Citation
Isolation rearing alters behavior and expression of brain-derived neurotrophic factor and the immediate early gene arc in the prefrontal cortex and amygdala of male and female rats

Material Information

Title:
Isolation rearing alters behavior and expression of brain-derived neurotrophic factor and the immediate early gene arc in the prefrontal cortex and amygdala of male and female rats
Creator:
Wall, Vanessa L
Publication Date:
Language:
English
Physical Description:
1 electronic file : ;

Subjects

Subjects / Keywords:
Stress (Physiology) ( lcsh )
Sensory deprivation ( lcsh )
Sensory deprivation ( fast )
Stress (Physiology) ( fast )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Review:
Early life adversity has been identified as a risk factor for the development of psychopathology later in life. One way that adverse experiences early in life may have deleterious effects on an individual is by producing brain and behavior abnormalities in stress responsivity, particularly in regard to stressors involving other people and social interactions. Abnormalities in stress responses and social interactions in individuals with a history of early life adversity may be partially explained by changes in brain regions related to stress regulation. The medial prefrontal cortex (mPFC) and amygdala have been implicated in stress regulation and emotional and cognitive functioning. The present study examined the effects of an animal model of early life adversity, isolation rearing, on aggressive and non-aggressive behavior and activation of the protein products of the activity dependent genes, Arc and BDNF in the PFC and amygdala produced by exposure to a subsequent mild social stressor (e.g. exposure to a novel rat). Relationships between behavior during social exposure and gene activation were also examined. Exposure to a novel, same-sex rat (conspecific) produced significant increases in Arc in group-housed animals, but this increase was blunted or absent in isolates. Social exposure produced small increases in BDNF activation, but this was not dependent on housing condition. Differences in patterns of activation, as well as in relationships between behavior and gene activation, were observed in a sex and subregion dependent manner.
Bibliography:
Includes bibliographical references.
Statement of Responsibility:
by Vanessa L. Wall.

Record Information

Source Institution:
|University of Colorado Denver
Holding Location:
|Auraria Library
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
859780715 ( OCLC )
ocn859780715

Auraria Membership

Aggregations:
Auraria Library
University of Colorado Denver

Downloads

This item has the following downloads:


Full Text
ISOLATION REARING ALTERS BEHAVIOR AND EXPRESSION OF BRAIN-DERIVED
NEUROTROPHIC FACTOR AND THE IMMEDIATE EARLY GENE ARC IN THE
PREFRONTAL CORTEX AND AMYGDALA OF MALE AND FEMALE RATS
by
Vanessa L. Wall
B.A., University of Colorado, Colorado Springs, 2008
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Master of Arts
Clinical Psychology M.A. Program
2012


This thesis for the Master of Arts degree by
Vanessa L. Wall
has been approved for the
Clinical Psychology M.A. Program
by
Dr. Sondra T. Bland, Chair
Dr. Richard Allen
Dr. Jim Grigsby
Date
4/26/2012
ii


Wall, Vanessa L. (M.A., Clinical Psychology M.A. Program)
Isolation Rearing Alters Behavior and Expression of Brain-derived Neurotrophic Factor and the
Immediate Early Gene Arc in the Prefrontal Cortex and Amygdala of Male and Female Rats
Thesis directed by Dr. Sondra T. Bland.
ABSTRACT
Early life adversity has been identified as a risk factor for the development of psychopathology
later in life. One way that adverse experiences early in life may have deleterious effects on an
individual is by producing brain and behavior abnormalities in stress responsivity, particularly in
regard to stressors involving other people and social interactions. Abnormalities in stress
responses and social interactions in individuals with a history of early life adversity may be
partially explained by changes in brain regions related to stress regulation. The medial
prefrontal cortex (mPFC) and amygdala have been implicated in stress regulation and emotional
and cognitive functioning. The present study examined the effects of an animal model of early
life adversity, isolation rearing, on aggressive and non-aggressive behavior and activation of the
protein products of the activity dependent genes, Arc and BDNF in the PFC and amygdala
produced by exposure to a subsequent mild social stressor (e.g. exposure to a novel rat).
Relationships between behavior during social exposure and gene activation were also
examined. Exposure to a novel, same-sex rat (conspecific) produced significant increases in Arc
in group housed animals, but this increase was blunted or absent in isolates. Social exposure
produced small increases in BDNF activation, but this was not dependent on housing condition.
Differences in patterns of activation, as well as in relationships between behavior and gene
activation, were observed in a sex and subregion dependent manner.
The form and content of this abstract are approved. I recommend its publication.
Approved: Sondra T. Bland


TABLE OF CONTENTS
Chapter
1. Introduction...........................................................................1
1.1. Consequences of stress and adversity in humans................................2
1.1.1. Physiological effects of stress........................................3
1.1.2. Chronic stress, social support, and psychopathology....................4
1.1.3. Chronic stress experiences in adolescence..............................5
1.2. Isolation rearing in rats (a model of early life adversity)...................6
1.3. Social behavior following chronic stress......................................7
1.4. Neurobiological correlates of behavioral stress responses and implications for brain
plasticity.........................................................................9
1.4.1. Brain-derived neurotrophic factor (BDNF)...............................9
1.4.2. Activity-regulated cytoskeleton-associated protein (Arc)..............11
1.5. Sex Differences in Stress Responses and Psychopathology......................13
1.6. Brain regions involved in stress.............................................14
1.6.1. Medial prefrontal cortex (mPFC).......................................14
1.6.2. Amygdala..............................................................15
1.7. Rationale....................................................................16
1.8. Purpose and research questions...............................................16
1.9. Predictions..................................................................17
2. Methods..............................................................................19
2.1. Animals.....................................................................19
2.2. Social exposure procedure...................................................19
2.3. Tissue harvest..............................................................20
2.4. Immunohistochemistry........................................................20
2.5. Statistics..................................................................21
IV


3. Results
23
3.1. Arc...............................................................................
3.1.1. Medial prefrontal cortex stereology........................................
3.1.2. Prefrontal cortex density counts...........................................
3.1.3. Amygdala density counts....................................................
3.2. BDNF..............................................................................
3.2.1. Medial prefrontal cortex stereology........................................
3.2.2. Prefrontal cortex density counts...........................................
3.3. Behavior during novel social exposure.............................................
3.4. Relationships between behavior and Arc and BDNF expression........................
4. Discussion..................................................................................
4.1. Arc activation produced by exposure to a novel conspecific........................
4.1.1. Arc activation varies by sex and subregion of the PFC and amygdala.........
4.2. BDNF activation in the PFC........................................................
4.3. Isolation-reared rats exhibit increased aggressive and nonaggressive behavior....
4.4. Relationships between Arc and BDNF activation and behavior........................
4.5. Is novel social exposure stressful, rewarding, or both for isolation-reared rats?.
References................................................................................
23
23
23
24
27
27
27
29
29
31
31
34
35
36
37
38
40
v


LIST OF FIGURES
Figure
1. Prefrontal cortex subregions.....................................................21
2. Amygdala subregions..............................................................21
3. Arc stereology...................................................................23
4. Arc prefrontal cortex density counts.............................................25
5. Arc amygdala density counts......................................................26
6. BDNF stereology..................................................................27
7. BDNF prefrontal cortex density counts............................................28
8. Behavior.........................................................................30
VI


1. Introduction
Effective clinical treatment of psychological disorders is dependent on preclinical
research examining the mechanisms by which psychopathology develops. Early life adversity
and stress in humans has been implicated as a factor in the etiology of a number of
psychological disorders, including anxiety disorders, depression, schizophrenia, and substance
abuse (Sadock & Sadock, 2007; Holmes & Wellman, 2009; Romeo, 2010). Stress can also
affect somatic health and immune function (OLeary, 1990), thereby exacerbating mental health
issues or in the absence of psychopathology, reducing overall quality of life. Isolation rearing in
rats is an animal model of early life adversity and stress that produces anxiety-like behaviors
and other abnormalities in behavior, brain morphology, and neurochemistry (Hall, 1998; Fone &
Porkess, 2008). This model allows researchers to observe the effects of acute or chronic early
life stress (depending on the duration of the social isolation) under experimental conditions,
providing a wealth of information about abnormalities in behavior and neurobiology produced by
such adversity while limiting extraneous variables. Of particular interest is the effect that early
life adversity has on social behavior as many psychological stressors as well as protective
factors are related to social functioning and support (Sadock & Sadock, 2007). In addition to
understanding the behavioral effects and influences on social functioning of early life adversity
and stress, changes in neurobiological activation and/or inhibition produced by isolation rearing
must be examined. Behavioral changes that arise from ongoing early life stress are better
understood in the context of the neurobiological changes that accompany them. Adding to the
complexity of the effects of early life adversity, it is generally understood that males and females
have different behavioral and physiological responses to stress and adversity (Gallucci, 1993;
Young, Korszun, Figueiredo, Banks-Solomon, & Herman, 2008; Wright et al., 2009). These
differences in behavior and physiology may partially explain the different prevalence rates for
males and females of a number of psychological disorders, such as mood and anxiety
disorders, substance use disorders, and PTSD (Altemus, 2006; Sadock & Sadock). In order to
1


effectively meet the different needs of males and females in clinical populations, behavioral and
neurobiological sex differences must be thoroughly examined. The present study examined the
effects of isolation rearing in male and female rats on social behavior and neurobiology, as
measured by activation of the protein products of brain derived neurotrophic factor (BDNF) and
the immediate early gene (IEG) Arc in the medial prefrontal cortex (mPFC) and amygdala
produced by exposure to a novel same-sex rat (conspecific).
1.1 Consequences of stress and adversity in humans
Stress is an unavoidable part of life, and at moderate levels is adaptive because of its
role in helping individuals deal with threatening events (Sadock & Sadock, 2007). Extended
periods of stress, however, can cause the bodys natural stress-response systems to become
dysfunctional, thus reducing the adaptive value of such systems. Understanding the ways that
stress and adversity are risk factors for the development of physiological and psychological
problems in humans requires examination of the specific effects of chronic stress. Long periods
of stress and adversity can take a toll on an individuals health. Chronic stress has been found
to have different effects on behavior, neuroendocrine responses, and susceptibility to
developing mental illness in humans than acute stress (Malarkey & Mills, 2007; Hammen et al.,
2010). The lasting effects of chronic stress may attenuate behavioral and physical responses to
subsequent stressors.
The effects of chronic stress in humans range from the molecular level to the organism
level to the population level. Chronic stress is associated with neurochemical abnormalities,
specifically dysregulation of serotonin, dopamine, and norepinephrine (Harro & Oreland, 2001).
Individuals experiencing chronic stress often exhibit problems with mood (Tafet et al., 2001),
and memory and cognitive function (Diamond, Campbell, Park, & Vouimba, 2004) as a result of
these neurochemical changes. In addition, stress has been associated with dysfunction of the
hypothalamic-pituitary-adrenal (HPA) axis (Ostrander et al., 2009). Dysfunction of the HPA axis
2


can lead to long-term increased or decreased reactivity to subsequent stressors (Ehlert, Gaab,
& Heinrichs, 2001). Living with chronic stress can affect social interactions and relationships
and has been identified as a risk factor for morbidity at the individual and societal level (Stauder
et al., 2010). In an environment where many people are living with chronic stress, the
deleterious effects reach to the entire population. Furthermore, stress has been implicated as a
factor in a number of psychiatric illnesses (McEwen, 2000). Understanding the mechanisms by
which chronic stress affects behavior and physiology is crucial for reducing and preventing the
detrimental effects of stress.
1.1.1. Physiological effects of stress
Stress is often associated with somatic symptoms such as gastrointestinal upset,
changes in appetite, and signs of arousal such as heart palpitations and increased perspiration
(Vermetten & Bremner, 2002). Multiple physiological systems are associated with stress;
however, dysfunction of the HPA axis is one of the best-studied and most commonly addressed
physiological responses to stress. The HPA axis is involved in maintaining homeostasis
following exposure to stress. Upon exposure to stress, the hypothalamus releases corticotrophin
releasing factor (CRF), which causes release of adrenocorticotropin hormone (ACTH) from the
anterior pituitary. ACTH stimulates release of cortisol from the adrenal glands, which feeds back
to the hypothalamus in a negative feedback loop, suppressing release of CRF and ACTH
(Ehlert et al., 2001). Dysfunction of this negative feedback loop can lead to dysregulation at any
or all steps in the stress response. One commonly studied result of dysfunction of the HPA axis
is imbalance in cortisol levels, which can affect immune function and increases vulnerability to
disease (OLeary, 1990). Other brain regions have been implicated in direct and indirect
regulation of the HPA axis, including the prefrontal cortex (Figueiredo, Bruestle, Bodie, Dolgas,
& Herman, 2003; Romeo, 2010) and the amygdala (Ostrander et al., 2009; Romeo, 2010) but
further research is needed to clarify the ways in which these brain regions may be related to
stress responses. The homeostatic response of the HPA axis has been extensively studied in
3


animal models (Vermetten & Bremner, 2002), but other neurobiological changes related to
stress responses and associated long-term effects are not as well understood. The present
study examined the role of other mechanisms associated with chronic stress, including IEG
activation and behavioral changes, in order to gain a better understanding of the long-term
effects of chronic stress during adolescence.
1.1.2. Chronic stress, social support, and psychopathology
In addition to problems with immune function, mood, memory, and cognitive function,
chronic stress plays a role in psychiatric illnesses. It has been implicated as an etiologic,
maintenance, and relapse factor in many mental illnesses; including depression, posttraumatic
stress disorder (McEwen, 2000), anxiety (Romeo, 2010) and substance use disorders (Marinelli
& Piazza, 2002). In addition to the direct effect stress can have on an individuals development
of a psychiatric illness, stress can affect interpersonal relationships. Social support is an
important prognostic factor in mental illness. Poor social support can be a risk factor for the
development of psychiatric illness and may predict poorer outcomes (Sadock & Sadock, 2007).
Stress associated with mental illness can negatively impact interpersonal relationships even in
an individual with good premorbid social support. While good social support is indicative of a
better prognosis, the chronic stress that is associated with many mental illnesses can erode this
protective factor. For individuals with poor premorbid social support, seeking social support
while experiencing the deleterious effects of chronic stress may prove to be a significant
challenge. For an individual suffering from mental illness, social interaction may be experienced
as an additional stressor, especially if the social environment is new or unknown. It is essential
to understand the ways in which chronic stress can affect social interaction. This understanding
will aid in the development of preventative and treatment techniques that address abnormalities
in social functioning as they are related to psychopathology.
4


1.1.3. Chronic stress experiences in adolescence
Exposure to early life adversity and stress is a significant risk factor for future
development of various somatic and psychological problems (Romeo, 2010). As previously
noted, chronic stress can produce many maladaptive changes, and this is true across
developmental stages. However, when one considers that experiences of on-going stress affect
adolescents differently than adults due to the unique hormonal and behavioral changes that
occur during this developmental stage (Romeo, 2010), adversity during adolescence emerges
as a unique and important area for research, distinct from adversity during other developmental
stages.
Pubertal development and stress have been found to interact in ways that are unique to
this developmental stage. For example, puberty marks a time of changes in the amount and
duration of hormone release by the HPA axis in response to stress. These changes in HPA axis
functioning may partially explain increases in stress responsiveness and emotionality that occur
during adolescence (Walker, 2002; Romeo, 2010). Other neurochemical changes, however,
play a role in stress responsiveness and behavior during adolescence as well. Some of these
changes have been extensively studied, such as the physiological and behavioral changes
produced by increases in gonadal hormone secretion and activity of the hypothalamic-pituitary-
gonadal (HPG) axis (see Sisk & Zehr, 2005 for a review). Other mechanisms involved in stress
responses and behavior during this developmental stage, such as plasticity of brain regions that
coordinate emotional and social responses like the medial prefrontal cortex (mPFC) and the
amygdala, have not been studied as thoroughly and may further explain stress-induced
changes in behavior and in the brains of adolescents. The role of the prefrontal cortex in
response to stress and social situations during adolescence are of particular interest since the
prefrontal cortex is not fully mature during adolescence. Changes in this brain region continue to
occur throughout adolescence as many neuronal connections are pruned back and others are
strengthened due to repeated activity (Weinberger, Elvevag, & Giedd, 2005).
5


Although many of the hormonal, behavioral, and social changes that occur during
adolescence are considered to be normative, this developmental stage has been identified as a
time of heightened risk for the onset of psychopathology (Walker, 2002). This increased risk for
mental health problems and the heightened stress responsivity many adolescents experience
indicate that particular care must be taken to understand the ways in which adolescents
respond to different types of stressors and the ways these behavioral and neurobiological
responses can affect subsequent functioning. A preclinical model of chronic early life stress is
necessary in order to examine the behavioral effects of early life adversity under experimental
conditions and to examine the neurobiological changes with which these behavioral effects are
associated.
12. Isolation rearing in rats (a model of early life adversity)
Social isolation rearing has been widely used as an animal model of early life adversity
and stress (Hall, 1998; see Fone & Porkess, 2008 for a review). The early-life stressor of social
isolation rearing has been linked to a number of neurochemical changes, including alterations in
dopamine and serotonin function as well as changes in neuronal morphology (Fone & Porkess,
2008). Isolation rearing leads to abnormalities in PFC structure and function such as decreased
constitutive expression of immediate early genes (Day-Wilson et al., 2006; Levine et al., 2007),
and synaptic-associated proteins (Hermes et al., 2011), reductions in PFC volume (Day-Wilson
et al., 2006), and changes in dendritic spine morphology (Ferdman et al., 2007). These
alterations in neuron structure and function are likely responsible for many of the behavioral
changes produced by isolation rearing. The enduring behavioral changes produced by this
model include changes in reactivity to a novel environment, social interaction and aggression,
pain sensitivity, and deficits in conditioned learning and novel object discrimination (Fone &
Porkess, 2008). The precise mechanisms of many of these neurobiological and behavioral
changes, however, are not well understood. The present study was designed to build on these
6


findings of behavioral and neurochemical abnormalities to examine additional specific
neurobiological and behavioral effects of isolation rearing and the ways in which such changes
may be associated.
Social interaction has been found to be rewarding for adolescent rats (Douglas,
Varlinskaya, & Spear, 2004). Social deprivation, especially during adolescence, is thought to be
a stressful experience as indicated by changes in HPA axis function and increased anxiety
behaviors as measured in the elevated plus maze (Hall, 1998; Fone & Porkess, 2008), and
deprivation of social play during adolescence leads to changes in neuron morphology in the
mPFC (Bell et al., 2010). The utility of the isolation rearing model is based on the specific use of
the model to examine the effects of social deprivation on species that exhibit high levels of
social and affiliative behavior. Social deprivation has been found to have consistent behavioral
and neurochemical effects across species (Hall, 1998). While there are a number of limitations
inherent in the use of animals to extrapolate to human phenomena, isolation rearing is a useful
model to examine the effects of early life adversity on subsequent behavioral and
neurobiological responses to stress in species whose social structures are marked by high
levels of social interaction.
1.3. Social behavior following chronic stress
In humans, early-life stressors may lead genetically disposed individuals to develop a
number of psychiatric disorders (Fone & Porkess, 2008). While early-life adverse events can
contribute to the development of psychiatric disorders, not everyone who develops such a
disorder was exposed to severe stressors early in life. Likewise, many individuals who are
exposed to severe adversity early in life do not subsequently develop symptoms of mental
illness. It is likely that additional experiences later in life can contribute to the risk of developing
a mental illness. Among other factors, it may that be subsequent stressors are central in
producing abnormalities in behavior and neurobiology for individuals with a history of chronic
7


stress. The effects of chronic stress on behavioral and physiological reactions to subsequent
stressors are particularly important as many people, both with and without diagnosis of a
psychiatric illness, may feel that the stressors in their lives seem to pile up. Due to the central
role that social interaction plays in most individuals lives, understanding how stress and social
functioning interact is crucial. Stress can have an adverse effect on social functioning (Everhart
& Emde, 2006), and conversely, social interactions may contribute to stress, even when the
interactions are not particularly negative. Many individuals interact with a large number of
people on a daily basis, and a large percentage of these interactions may be with relative
strangers. Therefore, it is necessary to study the behavioral effects of chronic stress on
subsequent novel social interaction stressors. Combining a history of chronic stress with a
subsequent interaction with a novel rat may be a model for the unique changes that occur when
individuals living with chronic stress are faced with stress associated with novel social
interactions.
As mentioned above, on-going stressful experiences may affect social behavior and
relationships in humans, preventing the development of social relationships or negatively
affecting existing bonds. The social interaction test of anxiety has been extensively used to test
for anxiogenic and anxiolytic effects of environmental factors and exogenously administered
drugs and peptides (File and Seth, 2003). In this test, the dependent variable is the amount of
time one rat spends engaging in social interaction (e.g. grooming, sniffing) with another rat. An
increase in social interaction without an increase in overall motor activity is indicative of an
anxiolytic effect; while a decrease in time spend interacting without a concomitant decrease in
motor activity indicates an anxiogenic effect (File & Seth, 2003). The present study modified this
test in order to examine group differences in time spent interacting as well as other indications
of abnormalities in social functioning, such as changes in aggressive behavior and play
behaviors like pinning and chasing.
8


