanding of how age at the time of exposure to AAS inﬂuence
behavioral responses to AAS. The objective of this study was to directly
compare the immediate effects of AAS on sexual and aggressive behaviors in
adolescent and adult intact male Syrian hamsters. Because adolescent male
hamsters are more responsive than adults to the organizational effects of
endogenous testicular hormones on male social behaviors (Schulz and Sisk,
2006), we hypothesized that adolescents would also be more responsive to
activational effects of exogenous AAS, leading to the prediction that AAS will

elicit greater behavioral responses in adolescent compared to adult males.

Materials and Methods
Animals

Eighteen-day-old male Syrian hamsters (Mesocn'cetus auratus) were
obtained from Harlan Sprague—Dawley Laboratories (Madison, WI). Upon arrival
males were housed in groups of eight animals per cage (polycarbonate, 33 x 38
x 17 cm) with ad libitum access to food (T elkad Rodent Diet No. 8640, Harlan)
and water. Animal colony temperature was maintained at 22 i 2°C, and animals
were kept on a light-dark cycle of 14:10 L:D (lights off at 1600h EST). At 25 days
of age, animals were singly housed in polycarbonate cages (30.5 x 10 x 20 cm,
for Experiment 1; 33 x 38 x 17 cm, for Experiment 2). All animals were treated in

accordance with the NIH Guide for the Care and Use of Laboratory Animals, and

30

all protocols were approved by the Michigan State University All-University

Committee for Animal Use and Care.

AAS Treatment

A cocktail of AAS containing 0.8 mglml of testosterone cypionate (TC;
Sigma Aldrich, Inc., St. Louis, M0), 0.8 mglml of nandrolone decanoate (ND;
Sigma Aldrich, Inc.), and 0.4 mglml of boldenone undecylenate (BU; Steraloids,
New Port, Rhode Island) was dissolved in 2-hydroxypropyl-B-cyclodextrin

(Sigma-Aldrich, Inc.). The AAS cocktail was prepared the day before treatment

began for each age group in each experiment, and was stored at 4°C.

Experimental design

Two separate experiments were conducted. The experimental designs
were identical except that the ﬁnal behavioral test in Experiment 1 assessed
sexual behavior with a receptive female whereas in Experiment 2 it assessed
agonistic behaviors with an intruder male. At 27 days of age, gonad-intact male
hamsters were randomly divided into AAS-treated or vehicle-treated groups.
These males received a daily subcutaneous (sc) injection of either the AAS
cocktail (2 mglkg TC, 2 mglkg ND, 1 mglkg BU) or vehicle (2-hydroxypropyl-B-
cyclodextrin), either during adolescence (from 27-41 days of age) or during
adulthood (from 63 -77 days of age). Group sizes were originally n=10lage and
treatment group in Experiment 1 (assessment of sexual behavior) and n=15/age

and treatment group in Experiment 2 (assessment of aggressive behavior). Body

31

weight for each animal was obtained daily in order to calculate the injection
volume required to administer the desired dose. Volume ranged from 0.1 ml to
0.25 ml over the two-week course of administration across age groups. Tests for
either sexual or aggressive behaviors were conducted one day after the last
injection, at 42 days of age for males treated during adolescence and at 78 days
of age for males treated as adults. All behavioral interactions were videotaped
with a low-light color video camera and were scored blind using a computer
program that tags each behavioral code with an elapsed time (software
generously provided by Dr. Kim Wallen, Emory University, Atlanta, GA). This

system allows the calculation of counts, duration, and latency for each behavior.

Sexual Behavior Tests (Experiment 12

Males were sexually inexperienced prior to a standardized behavior test
with a receptive female. Tests were conducted between 1700 and 2030 h EST
under red-light illumination, as described in previous studies (Heise et al., 2003;
Romeo et al., 2002; Romeo and Sisk, 2001; Schulz et al., 2004). Following a 5
min acclimation to the test chamber (a 51 x 26 x 31.5 cm glass aquarium with a
mirror underneath), males interacted with a stimulus female for 15 min.
Ovariectomized stimulus females were hormonally primed to induce receptivity
with an estradiol benzoate injection (10 pg in 0.05 ml sesame oil) 48 h before,
and a progesterone injection (500 pg in 0.1 ml sesame oil) 4 h before behavior
testing. Before the 15 min behavior test with the subject male, stimulus females

were tested for receptivity with a sexually experienced stud male. Anogenital

32

investigation of the female, mounts, intromissions, and ejaculations were scored
and quantiﬁed as previously described (Schulz et al., 2004). Data from two
adolescent and two adult subjects were excluded because females did not
remain receptive throughout the 15 minute test. Data from three other
adolescent and adult males were excluded because of technical difﬁculties
during videotaping of the behavioral tests. Two adult AAS-treated animals died
during treatment. Thus, ﬁnal sample sizes for Experiment 1 were: adolescent
vehicle-treated, n=7; adolescent AAS-treated, n=8; adult vehicle-treated, n=9;

adult AAS-treated, n=7.

Agonistic behavior tests (Experiment 2]

Animals were tested for agonistic behaviors using a resident-intruder
paradigm. The socially na'I've males were tested between 1700 and 2030 h EST
in a red-light illuminated room. A plexiglass extension was placed in the home
cage of the subject (AAS- or vehicle-treated) in order to make the walls of the
cage taller. After a 5 min acclimation period, an age- and weight-matched
intruder male was placed in the treated male’s home cage. Intruder males were
purchased from the same vendor and arrived at the same time as the subject
males. Ten-min behavior tests were videotaped and scored for aggressive and

submissive behaviors.

The aggressive/dominant behaviors quantiﬁed during each behavior test

were deﬁned as follows:

33

(1) Flank marks: the subject rubbed its pigmented sebaceous ﬂank gland
against the wall of his home cage. In male-male social encounters, ﬂank marking
serves to communicate dominance status (Drickamer et al., 1973; Ferris et al.,
1987; Johnston, 1975).

(2) Total contact time: the length of time the subject male approached the
intruder male and showed interest by snifﬁng or remaining in close proximity.

(3) Attacks: the subject moved quickly toward the intruder male to bite or
to attempt to bite. The pair either tumbled around or the treated male chased the
intruder male.

(4) Bites: The subject male bit the intruder.

(5) Offensive posturing: the subject stood upright and faced the intruder
with his front paws raised.

Defensive/Submissive behaviors were deﬁned as follows:

(1) Defensive posturing: the two males approached each other and the
subject male twisted sideways with his paw or paws stretched toward the other
male to ward off an attack.

(2) Tail-up walking: the subject walked around the home cage with his tail
raised and back arched. This behavior was usually exhibited in response to the
intruder male snifﬁng or investigating the hindquarters and communicated
submissive status.

(3) Escape dashes: a rapid movement away from the intruder male, which
occurred only after some form of social interaction, indicating that the subject

was fearful of the intruder male.

34

To assess overall aggressiveness of each subject, a composite
aggression/dominance score was obtained by adding the number of ﬂank marks,

attacks and bites, and subtracting tail-up walking.

Two adult males were excluded from the statistical analyses of aggressive
behaviors because of extremely low levels of social interaction during the test;
they were determined to be outliers by the Dixon outlier test. In addition, two
males assigned to the adult vehicle-treated group were not used because of poor
health prior to the treatment period. Data from six other males were excluded
because of technical difﬁculties during videotaping of the behavioral tests. Thus,
ﬁnal sample sizes for Experiment 2 were: adolescent vehicle-treated, n = 13;
adolescent AAS-treated, n = 14; adult vehicle-treated, n = 11; adult AAS-treated,

n=12

Physiological Measures

Immediately following the behavior test, animals were administered an
overdose of sodium pentobarbital (130 mglkg i.p.) and a terminal blood sample
was taken via cardiac puncture. Testes and seminal vesicles were removed and
weighed. At the time of sacriﬁce, ﬂank gland diameters were assessed by
shaving the hindquarters and measuring the gland with a caliper.
Plasma testosterone concentrations were measured in duplicate using a
commercially available radioimmunoassay kit (Coat-a-Count Total Testosterone,

Diagnostic Products, Los Angeles, CA). This assay has been previously

35

validated for measuring endogenous plasma testosterone levels in hamsters in
our laboratory (Parﬁtt et al., 1999). The intra-assay coefﬁcient of variation (CV)
was 5.9%, and the limit of detectability was 0.1 ng/ml. We determined whether
the assay detected exogenously administered AAS by using the Coat-a-Count
Total Testosterone kit to assay known amounts (100 pl, 10 DI and 1pl) of the
cocktail. The assay detected only 0.15% of the anabolic steroids added to the

assay tube.

Plasma luteinizing hormone (LH) levels were determined in duplicate
samples by a double antibody RIA using reference preparation RP-3. Reagents
in the rat LH kit were obtained from the National Institute of Diabetes and
Digestive and Kidney Diseases (courtesy of Dr. A.F. Parlow). This assay has
been validated for measurement of LH in the Syrian hamster (Richardson et al.,
2002). The intra-assay CV was 8.8% and the lower limit of detectability was 0.8

ng/ml.

Statistics

2-by—2 ANOVAs were used to determine main effects and interactions between
age at treatment (adolescent or adult) and treatment (vehicle or AAS treated) on
aggressive and reproductive behaviors, hormone levels, testes weights, seminal
vesicle weights, and ﬂank gland diameters. To probe signiﬁcant interactions,
simple main effects analyses were performed to compare vehicle- and AAS-

treated groups within each age. The Dixon outlier test was used to determine

36

any behavioral outliers, and Student’s t-tests were used to compare treatment
groups within adolescent and adult males. Chi-square analyses were performed
to determine whether there were differences in the proportion of vehicle- and
AAS-treated males that showed defensive/submissive behaviors. All differences
were considered signiﬁcant if they resulted in a p s 0.05. Data in all ﬁgures are

reported as group means +/- SEM.

Results
Experiment 1: The effects of AAS on sexual behaviors in adolescent and adult
male Syrian hamsters

AAS did not affect the amount of time that either adolescents or adults
spent investigating the receptive female (data not shown). There was a
signiﬁcant interaction between treatment and age on the number of mounts [F (1,
27) = 17.172; P < 0.0002], intromissions [F (1, 27) = 7.449; P < 0.01], and
ejaculations [F (1, 27) = 15.005; P < 0.0006]. In general, AAS facilitated
reproductive behavior in adolescent hamsters and reduced reproductive behavior
in adults (Fig 2.1). Post-hoc analyses showed a trend toward increased number
of mounts and a signiﬁcant increase in the number of intromissions [F (1, 13) =
7.482; P = 0.01] and ejaculations [F(1, 13) = 7.316; P < 0.02] in AAS-treated
adolescents relative to vehicle-treated adolescents. In contrast, AAS-treated
adults showed signiﬁcantly fewer mounts [F (1, 14) = 17.256; P < 0.001],
intromissions [F (1, 14) = 5.716; P < 0.05] and ejaculations [F (1, 14); P < 0.015]
compared to vehicle-treated adults. The same pattern of results was found for

latencies to show sexual behavior in that AAS treatment reduced latencies in

37

adolescents and lengthened latencies in adults. Thus, there were signiﬁcant
interactions between treatment and age on latency to mount [F (1, 27) = 10.773;
P < 0.003], intromit [F (1, 27) = 14.332; P < 0.001], and ejaculate [F (1, 27) =

8.646; P <0.007] (data not shown).

Mment 2: The effects of AAS on agonistic behaviors in adolescent and adult
_m_ale Syrian hamsters
Aggressive/Dominant Behaviors

ANOVAs revealed a signiﬁcant interaction between age and treatment on
overall aggression scores [F (1,46) = 7.417; P < 0.009]. Post-hoc analyses
showed that AAS treatment signiﬁcantly increased the overall aggression score
in adolescents [F (1, 25) = 26.809; P < 0.001], but not in adults [F (1, 21) = 2.781;
P < 0.1] (Fig 2.2). Signiﬁcant interactions between age and treatment were also
observed when individual aggressive behaviors were analyzed [number of
attacks [F (1,46) = 5.573; P < 0.03], number of bites [F (1,46); P < 0.0001], and
attack duration [F (1, 45) = 6.510; P < 0.02]. Post-hoc tests determined that AAS
treatment signiﬁcantly increased the number of attacks [F (1, 25) = 31.750; P <
0.0001)], number of bites [ F (1, 25) = 27.629; P < 0.0001], and attack duration [F
(1, 25) = 17.240; P < 0.0003] in adolescents. In contrast, none of these

measures were signiﬁcantly increased by AAS treatment in adulthood (Fig 2.3).

A two-way ANOVA showed a signiﬁcant interaction between age and

treatment [F (1,46) = 7.089; P < 0.01] on ﬂank marking (Fig 2.4). Although both

38

AAS-treated adolescents [ F (1, 25) = 1.25; P , 0.5] and adults [ F (1, 20); P <
0.05] displayed signiﬁcantly more ﬂank marks compared to vehicle-treated
males, the signiﬁcant interaction was due to an apparently greater response by
AAS-treated adolescent males (Fig 2.4). Two-way ANOVA revealed a main
effect of age on offensive posturing [F (1, 46) = 4.528; P < 0.05]. This main
effect appeared to be driven by the greater display of offensive postures in AAS-

treated adults (Fig 2.5).

Defensive/Submissive Behaviors:

Two-way ANOVAs revealed no effects of treatment or age nor any
interactions on defensive behaviors. This was primarily because only
occasionally did AAS-treated adolescent males even show these behaviors, and
in addition, within group variability of the other treatment groups was high. None
of the AAS-treated adolescent males displayed any escape dashes or tail-up
walking and only two AAS-treated adolescents displayed defensive posturing
(Table 2.1). Therefore, chi-square analyses were performed to determine
whether the proportion of males exhibiting defensive behaviors was different in
AAS-treated adolescents compared with the other three groups. The proportion
of vehicle-treated adolescents and vehicle-treated adults displaying tail-up
walking (P < 0.05) and escape clashes (P < 0.05) was signiﬁcantly greater than
the proportion of AAS-treated adolescents (T able 2.1). Similarly, the proportion
of AAS-treated adults exhibiting tail-up walking (P < 0.05) and defensive postures

(P < 0.05) was greater than that of AAS-treated adolescents. Only two AAS-

39

treated adults displayed escape dashes, and therefore this group was not

signiﬁcantly different from AAS-treated adolescents on this measure.

Ph ysiolggical Measures in Experiments 1%

In Experiment 1 (sexual behavior test immediately before terminal blood
sample collection), we found that there was neither an effect of treatment nor age
on plasma LH levels (Fig 2.6A). In contrast, there was a main effect of treatment
on plasma T levels [F (1, 29) = 10.393; P < 0.003], where AAS treatment
signiﬁcantly reduced endogenous T levels in both adolescents and adults (Fig
2.6A). In Experiment 2 (aggression test immediately before terminal blood
sample collection), there were no effects of treatment or age on either plasma LH

or T concentrations (Fig. 2.68).

