Effect of Chronic Stress Exposure on Beta-adrenergic Receptor Signaling and Fear- Learning
A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy
By
Robert Michael Camp
June 2015
© Copyright
All rights reserved
Except for previously published materials Dissertation written by
Robert M. Camp
B.A. Kent State University, 2009 Ph.D. Kent State University, 2015
Approved by ______Dr. John D. Johnson, Advisor, Department of Biological Sciences
______Dr. Eric M. Mintz, Member, Department of Biological Sciences
______Dr. Heather K. Caldwell, Member, Department of Biological Sciences
______Dr. Aaron M. Jasnow, Outside member, Department of Psychology
______Dr. David C. Riccio, Graduate faculty representative, Department of Psychology
Accepted by ______Dr. Laura G. Leff, Chair, Department of Biological Sciences
______Dr. James L. Blank, Dean, College of Arts & Sciences
ii Table of Contents
Page
List of Figures………………………………………………………………………………….viii
List of Abbreviations…………………………………………………………………………….x
Acknowledgements…………………………………………………………………………….xii
Chapter I. Introduction………………………………………………………………………….1
Stress, defined…………………………………………………………………………..1
Stress and psychological disorder…………………………………………………….2
Selye and the GAS, Cannon and “Fight-or-Flight”...... 5
The neuroendocrine stress response………………………………………………...7
The central stress response………………………………………………………….11
Memory, stress, and norepinephrine………………………………………………...14
Repeated stress alters noradrenergic signaling……………………………………16
Focus of dissertation…………………………………………………………………..17
Chapter II. Strain- and context-dependent effects of repeated stressor exposure on the expression of depressive-like behavior in rats……………………………………………..22
Introduction…………………………………………………………………………….22
Methods and procedures……………………………………………………………..25
Subjects..……………………………………………………………………….25
Drug delivery…………………………………………………………………...26
Repeated stress protocol……………………………………………………..27
Sucrose preference measurements………………………………………....28
iii Behavioral measurements…………………………………………………....28
Organ weight and stress hormone measurement………………………….29
Statistical analysis……………………………………………………………..30
Study 1: effects of context on behavioral changes in animals exposed to
chronic stress……………………………………………………………….….30
Study 2: the role of β-ARs in enhanced contextual fear conditioning in
chronically stressed animals………………………………………………....30
Study 3: differential effects of chronic stress between central and
peripheral β-ARs……………………………………………………………....31
Study 4: disparity between laboratory rat strains in effects of chronic mild
stress exposure..………………………………………………………………31
Results………………………………………………………………………………….32
Study 1: effect of context on repeated stress-induced behavioral
changes………………………………………………………………………...32
Study 2: effect of propranolol on repeated stress-induced behavioral
changes………………………………………………………………………...37
Study 2: effect of propranolol on stress-induced changes in body and
organ weights..………………………………………………………………...43
Study 2: effect of propranolol on corticosterone levels following acute and
repeated stressor exposure………………………………………………….46
Study 3: effect of nadolol on repeated stress-induced behavioral
changes………………………………………………………………………...49
iv Study 3: effect of nadolol on spleen weight following repeated stressor
exposure…...…………………………………………………………………...54
Study 4: effects of repeated stress-induced behavioral changes in
Sprague-Dawley rats….……………………………………………………….54
Discussion………………………………………………………………………………58
Conclusion……………………………………………………………………………...62
Chapter III: Repeated stressor exposure enhances contextual fear memory in a beta- adrenergic receptor-dependent process and increases impulsivity in a non-beta receptor-dependent fashion...………………………………………………………………...64
Introduction……………………………………………………………………………..64
Methods and materials…….…………………………………………………………..66
Subjects…………………………………………………………………………66
Repeated stress protocol……………………………………………………...67
Behavioral testing………………………………………………………………68
Conditioned fear………………………………………………………..68
Open field...……………………………………………………………..69
Passive avoidance….………………………………………………….69
Beta-adrenergic receptor antagonist administration……………………….70
Statistical analysis……………………………………………………………..70
Experimental design…………………………………………………………..70
Results………………………………………………………………………………….71
Effect of CMS on freezing behavior………………………………………….71
v Effect of CMS on passive avoidance behavior……………………………..74
Effect of repeated stress on non-contextual anxiety-like behavior……….74
Discussion……………………………………………………………………………...76
Chapter IV: Effects of chronic mild stress on sensitization of beta-adrenergic receptor- stimulated intracellular signaling in amygdaloid and hippocampal tissue……………….80
Introduction……………………………………………………………………………..80
Methods and procedures……………………………………………………………...83
Subjects…………………………………………………………………………83
Chronic mild stress protocol……...…………………………………………...83
Beta-adrenergic receptor stimulation………………………………………...83
Cellular signaling analysis…………………………………………………….84
Statistical analysis……………………………………………………………..84
Experimental design………………………………………………….………..85
Results……………………………………………………………………….………….85
Effect of stress and propranolol on intracellular signaling in the
amygdala……………………………………………………………………….85
Effect of propranolol on intracellular signaling on intracellular signaling in
the hippocampus……………………………………………………………….89
Discussion………………………………………………………………………………92
Chapter V: Global discussion………………………………………………………………...95
Future directions……………………………………………………………………...103
Final thoughts…………………………………………………………………………105
vi References……………………………………………………………………………………107
vii List of figures
Page
Chapter I. Introduction
Fig. 1. The HPA axis and the sympathetic nervous system………………………………..8
Fig. 2. Hypothetical process of stress-induced enhancement of fear memory…………18
Fig. 3. Intracellular signaling at β-ARs………………………………………………………21
Chapter II. Strain- and context-dependent effects of repeated stressor exposure on the expression of depressive-like behavior in rats
Fig. 4. Effects of context on behaviors following repeated stressor exposure………….33
Fig. 5. Effects of chronic propranolol infusion on behaviors following repeated stressor exposure………………………………………………………………………………………..38
Fig. 6. Effects of chronic propranolol infusion and repeated stressor exposure on body and organ weights……………………………………………………………………………..44
Fig. 7. Effects of chronic propranolol infusion on corticosterone responses…………….47
Fig. 8. Effects of chronic nadolol infusion on behaviors following repeated stressor exposure………………………………………………………………………………………..50
Fig. 9. Effect of chronic nadolol infusion and repeated stressor exposure on spleen weight…………………………………………………………………………………………...55
Fig. 10. Behavioral effects of repeated stressor exposure in Sprague-Dawley rats.…..56
Chapter III: Repeated stressor exposure enhances contextual fear memory in a beta- adrenergic receptor-dependent process and increases impulsivity in a non-beta
viii receptor-dependent fashion
Fig. 11. Effects of chronic mild stress on contextual fear conditioning…………………..72
Fig. 12. Effects of chronic mild stress on latency in a passive avoidance test..….……..73
Fig. 13. Effects of chronic mild stress on open field behavior.……………………………75
Chapter IV. Effects of chronic mild stress on sensitization of beta-adrenergic receptor- stimulated intracellular signaling in amygdaloid and hippocampal tissue
Fig. 14. Effects on intracellular signaling in the amygdala following repeated stressor exposure………………………………………………………………………………………..86
Fig. 15. Effects on intracellular signaling in the hippocampus following repeated stressor exposure………………………………………………………………………………………..90
Chapter V. Global discussion
Fig. 16. Observed process of stress-induced enhancement of fear memory...…………96
Fig. 17. Additional components of signaling at β-ARs……………………………………102
ix List of abbreviations
ACTH - adrenocorticotropic hormone
AMPA(R) - α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (receptor)
β-AR - beta-adrenergic receptor
BDNF - brain-derived neurotrophic factor
BLA - basolateral nucleus of the amygdala
CAMK-IV - calmodulin kinase IV cAMP - 3’-5’-cyclic adenosine monophosphate
CMS - chronic mild stress
CORT - corticosterone or cortisol
CREB - cAMP response element-binding
CRH - corticotropin-releasing hormone
DEX - dexamethasone
EDTA - ethylenediaminetetraacetic acid
ELISA - enzyme-linked immunosorbent assay
ERK - extracellular signal-regulated kinase
GAD - generalized anxiety disorder
GAS - general adaptation syndrome
GR – glucocorticoid receptor
GluR1 - glutamate receptor 1
HCC – home cage controls
HPA - hypothalamic-pituitary-adrenal
x IL-1β - interleukin-1 beta
IL-6 - interleukin-6
LC - locus cœruleus
LTP - long-term potentiation
MAPK - mitogen-activated protein kinase
MHPG - 3-methoxy-4-hydroxyphenethylene glycol
NE - norepinephrine
NMDA - N-methyl-D-aspartate
NTS - nucleus tractus solitarius or nucleus of the solitary tract
PFC - prefrontal cortex
PKA - protein kinase A
PTSD - posttraumatic stress disorder
RT-PCR - real-time polymerase chain reaction
SNS - sympathetic nervous system
TMT - trimethylthiazoline
TNF-α - tumor necrosis factor alpha
xi Acknowledgments
I am terrible at this sentimental stuff, and anyone who really knows me will understand.
So I beg of you, everyone who will actually be reading this, to please bear with me in this awkwardness.
To my advisor, John: You introduced me to the world of real scientific research and you allowed me to head in my own direction, even as it diverged from the lab's original focus. But most importantly, thank you for your endless patience and understanding, especially towards the end. I know I have my demons, and I'm doing my best to beat them; I appreciate that you understand the difficulties I have faced in doing so and have tolerated me throughout, as I am sure it has been especially difficult in these final months.
To the members of my committee, Eric, Heather, and Aaron: Thank you for helping to realize that this process doesn't involve being thrown to the lions. My mind and body may still react that way to a certain degree, but at least I'm consciously aware that it isn't the case.
To Colleen: You weren't even on my committee and yet you were so enthusiastic and helpful with my ongoing job search. I hope your students realize how good they have it with you as an advisor.
To Veronica: You have been my unofficial mentor throughout my graduate career, even after you had to leave for other opportunities. I will always look to you when I might have a problem, but more importantly, when I don't.
To Jenny: Through all of this, you have been my rock, commiserating and
xii empathizing with the difficulties of being a graduate student and a parent, and the hours of conversation we have shared over the years have been a mindsaver.
To Steve: You have been so helpful, especially in the last six months, with all your advice and encouragement on the job hunt, and I wish you the best on your own new job.
To Sahana and Kristin: Both of you have been so fantastic with your undergrad research studies and you have been great helpers in my experiments. You are so talented and I know that you both will do spectacularly on your own.
To my parents, Victor and Dreena, and my sister, Jamey: Thanks for all of your help, especially in these last few years, whether with Eva, the seemingly endless car repair, or just believing I could get through it, thank you. I only hope I did right by all of you this time.
To my wife, Jen: For the last decade and a half, even through the darkest times, you have been with me where no one else would go. I will never understand why you have stayed with me all this time, nor will I ever understand what it is you saw in me that made you join me. If not for your devotion, I would have gone through this entirely alone, if I ever started in the first place; you supported me from the beginning and suffered for it for so long, now I hope I finally can give you the better life that you deserve. You and Eva are all I need, so long as I have you two, I still have a reason to go on.
Finally, to my daughter, Eva: I honestly don't know if the efforts I'm making will pay off and finally get us out of the gutter, but I can at least take us to the edge. If you
xiii stand on my shoulders, you can finally climb free. Please, don't grow up to be like your dad, you can do so much better.
xiv Chapter I
Introduction
Stress, defined
While the concept of stress is notoriously difficult to define, modern researchers agree that at its core, stress is a disruption in an organism’s homeostasis, the optimal state of biochemical equilibrium in which an organism lives (Chrousos & Gold, 1992). Stress can be any factor, internal or external, that disrupts this equilibrium, whether they are threats to survival like predation or disease, or benign events such as stage performance or verbal confrontation between siblings.
No matter the source, stress requires the organism to undergo a variety of physiological changes in order to return to its natural regulatory state, in a process called allostasis (McEwen, 1998). Such changes involve neuroendocrine and subsequent metabolic processes as a means to distribute energy as to most efficiently aid in the organism’s survival; these changes can also lead to altered behavioral states that serve a purpose in the immediate-to-short term preservation of life. There are times when conditions (e.g. an unexpected weather hazard imposes on an organism’s habitat) require an organism to temporarily suspend certain functions, such as seeking food or a
1 mate, in order to confront, escape, or otherwise manage the more immediate threat.
Once more acceptable conditions are in place, suspended functions resume and equilibrium is once again reached. In this way, stress is adaptive and beneficial, whether facing an acute stressor or just going through daily life (McEwen, 2007).
Yet when allowed to continue for extended periods of time, these changes in both physiology and behavior, results of allostatic load (McEwen, 2004, 2005), can prove detrimental as growth and development are stunted, reproductive function and fertility decreases, digestion is inhibited, resistance to disease diminishes, and, as will be discussed in greater detail, deleterious affective, behavioral, and cognitive changes develop (Sapolsky, 1992).
Stress and psychological disorder
Affective disorders are rapidly becoming more prominent in Western society, and are contributing more to increases in health care costs and losses of productivity. As stress is a significant contributing factor to the development of such disorders, it will likely be a persisting problem as incidence rates for the diseases continue to rise.
Our bodies will undergo the same neuroendocrine changes whether we are facing the end of a long-term intimate relationship, financial destitution, the aftermath of an earthquake, or the horrors of war. We suffer stress through social interaction, from strife at school or the workplace (Kaltiala-Heino, Fröjd, & Marttunen, 2010; Kaltiala-
Heino, Rimpelä, Marttunen, Rimpelä, & Rantanen, 1999; Kivimäki et al., 2003; Seals &
Young, 2003), maltreatment from a parent or spouse (Bradley et al., 2014; Cascardi,
O’Leary, & Schlee, 1999), or a breakup or divorce (Field, Diego, Pelaez, Deeds, &
2 Delgado, 2009; Hill & Hilton, 1999; Najib, Lorberbaum, Kose, Bohning, & George, 2004;
Stack & Scourfield, 2013). Sociological stressors, particularly poverty, are well- documented to be at the center of depression (Belle & Doucet, 2003; G. W. Brown &
Moran, 1997; Patel, Abas, Broadhead, Todd, & Reeler, 2001), through homelessness
(Goodman, Saxe, & Harvey, 1991), maternal stress (Bergman, Sarkar, Glover, &
O’Connor, 2010), and insufficient maternal or perinatal care (Buitelaar, Huizink, Mulder, de Medina, & Visser, 2003; Fish et al., 2004) and subsequent low infant birth weight
(Hack et al., 2002, 2004, 2009). Stress can also manifest physically, through natural disasters (Foa, Stein, & McFarlane, 2006; Ginexi, Weihs, Simmens, & Hoyt, 2000;
Nolen-Hoeksema & Morrow, 1991), war (Erickson, Wolfe, King, King, & Sharkansky,
2001; Ginzburg, Ein-Dor, & Solomon, 2010; Thabet, Abed, & Vostanis, 2004), or even through an individual’s own illnesses or injuries (Scaf-Klomp, Sanderman, Ormel, &
Kempen, 2003; Suhr & Gunstad, 2002; Turvey, Schultz, Beglinger, & Klein, 2009;
Yirmiya, 2000).