A number of factors affect behavior during social interactions, and a number of changes
happen in the brain during social interaction. In order to interpret how the effects of chronic early
life adversity may interact with effects of social interaction, these two procedures were
combined (e.g. exposure to chronic early life stress followed by exposure to a novel rat); and the
neurobiological correlates of behavioral effects were measured. The present study examined
these behavioral and physiological aspects by exposing rats with a history of chronic early life
adversity to novel rats with no history of stress. In addition, levels of Arc and BDNF protein
were measured to examine the neurobiological changes that go along with any observed
changes in behavior.
1.4. Neurobiological correlates of behavioral stress responses and implications for brain
plasticity
Stress activates a number of complex short and long-term neurobiological responses in
the brain. One of these responses is the expression of activity-dependent genes. These genes
are activated by the cascade of events that occur when sensory experience (in this case
exposure to novel stimuli) leads to the activation of neurons and release of excitatory
neurotransmitter. Calcium influx that occurs in response to membrane depolarization triggers a
number of changes within the neuron, including local and cell wide gene activation and protein
translation (Flavell & Greenberg, 2008). The nature of activity-dependent genes makes them
valuable markers of neuronal activity as well as potential mediators of the plastic changes that
occur in the brain in response to experience (Schinder & Poo, 2000).
1.4.1. Brain-derived neurotrophic factor (BDNF)
BDNF is an activity-dependent gene that is activated in response to neuronal
stimulation. This gene encodes a neurotrophin that is released at synapses and plays a role in
synaptic plasticity. Neurotrophins are a class of proteins that play an important part in the
survival, differentiation, growth, and maintenance of neurons (Branchi, Francia, & Alieva, 2004;
9


Hashimoto, Shimizu, & lyo, 2004). Production of neurotrophins has been found to be experience
dependent, indicating that these proteins may be partially responsible for the ways in which
environmental experience can affect development, gene expression, and brain function (Branchi
et al., 2004). For example, early life experiences such as maternal separation and exposure to
an enriched environment have been found to significantly affect production of BDNF and nerve
growth factor (NGF) and alter behavior (Branchi et al., 2004). In addition to the influence of
experience on neurotrophin levels during development, stress during adulthood has been found
to alter neurotrophin levels as well (Branchi, 2004; Bland et al., 2005).
BDNF is considered a synaptic morphogen due to the wide variety of effects it has on
neuron function and structure and the various circumstances under which it is activated (Branchi
et al., 2004). Importantly, BDNF activation has been found to have opposing effects depending
on a number of internal and external factors, making it a likely contributor to the modulation of
plasticity in the CNS (see Greenberg et al., 2009 for a review). It has been found to protect
neurons in the face of a chemical stressor (Spina, Squinto, Miller, Linsay, & Hyman, 1992) and
is necessary for survival and differentiation of developing neurons (Branchi et al., 2004). In
addition, it has been implicated in a number of psychological disorders including major
depressive disorder, bipolar disorder and posttraumatic stress disorder (Smith, Makino,
Kvetnansky, & Post, 1995; see Hashimoto, 2004 for a review). For example, it has been
proposed that BDNF is an important aspect of recovery from major depressive disorder due to
the increase in BDNF that various treatments for major depressive disorder produce, and the
BDNF gene has been identified as a potential risk gene for the development of bipolar disorder
(Hashimoto et. al., 2004). BDNF has been found to be a major component in the effects of
chronic stress and is regulated by stress, (Smith et al., 1995). Roceri and colleagues (2002)
found that rats that had undergone the early life stressor of maternal separation had decreased
BDNF in the hippocampus and prefrontal cortex in adulthood. In addition, BDNF expression in
response to an acute stress during adulthood was altered as a result of early maternal
10


separation, indicating long-term changes in function as a result of this specific early life stressor,
(Roceri et al., 2002).
BDNF has been found to be a major component of learning and memory as well (Lu,
Christian, and Lu, 2008; Cunha, Brambilla, & Thomas, 2010). This aspect of BDNF is important
for our model of early life adversity due to the role that memory and learned behaviors play in
the development and expression of various stress responses and psychological illnesses. For
example, one of the symptoms of posttraumatic stress disorder is recurrent and distressing
recollection of the traumatic event that was experienced (Sadock & Sadock, 2007). Many
anxiety disorders are marked by behaviors such as compulsions in the case of obsessive-
compulsive disorder and avoidance in the case of phobias and panic disorder, both of which are
behaviors that an individual with an anxiety disorder has learned are effective in reducing
anxiety in the short term (Sadock & Sadock, 2007). Additionally, proper social functioning and
behavior in rats is learned through play experiences, and deprivation of play behavior can affect
subsequent social functioning (Hoi et al., 1999).
One of the important functions of BDNF is its role in the activation of another type of
activity-dependent gene, the immediate early gene Arc. Neurotrophins are known to induce
expression of this sub-type of activity-dependent genes (Lyford et al., 1995). lEGs undergo
rapid and transient transcription, which can be induced by different types of stimuli, and can
occur within 5 minutes of neuronal stimulation depending on the IEG (Flavell & Greenberg,
2008). The proposed study will measure expression of BDNF and the IEG Arc, which is
activated by BDNF (Bramham, Worley, Moore, & Guzowski, 2008), in order to further elucidate
the cascade of events that occur in the brain as it changes and adapts in response to adversity
and stress.
1.4.2. Activity-regulated cytoskeieton-associated protein (Arc)
Arc has been implicated in multiple forms of synaptic plasticity (Bramham et al., 2008),
and is thought to be associated with reorganization or development of synapses as a result of
11


stressful experiences (Kozlovsky et al. 2008). Arc is a neuronal activation marker that plays a
role in changes in synaptic transmission through alterations at receptor sites in the postsynaptic
density (Pinaud, 2004; Bramham et al., 2008). Arc mRNA is transported rapidly to the dendrites
and accumulates at sites of synaptic activity. Arc protein also accumulates in dendrites, and like
Arc mRNA, is found specifically in areas of synaptic activity, indicating that the protein is
synthesized locally at the synapse (Bramham et al., 2008). Arc has been closely linked with the
strength of excitatory synapses and has been implicated in multiple forms of neuronal plasticity,
including long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity
(Bramham et al., 2008). LTP refers to a long-term increase in excitability of a neuron due to
repeated, high-frequency activation, and conversely, LTD refers to a long-term decrease in the
excitability of a neuron (Carlson, 2008). Homeostatic plasticity refers to mechanisms that keep
neuronal activity relatively stable in the face of the forces of LTP and LTD (Turrigiano & Nelson,
2004).
While other lEGs have been studied in relation to chronic stress in animals, Arc is an
important I EG to study because of its possible role in the synaptic plasticity underlying long-term
changes in functioning. An increase or decrease in Arc expression may have an effect on the
ability of the brain to modify and protect itself from stressors. If a history of chronic stress
changes the ability of Arc genes to be activated and/or changes the amount of Arc protein that
is synthesized in response to neuronal activation, this may partially explain the deleterious
emotional and behavioral effects of chronic stress. Kozlovsky and colleagues (2008) proposed a
resilience/recovery role for Arc in response to exposure to stress. They found that rats with a
highly disrupted behavioral response to a predator scent stressor had significantly lower levels
of Arc mRNA in the hippocampus compared to rats whose behavioral response was minimal
(Kozlovsky et al, 2008). Bland and colleagues (abstract, 2009) reported different levels of Arc
labeled cells in the mPFC following an acute restraint stressor in male and female rats reared in
social isolation compared to male and female group reared rats. The present study built on
12


these findings by using social isolation rearing coupled with a subsequent social interaction
stressor in order to determine how a history of early life adversity attenuates future responses to
social interactions with a novelty stress component.
1.5. Sex Differences in Stress Responses and Psychopathology
Prevalence rates of stress-related disorders are different for males and females, with
females suffering from panic disorder, specific phobia, posttraumatic stress disorder, and
generalized anxiety disorder at a higher rate than males and males having higher rates of
substance use disorders such as alcohol and cocaine abuse and dependence (Sadock &
Sadock, 2007). Additionally, in those disorders where lifetime prevalence is approximately equal
among men and women, such as schizophrenia, the onset, course, and prognosis of the
disorder often vary by sex (Sadock & Sadock, 2007). Research examining sex differences in
stress responses has demonstrated that males and females have different physiological
responses to stress, including differences in HPA axis responsiveness (Gallucci et al., 1993)
and differences in emotional processing when depressed (Wright et al., 2009). Despite such
clinical evidence of sex as a factor in the development of mental illness, much preclinical
research has only used male rats in models of stress and psychopathology. Those studies that
have examined males and females have found that there are a number of behavioral and
neurobiological sex differences in response to stress.
Bland and colleagues (2005) found that female rats have a more pronounced HPA
response (as measured by plasma corticosterone levels) and that males and females have
different levels and time courses of neuronal activation (as measured by expression of the I EG
c-fos) and expression of BDNF in the prefrontal cortex in response to acute uncontrollable
stress. Likewise, Romeo (2010) reports that female rats have a higher peak corticosterone
response than male rats and a quicker return to baseline following a stressor. In an experiment
examining sex differences in social interaction and expression of the IEG zif268, females were
13


found to exhibit higher anxiety-like behavior during exposure to a novel rat and had lower levels
of zif268 expression in subregions of the mPFC (Stack et al., 2010). Research examining sex
differences in response to stress and adversity will help to clarify the reasons why prevalence
rates for various mental disorders are different for men and women and will aid in the
development of efficacious and possibly sex-dependent treatments.
1.6. Brain regions involved in stress
The understanding of many brain regions directly involved in stress responses, such as
the hypothalamus (Smith, Makino, Kim & Kvetnansky, 1995; Joels et al., 2004; see Herman,
Prewitt, & Cullinan, 1996 for a review) and hippocampus (Smith et al., 1995; Roceri et al., 2002;
Joels et al., 2004; see Maggio & Segal, 2010 for a review) has become more complete in recent
years. In addition to their direct regulation of stress responses, these brain regions have
projections to and from other regions that are important targets for research if behavioral and
physiological responses to stress are to be fully understood. The long-term effects of adversity
involve many brain regions that undergo a number of changes that can alter responses to a
wide variety of stimuli, including stress responses. Two brain regions that are particularly
important for understanding the effects of stress and adversity due to their functions and
connectivity with other brain regions are the medial prefrontal cortex (mPFC) and the amygdala.
1.6.1. Medial prefrontal cortex (mPFC)
Proper functioning of the prefrontal cortex is necessary for carrying out executive
functions, such as the ability to attend to or ignore stimuli, process information, and plan and
carry out a response (Holmes & Wellman, 2009). Stress produces activation of the mPFC, and
this brain region plays a role in the processing of stressful or threatening stimuli and in
physiological, cognitive, and emotional responses to stressors (Vermetten & Bremner, 2002).
One function of the mPFC in stress responses is to regulate activity of the HPA axis, and this
regulation appears to vary depending on what kind of stressor is present (Figueiredo et al.,
2003). Chronic stress in rats results in a decrease in length and number of dendrites in the
14


mPFC (Radley et al. 2004; Cook & Wellman, 2005). This neuroanatomical outcome of chronic
stress could potentially affect not only mPFC functioning but also its ability to communicate with
other brain regions, a crucial role of the mPFC. The mPFC projects to and receives input from a
number of brain regions involved in stress and emotional responses, including the amygdala
(Gabbott, Warner, Jays, Salway, & Busby, 2005). It projects to the hypothalamus and
brainstem, creating a connection to brain regions involved in neuroendocrine and autonomic
responses to stress (Holmes & Wellman, 2009). It has bidirectional connections with the locus
coeruleus, dorsal raphe nucleus, and ventral tegmental area, three monoaminergic nuclei which
are activated by stress and modulate executive function via their connections with the mPFC
(Holmes & Wellman, 2009). Due to the role of the mPFC in regulating the HPA axis and
modulating other neurobiological and behavioral responses to stress and its larger role in proper
cognitive and emotional functioning, it is crucial to study changes in the mPFC that occur as a
result of early life adversity and stress.
1.6.2. Amygdala
One of the best known functions of the amygdala is as a major center for processing
emotional and socially relevant information. It is also important for formulating appropriate
responses to fearful or threatening stimuli and for appropriate social behavior (see Adolphs,
2010 for a review). This brain region is complex in structure and function, with numerous
subregions containing nuclei that project to various areas in the brain, including the mPFC,
hypothalamus, and brainstem (Holmes & Wellman, 2009). These connections with other brain
regions and its role in emotional processing indicate that the amygdala is an important brain
region for formulating healthy and adaptive stress responses and may be a target for study in
individuals who have undergone extended periods of stress. Abnormalities in structure and
function have been reported in the amygdala in individuals suffering from mental illness such as
anxiety and depression (Monk, 2008). The focus of the present study is to understand the ways
in which a history of chronic adversity affects social, behavioral, and neurobiological responses
15


to a subsequent social interaction stressor. Due to its connectivity to brain regions directly
involved in stress responses, such as the hypothalamus, and its role in appropriate processing
of and response to emotionally and socially relevant information, the amygdala warrants study
as a link between the experience of adversity early in life and the subsequent development of
abnormal functioning.
1.7. Rationale
Stress affects people in numerous ways, depending on the nature of the stressor, the
individuals history of stress and adversity, sex, and a number of other environmental and
genetic factors. More information regarding the circumstances under which adverse
experiences contribute to the development of psychopathology is needed in order to effectively
prevent and treat mental illnesses. The effects of early life adversity on behavior and
neurobiological responses during social interaction are particularly important due to the role of
social support as a positive prognostic factor for individuals with mental illness (Sadock &
Sadock, 2007), and the adverse effect stress can have on social functioning (Everhart & Emde,
2006). This study combined a history of early life adversity with a subsequent exposure to a
novel rat in order to model the unique changes that occur when individuals who have
experienced chronic stress early in life are faced with the stress of novel social interactions.
Behavior during exposure to a novel rat was examined in conjunction with expression of BDNF
and the IEG Arc in the mPFC and amygdala in order to determine possible associations
between changes in the brain and changes in behavior in male and female rats.
1.8. Purpose and Research Questions
The purpose of this study is to investigate the effects of early life adversity on social
behavior and the neurobiological changes with which it is associated in male and female rats.
16


Question 1: How does isolation rearing affect expression of the protein products of BDNF and
the immediate early gene Arc in the amygdala and the prefrontal cortex produced by exposure
to a novel conspecific?
Question 2: How does isolation rearing affect behavior during exposure to a novel conspecific?
Question 3: Is Arc and BDNF expression produced by exposure to a novel conspecific
associated with behavior during exposure?
Question 4: Do male and female rats differ in these effects?
1.9. Predictions
H1: Isolation rearing will produce a decrease in Arc and BDNF protein expression in the
amygdala and mPFC produced by exposure to a novel rat.
The isolation rearing procedure has been found to produce decreases in other lEGs and
synaptic-associated proteins, so it is expected for the plasticity related genes measured
here to have a similar downregulation in isolates.
H2: Isolation rearing will produce a decrease in overall social interaction with a novel rat as well
as a decrease in typical play behaviors.
Isolation rearing has been found to increase anxiety-like behaviors and neophobia, so it
is expected that a novel social exposure will likewise produce avoidance and a decrease
in social behaviors.
H3: Arc and BDNF activation will have a positive relationship with behavior.
Since low levels of Arc and BDNF and low levels of behavior in isolates are anticipated,
and conversely higher levels of both gene activation and behaviors in group housed rats,
a linear relationship between behavior and gene activation is expected.
H4: Male and female isolation-reared rats will have similar results, but the effects will be more
pronounced in females.
17


Females have higher anxiety-like behaviors in response to exposure to a novel rat and
more pronounced HP A activation in response to a stressor, so it is expected that
behavioral and neurobiological changes produced by the chronic stressor of isolation
rearing will be more pronounced in females.
18


2. Methods
2.1. Animals
Male (n = 32) and female (n = 32) Sprague-Dawley rats were purchased from Harlan
(Indianapolis, IN) at postnatal day 21 and housed in a temperature controlled vivarium with a
12:12 light:dark cycle and unlimited access to food and water. Rats were housed for 4 weeks
either individually (isolation) or in same-sex groups of 4 (GH) in Plexiglas cages under standard
housing conditions.
2.2. Social Exposure Procedure
After 4 weeks of isolation or group housing, rats were placed in a cage with a novel
same-sex rat (conspecific) for 15 minutes. Exposure to novel conspecifics occured in standard
Plexiglas cages. Novel conspecifics were group housed in order to control for potential effects of
abnormalities in stimulus animals affecting the behavior of experimental animals. Control rats
were left in their home cages and all rats were sacrificed 90 min later. This time point was
chosen as an optimal time point to measure the protein product of activity-dependent genes
based on the time of peak protein expression (90-120 min) following acute stimuli for the
prototypical immediate-early gene, c-fos (Kovacs, 2008). Social exposure was video recorded
and behavior was coded using the following definitions:
Social Interaction: Overall time the experimental rat spends actively interacting (e.g. sniffing,
following, grooming) with the novel conspecific (File & Seth, 2003).
Pinning: Standing over/holding down the novel conspecific while it is in a supine posture (Hurst
et al., 1999).
Chasing: Pursuit of the novel conspecific while it is running away from the experimental rat.
Aggressive Grooming: Vigorous grooming by the experimental rat of the novel conspecific when
it is standing, crouching, supine, or trying to escape (Hurst et al., 1999).
19


2.3. Tissue harvest
Rats were sacrificed 90 minutes following completion of exposure to a novel rat and
home cage control rats were sacrificed at the same time. Rats were deeply anesthetized with
sodium pentobarbital and transcardially perfused with 0.9% saline followed by 4%
paraformaldehyde in 0.01 M PBS and cryoprotected in 30% sucrose for three days, then quickly
frozen in -30C isopentane. Brains were sliced through the prefrontal cortex and amygdala using
the atlas of Paxinos and Watson (1998) as a guide. Sections were stored at 4C in
cryoprotectant until immunohistochemistry was performed.
2.4. Immunohistochemistry
Sections were labeled using primary antibodies directed against Arc (rabbit anti-Arc,
1:4,000, Synaptic Systems) and biotinylated goat anti-rabbit secondary antibody (1:200,
Jackson Labs) or BDNF (mouse anti-BDNF, 1:300, Chemicon) and biotinylated goat anti-mouse
secondary antibody (1:200, Jackson Labs) with a 3,3-diaminobenzidine (DAB) and nickel as
chromogens. Density counts were conducted for Arc and BDNF in subregions of the mPFC: the
anterior cingulate cortex (AC), the prelimbic cortex (PL), and the infralimbic cortex (IL), and in
the motor cortex (MC) and ventral orbital cortex (VO), (Fig. 1), as well as subregions of the
amygdala: basolateral (BLA), central (CeA), and medial (MeA), (Fig. 2), at40x using an
Olympus BX51 microscope and NewCAST software (VisioPharm).
Arc and BDNF positive cells were counted using a counting frame centered within each
subregion and normalized as cells per mm2. For each rat, 3-5 sections (6-10 individual
hemisphere measurements) were assessed and mean values calculated. Unbiased stereology
was also performed to obtain estimates of total Arc and BDNF labeled cells in the mPFC.
Random meander sampling was performed at 100x (oil) using sampling fractions of 1% for Arc
and BDNF.
20


Figure 1. Section containing prefrontal cortex subregions: the anterior
cingulate (AC), prelimbic (PL), infralimbic (IL), ventral orbital (VO),
and primary motor cortices (MC), (Paxinos & Watson, 1998)
Figure 2. Section containing amygdala subregions: the central nucleus
(CeA), basolateral nucleus (BLA), and medial nucleus (MeA) (Paxinos &
Watson, 1998).
2.5. Statistics
Expression of Arc and BDNF were compared between groups using a 2 (sex: female,
male) x 2 (adolescent housing: group, isolated) x 2 (acute social exposure: social exposure, no
social exposure) factorial ANOVA for each brain subregion (AC, PL, IL, MC, VO, CeA, BLA,
21