Main effects of both age and treatment were found on testes weight,
seminal vesicle weight and flank gland diameter (Table 2.2). As expected, in
both experiments, testes and seminal vesicle weights and ﬂank gland diameters
were all larger in adults compared with adolescents. In males tested for sexual
behaviors (Exp. 1), testes [ F (1, 29) = 22.053; P < 0.0001] and seminal vesicle [
F (1, 29) = 20.155; P < 0.0001] weights were less in AAS-treated than in vehicle-
treated males. In contrast, AAS treatment increased ﬂank gland diameter [F (1,
29) = 5.530; P < 0.03]. A similar pattern was seen in males tested for agonistic
behaviors (Exp 2), where AAS-treatment reduced testes [ F (1, 47) = 11.147; P <

0,002], seminal vesicle [ F (1,47) = 3.805; P < 0.05] weights, and led to a trend

40

towards increased ﬂank gland diameter [F (1,47) = 2.893; P < 0.1). Males were
randomly assigned to treatment groups and there were no statistical differences
in their body weights at the beginning of treatment. At the end of the treatment
period in Experiment 2, however, mean body weight of AAS-treated adolescent
and adults males was less than the corresponding vehicle-treated males at each

age (P < 0.005; Table 2.2).

Discussion

These experiments demonstrate that adolescent males treated with a
cocktail of testosterone cypionate, boldenone undecylenate and nandrolone
decanoate show increased levels of aggressive and reproductive behaviors when
compared to both vehicle-treated adolescents and AAS-treated adults. This is the
ﬁrst direct comparison of the behavioral effects of anabolic steroid exposure at
two different developmental stages, adolescence and adulthood, and in two
different social contexts, sexual interactions with a receptive female or agonistic
interactions with a male of similar age and weight. The different behavioral
responses to anabolic steroids in adolescence and adulthood most likely reﬂect
structural and functional differences in the neural Circuits underlying social

behaviors at these two stages of life.

Normally, the postnatal maturation of reproductive behaviors in male
Syrian hamsters is a function of the increase in circulating testicular hormones

that occurs with the onset of puberty. Previous work from this laboratory has

41

demonstrated that male reproductive behavior cannot be activated by
testosterone, dihydrotestosterone or estrogen in prepubertal animals (Meek et
al., 1997; Romeo et al., 2002) and that the pubertal rise in testosterone organizes
(further masculinizes and defeminizes) neural circuits to enhance or maximize
the activation of reproductive behavior in adulthood (Schulz et al., 2004). In the
present experiment, adolescent exposure to AAS resulted in levels of sexual
behavior comparable to those typically observed in gonad-intact adult hamsters,
demonstrating that behavioral responsiveness of adolescent males is greater
than that of prepubertal males. Adolescent males may also be more responsive
than adults to the activational effects of supraphysiological levels of androgens,
since in this experiment AAS treatment increased sexual behavior in
adolescents, but not adults. In addition, because endogenous testosterone
further masculinizes the nervous system during puberty (Schulz and Sisk, 2006),
it is possible that AAS “hypermasculinize” the adolescent brain, resulting in
enhanced activational responses in adolescent males compared with adults.
Organizational effects of AAS that enhance activational responses would not be
expected to occur in AAS-treated adults, since previous work has shown that the
potential for organization of sexual behavior is greatly diminished after puberty
and adolescence (Schulz and Sisk, 2006). In fact, we found that AAS in
adulthood decreases sexual behavior. Similar reductions in sexual behavior in
response to exposure to supraphysiological levels of androgens have been
reported in rats (Farrell and McGinnis, 2003) and hamsters (Meek et al., 1997).

The decrement in sexual behavior induced by high levels of exogenous androgen

42

in adulthood may be secondary to a general suppression of endogenous
hypothalamic-pituitary-gonadal axis function. If so, then the current experiments
indicate that the adolescent brain is either less responsive to these inhibitory
effects, or that there are other mechanisms that override them to result in

enhancement of sexual behavior by AAS in adolescence.

Aggressive behavior in Syrian hamsters is not under strong activational
control by endogenous testosterone. In spite of this fact, adolescent animals
treated with AAS in the present study showed higher levels of aggressive
behaviors relative to controls, and spent at least one-third of the 10 minute test
attacking their opponent, similar to previous reports of effects of AAS on
aggression in adolescent hamsters (Grimes et al., 2006; Melloni et al., 1997;
Melloni and Ferris, 1996). In addition, AAS-treated adolescent males in the
present study did not express submissive behaviors such as defensive posturing,
escape dashes, or tail-up walking. In contrast to the heightened aggression
observed in AAS-treated adolescent males, AAS treatment in adulthood did not
result in increased levels of overt aggression, consistent with our previous
ﬁndings that aggressive behavior is not facilitated by testosterone in adulthood
(Romeo et al., 2003). Testicular hormones organize ﬂank marking behavior
during adolescence in male Syrian hamsters (Schulz et al., 2006). Interestingly,
both adolescent and adult males showed increased levels of ﬂank marking
behavior after AAS-treatment suggesting that this behavior may be organized

and activated during early to mid adolescence. In general, AAS-treated adults

43

communicated dominance by expressing threatening behaviors (e.g., offensive
posturing) and ﬂank marking behavior, rather than by overt aggression. Rats
treated with an anabolic steroid throughout adolescence and tested for
aggressive behaviors during adulthood also display an increase in threatening
behaviors (Farrell and McGinnis, 2003). Thus, the neural mechanisms that in
adults favor display of communicative agonistic behaviors (offensive posturing,
ﬂank marking) over overt aggression (bites, attacks) appear to be either not
engaged or overridden in adolescent males, which in the presence of AAS show

heightened overt aggression.

The heightened sexual and aggressive behaviors observed with
adolescent exposure to AAS may be indicative of either hyper-responsivity to
steroids or to a relative lack of inhibition of the underlying neural circuits at this
stage of development (Forbes and Dahl, 2005; Yurgelun-Todd, 2007). The
neural circuitries underlying sexual and agonistic behaviors in the male hamster
have both overlapping and distinctive components (KolIack-Walker and Newman,
1995). The medial amygdala, lateral septum, and bed nucleus of the stria
terminalis (BNST) are common elements of the two circuits, while the medial
preoptic area and anterior hypothalamus are areas selectively linked to sexual or
agonistic behaviors, respectively (Ferris and Delville, 1994; Swann, 1997; Wood
and Coolen, 1997; Wood and Swann, 2005). Most of these structures have
direct or indirect connections with cognitive control and reward areas, e.g.

nucleus accumbens and prefrontal cortex (Cunningham et al., 2002; Ernst et al.,

44

2006; Yurgelun-Todd, 2007). This study suggests that adolescent males may
not be able to regulate the expression of their social behaviors and/or have
atypical expression of behavior after AAS exposure when compared to adult
males. Our results ﬁt with previously reported relationships between high levels
of endogenous testosterone and heightened aggression and the importance of
context in the display of social behaviors in human adolescents (Davey et al.,

2007; Rowe et al., 2004).

Endogenous testosterone exerts negative feedback on GnRH and
gonadotropin release (Sisk and Foster, 2004). Therefore, AAS would also be
expected to exert negative feedback effects on LH secretion, and in fact a
previous report found that 8 weeks of testosterone cypionate treatment reduced
LH and testosterone levels in adult male rats (Feinberg et al., 1997; McGinnis,
2004). Therefore, it was surprising that AAS cocktail used in the present studies,
which contained testosterone cypionate, nandrolone decanoate and boldenone
undecylenate, did not result in a general suppression of hormone levels. Instead,
the type of social interaction that occurred immediately before blood sample
collection appeared to inﬂuence hormone levels more robustly than either AAS or
age. Males tested with a receptive female had LH levels that were on average
3-4x higher and T levels that were 2-3x higher than those of males tested with an
intruder male. This finding suggests that the social interaction acutely overrides
direct effects of AAS on the h-p-g axis. In general, LH secretion increases in

adult male hamsters after exposure to female pheromones, so these ﬁndings

45

were not too surprising (Richardson et al., 2004). The fact that AAS treatment
was associated with reduced T levels in males tested with a female does provide
some evidence for negative feedback inﬂuences of AAS. This is further
supported by the reduction in weight of the testes and seminal vesicles in AAS-
treated males at both ages in both experiments. Thus, unlike their behavioral
responses, peripheral tissues appear to be similarly responsive to AAS in

adolescent and adult males.

Hormonal facilitation of reproductive and aggressive behaviors includes
intracellular actions of both androgens and estrogens (Bodo and Rissman, 2006;
Nelson and Trainor, 2007; Romeo et al., 2002; Vagell and McGinnis, 1998).
Therefore, the mechanisms by which AAS inﬂuence neurotransmitter systems
and behavior are likely to include regulation and activation of nuclear androgen
(AR) and estrogen receptors (ER). All three androgens in the cocktail used in
these experiments are aromatizable to estrogenic metabolites that are known to
have effects on both the central nervous system and the periphery (Clark and
Henderson, 2003). When given to adult male rats, this cocktail upregulates AR
immunoreactivity widely throughout the brain (Menard and Harlan, 1993). Self-
administration of testosterone induces AR and ER immunoreactivity in steroid-
sensitive regions such as the medial amygdala and preoptic area in adult male
Syrian hamsters (Dimeo and Wood, 2005). Given that these areas are receptor
rich and that there are differential effects of AAS on adolescent and adult males,

one can hypothesize that AR and ER expression may be differentially inﬂuenced

46

by anabolic steroids in adolescent and adult males. Further studies are needed

to determine the mechanisms by which these compounds affect social behaviors.

In the last decade, increased understanding that pubertal gonadal
hormones organize the adolescent brain has heightened interest in discovering
how AAS affect the adolescent brain and behavioral maturation. This study was
the ﬁrst to directly compare males treated with anabolic steroids during
adolescence and adulthood, and we found signiﬁcant differences in the direction
and magnitude of AAS effects on sexual and aggressive behavior. The particular
cocktail used in this study is comparable to what a heavy or chronic anabolic
steroid user would use to enhance muscle growth and athletic performance
(Menard and Harlan, 1993). An increase in body image concerns during
adolescence, along with a decrease in the perceived risk of taking AAS has
resulted in increased use of AAS among US teenagers. lmportantly, through
neuroimaging and behavioral studies early adolescence has been shown to be a
sensitive period of neural, cognitive and emotional development. Our data
underscore the need for further research on age-speciﬁc the effects of AAS on
brain circuitry and organization throughout development and whether AAS use

during adolescence has enduring consequences on social behaviors.

47

Number of Mounts

 

 

 

 

Adolescent
B 30 — *

Number of Intromissions
a:

 

 

 

Adolescent

*

Number of Ejaculations

 

 

Adolescent

 

 

 

 

Adult

I: Vehicle-treated
I AAS-treated

 

Adult

Figure 2.1 AAS affect male sexual behaviors differently in adolescent and adult
hamsters. Two-way ANOVA revealed age by treatment interactions in which,
relative to age-matched vehicle-treated adolescents (n=8) and vehicle-treated
adults (n=9), AAS-treated adolescents (n=7) displayed more (A) mounts, (B)
intromissions, and (C) ejaculations, whereas these behaviors were decreased in
AAS—treated adults (n=7). Asterisk indicates signiﬁcant difference (p<0.05)
between AAS- and vehicle treatment within an age as determined by post hoc

analysis.

48

 

45'
40'
35"
30‘
25‘
20‘
15‘
10

[:I Vehicle-treated
I AAS-treated

Aggression Score

 

 

   

 

 

 

Adolescent Adult

Figure 2.2 AAS treatment increases aggression in adolescents but not in adults.
Two-way ANOVA revealed an age by treatment interaction on a global
aggression score [(flank marks + attacks + bites) — (tail-up walking)]. Sample
sizes: vehicle-treated adolescent, n=13; AAS-treated adolescent, n=14; vehicle-
treated adult, n=11; AAS-treated adult, n=13. Asterisk indicates signiﬁcant
difference (p<0.05) between AAS- and vehicle treatment within an age as
determined by post hoc analysis.

49

 

   

 

 

 

 

 

 

 

*
A 16
‘9 14
0 12
g 10
"6 8
h
3 e
E 4
Z 2
Adolescent
B *
20 -
w u
.2 15 - CI Vehicle-treated
m
“5 I AAS-treated
h- 10 _
.8
E
2 5—
Adolescent Adult
C 3- *
E 2.5
E
.5 2'
g 1 5
Q
s 1-
III
3:.
< .5‘
Adolescent MU“

Figure 2.3 AAS affect male aggressive behaviors differently in adolescent and
adult hamsters. Two-way ANOVA revealed age by treatment interactions on (A)
attacks, (B) bites, and (C) attack durations (min). Asterisk indicates signiﬁcant
difference (p<0.05) between AAS- and vehicle treatment within an age as
determined by post hoc analysis.

E] Vehicle-treated
I AAS-treated

 

Number of Flank Marks
0)

    

 

 

 

Adolescent Adult

Figure 2.4 AAS increase ﬂank marking in both adolescent and adult males.
Two-way ANOVA revealed a signiﬁcant effect of treatment. Asterisk indicates
signiﬁcant difference (p<0.05) between AAS- and vehicle treatment within an age
as determined by post hoc analysis.

 

 

[:I Vehicle-treated
I AAS-treated

 

 

Number of Offensive Pastures

 

 

 

 

Adolescent Adult

Figure 2.5 Adults engage in more offensive posturing than adolescents during a
male-male social interaction. Two-way ANOVA revealed a signiﬁcant main effect
of age (asterisk).

51

Table 2.1 AAS-treated adolescent males were less likely to display submissive
behaviors than vehicle controls and adult males.

 

 

 

 

Submissive Adolescent Adolescent - Adult - Adult - AAS
Behavior — Vehicle AAS treated Vehicle treated
(n=13) E14) (n=11) m=12)
Tail Up 3:I:1.5 0 3.4:I:1.9 1.81:1.2
Walking (5/13) * (0/14) (3/1 1) * (3/12) *
Escape 3.5 i 1.8 0 6 :t 3.2 4.3 :I: 3.1
Dashes (4/13) * (0/14) (4/1 1) * (2/12)
Defensive 4.3 :I: 1.6 0.3 i 0.2 7 i 2 5.2 :I: 1.5
posturing (7/13) * (2/14) (5/1 1) (6/12)*

 

 

 

** Chi-square analysis, p < 0.05

52

A — Males tested for Reproductive Behaviors

 

 

    

9‘ 4 i '
0E 8‘ E 35
c \ -

7‘1
E? 23’ 3
v .
on 6 92
IE 5- cad)

Cd

or; In;
.E—l 4‘ 24
N ”In
'E‘“ 3- ID
.65 I—E
cum 4 1’0
:52 2 2
A]; 1_ D.

