University of Southern California Dissertations and Theses

Testosterone’s effect on reward, cognition, and myelination: rat brain and behavior

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Testosterone’s effect on reward, cognition, and myelination: rat brain and behavior

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TESTOSTERONE’S EFFECT ON REWARD, COGNITION, AND MYELINATION:
RAT BRAIN AND BEHAVIOR

by

Alexandra Donovan

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)

December 2021

Copyright 2021 Alexandra Donovan
ii

Acknowledgements

I would like to thank my mentor, Dr. Ruth Wood, for her guidance these last five years.
Her confidence in me as a scientist propped me up when my own was lacking. Thank you for
your patience, your humor, and your contagious excitement for the scientific process. Your
openness with me as your mentor about the struggles and successes in your own scientific
endeavors helped me fight against the ever-present impostor syndrome. Finally, seeing your
ability to head a lab, be involved in your local music community, and spend quality time with
your family showed me it is possible to be a professor and mentor while still nurturing an
identity outside of academia.
Thank you to the other members of my dissertation committee. Dr. Megan Herting, my
committee chair, and Dr. John Monterosso both added valuable contributions to the research
described herein, especially the portions based on human studies. Thank you for your support
and guidance throughout this process. Additionally, I would like to thank Dr. Lindsey Schier
and Dr. Nina Bradley for serving on my qualification committee.
I would like to thank my former office buddy, co-worker, researcher, all-around
entertainer, and best friend Dr. Lisa Dokovna. She helped me make the transition from protein
biochemistry to animal behavioral studies and was my number one cheerleader along the way.
She also assisted with testing animals and coding programs in Chapter 2. But more than that, she
taught me how to believe in my own abilities, how to advocate for myself, and how to keep
moving forward.
Thank you to the former members of Wood lab, including former lab technicians Grace
Li and Rebecka Serpa who assisted with animal testing and programming in Chapters 2 and 3.
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Thank you to our visiting researcher Malin Källström who assisted with animal testing in
Chapter 2. Finally, thank you to lab technician Roy Miller and USC undergraduate Brianna Sun
for assistance staining in Chapter 3.
I firmly believe I would not be in this program today were it not for the enthusiastic
support from USC Neuroscience Graduate Program Manager Deanna Solórzano and Director of
Student Services Dawn Burke. They were my first point of contact for USC when I met them at
a conference and overwhelmed them with questions about NGP faculty. They never fail to make
me laugh, and our program is truly lucky to have two people so devoted to their student’s
success.
On my journey to this PhD program, I have been inspired by many wonderful women.
Dr. Andrea Tenner at UCI gave me my first glimpse into neuroscientific research when she
allowed me to volunteer in her lab. My mentor for my master’s degree at CSULB, Dr. Vasanthy
Narayanaswami took a chance on me, and under her leadership I first began to think of myself as
a scientist.
Finally, I must thank my friends and family for the support and understanding, not just
during this program but throughout my life. This journey began with my mother’s story of her
decision to quit graduate school. Not because I wanted to pick up where she left off, but because
in that moment I recognized that sometimes the best thing you can do is quit. The support of my
friends, family, and husband gave me the ability to quit a full-time job in the middle of a
recession to go back to school. To all my family and friends, near and far, know I am grateful to
have you in my life. A piece of this degree belongs to each of you.

iv

Table of Contents

Acknowledgements..........................................................................................................................ii
List of Figures..................................................................................................................................v
Abstract...........................................................................................................................................vi
Chapter 1: Introduction to anabolic androgenic steroids use and impairment.................................1
Chapter 2: Effort-based decision making in response to high-dose androgens: role of dopamine
receptor..........................................................................................................................................29
Chapter 3: Chronic high-dose testosterone does not decrease myelination of corticostriatal tracts
in rat brain.….................................................................................................................................52
Chapter 4: Discussion of findings and their translational significance..........................................79
References List...............................................................................................................................91

v

List of Figures

Figure 1: Operant task for effort discounting, chamber and schedule…………………………34
Figure 2: Effort discounting: Percent large reward preference and omissions at baseline……38
Figure 3: Effort discounting: Percent large reward preference and omissions with D1
antagonist……………………………………………………………………………………...…40
Figure 4: Effort discounting: Percent large reward preference and omissions with D2
antagonist………………………………………………………………………………………...42
Figure 5. Anatomical regions measured for myelin (gray) and oligodendrocytes (green).….….60
Figure 6. Myelin staining as a percentage of hemispheric area in fiber tracts…………………61
Figure 7. Olig2+ immunoreactivity.……………………………………………………………63

vi

Abstract

Anabolic androgenic steroids (AAS) are taken to increase muscle mass and improve
athletic performance. While there are legitimate medical uses for AAS, illicit use occurs in
professional sports, collegiate athletics, and fitness centers. Use of AAS is linked to risky
behaviors such as drinking and driving, unprotected sex, and violent crime. To understand the
neurobiological basis behind this impairment in decision making, our lab uses a rat model of
human AAS use. Previous work has shown that chronic high-dose testosterone increases a rat’s
willingness to exert effort for a large reward in an effort discounting task. Chapter 2 extends this
finding, testing the effect of testosterone on dopamine receptor blockade on the choice of large
reward. Testosterone-treated rats did not increase effortful choice at baseline, instead displaying
an increased sensitivity to systemic blockade of dopamine D2-like receptors. Additionally, both
testosterone- and vehicle-treated rats decreased effortful choice in response to blockade of
dopamine D1-like receptors. This highlights a testosterone-induced sensitivity to dopamine D2-
like receptor activity during tasks requiring increased physical effort. Testosterone treatment has
also been observed to decrease behavioral inhibition and cognitive flexibility in rats performing
reversal learning and set-shifting tasks. These behaviors rely on executive function mediated by
the prefrontal cortex, while reward related behaviors rely on nucleus accumbens activity.
Connectivity and integrity of myelinated axons traveling between these two regions is inversely
correlated with testosterone levels, and decreased connectivity of this medial corticostriatal tract
has been observed among AAS users. In chapter 3, we determined whether the decrease in
connectivity could be the result of chronic high-dose testosterone decreasing myelination of the
rat medial corticostriatal tract. Surprisingly, we did not find any significant effect of
vii

testosterone. Taken together, these studies show that the behavioral effects of AAS are the result
of multiple subtle neurobiological changes.

1

Chapter 1. Introduction to anabolic androgenic steroids use and
impairment
1.0 Overview
The Summer Olympics of 2021 has brought doping scandals back into the news cycle. For
most people, this is the only time they are aware of anabolic androgenic steroids (AAS).
Compared to cocaine or heroin, widespread AAS abuse is a recent problem. However, these
drugs have been used and abused for almost a century. AAS are not studied as widely as more
common addictive substances, perhaps because they don’t provide a “high” like other illicit
drugs. However, AAS have negative physical and psychological effects. In this introduction, I
will summarize studies of AAS use and their influence on behavior. The following chapters will
explore gaps in our knowledge of the neurobiology behind these influences.

1.1 Anabolic Androgenic Steroids: Bigger, faster, stronger
AAS are synthetic or natural substances that function as androgens. Anabolic refers to
the substances’ ability to build muscle mass, while androgenic refers to their masculinizing
effects (Kanayama et al., 2009a). Steroids are derived from cholesterol and act as chemical
messengers in the body. These messengers bind to the androgen receptor (AR), forming a
complex which moves into the nucleus to modify gene expression (Cheskis, 2004). Some AAS
can also be aromatized into estradiol and act on estrogen receptors, generating estrogenic effects
(Tobiansky et al., 2018). AAS are typically prescribed to counteract muscle wasting, stimulate
2

appetite, or correct a hormone deficit (Shahidi, 2001). However, AAS are also misused by
athletes and others to increase muscle mass and enhance sports performance. To counteract the
widespread manufacture and abuse of AAS, United States Congress deemed AAS a Schedule III
controlled substance in 1990 (Pope et al., 2014a).
AAS were initially developed in 1930s Germany, where their ability to increase muscle
mass and enhance physical performance drew interest from beyond the medical field. First
adapted by the military and elite athletes, AAS use among amateur athletes and body builders
dramatically increased in the 1980s (Pope et al., 2014a). In 1981, the first edition of the
Underground Steroid Handbook was distributed through advertisements in fitness magazines
(Kanayama et al., 2009a). This how-to guide provided instructions on finding, purchasing, and
using AAS, making AAS accessible to the average person (Kanayama and Pope, 2018). This
sudden accessibility coincided with a shift in Hollywood’s portrayal of masculinity. Action
heroes like Arnold Schwarzenegger’s Conan the Barbarian and Sylvester Stallone’s Rambo
defined a new, heavily-muscled ideal male physique that is still evident in today’s top male
actors such as Dwayne “the Rock” Johnson.
Today AAS can be found in high schools and fitness centers. A 2018 U.S. survey of high
school seniors found the rate of steroid use (1.1%) in the past year to be twice that of
methamphetamines (0.5%), and higher than crack cocaine (0.9%) (Johnston et al., 2019). Teens
taking nutritional supplements to burn fat or build muscle have a higher likelihood of progressing
to AAS use, viewing them as a tool to improve health (Nicholls et al., 2017). Collegiate athletes
use AAS to improve athletic performance, but most users are non-competitive young adults
seeking to build muscle mass (Parkinson and Evans, 2006; Pope et al., 2014a).

3

1.2 AAS: Multiple drugs, multiple side effects
The term AAS include a variety of androgens: nandrolone, boldenone, testosterone
propionate, with varying anabolic to androgenic ratios (Shahidi, 2001). Users combine multiple
AAS in “stacks” via oral, intramuscular injection, and gel or cream formulations applied to the
skin, resulting in up to 100x the normal physiologic levels of androgens (Pope et al., 1996; Pope
and Katz, 1988). AAS are taken in “cycles,” multiple months or years of consistent use which
are followed by drug-free time periods to allow for recovery of endogenous androgen
production. Because abrupt cessation of AAS leads to withdrawal symptoms, users will often
“pyramid”, tapering drug use at the start and end of the cycle (Kanayama et al., 2009a).
Alternatively, some users adopt a “cruise and blast” program of persistent but alternating high
and low dose AAS to avoid withdrawal (Kanayama et al., 2020).
In addition to AAS, users take supplemental and illicit drugs to mitigate unwanted side
effects. Gynecomastia and fluid retention are estrogenic side effects that occur when androgens
are aromatized into estradiol. AAS users combat these effects by adding aromatase inhibitors or
estrogen receptor blockers during their cycle (Parkinson and Evans, 2006). Supplemental
diuretics and thyroid hormones increase loss of fat and water, while additional prescription
medications are taken for acne, balding and erectile dysfunction (Parkinson and Evans, 2006;
Pope et al., 2014b; Skarberg et al., 2009). In a recent study, nearly 50% of AAS users reported
taking opiates and analgesics to block pain associated with strenuous exercise and injuries, while
65% used benzodiazepines to improve sleep and combat irritability or anxiety (Skarberg et al.,
2009).
Some side effects resolve with drug abstinence, but AAS users risk damage to multiple
organ systems. Cardiovascular side effects include high blood pressure, increased cholesterol,
4

cardiomyopathy, increased risk of blood clots, atherosclerosis, and stroke (Kanayama et al.,
2008; Kaufman et al., 2019; Pope et al., 2014b). AAS users have increased rates of kidney
disease, hepatotoxicity, and formation of tumors in the liver (Davani-Davari et al., 2019;
Kanayama et al., 2008). Unsurprisingly, large doses of AAS suppress testicular function,
resulting in hypogonadism and decreased fertility. Androgen levels slowly return to normal with
abstinence, but 27% of former users continue to experience hypogonadism up to 10 years after
last use (Kanayama et al., 2008; Kaufman et al., 2019; Rasmussen et al., 2016).

1.3 Affect and addiction: psychological side effects
Along with the physiologic impact of AAS, there are common psychological side effects
such as aggression, mania, depression, and dependance. Studies of experienced and naïve AAS
users report increases in mania and aggression while on AAS (Perry et al., 2003; Pope et al.,
2000, 1996; Rashid, 2000; Su et al., 1993). Manic episodes occurred in 4-25% of study
participants, and extreme effects lead to removal of subjects for the safety of their partners and
co-workers (Kanayama et al., 2009c; Pope et al., 2000; Pope and Katz, 1988; Su et al., 1993).
Similarly, case studies report mood changes and increased aggression leading up to violent
crimes committed by AAS users with no previous history of violence (Christoffersen et al., 2019;
Pope et al., 2021, 1996; Pope and Katz, 1990; Rashid, 2000). Thus far, researchers have been
unable to identify any commonalities among subjects experiencing AAS-induced manic episodes
and extreme aggression. Comparisons of family and personal psychiatric history, age, previous
illicit substance use, or ethnicity failed to find an association (Pope et al., 2000, 1996; Su et al.,
1993).
5

While increases in mania and aggression are common during AAS use, cessation is
associated with depression (Ip et al., 2012; Pope et al., 2014b; Rasmussen et al., 2016).
Comparison of active, former, and non-users found the highest levels of depressive symptoms
among former users, at 24% (Perry et al., 2003; Pope and Katz, 1988; Pope and Katz, 1994;
Rasmussen et al., 2016). Additionally, a history of suicidal thoughts and attempts are 2-4x more
prevalent among AAS users than non-users, often during withdrawal (Börjesson et al., 2020;
Kanayama et al., 2010; Lindqvist et al., 2002; Middleman et al., 1995). Suicidality and
depression may be symptoms of preexisting mood or personality disorders, but even healthy,
naïve subjects experience an increase in depressive symptoms during withdrawal from AAS
(Börjesson et al., 2020; Ip et al., 2012; Perry et al., 2003; Su et al., 1993). To combat the effects
of withdrawal, users may quickly return to active AAS use or switch to a “cruise and blast”
method of continuous use (Kanayama et al., 2009a).
Continued use despite negative effects and using to avoid withdrawal are two criteria for
diagnosing substance dependence as outlined in the DSM-V (Center for Behavioral Health
Statistics and Quality, 2016; Kanayama et al., 2009c). A review of existing literature found an
average of 30% of AAS users developed dependence (Kanayama et al., 2009a). This dependent
population exhibits extreme patterns of use, with an average lifetime duration of 302 weeks on-
cycle. In contrast, non-dependent users averaged 25 weeks on-cycle across their lifetime
(Kanayama et al., 2009c). Likewise, dependent AAS users reported an average maximum
weekly dose of 1920 mg, whereas the non-dependent users averaged a maximum of 712mg.
Across studies, dependent participants take larger doses for longer duration than their non-
dependent counterparts, though whether this is a cause or effect of dependence is unknown (Ip et
al., 2012; Kanayama et al., 2009a). Dependent users also report more physiological and
6

psychological side effects from AAS, as would be expected given the extreme pattern of usage
(Hauger et al., 2020; Ip et al., 2012; Kanayama et al., 2009b).

1.4 Bad behavior: cognitive effects
In addition to the mood effects discussed above, AAS also negatively impact cognitive
function. Cognitive side effects include decreases in working memory, behavioral inhibition,
and cognitive flexibility (Hauger et al., 2020; Heffernan et al., 2015). These impairments hinder
everyday life, and can lead to an increase in risky behaviors such as drunk driving, unsafe sex,
and carrying a weapon (Hauger et al., 2020; Middleman et al., 1995). AAS users exhibit
decreases in visuospatial, retrospective, prospective, and working memory (Bjørnebekk et al.,
2019; Hauger et al., 2019; Heffernan et al., 2015; Hildebrandt et al., 2014; Kanayama et al.,
2013). For example, 56% of dependent users experience sufficient memory loss to impact their
daily lives (Hauger et al., 2019). The severity of these memory deficits are positively correlated
with lifetime exposure to AAS (Bjørnebekk et al., 2019; Kanayama et al., 2013). However, both
short-term users and AAS naïve study participants experience a significant increase in
forgetfulness (Börjesson et al., 2020; Heffernan et al., 2015; Su et al., 1993).
As with memory impairment, decreases in behavioral inhibition and cognitive flexibility
correlate with lifetime exposure (Bjørnebekk et al., 2019; Börjesson et al., 2020). Reduced
impulse control is reported by 27% of long-term (greater than 10 years) AAS users (Bjørnebekk
et al., 2019). Yet, all AAS users scored lower than non-users on cognitive tasks requiring action
inhibition, processing speed, and adapting to rule changes. Interestingly, behavioral inhibition
relating to emotional stimuli was significantly reduced while on AAS (Hildebrandt et al., 2014),
7

and this likely contributes to the interpersonal conflicts reported by AAS users (Börjesson et al.,
2020; Hauger et al., 2020; Kanayama et al., 2009c).
Working memory, behavioral inhibition, and cognitive flexibility all rely on the
prefrontal cortex (PFC), a region with bidirectional connections to limbic and motor regions of
the brain (Pessoa, 2009; Sousa et al., 2018; Sowell et al., 2003). Collectively, these regions
direct behavior, and dysfunction of these circuits is associated with lower levels of cognitive
control and increased aggression (Liston et al., 2006; Peper et al., 2015, 2013). Brain imaging
studies of AAS users observed decreased functional and structural connectivity between PFC and
limbic regions (Kaufman et al., 2015; Westlye et al., 2017). In addition, studies found
volumetric changes, with decreases in cognitive control regions and increases in limbic areas
such as the amygdala (Hauger et al., 2019; Kaufman et al., 2015). These observations match the
behavioral impairments and affective changes observed in AAS users. However, it is unclear
whether decreases in connectivity are a direct result of AAS use or representative of a risk-taking
phenotype more likely to use AAS.

1.5 Young and plastic: why age of onset matters
Teens and young adults are especially susceptible to the negative psychological effects of
AAS. Adolescent onset of AAS use (<19 years) generates greater reductions in behavioral
inhibition than adult onset. This is concerning, as over half of users began taking AAS before
age 25 (Hildebrandt et al., 2014; Parkinson and Evans, 2006; Pope et al., 2014a). Teen and
young adult brains are still developing, and AAS use may interfere with this process. The PFC
continues to mature from adolescence to young adulthood, shifting in volume as it refines
8

cortical and subcortical connections (Sousa et al., 2018). Importantly, this organizational process
is sensitive to androgens, generating sex differences mediated by AR (Paus et al., 2010; Peper et
al., 2011; Perrin et al., 2008). Additionally, endogenous testosterone levels are inversely
correlated with connectivity of cognitive and emotional brain regions. Lower connectivity
during adolescence predicted increased aggression, impulsivity and decreased working memory
in young adulthood (Karlsgodt et al., 2014; Peper et al., 2015; Ziegler et al., 2019). Introducing
AAS during this stage of development may exaggerate effects seen with high endogenous levels
of testosterone, resulting in permanent impairment of cognitive-emotional circuits.

1.6 From brain to behavior: Animal studies, critical pathways
Longitudinal studies can track the impact of AAS use over time, but it is still difficult to
examine the neurobiological basis of behavioral changes in humans. Human users take a variety
of AAS in multiple doses, supplement with legal or illicit drugs, have pre-existing
psychopathology, and experience variable sociocultural pressures (Blashill and Safren, 2014;
Kanayama et al., 2009c; Nicholls et al., 2017). However, human research can inform animal
models of AAS use.
Animal models allow for in-depth examination of the neurobiological changes guiding
maladaptive behavior seen in AAS users. While controlling for dose and type of AAS, behaviors
can be quantified through standardized tasks (Winstanley and Floresco, 2016). Further,
manipulation of neurobiological factors before and during behavior can be used to probe the
impact of AAS on behavior and its underlying circuits (Wood, 2008). Finally, animal models
9

allow for analysis of AAS-induced changes across multiple anatomic levels, from the proteome
to the connectome.

1.7 Animal insights: AAS-induced changes in mood
In rodent models, AAS induce behavioral changes that parallel those observed in human
users, such as increased aggression (Clark and Henderson, 2003; Mhillaj et al., 2015; Wood et
al., 2013). Aggressive behavior is rewarding, and rodents will repeatedly nose-poke to gain
access to a conspecific to fight (Couppis and Kennedy, 2008; Wood et al., 2013).
Supraphysiologic doses of AAS decrease latency to first attack and increase the number of
attacks when male rats are presented with another male (Ambar and Chiavegatto, 2009;
Namjoshi et al., 2016; Ricci et al., 2013; Wood et al., 2013). However, increasing serotonergic
activity through serotonin reuptake inhibition or receptor activation reverses the AAS-induced
increase in aggression (Wood, 2008). This suggests AAS increase aggression by decreasing
serotonin. Indeed, AAS decrease both the number of serotonergic fibers and serotonin receptor
expression in the hypothalamus and amygdala. These two regions are necessary for the
expression of aggressive behavior (Ambar and Chiavegatto, 2009; Bertozzi et al., 2018; Grimes
and Melloni, 2002; Hines et al., 1992).
Decreases in serotonergic activity also underlie the depressive-like behaviors seen in
rodent models of AAS use. Animal models of depression spend more time immobile during
forced swim and tail suspension tests, exhibiting learned helplessness also seen in depressed
human AAS users (Becker et al., 2021). Accordingly, AAS administration causes an increase in
rodent immobility time across both tests of learned helplessness (Joksimović et al., 2017;
10

Matrisciano et al., 2010; Selakovic et al., 2019; Tabor et al., 2020). However, this effect can be
blocked by co-administration of a serotonin and norepinephrine reuptake inhibitor, suggesting an
effect of either norepinephrine or serotonin (Matrisciano et al., 2010). Analysis of AAS-treated
rat PFC, striatum, and hippocampus found decreases in serotonin but not norepinephrine levels,
indicating that the increase in depressive-like behavior was generated by an AAS-induced
decrease in serotonin (Lindqvist et al., 2002; Tucci et al., 2011; Zotti et al., 2013).

1.8 Animal insights: AAS-induced changes in reward
In addition to exhibiting the increases in aggression and depression seen in human users,
rodent models of AAS use also exhibit dependence. AAS are reinforcing, and animals will self-
administer AAS even to the point of death (Peters and Wood, 2005; Wood, 2008). Multiple
studies found rodents develop a conditioned place preference for AAS (Bontempi and Bonci,
2020; Martínez-Rivera et al., 2015; Packard et al., 1998; Schroeder and Packard, 2000).
Therefore, AAS are both rewarding and reinforcing, yet they do not increase dopamine (DA) in
the nucleus accumbens (Acb) like cocaine or methamphetamine (Triemstra et al., 2008).
Incongruously, conditioned placed preference for AAS does not develop if either DA type 1
(D1R) or type 2 (D2R) receptors are blocked (Schroeder and Packard, 2000). Thus, the
rewarding effect of AAS does rely on dopaminergic activity, albeit through different
mechanisms than other drugs of abuse.
AAS are rewarding, but they also generate neurobiological changes in reward circuitry
which may cause the impaired decision making observed among human users. To test decision-
making skills, rodents are presented with a choice between a small reward and a large reward
11

associated with a cost (time delay, foot-shock, or physical effort). AAS-treated rodents are more
willing to wait, work, and endure punishment for a high-value food reward (Cooper et al., 2014;
Wallin et al., 2015; Wood et al., 2013). This effect is similar to drugs which either stimulate DA
release such as amphetamine, or block DA reuptake in Acb (Bardgett et al., 2009; Castrellon et
al., 2021; Floresco et al., 2008b). Yet, AAS act in the opposite direction, decreasing DA levels
in Acb and PFC (Tucci et al., 2011; Zotti et al., 2013). However, AAS increase D1R expression
in the amygdala and D2R expression in Acb and ventral tegmental area (Birgner et al., 2008;
Kindlundh et al., 2001; Martínez-Rivera et al., 2015). This increase in expression levels within
key regions of the reward pathway may increase sensitivity to DA, driving the changes in
reward-related behaviors observed in rat models of AAS use. In Chapter 2 I investigate this
relationship further, combining the behavioral effects of AAS and DA receptor suppression in an
animal model of effort-based choice.

