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Exercise and dopamine modulation of fear extinction

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Exercise and dopamine modulation of fear extinction
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Bouchet, Courtney Anne ( author )
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Exposure therapy ( lcsh )
Dopamine ( lcsh )
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Anxiety and trauma-related disorders are prevalent and debilitating, yet treatments lack long term efficacy. Behavioral therapies, such as extinction-based exposure therapy, are beneficial but highly susceptible to relapse— the return of fear following successful extinction. Exercise is emerging as a healthy, noninvasive, inexpensive means to augment fear extinction. Data collected for my Master’s thesis supports this notion, as physical exercise immediately following fear extinction both enhanced fear extinction memory and reduced the later relapse of fear in adult, male, Long Evans rats. Interestingly, the same effects were not observed in female Long Evans rats, a disparity that opens the door for further research. Exercise impacts a variety of peripheral and neurobiological systems, one being midbrain dopamine (DA) circuits. Viral-mediated transfer of a gene coding for a Designer Receptor Exclusively Activated by a Designer Drug (DREADD) allowed us to control activity of select populations of DA neurons with high specificity during fear extinction. Activating midbrain DA neurons during fear extinction mimicked the effects of exercise: rats displayed enhanced fear extinction memory and prevented fear renewal in a novel context; data paralleled by changes in neural activity in brain regions involved in fear and contextual processing. One consequence of DA neural activity is activation of DA-1 receptors (D1) in the dorsal striatum, and populations of dorsal striatum D1-expressing neurons are anatomically linked to fear circuits. Interestingly, pharmacological activation of D1 in the dorsal striatum during fear extinction impacted fear extinction memory in a context-specific manner. Activation of D1 receptors during fear extinction blocked fear renewal in a novel context, but had no effect on fear extinction memory when tested in the same context in which extinction was learned. These data suggest that activation of midbrain DA neurons is sufficient to reproduce the memory-modulating effects of exercise, and these effects are partly mediated by D1 signaling in the dorsal striatum.
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Thesis (M.S.)-- University of Colorado Denver
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by Courtner Anne Bouchet.

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EXERCISE AND DOPAMINE MODULATION OF FEAR EXTINCTION
by
COURTNEY ANNE BOUCHET B.A. University of Colorado Boulder, 2009
A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Biology Program
2017


This thesis for the Master of Science degree by Courtney Anne Bouchet has been approved for the Biology Program by
Benjamin N. Greenwood, Chair Erik Ole son John Swallow
Date: May 13, 2017