Depression is characterized by symptoms such as fatigue, low mood, low self- esteem, loss of interest and motivation, and feelings of hopelessness and worthlessness (American Psychiatric Association (APA), 2013; World Health
Organization (WHO), 1992). It affects 16.9% of American adults over a lifetime, 9.1% over a twelve-month period (Andrade et al., 2003; Centers for Disease Control and
Prevention (CDC), 2010) with 3.4% of depressed patients committing suicide. In fact, it is estimated that 60% of all people who committed suicide suffered from depression
(Barlow & Durand, 2005). As of 2005, suicide was the eighth leading cause of death
3 among all American males and is ranked higher when accounting for age; suicide is the fifth leading cause of death in males aged 5-14, third in males aged 15-24, and fourth in males aged 25-44 (National Center for Health Statistics (NCHS), 2007). Antidepressant use in physician office visits has more than doubled for both males and females in the period between 1995 and 2005, and is the most abundantly prescribed drug for females
(NCHS, 2007).
Stress is also associated with anxiety disorders, which are a group of disorders characterized by worry over future events and a fearful reaction about current events
(APA, 2013). The lifetime prevalence of anxiety disorders in the United States is 29%
(Kessler, Chiu, Demler, & Walters, 2005), and will affect 11-18% of American adults in a twelve-month period (Corner & Olfson, 2010); these are often comorbid with depression. One of these, generalized anxiety disorder (GAD), is marked with excessive irrational worry over everyday minor things and a variety of physical symptoms ranging from tension and muscle aches to irritability and restlessness (WHO, 1992); GAD has a comorbidity rate of 12% with major depressive disorder (Moffitt et al., 2007).
Post-traumatic stress disorder (PTSD) is likely to develop when one is exposed to an extremely adverse event, such as child or spousal abuse, terrorism, or natural disaster; likelihood is increased when the trauma is assault-based. Patients experience recurring flashbacks, memories of the traumatic event, hypervigilance, among other symptoms (APA, 2013). Twenty million American adults are diagnosed with PTSD over their lifetimes, women are more likely to develop it than men, and children are more resilient to trauma than adults. PTSD is much more common in veterans, with 30.9% of
4 all male Vietnam veterans being diagnosed (Kulka et al., 1988) and approximately 10-
14% of the total military population in more recent wars (Kang, Natelson, Mahan, Lee, &
Murphy, 2003; Tanielian & Jaycox, 2008).
Depression and anxiety disorders are responsible for increases in absenteeism from work and school, as well as the cost of healthcare; the national cost of mental health services topped $100 million in 2003 (NCHS, 2007). Depression alone is now responsible for more absenteeism than any other disease, with sufferers taking three times as many sick days as those without depression; this lost productivity costs employers $44 billion (Stewart, Ricci, Chee, Hahn, & Morganstein, 2003). Patients of these disorders often endure failed marriages, their social lives are impaired, and they can be prone to substance abuse. They have a more difficult time just getting by day to day; approximately 80% of those with depression reported some related functional impairment, while 27% reported serious difficulties in their everyday lives (Pratt & Brody,
2008).
Selye and the GAS, Cannon and “Fight-or-Flight”
Two major concepts of stress, as we study it today, were first described by Walter
Cannon, who first described the “Fight-or-Flight” response in the early twentieth century
(Cannon, 1929, 1932), and by Hans Selye, who introduced the concept of the General
Adaptation Syndrome (GAS) in 1936 and elaborated upon it in 1950.
The fight or flight response is a set of physiological reactions that occur in response to a threat to one’s survival. The reaction is initiated by the sympathetic nervous system in order to prepare the body to defend itself or to escape the threat, as
5 it facilitates important survival mechanisms such as energy liberation, enhanced analgesia, and cardiovascular tone (Cannon, 1932; Sapolsky, 1992). The onset of
Selye’s general adaptation syndrome occurs following “[severe damage] by acute non- specific agents” that include stressors such as physical injury, surgery, or drug intoxication; the resulting symptoms that develop do not necessarily have to be akin to the damage incurred by the organism, but are rather the organism’s biological response to it. There are three stages of progression to the GAS; in the first, the “alarm” stage, a number of symptoms arise, including the atrophy of the spleen, thymus glands, and lymph nodes, loss of muscle mass (tone, as Selye puts it), and a decrease in the production of adrenal cortical lipoids and chromaffin cells. The body will eventually begin to adapt to the physiological changes seen in the alarm stage. In this “resistance” phase, the thyroid and adrenals see a rebound in cell proliferation to compensate for what was lost in the alarm phase. This comes at the cost of production of other hormones such as gonadotropins, growth hormones, and prolactin, which suggests that the body prioritizes metabolic and adrenotropic hormones over those for growth and development. In the final stage, the body will build resistance to the stressor to the point that everything nearly returns to a state of equilibrium. The organism will also alter its behaviors accordingly as a means to avoid further stress, this is where anxiety- and depressive-like behaviors begin to develop. Should exposure to the stressor continue, however, this resistance fails and the organism will give in to symptoms similar to those of the alarm stage due to a depletion of biological resources; this is referred to as the
“exhaustion” phase. As the body diverts its resources toward survival mechanisms,
6 other physiological processes are essentially shut down, ignored. This final stage of the
GAS has numerous pathological consequences, in that stress causes or aggravates a wide array of conditions from hypertension and peptic ulcers, to diabetes, ischemia, or even cancer.
The neuroendocrine stress response
The stress response is governed by two major neuroendocrine systems (Fig. 1).
The hypothalamic-pituitary-adrenal (HPA) axis is a system of interactions and feedback loops between three major endocrine structures. As its name implies, the HPA axis is a circuit involving the hypothalamus, the pituitary gland, and the adrenal glands. In response to a stressor, neurons that lie in the paraventricular nucleus of the hypothalamus release corticotropin-releasing hormone (CRH) into the hypophyseal portal system, activating corticotropes in the anterior pituitary gland for release of adrenocorticotropic hormone (ACTH). This then leads to stimulation of cells in the zona fasciculata of the adrenal cortex to produce glucocorticoids, with CORT (cortisol in humans, corticosterone in animals frequently used in research) being the one most widely associated with stress. The HPA circuit is completed through a negative feedback loop activated by circulating CORT.
Excessive HPA axis activity is associated with affective dysfunction, and a lack of feedback response control in cortisol production following administration of dexamethasone (DEX), an exogenous glucocorticoid, is often associated with depression in humans. DEX binds to glucocorticoid receptors in the pituitary gland, activating a negative feedback loop and suppressing the expression of ACTH. A
7 8 Figure 1. (A–B) The HPA axis and the sympathetic nervous system. (A) During exposure to stress, CRH is secreted from the paraventricular nucleus of the hypothalamus, which passes through the median eminence to the anterior lobe of the pituitary. Here, corticotropes release ACTH into the bloodstream to activate CORT production in the adrenal cortex; CORT activates receptors in the brain to inhibit further HPA activity, acting as the end of a negative feedback circuit. (B) Through the activation of the sympathetic nervous system, stress affects numerous organs and processes throughout the body. As the purpose of these effects are to liberate energy where needed in an immediate threat and to otherwise conserve it, some bodily functions are enhanced while others are inhibited. Most effects of the sympathetic response are mediated by norepinephrine (highlighted in blue), with two exceptions mediated by acetylcholine (highlighted in orange).
Abbreviations: AHP -- adenohypophysis (anterior pituitary); CG -- celiac ganglion; IMG -- inferior mesenteric ganglion; PVN -- paraventricular nucleus; SCG -- superior cervical ganglion
Adapted from Herman et al., 2003 and from OpenStax CNX, 2013.
9 measure known as the dexamethasone suppression test, typically employed in diagnosing Cushing’s syndrome, has also been used experimentally to assess the dysfunctional HPA response in depressed patients (Carroll, 1982); a 1.0mg dose of DEX is given and blood CORT is measured the following day at three time periods. In healthy patients, DEX administration results in reduced cortisol levels; however, this suppression is impaired in 46.2% patients with depression, if their CORT is suppressed at all (Rush et al., 1996). In animal models, prolonged exposure to stress leads to elevated basal CORT levels and a sensitized CORT response to acute stress (Bielajew,
Konkle, & Merali, 2002; Katz, Roth, & Carroll, 1981), as well as an impairment in negative feedback to the HPA axis, as seen in elevated levels of ACTH during exposure to novel acute stressors (Aguilera, 1994; Odio & Brodish, 1990). HPA axis hyperactivity contributes to affective dysfunction in a number of ways, for instance, through neurotoxicity and dendritic atrophy (Reagan & McEwen, 1997; Sapolsky, 2000b) and suppression of neurogenesis (Gould, Beylin, Tanapat, Reeves, & Shors, 1999; Gould,
Cameron, Daniels, Woolley, & McEwen, 1992; Reagan & McEwen, 1997). The morphological changes facilitated by glucocorticoids, found particularly in the hippocampal formations, are associated with depression in humans (Bremner et al.,
2000; Sheline, Sanghavi, Mintun, & Gado, 1999; Sheline, Wang, Gado, Csernansky, &
Vannier, 1996).
The sympathetic nervous system (SNS) is a subdivision of the autonomic nervous system, the involuntarily acting portion of the peripheral nervous system. It is the primary mechanism that underlies the aforementioned fight-or-flight response.
10 Sympathetic activation leads to the physiological changes that facilitate heightened sensitivity (e.g. dilated pupils, tunnel vision and auditory exclusion and other such selective perceptual narrowing), awareness (e.g. enhanced memory processing in relation to emotion), and responsiveness (e.g. increased blood sugar, dilated respiratory tract for increased airflow, elevated heartbeat, vasodilation in skeletal muscles). The
“currency” of the SNS is norepinephrine, a monoamine released by neurons throughout the system. Again, in the short term, the SNS-mediated stress response is vital in managing danger, but can also be dangerous when allowed to continue unabated.
Associated with depression is an increase in sympathetic tone, which includes physiological hyper-reactivity to environmental stressors, increases in heart rate and blood pressure, as well as a decrease in heart rate variability (Grippo, Moffitt, &
Johnson, 2002). As such, much of the neuroendocrine mechanism behind stress- induced affective dysfunction that will be discussed in this thesis is mediated by norepinephrine and the activation of its receptors in certain regions of the brain.
The central stress response
Throughout the brain, the stress response is expressed in myriad different fashions, resulting in a multitude of behavioral effects. Stress manifests in limbic structures like the amygdala, the hippocampus, and the bed nucleus of the stria terminalis (Flavin &
Winder, 2013; Ventura-Silva et al., 2012; Walker, Toufexis, & Davis, 2003) through expression of fear as well as in both enhancement and deficits in memory. In the prefrontal cortex, decision-making processes and other executive functions can be altered, and stress affects dopamine release in one of the brain’s foremost centers of
11 motivation and reward, the nucleus accumbens (Abercrombie, Keefe, DiFrischia, &
Zigmond, 1989; Kalivas & Duffy, 1995; Y. L. Wu, Yoshida, Emoto, & Tanaka, 1999). In immediacy, these effects are vital to survival; however, after a prolonged period of duress, they become a detriment to everyday function. Enhanced emotional memory processing leads to exaggerated fear behavior and anxiety, and often results in rumination over perceived negative experiences. Irrational and impulsive behavior can develop, and when combined with a dysfunctional reward pathway, stress can lead to addiction.
As psychological stressors are perceived in part by the sensory organs, information is transmitted throughout the brain, including the cortex, to the nucleus tractus solitarius (NTS) and to the locus cœruleus (LC) (Ferry & Quirarte, 2012), two noradrenergic neuron-rich centers located in the brainstem. When activated, these regions synthesize norepinephrine within the brain, mediating such stress responses as anxiety, panic, and increased alertness; exposure to uncontrollable stress leads to a substantial depletion of norepinephrine in the LC, which is later associated with depressive behaviors (Weiss et al., 1981). The LC projects to several sites throughout the brain; among them are the prefrontal cortex, the bed nucleus of the stria terminalis, the amygdala, and the hippocampus, whose functions involve emotion and memory, and are key in behavioral control and the expression of anxiety; as such, they are major targets of psychopathological research.
Norepinephrine mediates many of the effects of stress in developing pathological behavioral expression. Norepinephrine release in amygdaloid nuclei, BNST, and
12 prefrontal cortex subsequent to acute restraint stress leads to anxiety-like behaviors in elevated plus maze and defensive burying behavioral assessments, as well as the development of social withdrawal (Morilak et al., 2005). The use of the adrenergic autoreceptor antagonist yohimbine has produced similar effects to endogenous norepinephrine release in rodent models (ibid.) and has also produced increases in self- reported anxiety and in autonomic symptoms in humans (Dennis S Charney, Heninger,
& Redmond, 1983), and have been blocked through the use of α- and β-receptor antagonists. Noradrenergic hyperactivity is implicated in other anxiety disorders such as panic disorder and social phobia, demonstrated through measures of autonomic activity such as skin conductance and responses as well as vagal withdrawal after isometric exercise (Sullivan, Coplan, Kent, & Gorman, 1999). Norepinephrine impairs conditioned fear extinction by enhancing reconsolidation in the amygdala (Dębiec, Bush, & LeDoux,
2011; Dębiec & LeDoux, 2006), which is mitigated with the use of propranolol (Koob,
1999); such research has suggested a role for norepinephrine in the pathological persistence of the traumatic memories of PTSD patients.
The amygdalae will receive special focus in this dissertation as they are particularly important in learning about aversive events and the anxiety that follows.
They are a bilateral pair of groups of nuclei located deep in the medial regions of the temporal lobes, involved in memory, decision-making, and most importantly, in emotional processing (Lanteaume et al., 2007). Chief among the emotions regulated by the amygdala is fear; electrical stimulation of the amygdala leads to behaviors typically associated with fear (Davis, 1992) and lesions to the region are known to reduce or
13 even eliminate behaviors motivated by fear (Feinstein, Adolphs, Damasio, & Tranel,
2011). The amygdala is also important as the site of fear memory consolidation through synaptic strengthening via long-term potentiation (LTP) (LeDoux, 2007; Sigurdsson,
Doyère, Cain, & LeDoux, 2007). The amygdala is also closely associated with stress- related depression in both rodent models of repeated stress and in humans (Sibille et al., 2009; Surget et al., 2009); a recent fMRI study shows amygdala reactivity to be a predictive biomarker for vulnerability to depression, generalized anxiety, panic, and
PTSD (Swartz, Knodt, Radtke, & Hariri, 2015).
The hippocampal formation, a seahorse-shaped extension of the cerebral cortex, is one of the brain's principal memory centers, especially concerning contextual memory. It is among the first regions of the brain to suffer the effects of Alzheimer’s disease (Hampel et al., 2008), and was the first discovered to show the mechanism of
LTP (Bliss & Lømo, 1973). The hippocampus is implicated in both anxiety (Kalisch et al.,
2006) and depression (Videbech & Ravnkilde, 2004), in the form of reduced volume; this is also a common morphological feature of PTSD (Delahanty, 2011). Contributing to this in part is a reduction in the expression of hippocampal brain-derived neurotrophic factor (BDNF), a deficit that is alleviated with the administration of antidepressants
(Kimpton, 2012; Masi & Brovedani, 2011). In addition to reducing cell proliferation, stress also increases the incidence of apoptosis in the hippocampus (Fuchs, Czéh,
Kole, Michaelis, & Lucassen, 2004).