 

     

 

 

Adolescent Adolescent Adult

B - Males tested for Aggressive Behavior

- re
d in N in

LuteInIzmg Hormone
Plasma Levels (ng/ml)
'u.

Testosterone
Plasma Levels (ng/ml)

 

Adolescent Adult

Figure 2.6 Plasma luteinizing hormone (LH) and testosterone (T) concentrations
immediately after each behavior test. (A) In males tested with a receptive female
(A), there were no effects of age or treatment on LH concentrations, but AAS
decreased T concentrations in both AAS-treated adolescent and adult males
(main effect of treatment, p < 0.05). (B) In males tested with another male for
aggressive behaviors, there were no effects of age or treatment on either LH or T
concentrations. Asterisk indicates signiﬁcant difference (p<0.05) between AAS-
and vehicle treatment within an age as determined by post hoc analysis.

53

Table 2. Peripheral measures in vehicle- and AAS-treated adolescent and adult
males.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Adolescent Males Adult Males
Vehicle AAS-treated Vehicle AAS-
Experiment treated (Mean +l- Treated treated
(Mean +/- SEM) (Mean +/- (Mean +/-
SEM) SEM) SEM)
Testes WeiLht (g)
1 2.31 :I: .04 2.0 i .04* 3.85 :t .13 3.29 :I: .10*
2 2.06 i: .07 1.70 i: .07* 3.86 :I: .09 3.67 t .07*
Seminal Vesicle Weight (g)
1 0.167 :I: .01 0.114 :I: .02* 0.344 :I: .02 0.273 d:
.02*
2 0.21 :I: .01 0.16 :t .01* 0.36 :I: .03 0.32 :I: .03"
Flank Gland Diameter (mm)
1 6.77 :I: .33 8.24 :I: .34* 7.6 :I: .40 7.75 :I: .19*
2 7 :I: .3 7.9 :I: .3 8.5 i .26 8.5 :I: .25
Body Weight - beginning of treatment period )
1 56.41 :I: 1.12 59.49 :t 2.0 106.14 :I: 4.17 107 :I: 2.03
2 49.67 :I: 1.0 47.23 1: 1.6 107.78 :I: 2.6 108.61 :t
1.9
Body Weight - end of treatment period (9)
1 93.36 :I: 2.33 90.10 :I: 1.81 113.32 :I: 4.83 111.39 1:
2.36
2 86.9 11.8 81.8 :I: 2.1* 120.53 i 2.9 116.0 :I:
2.1*

 

 

 

 

 

 

* main effect of treatment in two-way ANOVA, p<0.05

54

CHAPTER 3: ADOLESCENT ANDROGENIC ANABOLIC STEROID
EXPOSURE ENHANCES AGGRESSIVE BEHAVIORS AND REDUCES THE
POTENTIAL TO REACH SATIETY AFTER DISCONTINUATION OF
TREATMENT
Introduction

The transition from childhood to adulthood during puberty and
adolescence comprises reproductive, neural, and behavioral maturation (Lenroot
et al., 2007; Sisk and Foster, 2004). The brain undergoes dramatic remodeling
during adolescence, and endogenous steroid hormones inﬂuence this
developmental process (Nelson et al., 2005; Sisk and Zehr, 2005). Thus, the
adolescent brain may be particularly vulnerable to the adverse consequences of
anabolic steroid use. Anabolic androgenic steroids (AAS) are synthetic
derivatives of testosterone used to enhance athletic performance and physical
appearance; increased use has been reported among adolescents. AAS
inﬂuence mood and behavior, including heightened aggression and increased or
decreased libido in humans (Bahrke and Yesalis, 2004; Yesalis and Bahrke,
2000; Yesalis and Bahrke, 2002; Yesalis et al., 2000). Although some AAS can
be detected up to 12-wks after withdrawal, few studies have focused on

understanding the long-term effects of AAS on the adolescent brain and behavior

(Ballard and Wood, 2005).

Androgens play a critical role in the pubertal maturation of social
behaviors. The absence of gonadal hormones during puberty results in long-

lasting impairments in steroid-dependent social behaviors in adulthood (Schulz et

55

al., 2004), suggesting that steroid hormones in adolescence program the
expression of sexual behaviors in adulthood. In addition, sexual experience in
adulthood does not compensate for a lack of testosterone during adolescence to
bring forth adult-typical levels of behavior (Schulz et al., 2004). These ﬁndings
also generalize to the expression of ﬂank marking behavior in the male Syrian
hamster (Schulz, et al 2007), suggesting that androgens during adolescence are
critical for the expression of social behaviors in adulthood. While these studies
show that the absence of testosterone during adolescence impairs social
behaviors in adulthood, relatively little is known about the long term effects of
exposure to pharmacological levels of testosterone or other androgens on adult

male social behaviors.

Exposure of male hamsters to a cocktail of AAS during adolescence
increases the expression of sexual and aggressive behaviors immediately after
treatment (Farrell and McGinnis, 2003; Melloni et al., 1997). In addition, AAS
affect aggression and sexual behaviors in other rodent models, such as rats and
mice, with speciﬁc effects depending on the particular compound and dose used
and age at treatment (Clark and Henderson, 2003; McGinnis, 2004).
Collectively, these studies show that the adolescent brain responds differently
than the adult brain to exogenous hormones, suggesting that the adolescent

brain is still developing and vulnerable to perturbations in steroid milieu.

56

A direct comparison between adolescent and adult exposure of the long-
term consequences of AAS has yet to be done. Single AAS compounds, like
testosterone, nandrolone, and stanozolol, affect aggressive behaviors up to 17
wks after discontinued treatment and reproductive behaviors up to 9 wks in intact
male rats exposed throughout adolescence (Farrell and McGinnis, 2004).
However, 19 days after adolescent exposure with a cocktail, the effects of
treatment on offensive behaviors did not persist in intact male Syrian hamsters
(Grimes et al., 2006). These results suggest that at least some of the alterations
in androgen-dependent behaviors by adolescent AAS exposure can persist after

drug exposure.

The goal of these experiments was to directly compare the long-term
behavioral consequences of AAS exposure during adolescence or adulthood.
Therefore, sexual and aggressive behavior was assessed either two or four
weeks after discontinuation of treatment with AAS or vehicle, where treatment
occurred either during adolescence or in adulthood. Because the adolescent
brain is more responsive to organizational effects of testosterone than the adult
brain (Schulz, et al 2007), the tested hypothesis was that adolescent, but not
adult AAS exposure exerts long-lasting changes in the nervous system, leading
to the prediction that AAS exposure during adolescence, but not in adulthood, will

result in effects on behavior that persist beyond the period of exposure to AAS.

57

Materials and Methods
Animals

Eighteen-day-old male Syrian hamsters (Mesocricetus auratus) were
obtained from Harlan Sprague—Dawley Laboratories (Madison, WI). Upon arrival
males were housed in groups of eight animals per cage (polycarbonate, 33 x 38
x 17 cm) with ad libitum access to food (T elkad Rodent Diet No. 8640, Harlan)
and water. Animal colony temperature was maintained at 22 i 2°C, and animals
were kept on a light-dark cycle of 14:10 L:D (lights off at 1600h EST). At 25 days
of age, animals were singly housed in polycarbonate cages (30.5 x 10 x 20 cm,
for Experiment 1; 33 x 38 x 17 cm, for Experiment 2). All animals were treated in
accordance with the NIH Guide for the Care and Use of Laboratory Animals, and
all protocols were approved by the Michigan State University All-University

Committee for Animal Use and Care.

AAS Treatment

A cocktail of AAS containing 0.8 mglml of testosterone cypionate (TC;
Sigma Aldrich, Inc., St. Louis, M0), 0.8 mglml of nandrolone decanoate (ND;
Sigma Aldrich, Inc.), and 0.4 mg/ml of boldenone undecylenate (BU; Steraloids,
New Port, Rhode Island) was dissolved in 2-hydroxypropyl-B—cyclodextrin

(Sigma-Aldrich, Inc.). The AAS cocktail was prepared the day before treatment

began for each age group in each experiment, and was stored at 4°C.

58

Experimental design

Two separate experiments were conducted to understand the long-term
effects of AAS on males treated during adolescence or in adulthood. Experiment
1 tested treated males with a receptive female to assess behavioral changes in
sexual behavior; whereas in Experiment 2 treated males were tested with an

intruder male to test for agonistic behavior.

On 25 days of age males were singly housed. At 27 days of age, gonad-
intact male hamsters were randomly divided into AAS-treated or vehicle-treated
groups. These males received a daily subcutaneous (sc) injection of either the
AAS cocktail (2 mglkg TC, 2 mglkg ND, 1 mglkg BU) or vehicle (2-hydroxypropyl-
B-cyclodextrin), either during adolescence (from 27-41 days of age) or during
adulthood (from 63 -77 days of age). Group sizes were originally n= 8-10/age
and treatment group in Experiment 1 (assessment of sexual behavior) and n=13-

15/age and treatment group in Experiment 2 (assessment of agonistic behavior).

The volume of each daily injection was determined according to the male’s
body weight. The volume of the injections ranged from 0.1 ml to 0.25 ml over the
two-week course of administration across age groups. Tests for either sexual or
aggressive behaviors were conducted either two or four weeks after the last
injection, at 56 (two-wks) or 70 days (four-wks) of age for males treated during
adolescence and at 92 (two-wks) or 106 (four-wks) days of age for males treated

as adults. Separate groups of animals were tested 2 and 4 weeks after

59

discontinuation of AAS or vehicle treatment. A repeated measures design was
not used in order to avoid a potential confound and additional source of variability
from prior social experience. All behavioral interactions were videotaped with a
low-light color video camera and were scored blind using a computer program
that tags each behavioral code with an elapsed time (software generously
provided by Dr. Kim Wallen, Emory University, Atlanta, GA). This system allows

the calculation of counts, duration, and latency for each behavior.

Experiment 1: Sexual Behavior

Males were sexually inexperienced prior to a standardized behavior test
with a receptive female. Tests were conducted between 1700 and 2030 h EST,
one-hour after “lights-out”, under red-light illumination, as described in previous
studies (Heise et al., 2003; Romeo et al., 2002; Romeo and Sisk, 2001; Schulz et
al., 2004). Following a 5 min acclimation to the test chamber (a 51 x 26 x 31.5
cm glass aquarium with a mirror underneath), males interacted with a stimulus
female for 15 min. Males were tested either two- or four- wks after AAS or

vehicle treatment during adolescence (P56 or P70) or adulthood (P92 or P106).

Ovariectomized stimulus females were hormonally primed to induce
receptivity with an estradiol benzoate injection (10 pg in 0.05 ml sesame oil) 48 h
before, and a progesterone injection (500 pg in 0.1 ml sesame oil) 4 h before
behavior testing. Prior to the 15 min behavior test with the subject male, stimulus

females were tested for receptivity with a sexually experienced stud male.

60

Anogenital investigation of the female, mounts, intromissions, and ejaculations
were scored and quantiﬁed as previously described (Schulz et al., 2004).

Satiety and latency to reach satiety were also assessed by quantifying long
intromissions. These are deﬁned as multiple rhythmic thrusts, about 30 seconds
long, performed by subject males after ﬁve to eight ejaculations and followed by
genital grooming as they approach satiety (Arteaga and Morali, 1997; Arteaga et
al., 2000). The efﬁciency rate was also determined for each male by dividing the
number of ejaculations by the sum of mounts and intromissions. Data from three
males were excluded because no seminal vesicles were found the day of
behavior testing which would skew the data gathered from peripheral tissues.
Data from two adolescent and three adult subjects were excluded because
females did not remain receptive throughout the 15 minute test, which resulted in
atypical behavior of both male and female hamsters. Data from four other
adolescent and adult males were excluded because of technical difﬁculties
during videotaping of the behavioral tests (e.g. the tape ran out during testing).
Thus, a total of 68 males were tested, and the ﬁnal sample sizes in Experiment 1
were: adolescent vehicle-treated tested on P56, n=8, and tested on P70, n = 10;
adolescent AAS-treated tested on P56, n=7, and tested on P70, n=9; adult
vehicle-treated tested on P92. n=8, and tested on P106, n=9; adult AAS-treated

tested on P92, n=9, and tested on P106. n=8.

61

Exmriment 2: Agonistic Behavior

Animals were tested for agonistic behaviors using a resident-intruder
paradigm. The socially naive males were tested between 1700 and 2030 h EST
in a red-light illuminated room. A riser made by cutting out the bottom part of the
polycarbonate cages was placed in the home cage of the subject (AAS— or
vehicle-treated) to make the walls of the home cage taller. After a 5 min
acclimation period, an age- and weight-matched intruder male was placed in the
treated male’s home cage. Intruder males were purchased from the same
vendor and arrived at the same time as the subject males. Males were tested
two- or four- wks after AAS or vehicle treatment during adolescence (P56 or P70)
or adulthood (P92 or P106). Ten-min behavior tests were videotaped and scored
for aggressive and submissive behaviors, as previously reported (Salas-Ramirez,
et al. 2007). Agonistic behaviors included in the analysis were: (1)
aggressive/dominant behaviors (ﬂank marking behavior, total contact time,
attacks, bites, and offensive posturing) and (2) defensive/submissive behaviors
(defensive posturing, tail-up walking, and escape dashes). In this experiment a
distinction was made between snifﬁng and a risk-assessment measure called
stretch-attend. It was deﬁned and scored as an approach made by the subject in
which the body is stretched and lowered, the hind legs stay planted and the front
legs stretch forward, head is out, eyes are on the opponent and the subject is

snifﬁng in the opponent’s direction (Albers, et al 2002).

62

To assess overall aggressiveness of each subject, a composite
aggression/dominance score was obtained by adding the number of ﬂank marks,

attacks and bites, and subtracting tail-up walking.

A total of 106 males were tested for agonistic behaviors in this experiment.
Two males treated during adolescence and four males treated during adulthood
were excluded from the statistical analyses of aggressive behaviors because of
extremely low levels of social interaction during the test; they were determined to
be outliers by the Dixon outlier test. In addition, two males assigned to the adult
vehicle-treated group were not used because their body weights were too low to
test with a weight- and age- matched intruder. Data from six other males were
excluded because of technical difﬁculties during videotaping of the behavioral
tests (e.g. tape ran out, cameras were moved during a test). Thus, ﬁnal sample
sizes for Experiment 2 were: adolescent vehicle-treated tested on P56, n =12,
and tested on P70, n=10; adolescent AAS-treated tested on P56, n = 14, and
tested on P70, n=10; adult vehicle-treated tested on P92, n = 11, and tested on

P106, n=13; adult AAS-treated tested on P92, n = 12, and tested on P105, n=10.