1.9 Animal insights: AAS-induced changes in cognition
AAS decrease cognitive flexibility and behavioral inhibition in human and rodent models
of AAS use (Hauger et al., 2020; Heffernan et al., 2015; Hildebrandt et al., 2014; Wallin and
Wood, 2015; Wood and Serpa, 2020). Specifically, AAS-treated rats were less able to shift
strategies from a directional cue to a visual cue in order to obtain a food reward, indicating
reduced cognitive flexibility (Wallin and Wood, 2015). Within the same study, AAS-treated rats
had difficulty switching to an inversion of the rules during a reversal learning task, exhibiting
reduced behavioral inhibition. Similarly, a biconditional discrimination task showed AAS-
treated rats were less able to use contextual cues to determine the correct response when given
conflicting audio and visual cues (Wood and Serpa, 2020). Collectively, these studies show
12

AAS hinder suppression of old strategies and adaption of new strategies, while blunting the
ability to use environmental cues to modify behavior. But the neurobiological basis for these
impairments is unclear.

1.10 Animal insights: AAS-induced changes in circuitry
The behavioral impairments associated with AAS use reflect alterations of emotional and
cognitive processes. Combining behavioral and neurobiological approaches, animal studies have
determined afferent and efferent connections necessary for these cognitive and emotional
processes (Floresco et al., 2008b; Floresco and Magyar, 2006; Ghods-Sharifi and Floresco,
2010). Many of these connections lie within the mesocorticolimbic circuit, dopaminergic
projections that travel from the ventral tegmental area to the PFC, amygdala, and Acb
(Tobiansky et al., 2018). Additionally, both the amygdala and PFC send excitatory projections
onto the Acb, forming a DA-sensitive circuit (Floresco et al., 2008a). Importantly, the PFC
exerts control over Acb through glutamatergic neurons in a PFC-Acb fiber tract. This cortical
control over subcortical function integrates higher order cognitive processes with limbic regions,
guiding behaviors (Dalley et al., 2004).
Endogenous and exogenous AAS have an inverse relationship with cortico-subcortical
connectivity, such that higher levels of AAS are associated with lower connectivity of the
frontostriatal tracts (Peper et al., 2013, 2011; Perrin et al., 2008). Tract connectivity depends on
the proper organization and distribution of myelin along the axon, and disruption of this pattern
leads to poor tract integrity. Lower frontostriatal tract integrity is associated with behavioral
changes common among AAS users, including increased impulsivity, aggression, and risk-
13

taking, and reduced behavioral inhibition (Jacobus et al., 2013; Liston et al., 2006; Peper et al.,
2015, 2013; Ziegler et al., 2019).
In Chapter 3 I measure the effect of high dose AAS on the myelin content of the rat PFC
to Acb tract. Given the correlations found in human studies, the AAS-induced changes in
behavior observed in animal studies may be the result of decreased myelination in tracts
connecting frontal and striatal regions. Thus far, animal studies have shown physiologically-
relevant levels of androgens increase remyelination after a demyelinating injury (Abi Ghanem et
al., 2017; Bielecki et al., 2016; Hussain et al., 2013). Yet no study has examined the effect of
supraphysiologic doses of AAS on myelination of fiber tracts in rats. This is the question
addressed by my research in Chapter 3.

14

1.11 References
Abi Ghanem, C., Degerny, C., Hussain, R., Liere, P., Pianos, A., Tourpin, S., Habert, R.,
Macklin, W.B., Schumacher, M., Ghoumari, A.M., 2017. Long-lasting masculinizing
effects of postnatal androgens on myelin governed by the brain androgen receptor. PLoS
Genet. 13. https://doi.org/10.1371/journal.pgen.1007049
Ambar, G., Chiavegatto, S., 2009. Anabolic-androgenic steroid treatment induces behavioral
disinhibition and downregulation of serotonin receptor messenger RNA in the prefrontal
cortex and amygdala of male mice. Genes, Brain Behav. 8, 161–173.
https://doi.org/10.1111/j.1601-183X.2008.00458.x
Bardgett, M.E., Depenbrock, M., Downs, N., Points, M., Green, L., 2009. Dopamine modulates
effort-based decision making in rats. Behav. Neurosci. 123, 242–251.
https://doi.org/10.1037/a0014625
Becker, M., Pinhasov, A., Ornoy, A., 2021. Animal models of depression: what can they teach us
about the human disease? Diagnostics 11.
https://doi.org/10.3390/DIAGNOSTICS11010123
Bertozzi, G., Sessa, F., Albano, G.D., Sani, G., Maglietta, F., Roshan, M.H.K., Volti, G.L.,
Bernardini, R., Avola, R., Pomara, C., Salerno, M., 2018. The role of anabolic androgenic
steroids in disruption of the physiological function in discrete areas of the central nervous
system. Mol. Neurobiol. https://doi.org/10.1007/s12035-017-0774-1

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Bielecki, B., Mattern, C., Ghoumari, A.M., Javaid, S., Smietanka, K., Ghanem, C.A., Mhaouty-
Kodja, S., Ghandour, M.S., Baulieu, E.E., Franklin, R.J.M., Schumacher, M., Traiffort, E.,
2016. Unexpected central role of the androgen receptor in the spontaneous regeneration of
myelin. Proc. Natl. Acad. Sci. U. S. A. 113, 14829–14834.
https://doi.org/10.1073/pnas.1614826113
Birgner, C., Kindlundh-Högberg, A.M.S., Alsiö, J., Lindblom, J., Schiöth, H.B., Bergström, L.,
2008. The anabolic androgenic steroid nandrolone decanoate affects mRNA expression of
dopaminergic but not serotonergic receptors. Brain Res. 1240, 221–228.
https://doi.org/10.1016/j.brainres.2008.09.003
Bjørnebekk, A., Westlye, L.T., Walhovd, K.B., Jørstad, M.L., Sundseth, Ø.Ø., Fjell, A.M., 2019.
Cognitive performance and structural brain correlates in long-term anabolic-androgenic
steroid exposed and nonexposed weightlifters. Neuropsychology 33, 547–559.
https://doi.org/10.1037/neu0000537
Blashill, A.J., Safren, S.A., 2014. Sexual orientation and anabolic-androgenic steroids in US
adolescent boys. Pediatrics 133, 469–475. https://doi.org/10.1542/peds.2013-2768
Bontempi, L., Bonci, A., 2020. µ-Opioid receptor-induced synaptic plasticity in dopamine
neurons mediates the rewarding properties of anabolic androgenic steroids. Sci. Signal. 13,
1–12. https://doi.org/10.1126/scisignal.aba1169
Börjesson, A., Möller, C., Hagelin, A., Vicente, V., Rane, A., Lehtihet, M., Dahl, M.-L.,
Gårevik, N., Ekström, L., 2020. Male anabolic androgenic steroid users with personality
disorders report more aggressive feelings, suicidal thoughts, and criminality. Medicina (B.
Aires). 56. https://doi.org/10.3390/MEDICINA56060265
16

Castrellon, J.J., Meade, J., Greenwald, L., Hurst, K., Samanez-Larkin, G.R., 2021. Dopaminergic
modulation of reward discounting in healthy rats: a systematic review and meta-analysis.
Psychopharmacology (Berl). 238, 711–723. https://doi.org/10.1007/s00213-020-05723-5
Center for Behavioral Health Statistics and Quality, 2016. Impact of the DSM-IV to DSM-5
changes on the national survey on drug use and health. Subst. Abus. Ment. Heal. Serv.
Adminsitration Rockville, MD.
Cheskis, B.J., 2004. Regulation of cell signalling cascades by steroid hormones. J. Cell.
Biochem. 93, 20–27. https://doi.org/10.1002/jcb.20180
Christoffersen, T., Andersen, J.T., Dalhoff, K.P., Horwitz, H., 2019. Anabolic-androgenic
steroids and the risk of imprisonment. https://doi.org/10.1016/j.drugalcdep.2019.04.041
Clark, A.S., Henderson, L.P., 2003. Behavioral and physiological responses to anabolic-
androgenic steroids. Neurosci. Biobehav. Rev. https://doi.org/10.1016/S0149-
7634(03)00064-2
Cooper, S.E., Goings, S.P., Kim, J.Y., Wood, R.I., 2014. Testosterone enhances risk tolerance
without altering motor impulsivity in male rats. Psychoneuroendocrinology 40, 201–212.
https://doi.org/10.1016/j.psyneuen.2013.11.017
Couppis, M.H., Kennedy, C.H., 2008. The rewarding effect of aggression is reduced by nucleus
accumbens dopamine receptor antagonism in mice. Psychopharmacology (Berl). 197, 449–
456. https://doi.org/10.1007/s00213-007-1054-y

17

Dalley, J.W., Cardinal, R.N., Robbins, T.W., 2004. Prefrontal executive and cognitive functions
in rodents: neural and neurochemical substrates, in: Neuroscience and Biobehavioral
Reviews. pp. 771–784. https://doi.org/10.1016/j.neubiorev.2004.09.006
Davani-Davari, D., Karimzadeh, I., Khalili, H., 2019. The potential effects of anabolic-
androgenic steroids and growth hormone as commonly used sport supplements on the
kidney: A systematic review. BMC Nephrol. https://doi.org/10.1186/s12882-019-1384-0
Floresco, S.B., Magyar, O., 2006. Mesocortical dopamine modulation of executive functions:
beyond working memory. Psychopharmacology (Berl). https://doi.org/10.1007/s00213-006-
0404-5
Floresco, S.B., St. Onge, J.R., Ghods-Sharifi, S., Winstanley, C.A., 2008a. Cortico-limbic-
striatal circuits subserving different forms of cost-benefit decision making. Cogn. Affect.
Behav. Neurosci. https://doi.org/10.3758/CABN.8.4.375
Floresco, S.B., Tse, M.T.L., Ghods-Sharifi, S., 2008b. Dopaminergic and glutamatergic
regulation of effort- and delay-based decision making. Neuropsychopharmacology 33,
1966–1979. https://doi.org/10.1038/sj.npp.1301565
Ghods-Sharifi, S., Floresco, S.B., 2010. Differential effects on effort discounting induced by
inactivations of the nucleus accumbens core or shell. Behav. Neurosci. 124, 179–191.
https://doi.org/10.1037/a0018932
Grimes, J.M., Melloni, R.H., 2002. Serotonin modulates offensive attack in adolescent anabolic
steroid-treated hamsters. Pharmacol. Biochem. Behav. 73, 713–721.
https://doi.org/10.1016/S0091-3057(02)00880-8
18

Hauger, L.E., Westlye, L.T., Bjørnebekk, A., 2020. Anabolic androgenic steroid dependence is
associated with executive dysfunction. Drug Alcohol Depend. 208, 107874.
https://doi.org/10.1016/j.drugalcdep.2020.107874
Hauger, L.E., Westlye, L.T., Fjell, A.M., Walhovd, K.B., Bjørnebekk, A., 2019. Structural brain
characteristics of anabolic–androgenic steroid dependence in men. Addiction 114, 1405–
1415. https://doi.org/10.1111/add.14629
Heffernan, T., Battersby, L., Bishop, P., O’Neill, T., 2015. Everyday memory deficits associated
with anabolic-androgenic steroid use in regular gymnasium users. Open Psychiatr. J. 9, 1–6.
Hildebrandt, T., Langenbucher, J.W., Flores, A., Harty, S., Berlin, H.A., 2014. The influence of
age of onset and acute anabolic steroid exposure on cognitive performance, impulsivity, and
aggression in men. Psychol Addict Behav 28, 1096–1104. https://doi.org/10.1037/a0036482
Hines, M., Allen, L.S., Gorski, R.A., 1992. Sex differences in subregions of the medial nucleus
of the amygdala and the bed nucleus of the stria terminalis of the rat. Brain Res. 579, 321–
326. https://doi.org/10.1016/0006-8993(92)90068-K
Hussain, R., Ghoumari, A.M., Bielecki, B., Steibel, J., Boehm, N., Liere, P., MacKlin, W.B.,
Kumar, N., Habert, R., Mhaouty-Kodja, S., Tronche, F., Sitruk-Ware, R., Schumacher, M.,
Ghandour, M.S., 2013. The neural androgen receptor: a therapeutic target for myelin repair
in chronic demyelination. Brain 136, 132–146. https://doi.org/10.1093/brain/aws284
Ip, E.J., Lu, D.H., Barnett, M.J., Tenerowicz, M.J., Vo, J.C., Perry, P.J., 2012. Psychological and
physical impact of anabolic-androgenic steroid dependence. Pharmacotherapy 32, 910–919.
https://doi.org/10.1002/j.1875-9114.2012.01123
19

Jacobus, J., Thayer, R.E., Trim, R.S., Bava, S., Frank, L.R., Tapert, S.F., 2013. White matter
integrity, substance use, and risk taking in adolescence. Psychol. Addict. Behav. 27, 431–
442. https://doi.org/10.1037/a0028235
Johnston, L.D., Miech, R.A., O’Malley, P.M., Bachman, J.G., Schulenberg, J.E., Patrick, M.E.,
2019. Monitoring the Future national survey results on drug use 1975-2018: overview, key
findings on adolescent drug use. Ann Arbor Inst. Soc. Res. Univ. Michigan.
https://doi.org/10.1176/foc.1.2.213
Joksimović, J., Selaković, D., Jakovljević, V., Mihailović, V., Katanić, J., Boroja, T., Rosić, G.,
2017. Alterations of the oxidative status in rat hippocampus and prodepressant effect of
chronic testosterone enanthate administration. Mol. Cell. Biochem. 433, 41–50.
https://doi.org/10.1007/s11010-017-3014-0
Kanayama, G., Brower, K.J., Wood, R.I., Hudson, J.I., Pope, H.G., 2010. Treatment of anabolic-
androgenic steroid dependence: emerging evidence and its implications. Drug Alcohol
Depend. https://doi.org/10.1016/j.drugalcdep.2010.01.011
Kanayama, G., Brower, K.J., Wood, R.I., Hudson, J.I., Pope, H.G., 2009a. Anabolic-androgenic
steroid dependence: an emerging disorder. Addiction 104, 1966–1978.
https://doi.org/10.1111/j.1360-0443.2009.02734.x
Kanayama, G., Brower, K.J., Wood, R.I., Hudson, J.I., Pope, H.G., 2009b. Issues for DSM-V:
clarifying the diagnostic criteria for anabolic-androgenic steroid dependence. Am. J.
Psychiatry 166, 642–645. https://doi.org/10.1176/appi.ajp.2009.08111699
Kanayama, G., Hudson, J.I., Pope, H.G., 2020. Anabolic-androgenic steroid use and body image
in men: a growing concern for clinicians. https://doi.org/10.1159/000505978
20

Kanayama, G., Hudson, J.I., Pope, H.G., 2009c. Features of men with anabolic-androgenic
steroid dependence: a comparison with nondependent AAS users and with AAS nonusers.
Drug Alcohol Depend. 102, 130–137. https://doi.org/10.1016/j.drugalcdep.2009.02.008
Kanayama, G., Hudson, J.I., Pope, H.G., 2008. Long-term psychiatric and medical consequences
of anabolic-androgenic steroid abuse: a looming public health concern? Drug Alcohol
Depend. 98, 1–12. https://doi.org/10.1016/j.drugalcdep.2008.05.004
Kanayama, G., Kean, J., Hudson, J.I., Pope, H.G., 2013. Cognitive deficits in long-term
anabolic-androgenic steroid users. Drug Alcohol Depend. 130, 208–214.
https://doi.org/10.1016/j.drugalcdep.2012.11.008
Kanayama, G., Pope, H.G., 2018. History and epidemiology of anabolic androgens in athletes
and non-athletes. Mol. Cell. Endocrinol. 464, 4–13.
https://doi.org/10.1016/j.mce.2017.02.039
Karlsgodt, K.H., Bato, A.A., Blair, M.A., DeRosse, P., Szeszko, P.R., Malhotra, A.K., 2014.
White matter microstructure in the executive network associated with aggression in healthy
adolescents and young adults. Soc. Cogn. Affect. Neurosci. 10, 1251–1256.
https://doi.org/10.1093/scan/nsv015
Kaufman, M.J., Janes, A.C., Hudson, J.I., Brennan, B.P., Kanayama, G., Kerrigan, A.R., Jensen,
J.E., Pope, H.G., 2015. Brain and cognition abnormalities in long-term anabolic-androgenic
steroid users. Drug Alcohol Depend. 152, 47–56.
https://doi.org/10.1016/j.drugalcdep.2015.04.023

21

Kaufman, M.J., Kanayama, G., Hudson, J.I., Pope, H.G., 2019. Supraphysiologic-dose anabolic–
androgenic steroid use: a risk factor for dementia? Neurosci. Biobehav. Rev. 100, 180–207.
https://doi.org/10.1016/j.neubiorev.2019.02.014
Kindlundh, A.M.S., Lindblom, J., Bergström, L., Wikberg, J.E.S., Nyberg, F., 2001. The
anabolic-androgenic steroid nandrolone decanoate affects the density of dopamine receptors
in the male rat brain. Eur. J. Neurosci. 13, 291–296. https://doi.org/10.1046/j.0953-
816X.2000.01402.x
Lindqvist, A.-S., Johansson-Steensland, P., Nyberg, F., Fahlke, C., 2002. Anabolic androgenic
steroid affects competitive behaviour, behavioural response to ethanol and brain serotonin
levels. Behav. Brain Res. 133, 21–29.
Liston, C., Watts, R., Tottenham, N., Davidson, M.C., Niogi, S., Ulug, A.M., Casey, B.J., 2006.
Frontostriatal microstructure modulates efficient recruitment of cognitive control. Cereb.
Cortex 16, 553–560. https://doi.org/10.1093/cercor/bhj003
Martínez-Rivera, F.J., Natal-Albelo, E.J., Martínez, N.A., Orozco-Vega, R.A., Muñiz-Seda,
O.A., Barreto-Estrada, J.L., 2015. The effect of the anabolic steroid, nandrolone, in
conditioned place preference and D1 dopamine receptor expression in adolescent and adult
mice. Behav. Processes 113, 81. https://doi.org/10.1016/J.BEPROC.2015.01.008
Matrisciano, F., Modafferi, A.M.E., Togna, G.I., Barone, Y., Pinna, G., Nicoletti, F.,
Scaccianoce, S., 2010. Repeated anabolic androgenic steroid treatment causes
antidepressant-reversible alterations of the hypothalamic - pituitary - adrenal axis, BDNF
levels and behavior. Neuropharmacology 58, 1078–1084.
https://doi.org/10.1016/j.neuropharm.2010.01.015
22

Mhillaj, E., Morgese, M.G., Tucci, P., Bove, M., Schiavone, S., Trabace, L., 2015. Effects of
anabolic-androgens on brain reward function. Front. Neurosci.
https://doi.org/10.3389/fnins.2015.00295
Middleman, A.B., Faulkner, A.H., Woods, E.R., Emans, S.J., DuRant, R.H., 1995. High-risk
behaviors among high school students in Massachusetts who use anabolic steroids.
Pediatrics 96, 268–272.
Namjoshi, D.R., Cheng, W.H., Carr, M., Martens, K.M., Zareyan, S., Wilkinson, A., McInnes,
K.A., Cripton, P.A., Wellington, C.L., 2016. Chronic exposure to androgenic-anabolic
steroids exacerbates axonal injury and microgliosis in the CHIMERA mouse model of
repetitive concussion. PLoS One 11, e0146540.
https://doi.org/10.1371/journal.pone.0146540
Nicholls, A.R., Cope, E., Bailey, R., Koenen, K., Dumon, D., Theodorou, N.C., Chanal, B.,
Laurent, D. Saint, Müller, D., Andrés, M.P., Kristensen, A.H., Thompson, M.A., Baumann,
W., Laurent, J.F., 2017. Children’s first experience of taking anabolic-androgenic steroids
can occur before their 10th birthday: a systematic review identifying 9 factors that predicted
doping among young people. Front. Psychol. 8. https://doi.org/10.3389/fpsyg.2017.01015
Packard, M.G., Schroeder, J.P., Alexander, G.M., 1998. Expression of testosterone conditioned
place preference is blocked by peripheral or intraaccumbens injection of α-flupenthixol.
Horm. Behav. 34, 39–47. https://doi.org/10.1006/hbeh.1998.1461
Parkinson, A.B., Evans, N.A., 2006. Anabolic androgenic steroids: a survey of 500 users. Med.
Sci. Sports Exerc. 38, 644–651. https://doi.org/10.1249/01.mss.0000210194.56834.5d

23

Paus, T., Nawaz-Khan, I., Leonard, G., Perron, M., Pike, G.B., Pitiot, A., Richer, L., Susman, E.,
Veillette, S., Pausova, Z., 2010. Sexual dimorphism in the adolescent brain: role of
testosterone and androgen receptor in global and local volumes of grey and white matter.
Horm. Behav. 57, 63–75. https://doi.org/10.1016/j.yhbeh.2009.08.004
Peper, J.S., De Reus, M.A., Van Den Heuvel, M.P., Schutter, D.J.L.G., 2015. Short fused?
Associations between white matter connections, sex steroids, and aggression across
adolescence. Hum Brain Mapp 36, 1043–1052. https://doi.org/10.1002/hbm.22684
Peper, J.S., Mandl, R.C.W., Braams, B.R., De Water, E., Heijboer, A.C., Koolschijn, P.C.M.P.,
Crone, E.A., 2013. Delay discounting and frontostriatal fiber tracts: a combined DTI and
MTR study on impulsive choices in healthy young adults. Cereb. Cortex 23, 1695–1702.
https://doi.org/10.1093/cercor/bhs163
Peper, J.S., van den Heuvel, M.P., Mandl, R.C.W., Pol, H.E.H., van Honk, J., 2011. Sex steroids
and connectivity in the human brain: A review of neuroimaging studies.
Psychoneuroendocrinology. https://doi.org/10.1016/j.psyneuen.2011.05.004
Perrin, J.S., Hervé, P.Y., Leonard, G., Perron, M., Pike, G.B., Pitiot, A., Richer, L., Veillette, S.,
Pausova, Z., Paus, T., 2008. Growth of white matter in the adolescent brain: role of
testosterone and androgen receptor. J. Neurosci. 28, 9519–9524.
https://doi.org/10.1523/JNEUROSCI.1212-08.2008
Perry, P.J., Kutscher, E.C., Lund, B.C., Yates, W.R., Holman, T.L., Demers, L., 2003. Measures
of aggression and mood changes in male weightlifters with and without androgenic anabolic
steroid use. J. Forensic Sci. 48, 2002240. https://doi.org/10.1520/jfs2002240

24

Pessoa, L., 2009. How do emotion and motivation direct executive control? Trends Cogn. Sci.
13, 160–166. https://doi.org/10.1016/j.tics.2009.01.006
Peters, K.D., Wood, R.I., 2005. Androgen dependence in hamsters: overdose, tolerance, and
potential opioidergic mechanisms. Neuroscience 130, 971–981.
https://doi.org/10.1016/j.neuroscience.2004.09.063
Pope, H.G., Kanayama, G., Athey, A., Ryan, E., Hudson, J.I., Baggish, A., Pope Jr, H.G.,
Kanayama, G., Athey, A., Ryan, E., Hudson, J.I., Baggish, A., 2014a. The lifetime
prevalence of anabolic-androgenic steroid use and dependence in Americans: current best
estimates, American Journal on Addictions. Wiley-Blackwell Publishing Ltd.
https://doi.org/10.1111/j.1521-0391.2013.12118.x
Pope, H.G., Kanayama, G., Hudson, J.I., Kaufman, M.J., 2021. Review article: anabolic-
androgenic steroids, violence, and crime: two cases and literature review. Am. J. Addict.
https://doi.org/10.1111/ajad.13157
Pope, H.G., Katz, D.L., 1990. Homicide and near-homicide by anabolic steroid users. J. Clin.
Psychiatry 51, 28–31.
Pope, H. G., Katz, D.L., 1988. Affective and psychotic symptoms associated with anabolic
steroid use. Am. J. Psychiatry. https://doi.org/10.1176/ajp.145.4.487
Pope, H.G., Kouri, E.M., Hudson, J.I., 2000. Effects of supraphysiologic doses of testosterone on
mood and aggression in normal men: A randomized controlled trial. Arch. Gen. Psychiatry
57, 133–140. https://doi.org/10.1001/archpsyc.57.2.133