Bouchet, Courtney Anne (M.S., Biology Program)
Exercise and dopamine modulation of fear extinction Thesis directed by Assistant Professor Benjamin N. Greenwood
ABSTRACT
Anxiety and trauma-related disorders are prevalent and debilitating, yet treatments lack long term efficacy. Behavioral therapies, such as extinction-based exposure therapy, are beneficial but highly susceptible to relapse the return of fear following successful extinction. Exercise is emerging as a healthy, non-invasive, inexpensive means to augment fear extinction. Data collected for my Masters thesis supports this notion, as physical exercise immediately following fear extinction both enhanced fear extinction memory and reduced the later relapse of fear in adult, male, Long Evans rats. Interestingly, the same effects were not observed in female Long Evans rats, a disparity that opens the door for further research. Exercise impacts a variety of peripheral and neurobiological systems, one being midbrain dopamine (DA) circuits. Viral-mediated transfer of a gene coding for a Designer Receptor Exclusively Activated by a Designer Drug (DREADD) allowed us to control activity of select populations of DA neurons with high specificity during fear extinction. Activating midbrain DA neurons during fear extinction mimicked the effects of exercise: rats displayed enhanced fear extinction memory and prevented fear renewal in a novel context; data paralleled by changes in neural activity in brain regions involved in fear and contextual processing. One consequence of DA neural activity is activation of DA-1 receptors (Dl) in the dorsal striatum, and populations of dorsal striatum Dl-expressing neurons are anatomically linked to fear circuits. Interestingly, pharmacological activation of Dl in the dorsal striatum during fear extinction impacted fear extinction memory in a context-specific manner. Activation of D1 receptors during fear extinction blocked fear renewal in a novel context, but had no effect on fear extinction memory when tested in the same context in which extinction was learned. These data suggest that activation of midbrain DA neurons is sufficient to reproduce the memory-modulating effects of exercise, and these effects are partly mediated by Dl signaling in the dorsal striatum.
The form and content of this abstract are approved. I recommend its publication.
Approved: Benjamin N. Greenwood
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DEDICATION
Mom, Dad, and Chris Your unwavering support throughout my academic endeavors means the world to me. Mom, thanks for answering my phone calls at midnight when I was walking home from campus after
a late experiment or late night in the lab.
Granddaddy Ever since I was little you have inspired me to be curious. Your passion for knowledge and
understanding has always been an inspiration.
Jeff Randall Thank you for being my rock and for learning way more about fear extinction and dopamine than you ever anticipated. Your support, enthusiasm, and kindness makes me so happy.
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ACKNOWLEDGEMENTS
Dr. Benjamin N. Greenwood -1 certainly would not be where I am today without you. Thank you for your support and guidance. Your enthusiasm and curiosity for research is contagious. I appreciate all that you
have done for me.
Dr. Aggie Mika -1 feel so lucky to have been mentored by you. Thank you for your guidance throughout my scientific career. Thank you for reminding me that science is fun and enjoyable.
The Greenwood Lab Thank you all for everything that you do. These experiments took a lot of time and effort, your help was much appreciated and necessary and did not go unnoticed. Specific authors for publications are identified in chapters.
Dr. Hannah Anchordoquy Thank you for your mentorship, you molded me into the teacher that I am today. Your guidance and support was incredibly helpful. I am forever grateful for it and for you.
Dr. Erik Oleson and Dr. John Swallow Thank you for making such a great and supportive committee.
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TABLE OF CONTENTS
I. INTRODUCTION.............................................................................1
Factors that Modulate Fear Extinction....................................................2
II. ACUTE EXERCISE ENHANCES THE CONSOLIDATION OF FEAR EXTINCTION MEMORY
AND REDUCES CONDITIONED FEAR RELAPSE IN A SEX-DEPENDENT MANNER..............................7
Introduction.............................................................................8
Materials and Methods...................................................................10
Subjects..............................................................................10
Estrus Cycle Monitoring...............................................................10
Behavioral Analyses...................................................................11
Post-Extinction Wheel Running.........................................................13
Procedures............................................................................13
Statistical Analysis..................................................................16
Results.................................................................................17
Experiment 1: Exercise During Consolidation of Contextual Fear Extinction Improves Long-Term
Memory of Fear Extinction in Males....................................................17
Experiment 2: Exercise Dining Consolidation of Auditory Fear Extinction Improves Fear Extinction
Memory and Reduces Fear Renewal in Males..............................................19
Experiment 3: Exercise during consolidation of auditory fear extinction fails to improve fear
extinction memory or reduce fear renewal in females...................................21
Discussion..............................................................................25
III. ACTIVATION OF NIGROSTRIATAL DOPAMINE NEURONS DURING FEAR EXTINCTION
PREVENTS THE RENEWAL OF FEAR...............................................................31
Abstract................................................................................31
Significance Statement..................................................................32
Introduction............................................................................32
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Materials and Methods
33
Animals...............................................................................33
Surgeries.............................................................................34
Microinjections.......................................................................35
Drugs.................................................................................35
Behavioral Analyses...................................................................36
Double-label fluorescent in situ hybridization........................................37
Immunohistochemistry..................................................................39
Statistical Analyses..................................................................40
Results..................................................................................40
Activation of SNc DA neurons enhances fear extinction memory and blocks fear renewal..40
Gq-DREADD activates target D1-expressing neurons in the DS............................43
Gq-DREADD-induced SNc DA activation during fear extinction alters brain activation patterns
during renewal........................................................................45
Activation of DS D1 receptors during fear extinction blocks fear renewal without enhancing fear
extinction memory.....................................................................47
Discussion...............................................................................50
Acknowledgements.........................................................................54
IV. CONCLUSIONS............................................................................55
REFERENCES..................................................................................56
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CHAPTERI
INTRODUCTION
Anxiety disorders are the most prevalent mental health disorders in developed countries with a lifetime prevalence of 28% (Kessler et al 2012, Kessler et al 2005) and yet, treatments for such disorders have limited availability and efficacy (Hoffman & Mathew 2008, Stein et al 2009, Steinberg et al 2009). Mechanisms underlying fear conditioning are thought to go awry in anxiety disorders (Elzinga & Bremner 2002). Current behavioral therapies to treat such disorders, such as exposure therapy, focus on fear extinction. Extinction is the decay of the fear response following repeated presentations of the fearful stimulus in the absence of an aversive event. Even after successful exposure therapy, rates of relapse remain high (Boschen et al 2009, Bouton 1993, Goode & Maren 2014). The three main types of fear relapse are 1) the return of fear in context different from which the extinction was learned (renewal; (Bouton & Ricker 1994b), 2) the return of fear after a stressful event (reinstatement; (Rescorla & Heth 1975), and 3) the return of fear over time (spontaneous recovery; (Goode & Maren 2014).
Fear conditioning is a Pavlovian associative learning phenomenon during which a previously neutral stimulus (conditioned stimulus; CS) comes to elicit a fear response because of its association with an aversive stimulus (unconditioned stimulus; US; (Pavlov 1927). Repeated exposures to the CS in the absence of the US extinguishes the fear associated with the CS, but this extinction is context-dependent. The relapse of fear following successful extinction suggests that extinction does not erase the fear memory, but that the extinction memory is a separate entity from the fear memory that suppresses the fear memory (for reviews see (Bouton 2004, Myers & Davis 2007). Manipulations that modify fear extinction learning or consolidation could produce an extinction memory that is resistant to contextual modulation, thus reducing fear renewal.
Considerable work has been done to elucidate the neural circuitry involved in fear extinction (Herry et al 2010, Knapska et al 2012, Mamiya et al 2009, Quirk & Mueller 2008, Sierra-Mercado et al 2011) and factors capable of modulating fear extinction learning and memory (Singewald et al 2015). Critical regions involved in fear and its extinction include the basolateral (BLA; comprised of the basal, lateral, and ba-somedial amygdala nuclei) and central nucleus of the amygdala (CeA), the hippocampus, and the medial
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prefrontal cortex (mPFC), specifically the prelimbic (PL) and infralimbic (IL) regions. These regions of the mPFC are thought to play opposing roles in the expression of fear. The PL, through excitatory projections to the CeA, drives the fear response during exposure to fear-eliciting events or cues (Burgos-Robles et al 2009, Corcoran & Quirk 2007, Fendt & Fanselow 1999, LeDoux et al 1988). In contrast, through neural projections to the inhibitory intercalated intemeurons of the amygdala, the IL can inhibit the expression of fear by inhibiting output of the CeA (Quirk et al 2006). Recent work, however, suggests there is limited connectivity between the IL and the intercalated intemeurons of the amygdala. This observation is consistent with, and could partially explain, the transient nature of fear extinction memories relative to fear memories (Giustino & Maren 2015). The BLA has been implicated in both fear conditioning and extinction with distinct cell populations contributing to high- and low-fear states (Goosens et al 2003, Herry et al 2008, Hobin et al 2003).The CeA is thought to function as an output station and is critically involved in the fear response through projections to the brain stem, hypothalamus, and periaqueductal gray (PAG), which together initiate the various aspects of the fear response including freezing (Fanselow & Gale 2003, Pare et al 2004). Contextual information pertaining to fear and extinction memories, which is an important contributor to the renewal of fear (Bouton 2004, Bouton et al 2006b), is encoded by the hippocampus (Corcoran & Maren 2001). For example, Jin and Maren (2015) found that dorsal hippocampal collateral projections to the IL and basal nucleus of the amygdala are preferentially activated during fear renewal, and inactivation of the hippocampus prevents renewal (Jin & Maren 2015). Huge strides have been made in understanding the circuitry underlying fear and its extinction; however, less is known about factors that modulate fear extinction and relapse.
Factors that Modulate Fear Extinction
Many factors are capable of modulating fear extinction learning and memory (see (Fitzgerald et al 2014, Singewald et al 2015) for reviews). These manipulations differ in their ease of use and success at reducing relapse in a clinical setting. For example, massive extinction, or a large number of extinction trials, reduces fear renewal in rodents (Denniston et al 2003); however, it is unlikely that patients will be willing to commit to the immense time commitment that mass extinction requires. Another behavioral manipulation aimed at reducing relapse involves performing exposure therapy in multiple contexts in order to
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reduce the contextual dependency of extinction memories. However, reports on the effects of extinction in multiple contexts on later fear relapse are mixed, with some studies reporting promising results (Chelonis et al 1999, Gunther et al 1998) and others not (Bouton et al 2006a). Extinction in multiple contexts also brings clinical challenges as it requires therapy sessions to occur in multiple different clinical environments. Some pharmacological manipulations could be simple to implement in a clinical setting, but not all pharmacological means to enhance extinction result in a successful reduction of relapse. Activation of glutamate NMDA receptors with D-cycloserine (DCS) during fear extinction can strengthen extinction learning; however, there are limitations to DCS treatment (Graham et al 2011). One limitation is that DCS also strengthens memory re-consolidation (Lee et al 2006) and is not specific to the extinction memory, thereby allowing the possibility that DCS during re-exposure to the fearful stimulus during extinction could actually strengthen the fear memory instead of extinguishing it. Indeed, fear extinction augmented with intra-IL or intra-BLA injections of DCS remains susceptible to fear renewal (Woods & Bouton 2006). Glucocorticoids are another modulatory system that can enhance fear extinction in rats (Bentz et al 2010, de Bitencourt et al 2013, Soravia et al 2014, Yang et al 2006). Clinical glucocorticoid therapy could be problematic, however, because acute glucocorticoid treatment has the potential to increase anxiety (Mitra & Sapolsky 2008), which could be counter-productive to therapy.
Despite potential limitations associated with the aforementioned strategies, several manipulations with the potential to reduce fear relapse after extinction remain. Among those most likely to be involved in the effects of exercise are neuromodulatory systems such as the endocannabinoid system, growth factors, such as Fibroblast growth factor-2 (FGF2; (Graham & Richardson 2011a)) and BDNF (Baker-Andresen et al 2013), as well as signaling through specific monoaminergic circuits. The endocannabinoid system is implicated in fear extinction learning (Papini et al 2015), is sensitive to physical activity, and has been implicated in anxiolytic effects of exercise (Fuss et al 2015). Endocannabinoids such as 2-arachidonoyl glycerol (2-AG) are increased in the BLA during fear extinction (Marsicano et al 2002), and blocking effects of endocannabinoids by receptor antagonists (Marsicano et al 2002) or genetic knockout of synthetic enzymes of 2-AG (Jenniches & Zimmer 2016) impairs fear extinction learning. Growth factors such as FGF2 are critically involved in the molecular mechanisms of long term memory (Graham & Richardson 2009b) and,
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if increased during extinction consolidation, can enhance fear extinction and reduce reinstatement (Graham & Richardson 2009a) and renewal (Graham & Richardson 2011b) of fear. Brain-derived neurotrophic factor (BDNF) is also intimately involved in memory and neuroplasticity. BDNF signaling in the BLA during fear extinction is necessary for consolidation of extinction memories (Chhatwal et al 2006), while activation of BDNF signaling pathways reduces the renewal of fear in female mice (Baker-Andre sen et al 2013). Additionally, extinction enhanced by BDNF produces a stronger long-term extinction memory ((Bredy et al 2007); for a recent review of the role of BDNF in fear extinction augmented by exercise see (Powers et al 2015)). Another neurotransmitter, dopamine (DA), classically associated with movement, reward, and reinforcement, is also an important memory modulator. Growing literature implicates DA as a promising potential modulator for fear extinction memory (see (Abraham et al 2014a) for a recent review). One, or many, of these factors could potentially be involved in the mechanisms by which acute exercise strengthens fear extinction learning and reduces the relapse of fear.
Three main dopaminergic pathways within the central nervous system are 1) the mesocortical pathway originating in the ventral tegmental area (VTA) and terminating in the PFC, 2) the mesolimbic pathway originating in the VTA and terminating in the nucleus accumbens (NA), and 3) the nigrostriatal pathway originating in the substantia nigra (SN) and terminating in the dorsal striatum (DS). Of course, midbrain DA neurons project to many other regions potentially involved in fear extinction, including the hippocampus and AMG. Recruitment of one or more of these DA pathways during acute exercise could potentially strengthen extinction memory.
DA systems could be recruited during exercise in order to promote locomotor activity or instrumental / goal-directed processes such as learning to run or choosing to run, or could be recruited due to the rewarding effects of exercise (Knab & Lightfoot 2010). Studies investigating the effects of exercise on extracellular DA have utilized microdialysis during forced treadmill training. These studies indicate that forced exercise increases extracellular DA in various brain regions including the hippocampus (Goekint et al 2012), hypothalamus (Ishiwata et al 2001), and striatum (Meeusen et al 1997). The effect of voluntary exercise on DA efflux remains unknown. Studies using immediate early genes to indirectly assess activity of DA cell bodies suggest that both forced and voluntary exercise activate midbrain DA neurons (Herrera
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et al 2016), an observation consistent with neural adaptations in the striatum suggestive of repeated DA activity during voluntary exercise (Clark et al 2014, Foley & Fleshner 2008, Greenwood et al 2011, Herrera et al 2016, Meeusen & De Meirleir 1995).
The fact that exercise recruits DA systems allows the possibility that DA could be involved in the mechanisms by which acute exercise augments fear extinction. The role of DA in fear extinction, however, is far from resolved (reviewed in (Abraham et al 2014b, de la Mora et al 2010, Singewald et al 2015). A study using fast-scan cyclic voltammetry revealed that phasic DA release in the nucleus accumbens core is initially suppressed by presentation of a fear-evoking CS during extinction, however; as extinction progresses and, presumably, as a prediction error occurs due to the absence of the expected US, phasic DA release in the NAc core increases (Badrinarayan et al 2012). These data suggest that DA systems are recruited during fear extinction, and allow the possibility that DA signaling could be involved in fear extinction learning. Other studies suggest that augmenting DA signaling can enhance fear extinction. Increasing extracellular DA by blocking DA re-uptake immediately following extinction enhances consolidation of fear extinction memory in mice (Abraham et al 2012). Human carriers of a polymorphism in the DA transporter gene, DAT1, which is predominantly expressed in the striatum and confers enhanced phasic DA release, show faster learning rates of fear extinction (Raczka et al 2011). Additionally, systemic administration of L-DOPA during the acquisition or consolidation phase of fear extinction learning reduces spontaneous recovery and reinstatement of conditioned fear in both mice & humans (Haaker et al 2013). Importantly, the reduction in fear relapse produced by L-DOPA is similar to that observed following acute exercise (Mika et al and Figure 1).
Despite these promising results, other studies report impairments (Borowski & Kokkinidis 1998, Mueller et al 2009), or no effect (Carmack et al 2010, Fiorenza et al 2012) of systemic DA manipulations on fear extinction. Inconsistent results could at least partly be due to differences in specific DA receptors manipulated, and/or the inability of pharmacological approaches to target specific neural circuits. Consistent with the former possibility, systemic administration of a Dl-like receptor agonist SKF 81297 during either the acquisition or consolidation phases of fear extinction learning enhances recall of both contextual and cued fear extinction (Abraham et al 2016). In contrast, systemic administration of the DA 2
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(D2)-like receptor agonist, quinpirole, partially blocks extinction memory, and systemic administration of the D2 antagonist, sulpiride, facilitates fear extinction memory (Ponnusamy et al 2005). These data suggest opposing roles of Dl and D2-like DA receptors in fear extinction. However, blockade of the D2 receptor by microinjection of haloperidol directly into the NAc during the acquisition of fear extinction was reported to impair fear extinction learning (Holtzman-Assif et al 2010b). Thus, the specific roles of various DA receptors in fear extinction likely depend on the brain region targeted. Indeed, one study reported that DA in the hippocampus can enhance fear extinction (Fiorenza et al 2012), while another indicated that DA in the IL inhibits the circuitry supporting fear extinction memory through a mechanism involving Dl receptors (Hitora-Imamura et al 2015).
We hypothesize that acute exercise immediately following fear extinction learning, during the consolidation period, will modulate fear extinction memory in such a way to enhance fear extinction memory and block fear renewal. Further, we hypothesize that nigrostriatal DA is involved in this process and, thus, we will be able to mimic the effects of exercise by activating nigrostriatal DA. To test these hypothesizes, we used a design that allowed male and female rats to run on voluntary running wheels immediately following either auditory or contextual fear extinction. In a separate set of experiments, we used Gq-DREADD to induce DA phasic release during fear extinction or a D1 receptor agonist to activate the Dl receptor in the DS.
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CHAPTER II
ACUTE EXERCISE ENHANCES THE CONSOLIDATION OF FEAR EXTINCTION MEMORY AND REDUCES CONDITIONED FEAR RELAPSE IN A SEX-DEPENDENT MANNER
Running title: Exercise and fear extinction
Courtney A. Bouchet1, Brian A. Lloyd1, Esteban C. Loetz1, Caroline E. Farmer1, Mykola Ostrovskyy1, Natalie Haddad1, Rebecca M. Foright2, Benjamin N. Greenwood1*
'University of Colorado, Denver. Department of Psychology 2University of Colorado, Denver. School of Medicine, Anschutz Medical Campus.
* Corresponding author
Keywords: wheel running, fear conditioning, fear renewal, anxiety Accepted with revisions: Learning and Memory. 3/10/17 Abstract
Fear extinction-based exposure therapy is the most common behavioral therapy for anxiety and trauma-related disorders, but fear extinction memories are labile and fear tends to return even after successful extinction. The relapse of fear contributes to the poor long-term efficacy of exposure therapy. A single session of voluntary exercise can enhance the acquisition and consolidation of fear extinction in male rats, but the effects of exercise on relapse of fear after extinction is not well understood. Here, we characterized the effects of 2 h of voluntary exercise during the consolidation phase of contextual or auditory fear extinction learning on long-term fear extinction memory and renewal in adult, male and female, Long-Evans rats. Results indicate that exercise enhances consolidation of fear extinction memory and reduces fear relapse after extinction in a sex-dependent manner. These data suggest that brief bouts of exercise could be used as an augmentation strategy for exposure therapy, even in previously sedentary subjects. Fear memories of discrete cues, rather than of contextual ones, may be most susceptible to exercise-augmented extinction, especially in males. Additionally, exercise seems to have the biggest impact on fear relapse phenomena, even if fear extinction memories themselves are only minimally enhanced.
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Introduction
Fear conditioning is an associative learning phenomenon during which a previously innocuous cue (conditioned stimulus; CS) comes to elicit an emotional fear response because of its association with an aversive stimulus (unconditioned stimulus; US). Mechanisms underlying fear conditioning are thought to go awry in anxiety and trauma-related disorders (Elzinga & Bremner 2002). Fear extinction, the decay of a fear response to a CS following repeated presentation of the fear-evoking CS in the absence of the aversive US (Pavlov 1927), forms the basis of exposure therapy, the behavioral therapy of choice for anxiety and trauma-related disorders. Unfortunately, extinction memories are labile and susceptible to relapse phenomena such as renewal (Bouton 1988), spontaneous recovery (Pavlov 1927) and reinstatement (Rescorla & Heth 1975); which together contribute to the poor long-term efficacy of exposure therapy (Boschen et al 2009, Neumann & Kitlertsirivatana 2010). Identification of novel means to render fear extinction memories impervious to relapse is of utmost importance to mental health. One factor that enhances many learning and memory processes and could potentially modulate fear extinction is physical activity.
Despite the well-established benefits of chronic exercise on cognition and learning processes (Cassilhas et al 2015, Cotman & Berchtold 2002, Hillman et al 2014, Hillman et al 2003, Prakash et al 2015), the lingering changes in the brain produced by habitual exercise are not sufficient to enhance fear extinction (Greenwood et al 2009). Moreover, maintenance of chronic exercise is essential for use in a clinical setting, yet maintaining regular exercise is a constant challenge and long-term exercise adherence rates are low (Dishman 1982, Hogg et al 2012, Zuckoff 2012) even when initial motivation is high (Van Roie et al 2015). In contrast to weeks of repeated exercise, individuals may be more likely to adhere to a recommendation of a relatively few sessions of exercise. A few acute bouts of exercise could be more practical to implement in clinical settings than repeated exercise. Even so, prior research investigating the effects of a single, recent bout of exercise (acute exercise) as a treatment for anxiety or trauma-related disorders is limited. Investigating the effects of acute exercise within the context of fear extinction learning and memory in an animal model is an initial step toward utilization of acute exercise in a clinical setting.
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Fear extinction learning incorporates two critical phases: the acquisition phase and the consolidation phase. During the acquisition phase, the association between the fear-eliciting CS and the lack of the predicted aversive event is initially encoded. Later, during the consolidation phase, molecular processes take place in the neural circuits responsible for forming the long-term memory of fear extinction. Previous research investigating the effects of chronic exercise on subsequent contextual (Greenwood et al 2009) or auditory (Dubreucq et al 2015) fear extinction report negative results, possibly due to the inability of prior chronic exercise to target a distinct extinction learning phase. Exercise initiates a plethora of physiological and neurobiological events; the timing of which in relation to a specific phase of fear extinction learning could be critical for the ability of exercise to modulate fear extinction.
There is evidence that acute exercise, within a small temporal window in relation to fear extinction learning, can modulate later fear extinction memory and relapse. Siette et al. (2014) reported that previously sedentary male rats allowed 3 h of voluntary exercise either immediately before or after contextual fear extinction displayed improved retention of fear extinction memory when tested the next day. However, if rats were allowed the same 3 h of acute exercise 6 h after fear extinction, presumably after the consolidation phase, exercise had no effect on extinction memory retention (Siette et al 2014). A pilot study in humans revealed a similar effect. Powers and colleagues (Powers et al 2015) allowed mostly female patients suffering from post-traumatic stress disorder (PTSD) to engage in moderate intensity treadmill exercise for 30 min immediately prior to each of 12 sessions of exposure therapy. Those patients who exercised prior to exposure therapy showed significant improvement of PTSD symptoms immediately following the final therapeutic session, relative to patients who had 90 min of exposure therapy without exercise. Further, Mika et al. (2015) found that male rats allowed to run in wheels during the acquisition of auditory fear extinction, relative to rats extinguished in locked wheels, demonstrated reduced fear when re-exposed to the CS one wk later in a novel context.
Although prior data suggest that acute exercise can enhance the acquisition or consolidation of fear extinction in such a way as to reduce fear relapse, critical questions remain unanswered. Siette et al. (2014) did not investigate the effects of acute exercise on relapse of contextual fear conditioning following extinction, and the experimental design used in Mika et al. (2015) did not allow the differentiation of the
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effects of acute exercise on auditory fear extinction memory from its relapse. Further, none of these previous experiments have investigated the effects of voluntary wheel running on fear extinction learning, memory and relapse in female rats. This is a critical oversight, as females can have impaired fear extinction depending on their estrus cycle phase (Milad et al 2009) and, relative to males, have a higher incidence of anxiety (McLean et al 2011) and are more likely to develop PTSD (Breslau et al 1997). The goal of the current studies was to further characterize effects of acute, voluntary exercise on consolidation and relapse of contextual and auditory conditioned fear in both males and cycling females. It is hypothesized that acute exercise during the consolidation phase of fear extinction learning will enhance fear extinction memory and reduce later fear relapse in both sexes.
Materials and Methods
Subjects
Adult, male (N=71, p~54 on date of arrival) or female (N=40, p~54 on date of arrival) Long Evans rats were used for all experiments. Animals were pair-housed in ventilated rat cages (45 W x 25.2 D x 14.7 H cm) with ad libitum access to food (standard Rat Chow) and water. The housing room was kept on a 12 12 h light-dark cycle with lights on from 0700 to 1900 and temperature was maintained at 25C. All procedures took place in the University of Colorado Denver Auraria campus animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Animals were allowed 1 wk to acclimate to their housing conditions before start of experimental manipulations. All rats were gently handled once daily during the last 4 days of this acclimation week. Care was taken to minimize pain and all protocols were approved by the University of Colorado Denver Institutional Animal Care and Use Committee.
Estrus Cycle Monitoring
After 1 wk of acclimation to the vivarium, vaginal lavages were conducted every 24 h for 5 consecutive days in order to establish cycle and habituate rats to lavage. Briefly, a sterile, blunt-tip eyedropper was used to flush the vagina with approximately 0.5 mL sterile filtered 0.2% PBS-Brij solution (Brij 35 Solution 30%; Sigma, B4184) using the ventral method (Becker et al 2005) to obtain vaginal epithelial cells. The fluid collected from lavages was transferred to a microscope slide and tissue was analyzed under
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bright-field at 400X (Olympus BX53). A single lavage was again collected ~ 6 h prior to fear extinction learning, immediately following the fear renewal test, and immediately following the long-term extinction memory test to confirm phase of estrus cycle on these days. Estrus cycle phase was determined by the morphology of the cells collected during vaginal lavages as previously described (Cora et al 2015).
Briefly, proestrus consists of small, round, often clumped mononucleated cells of relatively the same size (Figure 5F), the estrus phase is predominated by anucleated keratinized epithelial cells (Figure 5G), metes-trus consists of anucleated keratinized epithelial cells interspersed with leukocytes (Figure 5H), and die-strus is predominated by leukocytes and nucleated epithelial cells with a decrease in anucleated keratinized epithelial cells (Figure 51).
Wheel Running
At the start of all experiments, rats were placed into pre-assigned running wheels (1.1m circumference; Fafayette Instruments, Fafayette, IN, USA) for the duration of the dark (active) cycle for 4 consecutive nights. On alternating nights, the wheels were rendered immobile so that, in total, rats had 2 nights of voluntary running in mobile running wheels and 2 nights in immobile (locked) running wheels. The purpose of this familiarization procedure was two-fold: (1) to ensure that both the mobile and locked wheel environments were equally familiar and (2) to increase running behavior after extinction, as in our experience rats lacking prior experience with a running wheel run minimally. All wheel running activity was recorded with Activity Wheel Monitor software (Fafayette Instruments; Fafayette, IN, USA). Behavioral Analyses Fear Conditioning
Between 0800 and 1100, rats were placed into custom, rectangular conditioning chambers (20W x 10D x 12H; Context A) with a shock grid floor (Coulbourn Instruments, Allentown, PA) housed inside individual sound-attenuating cabinets. Rats were transported to Context A in their home cages. A fan located near the floor of the cabinets provided ventilation and background noise and bright white lights illuminated the chambers. For contextual fear conditioning (Experiment 1), rats were allowed 5 min to explore the context, after which 3 foot shock US (Is, 0.8mA) were delivered with a 1 min ITI. For auditory fear conditioning (Experiments 2 and 3), rats were allowed to explore the context for 3 min, followed by 4
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exposures to an auditory CS (10s, 80dB, 2kH), each co-terminating with a Is, 0.08 mA foot shock US delivered on a 1 min ITI. Auditory stimuli and foot shocks were delivered through Coulbourn tone generators and shock scramblers controlled via Noldus EthoVision XT software (Tacoma, WA) through a custom interface. All rats remained in the conditioning chamber for 1 min after the last shock before being transported back to their home cages. Chambers were cleaned with water between rats. Freezing behavior, an innate fear response, was defined as the absence of all movement except that required for respiration (Fan-selow 1980) and was used as the measure of fear in all behavior tests.
Fear Extinction
Each fear extinction training session took place near the start of the active (dark) cycle, to maximize running behavior of rats in the Mobile group (Greenwood et al 2011). Rats exposed to contextual fear extinction (Experiment 1) were placed into context A for 15 min in the absence of the shock US. All transport, lighting, and cleaning conditions were identical to fear conditioning. Rats exposed to auditory fear extinction (Experiments 2 and 3) were placed into a novel Context B that was either a custom Plexiglas rectangular chamber (15 W x 15 D x 20 H) with a textured floor or a custom Plexiglas triangular chamber (15 sides x 20 H) with a smooth floor. Rectangular and triangular Context B chambers were counterbalanced so half the rats were exposed to fear extinction training in the rectangular chambers and the other half in the triangular chambers. Context B was housed in the same sound-attenuating cabinets used for conditioning, but all other contextual features and discrete cues differed between contexts. Rats were transported to the sound attenuating chambers, which included vanilla scent, in their assigned Context B custom Plexiglas chamber. The fan within the chamber was turned off and the room was dimly lit by a lamp outside of the sound attenuating chambers. Context B was cleaned with 10% ethanol between rats. After a 3 min exploration period, the auditory CS was administered 30 times (1 min ITI) in the absence of the foot shock US. Similar auditory extinction parameters have been used previously in Long Evans rats (Maren 2014).
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Fear Renewal Test
Between 0800-1100, rats exposed to auditory fear conditioning and extinction were re-exposed to the auditory CS in either the same context used for extinction training (Context B; Same) or a novel Context C (Different). Rats were assigned to Same or Different contexts based on freezing levels during fear extinction, such that freezing levels during extinction were balanced between groups. Rats assigned to the Different group were transported to Context C in a novel inner Plexiglass chamber, so that rats extinguished in the square Plexiglass chamber were now placed into the triangle, and vise-versa. Context C consisted of a raspberry scent, red box lights, a fan near the top of the behavior cabinets was turned on, and chambers were cleaned with 1% acetic acid between tests. After a 3 min exploration period, the auditory CS was presented 4 times (1 min ITI) in the absence of the foot-shock US. Because fear renewal is the return of fear in contexts different from where fear extinction was learned (Bouton & Ricker 1994a), the difference in freezing between the Same and Different contexts was considered fear renewal.
Post-Extinction Wheel Running
Immediately following contextual or auditory fear extinction, rats were transferred to their familiar running wheels that were either rendered immobile (Locked) or freely mobile (Mobile). Rats were assigned to Locked or Mobile conditions based on freezing levels during fear conditioning, such that freezing levels during conditioning were balanced between wheel assignments. Rats were returned to their home cages following 2 h of exposure to the locked or mobile wheels. Rats in the No Extinction group were placed in their assigned Locked or Mobile wheels for 2 h / night, but were not exposed to fear extinction.
Procedures
Experiment 1: Effects of Acute Exercise on Extinction of Contextual Fear Conditioning
A timeline for the experiment is shown in Figure 1 A. Prior to fear conditioning, rats (N=32 males) were exposed to the running wheel familiarization procedure, so each rat spent 2 active cycles in mobile,
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A. Experiment 1
Running Wheel Familiarization
Conditioning
Extinction Day 1
Extinction
consolidation
Extinction Day 2
Extinction
consolidation
Extinction Day 3
Extinction
consolidation
No
Extinction
No
Extinction
No
Extinction
Extinction Memory Day 10
3 US 36 h ^ 15 Min 15 Min 15 Min 15 Min
Context A \ Context A / \ Context A / \ Context A / Context A
B. Experiment 2
Conditioning
Extinction Day 1
Fear Renewal Test
Running Wheel Familiarization
Figure 1. Experimental design. (A) All rats were placed into unlocked and locked wheels on alternating nights for 4 nights to equally familiarize rats with mobile and locked running wheels. Three days following the last running opportunity, rats were exposed to contextual fear conditioning in Context A by administering an un-signaled 1 s, 0.8 mA foot shock unconditioned stimulus (US) 3 times with a 1 min inter-trial interval (ITI). The following evening, all rats were placed back into the familiar conditioning Context A for 15 min immediately followed by 2 h in either locked or mobile running wheels. Contextual fear extinction followed by 2 h in locked or mobile wheels was repeated the following 2 evenings. One week following the last extinction trial, rats were placed into the conditioning Context A to assess long-term fear extinction memory. (B) All rats were placed into mobile and locked wheels on alternating nights for 4 nights to equally familiarize rats with mobile and locked running wheels. Three days following the last running opportunity, rats were conditioned to fear a tone in Context A by co-terminating a 10 s, 80 dB tone conditioned stimulus (CS) with a 1 s, 0.8 mA foot shock US four times with a 1 min inter-trial interval. The following evening, all rats were placed into a novel extinction Context B and exposed to the auditory CS 30 times in the absence of the foothsock US (1-min ITI). Immediately following fear extinction, rats were placed into either mobile or locked running wheels for 2 h. The fear extinction and running procedure was repeated the following evening, for a total of 2 nights of fear extinction following by either mobile or locked conditions. The morning after the second fear extinction trial, half of the rats were placed into the familiar, extinction Context B and the other half were placed into a novel Context C to test for fear renewal. (C). Experiment 2 was repeated with female rats with extinction consisting of 20 CS presentations instead of the 30 CS presentations as in experiment 2. One week following the fear renewal test, rats were placed back into Context B to assess long-term fear extinction memory.
voluntary running wheels alternating with 2 active cycles in the same locked running wheel. Three days following the second running opportunity, rats underwent contextual fear conditioning in Context A. During the following 3 evenings, immediately prior to the dark (active) cycle, rats were re-exposed to Context A for 15 min in the absence of shock, in order to extinguish contextual fear. Each of the three, 15 min extinction training sessions was immediately followed by 2 h in familiar Locked (n =11) or Mobile (n = 11) running wheels, so that rats in the Mobile group ran in wheels during the consolidation phase of contextual
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fear extinction. Rats were again exposed to Context A 1 wk following the third contextual fear extinction training session in order to assess the effects of post-extinction wheel running on long-term extinction memory / spontaneous recovery of fear. An additional cohort of rats were conditioned and placed into their assigned mobile or locked running wheels at the same time as the other rats, but were not exposed to fear extinction training. This subset of Locked (n=5) or Mobile (n=5) rats was placed into Context A on the day of the long-term extinction memory / spontaneous recovery test to assess potential effects of running in the absence of fear extinction training on subsequent freezing behavior.
Experiment 2: Effects of Acute Exercise on Extinction and Renewal of Auditory Conditioned Fear in Males
A timeline for the experiment is shown in Figure IB. Three days following the second familiarization running opportunity, rats (N=39 males) underwent auditory fear conditioning in Context A. The following 2 evenings, immediately prior to the dark (active) cycle, rats were exposed to auditory fear extinction training in Context B immediately followed by placement for 2 h into their familiar Locked (n=20) or Mobile (n=19) running wheels, so that rats assigned to the Mobile condition had the opportunity to run during the fear extinction consolidation period. The morning after the second fear extinction training session, rats were again exposed to the CS either in the extinction Context B (Locked Same n = 10; Mobile Same n = 10) or in a novel Context C (Locked Different n = 10; Mobile Different n = 9) to assess fear renewal.
Experiment 3: Effects of Acute Exercise on Extinction and Renewal of Auditory Fear Conditioning in Females
A timeline for the experiment is shown in Figure 1C. Experiment 2 was repeated except cycling females (n = 40 females) were used instead of males. Since males and females have been reported to similarly acquire auditory fear conditioning (Maren et al 1994), female rats in experiment 3 were administered the same number of CS-US pairings during conditioning as male rats in experiment 2. Pilot studies revealed that females displayed less freezing behavior during fear extinction memory tests than males. Therefore, to help avoid floor effects on freezing, females were exposed to 20 CS presentations during fear extinction training. A long-term fear extinction memory / spontaneous recovery test was added 1 wk after the fear renewal test.
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All behavioral tests were recorded with overhead cameras and freezing was scored both by multiple experimenters blind to the experimental conditions of the animals and by automated behavioral analyses software (Noldus Ethovision XT) during both the CS and the ITIs. Because regularly-scheduled ITIs can become part of the CS, and analyses indicated a lack of differential effects of exercise on freezing during ITIs and CS, freezing during each CS and subsequent ITI were combined and expressed as freezing during a trial, as in prior work (Fitzgerald et al 2015, Goode et al 2015, Mika et al 2015).
Statistical Analysis
Running distances were calculated using the Lafayette Instruments Activity Wheel Monitor data management add-on for Microsoft Excel, then total running distances were analyzed using ANOVA. Percent time spent freezing was calculated by averaging freezing data from individual experimenters blind to treatment condition of the animals with immobility times obtained from Noldus Ethovision XT. Pre-shock freezing for each test was averaged and group differences were analyzed with ANOVA. Average freezing across minutes during conditioning (Experiment 1) or across trials during conditioning and extinction (Experiments 2 and 3) were analyzed using repeated-measures ANOVA. Freezing scores within each extinction session in Experiment 1 were averaged and group differences compared using ANOVA. For renewal, freezing across trails was averaged and compared using 2-way ANOVA with Exercise and Context conditions as factors. For long-term memory / spontaneous recovery tests, overall group means were analyzed using ANOVA. Estrus phase was added as a factor in Experiment 3; therefore, 2-way repeated measures ANOVAs (Exercise x Estrus Phase) were used to analyze freezing during extinction days, and a 3-way ANOVA (Exercise x Context x Estrus Phase) was used to compare group means during the renewal test. Simple regressions were run to determine whether running distance during the running familiarization phase or after fear extinction predicted freezing levels during subsequent behavioral tests. Fishers least significant differences post hoc analyses were performed when appropriate. Group differences were considered different whenp < 0.05.
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Results
Experiment 1: Exercise During Consolidation of Contextual Fear Extinction Improves Long-Term Memory of Fear Extinction in Males
Rats used in experiment 1 ran equal amounts during the running familiarization period regardless of subsequent exposure to fear extinction (Figure 2A). Rats exhibited low levels of freezing prior to the first US during contextual fear conditioning (pre; Figure 3A). Freezing levels increased throughout US presentations (F(2,56) = 62.25, p < 0.0001) and did not differ between rats subsequently assigned to Locked or Mobile conditions, nor between No Extinction (n =10) and Extinction (Figure 3 A). There was indication of a trend toward a positive correlation between average distance run during the wheel running familiarization period and freezing during fear conditioning (R = 0.299, F(l,29) = 2.86; p 0.1: Figure 6A); an observation that would be consistent with prior reports indicating that wheel running enhances contextual
fear conditioning (Burghardt et al 2006, Greenwood et al 2009, Kolunan et al 2012, Van Hoomissen et al 2004).
^.Running wheel familiarization Running after extinction
Figure 2. Running distances. Female rats in experiment 3 (Exp 3) ran more than male rats used in experiment 1 (Exp 1) and experiment 2 (Exp 2), both (A) during the running familiarization phase and (B) during the 2 h running bouts after fear extinction training. Data displayed represent mean SEM. *** Exp 3 different from all other groups (p < 0.0001); Exp 3 day 2 different from Exp 3 day 1 (p < 0.01); # Exp 2 day 2 different from Exp 2 day 1 (p < 0.01): Q Exp 1 Extinction day 2 different from Exp 1 Extinction day 1 ft) <0.01).
Freezing levels during the first fear extinction training session did not differ between rats subsequently placed into Locked or Mobile wheels (Figure 3B), and distance run during the running familiarization period did not predict the amount of freezing during the first extinction training session. Running distances during the 2 h following the first and subsequent fear extinction training sessions are shown in Figure 2B. Exercise immediately following contextual fear extinction training had no effect on fear extinction
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memory. Rats in Locked and Mobile conditions displayed similar levels of freezing and within-session extinction (not shown) during the second (Figure 3C) and third (Figure 3D) fear extinction training sessions, which also served as fear extinction memory tests. Distance run during the consolidation of the first and second contextual fear extinction sessions was; however, negatively correlated to freezing levels during the second and third fear extinction memory tests (R = 0.60; F(l,9) = 5.07, p = 0.05; Figure 6C).
Rats were again re-exposed to Context A 1 wk after the third fear extinction training session in order to determine if post-extinction wheel running facilitated long-term memory of contextual fear extinction. Because freezing levels a week after fear extinction training are modulated both by the strength of the extinction memory and the spontaneous recovery of fear (Bouton 1993, Ponnusamy et al 2016), this memory test at day 10 represented both a long-term extinction memory test and a test of spontaneous recovery. However, freezing levels displayed by all rats exposed to fear extinction training remained at low levels during the extinction memory test at day 10 (Figure 3E), indicating minimal spontaneous recovery. The lack of spontaneous recovery could be due to rats receiving 3 sessions of fear extinction training, thus producing a strong fear extinction memory. Despite this, rats that ran after fear extinction displayed significantly less fear during the long-term extinction memory test than rats placed into locked wheels after fear extinction (p < 0.05); further, this effect of exercise was dependent on exercise being contingent with fear extinction (interaction between Exercise and Extinction: F(l,28) = 6.47, p = 0.01; Figure 3E). Rats that ran similar amounts (Figure 2) but were not exposed to fear extinction (Mobile-No Ext), displayed levels of freezing greater than that expressed by rats exposed to extinction (p < 0.05), and no different from Locked rats not exposed to fear extinction (Locked-No Ext, Figure 3E). These data indicate that exercise during the consolidation of contextual fear extinction facilitates retrieval of fear extinction memory at long-term time points.
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Locked Ext v Mobile Ext Locked
Locked No Ext O Mobile No Ext i i Mobile
Figure 3. Running after contextual fear extinction, but not running alone, enhances long-term extinction memory. (A) Freezing levels increased throughout US presentations during contextual fear conditioning (3, 1 s, 0.8mA foot shocks, 1 min ITI), regardless of subsequent group assignments. (B) There was no significant difference in freezing during the first fear extinction training session 1 day after conditioning between rats subsequently placed in either locked (Locked) or mobile (Mobile) running wheels. (C-D) Wheel running during the consolidation of contextual fear extinction had no impact on fear extinction memory during memory tests on days 2 and 3. (E) Rats that ran after extinction exhibited significantly less fear than those placed into locked wheels after extinction. Both Locked and Mobile rats that went through fear extinction (Ext) froze significantly less than those that were not exposed to fear extinction (No Ext). Data displayed represent mean SEM; p < 0.01. *** p < 0.0001
Experiment 2: Exercise During Consolidation of Auditory Fear Extinction Improves Fear Extinction Memory and Reduces Fear Renewal in Males
Rats exhibited low levels of freezing prior to the first CS-US pairing (pre; Figure 4A). Freezing levels increased throughout conditioning (F(3,l 11) = 58.20, p < 0.0001) and did not differ between rats subsequently assigned to Locked or Mobile conditions. Rats in Experiment 2 ran distances similar to rats in Experiment 1 during the running familiarization period (Figure 2A). Interestingly, the average running distance during the wheel running familiarization period negatively correlated with freezing levels during fear conditioning (R = -0.48; F(l,36) = 10.699; p < 0.01; Figure 6A). These data suggest that unlike chronic wheel running which has been reported to have no effect on the acquisition of auditory fear conditioning (Baruch et al 2004), brief access to running wheels could interfere with auditory fear conditioning learning. One wheel did not record running distance during the wheel running familiarization period, so this rat was omitted from this correlational analysis.
All rats exhibited negligible amounts of freezing prior to the first CS during day 1 of auditory fear
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extinction training in Context B, indicating that minimal fear transferred from the conditioning Context A to the fear extinction Context B (not shown). Freezing levels decreased across trials (F(5,185) = 147.12, p < 0.0001) and did not differ between rats subsequently placed into Locked or Mobile wheels (Figure 4B). The average running distance during the familiarization period negatively correlated with freezing levels during the first 4 trials of the first fear extinction training session (R = -0.34; F(l,36) = 4.60; p < 0.05; Figure 6B), consistent with the negative relationship between running distance and the initial acquisition of auditory fear conditioning (Figure 6A).
Running distances during the 2 h following the auditory fear extinction training sessions are shown in Figure 2B. Post-extinction wheel running facilitated fear extinction memory. When re-exposed to the extinction Context B the evening following post-fear extinction running, Mobile rats displayed significantly less freezing than locked rats across the duration of the fear extinction training session, which also served as a fear extinction memory test (F(l,37) = 6.10. p = 0.01: Figure 4C). Locked and Mobile rats displayed equivalent within-session extinction, as freezing levels decreased over time in both groups (F(5,185) = 43.67; p < 0.0001; Figure 4C). Distance rim after the first auditory fear extinction training session did not predict levels of freezing during the second fear extinction training session.
Rats were again placed into their locked or mobile wheels for 2 h following the second fear extinction training session. Then, to determine the effect of wheel running during consolidation of auditory fear extinction on renewal of auditory conditioned fear, rats were re-exposed to the extinguished CS in either Context B (Same) or a novel Context C (Different) the following day. Locked rats displayed typical fear renewal, as Locked rats re-exposed to the extinguished CS in a context different from where fear extinction was learned exhibited more fear than Locked rats re-exposed to the CS in the same extinction context (p < 0.05). Rats allowed to exercise during the consolidation of auditory fear extinction; however, were protected from fear renewal (interaction between Exercise and Context: F(l,35) = 5.11,/) < 0.05). Distance run after the second fear extinction training session did not predict the levels of freezing in either context during the renewal test on day 3.
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7rja| Block of 5 Trials Block of 5 Trials
Figure 4. Exercise enhances auditory fear extinction memory and blocks renewal in males. (A) Fear increased throughout conditioning trials (4 pairings of 10s, 80dB, 2 KHz auditory CS co-terminating with Is, 0.8mA foot shock US), and was not different between rats subsequently assigned to locked (Locked) or mobile (Mobile) wheel conditions. (B) Rats were placed into a novel extinction context and exposed to 30 CS presentations (I min ITI) in the absence of the US. Freezing decreased throughout the extinction trials and did not differ between rats subsequently assigned to locked or mobile conditions. (C) When re-exposed to the CS in the extinction context, rats in the Mobile group displayed significantly less fear behavior than those in the Locked group. (D) Locked rats placed into a context different from that in which extinction was learned (Different) displayed typical fear renewal. Rats placed into Mobile wheels after fear extinction were protected from fear renewal. Data displayed represent mean SEM; p < 0.01
Experiment 3: Exercise during consolidation of auditory fear extinction fails to improve fear extinction memory or reduce fear renewal in females
Analyses of the freezing data during the renewal test on day 3 was consistent with prior work (Lebron-Milad & Milad 2012) suggesting that estrus phase during fear extinction learning modulates later retrieval of fear extinction. Specifically, levels of freezing expressed dining the renewal test by rats that were in metestrus and diestrus (Met&Di; n = 17) during initial fear extinction learning on extinction day 1 differed from rats that were in proestrus and estrus (Pro&E; n = 23) on extinction day 1. In contrast, estrus phase during conditioning or renewal had no impact on freezing levels during any behavioral test. Therefore, females were separated into groups according to phase of the estrus cycle on the first day of fear extinction training (Extinction Day 1). Photomicrographs of vaginal cytology defining the estrus phases are shown in Figure 5F-I.
Overall distance run on the second wheel running familiarization day increased significantly from
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the first wheel familiarization opportunity (main effect of time: F(l,105) = 54.297; p < 0.0001), with female rats running significantly further than male rats (main effect of experiment: F(3,105) = 17.456; p < 0.0001; Figure 2A). Unlike the males, the distance run during the wheel running familiarization period did not predict levels of freezing during fear conditioning in female rats. Rats exhibited low levels of fear prior to the first CS-US pairing during conditioning (pre; Figure 5A). Freezing levels increased throughout CS-US pairings (F(3,108)= 45.63,< 0.0001; Figure 5A) for all rats regardless of subsequent estrus cycle phase or subsequent wheel assignment.
Freezing levels in all groups were negligible prior to the first CS during day 1 of auditory fear extinction training in Context B (data not shown). Freezing levels decreased across trials (F(4,144)= 76.28, p < 0.0001; Figure 5B) and did not differ between estrus phases or between rats subsequently placed into Locked or Mobile wheels. The average running distance during wheel familiarization did not predict levels of freezing during the first fear extinction training session.
Rats were placed into either locked or mobile wheels immediately following fear extinction. Postfear extinction running distances did not differ between rats at different stages of the estrus cycle, so running distances were averaged across all females (Figure 2B). Female rats ran significantly more than male rats during the 2 h running opportunities following fear extinction (F(3,48) = 29.09; p < 0.0001). Additionally, female rats running distance escalated across nights (p < 0.05), whereas the males maintained running distances across subsequent post-extinction running opportunities (Figure 2B). Despite the greater distances run by females, post-extinction wheel running did not facilitate auditory fear extinction memory in females. When re-exposed to the auditory CS in the extinction Context B the evening following post-fear extinction running, freezing levels were similar between groups and all rats demonstrated similar within-session extinction (F(4, 144)= 8.91. p < 0.0001; Figure 5C). Freezing within the first few trials of the second fear extinction training session is considered extinction memory retrieval (Bukalo et al 2015, Do-Monte et al 2015). Thus, to verify that post-extinction running did not impact fear extinction memory, freezing during the first 4 trials of extinction day 2 was analyzed separately. No differences between groups were observed during the first 4 trials (data not shown). Distance run after the first auditory fear
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extinction training session did not predict levels of freezing during the second fear extinction training session.
Rats in the Mobile group were again allowed to run for 2 h following the second fear extinction training session. Then, to determine the effect of wheel running during consolidation of auditory fear extinction on renewal of auditory conditioned fear, rats were re-exposed to the extinguished CS in either Context B (Sam) or a novel Context C (Different) the following day. Results are shown in Figure 5D. Rats placed into the Different context the next day displayed more freezing behavior than rats placed into the Same context (F(l,32) = 14.40, p < 0.001), indicating renewal of fear in the Different context. Rats in the Met&Di group displayed significantly higher freezing levels compared to Pro&E rats (F(l,32) = 6.54, p = 0.01), regardless of context. Moreover, ANOVA revealed an interaction between context and estrus phase (F(l,32) = 5.22, p < 0.05), indicating that rats in the Met&Di group displayed significantly more fear renewal than rats in the Pro&E group. Unlike males, wheel running during the consolidation of auditory fear extinction had no effect on freezing levels during the renewal test in females. Interestingly; though, the average running distance after both extinction training sessions negatively correlated with freezing levels in the Same context, but this association just missed significance (R = -0.58, F(l,8) = 4.14, p = 0.07; Figure 6D). No other significant correlations between freezing and running distance were found.
Rats were again re-exposed to Context B 1 wk after the fear renewal test to determine if post-extinction wheel running facilitated long-term memory of auditory fear extinction. Neither context on day 3 of testing (Same vs Different), phase of estrus cycle during day 1 of fear extinction training (Met&Di vs Pro&E), nor wheel running (Locked vs Mobile) impacted freezing levels when re-exposed to the CS 1 week later (Figure 5E).
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Trial Blocks of 4 trials Blocks of 4 trials Locked Mobile
Figure 5. Phase of estrus cycle, but not exercise, modulates fear renewal in female rats. Rats were grouped by phase of estrus cycle during fear extinction learning (fear extinction day 1). (A) Fear increased throughout conditioning trials (4 pairings of 10s, 80dB, 2 KHz auditory CS co-terminating with Is, 0.8mA foot shock US), and was not different between rats subsequently assigned to locked (Locked) or mobile (Mobile) wheel conditions. (B) Rats were placed into a novel extinction context and exposed to 20 CS presentations (1 min ITI) in the absence of the US. Freezing decreased throughout the extinction trials and did not differ between rats in metestrus and diestrus (Met&Di) or proestrus and estrus (Pro&E) phases of the estrus cycle, or subsequently assigned to Locked or Mobile conditions. (C) Freezing levels were similar between all groups and all rats demonstrated similar within-session extinction regardless of estrus phase or exercise condition. (D) Rats that were in Met&Di during fear extinction Day 1 displayed typical fear renewal when re-exposed to the CS in a context different from where extinction was learned (Different). Rats that were in Pro&E during fear extinction Day 1 were protected against the renewal of fear. (E) No differences between groups were observed when rats were re-exposed to the CS in Context B 1 wk after the fear renewal test. (F) Photomicrograph of vaginal cytology showing keratinized epithelial cells (white arrow) and leukocytes (black arrow) characteristic of metestrus. (G) Photomicrograph of vaginal cytology showing predominately leukocytes (white arrow) and nucleated epithelial cells (black arrow) with a decrease in anucleated keratinized epithelial cells. (H) Photomicrograph of vaginal cytology showing small, round, often clumped mononucleated cells (arrow) of relatively the same size. (I) Photomicrograph of vaginal cytology showing predominately anucleated keratinized epithelial cells. Data displayed represent mean + SEM; ** p < 0.001
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A. B.
Running distance Average running distance
after extinction day 2 (m) after extinction days 1 and 2 (m)
Running distance Average running distance
after extinction day 2 (m) after extinction days 1 and 2 (m)
v Exp 1....... Exp2------------ "Exp 3 -
Figure 6. Correlations between running and freezing levels. (A) Average running distance during running familiarization did not predict freezing during contextual fear conditioning in experiment 1 (Exp 1), but negatively correlated with freezing during auditory fear conditioning in experiment 2 (Exp 2). (B) The average running distance during running familiarization negatively correlated with freezing during the first 4 tones of fear extinction day 1 in Exp 2. (C) There was a trend for the average running distance following the auditory fear extinction sessions to negatively correlate with freezing in the Same context during the fear renewal test in females in experiment 3 (Exp 3). (D) Average running distance after the first and second fear extinction training sessions negatively correlated with average freezing levels during the second and third fear extinction training sessions (Exp 1).
Discussion
Here we report the novel findings that a brief increase in physical activity during the consolidation phase of fear extinction learning can enhance fear extinction and render fear extinction memory resistant to relapse. In males, wheel running during consolidation of contextual fear extinction improved long-term memory of fear extinction, recall of which is influenced by spontaneous recovery. Similarly, wheel running during the consolidation of auditory fear extinction improved fear extinction memory and prevented the renewal of fear. In both cases, rats that ran the greatest distance during the consolidation phase of fear extinction learning also tended to have the strongest extinction memory. Exercise, however, only improved fear extinction in males. In females, wheel running had no impact on either fear extinction consolidation or renewal of fear. However, females that ran the greatest distance during auditory fear extinction
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consolidation did tend to have the strongest fear extinction memory. Interestingly, females that learned fear extinction during proestrus and estrus phases of the estrus cycle were protected against fear renewal. Along with prior data (Mika et al 2015, Powers et al 2015, Siette et al 2014), these results suggest that brief exercise bouts could be used as an augmentation strategy for exposure therapy, even in previously sedentary subjects. Fear memories of discrete cues, rather than of contextual ones, may be most susceptible to exercise-augmented extinction, especially in males. Moreover, exercise seems to have the biggest impact on fear relapse phenomena, even if fear extinction memories themselves are only minimally enhanced, as is the case with contextual fear extinction. Further research is warranted to determine the effectiveness of exercise-augmentation of fear extinction in a clinical setting.
In experiment 1, post-extinction exercise only enhanced consolidation of contextual fear extinction when memory was assessed 1 wk after the final extinction training session. This observation differs from that of Siette et al. (2014), who observed that Wistar rats allowed to run during consolidation of contextual fear extinction had improved recall of fear extinction the next day. One difference between our experiment and the Siette et al. (2014) study is that rats in the current experiment had access to running wheels for 2 active cycles prior to fear conditioning, whereas the rats in the Siette (2014) study were naive to running wheels. This prior brief access to running wheels may have made the contextual fear memory more difficult to extinguish. Indeed, only 2 days of wheel running increases BDNF mRNA in the hippocampus (Neeper et al 1995, Neeper et al 1996) and it is well established that wheel running prior to conditioning enhances contextual fear conditioning (Burghardt et al 2006, Greenwood et al 2009, Kohman et al 2012, Van Hoomissen et al 2004) and makes it more difficult to extinguish (Greenwood et al 2009). Nevertheless, we did observe a negative correlation between the distance run after extinction training sessions 1 and 2 and freezing during the extinction memory tests on days 2 and 3, as well as a significant improvement in contextual fear extinction recall a week later. These observations suggest that exercise can enhance the consolidation of contextual fear extinction memory, even if the effect was not large enough to be significantly different from locked controls during the initial memory tests. That exercise enhances recall of contextual fear extinction memory a week after the third fear extinction training session is especially interesting, as fear expression at long-term timepoints can be influenced by a number of factors in addition
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to the strength of the fear extinction memory, including generalization of the original fear memory (Wiltgen & Silva 2007), and the spontaneous recovery of fear (Bouton 1993). Thus, our data suggest that exercise during consolidation of contextual fear extinction may render fear extinction memory resistant to processes that tend to increase conditioned fear responding over time, even in the absence of improving short-term extinction memory, per se. This effect of exercise may be especially relevant to the long-term efficacy of extinction-based exposure therapy.
Chronic wheel running begun after contextual fear conditioning has been reported to reduce conditioned fear responding, even in the absence of fear extinction (Akers et al 2014, Ishikawa et al 2016). It was therefore important to determine whether the reduction in freezing observed on day 10 in rats that ran after contextual fear extinction was dependent on wheel running being contingent with the consolidation of contextual fear extinction. Mobile rats not exposed to fear extinction, but that ran an equivalent time and distance to Mobile rats exposed to fear extinction, displayed freezing behavior equal to Locked rats not exposed to fear extinction (Figure 3E), indicating that brief exercise sessions did not themselves alter the later expression of conditioned freezing. The reduction in freezing displayed by the Mobile rats on Day 10 is therefore dependent on exercise being contingent with the consolidation of contextual fear extinction.
We have previously reported that wheel running during the acquisition of auditory fear extinction reduces freezing to the CS in a novel context one wk later (Mika et al 2015). The design that was used, however, rendered fear extinction memory indistinguishable from renewal and spontaneous recovery. Experiment 2 thus investigated whether exercise during the consolidation of auditory fear extinction could both enhance fear extinction memory and reduce fear renewal. We found that exercise during the consolidation phase of auditory fear extinction both enhances fear extinction memory and blocks fear renewal one day following fear extinction. Rats that ran in the absence of extinction were excluded from this experiment because Experiment 1 revealed that brief running bouts in the absence of fear extinction learning have no effect on freezing (Figure 4).
Interestingly, running distance during the wheel familiarization period prior to conditioning was negatively correlated to freezing during the acquisition of auditory conditioned fear (Figure 6A). Con-
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sistent with this observation, distance run during the wheel familiarization period was also negatively correlated to the strength of the fear conditioning memory assessed by freezing during the first 4 trials of the first fear extinction training session (Figure 6B). While short-term exercise has profound effects on the hippocampus (Molteni et al 2002, Neeper et al 1995, Neeper et al 1996), a structure important for contextual fear conditioning (Chang & Liang 2016, Matus-Amat et al 2004, Rudy et al 2002), less is known regarding exercise effects on the amygdala, a region critical for the CS-US association formed during auditory fear conditioning (Bergstrom 2016, Huff & Rudy 2004). Moreover, the effects of exercise on auditory fear conditioning are not as well characterized as contextual fear conditioning (Baruch et al 2004, Falls et al 2010). To our knowledge, no prior research has investigated effects of only a few days of wheel running on auditory fear conditioning. Investigation of this question is warranted given the current results.
Rats that ran after the first day of auditory fear extinction displayed significantly less fear during re-exposure to the auditory CS the following day. This is the first time, to our knowledge, that brief exercise has been reported to improve the consolidation of auditory fear extinction. Interestingly, the effect of exercise observed on extinction training day 2 was not observed again the next day in the same context. Mobile rats did not freeze any less than Locked rats when re-exposed to the CS in the extinction Context B (Same) during the fear renewal test on day 3. This could be because all rats had, by this time, been exposed to 2 prior fear extinction training sessions, thus enabling Locked rats ample opportunity to learn fear extinction in Context B and reducing the difference between Locked and Mobile groups. Nevertheless, the fear extinction memory strengthened by exercise was resistant to renewal. When re-exposed to the CS in an environment different from the extinction context, fear renewed in the rats placed into locked, but not mobile, wheels after extinction (Figure 4D).
The mechanisms by which brief exercise could enhance the consolidation of fear extinction are unknown, but could involve several factors sensitive to brief exercise sessions, including increases in glucocorticoids, endocannabinoids, glutamatergic, noradrenergic, and / or dopaminergic signaling. Indeed, acute increases in each of these factors can enhance fear extinction (for reviews see (Fitzgerald et al 2014, Myskiw et al 2014, Singewald et al 2015)), although in some cases the effects on fear relapse is not well established. In our previous study (Mika et al 2015), we observed that the recall of relapse-resistant fear
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extinction in rats that ran during fear extinction acquisition was associated with potentiated activity of direct-pathway neurons in the striatum. This activity was above that which would be expected by locomotor activity or fear memory recall, suggesting that it was the recall of the fear extinction memory and not the reduction in freezing or fear response itself that was driving activation of striatal direct-pathway neurons. These data suggest that striatal direct pathway neurons could be involved in the mechanisms by which brief exercise facilities fear extinction learning and reduces relapse. Although striatal direct pathway neurons are typically considered in the context of locomotor activity, recent technical advances have revealed roles for these neurons in emotional behavior (Kravitz & Kreitzer 2012, Lenz & Lobo 2013). Further research is required to determine how these neurons could interact with other exercise signals and the canonical fear circuitry.
Because exercise had a more robust effect on auditory fear extinction compared to contextual fear extinction, we used auditory fear extinction to determine the effects of brief exercise on fear extinction and relapse in females. Consistent with the literature (Kandasamy et al 2016, Venezia et al 2016), female rats ran significantly more than male rats during both the familiarization and post-extinction phases (Figure 2). Despite this vigorous wheel running, exercise neither enhanced the consolidation of auditory fear extinction nor prevented fear renewal in females. These data suggest that exercise might be a more useful strategy for augmentation of exposure therapy in males than in females.
It is well established that fear extinction learning and memory is dependent on the phase of the estrus / menstrual cycle in which female rats and humans initially learn extinction (Daviu et al 2014, Milad et al 2009). Consistent with these prior data, we observed that female rats that initially learned fear extinction in proestrus and estrus, phases of the estrus cycle during which levels of estradiol are highest, were protected against fear renewal (Figure 5D). Relatively high levels of estradiol during proestrus and estrus phases could have contributed to this effect. Indeed, women with naturally low or experimentally-reduced estrogen have impaired fear extinction (Graham & Milad 2013, Zeidan et al 2011), and memory deficits present when rats learn fear extinction during phases of low estradiol can be prevented by pre-extinction estrogen receptor agonist, or rescued by post-extinction estradiol (Chang et al 2009, Zeidan et al 2011). Activation of estrogen receptor B in the hippocampus 24 h prior to passive avoidance learning can increase
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the generalization of the memory of the context in which passive avoidance was learned to a neutral context (Lynch et al 2014, Lynch et al 2016). It is possible that high estrogen levels during fear extinction learning could similarly promote later generalization of the extinction memory, therefore reducing the contextual dependency of fear extinction.
Collectively, the current data suggest that brief exercise sessions could be a useful strategy to augment treatment for anxiety- and trauma-related disorders to help prevent relapse. This set of experiments further emphasizes the importance of investigating manipulations in females in addition to males, as we found that exercise differentially impacts auditory fear extinction learning and memory in male and female rats. These experiments expand our knowledge of factors able to modulate fear extinction learning and memory and provide building blocks upon which to further characterize the mechanisms by which exercise modulates fear extinction.
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CHAPTER III
ACTIVATION OF NIGROSTRIATAL DOPAMINE NEURONS DURING FEAR EXTINCTION
PREVENTS THE RENEWAL OF FEAR
Abbreviated title: Nigrostriatal dopamine facilitates fear extinction Author list: Courtney A. Bouchet, Megan Miner, Esteban C. Loetz, Holly S. Hake, Adam J. Rosberg, Caroline Farmer, Mykola Ostrovskyy, Nathan Gray, Benjamin N. Greenwood University of Colorado, Denver. Department of Psychology.
Abstract
Manipulations that increase dopamine (DA) signaling have been reported to enhance fear extinction, but the circuits involved remain unknown. DA neurons originating in the substantia pars compacta (SNc) projecting to the dorsal striatum (DS) are traditionally viewed in the context of motor behavior, but growing data implicate this nigrostriatal circuit in emotion. Here we investigated the role of nigrostriatal DA in fear extinction. Activation of SNc DA neurons with designer receptors exclusively activated by designer drugs (DREADD) during fear extinction had no effect on fear extinction acquisition, but enhanced fear extinction memory and blocked the renewal of fear in a novel context. D1 receptors in the DS are a likely target mediating the effects of SNc DA activation. Indeed, D 1-expressing neurons in the medial DS (DMS) were recruited during fear extinction, and DREADD-induced DA potentiated activity of D1-expressing neurons in both the DMS and the lateral DS. Rats whose SNc DA neurons were activated by DREADD during prior fear extinction displayed potentiated renewal-induced cFos expression in the CA1 of the hippocampus, a region important for contextual processing. Pharmacological activation of D1 receptors in the DS did not impact fear extinction acquisition or memory, but blocked fear renewal. These data suggest that activation of SNc DA neurons and DS D1 receptors could alter contextual processing during fear extinction, thus reducing the context specificity of the memory and preventing fear renewal. Nigrostriatal DA thus represents a novel target to enhance long-term efficacy of extinction-based therapies for anxiety and trauma-related disorders.
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Significance Statement
High relapse rates impede the efficacy of extinction-based therapies for anxiety and trauma-related disorders, thus identification of novel strategies to reduce fear relapse after extinction is of utmost importance to mental health. We found that activation of nigrostriatal dopamine during extinction of conditioned fear prevents the return of fear in a context different from where fear extinction was learned, a form of relapse termed renewal. These data add fear extinction to the purview of nigrostriatal DA functions and suggest that nigrostriatal DA could be a novel target for increasing the long-term efficacy of extinction-based therapies.
Introduction
Extinction of traumatic memories is a major goal of therapeutic strategies for anxiety and trauma-related disorders, but extinction memories are labile and fear associated with traumatic memories tends to resurface even following successful extinction. Identification of novel means to enhance fear extinction to prevent the relapse of fear after extinction is of utmost importance to mental health.
Dopamine (DA) is a memory modulator implicated in fear extinction. Recent theories proffer that fear extinction could involve learning a new association between the conditioned stimulus (CS) and a positive-affective state stemming from the relief that the predicted unconditioned stimulus (US) no longer follows the CS (prediction error; (Huh et al 2009); an association that could be supported by DA. Indeed, high-frequency (phasic) DA release in the striatum encodes both reward-value (Flagel et al 2011, Howe et al 2013) and prediction error (Schultz 2016). These functions of DA are traditionally thought to involve the mesolimbic DA pathway, originating in the ventral tegmental area (VTA) and terminating in the nucleus accumbens (NAc), in the ventral striatum. Consistent with this view, phasic DA release in the NAc increases during fear extinction (Badrinarayan et al 2012), and D2 receptor signaling in the NAc has been reported to be necessary for fear extinction (Holtzman-Assif et al 2010b). Emerging data indicate that functions of the nigrostriatal DA pathway, originating in the substantia nigra pars compacta (SNc) and terminating in the dorsal striatum (DS), overlap with those of mesolimbic DA (Kravitz & Kreitzer 2012,
Wise 2009). Indeed, although nigrostriatal DA is traditionally viewed in the context of motor behavior, recent data reveal a role for the DS, particularly DS D1-expressing neurons preferentially responsive to
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phasic DA release (Dreyer et al 2010, Schultz 2007), in reinforcement (Kravitz et al 2012) and emotional behavior (Lenz & Lobo 2013). Despite potential involvement of multiple DA pathways in fear extinction, nigrostriatal DA has not before been considered in fear extinction research.
The goal of the current set of experiments was to begin to elucidate the role of nigrostriatal DA in fear extinction. A combined, viral-mediated expression of designer receptors exclusively activated by designer drugs (DREADD) and pharmacological approach was used to test the hypothesis that activation of SNc DA neurons and subsequent DS D1 receptor signaling can enhance fear extinction learning. Results indicate that activation of SNc DA neurons dining fear extinction can facilitate fear extinction memory in a manner that resists the return of fear in a novel context (renewal; (Bouton 1988). Consistent with D1 receptors in the DS being a target of Gq-DREADD-induced DA, D 1-expressing neurons in the DS are recruited during fear extinction, and their activity is augmented by Gq-DREADD-induced DA. Pharmacological activation of DS D1 receptors during extinction prevents the renewal of fear without impacting fear extinction memory tested in the fear extinction context. These data suggest that nigrostriatal DA and DS D1 receptors could be a novel target for the prevention of fear relapse after extinction.
Materials and Methods
Animals
A total of 43 adult, male Tg(TH-Cre)3.1Deis rats on a Long-Evans background (TH-Cre; (Witten et al 2011) were supplied by Karl Deisseroth via the NIH Rat Resource and Research Center (Columbia, MO). A total of 88 adult, male wildtype (WT) Long Evans rats were purchased from Charles River. Rats were pair-housed in ventilated Nalgene Plexiglass cages (45.5 W x 24 D x 21 H cm) with ad libitum access to food (Standard Lab Chow) and water. All animals were well handled by experimenters once daily for at least 5 days prior to start of behavioral tests. Animals were kept on a 12 12 h light dark cycle with lights on from 0700 to 1900 in a temperature (22C) and humidity-controlled facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care located on the University of Colorado Denver Auraria campus. Care was taken to minimize discomfort in all procedures and all protocols were approved by the University of Colorado Denver Institutional Animal Care and Use Committee.
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Surgeries
All surgical procedures were performed under Ketamine (75.0 mg / kg i.p.) and Medetomidine (0.5 mg / kg i.p.) anesthesia. Carprofen (5 mg / kg s.c.) was administered for pain management at induction and then every 24 h for 72 h post-surgery. Atipamezole (0.5 mg/kg i.p.) was used to reverse the effects of Metetomidine to speed the recovery from anesthesia. Penicillin G (22,000 IU/rat, s.c.) was given every 24 h for 72 h following surgery to avoid infection.
Viral Transfection
AAV5/hSyn-DIO-hm3D(Gq)-mCherry (Gq-DREADD) viral vector was provided by Dr. Bryan Roth via the University of North Carolina viral core (Gene Therapy Center Vector Core; Chapel Hill, NC; (Zhu & Roth 2014). The virus was kept on wet ice during surgery and loaded into lOpl Hamilton syringes back-filled with silicon oil immediately before microinjection. After drilling holes into the skull, 1 pL of undiluted virus was injected bilaterally into the SNc (from Bregma: 5.4mm anterior, 2.3mm lateral, -8.8mm ventral from top of the skull) at a rate of 0.1 pL/min using a Micro4 Microsyringe Pump Controller (World Precision Instruments, Inc. Sarasota, FL, USA). The bevel on the tip of the Hamilton was oriented laterally to minimize spread of virus medially toward the VTA. The syringe was left in place for 10 min after the injection to avoid spreading of virus. The incision was closed with Woundclips. Behavior tests were conducted at least 1 month after viral injection to allow ample time for viral expression.
Location of viral transfection in the midbrain was verified in all rats by inspection of mCherry expression under 200X magnification on an Olympus BX53. Relative expression of mCherry in various projection regions of midbrain DA neurons was quantified with densitometry as previously described (Lloyd et al. 2016). Images through the SNc, dorsal medial striatum (DMS), dorsal lateral striatum (DLS), nucleus accumbens (NAc), medial prefrontal cortex (mPFC), dentate gyrus region of the hippocampus (DG) and basolateral amygdala (BLA) were captured at 200X magnification from at least 4 sections per brain region per animal, and intensity of signal above background (densitometry) was calculated with Cell Sense software (Olympus).
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Cannulae implantation
Bilateral 26-gauge guide cannula (4.6mm below pedestal; Plastics One) were implanted into the DS (from Bregma: +5.Omm anterior, +3.0mm lateral, -4.5mm ventral from top of the skull) following our published protocols (Greenwood et al 2012, Strong et al 2011). The guide cannulae were held in place by flowing dental acrylic around the cannulae and 4 anchoring screws. Dummy cannulae (Plastics One) were inserted into the guide cannulae to avoid clogging of the cannula. Rats were allowed at least 1 week of recovery before behavior testing.
Microinjections
Rats were held gently and microinjectors that extended 1 mm past the tip of the guide cannulae (PlasticsOne) were inserted through the guide cannulae into the DS. SKF38393 (lpg / hemisphere;
(Agnoli et al 2013) SKF 81297 (O.lpg / hemisphere; (Larkin et al 2016, Pezze et al 2015) or saline (1 pi) were injected at a rate of 0.5 pl/min bilaterally through the microinjector controlled by a Micro4 pump.
The injector was left in place for 2 min following injection to allow for diffusion and avoid backflow up the guide cannulae. D1 agonists or saline were injected 10 min prior to fear extinction training trials.
Drugs
Clozapine-N-Oxide (CNO; provided by National Institute of Mental Health Chemical Synthesis and Drug Supply Program, Bethesda, MD) was stored at -20C and CNO solution was prepared immediately before use. CNO was dissolved in sterile saline with 0.6% DMSO and administered i.p. at a dose of 1 mg/kg (Boekhoudt et al 2016). Vehicle-treated rats received equal volume 0.6% DMSO in saline (i.p.). Vehicle or CNO was administered 30 min before each of 2 fear extinction training sessions. This timing of CNO administration relative to behavior has been used previously with success (MacLaren et al 2016). Two different D1-like (D1 and D5, referred to here as D1 receptors) receptor agonists were used for this study: SKF38393 (2.3.4.5.-tctrahvdro-7.8-dihvdro.xy-1 -phenyl-1/7-3-bcn/a/cpinc hydrochloride) and SKF81297 (6-cliloro-7.8-dihydroxy-1 -pheny 1-2.3.4.5-tetrahydro-1/7-3-bcnzazcpinc hydrobromide). Both SKF38393 and SKF81297 have very high binding affinities for D1 receptors in the striatum (Mannoury la Corn et al 2007). SKF3839 (Sigma-Aldrich) was dissolved in sterile saline to a final concentration of
35