Memory, stress, and norepinephrine
Memories are more persistent when associated with strong emotions, and those of
14 stressful events tend to be more perseverant than others (Cahill & McGaugh, 1998;
McGaugh, 2004). Memory performance is known to increase with exposure to acute stressors (Henckens, Hermans, Pu, Joëls, & Fernández, 2009; Yuen et al., 2009). As some studies indicate that acute stress is an impediment to memory (LeBlanc, 2009;
McGaugh & Roozendaal, 2002), explanations have surfaced emphasizing the importance of timing, physical location, and emotional state -- i.e. context -- in enhanced memory processing (Joëls, Pu, Wiegert, Oitzl, & Krugers, 2006; Smeets, Giesbrecht,
Jelicic, & Merckelbach, 2007). In any event, neuroendocrine factors are highly implicated in emotionally-enhanced memory processing (Beylin & Shors, 2003;
McReynolds et al., 2010), occurring both directly (McGaugh, 2004; Roozendaal &
McGaugh, 2011) and indirectly in the amygdala (Ferry & Quirarte, 2012).
Infusion of norepinephrine (Roozendaal, McEwen, & Chattarji, 2009) or a β- adrenergic receptor (β-AR) agonist (Ferry, Roozendaal, & McGaugh, 1999) alone is implicated in increasing behaviors associated with learned fear in an passive avoidance test; meanwhile, decreasing or blocking norepinephrine release in the basolateral nucleus of the amygdala (BLA) has been documented to reduce the region's memory functioning capabilities as well, through the use of drugs such as β-endorphin (Quirarte,
Galvez, Roozendaal, & McGaugh, 1998), zinterol (Quirarte, Roozendaal, & McGaugh,
1997), and propranolol (Hatfield & McGaugh, 1999; Quirarte et al., 1997). The ability of norepinephrine to enhance memory formation could possibly be due to an increase in
LTP (Whitlock, Heynen, Shuler, & Bear, 2006), occurring through a signal transduction pathway beginning with the production of 3’-5’-cyclic adenosine monophosphate
15 (cAMP). While this is typically initiated by Ca2+ flowing through NMDA receptors, it is thought that LTP can be enhanced by the activation of β-ARs (Straube & Frey, 2003;
Straube, Korz, Balschun, & Frey, 2003), occurring through a slow Ras/MAPK pathway converging with a rapid calmodulin kinase IV (CAMK-IV) pathway (Watabe, Zaki, &
O’Dell, 2000; G.-Y. Wu, Deisseroth, & Tsien, 2001). Activation of beta-receptors by noradrenergic neurons activates ERK, which in turn phosphorylates cAMP response element-binding (CREB) protein, a nuclear transcription factor that facilitates expression of a number of genes. These include GRIA1-4, which encode for AMPA receptors, the production of which contributes to increasing synaptic strength, the central mechanism in LTP and synaptic plasticity. Working in tandem with CRH and glucocorticoids, beta- adrenergic receptor activation and subsequent cAMP-PKA-CREB signaling influences memory consolidation in the BLA (Hubbard, Nakashima, Lee, & Takahashi, 2007;
Roozendaal, Quirarte, & McGaugh, 2002; Roozendaal, Schelling, & McGaugh, 2008).
Evidence of this lies in increases in memory consolidation produced by glucocorticoid receptor (GR) agonist RU28362 being blocked with a concurrent infusion of the beta- blocker atenolol into the BLA (Quirarte et al., 1997), and as infusions of the GR antagonist RU38486 following a passive avoidance training task dampened the memory-enhancing effects of the β-receptor agonist clenbuterol (Roozendaal, Quirarte, et al., 2002).
Repeated stress alters noradrenergic signaling
Repeated exposure to stress profoundly alter the adrenergic system at multiple sites in the brain. As exposure to stress elevates norepinephrine levels and increases its
16 metabolism, a mechanism of neural adaptation is activated to counteract the increased noradrenergic receptor activity that comes with the stress (Seo, Shin, Seung, Han, &
Ko, 1999; Woo, Wilson, Sullivan, & Leon, 1996). To attenuate the adrenergic response with repeated exposure to stress, β-adrenergic receptor expression is downregulated in the hypothalamus, amygdala, and hippocampus, and β-AR binding affinity is decreased in the hypothalamus as well (Porterfield, Gabella, Simmons, & Johnson, 2012). In addition to these effects on β-ARs, stress is reported to reduce binding affinity in α2- adrenergic receptors in the amygdala, and in norepinephrine transporters in the amygdala and hippocampal formations, of Wistar Kyoto rats (Tejani-Butt, Paré, & Yang,
1994). Despite the neural adaptation that occurs following repeated stressor exposure there is recent evidence to suggest that β-AR signaling in the amygdala is enhanced following repeated stressor exposure, as there is a stress-induced increase in IL-1β production in the region (Veronica M Porterfield et al., 2012). It is yet unclear if the changes in β-AR activation within the amygdala following repeated stressor exposure affect the formation of fear memories.
Focus of dissertation
My graduate study began with my interest in the etiology of affective disorders, primarily depression and dysthymic disorders and generalized anxiety. I set out to establish an animal model of chronic mild stress (CMS) for use in psychoneuroimmunological studies. Studies performed in Chapter II were initially designed to test the initial hypothesis that the depressive behaviors that manifest as a consequence of repeated stress exposure are mediated by beta receptor stimulation, which can then be
17 Figure 2. Hypothetical process of stress-induced enhancement of fear memory. CMS will sensitize β-ARs, whose stress-induced activation will then enhance fear learning in context, leading to the development of anxiety-like behaviors. With no contextual cues present, no such behaviors will manifest.
18 attenuated or blocked with the use of beta blockers. After further experimentation and circumstances within the laboratory, another hypothesis was developed that some stress-associated behaviors are actually a result of contextual learning and association of stressors with environmental cues, and are more aligned with fear and anxiety than with depression; without these cues, stress-induced anxiety-like behaviors do not develop.
Chapter III presents a set of studies that focus more specifically examining the effects of chronic stress on learning and memory. While these first studies are primarily behavioral in nature, they do include elements that serve to answer a question about neuroendocrine function through the use of receptor antagonists. In these studies, the focus is explicitly on behaviors associated with fear and anxiety. As discussed above, there is substantial evidence that β-AR stimulation, with direct central infusions of norepinephrine or receptor agonists, enhance fear conditioning. As exposure to stress stimulates β-ARs, I predict that I can achieve similar results. My hypothetical expectations are thus threefold. First, subjects exposed to a repeated variable stress paradigm will show enhanced associative learning demonstrated by increased fear behaviors in a contextual fear conditioning test and by increased retention latency in a passive avoidance test. Second, the fear behaviors will not be expressed in an environment with no cues with which stress can be associated (e.g. open field). Finally, as with the previous set of experiments, the development of stress-induced fear behaviors are mediated through β-AR stimulation and will thus be blocked with beta- receptor antagonists.
19 The third part of this dissertation, discussed in Chapter IV, aims to examine intracellular signaling in the amygdala and hippocampus. I hypothesize that stress sensitizes beta-adrenoceptor activation and subsequent intracellular signaling, which mediates the resulting behavioral phenotypes. As β-ARs are G protein-coupled receptors, I propose that, as beta receptors are stimulated in relevant brain regions (i.e. amygdala, hippocampus) of subjects repeatedly exposed to stress, a greater amount of cell signaling molecules (i.e. cyclic AMP, p44/42 MAPK, Fig. 3) will be expressed or phosphorylated than in the brains of subjects not exposed to stress. Ultimately, the goal of this battery of studies is to gain more knowledge and insight into how exposure to stress leads to some of the hallmarks of anxiety disorders, and perhaps also to give researchers cause to consider what precautions may be necessary in designing future studies on stress.
20 Figure 3. Intracellular signaling at β-ARs. Upon activation, the α-subunit of a beta- adrenergic receptor activates adenylate cyclase, catalyzing the conversion of ATP into cAMP. The cAMP facilitates a cascade that includes the phosphorylation of ERK1/2, and the later activation of the transcription factor CREB. Phosphorylated CREB then facilitates genetic expression, including that for AMPA receptors, which play a key role in LTP.
21 Chapter II
Strain- and context-dependent effects of repeated stressor exposure on the
expression of depressive-like behaviors in rats1
Introduction
Repeated exposure to laboratory stressors is often reported to result in behavioral changes and anhedonia (decreased ability to feel pleasure) in laboratory animals
[reviewed in Willner (2005)]. However, many laboratories have found it difficult to replicate these findings, suggesting there are variables contributing to stress-induced behavioral changes that remain unknown (Forbes, Stewart, Matthews, & Reid, 1996). A recent finding by Greenwood et al. demonstrates fear conditioning contributes to escape deficits in the learned helplessness model when the stressor and testing environments share common cues (Greenwood, Strong, & Fleshner, 2010). The potential for conditioned fear responses to inhibit behavioral responses when repeated stressor exposure and behavioral testing occur in the same environment has not been examined.
Fear conditioning is a well-characterized and widely studied phenomenon. It occurs when an animal is exposed to an aversive stimulus and an unconditioned fear
22 response becomes associated with an environmental cue such as the context in which the aversive stimuli occurred (Rau & Fanselow, 2009; Rudy, Huff, & Matus-Amat, 2004).
Fear conditioning is typically studied by placing an animal in a conditioning chamber
(e.g. operant box) where it is exposed to an aversive event (e.g. footshock), and then later testing the animals’ memory of the event by re-exposing the animal to the chamber and measuring their conditioned fear response (e.g. time spent freezing). It has been recognized that the representation an animal forms of the environment in which an aversive event occurs include both salient, proximal cues (e.g. grid floor of an operant box) and distal cues (e.g. lighting, temperature, and smell of the more general environment). If animals are tested shortly after conditioning, then small alterations in either proximal or distal cues dramatically reduce the conditioned fear response
(Biedenkapp & Rudy, 2007; Gordon, McCracken, Dess-Beech, & Mowrer, 1981; Zhou &
Riccio, 1996). Unlike classical fear conditioning, many chronic mild stress models expose animals to a variety of different stressors (e.g. foot shocks, restraint, strobe light, wet bedding, etc.) that do not all take place in a specific conditioning chamber (i.e. proximal cues change), but may, due to space restrictions, take place in the same procedural room (i.e. distal cues remain the same). It is unclear in such a model whether animals would develop a more generalized fear response to the room even if behavioral testing occurred in a novel box never previously paired with an aversive event.
During emotionally arousing events, like stressor exposure, elevations in stress hormones (e.g. epinephrine and glucocorticoids) enhance the consolidation and
23 retrieval of the memory (McGaugh, 2004). The mechanisms by which circulating stress hormones facilitate retention have been well characterized and occur via enhancing beta-adrenergic receptor signaling in limbic brain areas, particularly the amygdala and hippocampus (ibid.). This can occur either by stimulation of brainstem noradrenergic neurons or potentially direct actions at the synapse that enhance norepinephrine signaling (Liang, Juler, & McGaugh, 1986; McIntyre, McGaugh, & Williams, 2012;
Quirarte et al., 1997; Roozendaal, Hahn, Nathan, de Quervain, & McGaugh, 2004;
Williams, Men, & Clayton, 2000). In contrast, inactivation of noradrenergic neurons or direct beta-adrenergic receptor blockade impairs memory formation (King II & Williams,
2009; Liang, Chen, & Huang, 1995; Liang, 1998). While acute glucocorticoids facilitate memory formation, chronic elevations in glucocorticoids are considered important factors in the suppression of neurogenesis and synaptic plasticity that have been proposed to underlie the neurobiology of depression (E. S. Brown, Rush, & McEwen,
1999; Goshen et al., 2008; McEwen, Magariños, & Reagan, 2002; McEwen, 2001;
Radley et al., 2006; Sapolsky, 2000a). Following repeated stressor exposure both fear conditioning and chronic glucocorticoids could contribute to change an organism’s behavior. While blockade of central beta-adrenergic receptors should interfere with the formation of emotionally arousing memories, it is unclear how this might affect the glucocorticoid response and behavioral changes to repeated stressor exposure.
In the studies presented here we tested the hypothesis that repeated exposure to stressors in a complex environment will inhibit social interaction and exploratory behavior if animals are later tested in the same environment but not if animals are
24 tested in a novel environment. Furthermore, we hypothesize that chronic infusion of propranolol (a beta-adrenoceptor antagonist that readily crosses into the brain), but not nadolol (a beta-adrenoceptor antagonist that does not readily cross the blood–brain barrier), during the time of stressor exposure would attenuate behavioral changes dependent on fear conditioning but not affect behavioral responses dependent on elevated corticosteroid responses.
Methods and procedures
Subjects
Fischer 344 rats and Sprague-Dawley rats were purchased from Harlan
Laboratories (Indianapolis, IN) and weighed approximately 225–275 g at baseline.
Animals were individually housed in clear Plexiglas cages (40 cm ×20 cm ×20 cm) with food and water available ad libitum and kept on a 12:12 h light/dark cycle, with lights on at 07:00 h. Rats were given seven days to become accustomed to their environment and handled for an additional five days prior to experimentation. Studies were conducted in accordance with the guidelines of the PHS Guide to the Care and Use of
Laboratory Animals and approved by the Kent State University Institutional Animal Care and Use Committee.
As with humans, not all animals handle stress in the same manner, there are ranges of susceptibility and resilience to the effects of stress even between genetically varying groups of the same animal species. Corticosterone and epinephrine increases during 60min restraint stress are significantly greater in the more stress-responsive
Fischer-344 rats than in Sprague-Dawleys, as does stress-induced transcription of IL1B
25 and subsequent production of IL-1β protein (Dhabhar, McEwen, & Spencer, 1993;
Porterfield et al., 2011). Furthermore, the more resistant latter strain’s physiological responses more readily habituate to prolonged stress (Dhabhar, Miller, McEwen, &
Spencer, 1995); after 2h restraint stress, Fischers’ CORT levels are still significantly higher than controls, while the effect of stress has faded in Spragues (Porterfield et al.,
2011), and HPA activity in Fischers shows no sign of habituation even after repeated daily exposure to restraint stress (Dhabhar, McEwen, & Spencer, 1997; Uchida et al.,
2008). These physiological variations in multiple systems should play out to result in subsequent behavioral variations, with Fischer-344 rats more readily developing a depressive or anxiety-like behavioral phenotype, while Sprague-Dawley rats should foster little to no such behavioral changes.
Drug delivery
Rats were anesthetized using a mixture of isoflurane and oxygen. A small area on their dorsal side just posterior to the shoulder blades was shaven and treated with betadine. A small (approximately 2 cm) incision was made with a sterile scalpel blade and a 2ML2 osmotic minipump (Alzet, Cupertino, CA) implanted subcutaneously (2ML2 pumps deliver continuous drug at 5 μL/hour for two weeks). Surgical staples were used to close the wound and animals returned to their home cage. Immediately prior to implantation, osmotic minipumps were filled with sterile saline, propranolol, or nadolol.
Propranolol concentrations were calculated to give 0.5 mg/kg/day, 2 mg/kg/day, or 10 mg/kg/day. Nadolol concentration was calculated to give 2 mg/kg/day. These doses were chosen because chronic infusion of propranolol in rats within this range has
26 previously been shown to block beta-receptor mediated splenocyte responses to stress
(Exton et al, 2002), and nadolol administration to rats between 1 and 5 mg kg-1 are sufficient to decrease cardiovascular responses (Hicks, Seifen, Stimers, & Kennedy,
1998; McKernan & Calaresu, 1996). Drugs were purchased from Sigma–Aldrich (St.
Louis, MO) and dissolved in sterile, endotoxin-free saline.