Physiological Measures

Immediately following the behavior test, animals were administered an
overdose of sodium pentobarbital (130 mglkg i.p.). Fifteen to twenty min after the
injection, a terminal blood sample was taken from each male tested. Testes and

seminal vesicles were removed and weighed. At the time of sacriﬁce, ﬂank gland

63

diameters were assessed by shaving the hindquarters and measuring the gland

with a caliper.

Plasma testosterone concentrations were measured in duplicate using a
commercially available radioimmunoassay kit (Coat-a-Count Total Testosterone,
Diagnostic Products, Los Angeles, CA). This assay has been previously
validated for measuring endogenous plasma testosterone levels in hamsters in
our laboratory (Parﬁtt et al., 1999). The intra-assay CV was 4.72 % in Experiment
1 and 5.81% in Experiment 2, and the limit of detectability was 0.1 nglml for both

experiments.

Statistics

Three-way ANOVAs were used to determine main effects and interactions
between age of exposure (adolescent or adult), time of testing post-treatment
(two or four weeks after discontinuation of treatment) and treatment (vehicle or
AAS treated) on aggressive and reproductive behaviors, testes weights, seminal
vesicle weights, and ﬂank gland diameters. In this two experiments time of
testing post-treatment did not affect the expression of behaviors, therefore
groups were collapsed to understand how age of AAS exposure and treatment
affected each behavior. Simple main effects analyses were performed to
compare vehicle- and AAS-treated groups within each age. The Dixon outlier
test was used to determine any behavioral outliers, which only occurred when a

male was tested with a non-receptive female or displayed little behavior when

64

tested with an age-and weight-matched male. In addition, one-way ANOVAs
were used to determine any behavioral differences between vehicle treated

subjects throughout development.

Results
Experiment 1: The long term effects of AAS on sexual behaviors after exposure

during adolescence or adulthood

A. Sexuaﬂehavior:

a. General Development of Adult Behavior:

An ANOVA determined that there were no signiﬁcant differences in the
expression of mounts and intromissions in vehicle-treated males tested with a

receptive female on P56, P70, P92 and P 106 (Figure 3.1).

b. Treatment effects:

AAS did not affect the amount of time that males spent in anogenital
investigation of the receptive female, the number of mounts, intromissions, or
ejaculations (data not shown). Interestingly, there was an overall main effect of
AAS on satiety measures. AAS treatment signiﬁcantly reduced the number of
long intromissions [F (1, 60) = 5.975; P < 0.02) and increased the latency to
display a long intromission [F (1, 60) = 5.922; P < 0.02) (Figure 3.2). Across the
other two variables (age of exposure, time of testing), subjects treated with AAS

took longer to reach satiety and performed fewer long intromissions.

65

8. Ph zsiological Measures

There were no effects of AAS on testes weight, seminal vesicle weight or
ﬂank gland diameter. However, as expected, there was a main effect of age of
exposure on the weight and size of the peripheral tissues (p < 0.05) (Table 3.1).

There were no effects of AAS treatment on plasma testosterone levels.

Experiment 2: The long-tenn effects of AAS on agonistic behaviors alter

exmsure during adolescence or adulthood

A. General Development of Agonistic behaviors
Agonistic behaviors can be classiﬁed into aggressive and submissive
behaviors. Unlike sexual behaviors, aggressive behaviors decrease signiﬁcantly
with age. A one-way ANOVA on vehicle-treated subjects showed that the
number of attacks is signiﬁcantly reduced over development [Figure 3.3A; F (3,
42) = 6.09; P < 0.002]. Interestingly, when comparing vehicle treated males, tail-
up walking, a typical submissive behavior, increases signiﬁcantly with age [Figure

3.33; F (3,42) = 7.26; P < 0.0005].

B. Aggressive/Dominant Behaviors

Adolescent AAS exposure increases the number of attacks and bites even
4-wks after discontinuation of treatment. A three-way ANOVA revealed a main
effect of AAS treatment by increasing the number of attacks [Figure 3.4A; F (1,

84) = 4.579; P < 0.05] and number of bites [Figure 3.4B; F (1, 84) = 4.496; P <

66

0.05) on adult male Syrian hamsters. Additionally, a main affect of age of
exposure was also found on number of attacks [F (1, 84) = 31.998; P < 0.0001]
and bites [F (1, 84) = 36.798; P < 0.0001), showing that males that were treated
during adolescence display an increase in aggressive behavior. Since time of
testing post-treatment did not affect the expression of aggressive behaviors,
behavioral data were collapsed across this variable, and a 2-way ANOVA was
performed in order to probe the effects of age of exposure and treatment. AAS
treatment resulted in increased aggression over vehicle controls regardless of
age of treatment. Since behavioral testing in this experiment was performed two
weeks or four weeks after AAS exposure, these results show persistent effects of
AAS on aggression that last beyond the period of drug exposure.

A critical behavior for establishing dominance in male hamsters is ﬂank
marking behavior. Again, an increase in ﬂank marking behavior was observed
after AAS exposure. Main effects of age of exposure [F (1, 84) = 10.53; P <
0.002] and treatment [F (1, 84) = 6.13; P < 0.02] were found by a three-way
ANOVA (Figure 3.5A). Again, since time of testing post-treatment did not affect
ﬂank marking behavior, data were collapsed across this variable and a two way
ANOVA was performed. As with the three-way ANOVA, there was a signiﬁcant
main effect of age of exposure, in which males treated with vehicle or AAS during
adolescence showed more aggression that males treated during adulthood.
those groups were collapsed to analyze effects of age of exposure and
treatment. AAS treatment resulted in increased ﬂank marking behavior over

vehicle controls independent of age of AAS exposure. Since behavioral testing

67

in this experiment was performed two weeks or four weeks after AAS exposure,

these results show persistent effects of AAS on ﬂank marking behavior.

Overall, males that were treated with AAS exposure adolescence had a
higher aggression score than males that were treated in adulthood (Figure 3.5B).
A main effect of age of exposure was observed [F (1, 84) = 33.970; P < 0.0001]
in this experiment, along with a main effect of treatment [F (1, 84) = 6.16; P <
0.02]. Time of testing post-treatment did not have a main effect on the
expression of aggression, therefore, a 2-way ANOVA was run collapsing the
groups by age of exposure and treatment. The same main effects of age and
treatment persisted. Again, AAS treatment resulted in increased the composite
aggression score over vehicle controls independent of age of AAS exposure.

This signiﬁcant increase persisted four-weeks after exposure.

C. Defensive/Submissive Behaviors:

Tail-up walking, escape dashes and stretch-attend are all behaviors
categorized as defensive or submissive behaviors. Interestingly, males exposed
to AAS during adolescence did not express tail-up walking, escapes dashes or
stretch-attend (Figure 3.6). A main effect of age of exposure was found on the
number of tail-up walking [F (1, 84) = 23.312; P < 0.0001], escape dashes [F (1,
84) = 9.980; P < 0.002] and stretch-attend [F (1, 84) = 14.142; P < 0.0005]
showing that males treated as adults displayed a signiﬁcantly higher number of

submissive behaviors.

68

C. Physiological Measures

In this experiment a main effect of age of AAS exposure was observed on
testes weight [F (1, 84) = 24.294; P < 0.0001), seminal vesicle weight [F (1, 84) =
59.856; P < 0.0001] and ﬂank gland diameter [F (1, 84) = 19.470; P < 0.0001], as
expected and also shown in experiment 1 (Table 3.1). Males that were treated
during adolescence had signiﬁcantly smaller peripheral tissue measurements.
However, in this experiment, a main effect of treatment [F (1, 84) = 22.030; P <
0.0001] and of testing post-treatment [F (1, 84) = 14.003; P < 0.0003] was found
on testes weight. AAS treated males and males that were tested 4 wks after
treatment had signiﬁcantly smaller testes weights. A signiﬁcant interaction
between age of exposure and testing-post treatment was also found on ﬂank
gland diameter [F (1, 84) = 12.165; P < 0.0008], showing that males that were
treated during adolescence and tested 4 wks has reduced ﬂank gland
measurements. No signiﬁcant effects of treatment were found on testosterone
levels in this experiment, but a main effect of testing post-treatment was

determined by a 3-way ANOVA [F (1, 84) = 5.410; P < 0.02].

Discussion

The purpose of these experiments was to determine if AAS exposure
during adolescence or adulthood had long term consequences on social
behaviors in an animal model. Using a cocktail comparable to what a heavy or
chronic anabolic steroid user would administer, these experiments show long-

lasting consequences due to AAS exposure after adolescent and adult treatment

69

on both sexual and aggressive behaviors. Sexual satiety is signiﬁcantly
decreased after anabolic steroid exposure, independent of age of treatment.
Also, aggressive behaviors remained enhanced and submissive behaviors
suppressed only after adolescent AAS exposure, while AAS had mild effects on
the expression of aggressive behaviors after adult exposure. This is the ﬁrst
study to compare directly the long-term effects of AAS exposure during
development on adult social behaviors. Data from these experiments show that
anabolic steroid exposure affects speciﬁc behavioral characteristics in a social

interaction with male and female counterparts.

Androgens play a critical role in socio—behavioral changes that occur
during adolescence. Gonadal hormones during puberty exert long-lasting
changes in neural circuits responsible for the programming of activational
responses to steroids later in adulthood. A two-stage model for maturation of
male social behaviors is proposed: a perinatal critical period for sexual
differentiation of neural circuits, followed by the pubertal period, during which
gonadal steroids further organize the circuits to enhance behavioral
responsiveness to hormones in adulthood (Sisk et al., 2003). In support of this
hypothesis, the lack of gonadal hormones during adolescence leads to
detrimental effects on social behaviors in adulthood (Schulz et al., 2004).
Normative behaviors were quantiﬁed in the vehicle-treated groups. Like in other
studies, sexual maturation did not change throughout adult development (Meek

et al., 1997). On the other hand, aggressive behaviors were reduced throughout

70

development. Previous studies from this laboratory describe developmental
changes in agonistic behaviors. Prepubertal males show increased aggression
when compared to adult males (Romeo et al., 2003) and ﬂank marking behavior
is increases after puberty (Schulz et al., 2006). In this study aggressive and
submissive behaviors were quantiﬁed. After puberty adult aggressive behavior
also goes through developmental changes where in it decreases with age and
submissive behaviors increase. Adult male uses less overt aggression to
establish dominance hierarchies; however, expression of ﬂank marking behavior

and submissive behaviors remain consistent throughout adulthood.

In all treated groups AAS exposure affected the potential to reach satiety.
AAS exposure decreased the expression of long intromissions, which are
indicative of the onset of sexual satiety (Arteaga, 2000), in hamsters that were
exposed to AAS in either adolescence or adulthood. Pubertal AAS exposure
resulted in decreases in the expression of sexual behaviors up to 9 wks after
withdrawal in intact male rats (Farrell and McGinnis, 2004). These ﬁndings are
not easy to compare with the current study since there was a longer treatment
period (8 wks, throughout adolescence) of single synthetic compounds and
repeated testing within subjects. However, potential effects of anabolic steroids
on the dopaminergic systems could result in the inability to reach satiety. AAS
exposure in adulthood results in an increase of dopamine synthesis and an
increase in dopamine receptors in the nucleus accumbens, which plays a critical

role in the interpretation of rewarding stimuli and sexual satiety (Birgner et al.,

71

2007; Kindlundh et al., 2001; Kindlundh et al., 2003b; Kindlundh et al., 2004).
However, dopamine regulation in the nucleus accumbens is related to
anticipatory sexual behavior, whereas in the MPOA it’s related to copulatory rate
and sexual motivation (Dominguez and Hull, 2005; Pfaus and Phillips, 1991).
Dopamine agonists can reduce or facilitate rodent male sexual behaviors
depending on the dose (Bazzett et al., 1991; Mas et al., 1995b; Moses et al.,
1995). The inhibition of dopaminergic neurons within the mating circuit (Me,
BNST and MPOA) results in the disappearance of long intromissions (Arteaga et
al., 2002; Parﬁtt and Newman, 1998). In addition, copulation leading to sexual
exhaustion is associated with elevated levels of dopaminergic activity in the
MPOA (Mas et al., 1995a). Unfortunately, the effects of AAS exposure on
dopaminergic systems related to the mating circuit has not been explored;
however, AAS may regulate dopamine release and the expression of dopamine

receptors differentially in neural circuits in the mating and reward circuits.

A link between anabolic steroid use and increased aggression has been
well documented in both human subjects and animal models (Clark and
Henderson, 2003; Fischer et al., 2007; McGinnis, 2004; Rowe et al., 2004).

Intact male rats exposed to AAS during adolescence and adulthood have
increased composite aggression scores and intact adolescent male Syrian
hamsters show an increase in attacks, bites and attack duration immediately
after exposure (Melloni et al., 1997). Follow-up studies focusing on the long-term

effects of AAS show that adolescent AAS treatment in male rats can lead to

72

enhanced expression of aggressive behaviors up 17 wks post-treatment (Farrell
and McGinnis, 2004); however, these long term effects were not observed in
adolescent male Syrian hamsters, where aggressive behaviors were the same as
in control males 19 days after adolescent AAS exposure (Grimes et al., 2006;
Ricci et al., 2007). Housing conditions for the intruder males were not controlled
in the aforementioned studies. Unpublished observations from this laboratory
indicate that it’s optimal when resident and intruder males are singly-housed,
especially since male Syrian hamsters prefer to be socially isolated. Preliminary
studies presented in this dissertation also indicate that social context is a variable

that may increase or decrease the expression of aggressive behaviors.

This experiment found long term changes on agonistic behaviors due to
AAS exposure during adolescence. A signiﬁcant increase in number of bites,
attacks, ﬂank marking behavior and a composite aggression score in adolescents
and not in adults even after four weeks of AAS exposure provides new evidence
that the adolescent brain is more vulnerable to exogenous androgens.
Interestingly, the display of submissive and/or defensive behaviors is also
affected by adolescent AAS exposure. In a previous study, AAS-treated
adolescent males showed few or no submissive behaviors when compared to
vehicle control and adult-treated groups. Similarly, adolescent males exposed to
AAS did not show any escape dashes, tail-up walking or stretch attend, resulting

in a main effect of age exposure for all three behaviors. These results suggest

73

that AAS exposure during adolescence results in a lack of regulation of risk

assessment and attenuation of expression of submissive behaviors.