25

Pope, H.G., Kouri, E.M., Powell, K.F., Campbell, C., Katz, D.L., 1996. Anabolic-androgenic
steroid use among 133 prisoners. Compr. Psychiatry 37, 322–327.
https://doi.org/10.1016/S0010-440X(96)90013-9
Pope, H.G., Wood, R.I., Rogol, A., Nyberg, F., Bowers, L., Bhasin, S., 2014b. Adverse health
consequences of performance-enhancing drugs: an endocrine society scientific statement.
Endocr. Rev. 35, 341–375. https://doi.org/10.1210/er.2013-1058
Pope, H.G.J., Katz, D.L., 1994. Psychiatric and medical effects of anabolic-androgenic steroid
use. A controlled study of 160 athletes. Arch. Gen. Psychiatry 51, 375–82.
https://doi.org/10.1001/archpsyc.1994.03950050035004
Rashid, W., 2000. Testosterone abuse and affective disorders. J. Subst. Abuse Treat. 18, 179–
184. https://doi.org/10.1016/S0740-5472(99)00023-9
Rasmussen, J.J., Selmer, C., østergren, P.B., Pedersen, K.B., Schou, M., Gustafsson, F., Faber,
J., Juul, A., Kistorp, C., 2016. Former abusers of anabolic androgenic steroids exhibit
decreased testosterone levels and hypogonadal symptoms years after cessation: a case-
control study. PLoS One 11. https://doi.org/10.1371/journal.pone.0161208
Ricci, L.A., Morrison, T.R., Melloni, R.H., 2013. Adolescent anabolic/androgenic steroids:
aggression and anxiety during exposure predict behavioral responding during withdrawal in
syrian hamsters (mesocricetus auratus). Horm Behav 64, 770–780.
https://doi.org/10.1016/j.yhbeh.2013.10.002
Schroeder, J.P., Packard, M.G., 2000. Role of dopamine receptor subtypes in the acquisition of a
testosterone conditioned place preference in rats, Neuroscience Letters.
https://doi.org/10.1016/S0304-3940(00)00839-9
26

Selakovic, D., Joksimovic, J., Jovicic, N., Mitrovic, S., Mihailovic, V., Katanic, J., Milovanovic,
D., Pantovic, S., Mijailovic, N., Rosic, G., 2019. The impact of hippocampal sex hormones
receptors in modulation of depressive-like behavior following chronic anabolic androgenic
steroids and exercise protocols in rats. Front. Behav. Neurosci. 13.
https://doi.org/10.3389/fnbeh.2019.00019
Shahidi, N.T., 2001. A review of the chemistry, biological action, and clinical applications of
anabolic-androgenic steroids. Clin. Ther. 23, 1355–1390. https://doi.org/10.1016/S0149-
2918(01)80114-4
Skarberg, K., Nyberg, F., Engstrom, I., 2009. Multisubstance use as a feature of addiction to
anabolic-androgenic steroids. Eur. Addict. Res. 15, 99–106.
https://doi.org/10.1159/000199045
Sousa, S.S., Amaro, E., Crego, A., Gonçalves, Ó.F., Sampaio, A., 2018. Developmental
trajectory of the prefrontal cortex: a systematic review of diffusion tensor imaging studies.
Brain Imaging Behav. https://doi.org/10.1007/s11682-017-9761-4
Sowell, E.R., Peterson, B.S., Thompson, P.M., Welcome, S.E., Henkenius, A.L., Toga, A.W.,
2003. Mapping cortical change across the human life span. https://doi.org/10.1038/nn1008
Su, T.P., Pagliaro, M., Schmidt, P.J., Pickar, D., Wolkowitz, O., Rubinow, D.R., 1993.
Neuropsychiatric effects of anabolic steroids in male normal volunteers. JAMA 269, 2760–
4. https://doi.org/10.1001/jama.269.21.2760

27

Tabor, J., Wright, D.K., Christensen, J., Zamani, A., Collins, R., Shultz, S.R., Mychasiuk, R.,
2020. Examining the effects of anabolic–androgenic steroids on repetitive mild traumatic
brain injury (RmTBI) outcomes in adolescent rats. Brain Sci. 10.
https://doi.org/10.3390/brainsci10050258
Tobiansky, D.J., Wallin-Miller, K.G., Floresco, S.B., Wood, R.I., Soma, K.K., 2018. Androgen
regulation of the mesocorticolimbic system and executive function. Front. Endocrinol.
(Lausanne). https://doi.org/10.3389/fendo.2018.00279
Triemstra, J.L., Sato, S.M., Wood, R.I., 2008. Testosterone and nucleus accumbens dopamine in
the male syrian hamster. Psychoneuroendocrinology 33, 386–394.
https://doi.org/10.1016/j.psyneuen.2007.12.006
Tucci, P., Morgese, M.G., Colaianna, M., Zotti, M., Schiavone, S., Cuomo, V., Trabace, L.,
2011. Neurochemical consequence of steroid abuse: stanozolol-induced monoaminergic
changes. https://doi.org/10.1016/j.steroids.2011.12.014
Wallin, K.G., Alves, J.M., Wood, R.I., 2015. Anabolic-androgenic steroids and decision making:
probability and effort discounting in male rats. Psychoneuroendocrinology 57, 84–92.
https://doi.org/10.1016/j.psyneuen.2015.03.023
Wallin, K.G., Wood, R.I., 2015. Anabolic-androgenic steroids impair set-shifting and reversal
learning in male rats. Eur. Neuropsychopharmacol. 25, 583–590.
https://doi.org/10.1016/j.euroneuro.2015.01.002
Westlye, L.T., Kaufmann, T., Alnæs, D., Hullstein, I.R., Bjørnebekk, A., 2017. Brain
connectivity aberrations in anabolic-androgenic steroid users. NeuroImage Clin. 13, 62–69.
https://doi.org/10.1016/j.nicl.2016.11.014
28

Winstanley, C.A., Floresco, S.B., 2016. Deciphering decision making: variation in animal
models of effort- and uncertainty-based choice reveals distinct neural circuitries underlying
core cognitive processes. J. Neurosci. 36, 12069–12079.
https://doi.org/10.1523/JNEUROSCI.1713-16.2016
Wood, R.I., 2008. Anabolic-androgenic steroid dependence? Insights from animals and humans.
Front. Neuroendocrinol. https://doi.org/10.1016/j.yfrne.2007.12.002
Wood, R.I., Armstrong, A., Fridkin, V., Shah, V., Najafi, A., Jakowec, M., 2013. ’Roid rage in
rats? Testosterone effects on aggressive motivation, impulsivity and tyrosine hydroxylase.
Physiol. Behav. 110–111, 6–12. https://doi.org/10.1016/j.physbeh.2012.12.005
Wood, R.I., Serpa, R.O., 2020. Anabolic-androgenic steroid abuse and cognitive impairment:
testosterone impairs biconditional task performance in male rats. Behav. Brain Res. 379,
112339. https://doi.org/10.1016/j.bbr.2019.112339
Ziegler, G., Hauser, T.U., Moutoussis, M., Bullmore, E.T., Goodyer, I.M., Fonagy, P., Jones,
P.B., Lindenberger, U., Dolan, R.J., 2019. Compulsivity and impulsivity traits linked to
attenuated developmental frontostriatal myelination trajectories. Nat. Neurosci. 22, 992–
999. https://doi.org/10.1038/s41593-019-0394-3
Zotti, M., Tucci, P., Colaianna, M., Morgese, M.G., Mhillaj, E., Schiavone, S., Scaccianoce, S.,
Cuomo, V., Trabace, L., 2013. Chronic nandrolone administration induces dysfunction of
the reward pathway in rats. https://doi.org/10.1016/j.steroids.2013.10.005

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Chapter 2. Effort-based decision making in response to high-dose
androgens: role of dopamine receptors

2.1 Abstract
Anabolic androgenic steroids (AAS) are performance-enhancing drugs used by both
world-class and rank-and-file athletes. AAS abuse has been linked with risky decision-making,
ranging from drunk driving to abusing multiple drugs. Our lab uses operant behavior in rats to
test effects of AAS (testosterone) on decision making. In our previous study, testosterone caused
rats to work harder for food reward in an effort discounting task, where rats choose between a
small easy reward (1 lever press for 1 sugar pellet) and a large difficult reward (2, 5, 10, or 15
presses for 3 pellets). Compared with controls, rats treated chronically with high-dose
testosterone (7.5mg/kg) preferred the large reward lever. Effort discounting is sensitive to
dopamine in the nucleus accumbens, and AAS alter accumbens dopamine receptor expression.
Accordingly, we determined if testosterone increases sensitivity to dopamine D1-like
(SCH23390, 0.01 mg/kg) or D2-like (eticlopride, 0.06 mg/kg) receptor antagonists during effort
discounting. At baseline, testosterone- and vehicle-treated rats showed similar preference for the
large reward lever (at FR5, testosterone: 68.6±9.7%, vehicle: 85.7±2.5%). SCH23390 reduced
large reward preference significantly in both groups (at FR5, testosterone: 41.3±9.2%, vehicle:
49.1±8.2%, F(1,16)=17.7, p<0.05). Eticlopride decreased large reward preference in both groups,
but more strongly in testosterone-treated rats (at FR5: testosterone: 37.0±9.7%, vehicle:
56.3±7.8%, F(1,16)=35.3, p<0.05). This suggests that testosterone increases willingness to exert
effort for a large reward through increased sensitivity to dopamine D2-like receptor blockade.

30

2.2 Introduction
Anabolic-androgenic steroids (AAS) enhance athletic performance by increasing
musculature and aerobic power, and proponents endorse them as part of a healthy lifestyle
(Kanayama et al., 2009b). However, AAS can induce cardiovascular, hepatic, reproductive and
psychiatric dysfunction (Kaufman et al., 2019). Additionally, AAS users are more likely to
make decisions that put themselves and others at risk: domestic violence, drunk driving, assault
with a deadly weapon, and homicide (Choi and Pope, 1994; Middleman et al., 1995; Pope et al.,
1996). Though impaired decision-making and cognitive function have been observed in human
AAS users, it is difficult to distinguish the effects of AAS, as they are often taken in combination
with other drugs of abuse (Bjørnebekk et al., 2019; Kanayama et al., 2013). Compounding this,
any pre-existing psychopathology may itself alter decision making and risk assessment. Thus, it
is difficult to isolate the effects of AAS on decision making in human users.
Our laboratory employs a rat model of AAS use to study behavioral effects on decision
making. Chronic high-dose testosterone drive rats to work harder, wait longer, and accept more
punishment in exchange for a large reward during operant behavioral discounting tasks (Cooper
et al., 2014; Wallin et al., 2015; Wood et al., 2013). Especially, effort discounting (ED) requires
subjects to expend physical effort for a large reward (Winstanley and Floresco, 2016).
Testosterone-treated rats are more willing to expend the effort required for large rewards than
vehicle controls (Wallin et al., 2015). Further understanding of these testosterone-induced
behavioral effects requires examining the circuitry involved during decision making.
ED behavior depends on the mesocorticolimbic dopamine (DA) circuitry, as lesion or
disconnection of key nodes within this circuit significantly reduce effortful choice (Ghods-
31

Sharifi et al., 2009; Ghods-Sharifi and Floresco, 2010). Systemic administration of flupenthixol,
an antagonist to DA D1-like (D1R) and D2-like receptors (D2R), decreases large reward
preference and local antagonism of either receptor decreased effort exerted for a more palatable
food reward (Floresco et al., 2008b; Nowend et al., 2001). This is significant, as AAS
themselves alter dopamine receptor expression in subcortical portions of the DA circuit,
simultaneously increasing D2R and decreasing D1R (Kindlundh et al., 2001). We hypothesize
the testosterone-induced behavioral changes during ED are the result of altered DA receptor
activity.
To test this, we pretreated rats with the D2R antagonist eticlopride to block D2R activity
during ED, with the expectation that chronic high-dose testosterone would increase sensitivity to
D2R blockade. We also determined the effect of blocking D1R activity with SCH23390 (SCH).
It is somewhat more difficult to predict the effect of SCH on ED in testosterone-treated rats.
Both testosterone and systemic D1R stimulation have been shown to increase willingness to
work for a large reward, but the testosterone-induced decrease in subcortical D1R expression
may result in a reduced response to D1R blockade during ED (Bardgett et al., 2009; Wallin et al.,
2015). Ultimately, we expect to see a decrease in large reward preference in response to D1R
antagonist.

2.3 Methods
2.3.1 Animals
18 male Long-Evans rats (20 weeks of age, Charles River Laboratories, MA) received
either vehicle (n= 9) or chronic high-dose testosterone treatment (n=9). They remained gonad-
32

intact to approximate human AAS use. Rats were pair-housed with a partner from the same
treatment group and were tested daily (5 d/w) during the dark phase under a reversed 14:10D
photoperiod. Food (standard lab chow) was restricted to maintain a slow rate of growth (3-4 g/d)
and facilitate operant responding; water was available ad libitum (Cooper et al., 2014; Wallin et
al., 2015). Throughout the study, vehicle- and testosterone- treated rats did not differ in body
weight. Experimental procedures were approved by USC’s Institutional Animal Care and Use
Committee and were conducted in accordance with the Guide for the Care and Use of Laboratory
Animals, 8th Ed (National Research Council, National Academies Press, Washington DC; 2011).

2.3.2 Drug treatments
2.3.2.1 AAS: Rats received injections of testosterone (7.5 mg/kg Steraloids, RI) or vehicle [3%
ethanol and 13% cyclodextrin (RBI, MA)] s.c. 5 d/w immediately prior to placement in operant
chambers (Cooper et al., 2014; Wallin et al., 2015). Treatment began at the onset of training and
continued for the duration of the experiment. Testosterone is the most common performance-
enhancing substance detected in urine tests by World Anti-Doping Agency-accredited labs
(55.5%; (WADA, 2012)). The 7.5 mg/kg dose approximates heavy steroid use in humans and
has been used previously to test ED (Wallin et al., 2015).

2.3.2.2 Dopamine receptor antagonists: SCH (0.01mg/kg) or eticlopride (0.06 mg/kg; Sigma-
Aldrich, MO) were dissolved in 0.9% sterile saline, and administered i.p. immediately prior to
testing on selected days (Figure 1). These doses represent the minimal effective dose to decrease
large reward preference in rats during ED (Hosking et al., 2015). Sterile saline was used as a
33

vehicle control in equivalent volume. Injections were co-administered with daily testosterone or
vehicle treatment.

2.3.3 Training
Operant chambers were equipped with 2 retractable levers flanking a food cup attached to
a pellet dispenser (Figure 1A; Med-Associates, VT). Chambers had a house-light for
illumination and were in sound-attenuating boxes with fans for ventilation. Injections were
administered immediately prior to training and testing, as in Wallin et al (2017). Briefly,
testosterone- and vehicle-treated rats were trained to respond on a lever within 10 s to receive a
single 45-mg sucrose pellet (Bio-Serv Inc., NJ). Each trial began in darkness in the inter-trial
interval state (ITI); darkness and retracted levers, lasting for 20 s. The house-light was then
turned on, and 1 lever was inserted into the chamber. Front and back levers were each inserted
once per pair of trials in random order. Failure to respond within 10 s reverted the chamber to
ITI, and the trial was counted as an omission. Each trial lasted 20 s. Training continued until
there were <5 omissions in 90 trials.
34

Figure 1: A: Operant task for effort discounting. Rats choose between small/low effort reward
(1 pellet for 1 lever press) and large/high effort reward (3 pellets for 1, 2, 5, or 10 presses). B:
Timeline of antagonist treatments and behavioral testing for testosterone- and vehicle-treated
rats.

2.3.4 Reward Discrimination
Sessions consisted of 4 blocks of 16 trials each: 8 forced-choice trials followed by 8 free-
choice trials. In forced-choice trials, 1 lever was inserted per trial (4 trials/lever) so that rats
learned the lever associated with the large (3 pellets) and small (1 pellet) rewards. In free-choice
35

trials, both levers were inserted, and the rat could select either the lever associated with the small
reward or large reward (Figure 1A). Rats had 10 s to make a response before levers retracted,
and the trial was counted as an omission. All rats were required to complete 80 trials with >80%
selection of the large reward lever.

2.3.5 Effort Discounting
As for reward discrimination, ED testing consisted of 4 blocks of 16 trials each: 8 forced-
choice trials followed by 8 free-choice trials. A response on the small reward lever caused
retraction of both levers and delivery of 1 pellet. A response on the large reward lever caused
retraction of the small reward lever, and delivery of 3 pellets after meeting the response
requirement of 1, 2, 5, or 10 lever presses (Uban et al., 2012). Failure to complete the response
requirement within 15 s resulted in an incomplete trial, and no pellets were delivered. Rats were
tested for ED for 12 days until behavior stabilized. Stability was assessed by repeated-measures
analysis of variance (RM-ANOVA) on the last 5 days of testing, with day as the repeated
measure. When there was no effect of day on large reward preference the behavior was deemed
stable. The last 3 days of testing were averaged to establish a baseline.

2.3.6 Dopamine antagonists
The order of drug treatments is shown in Figure 1B. Testosterone- and vehicle-treated
rats were tested for ED with saline pretreatment for 3 d (Saline1). Next, they received 3 days of
ED testing with SCH pretreatment. Saline pretreatment was repeated for 3 d (Saline2), after
which eticlopride was administered for the final 3 d of testing.
36

2.3.7 Data analysis
For each rat on each day of testing, we determined the number of trial omissions,
responses on the large and small reward levers, and incomplete trials in each block. In free-
choice trials, percent omissions were calculated as the number of omitted trials in 8 trials/block.
Large reward preference was defined as the percent of free-choice trials/block (less trial
omissions) in which rats chose the large reward lever. Percent incomplete trials were calculated
from the number of incomplete responses when the rat chose the large reward. Individual rat
responses were averaged across 3 d of treatment and used to compare responses from
testosterone- and vehicle-treated groups. Data from SCH and eticlopride treatments of both
groups were compared to their respective 3 d of saline pretreatment (SCH with Saline1,
eticlopride with Saline2). Behavioral responses were analyzed by RM-ANOVA using JMP Pro
14. Testosterone was the between-subjects factor, and block and DA receptor antagonists were
repeated measures within-subject factors. Significance was set at p<0.05.

2.4 RESULTS
2.4.1 Baseline ED
With initial reward discrimination (FR1 across all 4 blocks), all rats maintained a strong
preference for the large reward (>85%), and there was no significant difference between
testosterone- and vehicle-treated rats (Figure 2A). However, with ED at baseline, there was a
significant effect of block (F(3,48)= 97.9, p<0.05, η
2
=0.88), with both groups of rats reducing large
reward preference as the response requirement increased (Figure 2B). This was accompanied by
a significant increase in omissions (F(3,48)= 54.4, p<0.05; Figure 2C) primarily in the final FR10
block, but with no effect of testosterone. Incomplete trials were negligible during FR2-FR5: in 3
37

days of testing with 20 rats there were only 9 incomplete trials out of 1,920 total trials (0.5%). At
FR10 testosterone- and vehicle-treated rats averaged 31.5%, but there was no difference between
groups (data not shown). In contrast to our previous study (Wallin et al., 2015), there was no
significant effect of testosterone on large reward preference during ED, and no testosterone x block
interaction. At FR 5, testosterone-treated rats chose large reward lever on 68.6±9.7% of trials,
while vehicle-treated rats chose the large reward on 85.7±2.5%. During subsequent pretreatment
with saline, large reward preference and the number of omissions were unchanged.

38

Figure 2: Percent large reward preference (A, B; mean ± SEM) and trial omissions (C) for
testosterone- (triangles) and vehicle-treated rats (circles) during 8 free-choice trials/block of (A)
reward discrimination at FR1 and (B, C) effort discounting. Asterisk indicates significant effect
of block (p<0.05) for effort discounting.

39

2.4.2 D1R antagonist: SCH23390
Figure 3 illustrates the effect of the D1R antagonist SCH on ED in testosterone- and vehicle-
treated rats. As at baseline (Figure 2B), there was a significant effect of block on large reward
preference [F(3,48)= 121.4, p<0.05; Fig 3A and B], but no effect of testosterone, and no interaction.
Furthermore, pretreatment with D1R antagonist SCH decreased large reward preference
significantly in both groups [F(1,16)=17.7, p<0.05, η
2
=0.92]. During free-choice trials in the FR5
block, testosterone-treated rats responded on the large reward lever in 71.8±5.2% of trials with
saline pretreatment, while SCH pretreatment decreased large reward preference to 41.3±9.2%.
Vehicle-treated rats showed a similar response: 79.6±3.4% preference with saline pretreatment at
FR5, and 49.1±8.2% preference following SCH. Trial omissions (Figures 3C & D) increased
significantly in response to both block [F(3,48)=43.3 p<0.05] and D1R antagonist [F(1,16)= 16.8
p<0.05]. As at baseline, incomplete trials were negligible during FR2-FR5: in 3 days of saline and
3 days of SCH with 20 rats, there were only 11 incomplete trials out of 3,840 total trials (0.3%).
At FR10, testosterone- and vehicle-treated rats averaged 21.4% incomplete trials, but there was no
effect of testosterone or SCH (data not shown).
40

Figure 3: Percent large reward preference (A, B; mean ± SEM) and trial omissions (C, D) for
testosterone- (A, C; triangles) and vehicle-treated rats (B, D; circles) in response to saline (open
symbols) or the dopamine D1 receptor antagonist SCH23390 (SCH, 0.01 mg/kg; closed symbols)
during 8 free-choice trials/block of effort discounting. Asterisks indicate significant effect of block
and SCH (p<0.05).

41

2.4.3 D2R antagonist: eticlopride
Figure 4 shows the effect of the D2R antagonist eticlopride on ED in testosterone- and
vehicle-treated rats. There was again a significant effect of block [F(3,48)= 90.4, p<0.05], and
pretreatment with eticlopride significantly decreased large reward preference [F(1,16)=35.3, p<0.05,
η
2
=0.93]. However, there was also a significant effect of testosterone [F(1,16)=6.9, p<0.05], and an
interaction among testosterone, eticlopride, and block [F(3,48)=4.6, p<0.05]. In particular,
eticlopride reduced large reward preference in testosterone-treated rats at FR1: from 92.3±3.5%
with saline pretreatment to 63.8±9.2% with eticlopride (Fig. 4A). By contrast, eticlopride had
minimal effect on large reward preference in vehicle-treated rats (94.9±2.8% and 89.6±3.9%,
respectively; Fig. 4B).
This pattern was repeated for trial omissions (Fig. 4C and D), which increased significantly
in response to block [F(3,48)=23.3, p<0.05], D2R antagonist [F(1,16)= 28.4,p<0.05], and
testosterone [F(1,16)= 2.3, p<0.05]. With eticlopride pretreatment, testosterone-treated rats failed
to respond on any lever in 46.3% of free-choice trials at FR2, vs 1.4% with saline. Furthermore,
incomplete trials (Fig. 4E) increased significantly in the final FR10 block in response to D2R
antagonist [F(1,16)= 2.2, p<0.05] with an interaction between testosterone and D2R antagonist
[F(1,17)= 5.9, p<0.05].
42

43

Figure 4: Percent large reward preference (A, B; mean ± SEM) and trial omissions (C, D) for
testosterone- (A, C; triangles) and vehicle-treated rats (B, D; circles) in response to saline (open
symbols) or the dopamine D2 receptor antagonist eticlopride (0.06 mg/kg; closed symbols)
during 8 free-choice trials/block of effort discounting. Percent incomplete trials (E) for
testosterone- (left) and vehicle-treated rats (right) during free-choice trials in response to saline
(white) or eticlopride (black) during final block of effort discounting. Asterisks indicate
significant effect of block, eticlopride, and/or testosterone (p<0.05). Cross indicates a
testosterone by eticlopride interaction.