Repeated stress protocol
Rats were exposed to a variety of laboratory stressors according to a set daily schedule or remained in their home cage to serve as non-stressed controls. On the morning (8:00–10:00 h) of day 1 of stressor exposure, rats were placed in Decapi-Cone rodent restrainers (Braintree Scientific, Braintree, MA). Animals were returned to their home cage after 60 min of restraint and food was removed from animal cages at 15:00 h for an 18:00 h period. On the morning of day 2, rats were placed in operant boxes
(Lafayette Instrument, Lafayette, IN) for 30 min. Two minutes after placement in the operant box a 1.5 mA shock was administered for 2 s, 2 min later a second 1.5 mA shock was administered for 2 s and animals remained in the operant box for the remainder of the 30 min. The night of day 2, rats were exposed to constant light during the rats’ normal dark cycle. On the morning of day 3, rats’ home cages were tilted approximately 30° for 180 min and at 1500 h 1 L of warm water was poured into their cages, leaving their bedding damp for an 18 h period. On the morning of day 4, animals were placed back into the operant boxes for 30 min and the footshock procedure was repeated, as on day 2. No stress was given on the night of day 4 during the sucrose preference test or in the morning on day 5 during behavioral testing. All stressors
27 occurred in procedure room A (except overnight food deprivation and wet bedding which occurred in the colony room).
Sucrose preference measurements
Baseline sucrose preference tests were given four days prior to the initiation of the stress protocol. On day 4 of the stress paradigm at 1500 h, rats were presented with a two-choice preference test consisting of two graduated drinking tubes, one containing regular tap water and the other a 1% sucrose solution. Content volume was recorded before presentation to the animals, and again after a period of 18 h. Consumption from each bottle was measured and the percentage of sucrose consumed was calculated from the total volume.
Behavioral measurements
Baseline behavioral tests were given four days prior to the initiation of the stress protocol and occurred in the same procedural room (either A or B) in which the rats were later returned to for testing following repeated stressor exposure. Context A was a rectangular 19’ × 29’ room cyan in color with wall and floor cabinets on three walls. The room temperature was approximately 21°C and the light intensity between 140 and 240 lx. Context B was a 19’ × 21’ room light yellow in color with wall and floor cabinets on two walls. The room temperature was approximately 25°C and the light intensity between 350 and 450 lx. Behavioral testing was always performed the morning following the sucrose preference test. Animals were transported in their home cage to the testing room where exploratory behavior was measured by introducing an animal to a novel Plexiglas cage with no bedding (60 cm ×30 cm × 24 cm) and their behavior
28 recorded for 10 min. Exploration was defined as total time, in seconds, spent moving, rearing, and sniffing; grooming or idling behaviors were not counted. Immediately following the exploratory test, a novel juvenile (4-week old) male of the same strain was introduced to the cage, and social behavior was recorded for 5 min. Social interaction was defined as the total time, in seconds, of contact between the subject’s nose and the juvenile. Contact between the juvenile’s nose and the subject was not counted. Videos were analyzed by an individual blind to the experimental treatments and randomly verified by a second reviewer.
Organ weight and stress hormone measurement
Twenty-four hours after behavioral testing, animals were euthanized via decapitation (day 6). Trunk blood was collected in EDTA vacutainer tubes (BD
Pharmingen, Franklin Lakes, NJ) and placed on ice during tissue collection; samples were centrifuged at 4°C for 10 min at 4000 rpm, then plasma collected and stored at
−80°C until later use. Baseline body weight was measured prior to initial exposure to stress, and post-stress weight was measured following behavioral observations. Upon euthanasia, adrenal glands and spleens were removed and weighed, and their weights normalized for total body weight. Body weights are expressed in g, and peripheral organ weights expressed in mg g−1. Glucocorticoid levels were measured in plasma samples diluted 1:50 and placed in a heated water bath at 70°C for 1 h to dissociate corticosterone from corticosterone-binding globulin. Corticosterone levels were determined using a commercially available enzyme-linked immunosorbent assay kit
(Enzo Life Sciences, Farmingdale, NY), following standard protocol. Concentrations are
29 expressed in μg dL−1.
Statistical analysis
A repeated measure ANOVA was used for analyzing behavioral data collected across multiple time-points. In some studies, due to slight differences in baseline behavioral measurements between groups we also calculated the percent change each animal showed from their individual baseline and analyzed percent change using a one- way ANOVA. Both raw data means and percent change means are presented. Fisher’s least significant difference multiple comparison test was used to determine significance between individual treatment groups. Alpha was set at 0.05 for all statistical measures.
Study 1: effects of context on behavioral changes in animals exposed to chronic stress
Baseline behavioral measurements were obtained from 12 Fischer rats in procedural room A (context A) and from 20 Fischer rats in procedural room B (context
B). Half the animals tested in context A and half the animals tested in context B were exposed to the repeated stressor protocol described above (all stressors occurred in procedure room A). On day 5, all rats underwent behavioral testing and were returned to the same procedural room where baseline behaviors were measured, either context A
(n = 6/group) or context B (n = 10/group).
Study 2: the role of β-ARs in enhanced contextual fear conditioning in chronically stressed subjects
Thirty-five Fischer rats were implanted with osmotic minipumps: 14 contained endotoxin-free saline, 7 contained propranolol that administered 0.5 mg/kg/day, 7 contained propranolol that administered 2 mg/kg/day, and 7 contained propranolol that
30 administered 10 mg/kg/day. Four days after implantation, baseline behavioral measurements were obtained in context A. One week after minipump implantation all animals underwent repeated stressor exposure except for 7 control animals implanted with saline minipumps. During restraint stress on day 1 blood samples were collected from a tail vein following 0, 30 and 60 min of restraint. Blood samples were centrifuged at 10,000 rpm for 10 min and plasma stored at −80°C. On day 5, behavioral measurements were again assessed in context A. On day 6, animals were euthanized and blood and organs collected. Animals were run in 4 cohorts with 2–3 animals per group represented in either cohort. Two animals in the 10 mg/kg/day propranolol group were euthanized prior to the end of the study because of reopening of the surgical wound during restraint stress.
Study 3: differential effects of chronic stress between central and peripheral β-ARs
Eighteen Fischer rats were implanted with osmotic minipumps: 12 contained endotoxin-free saline and 6 administered 2 mg/kg/day of nadolol, a beta-blocker that does not readily cross the blood-brain barrier. Four days after implantation, baseline behavioral measurements were obtained in context A. One week after minipump implantation all animals underwent repeated stressor exposure except for 6 control animals implanted with saline minipumps. On day 5, behavioral measurements were again assessed in context A. Animals were run in 3 cohorts with 2 animals per group represented in either cohort.
Study 4: disparity between laboratory rat strains in effects of chronic mild stress exposure
31 Baseline behavioral measurements were obtained from 16 Sprague-Dawley rats in procedural room A (context A). Half the animals were exposed to the repeated stressor protocol described above. For all animals, stressors occurred in procedure room A (except overnight food deprivation and wet bedding which occurred in the colony room). On day 5, controls and stressed rats were returned to either context A (n =
6/group) for behavioral testing.
Results
Study 1: effect of context on repeated stress-induced behavioral changes
The first study was designed to examine the contribution conditioned fear responses play in behavioral changes when repeated mild stressor exposure and behavioral testing occur in the same procedural room. Fischer rats were repeatedly exposed to stressors in context A over a four-day period. As shown in Figure 4, rats tested in context A showed decreased exploratory behavior and social interaction, while rats tested in context B showed no change in behavior compared to non-stressed controls. Neither group of animals showed significant changes in sucrose preference after four days of repeated stressor exposure. This was supported by a repeated measures ANOVA that revealed a significant interaction between treatment (stress vs. control) and time (baseline vs. test day) for exploratory behavior [F[1,10] = 16.9; p = 0.002] and social interaction [F[1,10] = 10.8; p = 0.008] when animals were tested in context A, but not when animals were tested in context B (p = 0.894 and p = 0.407, respectively).
Further analysis of behavioral data in context A showed that baseline behaviors between groups were not significantly different, but exploratory [F[1,10] = 36.58; p < 0.001]
32 33 34 35 Figure 4. (A–C) Effects of context on behaviors following repeated stressor exposure. Fischer rats were repeatedly exposed to stressors (CMS group) in context A or remained in their home cage to serve as controls (HCC group). Twenty-four hours following the last stressor (day 5) animals were either returned to context A or taken to a new environment (context B) where exploratory behavior (A) and social interaction (B) were tested. Sucrose preference (C) was examined by an overnight sucrose preference test conducted in the animals’ colony room. Baseline behavioral measurements were collected three days prior to the initiation of the repeated stress protocol. Symbols represent group mean ± SEM.
* Signify significant (p < 0.05) difference compared to control animals.
36 and social [F[1,10] = 22.93; p = 0.001] behavior significantly decreased when testing occurred in the same environment after stressor exposure.
Study 2: effect of propranolol on repeated stress-induced behavioral changes
Central beta-adrenergic receptor blockade has been shown to reduce contextual fear conditioning, thus we hypothesized that chronic infusion of propranolol (a beta- adrenoceptor antagonist that readily crosses into the brain) would attenuate the decrease in exploratory behavior and social interaction observed when animals are exposed to repeated stressors in the same environment where they are behavioral tested. Baseline behavioral testing was done four days after implantation of osmotic minipumps so any effect of drug treatment could be observed. As typically reported, propranolol by itself had no effect on any behavior measured. As shown in Fig. 5, repeated stressor exposure again did not significantly alter sucrose preference and it was not altered by propranolol treatment. While there appeared to be an increase in sucrose preference in control animals across time compared to stressed animals, whose preference remained unchanged, a one-way ANOVA comparing percent change in sucrose preference was not significant (p = 0.221). There was, however, a significant effect of treatment across time [F[4,28] = 4.14; p = 0.009] in exploratory behavior. Non- stressed control animals showed an increase in exploration (31.9%) compared to their baseline, while stressed animals receiving 0 mg/kg/day propranolol (saline), 0.5 mg/kg/day propranolol, or 10 mg/kg/day propranolol showed dramatic decreases
(−34.3%, −20.5%, −44.4%, respectively). Interestingly, animals receiving 2 mg/kg/day propranolol showed a slight increase (17.8%) resulting in this group not being
37 38 39 40 Figure 5. (A-C) Effects of chronic propranolol infusion on behaviors following repeated stressor exposure. Fischer control rats were implanted with minipumps containing saline and remained in their home cage, while other animals were implanted with pumps containing 0 mg/kg/day, 0.5 mg/kg/day, 2 mg/kg/day, or 10 mg/kg/day propranolol one week prior to repeated stressor exposure in context A. Twenty-four hours following the last stressor (day 5) animals were returned to context A where exploratory behavior (A) and social interaction (B) were tested. Sucrose preference (C) was examined by an overnight sucrose preference test conducted in the animals’ colony room. Baseline behavioral measurements were collected three days prior to the initiation of the repeated stress protocol. Raw behavioral data are presented on the left and percentchange data calculated from each individual animals’ baseline presented on the right. Symbols and bars represent group mean ± SEM.
* Signify groups significantly (p < 0.05) different from non-stressed control (Con) animals.
41 statistically different from non-stress controls. Due to the large number of groups, each with slightly different baseline levels of exploration, an ANOVA of percent change in exploration was performed. This reveal a significant effect of treatment [F[4,28]=3.48; p=0.02] and post hoc analyses showed stressed animals receiving 0 mg/kg/day propranolol (p = 0.011), 0.5 mg/kg/day propranolol (p = 0.038), and 10 mg/kg/day propranolol (p = 0.007) were significantly different from control animals; animals receiving 2 mg/kg/day propranolol were not different from controls (p = 0.563), but were different from vehicle-treated stressed animals (p=0.042). With regard to social behavior, there was no significant interaction between treatment and time using a repeated measure ANOVA (p = 0.16); however, there was variability in baseline behaviors between groups. When percent change in social interaction was calculated from each animals’ individual baselines, an AVOVA revealed the effects of treatment to be close to significance [F[4,28] = 2.58; p = 0.059]. Post hoc analyses revealed the a priori prediction to be correct, that saline-treated animals exposed to repeated stress demonstrate significantly less social behavior (−36.4%; p = 0.022) compared to non- stressed controls (−3.03%). There was no significant difference between control animals and stressed animals receiving 0.5 mg/kg/day propranolol (−15.4%; p = 0.376), 2 mg/kg/day propranolol (5.1%; p = 0.561), or 10 mg/kg/day propranolol (−13.7%; p =
0.487). However, the only significant statistical difference between vehicle- and drug- treated animals was found in subjects receiving the 2mg dose (p=0.034) indicating
2mg/kg/day propranolol-treatment blocked the stress-induced decrease in social behavior.
42 Study 2: effect of propranolol on stress-induced changes in body and organ weights
Repeated stressor exposure often results in changes in body and organ weights either as a consequence of either high circulating stress hormones or as a response to meet the demands for stress hormones. Here, we measured body weight before and after repeated stressor exposure in addition to spleen and adrenal weight following completion of the study. As shown in Fig. 6, four days of repeated stressor exposure was sufficient to significantly reduce body weight gain. An ANOVA revealed a significant effect between treatment groups [F[4,28] = 4.55; p = 0.006], and post hoc analyses determined that percent change in body weight was significantly reduced in all stress groups compared to controls (p = 0.002, p = 0.004, p = 0.001, p = 0.018 respectively for
0 mg/kg/day, 0.5 mg/kg/day, 2.0 mg/kg/day and 10 mg/kg/day propranolol-treated animals). Spleen weight was slightly reduced in stressed animals receiving 0 mg/kg/day or 0.5 mg/kg/day propranolol; while 2 mg/kg/day propranolol appeared to block this reduction and 10 mg/kg/day propranolol actually increased spleen weight. An ANOVA revealed a significant effect between treatment groups [F(4,26) = 4.983; p = 0.004] for spleen weight, and post hoc analyses determined that the decrease in spleen weight in stressed animals administered saline and 0.5 mg/kg/day propranolol did not reach significance compared to controls (p = 0.15 and p = 0.09, respectively), animals administered 2 mg/kg/day propranolol were no different than controls (p = 0.71) and animals administered 10 mg/kg/day propranolol had significantly larger spleens than controls (p = 0.03). Additionally, there appeared to be a slight overall increase in adrenal size in stressed animals regardless of propranolol treatment. An ANOVA revealed no
43 44 Figure 6. (A-C) Effects of chronic propranolol infusion and repeated stressor exposure on body and organ weights. Twenty-four hours following behavioral testing, animals were euthanized and organ weights measured from non-stress control rats and stressed rats administered 0 mg/kg/day, 0.5 mg/kg/day, 2 mg/kg/day, or 10 mg/kg/day propranolol. Presented are percent changes in body weight compared to baseline weight (A), spleen-to-body weight ratio (B), and adrenal-to-body weight ratio (C). Bars represent group mean ± SEM.
* Signify groups significantly (p < 0.05) different from non-stressed control (Con) animals.
45 significant differences in adrenal size between individual treatment groups (p = 0.11), however, there was a main effect of stressor exposure if all stress treatments were collapsed [F(1,30) = 8.226; p = 0.007].