0.5jLLg/|_il which is the most effective dose to activate D1 receptors (Agnoli et al 2013, Pezze et al 2007). SKF81297 (Tocris Biosciences) was dissolved in sterile saline to a final concentration of O.lpg/pl This dose has been recently reported to impair appetitive learning when injected into the PFC (Pezze et al
2015) , and systemic administration of SKF81297 can facilitate contextual fear extinction (Abraham et al
2016) .
Behavioral Analyses
Auditory Fear Conditioning
Rats were placed into custom, rectangular conditioning chambers (20 W x 10 D x 12 H cm; Context A) with a shock grid floor (Coulbourn Instruments, Allentown, PA) housed inside individual soundattenuating cabinets. Rats were transported to Context A in their home cages. A fan located near the floor of the cabinets provided ventilation and background noise and bright white lights illuminated the chambers. Chambers were cleaned with water between rats. Rats were allowed 3 min to explore the context, after which rats were exposed to 4 auditory CS (10s, 80dB, 2kH), each co-terminating with a foot shock US (Is, 0.8mA; 1 min ITI). The rats were left in the conditioning chamber for 30 s after the last shock before being returned to their home cages.
Auditory Fear Extinction
Approximately 24 h after fear conditioning, rats were placed into a novel Context B that was either a custom Plexiglas rectangular chamber (15W x 15D x 20H) with a smooth floor or a custom Plexiglas triangular chamber (15D sides x 20H) with a textured floor for fear extinction training. Rectangular and triangular Context B chambers were counterbalanced so half the rats were exposed to fear extinction training in the rectangular chambers and the other half in the triangular chambers. Context B was housed in the same sound-attenuating cabinets used for conditioning, but all other contextual features and discrete cues differed between contexts. Rats were transported to the sound attenuating chambers, which included vanilla scent, in their respective Context B custom Plexiglas chambers. The fan within the chamber was turned off and the room was dimly lit by a lamp outside of the sound attenuating chambers. Context B was cleaned with 10% ethanol between rats. After a 3 min exploration period, the tone CS was administered 20
36