MeA) and for the entire mPFC (stereology counts). Tukeys post hoc were used to examine any
main effects and interactions that were found. The groups are as follows:
Male Female
Group housed Isolation reared Group housed Isolation reared
social exposure n = 8 n = 8 n = 8 n = 8
no social exposure n = 8 n = 8 n = 8 n = 8
All 64 animals were used for the Arc AC, PL, IL, MC, VO, and mPFC stereology
analyses. Four animals were excluded from Arc amygdala subregion analyses and six from the
BDNF PFC subregion and mPFC stereology analyses due to damage to tissue during
immunohistochemistry processing. Overall social interaction, pinning, chasing, and aggressive
grooming were examined for all rats that were exposed to a novel rat using a 2 (sex: female,
male) x 2 (adolescent housing: group, isolated) factorial ANOVA with Tukeys post hoc to follow
up on main effects. All animals in the social exposure groups (n = 32) were including in behavior
analyses. Correlations were conducted using Pearsons rto examine relationships between
behavior and Arc and BDNF expression. All statistical analyses were conducted using PASW
Statistics 18.0.
22


3. Results
3.1. Arc
3.1.1. Medial Prefrontal Cortex Stereo logy
Social exposure produced large increases in total estimates of Arc positive cells in the
mPFC in male and female group housed rats, and to a lesser extent in female isolated rats (Fig.
3). There was a significant housing X social exposure interaction, F (1,56) = 8.70, p <.01, for
total estimates of Arc positive cells in the mPFC. Post hoc tests revealed that exposure to a
novel conspecific produces significant increases in Arc protein expression in the mPFC in
female and male group housed rats, p <.001, and in female isolated rats, p < .05, but not in
male isolated rats.
io
+
UJ
j/>
o
o
+
o
<
Group Isolation Group Isolation
female male
no social
social
Figure 3. Exposure to a novel conspecific produced robust increases in Arc protein in
the mPFC in female and male GH rats, p < .001, and to a lesser extent in female
isolates, p < .05.
3.1.2. Prefrontal Cortex Density Counts
Exposure to a novel conspecific produced increased Arc activation in subregions of the
PFC, primarily in group housed rats (Fig. 4). There was a significant housing X social exposure
interaction in the AC, F (1,56) = 5.66, p = .02. Post hoc tests revealed that exposure to a novel
conspecific produced significant increases in Arc in the AC in female, p < .001, and male,
p = .001, group housed rats, but not in female or male isolated rats. There was also a strong
trend for a significant difference between female group housed and female isolate rats, p = .051.
23


There was a significant housing X social exposure interaction in the PL, F (1,56) = 12.62, p =
.001. Post hoc tests revealed that exposure to a novel conspecific produced significant
increases in Arc in the PL in female, p = .01, and male, p < .001, group housed rats, but not in
female or male isolated rats. In the IL, there was a significant sex X housing X social exposure
interaction, F (1,56) = 6.02, p = .02. Post hoc tests revealed that exposure to a novel conspecific
produced significant increases in Arc in the IL in male, p < .001, group housed rats, but not in
female group housed rats or male or female isolated rats. In the MC, there was a significant
housing X social exposure interaction, F (1,56) = 4.83, p = .03. Post hoc tests revealed that
exposure to a novel conspecific produced significant increases in Arc in the MC in female, p <
.01, and male, p = .02, group housed rats, but not in male or female isolated rats. In the VO,
there was a significant housing X social exposure interaction, F (1,56) = 9.52, p < .01. Post hoc
tests revealed that exposure to a novel conspecific produced significant increases in Arc in the
VO in female, p < .001, and male, p < .001, group housed rats, and to a lesser extent in male, p
< .01, and female, p < .05, isolated rats.
3.1.3. Amygdala Density Counts
Exposure to a novel conspecific produced increased Arc activation in subregions of the
amygdala, particularly in group housed rats (Fig. 5). Factorial ANOVA revealed a significant
housing X social exposure interaction in the CeA, F (1,52) = 4.55, p < .05. Post hoc tests
revealed that exposure to a novel conspecific produced significant increases in Arc protein in
the CeA in female, p < .001, and male, p < .01, group housed rats, and in male isolated rats, p <
.05, but not in female isolated rats. There was also a significant housing X social exposure
interaction in the BLA, F (1,52) = 8.39, p < .01. Post hoc tests revealed that exposure to a novel
conspecific produced significant increases in Arc in the BLA in female, p < .01, and male,
p <.01, group housed rats, but not in female or male isolated rats. In the MeA, there was a
significant main effect of social exposure, F (1,52) = 13.25, p < .01; rats exposed to a novel
conspecific had increased Arc activation compared to non-social exposure controls.
24


A. Anterior Cingulate
E
E
O)
o
+
o
V-
<
Group Isolation Group Isolation
female male
no social
social
D. Motor Cortex
Group Isolation Group Isolation
female male
B. Prelimbic
no social
social
E. Ventral Orbital Cortex
CN < 400-
E £ 300
(/>
o < 100 0
Group Isolation Group Isolation
no social
social
female male
C. Infralimbic
no social
social
Figure 4. Exposure to a novel conspecific produced robust increases in Arc in the AC in female and
male GH rats, p < .001, but not in isolates. There was also a strong trend for a significant difference
between female GH and female isolate rats, p = .051, (A). Social exposure produced large increases
in Arc in the PL in female, p < .01 and male, p < .001, GH rats but not in isolates. Arc activation in the
PL also differed between male GH and male isolate rats, p < .01, (B). Social exposure produced a
robust increase in Arc in the IL only in male GH rats, p < .001. Male and female GH rats differed in
Arc in the IL, p < .05, (C). Exposure to a novel conspecific produced increases in Arc in female, p < .
01, and male, p < .05, GH rats in the MC, but not in isolates, (D). Social exposure produced robust
increases in Arc in the VO in female and male GH rats, p < .001, and to a lesser extent in female, p
< .05, and male, p < .01, isolates, (E).
25


Arc + cells (mmA2) Arc + cells (mmA2)
A. Central Nucleus of the Amygdala
* *
Group Isolation Group Isolation
female male
B. Basolateral Nucleus of the Amygdala
Group Isolation Group Isolation
female male
no social
social
C. Medial Nucleus of the Amygdala
20
CM
<
E
E
15
J2
flj
o
+
o
<
10
5
0
| | no social
Group Isolation Group Isolation
female male
Figure 5. Exposure to a novel conspecific produced large increases in female, p < .001, and male, p < .01 GH rats
and to a lesser extent in male isolates, p < .05 in the CeA, (A). Social exposure produced large increases in Arc in
female and male GH rats, p < .01, but not in isolates in the BLA, (B). Social exposure produced a significant
increase in Arc in all groups in the MeA, p < .01, (C).
26


3.2. BDNF
3.2.1. Medial Prefrontal Cortex Stereology
Female rats had overall higher levels of BDNF protein in the mPFC than male rats (Fig.
6). There was a significant main effect of sex, F (1,49) = 6.41, p < .05. There was also a strong
trend for a main effect of social exposure, F (1,49) = 3.24, p = .08.
no social
social
Figure 6. Females had more BDNF in the mPFC than any other group, p < .05. There
was also a strong trend for an effect of social exposure, p = .08.
3.2.2. Prefrontal Cortex Density Counts
Exposure to a novel conspecific produced increased BDNF activation in some
subregions of the mPFC, but not in the AC, MC or VO (Fig. 7). There was a significant sex X
housing interaction in the AC, F (1,50) = 6.53, p < .05. Post hoc tests collapsed across social
exposure revealed that female group housed rats had more BDNF activation than any other
group, p < .05. In the PL, there was a significant main effect of social exposure, F (1,50) = 6.61,
p < .05. There was also a strong trend for a main effect of sex in the PL, F (1,50) = 3.62, p =
.06. In the IL, there was a significant main effect of social exposure, F (1,50) = 5.86, p = .04.
There were no group differences in BDNF activation in the MC or VO.
27


BDNF + cells (mmA2) O BDNF + cells (mmA2) OT BDNF + cells (mmA2)
D. Motor Cortex
A. Anterior Cingulate
*
female male
CM
<
E
E
(A
*53
u
+
Q
m
Group Isolation Group Isolation
female male
Prelimbic
E. Ventral Orbital Cortex
Infralimbic
200
150
no social
J social
100
50
0
Group Isolation Group Isolation
female male
Figure 7. Female group housed rats had more BDNF in the AC than any other group, p < .
05, (A). Social exposure produced significant increases in BDNF in all groups in the PL, p
< .05, and there was a strong trend for an effect of sex, p = .063, (B). Social exposure
produced an increase in BDNF in the IL in all groups, p = .04, (C). There were no group
differences in BDNF activation in the MC or VO, (D, £).
28


3.3. Behavior during novel social exposure
Behavior during exposure to a novel conspecific differed according to sex and housing
condition (Fig. 8). For overall social interaction, there was a significant main effect of sex, F
(1.28) = 36.25, p < .001, males interacted with a novel conspecific more than females. There
was also a main effect of housing, F (1,28) = 44.34, p < .001, isolated rats interacted more with
a novel conspecific than group housed rats. There was a significant main effect of housing for
aggressive grooming, F (1,28) = 42.43, p < .001, isolated rats engaged in more aggressive
grooming than group housed rats. For chasing, there was a significant main effect of housing, F
(1.28) = 22.31, p < .001, as well as a strong trend for a sex X housing interaction, F (1,28) =
4.15, p = .051. Post hoc tests revealed that male isolated rats chased a novel conspecific more
than group housed rats, p < .01, but this increase in chasing behavior was not present in
females. Additionally, male isolated rats chased a novel conspecific more than did female
isolates, p = .04. There was a significant sex X housing interaction for pinning, F (1,28) = 6.99, p
< .05. Post hoc tests revealed that male isolated rats pinned a novel conspecific more than
group housed rats, p < .01 but this increase in pinning behavior was not present in females.
Additionally, male isolated rats pinned a novel conspecific more than female isolates, p < .001.
3.4. Relationships between behavior and Arc and BDNF expression
There were no significant correlations found between total estimates of Arc-positive cells
in the mPFC and any of the behaviors measured for males or females, though there was a trend
for a negative relationship between total mPFC Arc and aggressive grooming for males, r(16) =
-.466, p = .069. Females had a significant negative correlation between BDNF-positive cells in
the mPFC and overall social interaction, r(16) = -.57, p = .021.
29


Chase (seconds) (") Social Interaction (seconds)
A.
B.
female
male
Female Male
150
100
50
Group
Iso
D.
Group Iso
Figure 8. Isolated rats interacted with a novel conspecific more than GH rats, p < .001. Males also interacted with a
novel conspecific more than female rats, p < .001, (A). Isolated rats exhibited more aggressive grooming toward a
novel conspecific than GH rats, p < .001, (S). Male Isolates chased a novel conspecific more than male GH rats, p < .
01, or female isolates, p < .05, (C). Male isolates pinned a novel conspecific more than male GH rats, p < .01, or
female isolates, p < .001, (D).
30


4. Discussion
4.1. Arc activation produced by exposure to a novel conspecific
Exposure to a novel conspecific produced robust increases in Arc protein expression in
the PFC and amygdala in group housed animals, but this increase was largely lacking in
isolates. These findings indicate a general lower level of cortical and subcortical Arc activation in
isolation reared rats. This study adds to the existing knowledge of abnormalities produced by
early life adversity.
There are several possible ways that the decrease in Arc activation seen here could be
related to other changes produced by isolation rearing. It could be that group housed and
isolated animals are similar in number and projections of neurons in these brain regions, but
that a morphological or molecular abnormality in isolates results in either mRNA transcription or
protein translation problems, thereby leading to a decrease in the amount of Arc protein.
Another possibility is that isolates differ from group housed animals in connectivity in the brain
regions under study, and so do not have the same patterns of activation in response to
exposure to a novel conspecific as group housed rats (e.g. this stimulus does not recruit the
PFC and amygdala in isolates the way it does in GH rats and instead is activating brain regions
not examined here). A third possible explanation is that isolates have fewer neurons in the PFC
and amygdala compared to GH rats, making it so that even if their levels of activation are
proportionally the same, the numbers of Arc positive cells in the brains of isolates will be lower
since total cell numbers are lower.
It is helpful to look at the current findings along with previous research on isolation
rearing to piece together how Arc may be related to other abnormalities produced by isolation
rearing, and to examine how accurate the three interpretations of the observed decrease in Arc
presented above may be. For example, Bock and colleagues (2008) found that isolation rearing
alters dendritic length and complexity in the AC and orbitofrontal cortex, and Hermes and
colleages (2011) found that isolation rearing suppresses expression of synaptic-associated
31


proteins in the PFC. These findings support the first interpretation that decreased Arc in isolates
may be related to differences in the morphology of their neurons or ability to synthesize new
proteins that are needed to respond to a novel social stimulus. Since Arc is localized at sites of
synaptic activity, and appears to be responsible for local protein synthesis at these sites
(Bramham, 2008), a decrease in Arc may result in fewer or less efficient dendrites. Arc also
plays a role in trafficking AMPA receptors to the postsynaptic density, is known to be
preferentially activated in glutamatergic neurons (Vazdarjanova et al., 2006), and is found in the
postsynaptic density but not in presynaptic terminals or axons (Steward & Worley, 2001). Arc
appears to be partially responsible for the endocytosis of AMPA receptors in the postsynaptic
density, with increased Arc translation being related to increased AMPA endocytosis (Waung et
al., 2008). If the dendrites on cells that would normally express Arc mRNA and protein are not
receiving signals properly, possibly due to improper AMPA receptor trafficking in the
postsynaptic density, there could be a cyclic effect. In this case, a decrease in Arc could result
in a dysfunctional level of AMPA receptors, which could lead to abnormalities in these dendrites.
A change in dendrite morphology and function could then lead to a further reduction in Arc
activation. In order to examine how these factors may be related, Arc activation could be
measured along with dendritic spine length, morphology, and levels of AMPA receptors present
over several time points during the isolation period (e.g. at 2 weeks, 3 weeks, and 4 weeks).
This would show if changes in Arc expression are ongoing and progressive and accompanied
by changes in dendritic spine morphology and changes in levels of AMPA receptors in the
postsynaptic density.
The second interpretation of the observed decrease in Arc in the PFC and amygdala in
isolates is that the abnormality could be related to connectivity between these brain regions and
other brain regions involved in a social stress response. A novel social exposure produces
robust activation of Arc in the PFC and amygdala in group housed animals. Presumably this
activation occurs due to the roles of these brain regions in social activity, decision-making, and
32


emotional responses, all of which are likely involved in a response to a novel social exposure or
stressor. If a novel social exposure or stressor recruits different brain regions in isolates due to
abnormalities in connectivity during development as a result of no exposure to play, perhaps
there are other brain regions being activated instead of the PFC and amygdala in these rats.
This interpretation could be followed up on by looking at other brain regions such as the
hippocampus to see if there is a stronger learning and memory component than executive
function and emotional component of this stimulus for isolates. Another brain region of interest
is the hypothalamus to see if there may be a stronger HPA axis activation component for
isolates rather than a cortical response. Alternatively, fMRI could be used to measure activity in
various brain regions immediately following social exposure.
Finally, it is possible that morphology and connectivity of neurons in the PFC and
amygdala are normal in isolates, but that there are overall fewer neurons in these brain regions
in isolation reared rats. Day-Wilson and colleages (2006) found decreased cortical volume in
isolation reared rats, but no changes in neuron number. Cortical volume and neuron number
should be examined in male and female rats, as this group only used male rats. If it is the case
that isolation reared males and females do not differ from group reared rats, but do differ in
cortical volume, this could point to additional abnormalities in neuron morphology. There may be
decreases in white matter, indicating abnormal axon growth or connectivity. Additionally, fewer
or smaller dendrites or soma may contribute to lower cortical volume in isolates.
The limitations of this study make it difficult to determine what kinds of cellular or
molecular mechanisms are causing a decrease in Arc activation and what downstream effects
this decrease may have. Therefore, the best interpretation of the data from this study is that
isolates have lower IEG activation, indicating lower neuronal activation (hypofunction of the
mPFC and amygdala). These findings will need to be built upon to examine what this means for
brain function, e.g. what kinds of cells have decreases in activation and how cell signaling may
be disrupted. Ongoing projects include examining changes in dopamine and serotonin signaling
33


in isolation reared rats using in vivo microdialysis and examining other synaptic associated
proteins such as PSD95 to more fully clarify how neuron function is altered by early life
adversity. Future research to examine more precisely the role isolation rearing plays in
alterations in brain plasticity and physiological stress responses could include measuring
corticosterone responses, double labeling for Arc with neurotransmitters such as serotonin,
dopamine, GABA, or glutamate, and looking at Arc mRNA along with Arc protein to determine if
the abnormalities in isolates lie in transcription or translation, or both.
4.1.1. Arc activation varies by sex and subregion of the PFC and amygdala
The data presented here indicate that isolation reared rats in general have lower levels
of Arc activation in the PFC and amygdala produced by exposure to a novel conspecific. Arc
activation in some subregions also appears to be influenced by sex. Sex-specific patterns of
activation indicate that Arc activation may vary in males and females and depending on brain
region. The first sex-specific pattern of activation of notice is in the mPFC. There appears to be
a dorsal-ventral gradient in Arc expression for females, but not for males. The increase in Arc
activation produced by social exposure in female group housed rats is more pronounced in the
dorsal mPFC (AC), then becomes less pronounced in the more ventral regions (PL and IL).
Males have proportional levels of Arc activation produced by social exposure throughout the
mPFC. In fact, these differing patterns of activation are so pronounced that male group housed
rats have significantly higher levels of Arc than female group housed rats in the IL (Fig. 4). The
ventral portion of the mPFC is also the only cortical region where female group housed rats did
not have a significant increase in Arc produced by social exposure. Likewise, in the CeA, female
group housed rats did not have an increase in Arc produced by social exposure, while male
group housed rats did. In these two brain regions (IL and CeA), female group housed rats
resemble isolation reared females in that social exposure did not produce significant increases
in Arc activation, though in all other subregions they do not show similar patterns of activation
(or lack of activation) to isolates.
34


The MeA was the only subregion that did not have an effect of housing condition or sex;
male and female group housed and isolation rats all had an increase in Arc produced by novel
social exposure. It is possible that this brain region is not affected by isolation rearing the way
that the other regions examined are. Additionally, cell counts in this region were much lower
than in other regions, and may have been too low to reveal group differences.
4.2. BDNF activation in the PFC
In contrast to the general effect of isolation rearing on Arc activation produced by social
exposure in the PFC and amygdala, BDNF expression appears to be affected less by housing
condition than by sex. Females were found to have overall higher levels of BDNF in the mPFC.
When examined by subregion, it is seen that female group housed rats have more BDNF
expression in the AC than any other groups, (Fig. 7), and that BDNF expression in this
subregion is not affected by social exposure, e.g. rats who were exposed to a novel rat did not
have different levels of BDNF in the AC than home cage control rats. In the PL and IL, however,
BDNF activation seems to only be affected by social exposure, though in the PL there is also a
strong trend for a main effect of sex. This pattern of activation is interesting, as BDNF appears
to be essentially acting like an IEG in the more ventral mPFC subregions, as indicated by
increased activation produced by social exposure. In the AC, however, BDNF activation
appears to be reflecting a process that is not stimulus dependent, as social exposure does not
seem to increase BDNF activation in this brain region. BDNF has been found to act as an IEG in
some circumstances, depending on which of its promotor regions are bound and the
subsequent distinct transcripts that are made (Lauterborn 1996). It is possible that certain BDNF
transcripts are preferentially transcribed in some brain regions and that this could be due to the
need for a fast and transient upregulation of BDNF as opposed to more sustained BDNF protein
levels in other brain regions.
35