Study 2: effect of propranolol on corticosterone levels following acute and repeated stressor exposure
The elevations of glucocorticoids during stressor exposure contribute to the enhanced consolidation of emotionally arousing memories and repeated elevations in glucocorticoids suppress neurogenesis and synaptic plasticity that have been proposed to be a possible mechanism involved in the etiology of depression. To determine how blockade of beta-adrenergic receptors might affect acute and long-term changes in corticosterone levels, blood samples were collected from the tail vein of all animals during exposure to acute stress on day 1 (0, 30, 60 min during restraint stress) and trunk blood collected at the end of the study on day 6 (Fig. 7). An ANOVA revealed no significant differences in baseline corticosterone levels at 0 min between treatment groups (p = 0.375). There was a significant increase in circulating corticosterone after
30 min of restraint [F[4,25] = 6.395; p = 0.002], which was sustained through 60 min of restraint [F[4,25] = 25.21; p < 0.001] with levels in all stressed animals significantly greater than non-stressed control (p < 0.01 for all groups). Treatment with propranolol had no effect on the ability of animals’ to generate a corticosterone response following acute stressor exposure. Post hoc tests revealed no significant difference at either time-point between saline-treated stressed animals and those receiving different doses of propranolol. Corticosterone levels were also measured at the end of the study. There
46 47 Figure 7. (A and B) Effects of chronic propranolol infusion on corticosterone responses. Plasma corticosterone levels are measured by EIA from tail blood collected at 0, 30, and 60 min during the first stressor (restraint) on day 1 (A) and trunk blood collected in the morning on day 6 following completion of the stress protocol (B). Data are presented from non-stress control rats and stressed rats administered 0 mg/kg/day, 0.5 mg/kg/day, 2 mg/kg/day, or 10 mg/kg/day propranolol. Symbols and bars represent group mean ± SEM.
* Signify groups significantly (p < 0.05) different from non-stressed control (Con) animals.
48 was a general trend for basal corticosterone levels to be elevated on day 6 in animals exposed to repeated stress regardless of propranolol treatment. However, an ANOVA revealed no significant differences in corticosterone between treatment groups (p =
0.158), and even if all stress groups are collapsed the main effect of stress failed to reach significance (p = 0.075).
Study 3: effect of nadolol on repeated stress-induced behavioral changes
To verify the effects of propranolol were truly due to its ability to block central beta-adrenergic receptors, additional animals were implanted with osmotic minipumps filled with saline or 2 mg/kg/day nadolol (a beta-adrenoceptor antagonist that does not readily cross the blood–brain barrier), and the study repeated. Again, repeated stressor exposure decreased exploratory and social behavior when animals were tested in the same environment where they previously were exposed to stressors and no change in sucrose preference. In contrast to propranolol treatment, nadolol failed to block the stress-induced decreases in exploration and social interaction (Fig. 8). A repeated measure ANOVA comparing treatment groups revealed a significant interaction across time for exploratory behavior [F[2,15] = 5.23; p = 0.019] and social interaction [F[2,15] =
11.84; p = 0.001], but not for sucrose preference (p = 0.948). Similarly, a one-way
ANOVA of behavioral data represented as a percent change from individual animals’ baseline also revealed a significant effect between groups for exploratory behavior
[F[2,15] = 5.387; p = 0.017] and social behavior [F[2,15] = 17.02; p < 0.001]. Post hoc analyses revealed that both saline- and nadolol-treated animals exposed to repeated stress exhibited reduced exploratory behavior (p = 0.047 and p = 0.006, respectively)
49 50 51 52 Figure 8. (A-C) Effects of chronic nadolol infusion on behaviors following repeated stressor exposure. Fischer control rats were implanted with minipumps containing saline and remained in their home cage, while other animals were implanted with pumps containing 0 mg/kg/day or 2 mg/kg/day nadolol one week prior to repeated stressor exposure in context A. Twenty-four hours following the last stressor (day 5) animals were returned to context A where exploratory behavior (A) and social interaction (B) were tested. Sucrose preference (C) was examined by an overnight sucrose preference test conducted in the animals’ colony room. Baseline behavioral measurements were collected three days prior to the initiation of the repeated stress protocol. Raw behavioral data are presented on the left and percent change data calculated from each individual animals’ baseline presented on the right. Symbols and bars represent group mean ± SEM.
* Signify groups significantly (p < 0.05) different from non-stressed control (Con) animals.
53 and reduced social interaction (p < 0.001 for both groups) compared to non-stressed controls.
Study 3: effects of nadolol on spleen weight following repeated stressor exposure
In this study, only spleen weights were measured at the end of the study since this was the only measure previously affected by beta-adrenergic blockade. As shown in
Fig. 9, repeated stressor exposure reduced spleen weight and chronic administration of nadolol blocked the reduction. An ANOVA revealed a significant effect of treatment [F[2,15]
= 4.99; p = 0.05] and post hoc analyses demonstrated a significant reduction in spleen weight in stressed animals treated with saline (p = 0.024), but not those treated with 2 mg/kg/day nadolol (p = 0.564), compared to control animals.
Study 4: effects of repeated stress-induced behavioral changes in Sprague-Dawley rats
It has been reported that the magnitude of conditioned fear response exhibited by animals differs between strains (Heiman et al., 1997; Katzev & Mills, 1974; Pryce,
Lehmann, & Feldon, 1999; Tang, Yang, & Sanford, 2005). There is considerable literature demonstrating Sprague-Dawley rats have reduced stress responses compared to Fischer rats measured both by circulating stress hormones (e.g. corticosterone and catecholamines) (Dhabhar et al., 1997; Dhabhar et al., 1993, 1995;
Porterfield et al., 2011) and central brain responses (e.g. norepinephrine release)
(Porterfield). Since these are major factors previously identified to facilitate consolidation and retrieval of memories (McGaugh, 2004), we examined the generalizability of repeated stressor exposure to alter behavioral responses in Sprague-
Dawley rats. Sprague-Dawley rats were repeatedly exposed to stressors in context A
54 Figure 9. Effect of chronic nadolol infusion and repeated stressor exposure on spleen weight. Twenty-four hours following behavioral testing, animals were euthanized and spleen weights measured from non-stress control rats and stressed rats administered 0 mg/kg/day or 2 mg/kg/day nadolol. Bars represent group mean ± SEM of spleen-to- body weight ratios. * Signify groups significantly (p < 0.05) different from non-stressed control (Con) animals.
55 56 Figure 10. (A-C) Behavioral effects of repeated stressor exposure in Sprague-Dawley rats. Sprague-Dawley rats were repeatedly exposed to stressors in context A or remained in their home cage to serve as controls. Twenty-four hours following the last stressor (day 5) animals were returned to context A where exploratory behavior (A) and social interaction (B) were tested. Sucrose preference (C) was examined by an overnight sucrose preference test conducted in the animals’ colony room. Baseline behavioral measurements were collected three days prior to the initiation of the repeated stress protocol. Symbols represent group mean ± SEM.
57 over a four-day period. At 15:00 h on day 4 animals were given an overnight sucrose preference test in their colony room and the following day transported back to context A for behavioral testing. In contrast to Fischer rats, Sprague-Dawley rats exposed to repeated stressors showed no change in exploratory behavior (p = 0.595) or social interaction (p = 0.80) compared to non-stressed controls (Fig. 10). Like Fischer animals,
Sprague-Dawley rats showed no change in sucrose preference after four-days of stressor exposure.
Discussion
The results of the studies presented here demonstrate Fischer rats show decreased exploratory and social behavior when behavioral testing is done in a context where they had been exposed to repeated stressor exposure. The behavioral changes appear to be due to contextual cues present in the testing environment since no changes in exploratory or social behavior were observed when animals were tested in a novel context. This is the first study that demonstrates contextual fear conditioning can contribute to behavioral changes often observed in a repeated stress paradigm. The stress paradigm used in the current study did not result in anhedonic responses characterized by decreased sucrose preference. However, stressed animals did show many of the classic anatomical changes associated with repeated stressor exposure such as decreased gain in body weight, adrenal hypertrophy, and splenic atrophy.
Peripheral beta-adrenergic receptor blockade did not prevent the reduction in body weight gain or adrenal hypertrophy. However, both propranolol- and nadolol-treatment blocked stress-induced atrophy of the spleen, a process largely mediated by
58 sympathetic nervous system activation (Johnson et al., 2005; Okimura, Ogawa,
Yamauchi, & Sasaki, 1986), demonstrating the pharmacological doses used in the current studies were sufficient to block peripheral beta-adrenergic receptors.
Chronic infusion of propranolol, but not nadolol, was sufficient to prevent the decrease in exploration and social interaction following repeated stressor exposure.
These data support previous work demonstrating blockade of central, but not peripheral, beta-adrenergic receptors attenuate consolidation of emotionally arousing memories
(Do-Monte, Canteras, Fernandes, Assreuy, & Carobrez, 2008) and provide further support that contextual fear responses contributed to the suppression in exploratory and social behavior in rats tested in the same environment where they were previously exposed to stressors. Interestingly, the dose–response effects of propranolol were different for exploratory behavior compared to social interaction. Dose–response curves for both behaviors appears to be U-shaped with the 2 mg/kg/day dose of propranolol the most effective in blocking behavioral changes, however all propranolol doses significantly blocked the stress-induced decrease in social interaction while only the 2 mg/kg/day dose of propranolol blocked the decrease in exploratory behavior. There has been significant work examining stress-associated brain areas and their differential regulation of behavioral responses during stress (Stone, Quartermain, Lin, & Lehmann,
2007). For example, the bed nucleus of the stria terminalis (BNST) has been shown to regulate the acute stress-induced decrease of open arm entries on the elevated plus maze (M Cecchi, Khoshbouei, Javors, & Morilak, 2002), while central nucleus of the amygdala regulates acute stress-induced suppression in social interaction (Marco
59 Cecchi, Khoshbouei, & Morilak, 2002). Thus, it is likely that different brain areas mediate the changes in exploratory and social behavior observed in the present study. Since beta-adrenergic receptors are heterogeneously distributed throughout the brain
(Rainbow, Parsons, & Wolfe, 1984), it is not surprising that the effective dose of propranolol to block the stress-induced behavioral changes differed for each behavior. It should be noted that baseline behavioral testing was done four days following implantation of minipumps and no effects of propranolol or nadolol were observed, however, one limitation of the current study is that longer duration of drug administration was not examined in non-stressed controls.
Chronic administration of propranolol blocked stress-induced behavioral changes without affecting the acute or chronic rise in circulating corticosterone following stressor exposure. Acute elevations in circulating glucocorticoids can enhance memory consolidation (Barsegyan, Mackenzie, Kurose, McGaugh, & Roozendaal, 2010;
Miranda, Quirarte, Rodriguez-Garcia, McGaugh, & Roozendaal, 2008), while chronic or repeated elevations are thought to play a role in the etiology of depression (McEwen,
2001; Sapolsky, Krey, & McEwen, 1985; Sterner & Kalynchuk, 2010; Watanabe, Gould,
Cameron, Daniels, & McEwen, 1992; Woolley, Gould, & McEwen, 1990). Propranolol treatment had no effect on the acute elevation in circulating corticosterone as observed in blood samples collected 30 and 60 min following restraint stress on day 1 of the stress paradigm indicating beta-adrenergic receptors do not play a significant role in the acute activation of the hypothalamic–pituitary–adrenal (HPA) axis. The fact that elevated corticosterone levels were not sufficient to facilitate fear conditioning in the presence of
60 propranolol are consistent with work demonstrating glucocorticoids enhance consolidation of emotional memories via facilitating noradrenergic signaling (Quirarte et al., 1997). The fact that repeated stressor exposure failed to result in behavioral changes that were not context-dependent is likely due to the magnitude and duration of stressor exposure. Often 3–4 weeks of stress (Goshen et al., 2008; Rygula et al., 2005) or corticosterone administration (Goshen et al., 2008; Gourley, Kedves, Olausson, &
Taylor, 2009; S. A. Johnson, Fournier, & Kalynchuk, 2006) are necessary to result in the onset of depressive-like behaviors.
Interestingly, the inhibition of exploratory and social behavior following repeated stressor exposure was not observed in Sprague-Dawley rats. Sprague-Dawley rats are commonly used to study contextual fear conditioning so clearly this strain is capable of forming associations between contextual cues and aversive stimuli. However, previous studies demonstrate that rat strains differ in their ability to form associations between environmental cues and aversive events (Katzev & Mills, 1974; Pryce et al., 1999; Rex,
Sondern, Voigt, Franck, & Fink, 1996; Tang et al., 2005); the current paradigm may have been more difficult for animals to make associations since only distal (room) cues were consistently present during each stressor exposure while proximal cues (e.g. operant box, restraint apparatus, wet cage) continuously changed. Behavioral testing took place in a novel cage so changes in behaviors were the result of conditioned fear responses largely, or solely, based on distal cues. A subgroup of Fischer rats were exposed to 1 h restraint in context A and exploratory and social behavior was normal when tested 24 h later in the same environment (unpublished observation), suggesting
61 multiple learning trials are necessary to result in the conditioned fear responses observed in the present study. The fact that Fischer, but not Sprague-Dawley, rats demonstrated changes in behavior in the current model, indicates Fischer rats have a stronger tendency to form associations between distal cues and stressful experiences.
There are physiological differences between the strains that might contribute to differential ability to form memories of aversive experiences. For example, Fischer rats have greater peripheral corticosterone and epinephrine and greater central norepinephrine release following stressor exposure compared to Sprague-Dawley rats
(Dhabhar et al., 1997; Dhabhar et al., 1993, 1995; Porterfield et al., 2011; Uchida et al.,
2008), and as described above these hormones and neurotransmitters facilitate memory formation. Thus, magnitude of central beta-adrenergic receptor activation may be critical to facilitate the association of distal contextual cues with aversive events.
Additional factors may exist to contribute to Fischers' relatively greater tendency to make such associations, such as differential gene expression and protein synthesis, or small but significant differences in brain morphology or in synaptic plasticity, which is itself instrumental in learning. This is the first study to our knowledge that demonstrates
Fischer rats have a higher propensity toward making associations with distal environmental cues.
Conclusion
Repeated stressor exposure can result in behavioral changes that are due to contextual fear conditioning and not onset of depressive-like behaviors. Since many laboratories use multipurpose rooms due to limited space and the locations of behavioral testing and
62 stressor exposure are rarely reported, these data are important for investigators to consider when interpreting behavioral changes in paradigms that involve repeated stressor exposures.
1Previously published in Behavioural Brain Research 233(2), 536-44.
63 Chapter III
Repeated stressor exposure enhances contextual fear memory in a beta-
adrenergic receptor-dependent process and increases impulsivity in a non-beta
receptor-dependent fashion2
Introduction
Exposure to life stressors is a risk factor for development of psychopathological disorders including post-traumatic stress disorder (PTSD). One of the major diagnostic criteria for PTSD is the presence of recurrent distressing memories. In fact, the aim of behavioral treatments such as exposure therapy is to reduce the emotional and physiological responses associated with aversive memories. Acute exposure to severe stressors or repeated exposure to chronic mild stressors (CMSs) can both result in enhanced anxiety-like behaviors; however, it has not been well documented to what extent behavior changes are a result of enhanced associative learning processes.
Exposure to acute stress prior to classical conditioning trials facilitates learning and the formation of the memory (Cordero, Venero, Kruyt, & Sandi, 2003; Shors, Weiss, &
Thompson, 1992). Classical learning is the process of pairing a neutral cue (conditioned stimulus) with an aversive, unconditioned stimulus such that the conditioned stimulus
64 alone elicits a response (Blanchard & Blanchard, 1969). For example, exposure to tail shock prior to the pairing of white noise with a brief periorbital shock results in significantly more conditioned eyeblink responses when the white noise is presented alone during testing (Shors, 2001). Most evidence points to the release of norepinephrine and stimulation of central β-ARs within the amygdala as the mechanism by which stressor exposure facilitates memory formation. Evidence for this phenomenon is quite extensive and is summarized in multiple reviews (Ferry et al., 1999; Roozendaal et al., 2009; Roozendaal & McGaugh, 2011). It has also been shown that administration of β-AR agonists directly into the basolateral amygdala during training trials increases memory retention while blockade of β-ARs in the BLA reduces memory formation
(Dębiec & LeDoux, 2006; Hatfield & McGaugh, 1999; Introini-Collison, Saghafi, Novack,
& McGaugh, 1992; LaLumiere, Buen, & McGaugh, 2003; Quirarte et al., 1997).