times (1 min ITI) in the absence of the foot-shock US. One min after the last tone CS, rats were transported to the housing room in their extinction chambers and placed into their home cage.
Fear renewal test
Approximately 24 h following the second fear extinction training session, rats were exposed to the auditory CS (1 min ITI) in either the same context used for extinction training (Context B; same) or a novel Context C {different). Rats assigned to the different group were transported to Context C in a novel inner chamber, so that rats extinguished in the square were now placed into the triangle, and vise-versa. Context C consisted of a raspberry scent, red box lights, a fan near the top of the behavior cabinets was turned on, and chambers were cleaned with 1% acetic acid between tests. After a 3 min exploration period, the tone CS was presented 4 times (1 min ITI) in the absence of the foot-shock US. Because fear renewal is the return of fear in contexts different from where fear extinction was learned (Bouton and Ricker 1994), the difference in freezing between same and different contexts was considered fear renewal.
All behavioral tests were recorded with overhead cameras and freezing was scored both by multiple experimenters who were blind to the experimental conditions of the animals and by automated behavioral analyses software (Noldus Ethovision XT) during both the CS and the ITIs. Because regularly-scheduled ITIs can become a part of the CS, and analyses indicated a lack of differential effects of exercise on freezing during ITIs and CS, freezing during each CS and subsequent ITI were combined and expressed as freezing during a trial, as in prior work (Fitzgerald et al 2015, Goode et al 2015, Mika et al 2015). Each trial consists of a 10 s tone and a 60 s ITI.
Double-label fluorescent in situ hybridization
Two days after the renewal test, a subset of TH-Cre rats were injected i.p. with either CNO (1 mg/kg) or vehicle and, 30 min later, either exposed to fear extinction or left in their home cages (no extinction). Immediately after the extinction session or equivalent time in home cages, rats were rapidly decapitated and brains were removed, flash frozen with isopentane maintained between -20 and -30 C, then stored at -80 C. Brains were prepared for in situ hybridization per our previously published protocols (Herrera et al 2016, Mika et al 2015). Brains were sectioned using RNase-free techniques at 10pm onto
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Superfrost Plus slides (FisherBrand) then stored at -80C until in situ hybridization. Riboprobes compli-
mentary to Dl, D2 or cfos were fdtered through a G50-50-Saphadex column and enzymatically stabilized with 2M dithiothreitol (DTT). Mounted tissue from 1.7 to -0.8 mm Bregma were moved directly from -80 C into 4% paraformaldehyde (PFA) for 1 h. Following a wash in 2X saline-sodium citrate (SSC), slides were placed in 0.01% triethanolamine solution (triethanolamine in H20 pH to 8.0, 0.25% acetic anhydride) for 20 min then dehydrated with EtOH. The labeled probe was added to hybridization buffer (formamide, dextran sulfate, sterile water, 20X SSC, 50X Denharts solution, tRNA, 0.5 M sodium phosphate) and applied to the slide via coverslip. Slides were kept in humidified chambers overnight at 55C to promote hybridization. The following day, cover slips were removed in 2X SSC. Slides were then incubated in RNase digestion solution (Tris-HCl, NaCl in H20 pH to 8.0, RNase A) for 1 h to degrade remaining single stranded RNA. Slides were then rinsed with graded SSC washes followed by 0.1X SSC at 65 C for 60 min, after which they were transferred to a 0.05 M phosphate buffered saline (PBS) solution. Slides were incubated in 2% peroxide solution (30% H202 in 0.05 M PBS) for 30 min. Slides were then washed in IX Tris-buffered saline with 0.05% Tween-20 (TBS-T) and then incubated in blocking buffer (blocking reagent in IX TBS; Perkin Elmer) for 1 h. Digoxigen and fluorescein flurophores (Perkin Elmer kit; reconstituted in DMSO) were added under dim light as follows. All solutions were applied to tissue with co-verslips and incubated in humidified chambers. Slides were incubated with anti-digoxigen horseradish peroxidase for 30 min (anti-digoxigen in blocking buffer). Cover slips were removed in TBS-T then slides were washed in TBS-T. Slides were incubated with Cyanine 3 Amplification Reagent for 45 min (1:100 dilution in IX Amplification diluent) followed by TBS-T then TBS washes. Slides were then incubated with anti-Fluorescein-horseradish peroxidase for 2 h (1:100 anti-fluorescein-HRP in blocking buffer). Slides were washed in TBS-T then incubated with fluorescein fluorophore tyramide for 1 h (1:100 tyra-mide fluorophore in IX amplification diluent). Slides were washed in TBS-T then TBS, then cover-slipped with ProLong Gold antifade reagent with DAPI (Life Technologies) and stored in the dark to dry.
FISH image capture and analysis
Images for the DMS, DLS, NAcC and NAcS were captured at 200x magnification using a confo-cal fluorescent microscope (Axio Observer Zl; Zeiss Microscopy, Jena, Germany). Images were captured
38