Bland and colleagues (2005) found higher levels of BDNF mRNA in their control
unstressed females compared to control males. It may be that females that have experienced
normal social exposure during development have higher baseline BDNF levels. Hill and
colleagues (2011) indicate that BDNF-TrkB signaling during adolescence is modulated by sex
steroid hormones differently in male and female rats. Sohrabji and colleagues (1995) suggested
that estrogen might increase the availability of neurotrophins in the cortex, thereby regulating
BDNF transcription. This may partially explain why females seem to have constitutively higher
levels of BDNF in the PFC. This could be examined by measuring BDNF expression during
different stages of estrous could be measured to see if levels very according to levels of
estrogen.
4.3. Isolation-reared rats exhibit increased aggressive and nonaggressive behavior
Male and female isolation reared rats engaged in more overall social interaction, as well
as more aggressive grooming with a novel conspecific compared to group housed males and
females. Male rats as a whole, however, engaged in more social interaction and aggressive
grooming compared to females as a whole. Male isolates showed higher levels of pinning and
chasing than male group housed rats, but female isolates were no different in these behaviors
than their group housed counterparts. The observed increase in social interaction in males is
similar to that found by other groups (Ferdman et al., 2007; Meng et al., 2010), but the increase
in social interaction for female isolates differed from Ferdman and colleagues and from others
(Hermes, 2011) who have found that female isolates spend less time interacting with a novel
rat. These inconsistencies may be due to several factors. Ferdman et al. (2007) used Wistar
rats and performed social interaction testing far into adulthood, at P98. Additionally, the social
exposure test used in that study was only 3 minutes long, compared to 10 minutes of social
exposure used here. Likewise, the social interaction test used by Hermes et al. (2011) only
exposed the rats to a novel rat for 5 minutes, and rats were exposed to a rat from a similar
36


housing condition (e.g. isolates paired with isolates). Any of these factors could potentially affect
the observed behavior of the experimental rat. The social interaction test used here, where the
stimulus rat for both the group housed and isolation reared animals is a group housed or
normal rat, is a good model for examining what happens with a rat that has undergone chronic
early life adversity is faced with a normal social situation. Additionally, a longer social exposure
allows time for a full spectrum of behaviors to be observed, as the first few minutes may be
spent habituating to the situation and exploring the new cage.
One important point to keep in mind is that isolation rearing has consistently been found
to elevate locomotor activity, though this effect varies by strain and seems to be less
pronounced in Sprague-Dawley rats (Fone & Porkess, 2008). The increase in social interaction
may be related to an overall increase in locomotor activity. Future studies should measure
locomotor activity as well as the behaviors recorded here to examine whether males and
females have similar increases in locomotor activity, and whether levels of locomotor activity are
associated with social interaction.
4.4. Relationships between Arc and BDNF activation and behavior
The only relationship observed between gene activation and behavior was between
BDNF-positive cells in the mPFC and overall social interaction in females, where higher BDNF
expression was associated with lower levels of social interaction. This is an interesting finding in
light of the fact that females have higher levels of BDNF and interact less with a novel
conspecific. This suggests a possible role of BDNF in behavioral differences in male and female
rats. Isolation rearing has been found to decrease activation of lEGs such as Arc while
increasing exploratory behavior (Levine 2007) while other groups have found decreases in IEG
expression alongside deficits in social behavior and decreased exploratory behavior (Hermes,
2011). Despite the lack of statistically significant correlations between behavior and Arc in the
present study, the observed decrease in Arc activation and increased aggressive and non-
37


aggressive behavior in isolates seems to fall in line with the findings of Levine et al. (2007). This
group postulated that the decrease in gene activation was likely due to an adaptation to the
long-term effects of the isolation procedure and not due to an interference of the behavior test
with gene activation. This explanation seems plausible, as lEGs are activation upon stimulation
and are associated with exploratory behavior (Pace et al., 2005), and increased activation was
seen with the group housed rats in this study. Isolation reared rats appear to have abnormalities
in both social behavior and mPFC and amygdala activation. One possible reason for this is that
isolates are lacking the inhibition needed to suppress aggressive behavior or lacking the mPFC
and amygdala activation needed to modulate proper social behavior. Social responses are
complex and require a finely tuned level of input from many brain regions in order to maintain a
proper balance of inhibition and activation. An increase in aggressive and non-aggressive
behavior may be related to a decrease in I EG activation in a non-linear manner.
4.5. Is novel social exposure stressful, rewarding, or both for isolation reared rats?
Ongoing research in the lab is examining how rewarding social exposure is to group
housed and isolated rats. While a novel social exposure is thought to be somewhat stressful, it
may be that this experience is also rewarding, and therefore not reflective of a negative
stressor. Preliminary data suggest that very low levels of novel social exposure is enough to
induce conditioned place preference (CPP) in male group housed and isolated rats, but not in
females. Interestingly, when novel social exposure is paired with a single cocaine injection (2
mg/kg), male group housed and isolated rats again develop CPP, and so do female isolated rats
but not female group housed rats (Dayton et al., 2011). These data indicate that the novelty
stressor of social exposure might be a rewarding experience for rats, and that this may differ
according to sex and housing condition. This relates to the increases in social interaction in
isolates seen here and highlights the importance of caution in interpreting stress responses.
38


Desirable events may be experienced as stressful, but the behavioral and biochemical
responses to such events may differ greatly from responses to undesirable events.
It is clear that early life adversity can have long lasting behavioral and neurobiological
effects. As more data are gathered about how behavior and the brain change in response to
early life adversity, it will become possible to tailor treatments for people suffering from a variety
of mental illnesses related to abnormalities in stress responses. An individual who has
experienced chronic stress early in life may have different treatment needs than someone with a
different background; even if symptoms or diagnoses are similar. Males and females differ in
their behavioral and physiological stress responses, indicating that different treatments may be
called for. Additionally, the subjective experience of stress may differ according to ones
background and sex. This is an important consideration to keep in mind when addressing an
individuals behavioral and emotional stress responses. Further examination of the outcomes of
isolation rearing, and how those outcomes interact with social functioning in males and females,
will help to untangle the complex problem of behavioral and neurobiological deficits related to
chronic stress early in life.
39


REFERENCES
Adolphs, R. (2010). What does the amygdala contribute to social cognition? Ann N Y Acad Sci,
1191(1), 42-61.
Altemus, M. (2006). Sex differences in depression and anxiety disorders: Potential biological
determinants. Hormones and Behavior, 50, 534-538.
Bell, H. C., Pellis, S. M., & Kolb, B. (2010). Juvenile play experience and the development of the
orbitofrontal and medial prefrontal cortices. Behavioural Brain Research, 207, 7-13.
Bland, S. T., Beckley, J. T., Fischer, E. K., Imonega, O. I., Ortiz, N. C., Watkins, L. R., & Maier,
S. F. (2009, October). Isolation rearing and restraint stress produce sex-dependent
changes in medial prefrontal cortex immediate early gene expression. Poster session
presented at the Society for Neuroscience annual meeting, Chicago, IL.
Bland, S. T., Schmid, M. J., Der-Avakian, A., Watkins, L. R., Spencer, R. L., & Maier, S. F.
(2005). Expression of c-fos and BDNF mRNA in subregions of the prefrontal cortex of
male and female rats after acute uncontrollable stress. Brain Research, 1051, 90-99.
Bock, J., Murmu, R. P., Ferdman, N., & Braun, K. (2008). Refinement of dendritic and synaptic
networks in the rodent anterior cingulate and orbitofrontal cortex: critical impact of early
and late social experience. Developmental Neurobiology, 68(5), 685-695.
Bramham, C. R., Worley, P. F., Moore, M. J., & Guzowski, J. F. (2008). The immediate early
gene Arc/Arg3.1: Regulation, mechanisms, and function. The Journal of Neuroscience,
28(46), 11760-11767.
Branchi, I., Francia, N., & Alieva, E. (2004). Epigenetic control of neurobehavioral plasticity: The
role of neurotrophins. Behavioural Pharmacology, 15, 353-362.
Carlson, N. R. (2008). Foundations of physiological psychology. Boston, MA: Pearson
Education Inc.
Cook, S. C. & Wellman, C. L. (2004). Chronic stress alters dendritic morphology in rat medial
prefrontal cortex. Journal of Neurobiology, 60, 236-248.
40


Cunha, C., Brambilla, R., Thomas, K. L. (2010). A simple role for BDNF in learning and
memory? Frontiers in Molecular Neuroscience, 3(1), 1-14.
Goodell, D., Wall, V., Grotewold, S., & Bland, S.T. (2011). Sex-dependent effects of adolescent
social deprivation on combined social cue/drug conditioned place preference. Poster
presented at the Society for Neuroscience annual meeting, Washington, D.C.
Day-Wilson, K. M., Jones, D. N. C., Southam, E., Cilia, J., &Totterdell, S. (2006). Medial
prefrontal cortex volume loss in rats with isolation rearing-induced deficits in prepulse
inhibition of acoustic startle. Neuroscience, 141, 1113-1121.
Diamond, D. M., Campbell, A., Park, C. R., & Vouimba, R. (2004). Preclinical research on
stress, memory, and the brain in the development of pharmacotherapy for depression.
European Neuropsychopharmacology, 14, 491-495.
Douglas, L. A., Varlinskaya, E. I., Spear, L. P. (2004). Rewarding properties of social
interactions in adolescent and adult male and female rats: Impact of social versus isolate
housing of subjects and partners. Developmental Psychobiology, 45, 153-162.
Ferdman, N., Murmu, R. P., Bock, J., Braun, K., & Leshem, M. (2007). Weaning age, social
isolation, and gender, interact to determine adult explorative and social behavior, and
dendritic and spine morphology in prefrontal cortex of rats. Behavioural Brain Research,
180, 174-182.
File, S. E. & Seth, P. (2003). A review of 25 years of the social interaction test. European
Journal of Pharmacology, 463, 35-53.
Flavell, S. W. & Greenberg. M. E. (2008). Signaling mechanisms linking neuronal activity to
gene expression and plasticity of the nervous system. Annu Rev Neurosci, 31, 563-590.
Fone, K. C. F. & Porkess, M. V. (2008). Behavioural and neurochemical effects of post-weaning
social isolation in rodents-Relevance to developmental neuropsychiatric disorders.
Neuroscience and Biobehavioral Reviews, 32, 1087-1102.
41


Ehlert, U., Gaab, J. & Heinrichs, M. (2001). Psychoneuroendocrinological contributions to the
etiology of depression, posttraumatic stress disorder, and stress-related bodily disorders:
The role of the hypothalamus-pituitary-adrenal axis. Biological Psychology, 57, 141-152.
Everhart, K. & Emde, R. N. (2006). Perspectives on stress and self-regulatory processes. In H.
E. Fitzgerald, b. M. Lester, & B. Zuckerman (Eds.), The crisis in youth mental health:
Critical issues and effective programs, Vol 1: Childhood disorders (pp. 1-24). Westport,
CT: Praeger Publishers/Greenwood Publishing Group.
Figueieredo, H. F., Bruestle, A., Bodie, B., Dolgas, C. M., Herman, J. P. The medial prefrontal
cortex differentially regulated stress-induced c-fos expression in the forebrain depending
on type of stressor. European Journal of Neuroscience, 18, 2357-2364.
Gabbott, P. L. A., Warner, T. A., Jays, P. R. L., Salway, P. & Busby, S. J. (2005). Prefrontal
cortex in the rat: Projections to subcortical autonomic, motor, and limbic centers. The
Journal of Comparative Neurology, 492, 145-177.
Gallucci, W. T., Baum, A., Laue, L., Rabin, D. S., Chrousos, G. P., Gold, P. W., Kling, M. A.
(1993). Sex differences in sensitivity of the hypothalamic-pituitary-adrenal axis. Health
Psychology, 12(5), 420-425.
Goodell, D. J., Wall, V., Grotewold, S., & Bland, S. T. (2011). Sex-dependent effects of
adolescent social deprivation on combined social cue/drug conditioned place preference.
Poster presented at the Society for Neuroscience Annual Meeting, abstract #263.04.
Greenberg, M. E., Xu, B., Lu, B., & Hempstead, B. L. (2009). New insights in the biology of
BDNF synthesis and release: implications in CNS function. Journal of Neuroscience,
29(41), 12764-12767.
Hall, F. S. (1998) Social deprivation of neonatal, adolescent, and adult rats has distinct
neurochemical and behavioral consequences. Critical Reviews in Neurobiology, 12, 129-
162.
42


Hammen, C., Brennan, P. A., Keenan-Miller, D., Hazel, N. A., & Najman, J. M. (2010). Chronic
and acute stress, gender, and serotonin transporter gene-environment interactions
predicting depression symptoms in youth. Journal of Child Psychology and Psychiatry,
51(2), 180-187.
Harro, J. & Oreland, L. (2001). Depression as a spreading adjustment disorder of
monoaminergic neurons: A case for primary implication of the locus coeruleus. Brain
Research Reviews, 38, 79-128.
Hashimoto, K., Shimizu, E., & lyo, M. (2004). Critical role of brain-derived neurotrophic factor in
mood disorders. Brain Research Reviews, 45, 104-114.
Herman, J. P., Prewitt, C. M., & Cullinan, W. E. (1996). Neuronal circuit regulation of the
hypothalamic-pituitary-adrenocortical stress axis. Critical Reviews in Neurobiology, 10(3-
4), 371-394.
Hermes, G., Li, N., Duman, C., & Duman, R. (2011). Post-weaning chronic social isolation
produces profound behavioral dysregulation with decreases in prefrontal cortex synaptic-
associated protein expression in female rats. Physiology and Behavior, 104(2), 354-359.
Hill, R. A., Wu, Y. C., Kwek, P., & van den Buuse, M. (2011). Modulatory effects of sex steroid
hormone on BDNF-TrkB expression during adolescent development in C57BI/6 mice.
Journal of Neuroendocrinology, accepted article.
Hoi, T., Van den Berg, C. L., Van Ree, J. M., & Spruijt, B. M. (1999). Isolation during the play
period in infancy decreases adult social interactions in rats. Behavioural Brain Research,
100, 91-97.
Holmes, A. & Wellman, C. L. (2009). Stress-induced prefrontal reorganization and executive
dysfunction in rodents. Neuroscience and Biobehavioral Reviews, 33, 773-783.
Joels, M., Karst, H., Alfarez, D., Heine, V. M., Qin, Y., van Riel, E., Verkuyl, M., Lucassen, P. J.,
& Krugers, H. J. (2004). Effects of chronic stress on structure and cell function in rat
hippocampus and hypothalamus. Stress, 7(4), 221-231.
43


Kovacs, K. J. (2008). Measurement of immediate-early gene activation- c-fos and beyond.
Journal of Neuroendocrinology, 20, 665-672.
Kozlovsky, N., Matar, M. A., Kaplan, Z., Kotler, M., Zohar, J., & Cohen, H. (2008). The
immediate early gene Arc is associated with behavioral resilience to stress exposure in
an animal model of posttraumatic stress disorder. European Neuropsychopharmacology,
18, 107-116.
Lauterborn, J. C., Rivera, S., Stinis, C. T., Hayes, V. Y., Isackson, P. J., & Gall, C. M. (1996).
Differential effects of protein synthesis inhibition on the activity-dependent expression of
BDNF transcripts: Evidence for immediate-early gene responses from specific
promotors. The Journal of Neuroscience, 16{23), 7428-7436.
Levine, J. B., Youngs, R. M., MacDonaldm M. L., Chu, M., Leeder, A. D., Berthiaume, F., &
Konradi, C. (2007). Isolation rearing and hyperlocomotion are associated with reduced
immediate early gene expression levels in the medial prefrontal cortex. Neuroscience,
145, 42-55.
Lu, Y., Christian, K. & Lu, B. (2008). BDNF: A key regulator for protein synthesis-dependent
LTP and long-term memory? Neurobiology of Learning and Memory, 89, 312-323.
Lyford, G. L., Yamagata, K., Kaufmann, W. E., Barnes, C. A., Sanders, L. K., Copeland, N. G.,
Gilbert, D. J., Jenkins, N. A., Lanahan, A. A. & Worley, P. F. (1995). Arc, a growth factor
and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is
enriched in neuronal dendrites. Neuron, 14, 433-445.
Maggio, N. & Segal, M. (2010). Corticosteroid regulation of synaptic plasticity in the
hippocampus. Scientific World Journal, 10, 462-469.
Maier, S. F. (1984). Learned helplessness and animal models of depression. Prog
Neuropsychopharmacol Biol Psychiatry, 8(3), 435-446.
Malarkey, W. B. & Mills, P. J. (2007). Endocrinology: The active partner in PNI research. Brain,
Behavior, and Immunity, 21, 161-168.
44


Marinelli, M. & Piazza, P. V. (2002). Interaction between glucocorticoid hormones, stress and
psychostimulant drugs. European Journal of Neuroscience, 16(3), 387-394.
McEwen, B. S. (2000). The neurobiology of stress: From serendipity to clinical relevance. Brain
Research, 886, 172-189.
Meng, Q., Li, N., Han, X., Shao, F., & Wang, W. (2010). Peri-adolescence isolation rearing
alters social behavior and nociception in rats. Neuroscience Letters, 480, 25-29.
Monk, C. S. (2008). The development of emotion-related neural circuitry in health and
psychopathology. Development and Psychopathology, 20, 1231-1250.
OLeary, A. (1990). Stress, emotion, and human immune function. Psychological Bulletin,
108(3), 363-382.
Ostrander, M. M., Ulrich-Lai, Y. M., Choi, D. C., Flak, J. N., Richtand, N. M. & Herman, J. P.
(2009). Chronic stress produces enduring decreases in novel stress-evoked c-fos mRNA
expression in discrete brain regions of the rat. Stress, 12(6), 469-477.
Pace, T. W., Gaylord, R., Topczewski, F., Girotti, M., Rubin, B., & Spencer, R. L. Immediate-
early gene induction in hippocampus and cortex as a result of novel experience is not
directly related to the stressfulness of that experience. European Journal of
Neuroscience, 22(7), 1679-1690.
Paxinos, G. & Watson, C. (1998). The rat brain in stereotaxic coordinates, (4th Ed.). San Diego,
CA: Academic Press.
Pinaud, R. (2004). Experience-dependent immediate early gene expression in the adult central
nervous system: Evidence from enriched-environment studies. International Journal of
Neuroscience, 114, 321-333.
Radley, J. J., Sisti, H. M., Hao, J., Rocher, A. B., McCall, T., Hof, P. R., McEwen, B. S., &
Morrison, J. H. (2004). Chronic behavioral stress induces apical dendritic reorganization
in pyramidal neurons of the medial prefrontal cortex. Neuroscience, 125, 1-6.
45


Roceri, M., Hendriks, W., Racagni, G., Ellenbroek, B. A. & Riva, M. A. (2002). Early maternal
deprivation reduces the expression of BDNF and NMDA receptor subunits in rat
hippocampus. Molecular Psychiatry, 1, 609-616.
Romeo, R. D. (2010). Pubertal maturation and programming of the hypothalamic-pituitary-
adrenal reactivity. Frontiers in Neuroendocrinology, 31, 232-240.
Sadock, B. J. & Sadock, V. A. (2007). Kaplan & Sadocks synopsis of psychiatry: Behavioral
sciences/clinical psychiatry. Philadelphia, PA: Lippincott Williams & Wilkins.
Schinder, A. F. & Poo, M. (2000). The neurotrophin hypothesis for synaptic plasticity. Trends in
Neuroscience, 23(12), 639-645.
Sisk, C. L. & Zehr, J. L. (2005). Pubertal hormones organize the adolescent brain and behavior.
Frontiers in Neuroendocrinology, 26(3-4), 163-174.
Smith, M. A., Makino, S., Kvetnansky, R. & Post, R. M. (1995). Effects of stress on neurotrophic
factor expression in the rat brain. Ann N Y Acad Sci, 29, 234-239.
Smith, M. A., Makino, S., Kim, S. Y., & Kvetnansky, R. (1995). Stress induces brain-derived
neurotrophic factor messenger ribonucleic acid in the hypothalamus and pituitary.
Endocrinology, 136(9), 3745-3750.
Spina, M. B., Squinto, S. P., Miller, J., Lindsay, R. M., & Hyman, C. (1992). Brain-derived
neurotrophic factor protects dopamine neurons against 6-hydroxydopamine and N-
methyl-4-phenylpyridinium ion toxicity: involvement of the glutathione system. Journal of
Neurochemistry, 5(1), 99-106.
Stack, A., Carrier, N., Dietz, D., Hollis, F., Sorenson, J., & Kabbaj, M. (2010). Sex differences in
social interaction in rats: Role of the immediate-early gene zif268.
Neuropsychopharmacology, 35, 570-580.
Stauder, A., Thege, B. K., Kovacs, M. E., Balog, P., Williams, V. P., & Williams, R. B. (2010).
Worldwide stress: Different problems, similar solutions? Cultural adaptation and
46


evaluation of a standardized stress management program in Hungary. International
Journal of Behavioral Medicine, 17(1), 25-32.
Steward, O. & Worley, P.F. (2001). Selective targeting of newly synthesized Arc mRNA to active
synapses requires NMDA receptor activation. Neuron, 30, 227-240.
Tafet, G. E. Idoyaga-Vargas, V. P., Abulafia, D. P., Calandria, J. M., Roffman, S. S., Chiovetta,
A., & Shinitzky, M. (2001). Correlation between cortisol level and serotonin uptake in
patients with chronic stress and depression. Cognitive, Affective, and Behavioral
Neuroscience, 1(4), 388-393.
The National Campaign to Prevent Teen Pregnancy. (2005). The Adolescent Brain: A Work in
Progress. [Brochure], Washington, DC: Weinberger, D. R., Elvevag, B., & Giedd, J. N.
Turrigiano, G. G., & Nelson, S. B. (2004). Homeostatic plasticity in the developing nervous
system. Nature Reviews Neuroscience, 5, 97-107.
Vazdarjanova, A., Ramirez-Amaya, V., Insel, N., Plummer, T. K., Rosi, S., Chowdhury, S.,
Mikhael, D., Worley, P. F., Guzowski, J. F., & Barnes, C.A. (2006). Spatial exploration
induces ARC, a plasticity-related immediate-early gene, only in calcium/calmodulin-
dependent protein kinase ll-positive principal excitatory and inhibitory neurons of the rat
forebrain. J. Comp. Neurol., 498, 317-329.
Vermetten, E. & Bremner, J. D. (2002). Circuits and systems in stress. I. Preclinical studies.
Depression and Anxiety, 15, 126-147.
Walker, E. F. (2002). Adolescent neurodevelopment and psychopathology. Current directions in
psychological science, 11(1), 24-28.
Waung, M. W., Pfeiffer, B. W., Nosyreva, E. D., Ronesi, J. A., & Huber, K. M. 2008. Rapid
translation of Arc/Arg3.1 selectively mediates mGluR dependent LTD through persistent
increases in AMPAR endocytosis rate. Neuron, 59(1), 84-97.
47