Exposure to restraint stress enhances subsequent long-term potentiation (LTP) in the
BLA pathway induced in vitro, which can be prevented by prior administration of a β-AR antagonist (Sarabdjitsingh, Kofink, Karst, de Kloet, & Joëls, 2012). While glucocorticoids released by the adrenal cortex during times of stress contribute to enhanced memory formation, their effects largely appear to be mediated through augmenting adrenergic signaling (Hubbard et al., 2007; Quirarte et al., 1997; Roozendaal, Brunson, Holloway,
McGaugh, & Baram, 2002; Roozendaal, Quirarte, et al., 2002; Roozendaal et al., 2008).
Together, these data suggest that β-ARs are involved in a common final pathway in memory consolidation.
Repeated exposure to mild stressors enhances NE turnover in the amygdala
65 (Porterfield et al., 2012); however, the effect this has on behaviors dependent on associative learning has not been fully determined. We previously published that some behavioral changes (e.g. decreased exploratory behavior, social withdrawal) following exposure to CMS can also be attributed to distal environmental cues (lighting, ambient temperature, and olfactory cues in the procedural room). This is significant because others have published that chronic stress does not induce anxiety-like behaviors in an open field test or elevated-plus maze (D’Aquila, Brain, & Willner, 1994; Kompagne et al.,
2008; Mineur, Belzung, & Crusio, 2006) but does enhance freezing behavior in a contextual fear-conditioning test (Cordero et al., 2003).
Here, we tested the hypothesis that exposure to CMS would alter behavioral responses in tasks that depend on classical fear conditioning. Undisturbed home cage control (HCC) and CMS animals were tested in two behavioral tests that require associative learning (e.g. contextual fear conditioning in an operant box and retention latencies in a passive avoidance test) or in a third behavioral test that is not associative learning dependent (e.g. open field). We predicted that animals exposed to CMS would show increased freezing behavior in an operant box and increased retention latencies in a passive avoidance apparatus following conditioning, but no anxiety-like behaviors in the open field test. We also tested whether administration of propranolol prior to conditioning trials in the operant box and passive avoidance chamber would prevent or attenuate chronic stress-induced behavioral changes.
Methods and materials
Subjects
66 Thirty-two Fischer-344 rats were purchased from Harlan Laboratories
(Indianapolis, IN), weighing approximately 200–262 g (xx = 230.1 g; σ=14.5 g) at baseline. Animals were housed and handled as described in Chapter II, and studies were conducted in accordance with the guidelines of the PHS Guide to the Care and
Use of Laboratory Animals, and approved by the Kent State University Institutional
Animal Care and Use Committee.
Repeated stress protocol
Subjects were exposed to a series of stressors following an adaptation of an established protocol (Chapter II), or were left undisturbed as HCC animals. This four day repeated stress paradigm was chosen since we previously demonstrated that it results in increased NE turnover in the amygdala, sensitized β-AR mediated responses, and enhanced fear conditioning to distal contextual cues (ibid.). However, as behavioral observations of fear conditioning rely on the use of footshock, the following alterations had to be made to the protocol: on the mornings (0800–1000 h) of days 1 and 3, CMS rats were placed in DecapiCone rodent restrainers for 60 min; on day 2, CMS subjects were placed in novel habitats, each with a piece of filter paper containing 35 μL trimethylthiazoline (TMT, a component of fox feces) to simulate predator odor (Fendt,
Endres, & Apfelbach, 2003) (Contech Enterprises, Victoria, BC); finally, on day 4, subjects were exposed to forced swim for 5 min in glass cylinders measuring 49 × 18.7 cm (inner height and diameter, respectively) filled approximately to the 37.5 cm line with water at a temperature of 21 °C. On the evening of day 4 following the stressor, water bottles were removed from CMS home cages for 18 h and returned on the next
67 morning. Following CMS, and prior to behavioral and physiological observations, subjects weights were recorded.
Behavioral testing
Behavioral assays were performed to test associative learning (e.g. contextual fear conditioning and passive avoidance) and generalized anxiety (e.g. open field), with tests occurring 2 h apart. The order of tests was counterbalanced to protect against possible order effects. A second cohort of animals was tested in a passive avoidance task only.
Conditioned fear
One day following the stress protocol (day 5), subjects (n=32) were placed in an
8.5× 8.5″ ×11″ (21.59 × 21.59 × 27.94 cm) operant box (Lafayette Instrument Company,
Lafayette, IN) with a floor consisting of a series of electrically conductive steel bars; after 2 min, animals received two foot shocks (1.5 mA for 2 s), the second shock administered 2 min after the first. One minute following the second foot shock, animals were removed from the box and placed back into their home cages. Twenty-four hours later, subjects were placed back into the operant box and behavior was recorded for 15 min and freezing behavior was evaluated. Freezing behavior was defined as complete immobility, save for movements necessary for respiration. Scoring was performed by a trained researcher blind to group assignment. Scores were obtained by checking the video every 10 s for 15 min, and one point was assigned for each instance of freezing behavior,with a maximum possible score of 90 pts. Points were not assigned if at any time point within the ten second interval a subject showed any additional voluntary
68 movement beyond what was required for respiration.
Open field
Subjects were placed in a 60×60×60 cm Plexiglas box and spontaneous motor activity was recorded using a ceiling-mounted video camera for 10min. Movements within the open field were evaluated using EthoVision XT tracking software (v8.0,
Noldus Information Technology, Wageningen, NL). Total time spent in the center section of the open field was measured with lower values indicating an increase in anxiety-like behavior; measurements of locomotion – distance traveled and time spent in motion – were also recorded. The parameters of the open field were defined by creating a 10 ×
10 grid (100 subdivisions) on the space within the viewport in the tracking software; the inner 64% of these subdivisions (8 × 8) was designated as the center section.
Passive avoidance
A second group of animals (n=32) was tested in a passive avoidance apparatus.
The apparatus consisted of two chambers: an illuminated, transparent “safe” compartment and a darkened, opaque “shock” compartment. The compartments were divided by a wall with a door that closed upon entry into the opposing chamber. Both chambers sat atop a floor constructed of electrically conductive steel bars; the floor was tilted in the center, which triggered the closing of the door between the chambers when the subject had fully crossed the threshold. On day 5, subjects were placed in the “safe” compartment of the apparatus. After a 20 s delay, the door to the dark chamber was opened, and the subject was allowed to freely explore the apparatus. Once the subject fully entered the dark chamber (four paws and tail inside the compartment), the door
69 closed and after a two-second delay, the subject received a 1.5 mA foot shock for 2 s.
One minute following the footshock, subjects were removed from the apparatus and placed back into their home cages. Twenty-four hours later, subjects were returned to the apparatus and once again placed in the safe compartment. The latency for the subject to enter the dark chamber with all four paws was recorded. If the subject did not cross into the dark chamber after a maximum time of 538 s (8′58″), the door closed automatically and the test was terminated.
Beta-adrenergic receptor antagonist administration
One hour prior to contextual fear training, subjects were given an intraperitoneal
(i.p.) injection of either a β-adrenergic receptor antagonist, propranolol (Sigma-Aldrich,
St. Louis, MO) dissolved in sterile endotoxin- free saline at a dose of 2mL kg−1 or an equivalent volume of 0.9% saline vehicle. We have previously used propranolol to block stress-induced brain cytokines and behavioral responses since it readily crosses the blood–brain barrier (Chapter II, Johnson et al., 2005).
Statistical analysis
Comparisons between groups were made using two-way analyses of variance
(ANOVAs) followed by Tukey HSD post-hoc tests when necessary; α=0.05 for all measures, results are expressed as means ± SEM. Statistical analysis was performed and charts were made using the R programming language (R Foundation for Statistical
Computing, Vienna, Austria).
Experimental design
To test the effects of chronic stress exposure on contextual fear conditioning,
70 subjects were divided into two groups (n=16/group), one experimental group being exposed to CMS and a non-stressed HCC group. A second cohort was similarly divided into CMS and HCC control groups (n=16/group), and tested in the passive avoidance apparatus. To examine the role of β-ARs in stress-induced fear learning, the aforementioned CMS and HCC groups were subdivided further into drug and vehicle groups, resulting in a total of four groups,with eight subjects per group for each portion of the experiment.
Results
Effect of CMS on freezing behavior
Control animals previously exposed to foot shocks in an operant box showed freezing behavior approximately 45% of the time when returned to the same operant box (without shock) 24 h later. Animals exposed to CMS prior to conditioning showed significantly more freezing behavior, approximately 70% of the time, when tested 24 h after conditioning (Fig. 11). To examine if enhanced β-AR activation during conditioning contributes to altered behavioral changes in stressed animals, animals were given a 2 mg kg−1 i.p. injection of propranolol prior to conditioning. Propranolol had no effect on freezing behavior in control animals, but significantly decreased freezing behavior in stressed animals. A two-way ANOVA revealed a significant stress-by-drug interaction
(F[1,28]=5.507, p=0.026); using a Tukey HSD post-hoc, a significant effect of stress was observed when comparing vehicle-injected CMS versus HCC animals (p=0.037), while there was no difference in freezing behavior between control and stressed animals receiving propranolol (p=0.969).
71 * Indicates groups significantly different (p<0.05) from non-stressed (control) animals.
# Indicates a significant effect (p<0.05) of drug on stressed subjects.
72 * Indicates groups significantly different (p<0.05) from vehicle-treated non-stressed controls.
# Indicates a significant effect (p<0.05) of drug treatment from vehicle-treated controls.
73 Effect of CMS on passive avoidance behavior
In the passive avoidance apparatus, control animals exposed to footshock upon entering the dark chamber in the training trial showed a nearly 400 s latency to enter the dark chamber when tested 24 h later. Animals exposed to CMS prior to training had significantly shorter latencies (approximately 100 s) to enter the dark chamber compared to HCC rats (Fig. 12). This finding was supported by a two-way ANOVA that revealed a significant main effect of stress (F[1,28]=44.039, p<0.001). Propranolol treatment prior to the training trial reduced the latency to enter the dark chamber when tested 24 h later regardless of stress condition. A two-way ANOVA revealed a significant main effect of drug treatment (F[1, 28]=14.935, p<0.001). There was also a significant interaction between stress and drug (F[1, 28]=6.822, p=0.014) with post-hoc analyses revealing propranolol significantly reduced latencies to enter the dark chamber in control animals (p<0.001) but did not result in further reductions in latency in stressed animals
(p=0.812).
Effect of CMS on non-contextual anxiety-like behavior
In the open field there was no significant effect of CMS (F[1, 26]=0.015, p=0.903) on the amount of time spent in the center zone, nor was there an effect of propranolol treatment (F[1, 26]=1.409, p=0.246) in either condition (Fig. 13). Additionally, there were no significant effects of stress or receptor antagonism, respectively, on overall locomotor activity — either by distance traveled (F[1, 28]=1.755, p=0.196; F[1, 26]=1.448, p=0.239) or by time spent in motion (F[1, 28]=0.052, p=0.822; F[1, 26]=3.414, p=0.075).
74 75 Discussion
The data presented here partially support our hypothesis that repeated stress exposure alters behavioral responses due to enhanced classical fear conditioning. Exposure to four days of variable stressors prior to contextual fear conditioning in an operant box significantly enhanced freezing behavior tested 24 h later. The enhanced freezing was not due to overall increased anxiety since no differences in behaviors were observed when animals were exposed to the open field, a test of anxiety that does not depend on associative learning. These findings support previous reports that chronic stress enhances contextual fear conditioning (Cordero et al., 2003), and demonstrates this is likely not due to generalized anxiety. The lack of a generalized anxiety-like behavioral phenotype is consistent with our previous observation in the open-field test (Chapter II).
There is a degree of variability in the expression of anxiety-like behaviors after repeated exposure to stress, with some stating that, while chronic stress does consistently contribute to development of depressive behaviors, it is not necessarily anxiogenic
(D’Aquila et al., 1994; Kompagne et al., 2008; Mineur et al., 2006). We think that this variability in the expression of “classical” anxiety-like behaviors (e.g. elevated plus- maze, open field) can perhaps be attributed to the context in which the observations were made (Chapter II; Greenwood et al., 2010). Furthermore, for the first time we demonstrated that the enhanced freezing in chronically stressed animals is dependent on β-AR activation during the aversive experience since administration of propranolol during the conditioning trial reduced freezing behavior in stressed animals to near
‘normal’ levels observed in control animals.
76 Contrary to our initial hypothesis, repeated stressor exposure decreased, rather than increased latencies of animals to enter a dark chamber previously paired with footshock. We had predicted that enhanced association between the dark chamber and foot shock during conditioning would increase latencies to enter the dark chamber in stressed animals tested 24 h later. Previous work has shown that direct infusion of NE into the BLA following passive avoidance training significantly increases latency to enter a shock chamber (LaLumiere et al., 2003).The findings of the present study are contrary to this result when tested in animals subjected to repeated stressor exposure. This presents a clear dissociation between the effects of NE infusion and stress exposure. It is likely that NE infusion into the BLA enhances associative learning but does so without causing widespread neural changes that occur following repeated stressor exposure that result in more complex behavioral changes observed in the present study. We do not believe that animals exposed to CMS fail to associate the dark chamber with an aversive stimulus since animals displayed no impairment, but rather an enhancement, in contextual fear conditioning. Given that the stress protocols were identical for all CMS rats and that the time interval between conditioning and testing for the contextual fear conditioning and passive avoidance tests were also identical,we believe that these differences are due to the different neural pathways that regulate the behavioral responses to each test. In the contextual fear conditioning task, animals simply need to recognize the context that was previously paired with footshock and evoke a behavioral
(freezing) response, which is largely mediated by projections from the amygdala to the periaqueductal gray (Pare & Duvarci, 2012). Alternatively, in the passive avoidance test
77 animals need to recall the environment in which they were shocked and inhibit their natural tendency to move into the dark chamber. Avoidance behavior involves interconnections between the amygdala and areas of the prefrontal cortex that mediate behavioral control (Bravo-Rivera, Roman-Ortiz, Brignoni-Perez, Sotres-Bayon, & Quirk,
2014). Stressor exposure can impair prefrontal cortex function, thus, even though stressed animals demonstrate increased contextual fear learning, their responses to specific behavioral tasks reflect the effects of stress on multiple brain areas, not simply associative learning within the amygdala. We propose that the lower latencies to enter the dark chamber indicate decreased behavioral inhibiiton in stressed animals. It is well- known that stress can increase impulsive behaviors such as drug abuse (Baker, Piper,
McCarthy, Majeskie, & Fiore, 2004; Khantzian, 1985; Koob & Le Moal, 1997;
Schaumberg et al., 2014), risk taking behavior (Cooper, Russell, & Frone, 1990; Finy,
Bresin, Korol, & Verona, 2014; James, Strom, & Leskela, 2014; V. Johnson & Pandina,
1993), and food intake (Sinha & Jastreboff, 2013). In fact, enhanced formation of fearful memories and increased impulsivity (including risk taking behaviors, drug abuse, and emotional outburst) are both commonly observed in veterans repeatedly exposed to stressful environments (James et al., 2014).