Many studies focused on the regulation of aggression have identiﬁed
serotonin as key neurotransmitter that modulates this social behavior (Delville et
al., 2000; Ferris and Delville, 1994; Ferris et al., 1999; lnsel et al., 1990; Nelson
and Chiavegatto, 2001). Importantly, a decrease in serotonergic innervations in
the amygdala and anterior hypothalamus (Grimes and Melloni, 2002), in
serotonin receptors (Grimes and Melloni, 2006), and a decrease in serotonin
release in the hypothalamus (Keleta et al., 2007) have been observed after
adolescent anabolic steroid exposure. These neurochemical changes are long-
lasting (Grimes and Melloni, 2006) and can explain the enhanced overt

aggression after AAS exposure, even well after treatment.

In this experiment a signiﬁcant decrease in testes weight was observed,
suggesting cohort differences in the effects of AAS on peripheral tissues (Table
3.1). These results are consistent with previous studies showing a signiﬁcant
decrease in testes weight after AAS-treatment. However, since no other
peripheral measure was affected by treatment, it implies that the effects of AAS
on behavior are due to their effects on the central nervous system since the
periphery is able to recover from adverse effects on seminal vesicles and a
decrease in hormone levels exhibited immediately after AAS exposure (Feinberg

etaL,1997)

74

Adolescence is a time marked by impulsivity, risk-taking behaviors and a
social re-orientation (Nelson et al., 2005). Areas like the prefrontal cortex, the
nucleus accumbens and the amygdala are undergoing dramatic neural changes,
all of which may be affected by the anabolic steroid exposure (Ernst et al., 2006).
These studies contribute signiﬁcantly to the understanding that AAS exposure
during adolescence may cause permanent changes to adult-typical social
behaviors by increasing aspects of impulsivity and inhibiting neural systems
involved in the regulation of behavior, such as dopaminergic and serotonergic
circuitry. Further understanding of how anabolic steroids may cause
organizational neural changes in the brain is still needed; however these
profound changes in adult behavior after adolescent anabolic steroid exposure

require further investigation.

75

>

Numberof Mounts

Nu mber of In tromissions

Figure 3.1 Adult sexual behavior remains consistent from P 56 to P106. A one-

YVay ANOVA revealed no signiﬁcant differences in the number of mounts and

'ntl‘omissions displayed by vehicle-treated males tested on P56, P70, P72 and
06.

35'
30‘
25‘
20‘
15‘
10'

 

 

 

 

 

 

 

 

 

 

 

 

 

56

70

92

Postnatal Day

 

 

 

 

 

 

 

 

 

 

 

 

 

56

70

Postnatal Day

92

76

106

 

 

Number of Long lntromisslons

 

 

 

El Vehicle-treated

 

 

 

2 wks 4 wks 2 WkS 4 wks - AAS @ adolescence

I AAS @ adulthood

Long Intromisslon Latency (mlns)

  

 

 

 

 

l

2 wks 4 wks 2 wks 4 wks

 

 

Time of testing post-treatment
Figure 3.2. AAS exposure reduces the potential to reach satiety independently of
age of AAS exposure and time of post-treatment behavioral test. A 3-way
ANOVA was used to determine that AAS exposure during adolescence and
adulthood signiﬁcantly (p < 0.05) (A) decreases the number of long intromissions
and (b) increases long intromission latency.

77

>

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

181
s '- I
g 14; I
g .
E 10‘.
g ‘. L
3 6‘. *
z _

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0

56 70 92 106

B PostnatalDay *
2’ ‘.
'2 _
— 30 I
(B .
g .4 H . .z
o. . I,
3. ” " .
'— '--r'I-II,,III'
s j , , ,,
3E 101 I
3 I- "i '

0

56 70 92 106

Postnatal Day

Figure 3.3 Developmental changes were found in the expression of agonistic
behaviors. A one—way ANOVA revealed that the older the vehicle-treated males
were when tested, the fewer (A) attacks (*, p < .002) and the more (B) tail-up
walking (*, p < 0.005) they displayed.

78

 

.3

22.5-
20-
17.5-
15i
12.5'
10-
7.5-
5.
2.5-

—h
0|
1 A

  

Numger of Attacks
0

  
  
      
 

0|

 

 

 

 

Adolescent Adult
Age of Exposure

:i' I: Vehicle-treated

2wks 4wks 2wks 4wks - AAS@adolescence
- AAS@adulthood

 

 

N umber of Attacks

 

 

 

 

 

 

-32-“!

Number of Bites

- é

 

 

0‘s;

 

 

Number of Bites

Adolescent Adult
Age of Exposure

 

 

 

 

2 wks 4 wks 2 wks 4 wks

Time of testing post-treatment

Figure 3.4 A main effect of treatment was found on (A) attacks and (B) bites (p <
0.05). A three-way ANOVA also revealed a main effect of age of exposure on
the expression of both behaviors (p < 0.0001). No signiﬁcant interactions were
found. The insets are ANOVAs comparing age of exposure and treatment. A 2-
way ANOVA comparing age of exposure and treatment determined that
adolescent AAS exposure signiﬁcantly increased (A) attacks (*, p < 0.05) and (B)
bites (*, p < 0.05).

79

*

4
1
1
4
4

Adolescent Adm
Age of Exposure

[:1 Vehicle-treated
- AAS@adolescence
4wks

- AAS @ adulthood

8

 

N
0|

 

3

Numberof Flank Marks
2:

<3

25'

 

 

 

 

 

C3 00

 

   

15'

NunberchmkMa'ke
8

10‘

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2M6
B w 1*
' 50'
§ 60‘. 5 4o
1'; 50 - arr .
I 4 § 20
.8 40 ‘ a
a: w 1 §10‘
+ A 'l a
g3 20: ..
33 g 10: I -20 Adoleemnt Adult
+
g 1 AgeoIExposure
2 0., ..... ..
t I
5 -2o -
-30 - ﬁ . -
2M8 4wks 2wks 4wks

Time of toting pod-treamert

Figure 3.5 AAS exposure during adolescence signiﬁcantly increased ﬂank
marking behaviors and overall aggression. (A) A 3-way ANOVA showed that
ﬂank marking behavior was increased after AAS exposure during adolescence
and adulthood (p < 0.02). A main effect of age of AAS-exposure was observed
(p < 0.002). The inset is a 2-way ANOVA revealed that adolescent AAS
exposure signiﬁcantly increases ﬂank marking behavior when compared to
vehicle- and adult-treated males (*, p < 0.05). (B) A 3-way ANOVA showed that
AAS treatment increases aggression in males treated during adolescence and
adulthood (p < 0.02). A main effect of age was also observed (p < 0.001). The
inset is a 2-way ANOVA determined males exposed to AAS treatment during
adolescence had signiﬁcantly higher composite aggression scores than vehicle-
and adult-treated adult (*, p < 0.005).

80

35: *

 

 

25 j
20 j ___
15 ‘

10" I
0

2ka 4wks *

 

Number of Tail-Up Walking

 

 

 

 

 

 

 

 

20
17.5q
15'

12.5 ' ___.

Vehicle-treated

AAS @ adolescence

 

AAS @ adulthood

Number of Escape Dashes
N .‘l _-
or on at o

0 0
2 wks 4 wks

 

 

 

 

 

O

 

u—L

o-‘wammﬂmoo
|.n.1.j

 

1| .0. lo

2 wks 4 wks 2 wks 4 wks

Time of testing post-treatment
Figure 3.6 Males exposed to AAS during adolescence did not express tail-up
walking, escape dashes or stretch-attend. A three-way ANOVA determined a
main effect of age of exposure for (A) number of tail-up walking (p < 0.001), (b)
escape dashes (p < 0.02) and (c) stretch attend (p < 0.0005).

j

Number of Stretch attend

 

 

 

 

 

81

Table 3.1 Peripheral measures in males exposed to AAS during adolescence or

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

aduﬂhood.
Adolescent Males Adult Males
Vehicle AAS-treated Vehicle AAS-treated
treated (Mean +/- Treated (Mean (Mean +l-
(Mean +/- SEM) +/- SEM) SEM)
SEM)
Age of testing P56 P70 P56 P70 P92 P106 P92 P106
Testing post— 2 wks 4 wks 2 wks 4 wks 2 wks 4 wks 2 wks 4
treatment wks
Testes Weight (9)
Experiment 1 3.43 3.41 3.11 3.81 3.93 4.04 3.75 3.77
:l: 0.1 :t 0.7 :l: 0.3 :l: 0.6 :t 0.4 :l: 0.3 t 0.5 :t 0.8
Experiment 2 3.10 3.53 2.87 3.39 3.87 3.75 3.21 3.45
* 1: 0.2 :l: 0.2 :l: 0.3 :l: 0.3 :l: 0.2 :l: 0.2 i 0.6 :t 0.4
Seminal Vesicle Weight (9)
Experiment 1 0.19 0. 22 0.26 0.24 0.31 0.33 0.30 0.35
:l: 0.07 :l: 0.04 :t 0.03 :l: 0.05 :I: 0.04 :l: 0.06 :I: 0.05 :l:
0.07
Experiment 2 0.21 0.27 0.24 0.25 0.34 0.35 0.37 0.37
:l: 0.07 :l: 0.04 :l: 0.06 :l: 0.06 :l: 0.10 :l: 0.06 :l: 0.08 1:
0.10
Flank Gland Diameter (mm)
Experiment 1 8.48 8.35 7.77 8.59 8.88 8.63 9.48 8.74
10.6 :l:1.2 :l:2.1 :l:1.6 10.9 10.8 :l:1.6 :l:0.6
Experiment 2 7.04 7.87 7.02 7.58 8.73 7.54 8.70 8.30
11.11 :l: 0.62 i 1.32 :l: 0.60 :l: 1.07 :l:1.07 :l: 1.09 :l:
0.80
Testosterone plasma levels (nglml)
Experiment 1 2.43 1.71 2.38 2.30 2.03 1.77 2.21 1.74
:l: 0.52 :l: 0.18 :t 0.49 :l: 0.34 1: 0.29 :l: 0.25 :l: 0.20 :l:
0.21
Experiment 2 1.26 0.67 0.82 0.47 0.77 0.47 0.61 0.54
* i 0.36 :l: 0.21 :l: 0.22 :l: 0.09 1 0.13 :l: 0.10 :l: 0.08 :i:
0.13

 

 

 

 

 

 

 

 

 

 

A main effect of age of exposure was found on all measures (p < 0.05).

* A main effect of treatment was found on testes weight in Experiment 2 (p < 0.001 ).
’ A main effect of testing post-treatment was found on testosterone plasma levels in Experiment 2

m<oom.

82

 

CHAPTER 4: ADOLESCENT ANABOLIC STEROID EXPOSURE AF FECTS
MEDIAL AMYGDALA VOLUME AND CELL ADDITION IN THE MALE SYRIAN
HAMSTER

Introduction:

Pheromonal communication and hon'nonal signals are critical for the
expression of male social behaviors in rodents. The neural circuits that underlie
male reproductive behaviors [which includes the bed nucleus of the stria
terminalis (BNST) and the medial preoptic area (MPOA)] and inter-male
aggressive behaviors [which includes the anterior hypothalamus (AH), the BNST,
and the lateral septum (LS)] share efferent and afferent connections to and from
the medial amygdala (Me) (Ferris et al., 1987; Kollack-Walker et al., 1999;
Kollack-Walker and Newman, 1995; Potegal et al., 1996; Romeo and Sisk,
2001). The connections of the amygdala can be divided into three systems (1)
sensory processing through connections with the olfactory cortex, posterior
thalamus, and sensory association cortex, (2) visceral function in relation to the
emotional stimuli through connections with the hypothalamus, and (3) emotional
behavior and mood through connections that comprise the forebrain circuit
(Price, 2003). The Me is a complex structure that has been subdivided based on
cytoarchitectonic, synaptic and functional criteria, into four main nuclei:
anterodorsal (MeAD), anteroventral (MeAV), posterodorsal (MePD) and
posteroventral (MePV) (Alheid, 2003; Cooke and Simerly, 2005). The Me
subnuclei contribute to the interpretation of sensory information (Wood and

Coolen, 1997), for the regulation of intermale aggression and reproductive

83

behaviors, (Kollack-Walker and Newman, 1995) and to the interpretation of

emotionally loaded stimuli and memory modulation (Rasia-Filho et al., 2004).

Gonadal hormones play a role in neural plasticity during puberty in the
amygdala (Romeo and Sisk, 2001). The anterior medial amygdala (MeA) and
posterior medial amygdala (MeP) are steroid sensitive, and morphological
changes in these nuclei may reﬂect changes in the expression of male social
behaviors. The removal of gonadal hormones in adult males results in a
decrease in somal size of individual cells and/or overall volume of the Me.
interestingly, the MeA is signiﬁcantly larger in prepubertal male Syrian hamsters
when compared to adults; however the inverse in true about the MePD (Romeo
and Sisk, 2001). Androgen-dependent plasticity in the MePD has also been
observed by using Golgi-staining in male Syrian hamsters. In castrated males,
neuronal structure in the posterior, but not in the anterior, region of the Me
undergo structural changes such as increase dendritic pruning during adolescent
development and due to a rise in gonadal hormones (Gomez and Newman,
1992; Zehr et al., 2006). The MeA receives substantial projections from the
accessory olfactory bulb, and lesions to the MeA abolish chemoinvestigatory
behavior, suggesting the pubertal development may involve changes that allow
appropriate processing and integration of these cues (Gomez and Newman,
1992; Wood and Newman, 1995b). However, the MeP contains more androgen
(AR) and estrogen receptors (ER) than the MeA (Cooke et al., 2003; Wood and

Newman, 1995a), suggesting that it may retain the capacity for plasticity

84

throughout life so that the expression of male social behaviors is possible under

speciﬁc steroidal and/or environmental conditions (Romeo and Sisk, 2001 ).

There is a major gap in understanding how AAS affect the adolescent
brain, speciﬁcally the Me; however several studies have been focused on the
effects of AAS on the adult brain. In adult male rats androgen receptors are
upregulated in the Me after being treated for two weeks with a cocktail of AAS
(Lynch and Story, 2000; Menard and Harlan, 1993). A central injection of
testosterone in adult male Syrian hamsters increases Fos, expression, a marker
for neuronal activity, in the MePD after one day of exposure (Dimeo and Wood,
2005). These ﬁndings suggest that Me neurons are sensitive to acute and
immediate effects of AAS. Recently, AAS exposure during adolescence was
also shown to increase spine density in the MeA and MeP immediately after
treatment, however these changes in spine density did not persist into adulthood

(Cunningham et al., 2007).

Another amygdaloid nucleus involved in social communication and
cognition that may be signiﬁcantly affected by AAS exposure is the central
amygdala (CeA). CeA has been implicated in fear related behaviors (Wilensky et
al., 2006), physical aggression in rodents (Bedard and Persinger, 1995) and
scent marking in the hamsters (Bamshad et al., 1997). Adolescent AAS
exposure can alter developmental patterns of serotonin innervations to other the

lateral hypothalamus and the anterior hypothalamus, that are implicated in

85

aggression and social communication (Grimes and Melloni, 2002). However,

little is known about neural changes that occur during adolescence in the CeA.