Wright, S. L., Langenecker, S. A., Deldin, P. J., Rapport, L. J., Nielson, K. A., Kade, A. M., Own,
L. S., Akil, H., Young, E. A., Zubieta, J. K. (2009). Gender-specific disruptions in emotion
processing in younger adults with depression. Depression and Anxiety, 26, 182-189.
Young, E.A., Korszun, A., Figueiredo, H. F., Banks-Solomon, M., & Herman, J. P. (2008). Sex
differences in HPA axis regulation. In J. B. Becker, K. J. Berkley, N. Geary, E. Hampson,
J. P. Herman, & E. A. Young (Eds.) Sex Differences in the Brain (95-105). New York,
NY: Oxford University Press, Inc.
48


Full Text

PAGE 1

ISOLATION REARING ALTERS BEHAVIOR AND EXPRESSION OF BRAIN DERIVED NEUROTROPHIC FACTOR AND THE IMMEDIATE EARLY GENE ARC IN THE PREFRONTAL CORTEX AND AMYGDALA OF MALE AND FEMALE RATS by Vanessa L. Wall B.A., University of Colorado, Colorado Springs, 2008 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Arts Clinical Psychology M.A. Program 2012

PAGE 2

!! This thesis for the Master of Arts degree by Vanessa L. Wall has been approved for the Clinical Psychology M.A. Program by Dr. Sondra T. Bland, Chair Dr. Richard Allen Dr. Jim Grigsby Date __ 4/26/ 2012 _____

PAGE 3

" !!! Wall, Vanessa L. (M.A., Clinical Psychology M.A. Program ) Isolation Rearing Al ters Behavior and Expression of Brain derived Neurotrophic Factor and the Immediate Early Gene Arc in the Prefrontal Cortex and Amygdala of Male and Female Rats Thesis directed by Dr. Sondra T. Bland ABSTRACT Early life adversity has been identified as a risk factor for the development of psychopathology later in life. One way that adverse experiences early in life may have deleterious effects on an individual is by producing brain and behavior abnormalities in stress responsivity, particularly in regard to stressors involving other people and social interactions. Abnormalities in stress responses and social interactions in individuals with a history of early life adversity may be partially explained by changes in brain regions related to stress regulati on. The medial prefrontal cortex (mPFC) and amygdala have been implicated in stress regulation and emotional and cognitive functioning. The present study examined the effects of an animal model of early life adversity, isolation rearing, on aggressive and non aggressive behavior and activation of the protein products of the activity dependent genes, Arc and BDNF in the PFC and amygdala produced by exposure to a subsequent mild social stressor ( e.g. exposure to a novel rat). Relationships between b ehavior du ring social exposure and gene ac tivation were also examined Exposure to a novel, same sex rat (conspecific) produced signif icant increases in Arc in group housed animals, but this increase was blunted or absent in isolates. Social exposure produced small increases in BDNF activation, but this was not dependent on housing condition. Differences in patterns of activation, as well as in relationships between behavior and gene activation, were observed in a sex and subregion dependent manner. The form and content of this abstract are approved. I recommend its publication. Approved: Sondra T. Bland

PAGE 4

" !# TABLE OF CONTENTS Chapter 1. Introduction ................................ ................................ ................................ ................... 1 1.1. Consequences of stress and a dversity in humans ................................ .................... 2 1.1.1. Physiological effects of stress ................................ ................................ .......... 3 1.1.2. Chronic stress, social support, and psychopathology ................................ ...... 4 1.1.3. Chronic stress experiences in adolescence ................................ ..................... 5 1.2. Isolation rearing in rats (a model of early life adversity) ................................ ............. 6 1.3. Soc ial behavior following chronic stress ................................ ................................ .... 7 1.4. Neurobiological correlates of behavioral stress responses and implications for brain plasticity ................................ ................................ ................................ ............................ 9 1.4.1. Brain derived neurotrophic factor (BDNF) ................................ ....................... 9 1.4.2. Activity regulated cytoskeleton associated protein (Arc) ............................... 11 1.5. Sex Differences in Stress Responses and Psychopathology ................................ .. 13 1.6. Brain regions involved in stress ................................ ................................ ............... 14 1.6.1. Medial prefrontal cortex (mPFC) ................................ ................................ .... 14 1.6.2. Amygdala ................................ ................................ ................................ ....... 15 1.7. Rationale ................................ ................................ ................................ .................. 16 1.8. Purpose and research questions ................................ ................................ ............. 16 1.9. Predictions ................................ ................................ ................................ ............... 17 2. Method s ................................ ................................ ................................ ...................... 19 2.1. Animals ................................ ................................ ................................ .................... 19 2.2. Social exposure procedure ................................ ................................ ...................... 19 2.3. Tissue harvest ................................ ................................ ................................ .......... 20 2.4. Immunohistochemistry ................................ ................................ ............................. 20 2.5. Statistics ................................ ................................ ................................ ................... 21

PAGE 5

" # 3. Results ................................ ................................ ................................ ................................ .. 2 3 3.1. Arc ................................ ................................ ................................ ............................ 23 3.1. 1. Medial prefrontal cortex stereology ................................ ................................ 23 3. 1. 2. Prefrontal cortex density counts ................................ ................................ .... 23 3.1.3. Amygdala density counts ................................ ................................ ............... 24 3.2. BDNF ................................ ................................ ................................ ....................... 27 3.2.1. Medial prefrontal cortex stereology ................................ ................................ 27 3.2.2. Prefrontal cortex density counts ................................ ................................ .... 27 3.3. Behavior during novel social exposure ................................ ................................ .... 29 3.4. Relationships between behavior and Arc and BDNF expression ............................. 29 4. Discussion ................................ ................................ ................................ ............................... 31 4.1. Arc activation produced by exposure to a novel conspecific ................................ .... 31 4.1.1. Arc activation varies by sex and subregion of the PFC and amygdala .......... 34 4.2. BDNF activation in the PFC ................................ ................................ ..................... 35 4.3. Isolation reared rats exhibit increased aggressive and nonaggressive behavior .... 36 4.4. Relationships between Arc and BDNF activation and behavior ............................... 37 4.5. Is novel social exposure stressful, rewarding, or both for isolation reared rats? ..... 38 References ................................ ................................ ................................ .......................... 40

PAGE 6

" #! LIST OF FIGURES Figure 1. Prefrontal cortex subregions ................................ ................................ ....................... 21 2. Amygdala subregions ................................ ................................ ................................ 21 3. Arc stereology ................................ ................................ ................................ ............. 23 4. Arc prefrontal cortex density counts ................................ ................................ ........... 25 5. Arc amygdala density counts ................................ ................................ ...................... 26 6. BDNF stereology ................................ ................................ ................................ ........ 27 7. BDNF prefrontal cortex density counts ................................ ................................ ....... 28 8. Behavior ................................ ................................ ................................ ..................... 30

PAGE 7

" $ 1. Introduction Effective c linical treatment o f psychological disorders is dependent on pre clinical research examining the mechanisms by which psychopathology develops. Early life adversity and stress in humans has been implicated as a factor in the etiology of a number of psychological disorders, inc luding anxiety disorders, depression, schizophrenia, and substance abuse ( Sadock & Sadock, 2007; Holmes & Wellman, 2009; Romeo, 2010 ). Stress can also affect somatic health and immune function (O'Leary, 1990), thereby exacerbating mental health is sues or i n the absence of psychopathology reducing overall quality of life. Isolation rearing in rats is an animal model of early life adversity and stress that produces anxiety like b ehaviors and other abnormalities in behavior, brain morphology, and neurochemist ry ( Hall, 1998; Fone & Porkess 2008 ) This model allows researchers to observe the effects of acute or chronic early life stress ( depending on the d uration of the social isolation) u nder experimental conditions, providing a wealth of information about abn ormalities in behavior and neurobiology produced by such adversity while limiting extraneous variables. Of particular interest is the effect that early life adversity has on social behavior as many psychological stressors as well as protective factors are related to social functioning and support ( Sadock & Sadock, 2007 ) In addition to understanding the behavioral effects and influences on social functioning of early life adversity and stress, changes in neurobiological activation and/or inhibition produced by isolation rearing must be examined Behavioral changes that arise from ongoing early life stress are better understood in the contex t of the neurobiological changes that accompany them. Add ing to the complexity of the effects of early life adversity i t is generally understood that males and females have different behavioral and physiological responses to stress and adversity ( Gallucci, 1993; Young, Korszun, Figueiredo, Banks Solomon, & Herman, 2008; Wright et al., 2009 ) These differences in behavior a nd physiology may partially explain the different prevalence rates for males and females of a number of psychological disorders, such as mood and anxiety di sorders, substance use disorders and PTSD ( Altemus, 2006; Sadock & Sadock ). In order to

PAGE 8

" % effectively meet the different needs of males and females in clinical populations, behavioral and neurobiological sex differences must be thoroughly examined. The present study examined the effects of isolation rearing in male and female rats on social behavior and n eurobi ology, as measured by activation of the protein products of brain derived neurotrophic factor (BDNF) and the immediate early gene (IEG) Arc in the medial prefrontal cortex (mPFC) and amygdala produced by exposure to a novel same sex rat (conspecific) 1.1. Consequences of stress and adversity in humans Stress is an unavoidable part of life, and at moderate levels is adaptive because of its role in helping individuals deal with threatening events (Sadock & Sadock, 2007). Extended periods of stress, however, can cause the body's natural stress response systems to become dysfunctional, thus reducing the adaptive value of such systems. Understanding the ways that stress and adversity are risk factors for the development of physiological and psychologica l problems in humans requires examination of the specific effects of chronic stress. Long periods of stress and adversity can take a toll on an individual's health. Chronic stre ss has been found to have different effects on behavior neuroendocrine respons es, and susceptibility to developing mental illness in humans than acute stress ( Malarkey & Mills, 2007 ; Hammen et al 2010 ). The lasting effects of chronic stress may attenuate behavioral and physical responses to subsequent stressors. The effects of c hronic stress in humans range from the molecular level to the organism level to the population level. Chronic stress is associated with neurochemical abnormalities, specifically dysregulation of serotonin, dopamine, and norepinephrine (Harro & Oreland, 200 1). Individuals experiencing chronic stress often exhibit problems with mood (Tafet et al., 2001), and memory and cognitive function ( Diamond, Campbell, Park, & Vouimba, 2004) as a result of these neurochemical changes. In addition, stress has been associa ted with dysfunction of the hypothalamic pituitary adrenal (HPA) axis (Ostrander et al. 2009). Dysfunction of the HPA axis

PAGE 9

" & can lead to long term increased or decreased reactivity to subsequent stressors (Ehlert, Gaab, & Heinrichs, 2001). Living with chro nic stress can affect social interactions and relationships and has been identified as a risk factor for morbidity at the individual and societal level (Stauder et al. 2010). In an environment where many people are living with chronic stress, the deleteri ous effects reach to the entire population. Furthermore, stress has been implicated as a factor in a number of psychiatric illnesses (McEwen, 2000). Understanding the mechanisms by which chronic stress affects behavior and physiology is crucial for reducin g and preventing the detrimental effects of stress. 1.1.1. Physiological effects of stress Stress is often associated with somatic symptoms such as gastrointestinal upset, changes in appetite, and signs of arousal such as heart palpitations and increased perspiration (Vermetten & Bremner, 2002). Multiple physiological systems are associated with stress; however, dysfunction of the HPA axis is one of the best studied and most commonly addressed physiological responses to stress. The HPA axis is involved in maintaining homeostasis following exposure to stress. Upon exposure to stress, the hypothalamus releases corticotrophin releasing factor (CRF), which causes release of adreno corticotropin hormone (ACTH) from the anterior pituitary. ACTH stimulates release of cortisol from the adrenal glands which feeds back to the hypothalamus in a negative feedback loop, suppressing release of CRF and ACTH (Ehlert et al., 2001). Dysfunction of this negative feedback loop can lead to dysregulation at any or all steps in th e stress response. One commonly studied result of dysfunction of the HPA axis is imbalance in cortisol levels which can affect immune function and increases vulnerability to disease (O'Leary, 1990). Other b rain regions have been implicated in direct and i ndirect regulation of the HPA axis, including the prefrontal cortex (Figueiredo, Bruestle, Bodie, Dolgas, & Herman, 2003 ; Romeo, 2010 ) and the amygdala (Ostrander et al., 2009 ; Romeo, 2010 ) but further research is needed to clarify the ways in which these brain region s may be related to stress responses The homeostatic response of the HPA axis has been extensively studied in

PAGE 10

" animal models (Vermetten & Bremner 2002), but other neurobiological changes related to stress responses and associated long term eff ects are not as well understood. The present study examined the role of other mechanisms associated with chronic stress, including IEG activation and behavioral changes, in order to gain a better understanding of the long term effects of chronic stress dur ing adolescence. 1.1.2. Chronic stress social support, and psychopathology In addition to problems with immune function, mood, memory, and cognitive function, chronic stress plays a role in psychiatric ill nesses. It has been implicated as an etiologic, m aintenance, and relapse factor in many mental illnesses; including depression, posttraumatic stress disorder (McEwen, 2000), anxiety (Romeo, 2010) and substance use disorders (Marinelli & Piazza, 2002). In addition to the direct effect stress can have on a n individual's development of a psychiatric illness, stress can affect interpersonal relationships. Social support is an important prognostic factor in mental illness. Poor social support can be a risk factor for the development of psychiatric illness and may predict poorer outcomes (Sadock & Sadock, 2007). Stress associated with mental illness can negatively impact interpersonal relationships even in an individual with good premorbid social support. While good social support is indicative of a better progn osis, the chronic stress that is associated with many mental illnesses can erode this protective factor. For individuals with poor premorbid social support, seeking social support while experiencing the deleterious effects of chronic stress may prove to be a significant challenge. For an individual suffering from mental illness, social interaction may be experienced as an additional stressor, especially if the social environment is new or unknown. It is essential to understand the ways in which chronic str ess can affect social interaction. This understanding will aid in the development of preventative and treatment techniques that address abnormalities in social functioning as they are related to psychopathology.

PAGE 11

" ( 1.1.3. Chronic stress experiences in ado lescence Exposure to early life adversity and stress is a significant risk factor for future development of various somatic and psychological problems (Romeo, 2010 ) As previously noted, chronic stress can produce many maladaptive changes, and this is tr ue across developmental stages. However, when one c onsiders that e x periences of on going stress affect adolescents differently than adults due to the unique hormonal and behavioral changes that occur during this developmental stage (Romeo, 2010), adversit y during adolescence emerges as a unique and important area for research distinct from adversity during other developmental stages. Pubertal development and stress have been found to interact in ways that are unique to this developmental stage. For examp le, puberty marks a time of changes in the amount a nd duration of hormone release by the HPA axis in response to stress. These changes in HPA axis functioning may partially explain increases in stress responsiveness and emotionality that occur during adole scence (Walker 2002 ; Romeo, 2010 ) Other neurochemical changes, however, play a role in stress responsiveness and behavior during adolescence as well. S ome of these changes have been extensively studied such as the physiological and behavioral changes produced by increases in gonadal hormone secretion and activity of the hypothalamic pituitary gonadal (HPG) axis ( see Sisk & Zehr, 2005 for a review ) O ther mechanisms involved in stress responses and behavior during t his developmental stage such as plas ticity of brain regions that coordinate emotional and social responses like the medial prefrontal cortex (mPFC) and the amygdala have not been studied as thoroughly and may further explain stress induced changes in behavior and in the brains of adolescent s The role of the prefrontal cortex in response to stress and social situations during adolescence are of particular interest since the prefrontal cortex is not fully mature during adolescence. Changes in this brain region continue to occur throughout ado lescence as many neuronal connections are pruned back and others are strengthened due to repeated activity ( Weinberger, Elvevag, & Giedd, 2005).

PAGE 12

" ) Although many of the hormonal, behavioral, and social changes that occur during adolescence are considered to be normative, this developmental stage has been identified as a time of heightened risk for th e onset of psychopathology (Walk er, 2002). This increased risk for mental health problems and the heightened stress responsivity many adolescents experience indi cate that particular care must be taken to understand the ways in which adolescents respond to different types of stressors and the ways these behavioral and neurobiological responses can affect subsequent functioning. A pre clinical model of chronic early life stress is necessary in order to examine the behavioral effects of early life adversity under experimental conditions and to examine the neurobiological changes with which these behavioral effects are associated. 1.2. Isolation rearing in rats ( a mod el of early life adversity) Social isolation rearing has been widely used as an animal model of early life adversity and stress ( Hall, 1998; see Fone & Porkess 2008 for a review ) The early life stressor of social isolation rearing has been linked to a nu mber of neurochemical changes, including alterations in dopamine and serotonin function as well as changes in neuronal morphology (Fone & Porkess, 2008). Isolation rearing leads to abnormalities in PFC structure and function such as decreased constitutive expression of immediate early genes (Day Wilson et al., 2006; Levine et al., 2007) and synaptic associated proteins (Hermes et al., 2011) reductions in PFC volume (Day Wi lson et al ., 2006), and changes in dendritic spine morphology (Ferdman et al., 2007) These alterations in neuron structure and function ar e likely responsible for many of the behavioral changes produced by isolation rearing The enduring behavioral changes produced by this model include changes in reactivity to a novel environment, social interaction and aggression, pain sensitivity, and def icits in conditioned learning and novel object discrimination (Fone & Porkess, 2008). The precise mechanisms of many of these neurobiological and behavioral changes, however, are not well understood. The present study was designed to build on these

PAGE 13

" finding s of behavioral and neurochemical abnormalities to examine additional specific neurobiological and behavioral effects of isolation rearing and the ways in which such changes may be associated. Social interaction has been found to be rewarding for adoles cent rats (Douglas Varlinskaya, & Spear, 2004) S ocial deprivation, especially during adolescence, is thought to be a stressful experience as indicated by changes in HPA axis function and increased anxiety behaviors as measured in the elevated plus maze ( Hall, 1998 ; Fone & Por kess, 2008 ) and d eprivation of social play during adolescence leads to changes in neuron morphology in the mPFC (Bell et al ., 2010). The utility of the isolatio n rearing model is based on the specific use of the model to examine the effects of social deprivation on species that exhibit high levels of social and affiliative behavior. Social deprivation has been found to have consistent behavioral and neurochemical effects across species (Hall, 1998). While there are a number of limitat ions inherent in the use of animals to ext rapolate to human phenomena, isolation rearing is a useful model to examine the effects of early life adversity on subsequent behavioral and neurobiological responses to stress in species whose social structures ar e marked by high levels of social interaction 1.3. Social behavior following chronic stress In humans, early life stressors may lead genetically disposed individuals to develop a n umber of psychiatric disorders (Fone & Porkess, 2008). While early life a dverse events can contribute to the development of psychiatric disorders, not everyone who develops such a disorder was exposed to severe stressors early in life. Likewise, many individuals who are exposed to severe adversity early in life do not subsequen tly develop symptoms of mental illness. It is likely that additional experiences later in life can contribute to the risk of developing a mental illness. Among other factors, it may that be s ubsequent stressors are central in producing abnormalities in beh avior and neurobiology for individuals with a history of chronic

PAGE 14

" + stress. The effects of chronic stress on behavioral and physiological reactions to subsequent stressors are particularly important as many people, both with and without diagnosis of a psychia tric illness, may feel that the stressors in their lives seem to "pile up." Due to the central role that social interaction plays in most individual's lives, understanding how stress and social functioning interact is crucial. Stress can have an adverse ef fect on social functioning (Everhart & Emde 2006 ), and conversely, social interactions may contribute to stress even when the interactions are not particularly negative Many individuals interact with a large number of people on a daily basis, and a lar ge percentage of these interactions may be with relative strangers. Therefore, it is necessary to study the behavioral effects of chronic stress on subsequent novel social interaction stressors. Combining a history of chronic stress with a subsequent inter action with a novel rat may be a model for the unique changes that occur when individuals living with chronic stress are faced with stress associated with novel social interactions. As mentioned above on going stressful experience s may affect social behav ior and relationships in humans, preventing the development of social relationships or negat ively affecting existing bonds. The social interaction test of anxiety has been extensively used to test for anxiogenic and anxiolytic effects of environmental fac tors and exogenously administered drugs and peptides (File and Seth 2003 ) In this test, the dependent variable is the amount of time one rat spends engaging in social interaction (e.g. grooming, sniffing) with another rat. An increase in social interacti on without an increase in overall motor activity is indicative of an anxiolytic effect; while a decrease in time spend interacting without a concomitant decrease in motor activity indicates an anxiogenic effect (File & Seth, 2003) The present study modifi ed this test in order to examine group differences in time spent interacting as well as other indications of abnormalities in social functioning, such as changes in aggressive behavior and play behaviors like pinning and chasing.