2.5 Discussion
The present study tested the effect of chronic high-dose testosterone on ED and in response
to systemic pretreatment with a D1R or D2R antagonist. Blocking either D1R or D2R decreased
large reward preference and increased omissions in ED, while D2R blockade also increased
incomplete trials. However, testosterone-treated rats were significantly more sensitive than
vehicle-treated rats to the D2R antagonist eticlopride. This supports our hypothesis that
testosterone sensitizes rats to D2R blockade.
In addition to increases in Acb, AAS increased D2R in the amygdala (Birgner et al.,
2008; Kindlundh et al., 2001). As with Acb, inactivation of amygdala decreased effortful choice
(Ghods-Sharifi et al., 2009). These AAS-induced increases in D2R across regions modulating
effortful choice may serve to increase sensitivity of this circuit to D2R activity. Thus, we saw a
significant increase in effort discounting among testosterone-treated rats in our study. While
both groups decreased large reward preference in response to D2R blockade, the decrease seen in
testosterone-treated rats was much larger than that of vehicle-treated rats (FR2: 40% vs 90%,
respectively).
When faced with increased response requirements to receive a large reward, it would
seem logical for rats to switch to responding on the small reward lever. Instead, it was somewhat
44

surprising that they often failed to respond on either lever. The increased number of omissions
in response to testosterone and eticlopride may relate to deficits in cognitive flexibility. Set-
shifting is a test of cognitive flexibility, our lab has previously shown testosterone-treated rats
exhibit impairments in set-shifting (Wallin and Wood, 2015). A reduction in cognitive
flexibility may diminish the rats’ ability to shift responses from the large to the small reward
lever, thereby increasing omissions. Additionally, there was an increase in incomplete trials
observed among testosterone-treated rats in response to D2R blockade. Interestingly, D2R
antagonists increase response latency in studies of physical and cognitive effort, and this effect
could account for the increase in incomplete trials (Besson et al., 2010; Hosking et al., 2015;
Pattij et al., 2007). A slower rate of response may cause the trial to time out before the requisite
number of lever presses is reached. Though human AAS users do not display increased reaction
times during neuropsychological testing, potential interactions of AAS and D2R blockade on
response latency remain unexplored (Kanayama et al., 2013).
Additionally, AAS decrease D1R expression in Acb (Kindlundh et al., 2001). In the
current study, systemic blockade of either D1R or D2R decreased willingness to exert effort in
both testosterone- and vehicle-treated rats. We expected D1R blockade to reduce rat’s
willingness to exert effort, but it was unclear whether any testosterone-induced reductions in Acb
D1R might exaggerate this effect. Since testosterone-treated rats responded with the same
decrease in large reward preference as vehicle-treated rats, any reductions in D1R in Acb did not
alter susceptibility to D1R blockade. Furthermore, D1R blockade did not promote incomplete
trials in either testosterone- or vehicle- treated rats, even though omissions increased. This
suggests that D1R blockade alters choice behavior and that this effect is not altered by
testosterone treatment.
45

Although testosterone increases preference for a large reward in ED, it has the opposite
effect on response to reward uncertainty during probability discounting (PD; Wallin et al, 2015).
Compared with vehicle controls, testosterone-treated rats made fewer responses on the large
reward lever as the likelihood of reward omission increased. As with ED, inactivation of Acb
decreases selection of the large reward in PD (Ghods-Sharifi and Floresco, 2010). However,
AAS decreases D1R in Acb (Kindlundh et al., 2001; Martínez-Rivera et al., 2015). Therefore,
the opposing effects of AAS on D1R and D2R levels in the Acb mirror the opposing effects of
behavior observed in testosterone-treated rats. This suggests that AAS-induced D1R and D2R
expression changes in Acb drive testosterone-induced changes in ED and PD behavior.
The testosterone-induced sensitivity to D2R manipulation observed here was also seen in
PD. Stimulation of D1R non-significantly increased selection of the large uncertain reward in
both vehicle- and testosterone-treated rats, but the increase due to D2R stimulation reached
significance in testosterone-treated rats only, as vehicle-treated rats showed a non-significant
effect (Wallin-Miller et al., 2018). Again, this displays a sensitivity to D2R manipulations
among testosterone-treated rats, further confirming that high-dose testosterone differentially
alters decision making via changes in D2R activity.
Though the D2R sensitization effect of testosterone appears consistent across different
discounting studies, we were unable to replicate baseline testosterone effects on ED in the
current study. Importantly, the discounting curve for all rats in the present study was steeper
than that of Wallin et al (2015). At FR10, our rats selected the large reward lever on ca. 20% of
trials vs 80-90% in the previous ED study. This may reflect differences in caloric restriction
between the two studies, with a steeper discounting curve reflecting reduced motivation for a
large reward. Nonetheless, there was no difference between the studies in large reward
46

preference at FR1 during reward discrimination training, suggesting that all rats preferred the
large reward when effort costs for large and small rewards are equal. This argues that
differences between discounting curves in the two studies is not due to satiety.
The results of the present study help explain how AAS may alter cognitive function in
human users. Deficits in visuospatial, cognitive, memory, and problem-solving tasks among
AAS users suggest widespread neurobiologic effects (Bjørnebekk et al., 2019; Kanayama et al.,
2013). AAS do not acutely stimulate DA release in Acb like other drugs of abuse, therefore
cognitive deficits in human AAS users are likely the result of persistent changes in DA receptors
rather than phasic alterations of DA (Triemstra et al., 2008). Understanding how the long-term
effects of AAS on decision making translate to risky behavior, depression, and other
psychological consequences of AAS abuse can aid us in building the pathway from neurobiology
to behavior.

47

2.6 References
ACSM, 1987. American college of sports medicine position stand on: the use of anabolic-
androgenic steroids in sports. Med. Sci. Sports Exerc. 19, 534–539.
Bardgett, M.E., Depenbrock, M., Downs, N., Points, M., Green, L., 2009. Dopamine modulates
effort-based decision making in rats. Behav. Neurosci. 123, 242–251.
https://doi.org/10.1037/a0014625
Besson, M., Belin, D., McNamara, R., Theobald, D.E.H., Castel, A., Beckett, V.L., Crittenden,
B.M., Newman, A.H., Everitt, B.J., Robbins, T.W., Dalley, J.W., 2010. Dissociable control
of impulsivity in rats by dopamine D2/3 receptors in the core and shell subregions of the
nucleus accumbens. Neuropsychopharmacology 35, 560–569.
https://doi.org/10.1038/npp.2009.162
Birgner, C., Kindlundh-Högberg, A.M.S., Alsiö, J., Lindblom, J., Schiöth, H.B., Bergström, L.,
2008. The anabolic androgenic steroid nandrolone decanoate affects mRNA expression of
dopaminergic but not serotonergic receptors. Brain Res. 1240, 221–228.
https://doi.org/10.1016/j.brainres.2008.09.003
Bjørnebekk, A., Westlye, L.T., Walhovd, K.B., Jørstad, M.L., Sundseth, Ø.Ø., Fjell, A.M., 2019.
Cognitive performance and structural brain correlates in long-term anabolic-androgenic
steroid exposed and nonexposed weightlifters. Neuropsychology 33, 547–559.
https://doi.org/10.1037/neu0000537
Choi, P.Y.L., Pope, H.G., 1994. Violence toward women and illicit androgenic-anabolic steroid
use. Ann. Clin. Psychiatry 6, 21–25. https://doi.org/10.3109/10401239409148835
48

Cooper, S.E., Goings, S.P., Kim, J.Y., Wood, R.I., 2014. Testosterone enhances risk tolerance
without altering motor impulsivity in male rats. Psychoneuroendocrinology 40, 201–212.
https://doi.org/10.1016/j.psyneuen.2013.11.017
Floresco, S.B., Tse, M.T.L., Ghods-Sharifi, S., 2008. Dopaminergic and glutamatergic regulation
of effort- and delay-based decision making. Neuropsychopharmacology 33, 1966–1979.
https://doi.org/10.1038/sj.npp.1301565
Ghods-Sharifi, S., Floresco, S.B., 2010. Differential effects on effort discounting induced by
inactivations of the nucleus accumbens core or shell. Behav. Neurosci. 124, 179–191.
https://doi.org/10.1037/a0018932
Ghods-Sharifi, S., St. Onge, J.R., Floresco, S.B., 2009. Fundamental contribution by the
basolateral amygdala to different forms of decision making. J. Neurosci. 29, 5251–5259.
https://doi.org/10.1523/JNEUROSCI.0315-09.2009
Haluk, D.M., Floresco, S.B., 2009. Ventral striatal dopamine modulation of different forms of
behavioral flexibility. Neuropsychopharmacology 34, 2041–2052.
https://doi.org/10.1038/npp.2009.21
Hauber, W., Sommer, S., 2009. Prefrontostriatal circuitry regulates effort-related decision
making. Cereb. Cortex 19, 2240–2247. https://doi.org/10.1093/cercor/bhn241
Hosking, J.G., Floresco, S.B., Winstanley, C.A., 2015. Dopamine antagonism decreases
willingness to expend physical, but not cognitive, effort: a comparison of two rodent
cost/benefit decision-making tasks. Neuropsychopharmacology 40, 1005–1015.
https://doi.org/10.1038/npp.2014.285
49

Kanayama, G., Kean, J., Hudson, J.I., Pope, H.G., 2013. Cognitive deficits in long-term
anabolic-androgenic steroid users. Drug Alcohol Depend. 130, 208–214.
https://doi.org/10.1016/j.drugalcdep.2012.11.008
Kaufman, M.J., Kanayama, G., Hudson, J.I., Pope, H.G., 2019. Supraphysiologic-dose anabolic–
androgenic steroid use: a risk factor for dementia? Neurosci. Biobehav. Rev. 100, 180–207.
https://doi.org/10.1016/j.neubiorev.2019.02.014
Kindlundh, A.M.S., Lindblom, J., Bergström, L., Wikberg, J.E.S., Nyberg, F., 2001. The
anabolic-androgenic steroid nandrolone decanoate affects the density of dopamine receptors
in the male rat brain. Eur. J. Neurosci. 13, 291–296. https://doi.org/10.1046/j.0953-
816X.2000.01402.x
Middleman, A.B., Faulkner, A.H., Woods, E.R., Emans, S.J., DuRant, R.H., 1995. High-risk
behaviors among high school students in Massachusetts who use anabolic steroids.
Pediatrics 96, 268–272.
Nowend, K.L., Arizzi, M., Carlson, B.B., Salamone, J.D., 2001. D1 or D2 antagonism in nucleus
accumbens core or dorsomedial shell suppresses lever pressing for food but leads to
compensatory increases in chow consumption. Pharmacol. Biochem. Behav. 69, 373–382.
https://doi.org/10.1016/S0091-3057(01)00524-X
Pattij, T., Janssen, M.C.W., Vanderschuren, L.J.M.J., Schoffelmeer, A.N.M., Van Gaalen, M.M.,
2007. Involvement of dopamine D1 and D2 receptors in the nucleus accumbens core and
shell in inhibitory response control. Psychopharmacology (Berl). 191, 587–598.
https://doi.org/10.1007/s00213-006-0533-x

50

Pope, H.G., Kouri, E.M., Powell, K.F., Campbell, C., Katz, D.L., 1996. Anabolic-androgenic
steroid use among 133 prisoners. Compr. Psychiatry 37, 322–327.
https://doi.org/10.1016/S0010-440X(96)90013-9
St. Onge, J.R., Floresco, S.B., 2009. Dopaminergic modulation of risk-based decision making.
Neuropsychopharmacology 34, 681–697. https://doi.org/10.1038/npp.2008.121
Triemstra, J.L., Sato, S.M., Wood, R.I., 2008. Testosterone and nucleus accumbens dopamine in
the male syrian hamster. Psychoneuroendocrinology 33, 386–394.
https://doi.org/10.1016/j.psyneuen.2007.12.006
Uban, K.A., Rummel, J., Floresco, S.B., Galea, L.A.M., 2012. Estradiol modulates effort-based
decision making in female rats. Neuropsychopharmacology 37, 390–401.
https://doi.org/10.1038/npp.2011.176
WADA, 2012. Testing Figures Report. Montr. Canada.
Wallin-Miller, K.G., Kreutz, F., Li, G., Wood, R.I., 2018. Anabolic-androgenic steroids (AAS)
increase sensitivity to uncertainty by inhibition of dopamine D1 and D2 receptors.
Psychopharmacology (Berl). 235, 959–969. https://doi.org/10.1007/s00213-017-4810-7
Wallin, K.G., Alves, J.M., Wood, R.I., 2015. Anabolic-androgenic steroids and decision making:
probability and effort discounting in male rats. Psychoneuroendocrinology 57, 84–92.
https://doi.org/10.1016/j.psyneuen.2015.03.023
Wallin, K.G., Wood, R.I., 2015. Anabolic-androgenic steroids impair set-shifting and reversal
learning in male rats. Eur. Neuropsychopharmacol. 25, 583–590.
https://doi.org/10.1016/j.euroneuro.2015.01.002
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Walton, M.E., Bannerman, D.M., Rushworth, M.F.S., 2002. The role of rat medial frontal cortex
in effort-based decision making. J. Neurosci. 22, 10996–11003.
https://doi.org/10.1523/JNEUROSCI.22-24-10996.2002
Winstanley, C.A., Floresco, S.B., 2016. Deciphering decision making: variation in animal
models of effort- and uncertainty-based choice reveals distinct neural circuitries underlying
core cognitive processes. J. Neurosci. 36, 12069–12079.
https://doi.org/10.1523/JNEUROSCI.1713-16.2016
Wood, R.I., Armstrong, A., Fridkin, V., Shah, V., Najafi, A., Jakowec, M., 2013. ’Roid rage in
rats? Testosterone effects on aggressive motivation, impulsivity and tyrosine hydroxylase.
Physiol. Behav. 110–111, 6–12. https://doi.org/10.1016/j.physbeh.2012.12.005

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Chapter 3. Chronic high-dose testosterone does not decrease
myelination of corticostriatal tracts in rat brain

3.1 Abstract
Anabolic androgenic steroids are controlled substances. Despite this, professional and
high school athletes use AAS to improve performance. AAS are associated with risky behavior
and aggression. Increased risk taking is correlated with lowered connectivity between the
prefrontal cortex (PFC) and the nucleus accumbens via the medial corticostriatal tract. Because
AAS users engage in high-risk/high-reward behaviors, display impaired executive functions, and
show lowered white matter integrity of the medial corticostriatal tract, we hypothesized that AAS
reduce the myelination of fiber tracts connecting frontostriatal regions. Rats given chronic high-
dose testosterone were sacrificed and brains sectioned. Myelination in the medial corticostriatal
tract, the forceps minor of the corpus callosum, the midbody of the corpus callosum, and the stria
terminalis from 8 vehicle- and testosterone-treated rats was compared using
immunohistochemistry for Myelin Basic Protein (MBP) and Oligodendrocyte 2
+
(Olig2+) as an
oligodendrocyte-specific nuclear marker. Photomicrographs were analyzed using NIH ImageJ.
MBP measurements of fiber tract area showed no effect of testosterone. Olig2+ measurements
of normalized stained area, mean gray value, oligodendrocyte cell count, and integrated optical
density showed no effect of testosterone. AAS increase risk-taking and aggressive behavior.
Studies examining the neurobiological basis of these changes have previously focused on
neuronal effects of AAS. Our study explored myelination as a new potential mechanism.
53

Although we found no effect of testosterone on myelination at the light microscopic level,
examination at the ultrastructural level may reveal further effects.

3.2 Introduction
Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone used by
athletes and body builders to increase muscle mass and enhance athletic performance despite
their status as illicit drugs (ACSM, 1987; Kaufman et al., 2019). Users see AAS as a natural
supplement to a healthy lifestyle (Parkinson and Evans, 2006), but taking AAS at
supraphysiologic doses has detrimental effects on multiple organ systems, including the brain
(Kanayama et al., 2020). AAS users show increases in aggression and risky behavior, and
decreases in executive function, but the neurobiology behind these changes has yet to be
revealed (Hauger et al., 2020; Middleman et al., 1995; Perry et al., 2003).
In this regard, human imaging studies show a positive correlation between executive
function and frontostriatal connectivity (Liston et al., 2006). The frontal cortex is responsible for
behavioral inhibition, working memory, and cognitive flexibility (Best and Miller, 2010). The
striatum assesses risk/reward ratios and incorporates emotional information from the amygdala
into this assessment (Gruber and McDonald, 2012). Thus, impaired frontostriatal connectivity
can lead to an increase in emotionally-driven and high-risk, high-reward behaviors (Liston et al.,
2006; Tobiansky et al., 2018). Decreased frontostriatal connectivity in young adults is associated
with increased impulsive choice and increased aggression (Karlsgodt et al., 2014; Peper et al.,
2015, 2013). These associations are also observed in AAS users, who demonstrate white matter
abnormalities and poor executive function (Hauger et al., 2020; Seitz et al., 2017).
54

However, studies of human AAS users must contend with many variables. Human users
take a variety of different AAS in varying doses. They may have pre-existing psychopathology,
and abuse other substances (Kanayama et al., 2020). By contrast, rodent studies can control the
dosing and type of AAS to model the executive dysfunction in human users. Operant responding
tasks measure decision-making and executive function performance by requiring rats to press a
lever for a food reward. These tasks have revealed that chronic high-dose testosterone increases
a rat’s willingness to work and endure punishment for a high-value food reward, while
decreasing behavioral and cognitive flexibility (Cooper et al., 2014; Wallin and Wood, 2015;
Wood and Serpa, 2020). Like human users, testosterone-treated rats chose high-risk/high-reward
options and show deficits in executive function. Further, rat studies connect AAS use to
neurobiological changes. For example, AAS decrease dopamine D1-like receptor in the
striatum, and increase expression of the androgen receptor throughout the hippocampus
(Kindlundh et al., 2001; Selakovic et al., 2019). These studies provide the opportunity to
identify AAS-induced connectivity changes at the level of individual fiber tracts.
With regard to AAS effects on brain connectivity, testosterone at the normal physiologic
level promotes myelination and increases oligodendrocytes in rodent studies. Oligodendrocytes
synthesize myelin to insulate axons, thereby increasing the speed of neuronal communication
(Bunge, 1968). Female mouse pups treated with testosterone neonatally showed an increase in
oligodendrocyte density and myelination of the corpus callosum, which persisted into adulthood
(Abi Ghanem et al., 2017). Testosterone also increases myelination and oligodendrocyte number
after acute and chronic de-myelinating treatment (Bielecki et al., 2016; Hussain et al., 2013). At
first glance, this might appear to contradict the imaging studies in human AAS users. However,
it is important to keep in mind that AAS are abused at supraphysiologic doses, and users have up
55

to 100x the normal level of circulating androgens (NIDA, 2018). Chronic exposure to AAS at
high doses can have unexpected effects. For example, testosterone is protective against
apoptosis at low nanomolar concentrations (10nM), but higher nanomolar (100nM) and
micromolar concentrations increase apoptosis in both neuronal and oligodendrocyte cultures
(Caruso et al., 2004; Orlando et al., 2007; Zelleroth et al., 2019). Thus, chronic high-dose
testosterone administration modelling human AAS use may damage myelinated fiber tracts in the
rat. AAS-induced myelin damage could account for white matter abnormalities and decreased
frontostriatal connectivity observed in human AAS users.
Though studies have shown that AAS can be beneficial or detrimental to myelination and
oligodendrocytes, the mechanism of influence is still unclear. Recent investigations into axon-
myelin communication reveal metabolic and glutamatergic signaling occurring in the periaxonal
space between the axon and the myelin sheath (Bergaglio et al., 2021; Habermacher et al., 2019;
Micu et al., 2017). Excitotoxic or oxidative damage can spread to the myelinating
oligodendrocyte, which may impair the metabolic support and insulating functions provided to
the axon. In rat brain and in vitro, AAS amplify excitotoxicity and oxidative stress, leading to
apoptosis (Caruso et al., 2004; Cunningham et al., 2009; Estrada et al., 2006; Joksimović et al.,
2017; Orlando et al., 2007; Turillazzi et al., 2016; Zelleroth et al., 2019). Thus, neurons sensitive
to AAS communicate this sensitivity to their surrounding myelin, potentially leading to
demyelination (Bergaglio et al., 2021; Micu et al., 2017).
In light of this research, our study determined if chronic high-dose testosterone inhibits
myelin in fiber tracts within the rat brain. In the rat, the medialcorticostriatal fiber tract (mcs)
connects the frontal cortex to the striatum in a role analogous to human frontostriatal connections
(Berendse et al., 1992; Coizet et al., 2017; Gorelova and Yang, 1996). The mcs travels from the
56

frontal cortex through the medial portion of the forceps minor of the corpus callosum (fmi) on its
way to connect with the nucleus accumbens (Gorelova and Yang, 1996). Thus, the present study
evaluated myelination in mcs and medial fmi. For comparison, we also measured myelin in fiber
tracts connecting steroid-sensitive brain regions (stria terminalis, st) and in those linking brain
areas with limited steroid sensitivity (corpus callosum, cc). The stria links the medial amygdala
with the bed nucleus of the stria terminalis and the medial preoptic area of the hypothalamus.
These areas are significantly larger in male than in female rats, and both the medial preoptic area
and the medial amygdala decrease γ-aminobutyric acid receptor (GABA) subunit expression in
response to AAS use (Cooke et al., 1998; Hines et al., 1992; McIntyre et al., 2002). Conversely,
the midbody of cc is not sexually dimorphic, connects testosterone-insensitive motor and sensory
regions, and does not display a relationship to testosterone levels (Chura et al., 2010; Dubb et al.,
2003; Fabri et al., 2011). To make quantitative comparisons, rat brain tissue was stained for
myelin basic protein (MBP) and Olig2+. MBP is a myelin-specific protein expressed throughout
myelinating oligodendrocytes. Olig 2+ is a transcription factor and somatic marker for
oligodendrocytes (Abi Ghanem et al., 2017).
3.3 Methods
3.3.1 Animals
16 male Long-Evans rats (Charles River Laboratories, MA) were pair-housed under a
reversed 14L:10D photoperiod and given access to water ad libitum. Rats remained gonad-intact
and received injections sc of 7.5 mg/kg testosterone (n=8, Steraloids, RI) or vehicle [n=8, 3%
ethanol and 13% cyclodextrin (RBI, MA)] 5 d/wk beginning at 5 weeks of age. This dose of
testosterone has been used to model human neurobiological and behavioral changes in response
to AAS (Wood and Wallin 2015; Wood and Serpa 2020; Cooper and Wood 2014). USC’s
57

Institutional Animal Care and Use Committee approved all experimental procedures, which were
conducted in accordance with the Guide for the Care and Use of Laboratory Animals, 8
th
Ed
(National Research Council, National Academies Press, Washington DC, 2011).
3.3.2 Perfusion
At 12 weeks of age, rats were deeply anesthetized with ketamine/xylazine (100 mg/kg
and 10 mg/kg, ip), and perfused transcardially with 100 ml of 0.1M phosphate-buffered saline
(PBS, pH 7.4), followed by 200 ml of 4% paraformaldehyde in PBS. Brains were removed,
post-fixed in the perfusion fixative for 1h at room temperature (RT), and cryoprotected overnight
in 20% sucrose at 4°C. Fixed brains were sectioned coronally at 40 uM on a freezing
microtome, and stored at 4°C in PBS with 0.01% sodium azide as a preservative.
3.3.3 Staining
Every 4
th
section was stained for MBP using a mouse monoclonal antibody (1:500,
ab62631, Abcam, Cambridge, MA). Adjacent sections were stained for Olig 2+ using a rabbit
monoclonal antibody (1:200, ab109186, Abcam) with the addition of heat-induced epitope
retrieval before incubation in the primary antibody. Brains from vehicle- and testosterone-
treated rats were stained at the same time.
For MBP staining, free-floating sections were washed extensively (3 times for 5 minutes
each) with 0.1M phosphate buffer (PB) between incubations. Sections were incubated overnight
at RT in MBP primary antibody with 4% normal donkey serum and 0.3% Triton X-100 in PB.
The next day, sections were incubated in biotinylated donkey anti-mouse secondary antibody
(1:200, Jackson ImmunoResearch Laboratories, West Grove, PA), followed by the avidin-biotin
horseradish peroxidase complex (1:50, Vectastain ABC Elite kit, Vector Laboratories,
58