from 4 separate hemispheres from 1.7 to -0.8 mm from Bregma. Single cfos. Dl, D2 and double-labeled cells were counted using Zeiss Zen software by multiple experimenters blind to treatment conditions of the animals. mCherry expression was quantified in alternate brain sections not used for FISH. Immunohistochemistry
Ninety min after the fear renewal test, TH-Cre and WT rats not used for FISH were perfused transcardially and brains prepared for immunohistochemistry as previously described (Herrera et al 2016, Lloyd et al 2017). Thirty-five pm coronal slices through the PFC (3.2 to 1.7 mm Bregma), AMG / hippocampus (-1.8 to -4.3 mm Bregma) or midbrain (-4.5 to -7.0 mm Bregma) were sliced on a cryostat at -20 C and stored in cryoprotectant (glycerol, ethylene glycol, 0.1M PB at pH 7.4) at -20 C until use. Sections were incubated in rabbit anti-cFos (1:3000; Santa Cruz sc-52) or mouse anti-TH (1:1000; Iinmuno Star cat #22941) at 4 C for 48 h. cFos primary binding was visualized using biotinylated secondary (1:200; Jack-son ImmunoResearch), avidin-bioten complex (Vector Laboratories) and nickel-enhanced 3,3-diamino-benzidine. TH primary was bound with fluorescent Donkey anti-Mouse IgG (H&L) DyLight 650 Conjugate (1:2000; Immunoreagents, Inc).
Immunohistochemistry> quantification
For cFos quantification, images of serial sections spaced 200 pm apart were captured at 200X magnification on an Olympus BX53. Cells expressing cFos protein were counted within a counting frame (1.48 x 105 pm) by multiple experimenters blind to treatment conditions of animals. Regions counted included the infralimbic (IL) and prelimbic (PL) regions of the prefrontal cortex, the basolateral (BLA) and central (CeA) nuclei of the amygdala, and the dentate gyrus (DG), CA1, CA2, and CA3 regions of the hippocampus. At least 6 hemispheres were counted per region per animal, but the number varied due to tissue damage incurred during slicing or staining procedures (range 6 10). Cells containing dark-brown or black nuclei were considered cFos-positive and lightly stained cells were not counted. Laser scanning con-focal microscopy software (Axio Observer Z1; Zeiss Microscopy) was used to image TH (green; A488) and mCherry (red; A568) positive cells in the SNc at 200x. Both hemispheres of five representative rats were visualized, 3 sections each. Numbers of single TH+, mCherry+, or double-labeled cells were quantified.
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Statistical Analyses
Percent time spent freezing was calculated by averaging freezing data from individual experimenters blind to treatment condition of the animals with immobility times obtained from Noldus Etho-vision XT. Pre-shock freezing for each test was averaged and group differences were analyzed with ANOVA. Average freezing across trials during conditioning and extinction were analyzed using repeated-measures ANOVA with subsequent Viral Expression Site or Drug as factors. For renewal, freezing across trails was averaged and compared using 2 (same vs different) x 3 (Veh vs SN vs Off-Target OR Veh vs SKF38393 vs SKF81297). Group differences in cFos protein expression were analyzed with 2x3 ANOVA. Group differences in the total number of cfos or D1 mRNA-expressing neurons and % Dl-ex-pressing neurons containing cfos mRNA were also analyzed with a 2 x 2 ANOVA. Differences in mCherry density between brain regions was analyzed with ANOVA, Fishers protected least significant differences post hoc analyses were performed when appropriate. Group differences were considered different whenp < 0.05.
Results
Activation of SNc DA neurons enhances fear extinction memory and blocks fear renewal
Viral transfection was confirmed in all rats. Figure 1A depicts bilateral mCherry expression in the SNc. Rats with mCherry expression restricted to the SNc were assigned after the experiment to the SNc group. Of the SNc rats injected with CNO, 6 were observed to have bilateral SNc mCherry expression and 2 had unilateral SNc mCherry; for a total of 8 rats in the SNc CNO group. Another 6 SNc rats received vehicle. Rats with mCherry extending into the VTA were considered off-target and were assigned to the off-target group. These rats tended to have limited expression in the SNc. Twelve off-target rats were injected with CNO and 5 were injected with vehicle. No significant differences were found between SNc and off-target rats injected with vehicle, so these rats were combined into the Vehicle group for all analyses. mCherry expression in brain regions involved in fear extinction and known to receive DA innervation from midbrain DA neurons was quantified with densitometry in SNc rats. The highest expression of mCherry was observed in the SN and DFS, moderate expression observed in the DMS and NAc, and minor expression in the PF, IF, DG and AMG (Figure IB). A photomicrograph depicting terminal expression
40