Another interesting observation was the fact that propranolol did not affect contextual fear memory, but did affect passive avoidance behavior, in control animals suggesting that the level of norepinephrine release and β-AR stimulation (likely within the amygdala) in control animals was not sufficient to facilitate contextual fear memory formation during conditioning. Alternatively, in the passive avoidance test, which
78 requires both contextual fear memory and behavioral inhibition (suppression of an animal’s natural desire to move into the dark chamber), norepinephrine release in control animals was sufficient to alter behavior since propranolol administration reduced latency to escape in control animals. Since there was no effect of propranolol on contextual fear memory, it suggests that norepinephrine more likely plays a role in normal behavioral inhibition (mediated by the prefrontal cortex). Low-to-moderate levels of norepinephrine help stimulate prefrontal cortex and aid in behavioral control, too little or too much norepinephrine release in the prefrontal cortex results in poor behavioral inhibition—often described as impulsivity (Arnsten & Li, 2005; Arnsten, 2011; Ma, Qi,
Peng, & Li, 2003). Our data suggest that β-AR stimulation during conditioning is necessary for normal behavioral inhibition in the passive avoidance test.
In conclusion, repeated stress exposure results in complex behavioral responses.
While we provide evidence that stressor exposure facilitates associative learning processes via β-ARs in a contextual fear conditioning test, it failed to do so in a passive avoidance task that also requires associative learning. These data demonstrate that stressor exposure affects multiple brain areas resulting in complex behavioral responses, and researchers should be cautious when trying to extrapolate the effects of specific stress hormones on behavioral responses to those observed following stressor exposure.
2Previously published in Physiology & Behavior xxx(x), xxx-x.
79 Chapter IV
Effects of chronic mild stress on sensitization of beta-adrenergic receptor-
stimulated intracellular signaling in amygdaloid and hippocampal tissue
Introduction
Central norepinephrine turnover is increased in rats exposed to repeated stress, particularly in the amygdala, hypothalamus, and prefrontal cortex, indicated by increased levels of the norepinephrine metabolite, normetanephrine, (Porterfield et al.,
2012). Similarly, levels of the NE metabolite, 3-methoxy-4-hydroxyphenethylene glycol
(MHPG), increase in healthy human subjects with exposure to stress (Lader, 1974;
Sweeney, Maas, & Heninger, 1978). This elevated NE metabolism is associated with subsequent behavioral dysfunctions that resemble depressive and anxiety-like phenotypes, such as reductions in sucrose preference, social withdrawal, reduced open-arm exploration in the elevated-plus maze, and increased defensive burying behavior (ibid.; Morilak et al., 2005). This also coincides with elevated production of the inflammatory cytokine IL-1β (Porterfield et al., 2012), as well as IL-6 and tumor necrosis factor alpha (TNF-α; Tan et al., 2007; Wang et al., 2010). Such increases in the immune response are commonly associated with repeated exposure to stress and are also
80 found to be related to depression (Glaser & Kiecolt-Glaser, 2005; Glaser, Robles,
Sheridan, Malarkey, & Kiecolt-Glaser, 2003; Goshen et al., 2008; Maes, Ombelet, De
Jongh, Kenis, & Bosmans, 2001; Raison, Capuron, & Miller, 2006). Noradrenergic function is also implicated in panic disorder and PTSD; patients suffering from both disorders experience panic attacks upon administration of the α2-adrenergic autoreceptor antagonist yohimbine (D S Charney, Woods, Goodman, & Heninger, 1987;
D S Charney, Woods, Krystal, Nagy, & Heninger, 1992; Southwick et al., 1993). Similar anxiety effects were found using the β-AR agonist isoproterenol (Pohl et al., 1988), which are treatable with tricyclic antidepressants (Pohl, Yeragani, & Balon, 1990). These effects of stress occur despite negative feedback loops activated with increased receptor activation, which results in their down-regulation within the brain and binding affinity of the receptors is decreased as well. The reason for this has not been sufficiently elucidated, but a possible mechanism could be through stress-induced sensitization of intracellular signaling at β-ARs.
As discussed in Chapters II and III, prolonged exposure to stress enhances emotional memory processing, which can manifest in increased freezing behavior in fear conditioning exercises or in contextually triggered social withdrawal and decreases in exploratory behavior. The mechanism through which this occurs is in the stimulation of beta-adrenergic receptors most likely in the amygdala; pharmacological blockade via the administration of propranolol during fear conditioning prevents stress-induced behavioral changes from developing. However, it is plausible that, in addition to β-ARs fulfilling a mediatory purpose in affective dysfunction, they also play a modulatory role,
81 in that too little or too much receptor stimulation in certain brain regions, such as the prefrontal cortex, can lead to these other behaviors, whose expression was not considered in the initial hypothesis. One such effect is in the reduction of behavioral inhibition, as indicated by the impulsive behavior displayed by subjects in the passive avoidance task in Chapter III.
Regarding emotional memory enhancement, it is likely that the role played by β-
ARs is related to LTP, an important neural component of learning. Signaling from beta- adrenoceptors interact with cholinergic receptor signaling in enhancing LTP through the activation of p44/42 mitogen activated protein kinase (MAPK, also called extracellular signal-regulated 1 and 2, or ERK1/2; Watabe et al., 2000), and coactivation of β-ARs and glutamate receptors increases associative synaptic plasticity in the hippocampus through the activation of adenylate cyclase (Gereau & Conn, 1994). Furthermore, β-AR blockade with propranolol blocks novelty-induced reinforcement of early- to late-phase
LTP memory formation (Straube et al., 2003).
To determine how β-ARs contribute to the expression of CMS-induced behaviors despite their downregulation and decreased binding affinity, a time-course study was done to observe intracellular signaling in pertinent regions of the brain during fear conditioning. Our prediction was that, following repeated exposure to stress, intracellular signaling would be increased over non-stressed controls, which would be observed through elevations in levels of cAMP, ERK1/2, and phosphorylated CREB. These increases would be found primarily in the amygdala, where enhanced fear conditioning is stressed animals is likely mediated, and there may be increased activity in other
82 regions such as the hippocampus and prefrontal cortex. To show that signal sensitization occurs at β-adrenergic receptors, we made the hypothesis that the use of the beta-receptor antagonist propranolol would block or at least attenuate the predicted increases in the signaling cascade.
Methods and procedures
Subjects
Fischer-344 rats were either purchased from Harlan Laboratories (Indianapolis,
IN) or were bred in-house. They were single-housed in Plexiglas habitats measuring
40×20×20 cm, given food and water ad libitum, and kept on a 12:12 L:D cycle with lights on at 0700 h. Animals purchased from Harlan were given seven days to acclimate to their surroundings, all were handled for five days prior to commencement of the CMS protocol. Studies were conducted in accordance with the guidelines of the PHS Guide to the Care and Use of Laboratory Animals and approved by the Kent State University
Institutional Animal Care and Use Committee.
Chronic mild stress protocol
Animals were exposed to a battery of stressors following an established four-day protocol known to increase NE turnover, enhance fear conditioning to both proximal and distal environmental cues, and contribute to development of aberrant affective behaviors; another group was left as unstressed HCC subjects. The protocol used was the same as that illustrated in Chapter III.
Beta-adrenergic receptor stimulation
Twenty-four hours following the last day of stress, subjects of both groups were
83 given an intraperitoneal (i.p.) injection of either the beta-adrenergic receptor antagonist propranolol (Sigma, St. Louis, MO), dissolved in sterile endotoxin-free saline at a dose of 2mg kg-1 or an equivalent volume of 0.9% saline vehicle. One hour later, animals were placed in an 8.5×8.5×11” (21.59×21.59×27.94cm) operant box (Lafayette
Instrument Company, Lafayette, IN); after 2 min, a mild (1.5 mA) foot shock was applied for 2 s, which was repeated 2 min later. Animals were returned to their home cages one minute following the second shock. A separate group was administered drug or vehicle and euthanized by rapid decapitation without footshock, to collect trunk blood and brain tissue to analyze baseline cellular signaling, two groups after 10min and 20min to analyze cAMP levels, and a fourth group was euthanized after 60min to analyze pERK and pCREB levels. Tissue was collected from the amygdala and hippocampus.
Samples were flash-frozen in liquid nitrogen and subsequently frozen at -76°C for later use.
Cellular signaling analysis
Cyclic AMP levels were analyzed using a commercially available ELISA (Arbor
Assays), following standard protocol for the acetylated format. Phosphorylated CREB was measured with a commercially available sandwich ELISA kit (Cell Signaling
Technology, Danvers, MA). ERK was measured using the commercially available
[pThr202/Tyr204]ERK1/2 ELISA kit (Enzo Life Sciences, Farmingdale, NY). All ELISA data was normalized using an assay to quantify total protein (Bradford, 1976) and concentrations are thus expressed in pmol mg-1.
Statistical analysis
84 Comparisons between groups were made using a three-way ANOVA and a Tukey
HSD post hoc test was used where applicable; outliers were identified using Rosner’s
ESD. In all measures α=0.05 and results are expressed as means±SEM. Calculations were performed and results were plotted using the R programming language (R
Foundation for Statistical Computing, Vienna, Austria).
Experimental design
Tissue samples collected from 83 rats were analyzed; of these subjects, 42 were exposed to daily repeated stressors. Thirty-three subjects were given the beta-blocker and 50 the saline vehicle. Finally, the subject pool was divided into four groups to simulate an intracellular signaling time course; 26 rats were sacrificed without exposure to fear conditioning so as to obtain baseline measures, 16 at 10 min and 12 at 20 min, possible time points at which cAMP may be at its peak production, and another 29 at 60 min, when CREB and ERK1/2 phosphorylation may be most active.
Results
These studies were conducted in order to examine the effect of stress on intracellular signaling initiated by β-AR stimulation. Fischer rats were repeatedly exposed to stressors over a four-day period followed by fear conditioning by footshock. Rather than observing the subsequent behavior, tissue was collected from the amygdala and hippocampus; concentrations of cAMP, phosphorylated ERK, and phosphorylated
CREB were analyzed with ELISA.
Effect of stress and propranolol on intracellular signaling in the amygdala
In response to four days of CMS, a three-way ANOVA between Stress, Drug
85 86 87 Figure 14. (A-D) Effects on intracellular signaling in the amygdala following repeated stressor exposure. Fischer rats were repeatedly exposed to stressors or remained in their home cage to serve as controls. Twenty-four hours following the last stressor (day 5) animals were given either propranolol or a saline vehicle, then were either euthanized immediately, or placed in a fear conditioning task and euthanized 10 min or 20 min later. Brains were collected and cAMP levels at 10 min (A) and 20 min (B) as well as phosphorylation of ERK (B) and CREB (C) were analyzed in the amygdala. Bars represent group mean ± SEM.
# Signifies a significant (p < 0.05) difference compared to basal cAMP measure.
88 Treatment, and Time after Conditioning reveals significant effects of stress [F(1, 31)=4.832; p=0.03552] and of conditioning [F(2, 31)=7.18; p=0.00274] on cAMP levels in the amygdala, but no significant interactions. Further post hoc analysis shows that cAMP is elevated in the amygdalæ of vehicle-treated animals 10 min after conditioning, at an average of 37.21 pmol mg-1 compared to a baseline of 20.97 pmol mg-1 (Fig. 14A), and returns to basal levels after 20 min (Fig. 14B). While there was no statistical interaction, clearly the majority of increase in cAMP at 10min was due to the increase found in previously stressed animals. There was a significant increase in cAMP at 10min in stressed animals (p=0.04715) that was not significant in controls (p=0.7462). No significant increase in cAMP levels was found after 10 min in stressed animals given propranolol (p=0.9711).
A significant drug effect [F(1, 43)=8.374; p=0.00596] on amygdaloid ERK1/2 phosphorylation was revealed in a three-way ANOVA. Further post hoc analysis shows an overall reduction in pERK at 60min following propranolol administration (p=0.01941); however, no significant differences were found between any individual group. There were no effects of stress or of time (p=0.4762 and p=0.2513, respectively; Fig. 14C).
An ANOVA revealed a significant effect of time on CREB phosphorylation in the amygdala, with an overall increase at 60min (F(1, 43)=8.331; p=0.006). As with ERK, no differences were found between any individual groups. There were no effects of stress or of propranolol (p=0.5706 and p=0.6799, respectively; Fig. 14D).
Effect of stress and propranolol on intracellular signaling in the hippocampus
The three-way ANOVA reveals no effects of stress on cAMP production or on the
89 90 Figure 15. (A-C) Effects on intracellular signaling in the hippocampus following repeated stressor exposure. Fischer rats were repeatedly exposed to stressors or remained in their home cage to serve as controls. Twenty-four hours following the last stressor (day 5) animals were given either propranolol or a saline vehicle, then were either euthanized immediately, or placed in a fear conditioning task and euthanized 10 min or 20 min later. Brains were collected and cAMP levels (A) and phosphorylation of ERK (B) and CREB (C) were analyzed in the hippocampus. Bars represent group mean ± SEM.
91 phosphorylation of ERK or CREB (p=0.433, 0.8809, and 0.5411, respectively, shown in
Fig. 15) in the hippocampus. There is a significant drug effect on ERK phosphorylation
[F(1, 40)=4.266; p=0.0431); post hoc analysis shows that β-AR antagonism results in an overall reduction of pERK in animals given propranolol (p=0.0432), but no differences between any individual groups. Analysis shows a significant effect of time on phosphorylation of CREB [F(1, 44)=4.266; p=0.0448). Further analysis reveals the effect is an overall increase in pCREB after 60min (p=0.4552), regardless of stress or drug.
Discussion
The results of these studies do not conclusively demonstrate that repeated exposure to stress sensitizes intracellular signaling at beta-adrenergic receptors. While there is a trend supporting stress-induced β-AR sensitization exhibited by increases in adenylate cyclase activation, no evidence has been found thus far with phosphorylation of ERK1/2 or CREB. Lending credence to this trend is the lack of an increase in cAMP production over baseline after 10min in rats treated with propranolol; again, there was no drug effect on ERK or CREB.
Although beta receptors are important in LTP enhancement (Thomas, Moody,
Makhinson, & O’Dell, 1996), there is evidence that this occurs not just through β-AR activation alone but through co-activation with α1-ARs (Katsuki, Izumi, & Zorumski,
1997) and cholinergic receptors (Decker, Gill, & McGaugh, 1990; Ohno, Yoshimatsu,
Kobayashi, & Watanabe, 1997; Watabe et al., 2000), and that there are other mechanisms in the stress response that can contribute to LTP enhancement (Patterson et al., 2001; Yuen et al., 2011). It is also possible that, as these subjects were exposed
92 to stress over four days rather than being given direct pharmaceutical manipulation,
10min may not have been the most optimal time point. Having another group euthanized 20min after conditioning shows that cAMP levels return to those seen at baseline, but it may be helpful to observe cAMP production at a five-minute time point.
Next, when analyzing levels of ERK phosphorylation, we used tissue samples taken from subjects 60min after conditioning with footshock, which was the same time point used in our analysis of CREB. While ERK-mediated CREB phosphorylation is a relatively slow, sustained mechanism (Wu et al., 2001), this was likely too late a time point and would have been better analyzed instead at 30min.