Burlingame, CA), both in PB with 0.3% Triton X-100 for 1h at RT. Staining was visualized
using NiCl-enhanced 3,3’-diaminobenzidine (DAB, Sigma, St. Louis, MO). Sections were
mounted onto gelatin-coated slides, dehydrated in alcohols, cleared in xylenes, and cover slipped
with Permount (Fisher Scientific, Pittsburgh, PA).
For Olig 2+ staining, sections were first rinsed in 10mM Tris-sodium citrate buffer (pH
6.0, with 0.05% Tween 20) for 15 min at 75°C, followed by 1% sodium borohydride in PB for
15 min at RT. Staining was as above for MBP, with the appropriate biotinylated donkey anti-
rabbit secondary antibody.
3.3.4 Data analysis
Sections were viewed under bright-field illumination with an Olympus BH-2 microscope
using a 10x objective unless otherwise noted. Fiber tracts were identified using the rat brain
atlas of Paxinos and Watson (2013). Photomicrographs of the forceps minor of the corpus
callosum (fmi) and mcs (Gorelova, 97) were obtained just rostral to the genu of the corpus
callosum (Paxinos and Watson Plate 11; Fig. 5A). The midbody of the corpus callosum (cc) was
photographed at the rostral border of the anterior commissure (Plate 17; Fig. 5B). Images of the
stria terminalis (st) were obtained rostral to the hippocampus at the level of the fimbria (Plate 23;
Fig. 5C)).
The area of each fiber tract was measured from MBP-stained sections using a 4x
objective for fmi, cc, and a 10x objective for st. The mcs tract fibers spread out as they exit the
fmi and terminate in the nucleus accumbens. Due to this spreading, the fiber tract was too
diffuse for measurement of MBP stain. Because the mcs tract travels through the medial fmi
prior to ramification, the medial fmi was used instead of mcs fiber tract for MBP quantification.
59

To account for variation in brain size among individuals, stained areas were expressed as a
percentage of the total hemisphere area and averaged by treatment group.
Olig2+ immunostaining was measured using ImageJ 1.52a (NIH). Photomicrographs
were converted to grayscale at 600 x 400 pixels and a rectangular region of interest (ROI) was
selected within each fiber tract (fmi 0.0934 mm
2
,

mcs 0.124 mm
2
, cc 0.160 mm
2
,

st 0.0434 mm
2
).
The mcs tract was included here because the somatic marker Olig2+ resulted in punctate, clear
signal area. Each ROI was thresholded (see Figure 5D) using the triangle threshold algorithm
(Zack et al., 1977). From the thresholded images of Olig2+ staining, we recorded the stained
area (% of pixels above threshold), mean gray value (scale value where 0=white and 255=black),
and integrated optical density (IOD: stained area x mean gray value). The number of
immunostained cells in each ROI was counted using the Analyze Particles function. To compare
staining across the fiber tracts, stained area, IOD, and number of cells were normalized to
account for differences in ROI size between tracts. Data from testosterone- and vehicle-treated
rats were compared using an RM-ANOVA with fiber tract as the within-subjects measure and
testosterone or vehicle treatment as the between-subjects.
60

Figure 5. Anatomical regions measured for myelin (gray) and oligodendrocytes (green). A.
Representation of section used to assess fmi and mcs. B. Section used for analysis of the
midbody of the cc. C. Zoomed in image of the section used for st. D. Sample of oligodendrocyte
staining (cc), with corresponding threshold applied. Abbreviations: aca- anterior commissure
61

anterior part, ac- anterior commissure, fmb- fimbria of hippocampus, lv- lateral ventricle.
Adapted from Paxinos and Watson, 5
th
Ed.
3.4 Results
3.4.1 Myelinated fiber tract area
Figure 6 illustrates myelinated fiber tract area relative to the area of the hemisphere, as
determined by MBP staining. Not surprisingly, fiber tracts vary significantly in size (F2,28 =
455.4, p<0.05, η
2
=0.96), with the fmi occupying the largest portion of the total hemisphere area.
Generally, the medial fmi occupies 3.8% of the rostral hemisphere, the midbody of the corpus
callosum represented 2.1%, and the stria terminalis represented a mere 0.6% of its accompanying
hemisphere. However, there was no significant effect of testosterone on fiber tract area.

Figure 6. Myelin staining as a percentage of hemispheric area in fiber tracts labeled by myelin
basic protein. Means ±SEM for testosterone- (closed bars, n=8) and vehicle-treated (open bars,
n=8) rats. Asterisk indicates significant effect of fiber tract, p<0.05.
3.4.2 Oligodendrocyte immunoreactivity
Similar to the effect on fiber tract area as measured by MBP, a significant effect of fiber
tract was present across all oligodendrocyte data quantified using Olig2+ staining (Figure 7A-D),
while there was no significant effect of testosterone and no interaction of testosterone by tract.
*
62

As shown in Figure 7A there was a significant effect of fiber tract (F3,39 = 24.3, p<0.05, η
2
=0.76)
on Olig2+ staining as a percentage of the total area for each ROI. In mcs, Olig2+ staining
accounted for 3.3±0.5% and 3.4±1.1% of the ROI in testosterone- and vehicle-treated rats,
respectively. By contrast in st, Olig2+ immunoreactivity took up nearly one-fifth of the ROI
(testosterone: 21.9±4.1% and vehicle: 19.7±3.6%). The area of Olig2+ staining in fmi and cc
was intermediate between mcs and st, occupying around 9% of the ROI. For all fiber tracts, the
vehicle and testosterone values were not significantly different.
Figure 7B displays mean gray values for Olig2+ staining. Here, the values of the fiber
tracts were split between those at the bottom of the range (mcs and cc) and those at the top (fmi
and st), generating a significant effect of fiber tract (F3,39 = 12.04, p<0.05, η
2
=0.75). Across
fiber tracts the mean gray value was higher in vehicle- than testosterone-treated rats, particularly
in the st where the vehicle mean was 131.5±7.5 and the testosterone mean was 99.9±10.8. This
interaction between fiber tract and testosterone was slight but non-significant (F3,39 = 2.1,
p=0.11).
Figure 7C presents the IOD per unit area. Mcs had the lowest values (testosterone: 0.01 ±
0.00 and vehicle: 0.03 ± 0.02 IOD/unit area), while fmi, cc, and st were all clustered around 0.26.
Unsurprisingly, there was a significant effect of fiber tract (F3,39 =3.47, p<0.05, η
2
=0.60).
Though the values for fmi appear to be lower in vehicle- than testosterone-treated rats
(testosterone: 0.25 ± 0.15 and vehicle: 0.09 ± 0.06 IOD/unit area), there was no significant effect
of testosterone on IOD. This is in part due to the variability between animals within the same
experimental groups.
Finally, Figure 7D illustrates the number of oligodendrocytes per millimeter squared for
each fiber tract. Similar to Figure 7A, mcs had relatively few oligodendrocytes (testosterone:
844±113 and vehicle: 924±216 cells/mm
2
), while st had numerous Olig2+-positive cells
(testosterone: 3004±293 and vehicle: 3357±205 cells/mm
2
). Both fmi (testosterone: 2303±447
and vehicle: 2229±398 cells/mm
2
) and cc (testosterone: 2130±430 and vehicle: 2337±313
63

cells/mm
2
) were intermediate. There was a significant effect of fiber tract (F3,39 = 32.8, p<0.05,
η
2
=0.79) but no significant effect of testosterone.

Figure 7. Olig2+ immunoreactivity. A. Percent of stained area out of total ROI area. B.
Average gray area value. C. IOD per unit area. D. Number of oligodendrocytes per unit area.
Mean ±SEM for testosterone- (closed bars, n=8) and vehicle-treated (open bars, n=7) rats.
Asterisk indicates significant effect of fiber tract, p<0.05.

* *
* *
64

3.5 Discussion
3.5.1 Overview
Building on rodent and human studies, we measured the effect of chronic high-dose
testosterone on fiber tracts connecting regions governing behaviors dysregulated in human AAS
users. This study measured myelination of three fiber tracts in vehicle- and testosterone-treated
rat brain using MBP and Olig2+ immunostaining. Our study found an effect of fiber tract across
all measurements of myelination, and no effect of testosterone.

3.5.2 Testosterone and oligodendrocytes
The lack of effect of testosterone in our study is surprising, given that testosterone has
demonstrated both neuroprotective and neurotoxic effects. Physiologically relevant levels of
testosterone have been shown to increase myelination of axons in vivo. Accordingly, multiple
studies have found normal levels of testosterone in males promote myelination both during
development and after demyelinating lesions (Abi Ghanem et al., 2017; Bielecki et al., 2016;
Cerghet et al., 2006; Hussain et al., 2013). This might suggest that testosterone treatment would
increase myelination of the fiber tracts in our study. However, the relationship between
myelination and testosterone levels may not be linear. Because our study compared across two
groups of gonadally-intact males, the lack of difference between the two groups may be the
result of a ceiling effect, where supraphysiologic doses of testosterone do not generate
abnormally high levels of myelination.
The promyelinating effect of testosterone is only half of the picture. Supraphysiologic
doses of testosterone appear to be neurotoxic. Studies of high dose testosterone reveal an
65

increase in mitochondrial dysfunction and apoptosis of neurons in vitro (Caruso et al., 2004;
Estrada et al., 2006; Orlando et al., 2007). In vivo, these high doses increase excitotoxic injury
and oxidative damage in neurons (Joksimović et al., 2017). Thus, the neuroprotective or
neurotoxic effects of testosterone may rely on the dose administered, generating an inverted U
model of neuroprotective effect. In the present study, the amount and duration of testosterone
exposure was far above normal physiologic levels. Accordingly, we would have expected a
negative effect on the number of oligodendrocytes or the amount of myelin. However, most of
the studies revealing detrimental effects combined high levels of testosterone with other
oxidative or neurotoxic effects. Thus, supraphysiologic levels of testosterone may generate
damage when added to systems already modified by other toxic factors.
Although testosterone alters myelination in the CNS, it is notable that myelinating
oligodendrocytes lack androgen receptors (AR). Typically, testosterone binds to cytoplasmic
AR, after which the androgen-AR complex translocates to the nucleus and regulates gene
expression (Ghoumari et al., 2020). AR is present in neurons, astrocytes, and oligodendrocyte
precursor cells, but has not been found on myelinating oligodendrocytes of human or rat brains.
Nonetheless, functional AR is necessary for the testosterone-induced increases in remyelination
observed after rat brain injury (Bielecki et al., 2016; Hussain et al., 2013). How can this
apparent contrast be resolved?
One possible solution lies in the communication between the axon and its myelin.
Neurons communicate with their myelinating oligodendrocytes via a periaxonal space to alter
oligodendrocyte glutamate receptors and calcium channels (Habermacher et al., 2019; Micu et
al., 2017). In particular, axonal damage can be translated to the myelinating oligodendrocyte,
disrupting the metabolic and structural characteristics of the myelin sheath (Bergaglio et al.,
66

2021). Testosterone can generate a rapid response within the axon, increasing calcium
concentrations and activating signaling pathways (Estrada et al., 2006; Foradori et al., 2007).
Supraphysiological testosterone increases mitochondrial damage, lipid peroxidation, and
apoptosis in neurons, releasing excessive glutamate, calcium, and other deleterious molecules
into the periaxonal space (Bergaglio et al., 2021; Cunningham et al., 2009; Zelleroth et al.,
2019). Thus, although testosterone may not act directly on oligodendrocytes through AR, there
is potential for AR-positive neurons to influence myelination of their axons.

3.5.3 Human AAS use
We anticipated an effect of chronic high-dose testosterone on the myelination of rat fiber
tracts analogous to the human frontostriatal tract. In behavioral tests of the frontal and striatal
regions, rats treated with chronic high-dose testosterone displayed a decrease in executive
function and an increase in willingness to exert effort and endure pain for a large reward (Cooper
et al., 2014; Wallin et al., 2015; Wallin and Wood, 2015). This suggested a link between rat
behavioral response to supraphysiologic levels of testosterone, and human studies of AAS users
showing decreases in frontostriatal tract integrity and connectivity (Kaufman et al., 2015; Seitz et
al., 2017; Westlye et al., 2017). However, AAS do not work alone in altering the cognitive
abilities of users.
In humans, it can be difficult to disentangle the effects of neurodevelopment, endogenous
testosterone, white matter integrity, and risky behavior. For example, the typical AAS user
(young adult male, early 20s) is still maturing the frontal cortex, a process which involves
changes in white matter (Peper et al., 2011; Pope et al., 2014a; Sowell et al., 1999). For males at
67

this age, endogenous testosterone levels are negatively associated with frontostriatal tract
integrity (Peper et al., 2013). This age also represents the peak of risk-taking behavior, and
males with higher levels of testosterone displayed further increases in risk-taking than same age
peers (Peper et al., 2018). Thus, the typical AAS user is primed to display high-risk/high-reward
behaviors prior to the onset of use. To control for this priming effect, our study began chronic
high-dose testosterone administration in late adolescence, when frontal cortex is still maturing.
Additionally, imaging studies of human AAS users face complications from polydrug
use, including pre-existing or exacerbated damage from other substances (Ersche et al., 2013).
AAS users began using all drugs at an earlier age than non-AAS using counterparts, and used
higher total number of substances (Havnes et al., 2020). Concurrent and prior use of
methamphetamine, cocaine, opioids, and benzodiazepines have been recorded across multiple
studies (Dodge and Hoagland, 2011; Kanayama et al., 2009d; Skarberg et al., 2009). Thus,
observed changes in connectivity within AAS user populations must be interpreted carefully.
Our study in rats not only eliminated polydrug use complications, but also used a consistent
dose, schedule, and drug, factors which vary substantially across human AAS user populations
(Kanayama et al., 2020).
Finally, a certain personality type may be more susceptible to the negative effects of AAS
than others. Administration of testosterone to AAS-naïve males generated a hypomanic response
in 16% of users, while the other 84% showed little psychological response (Pope et al., 2000).
Other studies have recorded depression during withdrawal from AAS, but this is not present in
all users (Kanayama et al., 2020). The National Institute for Drug Abuse recognizes that for a
subset of the population, AAS can be addictive and require professional assistance for
withdrawal (NIDA, 2018). Aggression, violent behavior, mania, and body dysmorphia have all
68

been observed among AAS user populations (Kanayama et al., 2020). However, as with other
drugs of abuse, it is unclear whether AAS use is a cause or effect of existing psychopathology.

3.5.4 Future studies
Expanding the anatomical scale of our investigation would generate a more detailed
record of the interaction between testosterone and myelin. Application of diffusion tensor
imaging (DTI) techniques in live rats as in (Jito et al., 2008) may provide fiber tract
microstructural data that can be compared to human user data and verified histologically within
the rat brain. Alternatively, ultrastructural studies using confocal and electron microscopy would
allow for measurements of myelinated axons, axonal diameter, and myelin sheath thickness as in
(Abi Ghanem et al., 2017; Bielecki et al., 2016; Hussain et al., 2013). As an added benefit, these
techniques may ameliorate the difficulty in comparing across fiber tracts encountered in our
current study. Application of these techniques to our rat model of human AAS use may reveal
chronic high-dose testosterone effects not visible at the current level of resolution.

3.5.5 Conclusion
Our study connected links between previous in vitro and in vivo studies of high-dose
AAS. We determined no effect of testosterone on myelin at our anatomical level of examination.
Further studies combining microstructural imaging with ultrastructural analysis should be used to
define the biological correlates of observed behavioral and cognitive effects of AAS.

69

3.6 References
Abi Ghanem, C., Degerny, C., Hussain, R., Liere, P., Pianos, A., Tourpin, S., Habert, R.,
Macklin, W.B., Schumacher, M., Ghoumari, A.M., 2017. Long-lasting masculinizing
effects of postnatal androgens on myelin governed by the brain androgen receptor. PLoS
Genet. 13. https://doi.org/10.1371/journal.pgen.1007049
ACSM, 1987. American college of sports medicine position stand on: the use of anabolic-
androgenic steroids in sports. Med. Sci. Sports Exerc. 19, 534–539.
Berendse, H.W., Graaf, Y.G., Groenewegen, H.J., 1992. Topographical organization and
relationship with ventral striatal compartments of prefrontal corticostriatal projections in the
rat. J. Comp. Neurol. 316, 314–347. https://doi.org/10.1002/cne.903160305
Bergaglio, T., Luchicchi, A., Schenk, G.J., 2021. Engine failure in axo-myelinic signaling: a
potential key player in the pathogenesis of multiple sclerosis. Front. Cell. Neurosci. 15.
https://doi.org/10.3389/fncel.2021.610295
Best, J.R., Miller, P.H., 2010. A developmental perspective on executive function. Child Dev.
https://doi.org/10.1111/j.1467-8624.2010.01499.x
Bielecki, B., Mattern, C., Ghoumari, A.M., Javaid, S., Smietanka, K., Ghanem, C.A., Mhaouty-
Kodja, S., Ghandour, M.S., Baulieu, E.E., Franklin, R.J.M., Schumacher, M., Traiffort, E.,
2016. Unexpected central role of the androgen receptor in the spontaneous regeneration of
myelin. Proc. Natl. Acad. Sci. U. S. A. 113, 14829–14834.
https://doi.org/10.1073/pnas.1614826113

70

Bunge, R.P., 1968. Glial cells and the central myelin sheath. Physiol. Rev.
https://doi.org/10.1152/physrev.1968.48.1.197
Caruso, A., Di Giorgi Gerevini, V., Castiglione, M., Marinelli, F., Tomassini, V., Pozzilli, C.,
Caricasole, A., Bruno, V., Caciagli, F., Moretti, A., Nicoletti, F., Melchiorri, D., 2004.
Testosterone amplifies excitotoxic damage of cultured oligodendrocytes. J. Neurochem. 88,
1179–1185. https://doi.org/10.1046/j.1471-4159.2004.02284.x
Cerghet, M., Skoff, R.P., Bessert, D., Zhang, Z., Mullins, C., Ghandour, M.S., 2006.
Proliferation and death of oligodendrocytes and myelin proteins are differentially regulated
in male and female rodents. J. Neurosci. 26, 1439–1447.
https://doi.org/10.1523/JNEUROSCI.2219-05.2006
Chura, L.R., Lombardo, M. V., Ashwin, E., Auyeung, B., Chakrabarti, B., Bullmore, E.T.,
Baron-Cohen, S., 2010. Organizational effects of fetal testosterone on human corpus
callosum size and asymmetry. Psychoneuroendocrinology 35, 122–132.
https://doi.org/10.1016/j.psyneuen.2009.09.009
Coizet, V., Heilbronner, S.R., Carcenac, C., Mailly, P., Lehman, J.F., Savasta, M., David, O.,
Deniau, J.M., Groenewegen, H.J., Haber, S.N., 2017. Organization of the anterior limb of
the internal capsule in the rat. J. Neurosci. 37, 2539–2554.
https://doi.org/10.1523/JNEUROSCI.3304-16.2017
Cooke, B., Hegstrom, C.D., Villeneuve, L.S., Breedlove, S.M., 1998. Sexual differentiation of
the vertebrate brain: principles and mechanisms.

71

Cooper, S.E., Goings, S.P., Kim, J.Y., Wood, R.I., 2014. Testosterone enhances risk tolerance
without altering motor impulsivity in male rats. Psychoneuroendocrinology 40, 201–212.
https://doi.org/10.1016/j.psyneuen.2013.11.017
Cunningham, R.L., Giuffrida, A., Roberts, J.L., 2009. Androgens induce dopaminergic
neurotoxicity via caspase-3-dependent activation of protein kinase Cδ. Endocrinology 150,
5539–5548. https://doi.org/10.1210/en.2009-0640
Dodge, T., Hoagland, M.F., 2011. The use of anabolic androgenic steroids and polypharmacy: a
review of the literature, Drug and Alcohol Dependence. NIH Public Access.
https://doi.org/10.1016/j.drugalcdep.2010.11.011
Dubb, A., Gur, R., Avants, B., Gee, J., 2003. Characterization of sexual dimorphism in the
human corpus callosum. Neuroimage 20, 512–519. https://doi.org/10.1016/S1053-
8119(03)00313-6
Ersche, K.D., Williams, G.B., Robbins, T.W., Bullmore, E.T., 2013. Meta-analysis of structural
brain abnormalities associated with stimulant drug dependence and neuroimaging of
addiction vulnerability and resilience. Curr. Opin. Neurobiol. 23, 615–624.
https://doi.org/10.1016/j.conb.2013.02.017
Estrada, M., Varshney, A., Ehrlich, B.E., 2006. Elevated testosterone induces apoptosis in
neuronal cells. J. Biol. Chem. 281, 25492–25501. https://doi.org/10.1074/jbc.M603193200
Fabri, M., Polonara, G., Mascioli, G., Salvolini, U., Manzoni, T., 2011. Topographical
organization of human corpus callosum: an fMRI mapping study. Brain Res. 1370, 99–111.
https://doi.org/10.1016/j.brainres.2010.11.039
72

Foradori, C.D., Weiser, M.J., Handa, R.J., 2008. Non-genomic actions of androgens. Frontiers in
Neuroendocrinology. doi:10.1016/j.yfrne.2007.10.005
Ghoumari, A.M., Ghanem, C.A., Asbelaoui, N., Schumacher, M., Hussain, R., 2020. Roles of
progesterone, testosterone and their nuclear receptors in central nervous system myelination
and remyelination. Int. J. Mol. Sci. 21. https://doi.org/10.3390/ijms21093163
Gorelova, N., Yang, C.R., 1996. The course of neural projection from the prefrontal cortex to the
nucleus accumbens in the rat. Neuroscience 76, 689–706. https://doi.org/10.1016/S0306-
4522(96)00380-6
Gruber, A.J., McDonald, R.J., 2012. Context, emotion, and the strategic pursuit of goals:
Interactions among multiple brain systems controlling motivated behavior. Front. Behav.
Neurosci. https://doi.org/10.3389/fnbeh.2012.00050
Habermacher, C., Angulo, M.C., Benamer, N., 2019. Glutamate versus GABA in neuron–
oligodendroglia communication. Glia 67, 2092–2106. https://doi.org/10.1002/glia.23618
Hauger, L.E., Westlye, L.T., Bjørnebekk, A., 2020. Anabolic androgenic steroid dependence is
associated with executive dysfunction. Drug Alcohol Depend. 208, 107874.
https://doi.org/10.1016/j.drugalcdep.2020.107874
Havnes, I.A., Bukten, A., Rognli, E.B., Muller, A.E., 2020. Use of anabolic-androgenic steroids
and other substances prior to and during imprisonment - results from the Norwegian
offender mental health and addiction (NorMA) study. Drug Alcohol Depend. 217, 108255.
https://doi.org/10.1016/j.drugalcdep.2020.108255