of mCherry in the DS is shown in Figure 1C. Four rats had no visible mCherry and were excluded from all analyses. TH immunohistochemistry revealed that 92% of the mCherry was contained within TH-express-ing neurons (Figure 1D-F), indicating the majority of the viral transfection was restricted to DA neurons.
3 2000
B.
JL
n n
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00 O F "0 Z U O ffl CO r- >------------------------------------

co o
D. i V- E. t < W V. F f
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M -/ -.A z4/i p. 'JP '* ,# V, '.
' M r VMm
wkr'ifc-f *. J s -Irf k. r : t
t ~ TH mCherry £ Figure 1. Gq-DREADD selectively targeted the SNc and was selective to TH-expressing neurons. (A) Representative picture of bilateral SNc Gq- DREADD injection; (B) mCherry intensity in SNc and potential terminal regions; (C) Representative picture of mCherry in the DS; (D) TH-labeled neurons; (E) mCherry autofluorescence; (F) Merge of TH and mCherry. # denotes significantly different from BLA (0.002), DG (p = 0.005), IL (p = 0.01), PL (p = 0.01); *** denotes p < 0.0001 from all other groups.
To determine the effects of Gq-DREADD-induced SNc DA activation during fear extinction on subsequent fear extinction memory and fear renewal, rats were injected with either vehicle or CNO during 2 subsequent days of fear extinction training. Fear conditioning and fear renewal were conducted drug-free (see Figure 2A for experimental timeline). Freezing levels were negligible prior to the first CS-US presentation during conditioning (Figure 2B; pre). All rats acquired fear conditioning equally (F(3, 105) = 16.421,/) <0.0001); Figure 2B), regardless of subsequent group assignment. The following day, all groups displayed equivalent within-session fear extinction in the presence of vehicle or CNO (F(4,140) = 55.004, p < 0.0001; Figure 2C). Given that SNc DA neurons are closely associated with movement, cage crossings were counted during the first 3 min of the extinction training sessions, before the first CS, as a measure of locomotor activity. No differences in overall locomotion were observed between groups on extinction day 1 (Vehicle 9.29 1.04; Off Target CNO 8.08 1.33; SN CNO 11.00 3.32; p = 0.52) or extinction day 2
41


{Vehicle 9.23 0.93; Off Target CNO 10.50 2.20; 11.20 1.24; p = 0.74). When tested for fear extinction memory the following day, all rats displayed within-session fear extinction (F(4,140) = 26.45,/) < 0.0001; Figure 2D), but the SNc CNO group displayed significantly less freezing than rats in other groups (F (2,35) = 3.22, p = 0.05; Figure 2D), indicating enhanced fear extinction memory recall in rats whose SNc DA neurons were activated during fear extinction. The interaction between viral expression and time just missed significance (F (8,140) = 1.89,/) = 0.06).
Rats were again exposed to the CS the next day in either the same or a different context for assessment of fear renewal. Consistent with fear renewal, within the vehicle group, rats exposed to the CS in the different context froze more than rats exposed to the CS in the same context in which extinction was learned (main effect of context; F(l,32) = 9.20,/) = 0.005; Figure 2E). However, activation of SNc DA neurons during extinction blocked fear renewal. Indeed, post-hoc analysis revealed no difference between the SN CNO same and different groups. The reduction in freezing displayed by the SNc CNO group during fear extinction day 2 was not observed in the same context again during the renewal test (Figure 2E). This is likely because by this point all groups had received sufficient extinction training in this context; thus, levels of freezing were at a floor. It is interesting that activation of Gq-DREADD-expressing DA neurons with CNO had no impact on fear extinction or renewal in rats in which viral transfection was observed in the VTA (off-target; Figure 2D and 2E). This observation could be due to the simple fact that fewer SNc DA neurons were transfected in off-target rats, or it could reflect a lack of effect of VTA DA neurons on fear extinction.
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Jest
Veh CNO Veh CNO
2500-
Figure 2. Gq-DREADD activation of SNc DA neurons, but not off-target DA neurons, enhances fear extinction and blocks fear renewal. (A) Experimental design. (B) All rats conditioned equally, take note that rats had not yet received CNO or vehicle. (C) 30 min after injection of either vehicle or CNO, all groups extinguished equally. (D) Rats received Vehicle or CNO 30 min prior to a second fear extinction session. When placed back into the extinction context 24 hours, CNO rats whose mCherry was selective to the SN exhibited significantly less freezing behavior, indicative of enhanced fear extinction memory. Main effect of SN CNO p = 0.05. (E) When tested in a context different form the fear extinction context, rats in the Vehicle and Off-target CNO groups had a significant increase in freezing behavior. Rats in the SN CNO had significantly lower freezing than both different groups; fear did not return in a different context. Vehicle and Off-target CNO different groups are significantly different from SN CNO different group. denotes p = 0.05; ** denotes p < 0.01.
Gq-DREADD activates target Dl-expressing neurons in the DS
We used double D1 Icfos FISH to determine if the Gq-DREADD approach successfully activates target Dl-expressing neurons in the DS. Similar to prior reports (Bertran-Gonzalez et al 2008, Bertran-Gonzalez et al 2010) virtually no co-localization of D1 and D2 mRNAs were observed in the DS (Figure 3A). This is in contrast to the NAc, in which D1 and D2 receptors have been observed to be colocalized (Bertran-Gonzalez et al 2008, Shetreat et al 1996). Since almost all neurons in the DS express either D1 or
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D2 mRNA, cells expressing single cfos mRNA can be assumed to be putative D2-expressing neurons (Gerfen et al 1990).
Veh CNO
Figure 3. Double fluorescent in situ hybridization labeling cfos and D1 mRNA during fear extinction. (A) Representative pictme from DS showing D1 and D2 receptor mRNA with low levels of co-localization. White bar represents 20pm. (B) Representative pictme from the DS from a Vehicle No Extinction rat showing very little D1 cfos coexpression; insert is a coronal section modified from the Paxinos and Watson rat brain atlas (Paxinos & Watson 1998). (C) Representative picture from the DS CNO extinction rat showing high levels of D1 cfos coexpression. (D) Activation of D1-expressing neurons in the DS. Fear extinction alone recruits Dl-expressing neurons in the DMS, while CNO injection activates Dl-expressing neurons in the DMS and DLS. (E) Activation of Dl-expressing neurons in the NAc is not increased with fear extinction nor peripheral CNO injection. denotes Extinction is p < 0.05 from No Extinction; # denotes CNO p < 0.05 from Vehicle.
Representative photomicrographs showing D1 and cfos mRNAs in the DMS of rats exposed to no
extinction or extinction are depicted in Figure 3B and 3C. Extinction learning alone increased the % of Dl-expressing neurons containing cfos mRNA in the DMS (Main effect of extinction: F(l,14) = 5.548. p =
0.03; Figure 3D) and the number of single cfos mRNA-expressing cells in the DLS (Main effect of extinc-
tion: F(l,14) = 4.750, p = 0.04, Table 1). Fear extinction learning did not alter the number of single cfos mRNA-containing cells nor the % of Dl-expressing neurons containing cfos mRNA in the NAcC or NAcS
(Table 1), but extinction learning increased the number of neurons expressing D1 mRNA in the NAcC
(F(l, 14) = 4.634,p = 0.04; Table 1). Activation of Gq-DREADD with CNO increased both the number of cells expressing cfos mRNA (F(l. 14) = 4.474,/) = 0.05; Table 1) and the % of Dl-expressing neurons con-
taining cfos mRNA in the DMS (F(l,14) = 13.492, p = 0.003; Figure 3D) and the number of double-labeled D1 /cfos mRNA-expressing neurons in the DLS (F(l. 16) = 5.564, p = 0.03; Figure 3D), but had no
effect in the NAcC or NAcS (Table 1 and Figure 3E). CNO did not impact cfos or D1 mRNAs in any other
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region. These data suggest that fear extinction recruits D1-expressing neurons in the DMS more than in other regions of the striatum. Further, the Gq-DREADD approach successfully activates D1-expressing neurons in the DS.
Table 1
Brain Region Veh No Ext Mean (SEM) Veh Ext Mean (SEM) CNO No Ext Mean (SEM) CNO Ext Mean (SEM)
#of DMS 33.59 (2.57) 34.77 (6.84) 22.81 (4.81) 34.70 (2.42)
single D1 mRN DLS 28.50 (4.67) 27.90 (3.82) 23.67 (4.04) 29.66(1.34)
A positive cells NAcC* 29.78 (2.75) 38.08 (6.51) 25.49 (2.32) 39.79 (3.77)
NAcS 35.46 (3.96) 45.62 (7.17) 35.17(7.59) 37.87 (2.19)
#of single DMS 3.28 (0.65) 4.2 (0.86) 4.57(1.32) 7.03 (1.12)
cfos mRN A pos- DLS* 4.02 (0.59) 6.875 (1.367) 4.25 (1.32) 8.31 (2.50)
itive cells NAcC 4.65 (1.19) 6.24(1.66) 4.36 (2.45) 6.04(1.86)
NAcS 4.18 a-02) 6.39(1.39) 4.86 (1.84) 7.37(0.72)
*Main effect of extinction p < 0.05
Gq-DREADD-induced SNc DA activation during fear extinction alters brain activation patterns during renewal
To probe which brain regions in the fear extinction circuitry might be impacted by SNc DA activation, rats were sacrificed after the fear renewal test and cFos was quantified in the hippocampus, mPFC, and amygdala; regions known to be important for fear extinction and renewal (Bergstrom 2016, Giustino & Maren 2015, Hirsch et al 2015, Huff & Rudy 2004, Jin & Maren 2015, Knapska et al 2012, Knapska & Maren 2009, Maren 2014, Thompson et al 2010). See Figure 4A for a graphic depiction of where cFos was
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quantified. No differences in cFos were noted in the PL (Figure 4B). Similar to a prior report (Knapska & Maren 2009), rats re-exposed to the extinguished CS in the same context in which fear extinction was learned demonstrated slightly higher cFos in the IL than rats re-exposed to the extinguished CS in a different context, although the main effect of context just missed significance (F(l,22) = 3.876, p = 0.06; Figure 4B). The increase in cFos in the IL noted in the same context could reflect this regions role in fear extinction recall (Chang & Maren 2011, Thompson et al 2010). SNc DA activation had no significant impact on cFos expression in the IL, suggesting that the IL is unlikely to be the site of action mediating the effects of SNc DA activation. Both the PL region of the PFC and the CeA are thought to drive the fear response in response to conditioned cues (Burgos-Robles et al 2009, Corcoran & Quirk 2007, Fendt & Fanselow 1999, LeDoux et al 1988). No group differences in cFos expression were observed in the PL (Figure 4B). However, consistent with the fear renewal observed in the different context, cFos expression in the CeA was significantly higher in rats placed into the different context than those placed into the same context (F(l,22) = 5.784,/) = 0.02). Interestingly, though, cFos expression in the CeA of rats whose SNc DA neurons were activated during fear extinction was identical between contexts (Figure 4B); a pattern of data that parallels the freezing behavior. No significant differences were observed in the BLA, CA3, or CA2. In the DG; however, cFos expression was higher in rats re-exposed to the extinguished CS in the same context compared to the different context (F(l, 21) = 4.131, p = 0.05; Figure 4C). This observation in the DG is similar to a prior report (Knapska & Maren 2009) and could reflect the recognition of the familiar extinction context by the hippocampus. Interesting data observed in CA1, where greater cFos expression was observed in the CA1 of rats whose SNc DA neurons were activated during fear extinction compared to rats in other groups, regardless of the context in which rats were re-exposed to the extinguished CS (F(2,21) = 3.566,/) = 0.04; Figure 4C). Representative photomicrographs depicting cFos immunoreactivity in the CA1 are shown in Figures 4D and 4E.
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Vehicle Same v////n Vehicle Different
' ' Off-target CNO Same \S / / / /~7\ Off-target CNO Different
SN CNO Same *= SN CNO Different
Figure 4. Immunohistochemistry cFos protein expression within regions implicated in fear extinction during fear renewal. (A) Representative images of regions quantified; including regions of the hippocampus, mPFC, and amygdala modified from the Paxinos and Watson rat brain atlas (Paxinos & Watson 1998). (B) Regions within the mPFC and amygdala. cFos protein expression in the CeA mirrors the fear expression observed in the renewal behavioral test, with significantly higher activation in the different vs same groups for the Vehicle and Off-Target CNO groups but no significant difference between the same and different SN CNO groups. (C) Regions within the hippocampus; testing in the same context increases cFos protein in the DG. Significantly higher levels of cFos protein were observed in the CA1 region of SN CNO rats. No differences were observed in the CA3 or CA2 regions of the hippocampus. (D) Representative picture of the CA1 region with low cFos protein expression; (E) Representative picture of the CA1 region with high cFos protein expression. Scale bar represents 50pm. represents p < 0.05.
Activation of DS D1 receptors during fear extinction blocks fear renewal without enhancing fear extinction memory
To determine the contribution of D1 receptors in the DS to the observed effects of Gq-DREADD-induced activation of SNc DA neurons, we microinjected D1 agonists into the DS prior to fear extinction learning on 2 subsequent days, and fear renewal was assessed drug free 24 h later (see Figure 5A for experimental timeline). Given a recent report that systemic administration of SKF81297 and SKF38393 have
47


different effects on fear extinction; whereby SKF81297, but not SKF38393, administered prior to fear extinction enhances fear extinction memory (Abraham et al 2016, Borowski & Kokkinidis 1998), but see (Mannoury la Corn et al 2007), we microinjected both drugs into the DS in different cohorts of rats prior to fear extinction. Fifty-two out of 54 rats had successful bilateral DS cannulae and the locations of the cannulae tips are shown in Figure 5B. Exclusion of missed injections resulted in the following group sizes: Saline same = 12, Saline different =11, SKF38393 same = 8, SKF38393 different = 9, SKF81297 same = 6, SKF81297 different = 6.
Pre1 2 3 4 Pre 1 2 3 4 5 Pre1 2 3 4 5
Trial
SKF38393
Blocks of 4 Trials Blocks of 4 Trials