PAGE 15

" A number of factors affec t behavior during social interactions, and a number of changes happen in the brain during social interaction. In order to interpret how the effects of chronic early life adversity may interact with effects of social interaction, th ese two procedures were c ombined (e.g. exposure to chronic early life stress followed by exposure to a novel rat); and the neurobiological correlates of behavioral effects were measured The present study examined these behavioral and physiological aspects by exposing rats with a history of chronic early life adversity to novel rats with no history of stress. In addition, levels of Arc and BDNF protein were measured to examine the neurobiological changes that go along with any observed changes in behavior. 1.4. Neurobiological co rrel ates of behavioral stress responses and implications for brain plasticity Stress activate s a number of complex short and long term neurobiological responses in the brain One of these responses is the expression of activity dependent genes. These ge nes are activated by the cascade of events that occur when sensory experience (in this case exposure to novel stimuli) leads to the activation of neurons and release of excitatory neurotransmitter. Calcium influx that occurs in response to membrane depolar ization triggers a number of changes within the neuron, including local and cell wide gene activation and protein translation (Flavell & Greenbe rg, 2008) The nature of activity dependent genes makes them valuable markers of neuronal activity as well as p otential mediators of the plastic changes that occur in the brain in response to experience (Schinder & Poo, 2000) 1.4.1. Brain derived neurotrophic factor (BDNF) BDNF is an activity dependent gene that is activated in response to neuronal stimulation. This gene encodes a neurotrophin that is released at synapses and plays a role in synaptic plasticity. Neurotrophins are a class of proteins that play an important part in the survival, differentiation, growth, and maintenance of neurons ( Branchi, Francia, & Alleva, 2004;

PAGE 16

" $" Hashimoto, Shimizu, & Iyo, 2004). Production of neurotrophins has been found to be experience dependent, indicating that these proteins may be partially responsible for the ways in which environmental experience can affect development, gen e expression, and brain function (Branchi et al. 2004). For example, early life experiences such as maternal separation and exposure to an enriched environment have been found to significantly affect production of BDNF and nerve growth factor (NGF) and al ter behavior (Branchi et al., 2004) In addition to the influence of experience on neurotrophin levels during development, stress during adulthood has been found to alter neurotrophin levels as well (Branchi, 2004 ; Bland et al., 2005 ). BDNF is considered a synaptic morphogen due to the wide variety of effects it has on neuron function and structure and the various circumstances under which it is activated (Branchi et al., 2004). Importantly, BDNF activation has been found to have opposing effects depending on a number of internal and external factors, making it a likely contributor to the modulation of plasticity in the CNS (see Greenberg et al., 2009 for a review) It has been found to protect neurons in the face of a chemical stressor (Spina, Squinto, Mi ller, Linsay, & Hyman, 1992) and is necessary for survival and differentiation of developing neurons (Branchi et al., 2004). In addition, it has been implicated in a number of psychological disorders including major depressive disorder, bipolar disorder an d posttraumatic stress disorder (Smith Makino, Kvetnansky, & Post, 1995 ; see Hashimoto, 2004 for a review ) For example, it has been proposed that BDNF is an important aspect of recovery from major depressive disorder due to the increase in BDNF that var ious treatments for major depressive disorder produce and the BDNF gene has been identified as a potential risk gene for the development of bipolar disorder (Hashimoto et. al., 2004). BDNF has been found to be a major component in the effects of chronic s tress and is regula ted by stress, (Smith et al., 1995). Roceri and colleagues (2002) found that rats that had undergone the early life stressor of maternal separation had decreased BDNF in the hippocampus and prefrontal cortex in adulthood. In addition, BD NF expression in response to an acute stress during adulthood was altered as a result of early mater nal

PAGE 17

" $$ separation, indicating long term changes in function as a result of this specific early life stressor (Roceri et al., 2002) BDNF has been found t o be a major component of learning and memory as well (Lu, Christian, and Lu, 2008 ; Cunha, Brambilla, & Thomas, 2010 ). This aspect of BDNF is important for our model of early life adversity due to the role that memory and learned behaviors play in the deve lopment and expression of various stress responses and psychological illnesses. For example, one of the symptoms of posttraumatic stress disorder is recurrent and distressing recolle ction of the traumatic ev ent that was exper ienced (Sadock & Sadock, 2007). M any anxiety disorders are marked by behaviors such as compulsions in the case of obsessive compulsive disorder and avoidance in the cas e of phobias and panic disorder, both of which are behaviors that an individual with an anxiety disorder has learned ar e effective in reducing anxiety in the short term (Sadock & Sadock, 2007). Additionally, proper social functioning and behavior in rats is learned through play experiences, and deprivation of play behavior can affect subsequent social functioning (Hol et a l., 1999). One of the i mportant fu nction s of BDNF is its role in the activation of another type of activity dependent gene the immediate early gene Arc. Neurotrophins are known to induce expression of this sub type of activity dependent genes (Lyford et al., 1995). IEGs undergo rapid and transient transcription, which can be induced by different types of stimuli, and can occur within 5 minutes of neuronal stimulation depending on the IEG (Flavell & Greenberg, 2008). The proposed study will measure expres sion of BDNF and the IEG Arc, which is activated by BDNF (Bramham, Worley, Moore, & Guzowski, 2008), in order to further elucidate the cascade of events that occur in the brain as it changes and adapts in res ponse to adversity and stress 1.4.2. Activity regulated cytoskeleton associated protein (Arc) Arc has been implicated in multiple forms of synaptic plasticity (Bramham et al., 2008), and is thought to be associated with reorganization or development of synapses as a result of

PAGE 18

" $% stressful exper iences (K ozlovsky et al. 2008). Arc is a neuronal activation marker that plays a role in changes in synaptic transmission through alteration s at receptor sites in the post synaptic density (Pinaud, 2004; Bramham et al., 2008) Arc mRNA is transported rapidly to the dendrites and accumulates at sites of synaptic activi ty. Arc protein also accumulates in dendrites, and like Arc mRNA, is found specifically in areas of synaptic activity, indicating that the protein is synthesized locally at the synapse (Bramham et al., 2008) Arc has been closely linked with the strength of excitatory synapses and has been implicated in multiple forms of neuronal plasticity, including long term potentiation (LTP), long term depression (LTD), and homeostatic plasticity (Bramham et al., 2 008). LTP refers to a long term increase in excitability of a neuron due to repeated, high frequency activation, and conversely, LTD refers to a long term decrease in the excitability of a neuron (Carlson, 2008). Homeostatic plasticity refers to mechanisms that keep neuronal activity relatively stable in the face of the forces of LTP and LTD (Turrigiano & Nelson, 2004). While other IEGs have been studied in relation to chronic stress in animals, Arc is an important IEG to study because of its possible role in the synaptic plasticity underlying long term changes in functioning. An increase or decrease in Arc expression may have an effect on the ability of the brain to modify and protect itself from stressors. If a history of chronic stress changes the abilit y of Arc genes to be activated and /or changes the amount of Arc protein that is synthesized in response to neuronal activation this may partially explain the deleterious emotional and behavioral effects of chronic stress. Kozlovsky and colleagues (2008) p roposed a resilience/recovery role for Arc in response to exposure to stress. They found that rats with a highly disrupted behavioral response to a predator scent stressor had significantly lower levels of Arc mRNA in the hippocampus compared to rats whose behavioral response was minimal (Kozlovsky et al, 2008). Bland and colleagues (abstract, 2009) reported different levels of Arc labeled cells in the mPFC following an acute restraint stressor in male and female rats reared in social isolation compared to male and female group reared rats. The present study built on

PAGE 19

" $& these findings by using social isolation rearing coupled with a subsequent social interaction stressor in order to determine how a history of early life adversity attenuates future responses to social interactions with a novelty stress component 1.5. Sex Differences in Stress Responses and Psychopathology Prevalence rates of stress related disorders are different for males and females, with females suffering from panic disorder, specific ph obia, posttraumatic stress disorder, and generalized anxiety disorder at a higher rate than males and males having higher rate s of substance use disorders such as alcohol and cocaine abuse and dependence (Sadock & Sadock, 2007). Additionally, in those diso rders where lifetime prevalence is approximately equal among men and women, such as schizophrenia, the onset, course, and prognosis of the disorder often vary by sex (Sadock & Sadock, 2007) Research examining sex differences in stress responses has demons trated that males and females have different physiological responses to stress, including differences in HPA axis responsiveness (Gallucci et al., 1993) and differences in emotion al processing when d epressed (Wright et al., 2009). Despite such clinical evi dence of sex as a factor in the development of mental illness, much preclinical research has only used male rats in models of stress and psychopathology Those studies that have examined males and females have found that there are a number of behavioral an d neurobiological sex differences in response to stress Bland and colleagues (2005) found that female rats have a more pronounced HPA response (as measured by plasma corticosterone levels) and that males and females have different levels and time courses of neuronal activation (as measured by expression of the IEG c fos) and expression of BDNF in the prefrontal cortex in response to acute uncontrollable stress. Likewise, Romeo (2010) reports that female rats have a higher peak corticosterone response than male rats and a quicker return to baseline following a stressor. In an experiment examining sex differences in social interaction and expression of the IEG zif268, females were

PAGE 20

" $' found to exhibit higher anxiety like behavior during exposure to a novel rat a nd had lower levels of zif268 expression in subregions of the mPFC (Stack et al., 2010). Research examining sex differences in response to stress and adversity will help to clarify the reasons why prevalence rates for various mental disorders are differen t for men and women and will aid in the development of efficacious and possibly sex dependent treatments. 1.6. Brain regions involved in stress The u nderstanding of many brain regions directly involved in stress responses, such as the hypothalamus ( Smit h, Makino, Kim & Kvetnansky, 1995 ; Joels et al., 2004; see Herman, Prewitt, & Cullinan, 1996 for a review) and hippocampus ( Smith et al., 1995; Roceri et al., 2002; Joels et al., 2004 ; see Maggio & Segal, 2010 for a review ) has become more complete in rece nt years In addition to their direct regulation of stress responses, t hese brain regions have projections to and from other regions that are important targets for research if behavioral and physiological responses to stress are to be fully understood. The long term effects of adversity involve many brain regions that undergo a number of changes that can alter responses to a wide variety of stimuli including stress responses Two brain regions that are particularly important for understanding the effects o f stress and adversity due to their function s and connectivity with other brain regions are the medial prefrontal cort ex (mPFC) and the amygdala 1.6.1. Medial prefrontal cortex (mPFC) Proper functioning of the prefrontal cortex is necessary for carrying out executive functions such as the ability to attend to or ignore stimuli, process information, and plan and carry out a response (Ho l mes & Wellman, 2009). Stress produces activation of the mPFC, and this brain region plays a role in the processing of st ressful or threatening stimuli and in physiological, cognitive and emotional responses to stressors (Vermetten & Bremner, 2002). One function of the mPFC in stress responses is to re gulate activity of the HPA axis, and this regulation appears to vary dep ending on what kind of stressor is present (Figueiredo et al., 2003). Chronic stress in rats results in a decrease in length and number of dendrites in the

PAGE 21

" $( mPFC (Radley et al. 2004; Cook & Wellman, 2005) This neuroanatomical outcome of chronic stress coul d potentially affect not only mPFC functioning but also its ability to communicate with other brain regions a crucial role of the mPFC The mPFC projects to and receives input from a number of brain regions involved in stress and emotional responses, incl uding the amygdala (Gabbott, Warner, Jays, Salway, & Busby, 2005). It projects to the hypothalamus and brainstem, creating a connection to brain region s involved in neuroendocrine and autonomic responses to stress (Holmes & Wellman, 2009). It has bidirecti onal connections with the locus coeruleus, dorsal raphe nucl eus, and ventral tegmental area, three monoaminergic nuclei which are activated by stress and modulate executive function via their connections with the mPFC (Holmes & Wellman, 2009). Due to the role of the mPFC in regulating the HPA axis and modulating other neurobiological and behavioral responses to stress and its larger role in proper cognitive and emotional functioning it is crucial to study changes in the mPFC that occur as a result of earl y life adversity and stress 1.6.2. Amygdala One of the best known functions of the amygdala is as a major center for processing emotional and socially relevant information It is also important for formulating appropriate responses to fearful or threaten ing stimuli and for appropriate social behavior (see Adolphs, 2010 for a review). This brain region is complex in structure and function, with numerous subregions containing nuclei that project to various areas in the brain, including the mPFC, hypothalamu s, and brainstem (Holmes & Wellman, 2009). These connections with other brain regions and its role in emotional processing indicate that the amygdala is an important brain region for formulating healthy and adaptive stress responses and may be a target for study in individuals who have undergone extended periods of stress. Abnormalities in structure and function have been reported in the amygdala in individuals suffering from mental illness such as anxiety and depression (Monk, 2008). The focus of the prese nt study is to understand the ways in which a history of chronic adversity affects social, behavioral, and neurobiological responses

PAGE 22

" $) to a subsequent social interaction stressor. Due to its connectivity to brain regions directly involved in stress resp onses such as the hypothalamus, and its role in appropriate processing of and response to emotionally and socially relevant information, the amygdala warrants study as a link between the experience of adversity early in life and the subsequent development of a bnormal functioning. 1.7. Rationale Stress affects people in numerous ways, depending on the nature of the stressor, the individual's history of stress and adversity, sex, and a number of other environmental and genetic factors. More information regar ding the circumstances under which adverse experiences contribute to the development of psychopathology is needed in order to effectively prevent and treat mental illnesses. The effects of early life adversity on behavior and neurobiological responses duri ng social interaction are particularly important due to the role of social support as a positive prognostic factor for individuals with mental illness (Sadock & Sadock, 2007), and the adverse e ffect stress can have on social functioning (Everhart & Emde, 2 006). This study combined a history of early life adversity with a subsequent exposure to a novel rat in order to model the unique changes that occur when individuals who have experienced chronic stress early in life are faced with the stress of novel soci al interactions. Behavior during exposure to a novel rat was examined in conjunction with expression of BDNF and the IEG Arc in the mPFC and amygdala in order to determine possible associations between changes in the brain and changes in behavior in male a nd female rats. 1.8. Purpose and Research Questions The purpose of this study is to investigate the effects of early life adversity on social behavior and the neurobiological changes with which it is associated in male and female rats

PAGE 23

" $* Question 1 : How d oes isolation rearing affect expression of the protein product s of BDNF and the immediate early gene Arc in the amygdala and the prefrontal cortex produced by exposure to a novel conspecific? Question 2 : How does isolation rearing affect behavior during ex posure to a novel conspecific? Question 3 : Is Arc and BDNF expression produced by exposure to a novel conspecific associated with behavior during exposure? Question 4 : Do male and female rats differ in these effects? 1.9. Predictions H1: Isolation rearin g will produce a decrease in Arc and BDNF protein expre ssion in the amygdala and mPFC produced by exposure to a novel rat. The isolation rearing procedure has been found to produce decreases in other IEGs and synaptic associated proteins, so it is expected for the plasticity related genes measured here to have a similar downregulation in isolates. H2: Isolation rearing will produce a decrease in overall social interaction with a novel rat as well as a decrease in typical play behaviors. Isolation rearing h as been found to increase anxiety like behaviors and neophobia so it is expected that a novel social exposure will likewise produce avoidance and a decrease in social behaviors. H3: Arc and BDNF activation will have a positive relationship with behavior Since low levels of Arc and BDNF and low levels of behavior in isolates are anticipated and conversely higher levels of both gene ac tivation and behaviors in group housed rats, a linear relationship between behavior and gene activation is expected H4: Ma le and female isolation reared rats will have similar results, but the effects will be more pronounced in females.

PAGE 24

" $+ Females have higher anxiety like behaviors in response to exposure to a novel rat and more pronounced HPA activation in response to a stresso r, so it is expected that behavioral and neurobiological changes produced by the chronic stressor of isolation rearing will be more pronounced in females.

PAGE 25

" $, 2. Methods 2.1. Animals Male (n = 32) and female (n = 32) Sprague Dawley rats were purchased from Harlan (Indianapolis, IN) at postnatal day 21 and housed in a temperature controlled vivarium with a 12:12 light:dark cycle and unlimited access to food and water. Rats were housed for 4 weeks either individually ( isolation ) or in same sex groups of 4 (GH) in Plexiglas cages under standard housing conditions 2.2. Social Exposure Procedure After 4 weeks of isolation or group housing, rats were placed in a cage with a novel same sex rat ( conspecific ) for 15 minutes. Exposure to novel c onspecifics occured in standard Plexiglas cages. Novel con specifics were group housed in order to control for potential effects of abnormalities in stimulus animals affecting the behavior of experimental animals Control rats were left in their home cages and all rats were sacrificed 90 min later. This time point was chosen as an optimal time point to measure the protein product of activity dependent genes based on the time of peak protein expression (90 120 min) following acute stimuli for the prototypical immediate early gene, c fos (Kovacs, 2008). Social exposure was video recorded and behavior was coded using the following definitions: Social Interaction : Overall time the experimental rat spends actively interacting (e.g. sniffing, following, grooming) with the novel conspecific (File & Seth, 2003). Pinning : Standing over/holding down the novel conspecific while it is in a supine posture (Hurst et al., 1999). Chasing : Pursuit of the novel conspecific while it is running away from the experimental rat. Aggressive Grooming : Vigorous grooming by the experimental rat of the novel conspecific when it is standing, crouching, supine, or trying to escape (Hurst et al., 1999).

PAGE 26

" %" 2.3. Tissue harvest Rats were sacrificed 90 minutes following completion of exposu re to a novel rat and home cage control rats were sacrificed at the same time. Rats were deeply anesthetized with sodium pentobarbital and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.01M PBS and cryoprotected in 30% sucros e for three days, then quickly frozen in 30C isopentane. Brains were sliced through the prefrontal cortex and amygdala using the atlas of Paxinos and Watson (1998) as a guide. Sections were stored at 4C in cryoprotecta nt until immunohistochemistry was performed. 2.4. Immunohistoche mistry Sections were labeled using primary antibodies directed against Arc (rabbit anti Arc, 1:4,000, Synaptic Systems) and biotinylated goat anti rabbit secondary an tibody (1:200, Jackson Labs) or BDNF (mouse anti BDNF, 1:300, Chemicon) and biotinylated g oat anti mouse secondary antibody (1:200, Jackson Labs) with a 3,3' diaminobenzidine (DAB) and nickel as chromogens. Density counts were conducted for Arc and BDNF in subregions of the mPFC: the anterior cingulate cortex (AC), the prelimbic cortex (PL), an d the infralimbic cortex (IL), and in the motor cortex (MC) and ventral orbital cortex ( VO), (Fig. 1), as well as subregions of the amygdala: basolateral (BLA), central (CeA), and medial (MeA ), (Fig. 2), at 40x using an Olympus BX51 microscope and NewCAST software (VisioPharm). Arc and BDNF positive cells were counted using a counting frame centered within each subregion and normalized as cells per mm 2 For each rat, 3 5 sections (6 10 individual hemisphere measurements) were assessed and mean values calcu lated. Unbiased stereology was also performed to obtain estimates of total Arc and BDNF labeled cells in the mPFC Random meander sampling was performed at 100x (oil) using sampling fractions of 1% for Arc and BDNF.