Gonadal steroid hormones, like testosterone and estrogen, also play an
important role in the proliferation (Ormerod and Galea, 2001), survival (Leranth et
al., 2000) and activation (lnsel, 1990) of neurons. Studies in male rodents show
that gonadal steroid hormones inﬂuence the number of newly born cells in the

amygdala (Fowler et al., 2003; Huang and Bittman, 2002).

In order to understand how androgenic-anabolic steroids (AAS) affect
neurogenesis, a study examined the effect of nandrolone decanoate on rat
neural stem cells (NSC) in culture on the dentate gyrus in the rat hippocampus.
In vitro, NSC were stimulated by epidermal growth factor and nandrolone
reduced cell proliferation. Nandrolone also decreased neurogenesis (measured
by bromodeoxyuridine (BrdU) labeling) in the dentate gyrus of the rat
hippocampus in both adult males and females after 5 days of administration
(Brannvall et al., 2005). Other drugs of abuse, such as nicotine, cocaine, and
alcohol, cause a disruption in neurogenesis, inhibit cell survival, and generally
affect neural plasticity (Abrous et al., 2002; He et al., 2005; Kems et al., 2005;

Yamaguchi et al., 2004).

The goal of this experiment was to determine if adolescent AAS exposure

affects the number of new cells added to amygdaloid nuclei after treating male

86

Syrian hamsters with a cocktail of AAS and BrdU throughout two-wks in early
adolescence. Evidence from previous studies predicts that AAS will decrease cell
proliferation in the adolescent brain. In addition, evidence from other studies
predicts that the MePD would be particularly sensitive to the effects of adolescent

AAS-exposure.

Materials and Methods
W

Thirty-two male Syrian hamsters (Harlan, Madison WI) arrived on
postnatal day 18 (P18) without their dams. Upon arrival subjects were housed in
groups of eight animals per cage (polycarbonate, 33 x 38x 17 cm) with ad libitum
access to food (Telkad Rodent Diet No. 8640, Harlan) and water. They were
singly housed on postnatal day 25 in clear polycarbonate cages and randomly
selected for assignment to one of four treatment groups. Animal colony
temperature was maintained at 22 :l: 2°C, and animals were kept on a light-dark
cycle of 14:10 L:D (lights off at 1600h EST). All animals were treated in
accordance with the NIH Guide for the Care and Use of Laboratory Animals, and
all protocols were approved by the Michigan State University All-University

Committee for Animal Use and Care.

AAS Treatment
A cocktail of AAS containing 0.8 mglml of testosterone cypionate (TC;

Sigma Aldrich, Inc., St. Louis, M0), 0.8 mglml of nandrolone decanoate (ND;

87

Sigma Aldrich, Inc.), and 0.4 mg/ml of boldenone undecylenate (BU; Steraloids,
New Port, Rhode Island) was dissolved in 2-hydroxypropyl-B-cyclodextrin
(Sigma-Aldrich, Inc.). The AAS cocktail was prepared the day before treatment

began, and was stored at 4°C.

Expeﬁmenlﬂgesigg

In order to examine the effects of AAS during adolescence on cell
proliferation and cell survival, gonad-intact male hamsters were randomly divided
into four groups: (1 and 2) vehicle-treated or AAS-treated and perfused during
late adolescence on P42, and (3 and 4) vehicle-treated or AAS-treated and
perfused in adulthood on P70. Subjects received a daily subcutaneous (sc)
injection of either the AAS cocktail (2 mglkg TC, 2 mglkg ND, 1 mglkg BU) or
vehicle (2-hydroxypropyl-B-cyclodextrin) during adolescence (from 27-41 days of
age). Injection volume ranged from 0.1 ml to 0.25 ml throughout the two-week

course of administration, depending on subject weight.

The cell birth marker, 5’-bromo-2’-deoxyuridine (BrdU; cat. No. B-9285,
Sigma: St. Louis, MO) was used to identify proliferating cells during adolescence.
BrdU injections were given intraperitoneally (i.p.; 300 mg/Kg BW, 10mg lml BrdU
in 0.9% NaCl) to all subjects every other day from P28-42 for a total of 8
injections per subject. BrdU was made fresh every day of injection. Injection
volume ranged from 1.6 ml to 2.6 ml throughout the two-week course of

administration, due to the increase in body weight during development.

88

Groups treated with either vehicle or AAS were perfused on postnatal day
42, 24 hours after the last AAS-injection and 4 hours after their last BrdU
injection to assess for cell proliferation immediately after AAS exposure. The two
remaining groups were perfused in adulthood on postnatal day 70, 4 wks after
the last AAS and BrdU injections to assess cell survival after adolescent AAS

exposure.

Tissue Collection and Physiological Measures

On the day of sacriﬁce, males were administered an overdose of sodium
pentobarbital (130 mglkg i.p.) and a terminal blood sample was taken via cardiac
puncture. Testes and seminal vesicles were removed and weighed. Flank gland
diameters were assessed by shaving the subject’s side and measuring the left
gland with a caliper. Perfusions were performed intracardially with heparinized
phosphate-buffered saline rinse (~200 ml) followed by 4% paraforrnaldehyde
(~150 ml). The brains were removed, post-ﬁxed in 4% paraforrnaldehyde
overnight, and then stored in a 30% sucrose solution. All brains were sectioned
on a cryostat in four series at 40pm per section and stored in glycerol-based
cryoprotectant at —20°C, except for one series that was mounted fresh frozen
while sectioning. This set of sections was Nissl-stained and used to determine

the boundaries of cell groups within the medial and central amygdala.

Plasma testosterone concentrations were measured in duplicate using a

commercially available radioimmunoassay kit (Coat-a-Count Total Testosterone,

89

Diagnostic Products, Los Angeles, CA). This assay has been previously
validated for measuring endogenous plasma testosterone levels in hamsters in
our laboratory (Parﬁtt et al., 1999). The intra-assay CV was 4.7%, and the limit

of detectability was 0.1 nglml.

_Br_d_t_/ Immunohistochemistv

A set of sections was used to perform a single-label BrdU
immunohistochemistry according to previously established protocols (Ahmed, et
al.). In brief, free-ﬂoating sections were incubated in 50% fonnamide at 65°C for
2 h, rinsed in 2X standard sodium citrate (SSC), incubated in 2 N HCI for 30 min
at 37°C, and rinsed in 0.1 M borate buffer pH 8.5 for 10 min. After three rinses in
Tris-buffered saline (TBS), sections were blocked in TBS containing 0.1% Triton
X-100 and 3% donkey serum (Jackson lmmunoResearch Lab., Inc, West Grove,
PA) for 30 min. Sections were incubated overnight at 4°C with monoclonal rat
anti-BrdU (1 :1000; MCA2060, Serotec, Ltd, Oxford, UK) and incubated for 2
hours at room temperature with biotinylated donkey anti-rat (1:250; 712-065-150,
Jackson lmmunoResearch Lab.,lnc, West Grove, PA). Finally, sections were
incubated for 60 min at room temperature with ABC reagent (ABC Elite kit;
Vector Laboratories, Burlingame, CA) and reacted for 6 min in diaminobenzedine
(0.25 pg/ml in TBS with 0.01% H202; Sigma-Aldrich, St. Louis, MO). All solutions
used in this experiment were made fresh the day before the

immunohistochemistry was performed.

90

To generate positive control tissue, pregnant female rats were injected
with BrdU (50 mglkg body weight) on gestational days 13-15 and allowed to
deliver litters naturally. When litters were 21 days old, brains of pups exposed to
BrdU on embryonic days 13-15 were collected as described for the experimental
groups. Tissue from these animals showed substantial BrdU labeling throughout
the brain. To assess nonspeciﬁc immunoreactivity, sections from BrdU-injected
animals were processed in the absence of either primary or secondary antibody.
Omission of primary or secondary antibodies eliminated BrdU-labeling. These
histological controls were included in the immunohistochemical processing of the

experimental tissue.

Microscopic Analysis
Subregions of the medial amygdala (MeAD, MeAV, MePD, MePV) and the

central amygdala (CeA) were traced bilaterally in Nissl-stained sections (Figure
1). Regional boundaries were traced at 4X magniﬁcation using Neurolucida
(Version number 6.50.1, Microbrightﬁeld, lnc., Williston, VT). Cytoarchitectural
characteristics (e.i. cells in the ventral nuclei are darker and densely packed,
whereas cells in the dorsal nuclei are organized in a columnar, cone shape) and
structural landmarks (e.i. the shape of the optic tract, the location and shape of
the basolateral amygdala and the intercalated nuclei of the amygdala) for each
nuclei were used to determine contour boundaries and tracings. Total cross-
sectional area for each subregion was calculated by summing subregion areas

across sections. The volume for each amygdaloid nucleus was calculated by

91

summing the traced cross-sectional areas and multiplying by the distance
between sections (120 um). Tracings from Nissl sections were aligned with
adjacent BrdU-ir sections using the optic tract and section outline, along with a
notch on the left hemisphere to determine laterality. All BrdU-ir cells located
within the tracing boundaries were counted at 40X magniﬁcation (Figure 2).
Because the size of medial amygdala subregions varies with age and sex (Cooke
et al., 1997; Romeo & Sisk, 2001; Morris et al., 2006), the total number of BrdU-fr

cells was divided by the total cross-sectional area for each subregion.

'? {Ev-“Tr . .'~' 59:15.; say
I.

Statistics I
Two-way ANOVAs were used to determine the main effects of treatment

(AAS or vehicle), time since the last BrdU injection (4-hrs or 4-wks after), and the

interactions between these two factors on the mean number of BrdU-labeled

cells per unit area in the subdivisions of the medial amygdala and in the central

amygdala. Post-hoc Student’s T-tests were performed to determine differences

between vehicle and AAS-treated males within each age group if an age by

treatment interaction was determined. ,

Results

 

Number of BrdU-labeled cells and cell aroup volume
A two-way ANOVA revealed a main effect of age of perfusion (P42, 4-hrs
after treatment vs. P70, 4-wks after treatment) on the number of BrdU-labeled

cells per mm2 in the CeA [Fig. 1A; F (1, 23) = 48.4; P < 0.0001], MeAD [Fig. 2A;

92

F (1, 23) = 18.6; P < 0.003], MeAV [Fig. 3A; F (1, 23) = 48.1; P < 0.0001], MePD
[Fig.4A; F (1, 23) = 64.5; P < 0.0001] and MePV [Fig. 5A; F (1, 23) = 33.6; P <
0.0001]. Subjects that were perfused on P42 had signiﬁcantly more BrdU-
labeled calls in all amygdaloid nuclei than subjects perfused on P70. This result

indicates that not all cells born between P28-P42 survive until P70.

A two-way ANOVA revealed a signiﬁcant interaction between age and
treatment in BrdU-labeled cells per mm2 in MePD [Fig. 4A; F (1, 23) = 6.2; P <
0.02]. Speciﬁcally, the number of BrdU-labeled cells was significantly less in the
vehicle-treated males perfused 4-hrs after treatment than in AAS-treated males
[Fig.4A; F (1, 12) = 9.2; P < 0.01]. However, by 4-wks after treatment, there were
no differences in the number of BrdU-labeled cells between vehicle- and AAS-

treated males.

No effects of age or treatment were found on the volume of the CeA (Fig
1B), MeAD (Fig 28), MePD (Fig 4B) or MePV (Fig SB). However, the volume of
the MeAV was signiﬁcantly increased by AAS exposure during adolescence [Fig
38; F (1, 23) = 4.2; P < 0.05). This increase in volume in AAS-treated males

persisted even 4-wks after discontinuation of AAS treatment.

Physiological measures:

AAS exposure during adolescence had profound effects on plasma

testosterone (T) levels and testes weight. Main effects of age [F ig. 6; F (1, 23) =

93

7.7; P < 0.01] and treatment [Fig. 6; F (1, 23) = 7.3; P < 0.01] were found on T
plasma levels; however no significant interactions were found. T plasma levels
were reduced by adolescent AAS treatment, and T samples taken on P42 were
higher than samples taken on P70. A signiﬁcant interaction between age and
treatment was found on testes weight [F ig.6; F (1, 23) = 4.9; P < 0.05], where
AAS-treated males perfused 4-hrs after treatment had significantly reduced
testes weight than vehicle-treated males [Fig.6; F (1, 12) = 17; P < 0.001], but no
signiﬁcant reduction in testes weights was found in males perfused 4-wks after
treatment. Seminal vesicle weight and ﬂank gland diameter were not affected by

adolescent AAS-exposure.

Discussion

This study provides the ﬁrst evidence that new cells are added to the
hamster medial amygdala during adolescence and that approximately one-third
of them survive to adulthood. In general, this study found no effect of AAS
treatment on the addition of new cells during adolescence, with the exception of
a decrease in cell proliferation in the MePD. This ﬁnding is similar to that of a
previous study showing nandrolone decanoate decreases cell proliferation in the
dentate gyrus of adult rodents (Brannvall et al., 2005) . Adolescence AAS
treatment did result in an increase MeAV volume that persisted into adulthood.
Increased cell proliferation in intact animals has been reported in other brain

regions such as hippocampus (He and Crews, 2007).

94

 

AAS exposure during adolescence signiﬁcantly decreased the number of
BrdU-labeled cells in the MePD suggesting that this subdivision of the amygdala
is vulnerable to supraphysiological levels of synthetic androgens. The MePD has
been studied extensively because of its role in sensory-hormonal integration and
its reciprocal interconnections with the limbic system in order to control social
behaviors (Newman, 1999; Skuse et al., 2003). In male Syrian hamsters, the
MePD is involved in regulating the expression of sexual and agonistic behaviors
(Kollack-Walker and Newman, 1995). Through studies assessing neurogenesis,
spine density, dendritic arborizations, soma size and volume, the medial
amygdala has been shown to be very plastic, especially during adolescence and
after manipulation of gonadal hormones (Cooke, 2006; Fowler et al., 2003;
Rasia-Filho et al., 2004; Romeo and Sisk, 2001; Zehr et al., 2006). The MePD is
steroid receptor rich and an increase in androgen receptor immunoreactivity is
observed after testosterone treatment in the prepubertal medial amygdala when
compared to adults (Meek et al., 1997; Romeo et al., 2000; Wood et al., 1992).
Therefore MePD cells may be particularly sensitive to the central actions of AAS
exposure during adolescence. In addition, a recent study showed that spine
density was increased by AAS immediately after treatment in the MeP, however,
these changes did not persist 4-wks after withdrawal (Cunningham et. al., 2007).
Taken together, adolescent AAS exposure affects neural plasticity in the MePD,

however, these particular changes do not persist into adulthood.