sA
% s%
%

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Figure 5. D1 agonists injected into the DS during fear extinction block fear renewal without enhancing fear extinction memory. (A) Experimental design. (B) Cannula placement verification for C-F. Coronal sections modified from the Paxinos and Watson rat brain atlas (Paxinos & Watson 1998). (C) All groups were equally conditioned; (D) Saline, SKF38393, or SKF 81297 were injected 10 minutes prior to fear extinction training. Presence of a D1 agonist had no impact on fear extinction learning. (E) Saline, SKF38393, or SKF81297 was injected 10 minutes prior to the second day of fear extinction training. No difference between groups was observed. (F) Saline rats placed into a different context exhibited significantly higher freezing than those placed into the same context; however, no significant differences were observed between same and different groups if rats were injected with D1 agonists prior to fear extinction training. (G) Experimental design for drug free extinction memory test. (H) Cannula placements for drug free extinction experiment. (I) All rats were equally conditioned to fear the tone; (J) Rats received microinjections of Vehicle or SKF38393 prior to fear extinction learning. No difference in fear extinction learning was observed. (K) When tested drug-free, no differences between groups were observed. denotes p < 0.05 from same group.
Rats acquired auditory fear conditioning equally (Main effect of time: F(3,147) = 65.679. p < 0.0001; Figure 5C), regardless of subsequent group assignment. Neither agonist impacted freezing during the first fear extinction training session. All rats displayed within-session extinction (Main effect of time: F(4,196) = 56.950, p < 0.0001) that did not differ between groups (Figure 5D). Similar effects were observed during the second fear extinction training session the next day, during which all rats similarly acquired within-session extinction (Main effect of time: F(4,196) = 46.846. p < 0.0001; Figure 6E). Freezing to the first few CS during a fear extinction session represents memory of previously acquired fear extinction (Bukalo et al 2015, Do-Monte et al 2015), thus freezing during the first 4 trials were analyzed separately. Intra-DMS D1 agonist had no impact on fear extinction memory during the first few trials of the second fear extinction training session. Similarly, all rats displayed equivalent freezing in the same context when tested drug free the next day, although freezing levels in the same context were approaching a floor.
Despite the fact that D1 activation had no effect on fear extinction learning or memory in the extinction context, rats that received D1 agonist prior to fear extinction were protected against fear renewal tested in a novel environment 24 h after the second fear extinction training session (drug by context interaction; F(2,46) = 3.1,/? < 0.0001; Figure 5F). Post hoc analysis revealed that the saline different rats froze significantly more than all other groups (saline same p = 0.006, SKF38393 differentp = 0.008, SKF38393 same p = 0.02, SKF81297 differentp = 0.009, SKF81297 same p = 0.03), and neither the SKF38393 different or SKF81297 different groups different from their same counterparts (p > 0.05).
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The observation that Gq-DREADD-induced DA release both enhances fear extinction memory and reduces renewal, whereas selective activation of DS D1 with D1 agonists reduce fear renewal without enhancing fear extinction, is an interesting finding. To rule out the possibility that the presence of D1 agonist drug during the extinction memory test (extinction day 2) obscured a potential effect of prior D1 agonist on extinction memory, SKF38393 was injected into the DS prior to the first fear extinction training session in a different cohort of rats, and fear extinction memory was tested the next day drug free (see figure 5G for experimental design). Location of cannula tips are shown in figure 5H. All rats acquired fear conditioning throughout CS-US pairings (Main effect of time: F(3,42) = 25.201 ,P< o .0001) regardless of subsequent group assignments (Figure 51). The following day, all rats acquired within-session fear extinction (Main effect of time: F(5,70) = 72.155, p < 0.0001; Figure 5J) and there was no difference between rats that received intra-DS injections of vehicle (n = 8) or SKF38393 (n = 8). When tested the following day drug-free, we again observed that D1 receptor activation during extinction failed to enhance fear extinction memory (Main effect of time: F(5,70) = 15.102,p < 0.0001; Figure 5K). Therefore, although activation of SNc DA neurons and DS D1 receptors during fear extinction both prevent fear renewal, they seem to have different effects on fear extinction memory when tested in the extinction context.
Discussion
Data presented here reveal a novel role for nigrostriatal DA in fear extinction. Activation of SNc DA neurons with Gq-DREADD during fear extinction had no effect on fear extinction acquisition, but enhanced fear extinction memory and blocked the renewal of fear; a pattern of data paralleled by cFos expression in the CeA. Dl-expressing neurons in the DS are a likely target mediating at least part of the effect of SNc DA activation. Indeed, expression of terminal mCherry, indicating presence of Gq-DREADD, was highest in the DS, Dl-expressing neurons in the DMS were observed to be recruited during fear extinction, and Gq-DREADD-induced DA potentiated activity of Dl-expressing neurons in both the DMS and the DLS. Interestingly, rats whose SNc DA neurons were activated by Gq-DREADD during prior fear extinction displayed potentiated cFos expression in the CA1 of the hippocampus, a region important for contextual processing (Matus-Amat et al 2004). Pharmacological activation of D1 receptors in the DS had no impact on fear extinction acquisition or memory, but blocked fear renewal in a novel context. Together,
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these data suggest that activation of SNc DA neurons and DS D1 receptors could alter the contextual processing of the fear extinction memory, thus reducing the context specificity of the memory and preventing fear renewal.
Prior studies investigating the roles of specific DA circuits reveal inconsistent results. A recent study suggests that VTA DA neurons projecting to the mPFC can impair fear extinction (Hitora-Imamura et al 2015). However, other studies implicate roles for Dl, D2, & D4 receptors in the mPFC in fear extinction (Hikind & Maroun 2008, Mueller et al 2010, Pfeiffer & Fendt 2006). VTA DA neurons also provide DA innervation to other limbic structures including the BLA, hippocampus and NAc. In the BLA, DA manipulations have no effect on fear extinction (Fiorenza et al 2012, Hikind & Maroun 2008). In contrast, Dl activation in the hippocampus can enhance short-term fear extinction memory (Fiorenza et al 2012), while inhibition of D2 in the NAc has been reported to impair fear extinction (Holtzman-Assif et al 2010a). Notably, relapse was not investigated in these studies; therefore, the effects of these manipulation on fear renewal is unknown. No studies to our knowledge have investigated the involvement of SNc DA neurons and their primary target, the DS, in fear extinction.
Here we used a Gq-DREADD approach in TH-Cre rats to selectively increase phasic activity of SNc DA neurons (Urban & Roth 2015). This approach was selective at targeting DA neurons in the SNc (Figure 1) and produced a functional increase in activity of target neurons downstream of SNc DA neurons. Gq-DREADD-induced activation of SNc DA neurons resulted in an increase in cfos mRNA in Dl mRNA-expressing neurons in the DMS and DLS (Figure 4A), but not the NAc. This is not surprising, since the DS is the primary recipient of DA terminals originating from the SNc (Figure IB). The effects of SNc DA activation was not due to a non-specific effect DA on locomotor activity. Indeed, the 1 mg/kg dose of CNO did not alter general locomotor activity measured by spontaneous cage crossings in Context B prior to the first CS during either fear extinction training session, nor did CNO reduce freezing during the first extinction training day. These observations suggest that the reduced freezing displayed by the SNc CNO group during extinction training day 2 was not due to an effect of CNO on the expression of freezing per se. but rather to prior SNc DA facilitating the later recall of fear extinction memory. Since CNO was injected prior to fear extinction and has a 3h half-life, the effect of SNc DA activation on later extinction
51


memory recall could have been due to enhancements of either extinction acquisition or consolidation. The observation that CNO had no impact on fear extinction acquisition on fear extinction day 1 argues for an effect on consolidation, but the current experiment was not designed to directly address the role of DA in acquisition versus consolidation.
A critical question is where DA is acting to produce the observed effects of SNc DA activation. Although the SNc projects to regions other than just the DS, minimal terminal mCherry expression was observed in regions traditionally implicated in fear extinction, such as the mPFC, hippocampus, and AMG. Therefore, it is unlikely that the observed effects of SNc DA activation was mediated by DA in these regions, although a role for DA in these regions cannot be completely ruled out. Double FISH revealed that D 1-expressing neurons in the DMS are recruited during fear extinction learning (Figure 3D). DMS-corti-cal circuits are involved in guiding goal-directed behavior (Shiflett et al 2010, Yin et al 2005). Goal-directed strategies may guide behavior as the animal acquires extinction and begins to evaluate the environment. We have similarly observed potentiated activity of DMS Dl-expressing neurons during recall of re-lapse-resistant fear extinction in rats that ran in running wheels during fear extinction acquisition (Mika et al 2015). Together, these data suggest that DMS Dl-expressing neurons could represent a previously unidentified component of fear extinction, recruitment of which could be involved in the learning or recall of fear extinction. Additionally, although DLS Dl-expressing neurons were not recruited during fear extinction, Gq-DREADD-induced SNc DA activity resulted in increased cfos mRNA in these DLS neurons (Figure 3A). The DLS is involved in guiding inflexible behavioral strategies that can occur at the expense of goal-directed or spatial strategies (i.e. habit; (Lovinger 2010, Schwabe et al 2008, Yin & Knowlton 2006). These data therefore raise the possibility that DA in the DLS elicited by Gq-DREADD could have altered the learning strategy used during fear extinction acquisition to one involving the DLS. This is an intriguing possibility, as learning strategies involving the DLS may be less susceptible to the disruptive effects of contingency changes, such as context, during recall (Dias-Ferreira et al 2009, Schwabe et al 2008). More selective targeting of DMS vs. DLS DA circuits will be required to determine the role of DA in these regions in fear extinction. Similarly, how these DS regions communicate with canonical fear circuitry is currently unknown. The observation of altered cFos expression patterns during fear extinction
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memory recall in the CeA and hippocampus of rats whose SNc DA neurons were activated during fear extinction (Figure 4B and 4C) implicates these regions in the mechanisms by which nigrostriatal DA enhances fear extinction. Modulation of neural activity in the hippocampus, in particular, could reflect altered contextual processing by nigrostriatal DA; a prospect that is supported by the complete blockade of fear renewal elicited by both SNc DA activation (Figure 2E) and pharmacological activation of DS D1 receptors (Figure 5F). The current data are consistent with prior work implicating the nigrostriatal pathway in learning processes that are independent of the hippocampus & thus contextual modulation (Da Cunha et al 2003, Faure et al 2005).
D1 agonists injected into the DS prior to fear extinction prevented fear renewal without impacting extinction memory, even when extinction memory was assessed drug free (Figure 5K); a pattern of data differing from that of SNc DA activation. This discrepancy could be explained by phasic DA elicited by Gq-DREADD interacting with DA receptors differently than specific receptor agonists. For example, phasic DA activates both D1 and D2-expressing neurons (table 1), whereas the partial D1 agonist SKF38393 is selective to D1 (Conroy et al 2015). SKF81297; however, agonizes both D1 and D2 receptors (Rashid et al 2007a), as well as D1/D2 heteromers (Rashid et al 2007b). Another possibility is that DA in the DS could render extinction memories resistant to relapse (e.g. renewal), whereas DA in a different region could be responsible for enhancing extinction memory in the same context. Since we didnt target D1 agonists to the DMS or DLS selectively (Figure 6B), the effects of pharmacological D1 activation cannot shed light on where within the DS Gq-DREADD-induced DA could have been acting. However, the pharmacological data does support the idea that nigrostriatal DA frees extinction memory from its contextual modulation through a mechanism involving D1 receptors in the DS.
The current data implicate DS D1-expressing neurons in fear extinction and demonstrate for the first time that SNc DA neurons can facilitation fear extinction memory. Importantly, fear extinction supported by nigrostriatal DA is resistant to fear renewal. As relapse phenomena, including renewal, are a major barrier to the long-term success of current treatment strategies for anxiety and trauma-related disorders, the nigrostriatal DA pathway provides a promising target for the development of more effective therapeutic strategies.
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Acknowledgements
We would like to thank Dr. Michael Baratta and Dr. Erik Oleson for technical advice on the use of DREADDs.
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CHAPTER IV
CONCLUSIONS
Data presented in this thesis increase our understanding of fear extinction learning and memory, specifically techniques to modify fear extinction learning so as to reduce the renewal of fear. Exercise is a safe, healthy, inexpensive behavioral modification that can easily be integrated into exposure therapy sessions (Powers et al 2015). We found that voluntary exercise enhances auditory fear extinction memory and blocks the renewal of fear, as well as enhancing long term contextual fear extinction memory in male Long Evans rats. Interestingly, voluntary exercise integrated into fear extinction had no effect on fear extinction memory or fear renewal in female Long Evans rats. This is an important finding, particularly because of established differences in anxiety- and trauma-disorders between males and females (Maeng & Milad 2015). Emerging sex differences in animal models can reveal pertinent knowledge gaps and change the way we address future experiments and future treatments. Further experiments are needed to further understand our observed sex differences in exercise manipulation of fear extinction learning and memory manipulated.
The finding that exercise can modulate fear extinction learning and memory could have interesting applications for the clinical setting; however, the underlying mechanisms are unknown. Exercise impacts many central and peripheral systems, one of which is the DA system. The DA system provides an interesting intersection between reward and movement, which makes it an excellent target for the neurobi-ological basis of the effects of exercise on fear extinction learning and memory. We targeted the nigrostri-atal DA system using Gq-DREADD and pharmacological manipulations. We found that Gq-DREADD-induced phasic DA release enhances fear extinction memory and creates a memory that is resistant to relapse; however, this effect was only observed if Gq-DREADD activation was selective to the SNc. This observation mimicked the effects of exercise during fear extinction consolidation. Interestingly pharmacological activation of the D1 receptor in the DS, the main terminal region of SNc DA projections according to our mCherry data, blocked the renewal of fear without effecting fear extinction memory. These data suggest that different memory systems modulate fear extinction memory and fear renewal. Further, to the best of our knowledge, these data are the first to implicate the dorsal striatum in fear extinction learning
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and memory. Data presented in this thesis provide evidence that increased activation of the D1 receptor in the DS during fear extinction learning, either through DREADD activation of SNc DA neurons or pharmacological activation of the D1 receptor in the DS, reduces fear renewal. Further studies investigating the neural circuitry connecting the DS to the canonical fear circuitry are needed.
Given the similar effects of nigrostriatal DA during fear extinction and exercise during fear extinction consolidation, it is likely that nigrostriatal DA plays a role in the observed exercise effect from chapter 1. Results from this thesis show that nigrostriatal DA activation is sufficient to produce the effects of exercise during fear extinction consolidation; however, further experiments are needed to assess whether nigrostriatal DA is necessary for these effects. Inhibitory DREADD during exercise after extinction would be an interesting means to test this question. If this manipulation interferes with the ability of exercise to enhance fear extinction memory and block renewal, then it could be concluded that an increase in nigrostriatal DA is both necessary and sufficient for the effects. It is possible; however, that inhibitory DREADD during exercise could have an adverse effect on movement and impair the rats ability to exercise.
Data presented in this thesis identify a behavioral manipulation, voluntary exercise, that can enhance fear extinction learning and memory and block the renewal of fear. Further we show that activation of SNc DA neurons, resulting in phasic DA release, during fear extinction produces the same behavioral outcome enhanced fear extinction and lack of fear renewal. Fear extinction is the basis of exposure therapy, a common behavioral therapy used for the treatment of PTSD- and anxiety-related disorders. Enhancing our understanding of the neural circuitry underlying fear extinction, and factors that can modulate fear extinction learning and memory, can have useful clinical implications for use in improving the efficacy of exposure therapy.
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DISS_cat_code 0317
DISS_cat_desc Neurosciences
0307
Molecular biology
0719
Physiology
DISS_keyword D1 receptor, Dopamine, Dorsal Striatum, DREADD, Exercise, Fear Extinction
DISS_language en
DISS_content
DISS_abstract
DISS_para Anxiety and trauma-related disorders are prevalent and debilitating, yet treatments lack long term efficacy. Behavioral therapies, such as extinction-based exposure therapy, are beneficial but highly susceptible to relapse— the return of fear following successful extinction. Exercise is emerging as a healthy, noninvasive, inexpensive means to augment fear extinction. Data collected for my Master’s thesis supports this notion, as physical exercise immediately following fear extinction both enhanced fear extinction memory and reduced the later relapse of fear in adult, male, Long Evans rats. Interestingly, the same effects were not observed in female Long Evans rats, a disparity that opens the door for further research. Exercise impacts a variety of peripheral and neurobiological systems, one being midbrain dopamine (DA) circuits. Viral-mediated transfer of a gene coding for a Designer Receptor Exclusively Activated by a Designer Drug (DREADD) allowed us to control activity of select populations of DA neurons with high specificity during fear extinction. Activating midbrain DA neurons during fear extinction mimicked the effects of exercise: rats displayed enhanced fear extinction memory and prevented fear renewal in a novel context; data paralleled by changes in neural activity in brain regions involved in fear and contextual processing. One consequence of DA neural activity is activation of DA-1 receptors (D1) in the dorsal striatum, and populations of dorsal striatum D1-expressing neurons are anatomically linked to fear circuits. Interestingly, pharmacological activation of D1 in the dorsal striatum during fear extinction impacted fear
extinction memory in a context-specific manner. Activation of D1 receptors during fear extinction blocked fear renewal in a novel context, but had no effect on fear extinction memory when tested in the same context in which extinction was learned. These data suggest that activation of midbrain DA neurons is sufficient to reproduce the memory-modulating effects of exercise, and these effects are partly mediated by D1 signaling in the dorsal striatum.
DISS_supp_abstract
DISS_binary PDF Bouchet_ucdenver_0765N_10841.pdf
DISS_restriction
DISS_repository
DISS_version 2011-11-08 15:37:33
DISS_agreement_decision_date 2017-04-21 14:55:40
DISS_acceptance 1
DISS_delayed_release
DISS_access_option



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EXERCISE AND DOPAMINE MODULATION OF FEAR EXTINCTION by COURTNEY ANNE BOUCHET B.A. University of Colorado Boulder, 2009 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the require ments for the degree of Master of Science Biology Program 2017

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ii This thesis for the Master of Science degree by Courtney Anne Bouchet has been approved for the Biology Program by Benjamin N. Greenwood, Chair Erik Oleson John Swallow Date: Ma y 13, 201 7

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iii Bouchet, Courtney Anne (M.S., Biology Program) Exercise and dopamine modulation of fear extinction Thesis directed by Assistant Professor Benjamin N. Greenwood ABSTRACT Anxiety and trauma related disorders are prevalent and debilitating, y et treatments lack long term efficacy. Behavioral therapies such as extinction based exposure therapy are beneficial but highly susceptible to relapse the return of fear following successful extinction Exercise is emerging as a healthy, non invasive, i nexpensive means to augment fear extinction. Data collected for my Master's thesis supports this notion, as physical exercise immediately following fear extinction both enhanced fear extinction memory and reduced the later relapse of fear in adult, male, L ong Evans rats. Interestingly, the same effects were not observed in female Long Evans rats, a disparity that opens the door for further research. Exercise impacts a variety of peripheral and neurobiological systems, one being midbrain dopamine (DA) circui ts. Viral mediated transfer of a gene coding for a Designer Receptor Exclusively Activated by a Designer Drug (DREADD) allowed us to control activity of select populations of DA neurons with high specificity during fear extinction. Activating midbrain DA neurons during fear extinction mimicked the effects of exercise: rats displayed enhanced fear extinction memory and prevented fear renewal in a novel context; data paralleled by changes in neural activity in brain regions involved in fear and contextual pr ocessing. One consequence of DA neural activity is activation of DA 1 receptors (D1) in the dorsal striatum, and populations of dorsal striatum D1 expressing neurons are anatomically linked to fear circuits. Interestingly, pharmacological activation of D1 in the dorsal striatum during fear extinction impacted fear extinction memory in a context specific manner. Activation of D1 receptors during fear extinction blocked fear renewal in a novel context, but had no effect on fear extinction memory when tested i n the same context in which extinction was learned These data suggest that activation of midbrain DA neurons is sufficient to reproduce the memory modulating effects of exercise, and these effects are partly mediated by D1 signaling in the dorsal striatum The form and content of this abstract are approved. I recommend its publication. Approved: Benjamin N. Greenwood

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iv DEDICATION Mom, Dad, and Chris Y ou r unwavering support throughout my academic endeavors means the world to me Mom, thanks for answering m y phone calls at midnight when I was walking home from campus after a late experiment or late night in the lab. Granddaddy Ever since I was little you have inspired me to be curious. Y our passion for knowledge and understanding has always been an inspir ation. Jeff Randall Thank you for being my rock and for learning way more about fear extinction and dopamine than you ever anticipated Y our support, enthusiasm, and kindness makes me so happy.