PAGE 27

" %$ " .!/012"%3"4256!78"57869!8!8/"9:;/<9=9">0?12/!78>@"6A2"528619="805=20>" BC2DEF"?9>7=96219="805=20>"BGHDEF"98<":2"BI2DE"BJ9K!87>"L" M96>78F"$,,+E3 " .!/012"$3"4256!78"57869!8!8/"N12O17869="57162K">0?12/!78>@"6A2" 98621!71" 5!8/0=962"BDCEF"N12=!:?!5"BJHEF" !8O19=!:?!5" BPHE "F"#28619="71?!69="BQREF"" 98<"N1!:91;":7671"571 6!52>"BICEF""BJ9K!87>"L"M96>78F"$,,+E " 3 2.5. Statist ics Expression of Arc and BDNF were compared between groups using a 2 (sex: female, male) x 2 (adolescent housing: group, isolated) x 2 (acute social exposure: social exposure, no social exposure) factorial ANOVA for each brain sub region (A C, PL, IL, MC, VO, CeA, BLA,

PAGE 28

" %% MeA ) and for the entire mPFC (stereology counts). Tukey' s post hoc were used to examine any main effects and interactions that were found. The groups are as follows: Male Female Group housed Isolat ion reared Group housed Isolat ion reare d social exposure n = 8 n = 8 n = 8 n = 8 no social exposure n = 8 n = 8 n = 8 n = 8 All 64 animals were used for the Arc AC, PL, IL, MC, VO and mPFC stereology analyses. Four animals were excluded from Arc amygdala subregion analyses and six from the BDNF PFC subregion and mPFC stereology analyses due to damage to tissue during immunohistochemistry processing. Overall social interaction, pinning, chasing, and aggressive grooming were examined for all rats that were exposed to a novel rat using a 2 (se x: female, male) x 2 (adolescent housing: group, isolated) factorial ANOVA with Tukey's post hoc to follow up on main effects. All animals in the social exposure groups (n = 32) were including in behavior analyses. Correlations were conducted using Pearson 's r to examine relationships between behavior and Arc and BDNF express ion All statistical analyses were conducted using PASW Statistics 18.0.

PAGE 29

" %& 3. Results 3.1. Arc 3.1.1. Medial Prefrontal Cortex Stereology So cial exposure produced large increases in total estimates of Arc positive cells in the mPFC in male and female group housed rats, and to a lesser extent in female isolated r ats (Fig. 3). There was a significant housing X social exposure interaction, F (1,56) = 8.70, p <.01 for total estimates of Arc positive cells in the mPFC Post hoc tests revealed that exposure to a novel conspecific produces significant increases in Arc protein expression in the mPFC in female and male group housed rats, p <.001, and in female isolated rats, p < .05, but n ot in male isolated rats. 3.1.2. Prefrontal Cortex Density Counts Exposure to a novel conspecific produced increased Arc activatio n in subregions of the PFC, primarily in group housed rat s (Fig. 4). There was a significant housing X social exposure int eraction in the AC, F (1, 56) = 5.66 p = .02 Post hoc tests revealed that exposure to a novel conspecific produced significant increase s in Arc in the AC in fe male p < .0 0 1, and male p = .00 1, group housed rats but not in female or male isolated rats. There was also a strong trend for a significant difference between female group housed and female isolate rats p = .051

PAGE 30

" %' There was a significant housing X social exposure interaction in the PL, F (1,56) = 12.62 p = .0 0 1 P ost hoc tests revealed that exp osure to a novel conspecific produced significan t increases in Arc in the PL in female, p = .01, and male, p < .001, group housed rats, but not in female or male isolated rats. In the IL there was a significant sex X hous ing X social exposure interaction, F (1,56) = 6.02 p = 02 Post hoc tests revealed that exposure to a novel conspecific produced signi ficant increases in Arc in the I L in male, p < 0 01, group housed rats, but not in female group housed rats or male or female isolated rats. In the MC, th ere was a significant housing X social exposure interaction, F (1,56) = 4.83 p = .03. Post hoc tests revealed that exposure to a novel conspecific produced significant increases in Arc in the MC in fe male, p < .01, and male, p = .02 group housed rats, bu t not in male or female isolated rats. In the VO, th ere was a significant housing X social exposure interaction F (1, 56) = 9.52 p < .01 Post hoc tests revealed that exposure to a novel conspecific produced significant increases in Arc in t he VO in fe mal e, p < .0 0 1, and male, p < .0 0 1, group housed rats, and to a lesser extent in male, p < .01, and female, p < .05 isolated rats. 3.1.3. Amygdala Density Counts Exposure to a novel conspecific produced increased Arc activation in subregions of the amygdala particularly in group housed rats (Fig. 5). Factorial ANOVA r evealed a significant housing X social exposure interaction in the CeA, F (1,52) = 4.55, p < .05. Post hoc tests revealed that exposure to a novel conspecific produced significant increases in Arc protein in the CeA in female, p < 0 01, and male, p < .01 group housed rats, and in male isolated rats, p < .05 but not in fe male isolated rats. There was also a significant housing X social exposure interaction in the BLA, F (1,52) = 8.39, p < .01. Post hoc test s revealed that exposure to a novel conspecific produced significant increases in Arc in the BLA in female, p < .01, and male, p <.01 group housed rats, but not in female or male isolated rats. In the MeA, there was a significant ma in effect of social exposure, F (1,52) = 13.25, p < .01; rats exposed to a novel conspecific had increased Arc activation compared to non social exposure controls

PAGE 31

" %(

PAGE 32

" %)

PAGE 33

" %* 3.2. BDNF 3.2.1. Medial Prefrontal Cortex Stereology Female rats had overall higher levels of BDNF protein in the mPFC than male rats (Fig. 6). There was a significant main effect of sex, F (1, 49) = 6.41, p < .05. There was also a strong trend for a main effect of social exposure, F (1,49) = 3.24, p = .08. 3.2.2. Prefrontal Cortex De nsity Counts Exposure to a novel conspecific produced increased BDNF activation in some subregions of the m PFC, but not in the AC, MC or VO (Fig. 7). There was a significant sex X housing interaction in the AC, F (1, 50) = 6.53 p < .05 Post hoc tests col lapsed across social exposure revealed that female group housed rats had more BDNF activation than any other group, p < .05. In the PL, t here was a significant main effect of social exposure, F (1,50) = 6.61, p < .05 There was also a strong trend for a ma in effect of sex in the PL, F (1,50) = 3.62, p = .06. In the IL, there was a significant main effect of social exposure, F (1,50) = 5.86, p = .04 There were no group differences in BDNF activation in the MC or VO.

PAGE 34

" %+

PAGE 35

" %, 3.3. Behavior during novel social e xposure Behavior during exposure to a novel conspecific differed accord ing to sex and housing condition (Fig. 8). For overall social interaction, t here was a significant main effect of sex, F (1,28) = 36.25, p < .00 1 males interacted with a novel conspeci fic more than females. There was also a main effect of housing, F (1,28) = 44.34, p < .001, isolated rats interacted more with a novel conspecific than group housed rats. There was a significant main effect of housing for aggressive grooming, F (1,28) = 42 .43, p < .001, isolate d rats engaged in more aggressive grooming than group housed rats. For chasing, there was a significant main effect of housing, F (1,28) = 22.31, p < .001, as well as a strong trend for a sex X housing interaction, F (1,28) = 4.15, p = .051. Post hoc t ests revealed that male isolate d rats chased a novel conspecific more than group housed rats, p < 01 but this increase in chasing behavior was not present in females. Additionally, male isolate d rats chased a novel conspecific more than did female isolates, p = .04. There was a significant sex X housing interaction for pinning, F (1,28) = 6.99, p < .05. Post hoc tests revealed that male isolate d rats pinned a novel conspecific more than group housed rats, p < 01 but this increase in pin ning behavior was not present in females. Additionally, male isolate d rats pinned a novel conspecific more than female isolates, p < .001. 3.4. Relationships between behavior and Arc and BDNF expression There were no significant correlations found betwe en total estimates of Arc positive cells in the mPFC and any of the behaviors measured for males or females, though there was a trend for a negative relationship between total mPFC Arc and aggressive grooming for males, r(16) = .466, p = .069. Females had a significant negative correlation between BDNF positive cells in the mPFC and overall social interaction, r(16) = .57, p = .021.

PAGE 36

" &"

PAGE 37

" &$ 4. Discussion 4.1. Arc activation produced by exposure to a novel conspecific Exposure to a novel conspecific produced robust increases in Arc protein expression in the PFC and amygdala in group housed animals, but this increase was largely lacking in isolates. These findings indicate a general lower level of cortical and subcortical Arc activation in isolation re ared rats This study adds to the existing knowledge of abnormalities produced by early life adversity. There are several possible ways that the decrease in Arc activation seen here could be related to other changes produced by isolation rearing It could be that group housed and isolated animals ar e similar in number and projection s of neurons in these brain regions but that a morphological or molecular abnormality in isolates results in either mRNA transcription or protein translation problems thereby l ea ding to a decrease in the amount of Arc protein Another possibility is that isolates differ from group housed animals in connectivity in the brain regions under study, and so do not have the same patterns of activation in response to exposure to a novel conspecific as group housed rats (e.g. this stimulus does not recruit the PFC and amygdala in isolates the way it does in GH rats and instead is activating brain regions not examined here ) A third possible e xplanation is that isolates have fewer neurons in the PFC and amygdala compared to GH rats, making it so that e ven if their levels of activation are proportionally the same, the numbers of Arc positive cells in the brains of isolates will be lower since total cell numbers are lower. I t is helpful to look at the current findings along with previous researc h on isolation rearing to piece together how Arc may be related to other abnormalities produced by isolation rearing, and to examine how accurate the three interpretations of the observed decrease in Arc presented above may be For example, Bock and colleagues (2008) found that isolation rear ing alters dendritic length and complexity in the AC and orbitofrontal cortex and Hermes and colleages (2011) found that isolation rearing suppresses expression o f synaptic associated

PAGE 38

" &% proteins in the PFC. These findings support the first interpretation that decreased Arc in isolates may be related to differences in the morphology of their neurons or ability to synthesize new proteins that are needed to respond to a novel social stimulus. Since Arc is localized at sites of synaptic activity, and appears to be responsible for local protein synthesis at these sites (Bramham, 2008 ), a decrease in Arc may result in fewer or less efficient dendrites. Arc also plays a rol e in trafficking AMPA recepto rs to the post synaptic density, is known to be preferentially activated in glutamatergic neurons (Vazdarjanova et al., 2006) and is found in the postsynaptic density but not in presynaptic terminals or axons (Steward & Worley, 2001). Arc appears to be partially responsible for the endocytosi s of AMPA receptors in the post synaptic density, with increased Arc translation being related to increased AMPA endocytosis (Waung et al., 2008). If the dendrites on cells that would normall y express Arc mRNA and protein are not receiving signals properly, possibly due to improper AMPA receptor trafficking in the postsynaptic density, there could be a cyclic effect. In this case, a decrease in Arc could result in a dysfunctional level of AMPA receptors, which could lead to abnormalities in these dendrites A change in dendrite morphology and function could then lead to a further reduction in Arc activation In order to examine how these factors may be related, Arc activation could be measured along wi th dendritic spine length, morphology and levels of AMPA receptors present over several time points during the isolation period (e.g. at 2 weeks, 3 weeks, and 4 weeks) This would show if changes in Arc expression are ongoing and progressive and a ccompanied by changes in dendritic spine morphology and changes in levels of AMPA receptors in the postsynaptic density The second interpretation of the observed decrease in Arc in the PFC and amygdala in isolates is that the abnormality could be related to connectivity between these brain regions and other brain regions involved in a social stress response A novel social exposure produces robust activation of Arc i n the PFC and amygdala in group housed animals. Presumably this activation occurs due to t he roles of these brain regio ns in social activity, decision making, and

PAGE 39

" && emotional responses, all of which are likely involved in a response to a novel social exposure or stressor. If a novel social exposure or stressor recruits different brain regions in isolates due to abnormalities in connectivity during development as a result of no exposure to play, perhaps there are other brain regions being activated instead of the PFC and amygdala in these rats. This interpretation could be followed up on by looking at other brain regions such as the hippocampus to see if there is a stronger learning and memory component than executive function and emotional component of this stim ul us for isolates. Another brain region of interest is the hypothalamus to see if there may be a stronger HPA axis activation component for isolates rather than a cortical response. Alternatively, fMRI could be used to measure activity in various brain regions immediately following social exposure. Finally, it is possible that morphology and connectivity of neurons in the PFC and amygdala are normal in isolates, but that there are overall fewer neurons in these brain regions in isolation reared rats. Day Wilson and colleages (2006) found decreas ed cortical volume in isolation reared rats, but no changes in neuron number. Cortical volume and neuron number should be examined in male and female rats, as this group only used male rats. I f it is the case that isolation reared males and f emales do not differ from group reared rats, but do differ in cortical volume, this could point to additional abnormalities in neuron morphology. There may be decreases in white matter, indicating abnormal axon growth or connectivity. Additionally, fewer or smaller dendrites or soma may contribute to lower cortical v olume in isolates. The limitations of this study make it difficult to determine what kinds of cellular or molecular mechanisms are causing a decrease in Arc activation and what downstream effects this decrease may have. Therefore, the best interpretation of the data from this study is that isolates have lower IEG activation, indicating lower neuronal activation (hypofunction of the mPFC and amygdala). These findings will need to be built upon to examine what this means for brain function, e.g. what kinds o f cells have decreases in activation and how cell signaling may be disrupted. Ongoing projects include examining changes in dopamine and serotonin signaling

PAGE 40

" &' in isolation reared rats using in vivo microdialysis and examining other synaptic associated protei ns such as PSD95 to more fully clarify how neuron function is altered by early life adversity. Future research to examine more precisely the role isolation rearing plays in alterations in brain plasticity and physiological stress responses could include me asuring corticosterone responses, double labeling for Arc with neurotransmitters such as serotonin, dopamine, GABA, or glutamate, and looking at Arc mRNA along with Arc protein to determine if the abnormalities in isolates lie in transcription or translati on, or both. 4.1.1. Arc activation varies by sex and subregion of the PFC and amygdala The data presented here indicate that isolation reared rats in general have lower levels of Arc activation in the PFC and amygdala produced by exposure to a novel consp ecific. Arc activation in some subregions also appears to be influenced by sex. S ex specific patterns of activation indicate that Arc activation may vary in males and females and depending on brain region. The first sex specific pattern of activation of n otice is in the mPFC. There appears to be a dorsal ventral gradient in Arc expression for females, but not for males T he increase in Arc activation produced by social exposure in female group housed rats is more pronounced in the dorsal mPFC (AC), then be comes less pronounced in the more ventral regions (PL and IL). Males have proportional levels of Arc activation produced by social exposure throughout the mPFC. In fact, these differing patterns of activation are so pronounced that male group housed rats have significantly higher levels of Arc than female group housed rats in the IL (Fig. 4). The ventral portion of the mPFC is also the only cortical region where female group housed rats did not have a significant increase in Arc produced by social exposure Likewise, in the CeA, female group housed rats did not have an increase in Arc produced by social exposure, while male group housed rats did. In these two brain regions (IL and CeA), female group housed rats resemble isolation reared females in that soci al exposure did not produce significant increases in Arc activation though in all other subregions they do not show similar patterns of activation (or lack of activation) to isolates.

PAGE 41

" &( The MeA was the only subregion that did not have an effect of housing condition or sex; male and female group housed and isolation rats all had an increase in Arc produced by novel social exposure. It is possible that this brain region is not affected by isolation rearing the way that the other regions examined are. Additio nally, cell counts in this region were much lower than in other regions, and may have been too low to reveal group differences. 4.2. BDNF activation in the PFC In contrast to the general effect of isolation rearing on Arc activation produced by social e xposure in the PFC and amygdala, BDNF expression appears to be affected less by housing condition than by sex Females were found to have overall higher levels of BDNF in the mPFC. When examined by subregion, it is seen that female group housed rats have m ore BDNF expression in the AC than any other groups (Fig. 7), and that BDNF expression in this subregion is not affected by social exposure, e.g. rats who were exposed to a novel rat did not have different levels of BDNF in the AC than home cage control r ats In the PL and IL, however, BDNF activation seems to only be affected by social exposure, though in the PL there is also a strong trend for a main effect of sex. This pattern of activation is interesting, as BDNF appears to be essentially acting like an IEG in the more ventral mPFC subregions, as indicated by increased activation produced by social exposure. In the AC, however, BDNF activation appears to be reflecting a process that is not stimulus dependent, as social exposure does not se em to increa se BDNF activation in this brain region. BDNF has been found to act as an IEG in some circumstances, depending on which of its promotor regions are bound and the subsequent distinct transcripts that are made (Lauterborn 1996). It is possible that certain B DNF transcripts are preferentially transcribed in some brain regions and that this could be due to the need for a fast and transient upregulation of BDNF as opposed to more sustained BDNF protein levels in other brain regions.

PAGE 42

" &) Bland and colleagues (2005) found higher levels of BDNF mRNA in their control unstressed females compared to control males. It may be that females that have experienced normal social exposure during development have higher baseline BDNF levels. Hill and colleagues (2011) indicate that BDNF TrkB signal ing during adolescence is modulated by sex steroid hormones diffe rently in male and female rats. Sohrabji and colleagues (1995) suggested that estrogen might increase the availability of neurotrophins in the cortex, thereby regulating BDNF transcription T his may partially explain why females seem to have constitutively higher levels of BDNF in the PFC. This could be examined by measuring BDNF expression during different stages of estrous could be measured to see if levels very according to levels of estro gen. 4.3 Isolation reared rats exhibit increased aggressive and nonaggressive behavior Male and female isolation reared rats engaged in more overall social interaction as well as more aggressive grooming with a novel conspecific compared to group housed males and females. Male rats as a whole, however, engaged in more social interaction and aggressive grooming compared to females as a whole. Male isolates showed higher levels of pinning and chasing than male group housed rats, but female isolates were no different in these behaviors than their group housed counterparts. The observed increase in social interaction in males is similar to that found by other groups (Ferdman et al., 2007 ; Meng et al. 2010 ), but the increase in social interaction for female i solates differed from Ferdman and colleagues and from others (Hermes, 2011) who have found that female isolates spend less time interacting with a novel rat. These inconsistencies may be due to several factors. Fer dman et al. (2007) used Wistar rats and performed social interaction testing far into adulthood, at P98. Additionally, the social exposure test used in that study was only 3 minutes long, compared to 10 minutes of social exposure used here. Likewise, t he social interaction test used by Hermes et al (2011) only exposed the ra ts to a novel rat for 5 minutes and rats were exposed to a rat from a similar

PAGE 43

" &* housing condition (e.g. isolates paired with isolates) Any of these factors could potentially affect the observed behavior of the experimental rat. The social int eraction test used here, where the s timulus rat for both the group housed and isolati on reared animals is a group housed or "normal" rat, is a good model for examining what happens with a rat that has undergone chronic early life adversity is faced with a "normal" social situation. Additionally, a longer social exposure allows time for a full spectrum of behaviors to be observed, as the first few minutes may be spent habituating to the situation and exploring the new cage. One important point to keep in min d is that isolation rearing has consistently been found to elevate locomotor activity though this effect varies by strain and seems to be less pronounced in Sprague Dawley rats (Fone & P orkess 2008 ) The increase in social interaction may be related to a n overall increase in locomotor activity. Future studies should measure locomotor activity as well as the behaviors recorded here to examine whether males and females have similar increases in locomotor activity and whether levels of locomotor activity ar e associated with social interaction 4.4. Relationships between Arc and BDNF activation and behavior The only relationship observed between gene activation and behavior was between BDNF positive cells in the mPFC and overall social inter action in female s where higher BDNF expression was associated with lower levels of social interaction. This is an interesting finding in light of the fact that females have higher levels of BDNF and interact less with a novel conspecific. This suggests a possible role of BDNF in behavioral differences in male and female rats. Isolation rearing has been found to decrease activation of IEGs such as Arc while increasing exploratory behavior (Levine 2007) while other groups have found decreases in IEG expression alongside def icits in social behavior and decreased exploratory behavior (Hermes, 2011) Despite the lack of statistically significant correlations between behavior and Arc in the present study, the observed decrease in Arc activation and increased aggressive and non

PAGE 44

" &+ aggressive behavior in isolates seems to fall in line with the findings of Levine et al (2007) This group postulated that the decrease in gene activation was likely due to an adaptation to the long term effects of the isolat ion procedure and not due to a n interference of the behavior test with gene activation. This explanation seems plausible, as IEGs are activation upon stimulation and are associated with exploratory behavior (Pace et al., 2005) and increased act ivation was seen with the group housed ra ts in this study. Isolation reared rats appear to have abnormalities in both social behavior and mPFC and amygdala activation. One possible reason for this is that isolates are lacking the inhibition needed to suppress aggressive behavior or lacking the mP FC and amygdala activation needed to modulate proper social behavior. Social responses are complex and require a finely tuned level of input from many brain regions in order to maintain a proper balance of inhibition and activation. An increase in aggressi ve and non aggressive behavior may be related to a decrease in IEG activation in a non linear manner. 4.5. Is novel social exposure stressful, rewarding, or both for isolation reared rats? Ongoing research in the lab is examining how rewarding social ex posure is to group housed and isolated rats. While a novel social exposure is thought to be somewhat stressful, it may be that this experience is also rewarding, and therefore not reflective of a negative stressor. Preliminary data suggest that very low levels of novel social exposure is enough to induce conditioned place preference (CPP) in male group housed and isolated rats, but not in females. Interestingly, when novel social exposure is paired with a single cocaine injection (2 mg/kg), male group housed and isolated rats again develop CPP, and so do female iso lated rats but not female group housed rats (Dayton et al., 20 11) These data indicate that the novelty stressor of social exposure might be a rewarding experience for rats and that this may differ according to sex and housing condition This relates to the increases in social interaction in isolates seen here and highlights the importance of caution in interpreting stress responses.