Finally, it may perhaps have been beneficial to focus on other potential targets in the signaling cascade and in LTP, such as protein kinase A or AMPA receptors. It has been shown that there is increased trafficking of the latter through the phosphorylation of GluR1, which can be achieved through emotional stress and through infusion of NE, and ultimately enhances learning (Hu et al., 2007).
There are also possible methodological issues in this study. First, there may have been an unwanted effect of rapid decapitation on norepinephrine release; as a subject is euthanized, the ATP powering the monoamine transporters involved in neurotransmitter reuptake is lost, resulting in a sudden surge of, in this case, norepinephrine being released from neurons in the brain, leading to increased adrenergic receptor activation even in nonstressed controls. This may have also introduced a ceiling effect, and results may not have been accurately representative of the CMS model.
93 While literature on LTP is centered primarily on the hippocampal formations, studies on noradrenergic signaling-mediated enhancement of fear memory have focused particularly on the basolateral nucleus of the amygdala. My intention was to analyze intracellular signaling through the quantification of signaling cascade products such as cAMP and phosphorylated CREB; in doing so, however, I was only able to collect whole amygdalae rather than a specific portion of it, which may have resulted in diluting an otherwise statistically significant effect of stress. The most plausible solution would have been to microdissect even further the amygdala into its component subregions using tissue punching, though planning would be necessary to determine which specific neurons to collect. One possible, though not entirely perfect solution would be to evaluate possible localization of such targets through the use of immunohistochemistry. While IHC staining does not necessarily quantify target proteins, we can at least visually compare specific brain regions between stressed subjects and controls by analyzing the optical density of the stained area. Similarly, in situ hybridization could be used to assess increases in the transcription of GRIA1, which encodes for the GluR1 subunit found on certain AMPA receptors, which would hypothetically be upregulated with sensitized β-AR signaling. Until these issues have been rectified, I do not believe that a proper conclusion can be discussed.
94 Chapter V
Global discussion
It is well established that chronic exposure to stress is a major contributor to the development of many of the behaviors we see in mood and anxiety disorders. The studies presented here show chronic mild stress enhances context-dependent learning processes that may in turn play an important mediatory role in behavioral pathology. It is made clear that the enhanced emotional learning is facilitated by the activation of beta- adrenergic receptors in the brain; what is not yet clear, however, is how β-AR signaling remains so strong with repeated stress exposure, despite receptor downregulation and reductions in binding affinity resulting from negative feedback. Attempts were made to elucidate a possible mechanism by which this is possible, but results were unfortunately not conclusive (Fig. 16).
After experiencing some success and promise with the behavioral model I developed for chronic mild stress in rodents, it became evident that context plays a crucial role in facilitating the expression of the stress-induced behaviors we had been observing, when we began to expose subjects to stress in a procedural room separate from the one in which their behaviors were observed. Without environmental cues
95 Figure 16. Observed process of stress-induced enhancement of fear memory. Through β-AR activation, CMS enhances fear learning in context, leading to increased freezing behavior and other depressive and anxiety-like behaviors, but only if environmental cues are present. Rather than increasing retention latencies in the passive avoidance test, behavioral inhibition was decreased, leading to decreased latencies. Whether CMS does sensitize beta-receptors remains to be seen, as results were inconclusive.
96 present to remind them of the stressors they endured, subjects did not develop a depressive or anxiety-like behavioral phenotype, indicating that learning is important in behavioral dysfunction, and that prolonged stress exposure enhances that learning.
This occurs in part through stimulation of adrenergic receptors, as previous studies show and as my later study would confirm.
As discussed in Chapter II, the first question I set out to pursue with this CMS model was in how the central noradrenergic system is involved in stress-induced depressive behavior. With the use of beta-blockers in our paradigm, we were able to show that beta-adrenergic receptors appear to play a substantial role in the behavioral manifestation of stress, though the lack of efficacy presented by nadolol in behavioral rescue leads us to emphasize the importance of central receptors to stress-induced, contextual learning-mediated behavioral alteration. Through the use of multiple doses, we also saw that there is an optimal amount of β-AR stimulation necessary in keeping behavior stable, as doses too great or too small did not have the efficacy of those in the middle; furthermore, the varying dose-effect differentials on the different behaviors we observed also suggests that each behavior is the result of separate mechanisms that share alterations in the β-AR system as a common element.
This study also showed that the elevation of glucocorticoids that is the principal hallmark of stress physiology does not have the effect on behavior that it is commonly associated to have; as has been shown in Chapter II, greatly elevated levels of glucocorticoids, both during acute stress and at baseline, were present even in subject groups whose baseline behavior had been rescued with the use of the beta blocker.
97 That said, investigators have found in recent years that glucocorticoid receptors work in conjunction with adrenergic receptors in fear memory processing; if one receptor type is blocked with an antagonist, stimulation of the other does not produce the behavioral effects we typically expect to see. It is possible that while glucocorticoids are important in facilitating such behavioral changes, they are not necessary for their maintenance.
The next logical step was taken in Aim 2, as we examined explicitly the importance of context in NE-mediated affective dysfunction associated with stress.
Because subjects showing increased freezing behavior did not show similar anxiety-like behaviors in the open field, I concluded that anxiety-like behaviors that develop as a result of stress exposure do not arise in a generalized fashion, but rather through stress-induced enhancement of associative learning, just as had been shown obliquely in the prior study. While the previous study too showed that β-AR stimulation during stress appeared to contribute to these behavioral changes, we found in this study that this is due to receptor activation during the aversive experience itself -- the footshock, in context -- as propranolol was administered just one hour prior to operant box training.
That propranolol had no effect on any subject group’s behavior in the open field maze suggests further that what appears to be stress-induced anxiety-like behaviors are actually the consequences of a mediatory effect of enhanced emotional memory processing, initiated by stress-induced beta-receptor stimulation.
We predicted that, as with the freezing behavior of contextual fear conditioning, step-through latency into the darkened chamber of the apparatus would similarly increase, as subjects’ exposure to stress had enhanced ability to associate their
98 environment with aversive stimuli. What we observed, however, was diametrically opposed to this, and even in contrast to literature from which the hypothesis was based; animals repeatedly exposed to stress actually had lower retention latencies, on average only a quarter of those of non-stressed controls, suggesting that, rather than enhancing learning in this case, chronic stress led to a reduction in behavioral inhibition and an decrease in behavioral inhibition. This development has led to questions about how
CMS affects interactions between the amygdala and the prefrontal cortex, resulting in this reduced behavioral inhibition.
The PFC integrates cognitive and affective behavior. It modulates the stress response through regulation of the hypothalamus, and by extension, the HPA and the
SNS. Through impairment of LTP between the PFC and hippocampus, chronic stress has an architectural effect on the region, resulting in reduced synaptic plasticity and decreased volume (Arnsten, 2009), and leads to reduced activity, especially in the left hemisphere (Cerqueira, Mailliet, Almeida, Jay, & Sousa, 2007). With reduced activity of the PFC and damage to the hippocampus, there is accompanying hyperactivity of the amygdala, leading to increases in depressive and anxiety-like behavior and decreases in cognitive and behavioral flexibility (Cerqueira, Almeida, & Sousa, 2008; Liston,
McEwen, & Casey, 2009). Stress effects in the prefrontal cortex also lead to lowered behavioral inhibition and increased impulsivity in rodents (Mika et al., 2012) as well as in humans (Shackman, McMenamin, Maxwell, Greischar, & Davidson, 2009).
As I should have considered, the effects of stress are more complex than what would be observed when simply injecting animals with stress hormones. Where my
99 study differed from those illustrated in the literature was that, rather than directly facilitating β-AR stimulation in specific brain regions through the use of receptor agonists and other drugs (Ferry et al., 1999; Quirarte et al., 1997; Roozendaal et al.,
2009), my subjects were repeatedly exposed to stress. Direct infusion of drugs into subjects’ brains has its advantages, such as allowing investigators to observe the specific roles that neurotransmitters may play in the function of certain brain regions.
However, such an approach is not necessarily an ideal model of stress exposure, as stress affects the entire brain (as well as the periphery) and involves every neurochemical substrate coursing throughout the nervous system; ultimately, much consideration must be taken when designing studies examining certain physiological aspects of stress as they relate to behavior, as no single effect of stress takes place in a vacuum, and one must take care to see the forest for the trees.
In the third aim, I set out to determine how stress affects beta-receptor signaling so that it contributes to learning enhancement and subsequent behavioral changes, operating under the hypothesis that intracellular signaling is sensitized with chronic exposure to stress. However, my attempts to do so were less than optimal; before I am able to make any conclusive judgments and further hypotheses regarding other possible mechanisms or interactions between systems, I attribute the unclear results primarily to my choices in procedure.
There are a number of changes in protocol that would have to be considered, should I make further attempts at examining changes in intracellular activity. First, I would have to consider additional time points for certain signaling cascade elements
100 (e.g. an earlier time point for ERK1/2). It would be beneficial as well to include additional targets for analysis, such as the AMPA receptors typically produced through LTP, as well as PKA, Rap1, and B-Raf (Fig. 17), which are downstream signaling components of Gs- protein coupled receptor activation. Secondly, I would need to determine a method of tissue collection that would not result in a surge of norepinephrine release upon the subject’s death, and would also allow for the microdissection of more specific brain subregions, namely the basolateral nucleus of the amygdala and perhaps any relevant surrounding nuclei. Finally, I should have invested some time in learning how to conduct additional methods of analyzing my target proteins. While immunohistochemical techniques have their shortcomings in regard to protein quantification, IHC may be a good way to obtain at least preliminary evidence of protein increases in specific brain regions, eliminating the issue of tissue dissection. Similarly, in situ hybridization could have allowed me to find localized increases in GRIA1 transcription, indicating upregulation in AMPARs containing GluR1; while RT-PCR may more reliably quantify such increases, the diluting effect of using the entire amygdaloid tissue may blunt this advantage. Lastly, Western blotting could have been used as an alternative to ELISA, as it does have an advantage in that multiple target proteins could be analyzed with its use; one problem with an ELISA kit used in this study was that it did not actually quantify the target protein. Furthermore, ELISA can underestimate quantities as the technique only detects correctly folded proteins. However, as immunoblotting has disadvantages as well, being cumbersome and time-consuming in its use, I am not suggesting it replace ELISA as part of the protocol, only that it is taken into
101 Figure 17. Additional components of signaling at β-ARs. Between conversion of ATP into cAMP and phosphorylation of ERK1/2, protein kinase A, Rap1, and B-Raf could be targeted for analysis, as could be the AMPA receptor subunit glutamate receptor 1 and the gene that encodes it, GRIA1.
102 consideration. Until the procedures for this study are refined, I cannot say with certainty that chronic mild stress does not sensitize beta-adrenergic receptor signaling.
Future Directions
The most obvious next step to take in regard to fear conditioning is to examine forgetting (Riccio & Joynes, 2007) by extending the operant box CFC paradigm. I would like to test further the timing of propranolol administration (e.g. after training or forgetting) and perhaps to observe how extended stress exposure affects the forgetting of aversive stimuli. We have already seen in subsequent experiments that, interestingly, even a month-long delay between the training session and behavioral observation is not sufficient to extinguish the conditioned fear of the foot shock in the operant box, as animals that have been exposed to stress still spend 55% of their time freezing, compared to the one-third of the time that non-stressed controls spent frozen in place.
A hypothesis in Aim 2, with results discussed subsequently in Chapter III, was that repeated stress exposure does not contribute to generalized anxiety, that the anxiety-like behaviors that result from the stress are dependent upon context. However, fear is indeed generalizable, as it is common for PTSD patients to live normal lives until the they find themselves in situations that force them to recall, and ultimately relive, traumatic experiences, just as a veteran of combat may experience a flashback upon hearing a backfiring vehicle. It is because of this fear generalization that PTSD sufferers are prone to hyperarousal and highly elevated vigilance. Were I to continue this line of behavioral research, I would explore further the generalizability of contextual fear to different spaces with cues that are reminiscent of the original stressors. I would thus like
103 to observe how subjects behave when placed in locations that are separate from that where they were exposed to stress, but would feature similar environmental conditions, such as ambient temperature, lighting, odors, and sounds.
I hope to see further investigations into the mechanisms that lead to increased impulsive behavior. The passive avoidance tests we used to assess fear learning have shown us that, not only does chronic stress exposure lead to more behavioral changes than simply associative learning, but the mechanisms underlying these effect are also more complicated than we had anticipated, considering the results of the use of propranolol. To explore this would involve the manipulation of multiple individual regions of the brain and noradrenergic pathways, requiring a more complex and nuanced approach. Ironically, this may contradict my earlier statement regarding the focus on specific brain regions as a model of stress; however, I propose that future studies employ the use of neuromodulatory techniques like optogenetics or DREADDs to examine how these different regions and systems interact with one another to produce the effects we see develop from prolonged stress exposure.
An important early lesson I learned about stress research was in the individuality of predisposition to effects of stress and their consequent behavioral manifestation, as there are significant differences between different strains of rats used as subjects, particularly between the outbred Sprague-Dawley and inbred Fischer-344 strains used in our research, the latter of which being considerably more sensitive to stress in both physiology and behavior. In developing a behavioral CMS model in rodents, these disparities presented challenges that were influential in the development my interest in
104 resilience and vulnerability to stress as a research topic. As such, I would be interested in exploring factors that may be protective against the effects of contextual fear. These may involve introducing environmental enrichment paradigms, or may be based in social behavior, such as group housing or maternal care. I would like to know how such factors might work as prophylactic measures or in aiding in extinction of the fearful memories, and their physiological effects. Finally, I would also be interested in determining if these factors may have an effect on subjects’ offspring, whether through learned behavior or through alterations in the epigenome.
Final Thoughts
This thesis demonstrates to a certain degree the complexity of the stress response, that affective and behavioral dysfunctions do not develop due to any one mechanism on its own, that interactions between mechanisms can lead to unpredictable results, and that the behavioral consequences of chronic stress can be significantly affected when there are unforeseen oversights in experimental design.
The CMS paradigm is a valid and reliable approach to examining factors in the development of the stress-induced depressive phenotype. However, the environmental conditions of the procedural rooms are not often reported in the literature, leaving one to question certain logistical aspects of the protocols in use. I have some doubts that such conditions are considered so carefully in the broader field of stress research, and my experiences in it have come to show me that, because their effects are so profound and can fundamentally alter the direction of a study, they should not be ignored or even taken lightly.
105 Passive avoidance is frequently used in tests of contextual fear conditioning, and while passive avoidance applications have been used in studies of human behavioral inhibition (Farmer & Golden, 2009; Gremore, Chapman, & Farmer, 2005), it appears that it is rarely considered beyond studies of fear and memory in stress research, particularly in rodent models. However, passive avoidance looks to be a viable test for behavioral disinhibition because of the very nature of its execution; rats prefer to stay in dark, enclosed spaces when threatened, and must then inhibit their natural impulse to escape into the darkened chamber of the apparatus when presented with a new aversive experience. This element of inhibition in this task is equally present as that of learning and memory. It also translates well to stress research, as there is an aspect of impulsivity present in chronic stress-related behavior. Overeating, alcoholism, and drug abuse are all very common in affective and anxiety disorders. People have greater difficulty restraining themselves under heightened stress, they do not think as rationally, their executive functioning is dysfunctional. I hope that this discovery that my study has revealed will open up new ways of examining stress-induced impulsivity and that, overall, my findings will contribute to advances in stress research by causing investigators to think in new ways about how learning and emotion interact to influence behavior.
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