95

AAS treatment increased the volume of the MeAV immediately and 4-wks
after exposure, suggesting a permanent change in this particular subdivision of
the Me (Fig.2). The MeAD and MeAV are activated after general arousal,
especially in relationship to sexual behavior (Gomez and Newman, 1992;
Kollack-Walker and Newman, 1997). The MeA is also involved in the
interpretation of a social interaction resulting in a defensive behavior (Choi et al.,
2005). Studies from this laboratory have shown that adolescent AAS exposure
signiﬁcantly reduced or eliminated the expression of submissive and defensive
behaviors, even 4-wks after discontinuation of AAS treatment (see Chapters 2
and 3). These data suggest the AAS-induced changes in volume in the MeAV
may be a result of organizational changes at the cellular or molecular level that in

turn affect behavior.

The amygdaloid nuclei CeA, MeAD and MePV were not affected by
adolescent AAS exposure, however, they are all involved in the interpretation of
environmental cues and mediate social behaviors. The central amygdala has
been implicated in the coordinating actions of fear and anxiety like behaviors
(Jasnow et al., 2004). All subdivisions of the medial amygdala are activated
after sexual and agonistic behaviors (Kollack-Walker and Newman, 1995). Male
treated with this cocktail of AAS have long lasting changes in serotonergic
circuitry in the central and medial amygdala (Grimes and Melloni, 2002),
suggesting cell proliferation is not the only neural change responsible for the

effects on social behaviors induced by adolescent AAS exposure.

96

This experiment used BrdU to label cells added to the amygdaloid nuclei.
In the last decade several studies have been conducted to assess the BrdU
labels newly born cells. Different methods of delivery have been performed and
peripheral injections have consistently labeled cells that have undergone DNA
synthesis (Kokoeva et al., 2005; Rakic, 2002). Most studies of neurogenesis
used a commonly used dose of BrdU injection at a concentration of 50mg/kg. In
the present study BrdU was injected at a concentration of 300mg/kg because of
a recent report suggests that a higher dose of BrdU (300 mglkg) is a better
quantitative marker of proliferating cells of rats, as a lower dosages only label a
fraction of the S-phase cells (Cameron and McKay, 2001). In addition to single
label BrdU, fluorescent immunocytochemical techniques have been used to
identify mature and immature neurons, astrocytes and whether these new born
cells are functional (Huang and Bittman, 2002; Kokoeva et al., 2005; Kokoeva et
al., 2007). A natural follow-up to this study would be to assess whether the cells
that are affected in the MePD are neurons or glia. In this particular study, many
more BrdU-labeled cells were observed in the adolescent brain (immediately
after treatment) that the adult brain (cell survival). Previous ﬁndings demonstrate
a relationship between steroid hormones and neurogenesis in the medial

amygdala during in adulthood (Bernier et al., 2002; Fowler et al., 2007) .

Thus far, few studies have focused on organizational changes caused by
adolescent AAS exposure and the underlying mechanisms for cell addition to

neural structure during adolescence and adulthood. The views on neural

97

plasticity and organizational changes during adolescence are being modiﬁed clue
to recent imaging evidence (Giedd et al., 1999; Giedd et al., 2006; Lenroot et al.,
2007). It is important to continue to investigate the underlying mechanisms that
make the adolescent brain vulnerable to exogenous compounds and the
consequences of these. This study provides further evidence on how AAS affect
the adolescent brain and the effects of supraphysiological androgen levels on

neural plasticity.

   

'llh

Figure 4.1 Photomicrographs of Nissl-stained tissue at 4X of the amygdaloid
nuclei. Neurolucida tracings outline (A) central amygdala (CeA), anterodorsal
medial amygdala (MeAD), anteroventral medial amygdala (MeAV); (B) CeA,
posterodorsal medial amygdala (MePD), posteroventral medial amygdala

(MePV)].

98

   

P42 - 4 hrs after last BrdU injection P70- 4-wks after last BrdU injection

Figure 4.2 Photomicrographs at 40X of BrdU-labeled cells (arrows) in the
amygdala (scale bar = 500 pm). In general, a signiﬁcantly higher number of
BrdU-labeled cells was identiﬁed in males perfused 4-hrs after the last BrdU
injection (A) than in males perfused 4 wk after the last BrdU injection (B).
Approximately one third of the cells born during adolescence survive to
adulthood.

99

Central Amygdala (CeA)

 

>
A
8

- —_:

 

BrdU-labeled cells/ mm2

 

 

 

 

 

D vehicle

I AAS

 

III
_s

l
l“
g.
i
I

CeA volume (mm3)

 

 

 

 

 

 

P42 P70
4-h rs after treatment 4-wks after treatment

Age
Figure 4.3 AAS exposure during adolescence does not affect cell proliferation or
cell survival in the CeA. (A) A 2-way ANOVA revealed a main effect of age on
the number of BrdU-labeled cells/mm2 (*p < 0.0001). No effects of treatment
were found in BrdU-ir cells/mm2 or volume (mm3) (B).

 

100

Anterodorsal Media Amygdala (MeAD)
160 *
_ I

120‘

 

>

 

80‘

40‘

BrdU-ilabeled cells/ mm2

D vehicle

.AAS

 

 

 

 

 

.18,
.16:
.14:
.12:

.1:
.08:
.06:
.04:
.02:

MEAD Volume (mm3)

 

   

 

 

 

 

P42 P70
4-h rs after treatment 4-wks after treatment

Age
Figure 4.4 AAS exposure during adolescence does not affect cell proliferation or
cell survival in the MeAD. (A) A 2-way ANOVA revealed a main effect of age on
BrdU-labeled cells/mm2 (*p< 0.001). No effects of treatment were found on
BrdU-labeled cells/mm2 or volume (mm3) (B).

101

Anteroventral Medial Amydala (MeAV)
180 L

 

140 ‘

100 ‘

 

60'

BrdU labeled cells/ mm2 3’

20'

 

 

 

 

E] vehicle

.AAS

 

.25
.23 '
.2 ‘
.17 '
.15 ‘
.13 '
.1 ‘
.08 ‘
.05‘
.03 ‘

‘11—:rz-"ﬁ‘ “ ‘ "

MeAV Volume (mm3)

 

 

 

 

 

 

P42 P70
4-h rs after treatment 4-wks after treatment

Age

Figure 4.5. AAS exposure during adolescence does not affect cell proliferation or
cell survival in the MeAV, however AAS exposure significantly increases MeAV
volume even 4 wks after treatment. (A) A 2-way ANOVA revealed a main effect
of age on BrdU-ir cells/mm2 (* p<0.0001). (B) AAS-treatment increased MeAV
volume (mm3) in males perfused immediately after treatment and 4 wks after
treatment (* p < 0.05).

 

102

Posterodorsal Medial Amygdala (MePD)

>

 

450

Interaction age* treatment

350‘

250‘

BrdU labeled cells/ mm2

 

 

 

D vehicle

.AAS

 

 

III
.1;

war-HE“ *

.15‘
.1 ‘
.05‘

MePD Vol u me (mm3)

 

 

 

 

 

 

P42 P70
4-h rs after treatment 4-wks after treatment

Age
Figure 4.6 AAS exposure during adolescence decreases the number of cells
added to the MePD. (A) A signiﬁcant interaction between age and AAS-
treatment was found on BrdU-ir cells/mm2 (* p <0.05). AAS treated males had
significantly less cells added to the MePD than vehicle-treated animals when
perfused 4 hr after treatment but not when perfused 4 wk after treatment. (B) No
effects of age or treatment were found on MePD volume (mm3).

 

103

Posteroventral Media Amygdala (MePV)

 

 

 

 

 

 

 

 

 

 

 

 

A 350 ‘ *
N a
E —l:
E 1
E 250 ‘
E .
U -l
'O
2 .
g 150 ‘
32
3I _.
"E
m 50 ‘
- |:| vehicle

B 0 I AAS
A .3‘
(”DE _
E
“E’ .2-
3
E -
E .1 :
1D
2

0

P42 P70
4-h rs after treatment 4-wks after treatment
Age

Figure 4.7 AAS exposure during adolescence does not affect cell proliferation or
cell survival in the MePV. (A) A 2-way ANOVA revealed a main effect of age on
BrdU-ir cells/mm2 (p < 0.0001). No signiﬁcant of treatment was found on BrdU-ir
cells/mm2 or volume (mm3) (B).

104

 

Testosterone plasma levels and testes weight

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

E 2.5
B)
5
2 2'
(D
>
2 _
g 1.5
8 - *
a 1
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g I AAS

3-5 - Interaction Age* Treatment
A 3 _
9 j
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.9 2 _
o
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.8 1.5 ‘
<0 _
e 1 .

.5 j

0

P42 P70
4-h rs after treatment 4-wks after treatment

Age
Figure 4.8 AAS exposure during adolescence decreases plasma testosterone
levels and testes weight immediately after treatment. (A) A 2-way ANOVA
revealed a significant main effect of treatment (*p < 0.01) and age (p < 0.01) on
plasma testosterone levels. (B) A 2—way ANOVA revealed an interaction between
age and treatment (p < 0.05) on testes weight.

105

CHAPTER 5: IMPLICATIONS AND CONTRIBUTIONS TO UNDERSTANDING
THE DEVELOPMENT OF SOCIAL BEHAVIORS AND THE EFFECT OF
ANABOLIC STEROIDS ON THE ADOLESCENT BRAIN

In the last decade, adolescent maturation of brain, behavior and cognition
has received more attention than ever before. Most people understand that
adolescence is a time when secondary sexual characteristics develop; for
example, a male’s voice gets deeper. However, there is now an increasing
appreciation for the substantial neuroanatomical, emotional, and behavioral
changes that also occur during this developmental stage (Lenroot and Giedd,
2006; Nelson et al., 2005; Yurgelun-Todd, 2007). For example, cortical and
subcortical regions change in volume in human adolescents, and areas important
for decision making such as the prefrontal cortex develop late in adolescence
and young adulthood (Giedd et al., 2006). These developmental changes likely
underlie the changes in emotional regulation and risk taking behavior exhibited
by adolescent males and females (Ernst et al., 2006; Gardner and Steinberg,
2005; Steinberg, 2004). The environment of an adolescent is also in constant
ﬂux, and the socio-emotional behavioral changes occurring during adolescence
in turn inﬂuence their environment. For example, the behavior of teens varies
dramatically depending on the presence or absence of peers (Steinberg and
Monahan, 2007). Given the dramatic neurodevelopmental and socio—emotional
changes occurring during adolescence, it is not surprising that many
psychopathologies such as depression, schizophrenia and body image disorders
(e.g. anorexia, bulimia) ﬁrst emerge during adolescence (Choi et al., 2002;

Kanayama et al., 2006; Klump et al., 2007; Thompson et al., 2004; Zehr et al.,

106

 

 

2007). Furthermore, both human and animal research clearly demonstrate an
increased propensity for risk taking behavior and novelty seeking during
adolescence (Laviola et al., 2003; Spear, 2000), which undoubtedly contributes
to increased drug use during adolescence. Thus, in many ways, adolescence is
a period of developmental vulnerability (Crews et al., 2007).

Although drug use increases during the adolescent period, our
understanding of the neurodevelopmental and behavioral consequences of many
drugs are poorly understood. Adolescent anabolic steroid use, for example, is
rapidly becoming a health problem in the United States (Kanayama et al., 2006;
Wood, 2006). At least one million Americans use AAS to gain muscle mass
and/or lose body fat, and, unfortunately, the prevalence in AAS use among
adolescents has increased signiﬁcantly in the last 10—yrs. In general, AAS use
causes signiﬁcant medical problems such as suppression of neuroendocrine
function, hepatoxicity, myocardial dysfunction and adverse effects on
lipoproteins. In addition, AAS use also promotes psychiatric effects such as
major mood disorders (Pope and Katz, 1994), dependence syndrome (Brower,
2002), violent behavior (Daly et al., 2003; Midgley et al., 2001; Perry et al., 2003)
and possibly an increase in progressing to opioid abuse (Celerier et al., 2006;
Celerier et al., 2003; Peters and Wood, 2005). The experiments within this
dissertation sought to elucidate the some of the neurobiological and behavioral
consequences of AAS use during adolescence by 1) comparing the effects of
AAS treatment during adolescence and adulthood, and 2) determining both the

short and long-term effects of AAS use on the development of social behaviors

107

 

a'iﬂﬁ Q. “a .

 

(mating and aggression), and the key brain region regulating these behaviors,
the medial amygdala. The results of these experiments clearly demonstrate that
the adolescent brain is particularly sensitive to the effects of anabolic steroid

exposure.

ChapteLZ Summal

Experiments in Chapter 2 were designed to determine whether behavioral
responses to AAS, immediately following the cessation of treatment, differ
between adolescent and adult animals. Interestingly, AAS signiﬁcantly increased
sexual behavior in adolescent males, but signiﬁcantly decreased sexual behavior
in adult males. Aggressive behaviors were also signiﬁcantly increased in
adolescent males, but were relatively unaffected in adult males. Thus, clear
differences in behavioral responses to AAS were observed between adolescent
and adult animals.

The enhanced behavioral responses to AAS in adolescents could be due
to age-related differences in neurotransmitter release, receptors, or connectivity
within and among the brain regions comprising these behavioral circuits. For
example, several neurotransmitter systems within these areas mature during
adolescent brain development. These include dopamine (DA), serotonin (5-HT),
argenine vasopressin (AVP), and GABA (Clark and Henderson, 2003). Not only
are these systems different pre- and post-pubertally, but they are also in ﬂux
during adolescence and are steroid-sensitive. In other experimental paradigms,

AAS have been shown to increase 5HT1b receptor expression in the adolescent

108

male Syrian hamster (Ferris et al., 1997; Grimes and Melloni, 2002; Grimes and
Melloni, 2005; Grimes and Melloni, 2006; Ricci et al., 2005b; Ricci et al., 2006),
to increase DA receptor expression in the adult male rat (Kindlundh et al., 2002;
Kindlundh et al., 2001; Kindlundh et al., 2003b; Kindlundh et al., 2004), to
decrease vasopressin immunoreactivity in varicosities and terminals (DeLeon et
al., 2002; Ferris et al., 1996; Ferris et al., 1997; Grimes et al., 2006; Harrison et
al., 2000), to decrease GAD65 (Grimes et al., 2003; Ricci et al., 2005a), and to
alter GABAA subunit composition in pubertal mice (Clark et al., 2006; Henderson
et al., 2006; McIntyre et al., 2002; Penatti et al., 2005) . Furthermore, serotonin
decreases and dopamine increases in the rat hypothalamus, striatum and
amygdala after AAS-exposure (Keleta et al., 2007; Thiblin et al., 1999a). These
previous studies examined neurochemical responses in adulthood after AAS
treatment of various durations and times (during adolescence and adulthood),
and therefore are not directly comparable to the AAS treatments and age groups
in the present experiments. Nevertheless, they suggest that the dissimilar
behavioral responses observed here in adolescents and adults could be the

result of actions of AAS unique to particular stages of brain development.