73

Hines, M., Allen, L.S., Gorski, R.A., Hines, M., 1992. Sex differences in subregions of the
medial nucleus of the amygdala and the bed nucleus of the stria terminalis of the rat. Brain
Research. 579, 321-326. https://doi:10.1016/0006-8993(92)90068-K
Hussain, R., Ghoumari, A.M., Bielecki, B., Steibel, J., Boehm, N., Liere, P., MacKlin, W.B.,
Kumar, N., Habert, R., Mhaouty-Kodja, S., Tronche, F., Sitruk-Ware, R., Schumacher, M.,
Ghandour, M.S., 2013. The neural androgen receptor: a therapeutic target for myelin repair
in chronic demyelination. Brain 136, 132–146. https://doi.org/10.1093/brain/aws284
Jito, J., Nakasu, S., Ito, R., Fukami, T., Morikawa, S., Inubushi, T., 2008. Maturational changes
in diffusion anisotropy in the rat corpus callosum: comparison with quantitative histological
evaluation. J. Magn. Reson. Imaging 28, 847–854. https://doi.org/10.1002/jmri.21496
Joksimović, J., Selaković, D., Jakovljević, V., Mihailović, V., Katanić, J., Boroja, T., Rosić, G.,
2017. Alterations of the oxidative status in rat hippocampus and prodepressant effect of
chronic testosterone enanthate administration. Mol. Cell. Biochem. 433, 41–50.
https://doi.org/10.1007/s11010-017-3014-0
Kanayama, G., Hudson, J.I., Pope, H.G., 2020. Anabolic-androgenic steroid use and body image
in men: a growing concern for clinicians. Psychotherapy and Psychosomatics.
https://doi.org/10.1159/000505978
Karlsgodt, K.H., Bato, A.A., Blair, M.A., DeRosse, P., Szeszko, P.R., Malhotra, A.K., 2014.
White matter microstructure in the executive network associated with aggression in healthy
adolescents and young adults. Soc. Cogn. Affect. Neurosci. 10, 1251–1256.
https://doi.org/10.1093/scan/nsv015

74

Kaufman, M.J., Janes, A.C., Hudson, J.I., Brennan, B.P., Kanayama, G., Kerrigan, A.R., Jensen,
J.E., Pope, H.G., 2015. Brain and cognition abnormalities in long-term anabolic-androgenic
steroid users. Drug Alcohol Depend. 152, 47–56.
https://doi.org/10.1016/j.drugalcdep.2015.04.023
Kaufman, M.J., Kanayama, G., Hudson, J.I., Pope, H.G., 2019. Supraphysiologic-dose anabolic–
androgenic steroid use: a risk factor for dementia? Neurosci. Biobehav. Rev. 100, 180–207.
https://doi.org/10.1016/j.neubiorev.2019.02.014
Kindlundh, A.M.S., Lindblom, J., Bergström, L., Wikberg, J.E.S., Nyberg, F., 2001. The
anabolic-androgenic steroid nandrolone decanoate affects the density of dopamine receptors
in the male rat brain. Eur. J. Neurosci. 13, 291–296. https://doi.org/10.1046/j.0953-
816X.2000.01402.x
Liston, C., Watts, R., Tottenham, N., Davidson, M.C., Niogi, S., Ulug, A.M., Casey, B.J., 2006.
Frontostriatal microstructure modulates efficient recruitment of cognitive control. Cereb.
Cortex 16, 553–560. https://doi.org/10.1093/cercor/bhj003
McIntyre, K.L., Porter, D.M., Henderson, L.P., 2002. Anabolic androgenic steroids induce age-,
sex-, and dose-dependent changes in GABAA receptor subunit mRNAs in the mouse
forebrain. Neuropharmacology 43, 634–645. https://doi.org/10.1016/S0028-3908(02)00154-
5
Micu, I., Plemel, J.R., Caprariello, A. V, Nave, K., Stys, P.K., 2017. Axo-myelinic
neurotransmission: a novel mode of cell signalling in the central nervous system, Nature
Publishing Group. https://doi.org/10.1038/nrn.2017.128

75

Middleman, A.B., Faulkner, A.H., Woods, E.R., Emans, S.J., DuRant, R.H., 1995. High-risk
behaviors among high school students in Massachusetts who use anabolic steroids.
Pediatrics 96, 268–272.
NIDA, 2018. Steroids and other appearance and performance enhancing drugs (APEDs). Natl.
Inst. Drug Abus. 1–35.
Orlando, R., Caruso, A., Molinaro, G., Motolese, M., Matrisciano, F., Togna, G., Melchiorri, D.,
Nicoletti, F., Bruno, V., 2007. Nanomolar concentrations of anabolic-androgenic steroids
amplify excitotoxic neuronal death in mixed mouse cortical cultures.
https://doi.org/10.1016/j.brainres.2007.06.047
Parkinson, A.B., Evans, N.A., 2006. Anabolic androgenic steroids: a survey of 500 users. Med.
Sci. Sports Exerc. 38, 644–651. https://doi.org/10.1249/01.mss.0000210194.56834.5d
Peper, J.S., Braams, B.R., Blankenstein, N.E., Bos, M.G.N., Crone, E.A., 2018. Development of
multifaceted risk taking and the relations to sex steroid hormones: a longitudinal study.
Child Dev. 89, 1887–1907. https://doi.org/10.1111/cdev.13063
Peper, J.S., De Reus, M.A., Van Den Heuvel, M.P., Schutter, D.J.L.G., 2015. Short Fused?
Associations between white matter connections, sex steroids, and aggression across
adolescence. Hum Brain Mapp 36, 1043–1052. https://doi.org/10.1002/hbm.22684
Peper, J.S., Mandl, R.C.W., Braams, B.R., De Water, E., Heijboer, A.C., Koolschijn, P.C.M.P.,
Crone, E.A., 2013. Delay discounting and frontostriatal fiber tracts: a combined DTI and
MTR study on impulsive choices in healthy young adults. Cereb. Cortex 23, 1695–1702.
https://doi.org/10.1093/cercor/bhs163
76

Peper, J.S., van den Heuvel, M.P., Mandl, R.C.W., Pol, H.E.H., van Honk, J., 2011. Sex steroids
and connectivity in the human brain: a review of neuroimaging studies.
Psychoneuroendocrinology. https://doi.org/10.1016/j.psyneuen.2011.05.004
Perry, P.J., Kutscher, E.C., Lund, B.C., Yates, W.R., Holman, T.L., Demers, L., 2003. Measures
of aggression and mood changes in male weightlifters with and without androgenic anabolic
steroid use. J. Forensic Sci. 48, 2002240. https://doi.org/10.1520/jfs2002240
Pope, H.G., Kanayama, G., Athey, A., Ryan, E., Hudson, J.I., Baggish, A., Pope Jr, H.G.,
Kanayama, G., Athey, A., Ryan, E., Hudson, J.I., Baggish, A., 2014. The lifetime
prevalence of anabolic-androgenic steroid use and dependence in Americans: current best
estimates, American Journal on Addictions. Wiley-Blackwell Publishing Ltd.
https://doi.org/10.1111/j.1521-0391.2013.12118.x
Pope, H.G., Kouri, E.M., Hudson, J.I., 2000. Effects of supraphysiologic doses of testosterone on
mood and aggression in normal men: a randomized controlled trial. Arch. Gen. Psychiatry
57, 133–140. https://doi.org/10.1001/archpsyc.57.2.133
Seitz, J., Lyall, A.E., Kanayama, G., Makris, N., Hudson, J.I., Kubicki, M., Pope, H.G.,
Kaufman, M.J., 2017. White matter abnormalities in long-term anabolic-androgenic steroid
users: a pilot study. Psychiatry Res. - Neuroimaging 260, 1–5.
https://doi.org/10.1016/j.pscychresns.2016.12.003

77

Selakovic, D., Joksimovic, J., Jovicic, N., Mitrovic, S., Mihailovic, V., Katanic, J., Milovanovic,
D., Pantovic, S., Mijailovic, N., Rosic, G., 2019. The impact of hippocampal sex hormones
receptors in modulation of depressive-like behavior following chronic anabolic androgenic
steroids and exercise protocols in rats. Front. Behav. Neurosci. 13.
https://doi.org/10.3389/fnbeh.2019.00019
Sowell, E.R., Thompson, P.M., Holmes, C.J., Jernigan, T.L., Toga, A.W., 1999. In vivo evidence
for post-adolescent brain maturation in frontal and striatal regions [1]. Nat. Neurosci.
https://doi.org/10.1038/13154
Tobiansky, D.J., Wallin-Miller, K.G., Floresco, S.B., Wood, R.I., Soma, K.K., 2018. Androgen
regulation of the mesocorticolimbic system and executive function. Front. Endocrinol.
(Lausanne). https://doi.org/10.3389/fendo.2018.00279
Turillazzi, E., Neri, M., Cerretani, D., Cantatore, S., Frati, P., Moltoni, L., Busardò, F.P.,
Pomara, C., Riezzo, I., Fineschi, V., 2016. Lipid peroxidation and apoptotic response in rat
brain areas induced by long‐term administration of nandrolone: the mutual crosstalk
between ROS and NF‐kB. J. Cell. Mol. Med. 20, 601–612.
https://doi.org/10.1111/jcmm.12748
Wallin, K.G., Alves, J.M., Wood, R.I., 2015. Anabolic-androgenic steroids and decision making:
probability and effort discounting in male rats. Psychoneuroendocrinology 57, 84–92.
https://doi.org/10.1016/j.psyneuen.2015.03.023
Wallin, K.G., Wood, R.I., 2015. Anabolic-androgenic steroids impair set-shifting and reversal
learning in male rats. Eur. Neuropsychopharmacol. 25, 583–590.
https://doi.org/10.1016/j.euroneuro.2015.01.002
78

Westlye, L.T., Kaufmann, T., Alnæs, D., Hullstein, I.R., Bjørnebekk, A., 2017. Brain
connectivity aberrations in anabolic-androgenic steroid users. NeuroImage Clin. 13, 62–69.
https://doi.org/10.1016/j.nicl.2016.11.014
Wood, R.I., Serpa, R.O., 2020. Anabolic-androgenic steroid abuse and cognitive impairment:
testosterone impairs biconditional task performance in male rats. Behav. Brain Res. 379,
112339. https://doi.org/10.1016/j.bbr.2019.112339
Zack, G.W., Rogers, W.E., Latt, S.A., 1977. Automatic measurement of sister chromatid
exchange frequency. Journal of Histochemistry and Cytochemistry 25, 741-753.
doi:10.1177/25.7.70454
Zelleroth, S., Nylander, E., Nyberg, F., Grönbladh, A., Hallberg, M., 2019. Toxic impact of
anabolic androgenic steroids in primary rat cortical cell cultures. Neuroscience 397, 172–
183. https://doi.org/10.1016/j.neuroscience.2018.11.035

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Chapter 4. Discussion of findings and their translational significance
4.1 Summary
AAS use generates behavioral impairments, and the present studies further our
understanding of the causative neurobiological mechanisms. These studies used chronic high-
dose testosterone administration to generate a rat model of human AAS use. Chapter 2 assessed
the effect of both testosterone and D1R or D2R suppression on effort discounting behavior. The
main result was a marked decrease in willingness to exert effort among AAS treated rats after
suppression of D2R activity. Thus, testosterone treatment sensitized brain regions mediating
effortful choice to D2R blockade. Chapter 3 investigated the impact of testosterone
administration on measures of myelination. Analysis of fiber tracts showed no difference
between groups in any of the measurements of myelin and oligodendrocyte population,
suggesting AAS alone do not substantially modify these measures. By employing a combination
of behavioral and anatomic studies, my research highlights biologic changes relevant to
behavioral impairments.

4.2 Animal studies: Questions answered, questions posed
In my research, I aimed to better understand the specific biological changes occurring as
a result of AAS use, and how these changes translate into observed differences in behavior. The
existing literature on this topic is limited when compared to that of other drugs of abuse, such as
stimulants or opioids. However, this does not mean that the issue is any less complicated. In
addition to alterations across the dopaminergic pathways, AAS have been shown to alter
inhibitory GABA activity, and serotonergic pathways (Ambar and Chiavegatto, 2009; Bertozzi et
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al., 2018). Thus, there are multiple questions that could be asked when studying behavioral
effects of AAS.
The present body of work began with an investigation into dopaminergic effects of AAS.
AAS alter the dopaminergic reward pathway across multiple nodes. But, as with other
neuromodulators, it is not a broad increase or decrease. Changes in dopamine receptor
expression, dopamine release, and dopamine metabolism all occur as a result of AAS use and the
resulting behavior is the sum of these modifications. Behind all of these dopaminergic
modifications lies a larger question. Why do AAS make these changes?
For example, our study unmasked a testosterone-induced dependence on D2R activity. If
we consider the resulting behavior as an end point, working backwards leads us to some
mechanism relying on D2R activity to motivate physical effort. AAS-induced increases in D2R
expression observed across brain regions suggest a homeostatic response to decreased
dopaminergic activity. But are D2R receptors being upregulated due to a lack of ligand present,
or a reduced efficacy of the receptor on the cell? Furthermore, why do AAS generally
upregulate D2R and downregulate D1R? In a simplistic view, these receptors bind the same
ligand, dopamine, but have opposing downstream effects including alterations in neuronal
excitability (Jackson and Westlind-Danielsson, 1994). Exploring AAS-induced changes in
electrophysiologic properties of central dopaminergic neurons would build upon existing studies
in peripheral systems and provide valuable insight into mechanisms behind behavioral
impairments (Oberlander 2012). Moving research in this direction would be a valuable addition
to existing studies of the effect of AAS on GABAergic and serotonergic neuronal excitability
(Hildebrandt et al., 2018)
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The behavioral impairments seen among AAS users are not solely a factor of reward
systems. As reviewed earlier, AAS users exhibit behavior consistent with decreased cognitive
control. Impulsivity, aggression, and risk taking are inversely correlated with corticolimbic
connectivity, and AAS users have decreased integrity of these corticolimbic connections.
Additionally, the integrity of these connections are inversely correlated to endogenous
testosterone levels (Karlsgodt et al., 2014; Peper et al., 2015, 2013; Perrin et al., 2008; Westlye
et al., 2017). Finally, sexually dimorphic myelination of rat brain shows testosterone increases
myelin, and additional studies have shown testosterone can increase remyelination after
demyelinating injury (Abi Ghanem et al., 2017; Bielecki et al., 2016; Cerghet et al., 2006;
Hussain et al., 2013). Given the literature, there should have been some difference in
myelination between testosterone- and vehicle-treated rats. Why didn’t we find one?
As noted in Chapter 3, these results suggest two possible conclusions, both stemming
from differences between populations in the literature and in the study. Namely, our population
had not undergone a demyelinating injury, and we were not comparing between sexes. Instead,
we were comparing two groups of gonadally-intact uninjured male rats. Considering that the
majority of AAS users are male, comparing the effects of AAS on female rats would provide
results relevant to only a minor subset of AAS users. Pursuing the impact of the demyelinating
injury may yield more broadly applicable results.
While physiologic levels of AAS increased remyelination, larger micromolar
concentrations induced apoptosis in neuronal cultures (Cunningham et al., 2009; Estrada et al.,
2006; Zelleroth et al., 2019). Additionally, other studies revealed AAS treatment amplifies the
excitotoxic damage induced by a previous excitatory stimulation (Caruso et al., 2004; Orlando et
al., 2007). Thus, in addition to the concentration, the timing of AAS exposure also dictated
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whether the effects were positive or negative. If administered prior to an oxidative stress, AAS
are neuroprotective. However, if the oxidative stress precedes AAS, the result is neurotoxic
(Holmes et al., 2013).
This sequence of events accounts for both the positive and negative effects of AAS on
neuronal health. Negative effects of AAS use in humans likely depends on an initial stressor or
injury, onto which the additional weight of AAS use accelerates damage. Adding a preceding
stress or injury to rat models of AAS use may also increase the applicability of findings, as this
more closely replicates the complexities of the human experience.

4.3 Human experience: How heavy is your burden?
The magnitude of the behavioral response to AAS use depends on the state of the existing
framework onto which AAS acts. Though human studies cannot assign causative roles to pre-
existing psychological impairments or risk phenotypes when examining behavioral responses to
AAS use, the frequency with which these conditions are reported in the literature point to a
relevant interaction. Studies of AAS users differ in their inclusion of subjects with either
ongoing or past psychological diagnosis and non-AAS drug use. Though these factors obscure
the effects of AAS, studies examining common features of AAS, psychopathology, and drug
abuse may improve our understanding of the neurobiological changes resulting in behavioral
impairments.
For example, interviews of AAS users assessing personality disorders have found a
significant increase in antisocial personality disorder (Börjesson et al., 2020; Pope and Katz,
1994). Prevalence of antisocial personality disorder is further enriched in the dependent subset
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of AAS users, and who reported increased aggressive behaviors, suicidal thoughts, and
criminality (Kanayama et al., 2009d; Perry et al., 2003). Similarly, previous diagnosis of
depressive or anxiety disorders is increased among dependent AAS users (Ip et al., 2012;
Kanayama et al., 2009d). Thus, development of AAS dependence and impaired behavioral
control may be the result of environmental and genetic factors shared by these observed
disorders. The added burden of AAS use taxes systems already strained by environmental and
genetic influences, overwhelming compensatory mechanisms and resulting in exaggerated
behavioral responses to AAS.
Polydrug use is more common among AAS users than non-users, and the negative effects
of these additional drugs undoubtedly compound AAS-induced behavioral deficits (Dodge and
Hoagland, 2011). Thus far, it is not clear whether the increased association with substance abuse
is a cause or effect of AAS use. Users report substance abuse patterns antecedent to onset of
AAS use as well as additional drug use for the management of AAS-induced side effects such as
irritability, insomnia, and pain (Dodge and Hoagland, 2011; Ip et al., 2012; Kanayama et al.,
2009d; Skarberg et al., 2009). Given the widespread practice of polypharmacy among AAS
users, preclinical studies of the interactions between additional drugs of abuse and AAS yield
results with more real-world relevance.
Study of AAS use with the combined effects of environmental, genetic, and
pharmacological stressors may be particularly useful for investigating the neurodegenerative
properties of AAS. Users who began taking AAS in the 1980s are now leaving middle age, and
their risk of developing age-related neurodegenerative disorders is increasing (Kanayama et al.,
2008). Preclinical and human imaging studies have reported neurotoxic effects of AAS that are
likely involved in cortical thinning observed among long-term users (Bjørnebekk et al., 2019;
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Hauger et al., 2019; Pomara et al., 2014). Additionally, a recent review found multiple health
and behavioral similarities between AAS users and both at-risk and diagnosed Alzheimer’s
disease population (Kaufman et al., 2019). Thus, studying the effect of AAS use on vulnerable
neuronal systems will better predict neurodegenerative changes occurring in aging user
populations.

4.4 Conclusion
The myriad neurobiological effects of AAS use that result in observed increases in
aggression, depression, cognitive dysfunction and behavioral disinhibition represent the
complexity of human behavior. This complexity should inform public education, intervention,
and support systems for AAS users. Assessing and addressing pre-existing psychopathology and
comorbid substance abuse will decrease relapse and suicide among users seeking to quit AAS.
Finally, providing accessible mental health services and reducing the stigma associated with their
use would decrease the percent of young adults who choose to take AAS, lessening the burden
on public health.

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4.5 References
Abi Ghanem, C., Degerny, C., Hussain, R., Liere, P., Pianos, A., Tourpin, S., Habert, R.,
Macklin, W.B., Schumacher, M., Ghoumari, A.M., 2017. Long-lasting masculinizing
effects of postnatal androgens on myelin governed by the brain androgen receptor. PLoS
Genet. 13. https://doi.org/10.1371/journal.pgen.1007049
Ambar, G., Chiavegatto, S., 2009. Anabolic-androgenic steroid treatment induces behavioral
disinhibition and downregulation of serotonin receptor messenger RNA in the prefrontal
cortex and amygdala of male mice. Genes, Brain Behav. 8, 161–173.
https://doi.org/10.1111/j.1601-183X.2008.00458.x
Bertozzi, G., Sessa, F., Albano, G.D., Sani, G., Maglietta, F., Roshan, M.H.K., Volti, G.L.,
Bernardini, R., Avola, R., Pomara, C., Salerno, M., 2018. The role of anabolic androgenic
steroids in disruption of the physiological function in discrete areas of the central nervous
system. Mol. Neurobiol. https://doi.org/10.1007/s12035-017-0774-1
Bielecki, B., Mattern, C., Ghoumari, A.M., Javaid, S., Smietanka, K., Ghanem, C.A., Mhaouty-
Kodja, S., Ghandour, M.S., Baulieu, E.E., Franklin, R.J.M., Schumacher, M., Traiffort, E.,
2016. Unexpected central role of the androgen receptor in the spontaneous regeneration of
myelin. Proc. Natl. Acad. Sci. U. S. A. 113, 14829–14834.
https://doi.org/10.1073/pnas.1614826113
Bjørnebekk, A., Westlye, L.T., Walhovd, K.B., Jørstad, M.L., Sundseth, Ø.Ø., Fjell, A.M., 2019.
Cognitive performance and structural brain correlates in long-term anabolic-androgenic
steroid exposed and nonexposed weightlifters. Neuropsychology 33, 547–559.
https://doi.org/10.1037/neu0000537
86

Börjesson, A., Möller, C., Hagelin, A., Vicente, V., Rane, A., Lehtihet, M., Dahl, M.-L.,
Gårevik, N., Ekström, L., 2020. Male anabolic androgenic steroid users with personality
disorders report more aggressive feelings, suicidal thoughts, and criminality. Medicina (B.
Aires). 56, 265. https://doi.org/10.3390/medicina56060265
Caruso, A., Di Giorgi Gerevini, V., Castiglione, M., Marinelli, F., Tomassini, V., Pozzilli, C.,
Caricasole, A., Bruno, V., Caciagli, F., Moretti, A., Nicoletti, F., Melchiorri, D., 2004.
Testosterone amplifies excitotoxic damage of cultured oligodendrocytes. J. Neurochem. 88,
1179–1185. https://doi.org/10.1046/j.1471-4159.2004.02284.x
Cerghet, M., Skoff, R.P., Bessert, D., Zhang, Z., Mullins, C., Ghandour, M.S., 2006.
Proliferation and death of oligodendrocytes and myelin proteins are differentially regulated
in male and female rodents. J. Neurosci. 26, 1439–1447.
https://doi.org/10.1523/JNEUROSCI.2219-05.2006
Cunningham, R.L., Giuffrida, A., Roberts, J.L., 2009. Androgens induce dopaminergic
neurotoxicity via caspase-3-dependent activation of protein kinase Cδ. Endocrinology 150,
5539–5548. https://doi.org/10.1210/en.2009-0640
Dodge, T., Hoagland, M.F., 2011. The use of anabolic androgenic steroids and polypharmacy: a
review of the literature. Drug and Alcohol Dependence. NIH Public Access.
https://doi.org/10.1016/j.drugalcdep.2010.11.011
Estrada, M., Varshney, A., Ehrlich, B.E., 2006. Elevated testosterone induces apoptosis in
neuronal cells. J. Biol. Chem. 281, 25492–25501. https://doi.org/10.1074/jbc.M603193200

87

Hauger, L.E., Westlye, L.T., Fjell, A.M., Walhovd, K.B., Bjørnebekk, A., 2019. Structural brain
characteristics of anabolic–androgenic steroid dependence in men. Addiction 114, 1405–
1415. https://doi.org/10.1111/add.14629
Hildebrandt, T., Heywood, A., Wesley, D., Schulz, K., 2018. Defining the construct of synthetic
androgen intoxication: an application of general brain arousal. Front. Psychol. 9.
https://doi.org/10.3389/FPSYG.2018.00390
Holmes, S., Abbassi, B., Su, C., Singh, M., Cunningham, R.L., 2013. Oxidative stress defines the
neuroprotective or neurotoxic properties of androgens in immortalized female rat
dopaminergic neuronal cells. Endocrinology 154, 4281–4292.
https://doi.org/10.1210/en.2013-1242
Hussain, R., Ghoumari, A.M., Bielecki, B., Steibel, J., Boehm, N., Liere, P., MacKlin, W.B.,
Kumar, N., Habert, R., Mhaouty-Kodja, S., Tronche, F., Sitruk-Ware, R., Schumacher, M.,
Ghandour, M.S., 2013. The neural androgen receptor: a therapeutic target for myelin repair
in chronic demyelination. Brain 136, 132–146. https://doi.org/10.1093/brain/aws284
Ip, E.J., Lu, D.H., Barnett, M.J., Tenerowicz, M.J., Vo, J.C., Perry, P.J., 2012. Psychological and
physical impact of anabolic-androgenic steroid dependence. Pharmacotherapy 32, 910–919.
https://doi.org/10.1002/j.1875-9114.2012.01123
Jackson, D.M., Westlind-Danielsson, A., 1994. Dopamine receptors: molecular biology,
biochemistry and behavioural aspects. Pharmacol. Ther. 64, 291–370.
https://doi.org/10.1016/0163-7258(94)90041-8