PAGE 45

" &, Desirable events may be experienced as "stressful," but the behavioral and b iochemical responses to such events may differ greatly from responses to undesirable events. It is clear that early life adversity can have long lasting behavior al and neurobiological effects. As more data are gathered about how behavior and the brain change in response to early life adversity it will become possible to tailor treatments for p eople suffering from a variety of mental illnesses related to abnormalities in stress responses. An individual who has experienced chronic stress early in life may have different treatment needs than someone with a different background; even if symptoms or diagnoses are similar. Males and females differ in their behavioral and physiological stress responses, indicating that different treatments may be called for. Additionally, the subjective experience of stress may differ according to one's background and sex This is an important consideration to keep in mind when addressing an individual's behavioral and emotional stress responses. Further examination of the outcomes of isolation rearing, and how those outcomes interact with social functioning in males an d females, will help to untangle the complex problem of behavioral and neurobiological deficits related to chronic stress early in life.

PAGE 46

" '" REFERENCES Adolphs, R. (2010). What does the amygdala contribute to social cognition? Ann N Y Acad Sci, 1191 (1), 42 61. Altemus, M. (2006). Sex differences in depression and anxiety disorders: Potential biological determinants. Hormones and Behavior, 50 534 538. Bell, H. C., Pellis, S. M., & Kolb, B. (2010). Juvenile play experience and the development of the orbitofrontal and medial prefrontal cortices. Behavioural Brain Research, 207, 7 13. Bland, S. T., Beckley, J. T., Fischer, E. K., Imonega, O. I., Ortiz, N. C., Watkins, L. R., & Maier, S. F. (2009, October). Isolation rearing and restraint stress produce sex dependent changes in medial prefrontal cortex immediate early gene expression. Poster session presented at the Society for Neuroscience annual meeting, Chicago, IL. Bland, S. T., Schmid, M. J., Der Avakian, A., Watkins, L. R., Spencer, R. L., & Maier S. F. (2005). Expression of c fos and BDNF mRNA in subregions of the prefrontal cortex of male and female rats after acute uncontrollable stress. Brain Research, 1051 90 99. Bock, J., Murmu, R. P., Ferdman, N., & Braun, K. (2008). Refinement of dendrit ic and synaptic networks in the rodent anterior cingulate and orbitofrontal cortex: critical impact of early and late social experience. Developmental Neurobiology, 68 (5), 685 695. Bramham, C. R., Worley, P. F., Moore, M. J., & Guzowski, J. F. (2008). Th e immediate early gene Arc/Arg3.1 : Regulation, mechanisms, and function. The Journal of Neuroscience, 28 (46), 11760 11767. Branchi, I., Francia, N., & Alleva, E. (2004). Epigenetic control of neurobehavioral plasticity: The role of neurotrophins. Behaviou ral Pharmacology, 15, 353 362. Carlson, N. R. (2008). Foundations of physiological psychology Boston, MA: Pearson Education Inc. Cook, S. C. & Wellman, C. L. (2004). Chronic stress alters dendritic morphology in rat medial prefrontal cortex. Journal of Neurobiology, 60, 236 248.

PAGE 47

" '$ Cunha, C., Brambilla, R., Thomas, K. L. (2010). A simple role for BDNF in learning and memory? Frontiers in Molecular Neuroscience, 3 (1), 1 14. Goodell, D., Wall, V., Grotewold, S., & Bland, S.T. (2011). Sex dependent effects o f adolescent social deprivation on combined social cue/drug conditioned place preference. Poster presented at the Society for Neuroscience annual meeting, Washington, D.C Day Wilson, K. M., Jones, D. N. C., Southam, E., Cilia, J., & Totterdell, S. (2006). Medial prefrontal cortex volume loss in rats with isolation rearing induced deficits in prepulse inhibition of acoustic startle. Neuroscience, 141, 1113 1121. Diamond, D. M., Campbell, A., Park, C. R., & Vouimba, R. (2004). Preclinical research on stress, memory, and the brain in the development of pharmacotherapy for depression. European Neuropsychopharmacology, 14 491 495. Douglas, L. A., Varlinskaya, E. I., Spear, L. P. (2004). Rewarding properties of social interactions in adolescent and adult male a nd female rats: Impact of social versus isolate housing of subjects and partners. Developmental Psychobiology, 45, 153 162. Ferdman, N., Murmu, R. P., Bock, J., Braun, K., & Leshem, M. (2007). Weaning age, social isolation, and gender, interact to determi ne adult explorative and social behavior, and dendritic and spine morphology in prefrontal cortex of rats. Behavioural Brain Research, 180, 174 182. File, S. E. & Seth, P. (2003). A review of 25 years of the social interaction test. European Journal of Ph armacology, 463 35 53. Flavell, S. W. & Greenberg. M. E. (2008). Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system. Annu Rev Neurosci, 31, 563 590. Fone, K. C. F. & Porkess, M. V. (2008). Behavioural a nd neurochemical effects of post weaning social isolation in rodents Relevance to developmental neuropsychiatric disorders. Neuroscience and Biobehavioral Reviews, 32, 1087 1102.

PAGE 48

" '% Ehlert, U., Gaab, J. & Heinrichs, M. (2001). Psychoneuroendocrinological con tributions to the etiology of depression, posttraumatic stress disorder, and stress related bodily disorders: The role of the hypothalamus pituitary adrenal axis. Biological Psychology, 57 141 152. Everhart, K. & Emde, R. N. (2006). Perspectives on stres s and self regulatory processes. In H. E. Fitzgerald, b. M. Lester, & B. Zuckerman (Eds.), The crisis in youth mental health: Critical issues and effective programs, Vol 1: Childhood disorders (pp. 1 24). Westport, CT: Praeger Publishers/Greenwood Publishi ng Group. Figueieredo, H. F., Bruestle, A., Bodie, B., Dolgas, C. M., Herman, J. P. The medial prefrontal cortex differentially regulated stress induced c fos expression in the forebrain depending on type of stressor. European Journal of Neuroscience, 18, 2357 2364. Gabbott, P. L. A., Warner, T. A., Jays, P. R. L., Salway, P. & Busby, S. J. (2005). Prefrontal cortex in the rat: Projections to subcortical autonomic, motor, and limbic centers. The Journal of Comparative Neurology, 492, 145 177. Gallucci, W T., Baum, A., Laue, L., Rabin, D. S., Chrousos, G. P., Gold, P. W., Kling, M. A. (1993). Sex differences in sensitivity of the hypothalamic pituitary adrenal axis. Health Psychology, 12 (5), 420 425. Goodell, D. J., Wall, V., Grotewold, S., & Bland, S. T (2011). Sex dependent effects of adolescent social deprivation on combined social cue/drug conditioned place preference. Poster presented at the Society for Neuroscience Annual Meeting, abstract #263.04. Greenberg, M. E., Xu, B., Lu, B., & Hempstead, B. L. (2009). New insights in the biology of BDNF synthesis and release: implications in CNS function. Journal of Neuroscience, 29 (41), 12764 12767. Hall, F. S. (1998) Social deprivation of neonatal, adolescent, and adult rats has distinct neurochemical and behavioral consequences. Critical Reviews in Neurobiology, 12 129 162.

PAGE 49

" '& Hammen, C., Brennan, P. A., Keenan Miller, D., Hazel, N. A., & Najman, J. M. (2010). Chronic and acute stress, gender, and serotonin transporter gene environment interactions predicti ng depression symptoms in youth. Journal of Child Psychology and Psychiatry, 51 (2), 180 187. Harro, J. & Oreland, L. (2001). Depression as a spreading adjustment disorder of monoaminergic neurons: A case for primary implication of the locus coeruleus. Bra in Research Reviews, 38 79 128. Hashimoto, K., Shimizu, E., & Iyo, M. (2004). Critical role of brain derived neurotrophic factor in mood disorders. Brain Research Reviews, 45, 104 114. Herman, J. P., Prewitt, C. M., & Cullinan, W. E. (1996). Neuronal ci rcuit regulation of the hypothalamic pituitary adrenocortical stress axis. Critical Reviews in Neurobiology, 10 (3 4), 371 394. Hermes, G., Li, N., Duman, C., & Duman, R. (2011). Post weaning chronic social isolation produces profound behavioral dysregulat ion with decreases in prefrontal cortex synaptic associated protein expression in female rats. Physiology and Behavior, 104 (2), 354 359. Hill, R. A., Wu, Y. C., Kwek, P., & van den Buuse, M. (2011). Modulatory effects of sex steroid hormone on BDNF TrkB ex pression during adolescent development in C57BI/6 mice. Journal of Neuroendocrinology, accepted article Hol, T., Van den Berg, C. L., Van Ree, J. M., & Spruijt, B. M. (1999). Isolation during the play period in infancy decreases adult social interactions in rats. Behavioural Brain Research, 100, 91 97. Holmes, A. & Wellman, C. L. (2009). Stress induced prefrontal reorganization and executive dysfunction in rodents. Neuroscience and Biobehavioral Reviews, 33, 773 783. Joels, M., Karst, H., Alfarez, D., He ine, V. M., Qin, Y., van Riel, E., Verkuyl, M., Lucassen, P. J., & Krugers, H. J. (2004). Effects of chronic stress on structure and cell function in rat hippocampus and hypothalamus. Stress, 7 (4), 221 231.

PAGE 50

" '' Kovacs, K. J. (2008). Measurement of immediate e arly gene activation c fos and beyond. Journal of Neuroendocrinology, 20, 665 672. Kozlovsky, N., Matar, M. A., Kaplan, Z., Kotler, M., Zohar, J., & Cohen, H. (2008). The immediate early gene Arc is associated with behavioral resilience to stress exposur e in an animal model of posttraumatic stress disorder. European Neuropsychopharmacology, 18, 107 116. Lauterborn, J. C., Rivera, S., Stinis, C. T., Hayes, V. Y., Isackson, P. J., & Gall, C. M. (1996). Differential effects of protein synthesis inhibition o n the activity dependent expression of BDNF transcripts: Evidence for immediate early gene responses from specific promotors. The Journal of Neuroscience, 16 (23), 7428 7436. Levine, J. B., Youngs, R. M., MacDonaldm M. L., Chu, M., Leeder, A. D., Berthiaum e, F., & Konradi, C. (2007). Isolation rearing and hyperlocomotion are associated with reduced immediate early gene expression levels in the medial prefrontal cortex. Neuroscience, 145, 42 55. Lu, Y., Christian, K. & Lu, B. (2008). BDNF: A key regulator f or protein synthesis dependent LTP and long term memory? Neurobiology of Learning and Memory, 89, 312 323. Lyford, G. L., Yamagata, K., Kaufmann, W. E., Barnes, C. A., Sanders, L. K., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Lanahan, A. A. & Worle y, P. F. (1995). Arc, a growth factor and activity regulated gene, encodes a novel cytoskeleton associated protein that is enriched in neuronal dendrites. Neuron, 14, 433 445. Maggio, N. & Segal, M. (2010). Corticosteroid regulation of synaptic plasticity in the hippocampus. Scientific World Journal, 10 462 469. Maier, S. F. (1984). Learned helplessness and animal models of depression. Prog Neuropsychopharmacol Biol Psychiatry, 8 (3), 435 446. Malarkey, W. B. & Mills, P. J. (2007). Endocrinology: The act ive partner in PNI research. Brain, Behavior, and Immunity, 21, 161 168.

PAGE 51

" '( Marinelli, M. & Piazza, P. V. (2002). Interaction between glucocorticoid hormones, stress and psychostimulant drugs. European Journal of Neuroscience, 16 (3), 387 394. McEwen, B. S. (2000). The neurobiology of stress: From serendipity to clinical relevance. Brain Research, 886, 172 189. Meng, Q., Li, N., Han, X., Shao, F., & Wang, W. (2010). Peri adolescence isolation rearing alters social behavior and nociception in rats. Neuroscie nce Letters, 480, 25 29. Monk, C. S. (2008). The development of emotion related neural circuitry in health and psychopathology. Development and Psychopathology, 20, 1231 1250. O'Leary, A. (1990). Stress, emotion, and human immune function. Psychological B ulletin, 108 (3), 363 382. Ostrander, M. M., Ulrich Lai, Y. M., Choi, D. C., Flak, J. N., Richtand, N. M. & Herman, J. P. (2009). Chronic stress produces enduring decreases in novel stress evoked c fos mRNA expression in discrete brain regions of the rat. S tress, 12 (6), 469 477. Pace, T. W., Gaylord, R., Topczewski, F., Girotti, M., Rubin, B., & Spencer, R. L. Immediate early gene induction in hippocampus and cortex as a result of novel experience is not directly related to the stressfulness of that experie nce. European Journal of Neuroscience, 22 (7), 1679 1690. Paxinos, G. & Watson, C. (1998). The rat brain in stereotaxic coordinates, (4 th Ed.). San Diego, CA: Academic Press. Pinaud, R. (2004). Experience dependent immediate early gene expression in the a dult central nervous system: Evidence from enriched environment studies. International Journal of Neuroscience, 114, 321 333. Radley, J. J., Sisti, H. M., Hao, J., Rocher, A. B., McCall, T., Hof, P. R., McEwen, B. S., & Morrison, J. H. (2004). Chronic beh avioral stress induces apical dendritic reorganization in pyramidal neurons of the medial prefrontal cortex. Neuroscience, 125, 1 6.

PAGE 52

" ') Roceri, M., Hendriks, W., Racagni, G., Ellenbroek, B. A. & Riva, M. A. (2002). Early maternal deprivation reduces the ex pression of BDNF and NMDA receptor subunits in rat hippocampus. Molecular Psychiatry, 7, 609 616. Romeo, R. D. (2010). Pubertal maturation and programming of the hypothalamic pituitary adrenal reactivity. Frontiers in Neuroendocrinology, 31 232 240. Sad ock, B. J. & Sadock, V. A. (2007). Kaplan & Sadock's synopsis of psychiatry: Behavioral sciences/clinical psychiatry. Philadelphia, PA: Lippincott Williams & Wilkins. Schinder, A. F. & Poo, M. (2000). The neurotrophin hypothesis for synaptic plasticity. T rends in Neuroscience, 23 (12), 639 645. Sisk, C. L. & Zehr, J. L. (2005). Pubertal hormones organize the adolescent brain and behavior. Frontiers in Neuroendocrinology, 26 (3 4), 163 174. Smith, M. A., Makino, S., Kvetnansky, R. & Post, R. M. (1995). Effec ts of stress on neurotrophic factor expression in the rat brain. Ann N Y Acad Sci, 29, 234 239. Smith, M. A., Makino, S., Kim, S. Y., & Kvetnansky, R. (1995). Stress induces brain derived neurotrophic factor messenger ribonucleic acid in the hypothalamus and pituitary. Endocrinology, 136 (9), 3745 3750. Spina, M. B., Squinto, S. P., Miller, J., Lindsay, R. M., & Hyman, C. (1992). Brain derived neurotrophic factor protects dopamine neurons against 6 hydroxydopamine and N methyl 4 phenylpyridinium ion toxici ty: involvement of the glutathione system. Journal of Neurochemistry, 5 (1), 99 106. Stack, A., Carrier, N., Dietz, D., Hollis, F., Sorenson, J., & Kabbaj, M. (2010). Sex differences in social interaction in rats: Role of the immediate early gene zif268. N europsychopharmacology, 35 570 580. Stauder, A., Thege, B. K., Kovacs, M. E., Balog, P., Williams, V. P., & Williams, R. B. (2010). Worldwide stress: Different problems, similar solutions? Cultural adaptation and

PAGE 53

" '* evaluation of a standardized stress manag ement program in Hungary. International Journal of Behavioral Medicine, 17 (1), 25 32. Steward, O. & Worley, P.F. (2001). Selective targeting of newly synthesized Arc mRNA to active synapses requires NMDA receptor activation. Neuron, 30, 227 240. Tafet, G. E. Idoyaga Vargas, V. P., Abulafia, D. P., Calandria, J. M., Roffman, S. S., Chiovetta, A., & Shinitzky, M. (2001). Correlation between cortisol level and serotonin uptake in patients with chronic stress and depression. Cognitive, Affective, and Behavio ral Neuroscience, 1 (4), 388 393. The National Campaign to Prevent Teen Pregnancy. (2005). The Adolescent Brain: A Work in Progress. [Brochure]. Washington, DC: Weinberger, D. R., Elvevag, B., & Giedd, J. N. Turrigiano, G. G., & Nelson, S. B. (2004). Home ostatic plasticity in the developing nervous system. Nature Reviews Neuroscience, 5, 97 107. Vazdarjanova A., Ramirez Amaya, V., Insel, N., Plummer, T. K., Rosi, S., Chowdhury, S., Mikhael, D., Worley, P. F., Guzowski, J. F., & Barnes, C.A. (2006). Spatia l exploration induces ARC, a plasticity related immediate early gene, only in calcium/calmodulin dependent protein kinase II positive principal excitatory and inhibitory neurons of the rat forebrain. J. Comp. Neurol., 498, 317 329. Vermetten, E. & Bremner J. D. (2002). Circuits and systems in stress. I. Preclinical studies. Depression and Anxiety, 15, 126 147. Walker, E. F. (2002). Adolescent neurodevelopment and psychopathology. Current directions in psychological science, 11 (1), 24 28. Waung, M. W., P feiffer, B. W., Nosyreva, E. D., Ronesi, J. A., & Huber, K. M. 2008. Rapid translation of Arc/Arg3.1 selectively mediates mGluR dependent LTD through persistent increases in AMPAR endocytosis rate. Neuron, 59 (1), 84 97.

PAGE 54

" '+ Wright, S. L., Langenecker, S. A., D eldin, P. J., Rapport, L. J., Nielson, K. A., Kade, A. M., Own, L. S., Akil, H., Young, E. A., Zubieta, J. K. (2009). Gender specific disruptions in emotion processing in younger adults with depression. Depression and Anxiety, 26 182 189. Young, E.A., Ko rszun, A., Figueiredo, H. F., Banks Solomon, M., & Herman, J. P. (2008). Sex differences in HPA axis regulation. In J. B. Becker, K. J. Berkley, N. Geary, E. Hampson, J. P. Herman, & E. A. Young (Eds.) Sex Differences in the Brain (95 105). New York, NY: O xford University Press, Inc.