Chapter .3 Summary

The experiments within chapter 3 were aimed at determining whether the
immediate effects on social behavior observed in Chapter 2 are long-lasting.
Therefore, behavior was assessed two- and four— weeks after the discontinuation

of AAS treatment. In general there were no long term effects on the display of

109

 

sexual behaviors, regardless of age of exposure. However, AAS exposure
reduced the likelihood of reaching sexual satiety in treated males, measured by
the display and latency of long intromissions. This is a ﬁnding unique since
Farrell, et al. (2004) found there was an inhibition of sexual behaviors in rats
treated with anabolic steroids during adolescence. Satiety is deﬁned as a state
of feeling satisﬁed, and is mostly mediated by the reward circuit composed of the
nucleus accumbens and prefrontal cortex. Since satiety was the only measure
decreased when males were tested with a receptive female, the data presented
in this dissertation suggest that anabolic steroids may alter brain areas that
reward-related associative behaviors. Studies done in rats and mice have shown
an increase is dopamine synthesis immediately after exposure and 02 receptors
are down-regulated in the key structures in the reward circuit (e.g. nucleus
accumbens) (Kindlundh et al., 2001; Kindlundh et al., 2003b; Kurling et al., 2005;
Thiblin et al., 1999a). All these changes will alter the potential to reach satiety,
therefore delaying the display of long intromissions within a 15-minute interaction
of a male and female.

Adolescent AAS exposure increases aggressive behaviors throughout
development. Males tested with an age and weight matched opponent displayed
signiﬁcantly more attacks, bites and ﬂank marks immediately after treatment and
long after. Interestingly, males exposed to AAS during adolescence did not
display any submissive behaviors. This was not the case in males treated in
adulthood. In general there was more variability in an intermale interaction

between adults. After AAS exposure, adults tended to display more overt

110

 

 

aggression when compared to vehicle-treated adults. Over development a
signiﬁcant increase in the expression of submissive behaviors was observed;
however, AAS treated adults displayed less submissive behaviors than vehicle-
treated adults. These ﬁndings are comparable to human and rodent studies
done to assess the long-term effects of AAS on aggression when aggressive
behaviors remain elevated up to 17 wks after discontinuation of use (Farrell and
McGinnis, 2004; McGinnis, 2004). In addition, even male rats that have been
subjected to a subordinate situation tended to display less submissive behaviors
after anabolic steroid treatment (Steensland et al., 2005).

Table 5.1 Short Term vs. Long Term Effects: Summary of the Effects of AAS
on Social Behaviors Compared to Adult-Typical Social Behaviors

 

        

 

Vehicle - AAS-treated Vehicle- AAS treated
treated adolescents treated adults adults
adolescents
Immediate effects
Sexual SigniﬁCantly Signiﬁcantly Adult-typlcal Signiﬁcantly '
behavior less than' " increaSed educed , *
, . adults ' ‘
Aggressive
behavior
Submissive Adult-typical “ “ .N - ‘ ! Adult-typical . Adult-typiCal
behavior . " ‘ .
Lon- -term Effects
Sexual . Adult-typical ﬁReduced ‘ Adult-typical Reduced
behavior ' ‘ satiety ‘ satiety
Aggressive
behavior
Submissive (Adult-typical .. ' N/A. ‘, Adult—typical . Reduced‘
behavior “ . ’ , ' compared to

vehicle
treated adults

111

Chapter 4 Summa_ry

The aim of the experiment in Chapter 4 was to determine whether AAS
treatment alters cell proliferation within the medial amygdala, a key brain region
mediating social behavior. Although a great deal of immunocytochemical work
has been conducted investigating changes in serotonin and vasopressin
immunoreactivity following AAS exposure, no work has previously investigated
whether changes in amygdala cell proliferation occur following AAS exposure.
The amygdala contains many androgen and estrogen sensitive neurons that are
responsible for processing sensory cues into behaviors and neuroendocrine
events which result in the display of social behaviors. The amygdala also
exhibits hormone-dependent and hormone-independent changes during
adolescence. In male Syrian hamsters, for example, the MePD volume
increases signiﬁcantly during puberty, but this is not due to having more neurons
because neuron number does not change. Instead, these morphological
changes in the MePD are due to an increased dendritic length and an increase in
cell soma size (Cooke et al., 2007). In contrast, the MeAV decreases during
adolescence, but this decrease does not appear to be dependent on circulating
gonadal hormones (Romeo and Sisk, 2001). Interestingly, the experiments in
chapter 4 found that changes in cell proliferation following AAS exposure were
localized to the MePD. Adolescent but not adult AAS exposure signiﬁcantly
decreased cell proliferation in the MePD, whereas the volume of this region was

unaffected. This suggests that although testicular secretions of testosterone

112

'l‘J.Q-.IL

L-‘ul . I

 

 

normally increase the MePD volume, AAS affects cell proliferation independently
of regional volume.

Surprisingly, volume changes within the medial amygdala were limited to
the MeAV, a region that normally does not exhibit steroid-dependent plasticity.
Thus, during the course of normal adolescence, this region usually decreases in
size, but AAS exposure caused this region to increase in volume and remained
larger than normal. The MeAD and MeAV are activated after general arousal,
especially in relationship to sexual behavior (Gomez and Newman, 1992;
Kollack-Walker and Newman, 1997). The MeA is also involved in the
interpretation of a social interaction that results in a defensive behavior (Choi et
al., 2005). Studies from this laboratory have shown that adolescent AAS
exposure signiﬁcantly reduced or eliminated the expression of submissive and
defensive behaviors, even 4-wks after discontinuation of AAS treatment (see
Chapters 2 and 3). These data suggest the AAS-induced changes in volume in
the MeAV may be a result of organizational changes at the cellular or molecular

level that in turn affect behavior.

All of the AAS used in the cocktail are aromatizable and can be
enzymatically converted to androgenic and estrogenic metabolites, in addition to
upregulating androgen receptors and estrogen receptors in the brain (Bartsch,
1993; Clark and Henderson, 2003; Menard and Harlan, 1993; Roselli, 1998). I
Androgens and estrogens mediate cell proliferation and cell survival in voles and
rats in the hippocampus and amygdala in adult animals (Fowler et al., 2003;

Fowler et al., 2005; Spritzer and Galea, 2007). In turn, circulating hormones can

113

also affect neurotrophic factors and growth factors like nerve growth factor
(NGF), brain derived neurotrophic factor (BDNF) and insulin growth factor (IGF-
1), which have also been shown to mediate neurogenesis in the hippocampus,
amygdala and hypothalamus in rodent models throughout development (F iore et
al., 2003; Fowler et al., 2007; He and Crews, 2007). However, further studies
are needed to elucidate how AAS affect the nerve growth factors in order to
understand how AAS can affect cell proliferation. In addition, androgens, as well
as AAS, have also been reported to have affects on different neurotransmitters
systems, such as serotonin (Grimes and Melloni, 2002; Grimes and Melloni,
2005; Keleta et al., 2007; Ricci et al., 2005b; Ricci et al., 2006; Thiblin et al.,
1999a). A decrease in serotonergic activity results in a decrease in cell
proliferation (Fowler et al., 2007). These data indicate that speciﬁc neuronal
subtypes may be affected by AAS. Because cell proliferation and survival rely
on these neural support networks and environmental cues, it suggests that the
adolescent brain can compensate for the inhibitory effects of AAS on cell

proliferation after discontinuation of treatment.

Normal Development of Social Behaviors:

Although the overarching purpose of the studies described here was to
elucidate the effects of AAS treatment on adolescent brain and behavioral
development, many interesting developmental changes were also found in the
vehicle-treated controls. We found that the trajectory of the maturation of social

behaviors is completely different for sexual behaviors compared with aggressive

114

Y'leu._.r—
-"Ir.'

 

i~

 

and submissive behaviors. In these studies males were tested with a receptive
female or an age, weight-matched opponent in order to assess sexual and
agonistic behaviors. As described previously, the display of sexual behaviors,
such as mounts, intromissions and ejaculations was higher in males tested as
adults than males tested during adolescence. When males reach adulthood, the
display of sexual behaviors remains consistent. Developmental analyses of both
aggressive and submissive behaviors across adolescence and adulthood has not
previously been conducted. When males were tested during adolescence with
an opponent, overt aggression remained high, however, the older the male was,
the less overt aggression was displayed. For the most part, adult males used
ﬂank marking behavior to establish dominance and displayed submissive
behaviors readily. Submissive behaviors increased with age suggesting that an
adult male can regulate their aggressive behaviors and will only display the
behavior needed to communicate with their opponent to establish a dominance

hierarchy, where an adolescent animal may “ﬁght” to establish dominance.

Chapters two and three of this dissertation focused on understanding the
effects of anabolic steroids on behavior and how it compared to adult AAS
exposure. In addition, insights were gained into the normal development of these
behaviors from adolescence to adulthood. In summary, adolescents treated with
anabolic steroids show an increase in sexual behaviors immediately after testing;
however these changes do not persist. They also show a signiﬁcant increase in

overt aggression, and do not display submissive behaviors. The behavioral

115

 

d
4
.1
,
0
Fl

i

 

changes on agonistic behaviors persist after adolescent AAS exposure. Adults
display differential behavioral responses after AAS exposure. Immediately after
treatment sexual behaviors is signiﬁcantly reduced, and these changes are not
permanent. However, the same effects on satiety are observed after adult
exposure. With respect to agonistic behaviors, there is a mild increase in
aggressive behaviors throughout development; however there is more variability
within adult social interactions with another male. These results suggest that the
adolescent brain is particularly vulnerable to the adverse effects of AAS and the

regulation of aggressive behaviors may be affected permanently.

 

  

 

 

 

9 -~——— Sexual Behaviors
.9 —— Aggressive Behaviors
B ------ Submissive Behaviors
.c
G)
(D
E
o
O
(D
H—
O
>.
L“ .......
o.
.9
Q
Adolescence Mid-adolescence Early Adulthood Adulthood
(P28) (P42) (p56, p70) (P78, P92, P106)
Age

Figure 1. Depiction of the development of male social behavior from adolescence
to adulthood.

116

Haj. AW"
J

 

Are the changes caused by AAS activational, organizational or both?

Deﬁnitions for activational and organizational changes due to gonadal
hormones were introduced in the ﬁrst chapter. These studies, which were
focused on the effects of AAS on social behaviors and amygdaloid neural
plasticity, do not directly address activational affects of gonadal hormones since
the males were intact and were administered an exogenous steroid. However,
these studies do suggest that AAS may cause organizational changes in sexual
behaviors, by their affects on long intromissions, and on aggressive behaviors on
adolescents, since they maintain high levels of aggressive behaviors well after
discontinuation of treatment. These data also provide some evidence that
adolescent AAS exposure causes organizational changes on the brain by

increasing MeAV volume.

Implications and Contributions to UnderstandinQLHumaﬁ Ana_bolic Steroid Use

Interest in understanding how gonadal hormones organize the adolescent
brain has increased and has caused a heightened concern in exploring how drug
use can affect the adolescent brain and behavioral maturation. In this
dissertation the effects of exogenous synthetic androgens are explored. AAS are
used to treat malnutrition and mood disorders (Kopera, 1993; Perry et al., 2002);
however, the advantages they have for treating physiological and psychological

disorders may become disadvantages when they are used inappropriately (Blue

117

~Tﬂnfe¢uu auction-ﬂ”

 

and Lombardo, 1999; Corcoran and Longo, 1992). These studies are the ﬁrst to
directly compare males treated with anabolic steroids during adolescence and
adulthood in order to explore immediate and long term effects on social
behaviors. Most studies exploring human anabolic steroid use are
epidemiological or limited to focusing on adult use. The particular cocktail of
anabolic steroids used, which included testosterone cypionate, nandrolone
decanoate and boldenone undecylenate, is comparable to what a heavy or
chronic anabolic steroid user would combine for effective muscle and
performance enhancing. Athletes and non-athletes of different ages use doses
of AAS 10-100 times higher than doses clinically prescribed for cosmetic and
anabolic purposes (Bahrke and Yesalis, 2004; Thiblin and Petersson, 2005).
Unfortunately, an increase in body image issues during adolescence, along with
a decrease in the perceived risk of taking AAS, have resulted in increased use of
AAS among this age group. First— time AAS use has been reported as early as
age 10, and use increases with age. Anecdotally, and through interviews with
school counselors and teachers, adolescent AAS users are described as
impulsive, spontaneous, and engaging in more risk-taking behaviors when
compared to a non-user their age. Interestingly, the enhanced social behaviors
displayed by AAS treated adolescent hamsters suggest higher levels of
impulsivity and lack of regulation of certain behavioral responses to a stimulus.
In human adult males, especially after long-term use, they are described as
explosive, aggressive and reckless; however many times the expression of this

behavior is context dependent. Moreover, if they cease to use these

118

compounds, subjects can experience withdrawal symptoms, just as if they were
consuming opiates or other chronic drugs. Additionally, users can also
experience symptoms of clinical depression and have suicidal tendencies, which
enhance the concern of mental health in AAS users, at any age. Physically, AAS

can cause liver problems, heart disease and body acne after long term use.

The male Syrian hamster has been a great animal model for studying how

different hormone treatments affect the expression and aspects of complex social

behaviors throughout development. The advantage of using this animal model

 

and this treatment regimen is that the basic knowledge on its neuroendocrine

mud 1: L" a
.

proﬁle and patterns of social behaviors along with having some evidence of the
neurochemical effects of AAS on offensive behaviors (McGinnis, 2004; Schulz
and Sisk, 2006b) . There are three main points that this work can address: (1)
adolescent anabolic steroid exposure increases the expression of social
behaviors, (2) there are permanent effects on enhanced aggressive and
suppressed submissive behaviors after adolescent exposure and (3) the
adolescent amygdala is vulnerable to morphological changes after drug

exposure.

 

119

:" " Sexual Behaviors
— Aggressive Behaviors
» Submissive Behaviors

 

- .0... 4.....1..__ .a-r-...—~ h..."
.0".

    

 

 

Display of Socnal Behavrors
Dlsplay of Soc1al BehaVIors

M... p.94 .. r v. .-. .u , e... .... .. .... . ... ... .. .. ..

 

Adolescence Mid-adolescence EarlyAdulthood EarlAdulthood Adulthood
(P28) (P42) (p56. p70) (P63) (P78, 92, 106)

Age Age

 

Figure 5.2 Depiction of the display of social behaviors after adolescent and adult
AAS exposure.

120

 

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121

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