88

Kanayama, G., Hudson, J.I., Pope, H.G., 2009. Features of men with anabolic-androgenic steroid
dependence: a comparison with nondependent AAS users and with AAS nonusers. Drug
Alcohol Depend. 102, 130–137. https://doi.org/10.1016/j.drugalcdep.2009.02.008
Kanayama, G., Hudson, J.I., Pope, H.G., 2008. Long-term psychiatric and medical consequences
of anabolic-androgenic steroid abuse: a looming public health concern? Drug Alcohol
Depend. 98, 1–12. https://doi.org/10.1016/j.drugalcdep.2008.05.004
Karlsgodt, K.H., Bato, A.A., Blair, M.A., DeRosse, P., Szeszko, P.R., Malhotra, A.K., 2014.
White matter microstructure in the executive network associated with aggression in healthy
adolescents and young adults. Soc. Cogn. Affect. Neurosci. 10, 1251–1256.
https://doi.org/10.1093/scan/nsv015
Kaufman, M.J., Kanayama, G., Hudson, J.I., Pope, H.G., 2019. Supraphysiologic-dose anabolic–
androgenic steroid use: a risk factor for dementia? Neurosci. Biobehav. Rev. 100, 180–207.
https://doi.org/10.1016/j.neubiorev.2019.02.014
Orlando, R., Caruso, A., Molinaro, G., Motolese, M., Matrisciano, F., Togna, G., Melchiorri, D.,
Nicoletti, F., Bruno, V., 2007. Nanomolar concentrations of anabolic-androgenic steroids
amplify excitotoxic neuronal death in mixed mouse cortical cultures.
https://doi.org/10.1016/j.brainres.2007.06.047
Peper, J.S., De Reus, M.A., Van Den Heuvel, M.P., Schutter, D.J.L.G., 2015. Short Fused?
Associations between white matter connections, sex steroids, and aggression across
adolescence. Hum Brain Mapp 36, 1043–1052. https://doi.org/10.1002/hbm.22684

89

Peper, J.S., Mandl, R.C.W., Braams, B.R., De Water, E., Heijboer, A.C., Koolschijn, P.C.M.P.,
Crone, E.A., 2013. Delay discounting and frontostriatal fiber tracts: a combined DTI and
MTR study on impulsive choices in healthy young adults. Cereb. Cortex 23, 1695–1702.
https://doi.org/10.1093/cercor/bhs163
Perrin, J.S., Hervé, P.Y., Leonard, G., Perron, M., Pike, G.B., Pitiot, A., Richer, L., Veillette, S.,
Pausova, Z., Paus, T., 2008. Growth of white matter in the adolescent brain: role of
testosterone and androgen receptor. J. Neurosci. 28, 9519–9524.
https://doi.org/10.1523/JNEUROSCI.1212-08.2008
Perry, P.J., Kutscher, E.C., Lund, B.C., Yates, W.R., Holman, T.L., Demers, L., 2003. Measures
of aggression and mood changes in male weightlifters with and without androgenic anabolic
steroid use. J. Forensic Sci. 48, 2002240. https://doi.org/10.1520/jfs2002240
Pomara, C., Neri, M., Bello, S., Fiore, C., Riezzo, I., Turillazzi, E., 2014. Neurotoxicity by
synthetic androgen steroids: oxidative stress, apoptosis, and neuropathology: a review. Curr.
Neuropharmacol. 13, 132–145. https://doi.org/10.2174/1570159x13666141210221434
Pope, H.G.J., Katz, D.L., 1994. Psychiatric and medical effects of anabolic-androgenic steroid
use. A controlled study of 160 athletes. Arch. Gen. Psychiatry 51, 375–82.
https://doi.org/10.1001/archpsyc.1994.03950050035004
Skarberg, K., Nyberg, F., Engstrom, I., 2009. Multisubstance use as a feature of addiction to
anabolic-androgenic steroids. Eur. Addict. Res. 15, 99–106.
https://doi.org/10.1159/000199045

90

Westlye, L.T., Kaufmann, T., Alnæs, D., Hullstein, I.R., Bjørnebekk, A., 2017. Brain
connectivity aberrations in anabolic-androgenic steroid users. NeuroImage Clin. 13, 62–69.
https://doi.org/10.1016/j.nicl.2016.11.014
Zelleroth, S., Nylander, E., Nyberg, F., Grönbladh, A., Hallberg, M., 2019. Toxic impact of
anabolic androgenic steroids in primary rat cortical cell cultures. Neuroscience 397, 172–
183. https://doi.org/10.1016/j.neuroscience.2018.11.035

91

References List

Abi Ghanem, C., Degerny, C., Hussain, R., Liere, P., Pianos, A., Tourpin, S., Habert, R.,
Macklin, W.B., Schumacher, M., Ghoumari, A.M., 2017. Long-lasting masculinizing
effects of postnatal androgens on myelin governed by the brain androgen receptor. PLoS
Genet. 13. https://doi.org/10.1371/journal.pgen.1007049
ACSM, 1987. American college of sports medicine position stand on: the use of anabolic-
androgenic steroids in sports. Med. Sci. Sports Exerc. 19, 534–539.
Ambar, G., Chiavegatto, S., 2009. Anabolic-androgenic steroid treatment induces behavioral
disinhibition and downregulation of serotonin receptor messenger RNA in the prefrontal
cortex and amygdala of male mice. Genes, Brain Behav. 8, 161–173.
https://doi.org/10.1111/j.1601-183X.2008.00458.x
Bardgett, M.E., Depenbrock, M., Downs, N., Points, M., Green, L., 2009. Dopamine modulates
effort-based decision making in rats. Behav. Neurosci. 123, 242–251.
https://doi.org/10.1037/a0014625
Becker, M., Pinhasov, A., Ornoy, A., 2021. Animal models of depression: what can they teach us
about the human disease? Diagnostics 11.
https://doi.org/10.3390/DIAGNOSTICS11010123
Berendse, H.W., Graaf, Y.G., Groenewegen, H.J., 1992. Topographical organization and
relationship with ventral striatal compartments of prefrontal corticostriatal projections in the
rat. J. Comp. Neurol. 316, 314–347. https://doi.org/10.1002/cne.903160305

92

Bergaglio, T., Luchicchi, A., Schenk, G.J., 2021. Engine failure in axo-myelinic signaling: a
potential key player in the pathogenesis of multiple sclerosis. Front. Cell. Neurosci. 15.
https://doi.org/10.3389/fncel.2021.610295
Bertozzi, G., Sessa, F., Albano, G.D., Sani, G., Maglietta, F., Roshan, M.H.K., Volti, G.L.,
Bernardini, R., Avola, R., Pomara, C., Salerno, M., 2018. The role of anabolic androgenic
steroids in disruption of the physiological function in discrete areas of the central nervous
system. Mol. Neurobiol. https://doi.org/10.1007/s12035-017-0774-1
Besson, M., Belin, D., McNamara, R., Theobald, D.E.H., Castel, A., Beckett, V.L., Crittenden,
B.M., Newman, A.H., Everitt, B.J., Robbins, T.W., Dalley, J.W., 2010. Dissociable control
of impulsivity in rats by dopamine D2/3 receptors in the core and shell subregions of the
nucleus accumbens. Neuropsychopharmacology 35, 560–569.
https://doi.org/10.1038/npp.2009.162
Best, J.R., Miller, P.H., 2010. A developmental perspective on executive function. Child Dev.
https://doi.org/10.1111/j.1467-8624.2010.01499.x
Bielecki, B., Mattern, C., Ghoumari, A.M., Javaid, S., Smietanka, K., Ghanem, C.A., Mhaouty-
Kodja, S., Ghandour, M.S., Baulieu, E.E., Franklin, R.J.M., Schumacher, M., Traiffort, E.,
2016. Unexpected central role of the androgen receptor in the spontaneous regeneration of
myelin. Proc. Natl. Acad. Sci. U. S. A. 113, 14829–14834.
https://doi.org/10.1073/pnas.1614826113

93

Birgner, C., Kindlundh-Högberg, A.M.S., Alsiö, J., Lindblom, J., Schiöth, H.B., Bergström, L.,
2008. The anabolic androgenic steroid nandrolone decanoate affects mRNA expression of
dopaminergic but not serotonergic receptors. Brain Res. 1240, 221–228.
https://doi.org/10.1016/j.brainres.2008.09.003
Bjørnebekk, A., Westlye, L.T., Walhovd, K.B., Jørstad, M.L., Sundseth, Ø.Ø., Fjell, A.M., 2019.
Cognitive performance and structural brain correlates in long-term anabolic-androgenic
steroid exposed and nonexposed weightlifters. Neuropsychology 33, 547–559.
https://doi.org/10.1037/neu0000537
Blashill, A.J., Safren, S.A., 2014. Sexual orientation and anabolic-androgenic steroids in US
adolescent boys. Pediatrics 133, 469–475. https://doi.org/10.1542/peds.2013-2768
Bontempi, L., Bonci, A., 2020. µ-Opioid receptor-induced synaptic plasticity in dopamine
neurons mediates the rewarding properties of anabolic androgenic steroids. Sci. Signal. 13,
1–12. https://doi.org/10.1126/scisignal.aba1169
Börjesson, A., Möller, C., Hagelin, A., Vicente, V., Rane, A., Lehtihet, M., Dahl, M.-L.,
Gårevik, N., Ekström, L., 2020. Male anabolic androgenic steroid users with personality
disorders report more aggressive feelings, suicidal thoughts, and criminality. Medicina (B.
Aires). 56, 265. https://doi.org/10.3390/medicina56060265
Bunge, R.P., 1968. Glial cells and the central myelin sheath. Physiol. Rev.
https://doi.org/10.1152/physrev.1968.48.1.197

94

Caruso, A., Di Giorgi Gerevini, V., Castiglione, M., Marinelli, F., Tomassini, V., Pozzilli, C.,
Caricasole, A., Bruno, V., Caciagli, F., Moretti, A., Nicoletti, F., Melchiorri, D., 2004.
Testosterone amplifies excitotoxic damage of cultured oligodendrocytes. J. Neurochem. 88,
1179–1185. https://doi.org/10.1046/j.1471-4159.2004.02284.x
Castrellon, J.J., Meade, J., Greenwald, L., Hurst, K., Samanez-Larkin, G.R., 2021. Dopaminergic
modulation of reward discounting in healthy rats: a systematic review and meta-analysis.
Psychopharmacology (Berl). 238, 711–723. https://doi.org/10.1007/s00213-020-05723-5
Center for Behavioral Health Statistics and Quality, 2016. Impact of the DSM-IV to DSM-5
changes on the national survey on drug use and health. Subst. Abus. Ment. Heal. Serv.
Adminsitration Rockville, MD.
Cerghet, M., Skoff, R.P., Bessert, D., Zhang, Z., Mullins, C., Ghandour, M.S., 2006.
Proliferation and death of oligodendrocytes and myelin proteins are differentially regulated
in male and female rodents. J. Neurosci. 26, 1439–1447.
https://doi.org/10.1523/JNEUROSCI.2219-05.2006
Cheskis, B.J., 2004. Regulation of cell signalling cascades by steroid hormones. J. Cell.
Biochem. 93, 20–27. https://doi.org/10.1002/jcb.20180
Choi, P.Y.L., Pope, H.G., 1994. Violence toward women and illicit androgenic-anabolic steroid
use. Ann. Clin. Psychiatry 6, 21–25. https://doi.org/10.3109/10401239409148835
Christoffersen, T., Andersen, J.T., Dalhoff, K.P., Horwitz, H., 2019. Anabolic-androgenic
steroids and the risk of imprisonment. https://doi.org/10.1016/j.drugalcdep.2019.04.041

95

Chura, L.R., Lombardo, M. V., Ashwin, E., Auyeung, B., Chakrabarti, B., Bullmore, E.T.,
Baron-Cohen, S., 2010. Organizational effects of fetal testosterone on human corpus
callosum size and asymmetry. Psychoneuroendocrinology 35, 122–132.
https://doi.org/10.1016/j.psyneuen.2009.09.009
Clark, A.S., Henderson, L.P., 2003. Behavioral and physiological responses to anabolic-
androgenic steroids. Neurosci. Biobehav. Rev. https://doi.org/10.1016/S0149-
7634(03)00064-2
Coizet, V., Heilbronner, S.R., Carcenac, C., Mailly, P., Lehman, J.F., Savasta, M., David, O.,
Deniau, J.M., Groenewegen, H.J., Haber, S.N., 2017. Organization of the anterior limb of
the internal capsule in the rat. J. Neurosci. 37, 2539–2554.
https://doi.org/10.1523/JNEUROSCI.3304-16.2017
Cooke, B., Hegstrom, C.D., Villeneuve, L.S., Breedlove, S.M., 1998. Sexual differentiation of
the vertebrate brain: principles and mechanisms.
Cooper, S.E., Goings, S.P., Kim, J.Y., Wood, R.I., 2014. Testosterone enhances risk tolerance
without altering motor impulsivity in male rats. Psychoneuroendocrinology 40, 201–212.
https://doi.org/10.1016/j.psyneuen.2013.11.017
Couppis, M.H., Kennedy, C.H., 2008. The rewarding effect of aggression is reduced by nucleus
accumbens dopamine receptor antagonism in mice. Psychopharmacology (Berl). 197, 449–
456. https://doi.org/10.1007/s00213-007-1054-y
Cunningham, R.L., Giuffrida, A., Roberts, J.L., 2009. Androgens induce dopaminergic
neurotoxicity via caspase-3-dependent activation of protein kinase Cδ. Endocrinology 150,
5539–5548. https://doi.org/10.1210/en.2009-0640
96

Dalley, J.W., Cardinal, R.N., Robbins, T.W., 2004. Prefrontal executive and cognitive functions
in rodents: neural and neurochemical substrates, in: Neuroscience and Biobehavioral
Reviews. pp. 771–784. https://doi.org/10.1016/j.neubiorev.2004.09.006
Davani-Davari, D., Karimzadeh, I., Khalili, H., 2019. The potential effects of anabolic-
androgenic steroids and growth hormone as commonly used sport supplements on the
kidney: A systematic review. BMC Nephrol. https://doi.org/10.1186/s12882-019-1384-0
Dodge, T., Hoagland, M.F., 2011. The use of anabolic androgenic steroids and polypharmacy: a
review of the literature, Drug and Alcohol Dependence. NIH Public Access.
https://doi.org/10.1016/j.drugalcdep.2010.11.011
Dubb, A., Gur, R., Avants, B., Gee, J., 2003. Characterization of sexual dimorphism in the
human corpus callosum. Neuroimage 20, 512–519. https://doi.org/10.1016/S1053-
8119(03)00313-6
Ersche, K.D., Williams, G.B., Robbins, T.W., Bullmore, E.T., 2013. Meta-analysis of structural
brain abnormalities associated with stimulant drug dependence and neuroimaging of
addiction vulnerability and resilience. Curr. Opin. Neurobiol. 23, 615–624.
https://doi.org/10.1016/j.conb.2013.02.017
Estrada, M., Varshney, A., Ehrlich, B.E., 2006. Elevated testosterone induces apoptosis in
neuronal cells. J. Biol. Chem. 281, 25492–25501. https://doi.org/10.1074/jbc.M603193200
Fabri, M., Polonara, G., Mascioli, G., Salvolini, U., Manzoni, T., 2011. Topographical
organization of human corpus callosum: an fMRI mapping study. Brain Res. 1370, 99–111.
https://doi.org/10.1016/j.brainres.2010.11.039
97

Floresco, S.B., Magyar, O., 2006. Mesocortical dopamine modulation of executive functions:
beyond working memory. Psychopharmacology (Berl). https://doi.org/10.1007/s00213-006-
0404-5
Floresco, S.B., St. Onge, J.R., Ghods-Sharifi, S., Winstanley, C.A., 2008a. Cortico-limbic-
striatal circuits subserving different forms of cost-benefit decision making. Cogn. Affect.
Behav. Neurosci. https://doi.org/10.3758/CABN.8.4.375
Floresco, S.B., Tse, M.T.L., Ghods-Sharifi, S., 2008b. Dopaminergic and glutamatergic
regulation of effort- and delay-based decision making. Neuropsychopharmacology 33,
1966–1979. https://doi.org/10.1038/sj.npp.1301565
Foradori, C.D., Weiser, M.J., Handa, R.J., 2007. Non-genomic Actions of Androgens.
https://doi.org/10.1016/j.yfrne.2007.10.005
Ghods-Sharifi, S., Floresco, S.B., 2010. Differential effects on effort discounting induced by
inactivations of the nucleus accumbens core or shell. Behav. Neurosci. 124, 179–191.
https://doi.org/10.1037/a0018932
Ghods-Sharifi, S., St. Onge, J.R., Floresco, S.B., 2009. Fundamental contribution by the
basolateral amygdala to different forms of decision making. J. Neurosci. 29, 5251–5259.
https://doi.org/10.1523/JNEUROSCI.0315-09.2009
Ghoumari, A.M., Ghanem, C.A., Asbelaoui, N., Schumacher, M., Hussain, R., 2020. Roles of
progesterone, testosterone and their nuclear receptors in central nervous system myelination
and remyelination. Int. J. Mol. Sci. 21. https://doi.org/10.3390/ijms21093163

98

Gorelova, N., Yang, C.R., 1996. The course of neural projection from the prefrontal cortex to the
nucleus accumbens in the rat. Neuroscience 76, 689–706. https://doi.org/10.1016/S0306-
4522(96)00380-6
Grimes, J.M., Melloni, R.H., 2002. Serotonin modulates offensive attack in adolescent anabolic
steroid-treated hamsters. Pharmacol. Biochem. Behav. 73, 713–721.
https://doi.org/10.1016/S0091-3057(02)00880-8
Gruber, A.J., McDonald, R.J., 2012. Context, emotion, and the strategic pursuit of goals:
Interactions among multiple brain systems controlling motivated behavior. Front. Behav.
Neurosci. https://doi.org/10.3389/fnbeh.2012.00050
Habermacher, C., Angulo, M.C., Benamer, N., 2019. Glutamate versus GABA in neuron–
oligodendroglia communication. Glia 67, 2092–2106. https://doi.org/10.1002/glia.23618
Hauger, L.E., Westlye, L.T., Bjørnebekk, A., 2020. Anabolic androgenic steroid dependence is
associated with executive dysfunction. Drug Alcohol Depend. 208, 107874.
https://doi.org/10.1016/j.drugalcdep.2020.107874
Hauger, L.E., Westlye, L.T., Fjell, A.M., Walhovd, K.B., Bjørnebekk, A., 2019. Structural brain
characteristics of anabolic–androgenic steroid dependence in men. Addiction 114, 1405–
1415. https://doi.org/10.1111/add.14629
Havnes, I.A., Bukten, A., Rognli, E.B., Muller, A.E., 2020. Use of anabolic-androgenic steroids
and other substances prior to and during imprisonment - Results from the Norwegian
Offender Mental Health and Addiction (NorMA) study. Drug Alcohol Depend. 217,
108255. https://doi.org/10.1016/j.drugalcdep.2020.108255
99

Heffernan, T., Battersby, L., Bishop, P., O’Neill, T., 2015. Everyday memory deficits associated
with anabolic-androgenic steroid use in regular gymnasium users. Open Psychiatr. J. 9, 1–6.
Hildebrandt, T., Heywood, A., Wesley, D., Schulz, K., 2018. Defining the construct of synthetic
androgen intoxication: an application of general brain arousal. Front. Psychol. 9.
https://doi.org/10.3389/FPSYG.2018.00390
Hildebrandt, T., Langenbucher, J.W., Flores, A., Harty, S., Berlin, H.A., 2014. The influence of
age of onset and acute anabolic steroid exposure on cognitive performance, impulsivity, and
aggression in men. Psychol Addict Behav 28, 1096–1104. https://doi.org/10.1037/a0036482
Hines, M., Allen, L.S., Gorski, R.A., 1992. Sex differences in subregions of the medial nucleus
of the amygdala and the bed nucleus of the stria terminalis of the rat. Brain Res. 579, 321–
326. https://doi.org/10.1016/0006-8993(92)90068-K
Holmes, S., Abbassi, B., Su, C., Singh, M., Cunningham, R.L., 2013. Oxidative stress defines the
neuroprotective or neurotoxic properties of androgens in immortalized female rat
dopaminergic neuronal cells. Endocrinology 154, 4281–4292.
https://doi.org/10.1210/en.2013-1242
Hosking, J.G., Floresco, S.B., Winstanley, C.A., 2015. Dopamine antagonism decreases
willingness to expend physical, but not cognitive, effort: a comparison of two rodent
cost/benefit decision-making tasks. Neuropsychopharmacology 40, 1005–1015.
https://doi.org/10.1038/npp.2014.285

100

Hussain, R., Ghoumari, A.M., Bielecki, B., Steibel, J., Boehm, N., Liere, P., MacKlin, W.B.,
Kumar, N., Habert, R., Mhaouty-Kodja, S., Tronche, F., Sitruk-Ware, R., Schumacher, M.,
Ghandour, M.S., 2013. The neural androgen receptor: a therapeutic target for myelin repair
in chronic demyelination. Brain 136, 132–146. https://doi.org/10.1093/brain/aws284
Ip, E.J., Lu, D.H., Barnett, M.J., Tenerowicz, M.J., Vo, J.C., Perry, P.J., 2012. Psychological and
physical impact of anabolic-androgenic steroid dependence. Pharmacotherapy 32, 910–919.
https://doi.org/10.1002/j.1875-9114.2012.01123
Jackson, D.M., Westlind-Danielsson, A., 1994. Dopamine receptors: molecular biology,
biochemistry and behavioural aspects. Pharmacol. Ther. 64, 291–370.
https://doi.org/10.1016/0163-7258(94)90041-8
Jacobus, J., Thayer, R.E., Trim, R.S., Bava, S., Frank, L.R., Tapert, S.F., 2013. White matter
integrity, substance use, and risk taking in adolescence. Psychol. Addict. Behav. 27, 431–
442. https://doi.org/10.1037/a0028235
Jito, J., Nakasu, S., Ito, R., Fukami, T., Morikawa, S., Inubushi, T., 2008. Maturational changes
in diffusion anisotropy in the rat corpus callosum: comparison with quantitative histological
evaluation. J. Magn. Reson. Imaging 28, 847–854. https://doi.org/10.1002/jmri.21496
Johnston, L.D., Miech, R.A., O’Malley, P.M., Bachman, J.G., Schulenberg, J.E., Patrick, M.E.,
2019. Monitoring the Future national survey results on drug use 1975-2018: overview, key
findings on adolescent drug use. Ann Arbor Inst. Soc. Res. Univ. Michigan.
https://doi.org/10.1176/foc.1.2.213

101

Joksimović, J., Selaković, D., Jakovljević, V., Mihailović, V., Katanić, J., Boroja, T., Rosić, G.,
2017. Alterations of the oxidative status in rat hippocampus and prodepressant effect of
chronic testosterone enanthate administration. Mol. Cell. Biochem. 433, 41–50.
https://doi.org/10.1007/s11010-017-3014-0
Kanayama, G., Brower, K.J., Wood, R.I., Hudson, J.I., Jr, H.G.P., 2009a. Anabolic – androgenic
steroid dependence : an emerging disorder 1966–1978. https://doi.org/10.1111/j.1360-
0443.2009.02734.x
Kanayama, G., Brower, K.J., Wood, R.I., Hudson, J.I., Pope, H.G., 2010. Treatment of anabolic-
androgenic steroid dependence: emerging evidence and its implications. Drug Alcohol
Depend. https://doi.org/10.1016/j.drugalcdep.2010.01.011
Kanayama, G., Brower, K.J., Wood, R.I., Hudson, J.I., Pope, H.G., 2009b. Anabolic-androgenic
steroid dependence: an emerging disorder. Addiction 104, 1966–1978.
https://doi.org/10.1111/j.1360-0443.2009.02734.x
Kanayama, G., Brower, K.J., Wood, R.I., Hudson, J.I., Pope, H.G., 2009c. Issues for DSM-V:
clarifying the diagnostic criteria for anabolic-androgenic steroid dependence. Am. J.
Psychiatry 166, 642–645. https://doi.org/10.1176/appi.ajp.2009.08111699
Kanayama, G., Hudson, J.I., Pope, H.G., 2020. Anabolic-androgenic steroid use and body image
in men: a growing concern for clinicians. Psychother. Psychosom.
https://doi.org/10.1159/000505978
Kanayama, G., Hudson, J.I., Pope, H.G., 2009d. Features of men with anabolic-androgenic
steroid dependence: a comparison with nondependent AAS users and with AAS nonusers.
Drug Alcohol Depend. 102, 130–137. https://doi.org/10.1016/j.drugalcdep.2009.02.008
102

Kanayama, G., Hudson, J.I., Pope, H.G., 2008. Long-term psychiatric and medical consequences
of anabolic-androgenic steroid abuse: a looming public health concern? Drug Alcohol
Depend. 98, 1–12. https://doi.org/10.1016/j.drugalcdep.2008.05.004
Kanayama, G., Kean, J., Hudson, J.I., Pope, H.G., 2013. Cognitive deficits in long-term
anabolic-androgenic steroid users. Drug Alcohol Depend. 130, 208–214.
https://doi.org/10.1016/j.drugalcdep.2012.11.008
Kanayama, G., Pope, H.G., 2018. History and epidemiology of anabolic androgens in athletes
and non-athletes. Mol. Cell. Endocrinol. 464, 4–13.
https://doi.org/10.1016/j.mce.2017.02.039
Karlsgodt, K.H., Bato, A.A., Blair, M.A., DeRosse, P., Szeszko, P.R., Malhotra, A.K., 2014.
White matter microstructure in the executive network associated with aggression in healthy
adolescents and young adults. Soc. Cogn. Affect. Neurosci. 10, 1251–1256.
https://doi.org/10.1093/scan/nsv015
Kaufman, M.J., Janes, A.C., Hudson, J.I., Brennan, B.P., Kanayama, G., Kerrigan, A.R., Jensen,
J.E., Pope, H.G., 2015. Brain and cognition abnormalities in long-term anabolic-androgenic
steroid users. Drug Alcohol Depend. 152, 47–56.
https://doi.org/10.1016/j.drugalcdep.2015.04.023
Kaufman, M.J., Kanayama, G., Hudson, J.I., Pope, H.G., 2019. Supraphysiologic-dose anabolic–
androgenic steroid use: a risk factor for dementia? Neurosci. Biobehav. Rev. 100, 180–207.
https://doi.org/10.1016/j.neubiorev.2019.02.014

103

Kindlundh, A.M.S., Lindblom, J., Bergström, L., Wikberg, J.E.S., Nyberg, F., 2001. The
anabolic-androgenic steroid nandrolone decanoate affects the density of dopamine receptors
in the male rat brain. Eur. J. Neurosci. 13, 291–296. https://doi.org/10.1046/j.0953-
816X.2000.01402.x
Lindqvist, A.-S., Johansson-Steensland, P., Nyberg, F., Fahlke, C., 2002. Anabolic androgenic
steroid affects competitive behaviour, behavioural response to ethanol and brain serotonin
levels. Behav. Brain Res. 133, 21–29.
Liston, C., Watts, R., Tottenham, N., Davidson, M.C., Niogi, S., Ulug, A.M., Casey, B.J., 2006.
Frontostriatal microstructure modulates efficient recruitment of cognitive control. Cereb.
Cortex 16, 553–560. https://doi.org/10.1093/cercor/bhj003
Martínez-Rivera, F.J., Natal-Albelo, E.J., Martínez, N.A., Orozco-Vega, R.A., Muñiz-Seda,
O.A., Barreto-Estrada, J.L., 2015. The effect of the anabolic steroid, nandrolone, in
conditioned place preference and D1 dopamine receptor expression in adolescent and adult
mice. Behav. Processes 113, 81. https://doi.org/10.1016/J.BEPROC.2015.01.008
Matrisciano, F., Modafferi, A.M.E., Togna, G.I., Barone, Y., Pinna, G., Nicoletti, F.,
Scaccianoce, S., 2010. Repeated anabolic androgenic steroid treatment causes
antidepressant-reversible alterations of the hypothalamic - pituitary - adrenal axis, BDNF
levels and behavior. Neuropharmacology 58, 1078–1084.
https://doi.org/10.1016/j.neuropharm.2010.01.015

104

McIntyre, K.L., Porter, D.M., Henderson, L.P., 2002. Anabolic androgenic steroids induce age-,
sex-, and dose-dependent changes in GABAA receptor subunit mRNAs in the mouse
forebrain. Neuropharmacology 43, 634–645. https://doi.org/10.1016/S0028-3908(02)00154-
5
Mhillaj, E., Morgese, M.G., Tucci, P., Bove, M., Schiavone, S., Trabace, L., 2015. Effects of
anabolic-androgens on brain reward function. Front. Neurosci.
https://doi.org/10.3389/fnins.2015.00295
Micu, I., Plemel, J.R., Caprariello, A. V, Nave, K., Stys, P.K., 2017. Axo-myelinic
neurotransmission: a novel mode of cell signalling in the central nervous system, Nature
Publishing Group. https://doi.org/10.1038/nrn.2017.128
Middleman, A.B., Faulkner, A.H., Woods, E.R., Emans, S.J., DuRant, R.H., 1995. High-risk
behaviors among high school students in Massachusetts who use anabolic steroids.
Pediatrics 96, 268–272.
Namjoshi, D.R., Cheng, W.H., Carr, M., Martens, K.M., Zareyan, S., Wilkinson, A., McInnes,
K.A., Cripton, P.A., Wellington, C.L., 2016. Chronic exposure to androgenic-anabolic
steroids exacerbates axonal injury and microgliosis in the CHIMERA mouse model of
repetitive concussion. PLoS One 11, e0146540.
https://doi.org/10.1371/journal.pone.0146540

105

Nicholls, A.R., Cope, E., Bailey, R., Koenen, K., Dumon, D., Theodorou, N.C., Chanal, B.,
Laurent, D. Saint, Müller, D., Andrés, M.P., Kristensen, A.H., Thompson, M.A., Baumann,
W., Laurent, J.F., 2017. Children’s first experience of taking anabolic-androgenic steroids
can occur before their 10th birthday: a systematic review identifying 9 factors that predicted
doping among young people. Front. Psychol. 8. https://doi.org/10.3389/fpsyg.2017.01015
NIDA, 2018. Steroids and other appearance and performance enhancing drugs (APEDs). Natl.
Inst. Drug Abus. 1–35.
Nowend, K.L., Arizzi, M., Carlson, B.B., Salamone, J.D., 2001. D1 or D2 antagonism in nucleus
accumbens core or dorsomedial shell suppresses lever pressing for food but leads to
compensatory increases in chow consumption. Pharmacol. Biochem. Behav. 69, 373–382.
https://doi.org/10.1016/S0091-3057(01)00524-X
Orlando, R., Caruso, A., Molinaro, G., Motolese, M., Matrisciano, F., Togna, G., Melchiorri, D.,
Nicoletti, F., Bruno, V., 2007. Nanomolar concentrations of anabolic-androgenic steroids
amplify excitotoxic neuronal death in mixed mouse cortical cultures.
https://doi.org/10.1016/j.brainres.2007.06.047
Packard, M.G., Schroeder, J.P., Alexander, G.M., 1998. Expression of testosterone conditioned
place preference is blocked by peripheral or intraaccumbens injection of α-flupenthixol.
Horm. Behav. 34, 39–47. https://doi.org/10.1006/hbeh.1998.1461
Parkinson, A.B., Evans, N.A., 2006. Anabolic androgenic steroids: a survey of 500 users. Med.
Sci. Sports Exerc. 38, 644–651. https://doi.org/10.1249/01.mss.0000210194.56834.5d

106

Pattij, T., Janssen, M.C.W., Vanderschuren, L.J.M.J., Schoffelmeer, A.N.M., Van Gaalen, M.M.,
2007. Involvement of dopamine D1 and D2 receptors in the nucleus accumbens core and
shell in inhibitory response control. Psychopharmacology (Berl). 191, 587–598.
https://doi.org/10.1007/s00213-006-0533-x
Paus, T., Nawaz-Khan, I., Leonard, G., Perron, M., Pike, G.B., Pitiot, A., Richer, L., Susman, E.,
Veillette, S., Pausova, Z., 2010. Sexual dimorphism in the adolescent brain: role of
testosterone and androgen receptor in global and local volumes of grey and white matter.
Horm. Behav. 57, 63–75. https://doi.org/10.1016/j.yhbeh.2009.08.004
Peper, J.S., Braams, B.R., Blankenstein, N.E., Bos, M.G.N., Crone, E.A., 2018. Development of
multifaceted risk taking and the relations to sex steroid hormones: a longitudinal study.
Child Dev. 89, 1887–1907. https://doi.org/10.1111/cdev.13063
Peper, J.S., De Reus, M.A., Van Den Heuvel, M.P., Schutter, D.J.L.G., 2015. Short Fused?
Associations between white matter connections, sex steroids, and aggression across
adolescence. Hum Brain Mapp 36, 1043–1052. https://doi.org/10.1002/hbm.22684
Peper, J.S., Mandl, R.C.W., Braams, B.R., De Water, E., Heijboer, A.C., Koolschijn, P.C.M.P.,
Crone, E.A., 2013. Delay discounting and frontostriatal fiber tracts: a combined DTI and
MTR study on impulsive choices in healthy young adults. Cereb. Cortex 23, 1695–1702.
https://doi.org/10.1093/cercor/bhs163
Peper, J.S., van den Heuvel, M.P., Mandl, R.C.W., Pol, H.E.H., van Honk, J., 2011. Sex steroids
and connectivity in the human brain: a review of neuroimaging studies.
Psychoneuroendocrinology. https://doi.org/10.1016/j.psyneuen.2011.05.004

107

Perrin, J.S., Hervé, P.Y., Leonard, G., Perron, M., Pike, G.B., Pitiot, A., Richer, L., Veillette, S.,
Pausova, Z., Paus, T., 2008. Growth of white matter in the adolescent brain: role of
testosterone and androgen receptor. J. Neurosci. 28, 9519–9524.
https://doi.org/10.1523/JNEUROSCI.1212-08.2008
Perry, P.J., Kutscher, E.C., Lund, B.C., Yates, W.R., Holman, T.L., Demers, L., 2003. Measures
of aggression and mood changes in male weightlifters with and without androgenic anabolic
steroid use. J. Forensic Sci. 48, 2002240. https://doi.org/10.1520/jfs2002240
Pessoa, L., 2009. How do emotion and motivation direct executive control? Trends Cogn. Sci.
13, 160–166. https://doi.org/10.1016/j.tics.2009.01.006
Peters, K.D., Wood, R.I., 2005. Androgen dependence in hamsters: overdose, tolerance, and
potential opioidergic mechanisms. Neuroscience 130, 971–981.
https://doi.org/10.1016/j.neuroscience.2004.09.063
Pomara, C., Neri, M., Bello, S., Fiore, C., Riezzo, I., Turillazzi, E., 2014. Neurotoxicity by
synthetic androgen steroids: oxidative stress, apoptosis, and neuropathology: a review. Curr.
Neuropharmacol. 13, 132–145. https://doi.org/10.2174/1570159x13666141210221434
Pope, H.G., Kanayama, G., Athey, A., Ryan, E., Hudson, J.I., Baggish, A., Pope Jr, H.G.,
Kanayama, G., Athey, A., Ryan, E., Hudson, J.I., Baggish, A., 2014a. The lifetime
prevalence of anabolic-androgenic steroid use and dependence in Americans: current best
estimates, American Journal on Addictions. Wiley-Blackwell Publishing Ltd.
https://doi.org/10.1111/j.1521-0391.2013.12118.x

108

Pope, H.G., Kanayama, G., Hudson, J.I., Kaufman, M.J., 2021. Review article: anabolic-
androgenic steroids, violence, and crime: two cases and literature review. Am. J. Addict.
https://doi.org/10.1111/ajad.13157
Pope, H.G., Katz, D.L., 1990. Homicide and near-homicide by anabolic steroid users. J. Clin.
Psychiatry 51, 28–31.
Pope, H. G., Katz, D.L., 1988. Affective and psychotic symptoms associated with anabolic
steroid use. Am. J. Psychiatry. https://doi.org/10.1176/ajp.145.4.487
Pope, Harrison G, Katz, D.L., 1988. Affective and Psychotic Symptoms Associated With
Anabolic Steroid Use, Am J Psychiatry.
Pope, H.G., Kouri, E.M., Hudson, J.I., 2000. Effects of supraphysiologic doses of testosterone on
mood and aggression in normal men: a randomized controlled trial. Arch. Gen. Psychiatry
57, 133–140. https://doi.org/10.1001/archpsyc.57.2.133
Pope, H.G., Kouri, E.M., Powell, K.F., Campbell, C., Katz, D.L., 1996. Anabolic-androgenic
steroid use among 133 prisoners. Compr. Psychiatry 37, 322–327.
https://doi.org/10.1016/S0010-440X(96)90013-9
Pope, H.G., Wood, R.I., Rogol, A., Nyberg, F., Bowers, L., Bhasin, S., 2014b. Adverse health
consequences of performance-enhancing drugs: an endocrine society scientific statement.
Endocr. Rev. 35, 341–375. https://doi.org/10.1210/er.2013-1058
Pope, H.G.J., Katz, D.L., 1994. Psychiatric and medical effects of anabolic-androgenic steroid
use. A controlled study of 160 athletes. Arch. Gen. Psychiatry 51, 375–82.
https://doi.org/10.1001/archpsyc.1994.03950050035004
109

Rashid, W., 2000. Testosterone abuse and affective disorders. J. Subst. Abuse Treat. 18, 179–
184. https://doi.org/10.1016/S0740-5472(99)00023-9
Rasmussen, J.J., Selmer, C., Østergren, P.B., Pedersen, K.B., Schou, M., Gustafsson, F., Faber,
J., Juul, A., Kistorp, C., 2016. Former abusers of anabolic androgenic steroids exhibit
decreased testosterone levels and hypogonadal symptoms years after cessation: a case-
control study. PLoS One 11. https://doi.org/10.1371/journal.pone.0161208
Ricci, L.A., Morrison, T.R., Melloni, R.H., 2013. Adolescent anabolic/androgenic steroids:
aggression and anxiety during exposure predict behavioral responding during withdrawal in
syrian hamsters (mesocricetus auratus). Horm Behav 64, 770–780.
https://doi.org/10.1016/j.yhbeh.2013.10.002
Schroeder, J.P., Packard, M.G., 2000. Role of dopamine receptor subtypes in the acquisition of a
testosterone conditioned place preference in rats, Neuroscience Letters.
https://doi.org/10.1016/S0304-3940(00)00839-9
Seitz, J., Lyall, A.E., Kanayama, G., Makris, N., Hudson, J.I., Kubicki, M., Pope, H.G.,
Kaufman, M.J., 2017. White matter abnormalities in long-term anabolic-androgenic steroid
users: a pilot study. Psychiatry Res. - Neuroimaging 260, 1–5.
https://doi.org/10.1016/j.pscychresns.2016.12.003
Selakovic, D., Joksimovic, J., Jovicic, N., Mitrovic, S., Mihailovic, V., Katanic, J., Milovanovic,
D., Pantovic, S., Mijailovic, N., Rosic, G., 2019. The impact of hippocampal sex hormones
receptors in modulation of depressive-like behavior following chronic anabolic androgenic
steroids and exercise protocols in rats. Front. Behav. Neurosci. 13.
https://doi.org/10.3389/fnbeh.2019.00019
110

Shahidi, N.T., 2001a. A review of the chemistry, biological action, and clinical applications of
anabolic-androgenic steroids. Clin. Ther. 23, 1355–1390. https://doi.org/10.1016/S0149-
2918(01)80114-4
Shahidi, N.T., 2001b. A review of the chemistry, biological action, and clinical applications of
anabolic-androgenic steroids.
Skarberg, K., Nyberg, F., Engstrom, I., 2009. Multisubstance use as a feature of addiction to
anabolic-androgenic steroids. Eur. Addict. Res. 15, 99–106.
https://doi.org/10.1159/000199045
Sousa, S.S., Amaro, E., Crego, A., Gonçalves, Ó.F., Sampaio, A., 2018. Developmental
trajectory of the prefrontal cortex: a systematic review of diffusion tensor imaging studies.
Brain Imaging Behav. https://doi.org/10.1007/s11682-017-9761-4
Sowell, E.R., Peterson, B.S., Thompson, P.M., Welcome, S.E., Henkenius, A.L., Toga, A.W.,
2003. Mapping cortical change across the human life span. https://doi.org/10.1038/nn1008
Sowell, E.R., Thompson, P.M., Holmes, C.J., Jernigan, T.L., Toga, A.W., 1999. In vivo evidence
for post-adolescent brain maturation in frontal and striatal regions [1]. Nat. Neurosci.
https://doi.org/10.1038/13154
Su, T.P., Pagliaro, M., Schmidt, P.J., Pickar, D., Wolkowitz, O., Rubinow, D.R., 1993.
Neuropsychiatric effects of anabolic steroids in male normal volunteers. JAMA 269, 2760–
4. https://doi.org/10.1001/jama.269.21.2760

111

Tabor, J., Wright, D.K., Christensen, J., Zamani, A., Collins, R., Shultz, S.R., Mychasiuk, R.,
2020. Examining the effects of anabolic–androgenic steroids on repetitive mild traumatic
brain injury (RmTBI) outcomes in adolescent rats. Brain Sci. 10.
https://doi.org/10.3390/brainsci10050258
Tobiansky, D.J., Wallin-Miller, K.G., Floresco, S.B., Wood, R.I., Soma, K.K., 2018. Androgen
regulation of the mesocorticolimbic system and executive function. Front. Endocrinol.
(Lausanne). https://doi.org/10.3389/fendo.2018.00279
Triemstra, J.L., Sato, S.M., Wood, R.I., 2008. Testosterone and nucleus accumbens dopamine in
the male syrian hamster. Psychoneuroendocrinology 33, 386–394.
https://doi.org/10.1016/j.psyneuen.2007.12.006
Tucci, P., Morgese, M.G., Colaianna, M., Zotti, M., Schiavone, S., Cuomo, V., Trabace, L.,
2011. Neurochemical consequence of steroid abuse: stanozolol-induced monoaminergic
changes. https://doi.org/10.1016/j.steroids.2011.12.014
Turillazzi, E., Neri, M., Cerretani, D., Cantatore, S., Frati, P., Moltoni, L., Busardò, F.P.,
Pomara, C., Riezzo, I., Fineschi, V., 2016. Lipid peroxidation and apoptotic response in rat
brain areas induced by long‐term administration of nandrolone: the mutual crosstalk
between ROS and NF ‐ kB. J. Cell. Mol. Med. 20,
601–612. https://doi.org/10.1111/jcmm.12748
Uban, K.A., Rummel, J., Floresco, S.B., Galea, L.A.M., 2012. Estradiol modulates effort-based
decision making in female rats. Neuropsychopharmacology 37, 390–401.
https://doi.org/10.1038/npp.2011.176
WADA, 2012. Testing Figures Report. Montr. Canada.
112

Wallin-Miller, K.G., Kreutz, F., Li, G., Wood, R.I., 2018. Anabolic-androgenic steroids (AAS)
increase sensitivity to uncertainty by inhibition of dopamine D1 and D2 receptors.
Psychopharmacology (Berl). 235, 959–969. https://doi.org/10.1007/s00213-017-4810-7
Wallin, K.G., Alves, J.M., Wood, R.I., 2015. Anabolic-androgenic steroids and decision making:
probability and effort discounting in male rats. Psychoneuroendocrinology 57, 84–92.
https://doi.org/10.1016/j.psyneuen.2015.03.023
Wallin, K.G., Wood, R.I., 2015. Anabolic-androgenic steroids impair set-shifting and reversal
learning in male rats. Eur. Neuropsychopharmacol. 25, 583–590.
https://doi.org/10.1016/j.euroneuro.2015.01.002
Westlye, L.T., Kaufmann, T., Alnæs, D., Hullstein, I.R., Bjørnebekk, A., 2017. Brain
connectivity aberrations in anabolic-androgenic steroid users. NeuroImage Clin. 13, 62–69.
https://doi.org/10.1016/j.nicl.2016.11.014
Winstanley, C.A., Floresco, S.B., 2016. Deciphering decision making: variation in animal
models of effort- and uncertainty-based choice reveals distinct neural circuitries underlying
core cognitive processes. J. Neurosci. 36, 12069–12079.
https://doi.org/10.1523/JNEUROSCI.1713-16.2016
Wood, R.I., 2008. Anabolic-androgenic steroid dependence? Insights from animals and humans.
Front. Neuroendocrinol. https://doi.org/10.1016/j.yfrne.2007.12.002
Wood, R.I., Armstrong, A., Fridkin, V., Shah, V., Najafi, A., Jakowec, M., 2013. ’Roid rage in
rats? Testosterone effects on aggressive motivation, impulsivity and tyrosine hydroxylase.
Physiol. Behav. 110–111, 6–12. https://doi.org/10.1016/j.physbeh.2012.12.005
113

Wood, R.I., Serpa, R.O., 2020. Anabolic-androgenic steroid abuse and cognitive impairment:
testosterone impairs biconditional task performance in male rats. Behav. Brain Res. 379,
112339. https://doi.org/10.1016/j.bbr.2019.112339
Zack, G.W., Rogers, W.E., Latt, S.A., 1977. Automatic measurement of sister chromatid
exchange frequency. J. Histochem. Cytochem. 25, 741–753.
https://doi.org/10.1177/25.7.70454
Zelleroth, S., Nylander, E., Nyberg, F., Grönbladh, A., Hallberg, M., 2019. Toxic impact of
anabolic androgenic steroids in primary rat cortical cell cultures. Neuroscience 397, 172–
183. https://doi.org/10.1016/j.neuroscience.2018.11.035
Ziegler, G., Hauser, T.U., Moutoussis, M., Bullmore, E.T., Goodyer, I.M., Fonagy, P., Jones,
P.B., Lindenberger, U., Dolan, R.J., 2019. Compulsivity and impulsivity traits linked to
attenuated developmental frontostriatal myelination trajectories. Nat. Neurosci. 22, 992–
999. https://doi.org/10.1038/s41593-019-0394-3
Zotti, M., Tucci, P., Colaianna, M., Morgese, M.G., Mhillaj, E., Schiavone, S., Scaccianoce, S.,
Cuomo, V., Trabace, L., 2013. Chronic nandrolone administration induces dysfunction of
the reward pathway in rats. https://doi.org/10.1016/j.steroids.2013.10.005

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Creator Donovan, Alexandra (author)

Core Title Testosterone’s effect on reward, cognition, and myelination: rat brain and behavior

School College of Letters, Arts and Sciences

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Degree Conferral Date 2021-12

Publication Date 11/02/2021

Defense Date 09/08/2021

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