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v ACKNOWLEDGEMENTS Dr. Benjamin N. Greenwood I cer tainly would not be where I am today without you. Thank you for your support and guidance. Your enthusiasm and curiosity for research is contagious. I ap preciate all that you have done for me. Dr. Aggie Mika I feel so lucky to have been mentored by you. Thank you for your guidance throughout my s cientific career. Thank you for reminding me that science is fun and e njoyable. The Greenwood Lab Thank you all for everything that you do. These experiments took a lot of time and effort your help was much a ppreciated and necessary and did not go unnoticed. Specific authors for publications are identified in chapters. Dr. Hannah Anchordoquy Thank you for your mentorshi p, y ou m olded me into the teacher that I am today. Your guidance and support was incredib ly helpful I am forever grateful for it and for you Dr. Erik Oleson and Dr. John Swallow Thank you for making such a great and supportive committee.

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vi TABLE OF CONTENTS I. INTRODUCTION ................................ ................................ ................................ ................................ ....... 1 Factors that Modulate Fear Extinction ................................ ................................ ................................ ....... 2 II. ACUTE EXERCISE ENHANCES THE CONSOLIDATION OF FEAR EXTINCTION MEMORY AND REDUCES CONDITIONED FEAR RELAPSE IN A SEX DEPENDENT MANNER ...................... 7 Introduction ................................ ................................ ................................ ................................ ................ 8 Materials and Methods ................................ ................................ ................................ ............................. 10 Subjects ................................ ................................ ................................ ................................ ................ 10 Estrus Cycle Monitoring ................................ ................................ ................................ ...................... 10 Behavioral Analyses ................................ ................................ ................................ ............................ 11 Post Extinction Wheel Running ................................ ................................ ................................ .......... 13 Procedures ................................ ................................ ................................ ................................ ............ 13 Statistical Analysis ................................ ................................ ................................ ............................... 16 Results ................................ ................................ ................................ ................................ ...................... 17 Experiment 1: Exercise During Consolidation of Contextual Fear Extinction Improves Long Term Memory of Fear Extinction in Males ................................ ................................ ................................ ... 17 Experiment 2: Exercise During Consolidation of Auditory Fear Extinction Improves Fear Extinction Memory and Reduces Fear Renewal in Males ................................ ................................ .................... 19 Experiment 3: Exercise dur ing consolidation of auditory fear extinction fails to improve fear extinction memory or reduce fear renewal in females ................................ ................................ ......... 21 Discussion ................................ ................................ ................................ ................................ ................ 25 III. ACTIVATION OF NIGROSTRIATAL DOPAMINE NEURONS DURING FEAR EXTINCTION PREVENTS THE RENEWAL OF FEAR ................................ ................................ ................................ .... 31 Abstract ................................ ................................ ................................ ................................ .................... 31 Signif icance Statement ................................ ................................ ................................ ............................. 32 Introduction ................................ ................................ ................................ ................................ .............. 32

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vii Materials and Methods ................................ ................................ ................................ ............................. 33 Animal s ................................ ................................ ................................ ................................ ................ 33 Surgeries ................................ ................................ ................................ ................................ .............. 34 Microinjections ................................ ................................ ................................ ................................ .... 35 Drugs ................................ ................................ ................................ ................................ .................... 35 Behavioral Analyses ................................ ................................ ................................ ............................ 36 Double label fluorescent in situ hybridization ................................ ................................ .................... 37 Immunohistochem istry ................................ ................................ ................................ ........................ 39 Statistical Analyses ................................ ................................ ................................ .............................. 40 Results ................................ ................................ ................................ ................................ ...................... 40 Activation of SNc DA ne urons enhances fear extinction memory and blocks fear renewal ............... 40 Gq DREADD activates target D1 expressing neurons in the DS ................................ ....................... 43 Gq DREADD induced SNc DA activation during fear extinction alters brain activation patterns during renewal ................................ ................................ ................................ ................................ ..... 45 Activation of DS D1 receptors during fear extinction blocks fear renewal without enhan cing fear extinction memory ................................ ................................ ................................ ............................... 47 Discussion ................................ ................................ ................................ ................................ ................ 50 Acknowledgements ................................ ................................ ................................ ................................ .. 54 IV. CONCLUSIONS ................................ ................................ ................................ ................................ .... 55 REFERENCES ................................ ................................ ................................ ................................ ............. 56

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1 CHAPTER I INTRODUCTION Anxiety disorders are the most prevalent mental health disorders in developed countries with a lifetime prevalence of 28% (Kessler et al 2012, Kessler et al 2005) and yet, treatments for such disorders have limited availability and efficacy (Hoffman & Mathew 2008, Stein et al 2009, Steinberg et al 2009) Mechanisms underlying fear conditioning are thought to go awry in anxiety disorders (Elzinga & Bremner 2002) Current behavioral therapies to treat such disorders, such as exposure therapy, focus on fear extinction. Extinction is the decay of the fear response following repeated presentations of the fearful stimulus in the absence of an aversive event. Even after success ful exposure therapy, rates of relapse remain high (Boschen et al 2009, Bouton 1993, Goode & Maren 2014) The three main types of fear relapse are 1) the return of fear in context different from which the extincti on was learned (renewal; (Bouton & Ricker 1994b) 2) the return of fear after a stressful event (reinstatement; (Rescorla & Heth 1975) and 3) the return of fear over time (spontaneous recovery; (Goode & Maren 2014) Fear conditioning is a Pavlovian associative learning phenomenon during which a previously neutral stimulus (conditioned stimulus; CS) comes to elicit a fear response because of its association with an aversive stimulus (unconditioned stimulus; US; (Pavlov 1927) Repeated exposures to the CS in the absence of the US extinguishes the fear associated with the CS, but this extinction is context dependent The relapse of fear following successful extinction suggests that extinction does not erase the fear memory, but that the extinction memory is a separate entity from the fear memory that suppresses the fear memory (for reviews see (Bouton 2004, Myers & Davis 2007) Manipulations that modify fear extinction learning or consolidation could produce an extinction memory that is resistant to contextual modulation, thus reducing fear renewal. Considerable work has bee n done to elucidate the neural circuitry involved in fear extinction (Herry et al 2010, Knapska et al 2012, Mamiya et al 2009, Quirk & Mueller 2008, Sierra Mercado et al 2011) and factors capable of modulating fear extinction learning and memory (Singewald et al 2015) Critical regions involved in fear and its extinction include the basolateral (BLA; comprised of the basal, lateral, and basomedial amygdala nuclei) and centra l nucleus of the amygdala (CeA), the hippocampus, and the medial

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2 prefrontal cortex (mPFC), specifically the prelimbic (PL) and infralimbic (IL) regions. These regions of the mPFC are thought to play opposing roles in the expression of fear. The PL, through excitatory projections to the CeA, drives the fear response during exposure to fear eliciting events or cues (Burgos Robles et al 2009, Corcoran & Quirk 2007, Fendt & Fanselow 1999, LeDoux et al 1988) In contrast through neural projections to the inhibitory intercalated interneurons of the amygdala, the IL can inhibit the expression of fear by inhibiting output of the CeA (Quirk et al 2006) Recent work, however, suggests there is limited connectivity betwee n the IL and the intercalated interneurons of the amygdala. This observation is consistent with, and could partially explain, the transient nature of fear extinction memories relative to fear memories (Giustino & Maren 2015) The BLA has been implicated in both fear conditioning and extinction with distinct cell populations contributing to high and low fear states (Goosens et al 2003, Herry et al 2008, Hobin et al 2003) .The CeA is thought to f unction as an output station and is critically involved in the fear response through projections to the brain stem, hypothalamus, and periaqueductal gray (PAG), which together initiate the various aspects of the fear response including freezing (Fanselow & Gale 2003, Pare et al 2004) Contextual information pertaining to fear and extinction memories, which is an important contributor to the renewal of fear (Bouton 2004, Bouton et al 2006b) is encoded by the hippocampus (Corcoran & Maren 2001) For example, Jin and Maren (2015) found that dorsal hippocampal collateral projections to the IL and basal nucleus of the amygdala are preferentially activated during fear renewal, and inactivation of the hippocampus preve nts renewal (Jin & Maren 2015) Huge strides have been made in understanding the circuitry underlying fear and its extinction; however, less is known about factors that modulate fear extinction and relapse. Factors that Modulate Fear Extinction Many factors are capable of modulating fear extinction learning and memory (see (Fitzgerald et al 2014, Singewald et al 2015) for reviews). These manipulations differ in their ease o f use and success at reducing relapse in a clinical setting. For example, massive extinction, or a large number of extinction trials, reduces fear renewal in rodents (Denniston et al 2003) ; however, it is unlikely that patients will be willing to commit to the immense time commitment that mass extinction requires. Another behavioral manipulation aim ed at reducing relapse involves performing exposure therapy in multiple contexts in order to

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3 reduce the contextual dependency of extinction memories. However, reports on the effects of extinction in multiple contexts on later fear relapse are mixed, with s ome studies reporting promising results (Chelonis et al 1999, Gunther et al 1998) and others not (Bouton et al 2006a) Extinction in multiple contexts also brings clinical challenges as it requires therapy sessions to occur in multiple different clinical environments. Some pharmacological manipulations could be simple to implement in a clinical set ting, but not all pharmacological means to enhance extinction result in a successful reduction of relapse. Activation of glutamate NMDA receptors with D cycloserine (DCS) during fear extinction can strengthen extinction learning; however, there are limitat ions to DCS treatment (Graham et al 2011) One limitation is that DCS also strengthens memory re consolidation (Lee et al 2006) and is not specific to the extinction memory, thereby allowing the possibility that DCS during re exposure to the fearful stimulus during extinction could actually strengthen the fear memory instead of extinguishing it. Indeed, fear extinction augmented with intra IL or intra BLA injections of DCS remains susceptible to fear renewal (Woods & Bouton 2006) Glucocorticoids are another m odulatory system that can enhance fear extinction in rats (Bentz et al 2010, de Bitencourt et al 2013, Soravia et al 2014, Yang et al 2006) Clinical glucocorticoid therapy could be problematic, however, because ac ute glucocorticoid treatment has the potential to increase anxiety (Mitra & Sapolsky 2008) which could be counter productive to therapy. Despite potential limitations associated with the aforementioned strategies, several manipulations with the potential to reduce fear relapse after extinction remain. Among those most likely to be invol ved in the effects of exercise are neuromodulatory systems such as the endocannabinoid system, growth factors, such as Fibroblast growth factor 2 (FGF2; (Graham & Richardson 2011a) ) and BDNF (Baker Andresen et al 2013) as well as signaling throu gh specific monoaminergic circuits. The endocannabinoid system is implicated in fear extinction learning (Papini et al 2015) is sensitive to physical activity, and has been implicated in anxiolytic effects of exer cise (Fuss et al 2015) Endocannabinoids such as 2 arachidonoyl glycerol (2 AG) are increased in the BLA during fear extinction (Marsicano et al 2002) and blocking effects of endocannabinoids by receptor antagonists (Marsicano et al 2002) or genetic knockout of synthetic enzymes of 2 AG (Jenniches & Zimmer 2016) impairs fear extinction learning. Growth factors such as FGF2 are critically involved in the molecular mechanisms of long term memory (Graham & Richardson 2009b) and,

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4 if increased during extinction consolidation, can enhance fear extinction and reduce reinstatement (Graham & Richardson 2009a) and renewal (Graham & Richardson 2011b) of fear. Brain derived neurotrophic factor (BDNF) is also intimately involved in memory and neuroplasticity. BDNF signaling in the BLA during fear extinction is neces sary for consolidation of extinction memories (Chhatwal et al 2006) while activation of BDNF signaling pathways reduces the renewal of fear in female mice (Baker Andresen e t al 2013) Additionally, extinction enhanced by BDNF produces a stronger long term extinction memory ( (Bredy et al 2007) ; for a recent review of the role of BDNF in fear extinction augmented by exercise see (Powers et al 2015) ). Another neurotransmitter, dopamine (DA), classically associated with movement, reward, and reinf orcement, is also an important memory modulator. Growing literature implicates DA as a promising potential modulator for fear extinction memory (see (Abraham et al 2014a) for a recent review). One, or many, of thes e factors could potentially be involved in the mechanisms by which acute exercise strengthens fear extinction learning and reduces the relapse of fear. Three main dopaminergic pathways within the central nervous system are 1) the mesocortical pathway orig inating in the ventral tegmental area (VTA) and terminating in the PFC, 2) the mesolimbic pathway originating in the VTA and terminating in the nucleus accumbens (NA), and 3) the nigrostriatal pathway originating in the substantia nigra (SN) and terminatin g in the dorsal striatum (DS). Of course, midbrain DA neurons project to many other regions potentially involved in fear extinction, including the hippocampus and AMG. Recruitment of one or more of these DA pathways during acute exercise could potentially strengthen extinction memory. DA systems could be recruited during exercise in order to promote locomotor activity or instrumental / goal directed processes such as learning to run or choosing to run, or could be recruited due to the rewarding effects of e xercise (Knab & Lightfoot 2010) Studies investigating the effects of exercise on extracellular DA have utilized microdialysis during forced treadmill training. These studies indicate that forced exercise increases extracellular DA in various brain regions including the hippocampus (Goekint et al 2012) hypothalamus (Ishiwata et al 2001) and striatum (Meeusen et al 1997) T he effect of voluntary exercise on DA efflux remains unknown. Studies using immediate early genes to indirectly assess activity of DA cell bodies suggest that both forced and voluntary exercise activate midbrain DA neurons (Herrera

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5 et al 2016) an observat ion consistent with neural adaptations in the striatum suggestive of repeated DA activity during voluntary exercise (Clark et al 2014, Foley & Fleshner 2008, Greenwood et al 2011, Herrera et al 2016, Meeusen & De Me irleir 1995) The fact that exercise recruits DA systems allows the possibility that DA could be involved in the mechanisms by which acute exercise augments fear extinction. The role of DA in fear extinction, however, is far from resolved (reviewed in (Abraham et al 2014b, de la Mora et al 2010, Singewald et al 2015) A study using fast scan cyclic voltammetry revealed that phasic DA release in the nucleus accumbens core is initially suppressed by presentation of a fear evoking CS during extinction, however; as extinction progresses and, presumably, as a prediction error occurs due to the absence of the expected US, phasic DA release in the NAc core increases (Badrinarayan et al 2012) These data suggest that DA systems are recruited during fear extinction, and allow the possibility that DA signaling could be involved in fear extinction lear ning. Other studies suggest that augmenting DA signaling can enhance fear extinction. Increasing extracellular DA by blocking DA re uptake immediately following extinction enhances consolidation of fear extinction memory in mice (Abraham et al 2012) Human carriers of a polymorphism in the DA transporter gene, DAT1, which is predominantly expressed in the striatum and confers enha nced phasic DA release, show faster learning rates of fear extinction (Raczka et al 2011) Additionally, systemic administration of L DOPA during the acquisition or consolidation phase of fear extinction learning r educes spontaneous recovery and reinstatement of conditioned fear in both mice & humans (Haaker et al 2013) Importantly, the reduction in fear relapse produced by L DOPA is similar to that observed following acute exercise (Mika et al and Figure 1). Despite these promising results, other studies report impairments (Borowski & Kokkinidis 1998, Mueller et al 2009) or no effect (Carma ck et al 2010, Fiorenza et al 2012) of systemic DA manipulations on fear extinction. Inconsistent results could at least partly be due to differences in specific DA receptors manipulated, and/or the inability of pharmacological approaches to target specif ic neural circuits. Consistent with the former possibility, systemic administration of a D1 like receptor agonist SKF 81297 during either the acquisition or consolidation phases of fear extinction learning enhances recall of both contextual and cued fear e xtinction (Abraham et al 2016) In contrast, systemic administration of the DA 2

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6 (D2) like receptor agonist, quinpirole, partially blocks extinction memory, and systemic administration of the D 2 antagonist, sulpi ride, facilitates fear extinction memory (Ponnusa my et al 2005) These data suggest opposing roles of D1 and D2 like DA receptors in fear extinction. However, blockade of the D2 receptor by microinjection of haloperidol directly into the NAc during the acquisition of fear extinction was reported to imp air fear extinction learning (Holtzman Assif et al 2010b) Thus, the specific roles of various DA receptors in fear extinction likely depend on the brain region targeted. Indeed, one study reported that DA in the hippocampus can enhance fear extinction (Fiorenza et al 2012) while another indicated that DA in the IL inhibits the circuitry supporting fear extinction memory through a mechanism involving D 1 receptors (Hitora Imamura et al 2015) We hypothesize that acute exercise immediately following fear extinction learning, during the consolidation period, will modulate fear extinction memory in such a way to enhance fear extinction memory and block fear renewal. Further, w e hypothesize that nigrostriatal DA is involved in this process and thus, we will be able to mimic the effects of exercise by activating nigrostriatal DA To test these hypothesizes, we used a design that allowed male and female rats to run on voluntary running wheels immediately following either auditory or contextual f ear extinction. In a separate set of experiments, we used G q DREADD to induce DA phasic release during fear extinction or a D1 receptor agonist to activate the D1 receptor in the DS.

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7 CHAPTER I I ACUTE EXERCISE ENHANCES THE CONSOLIDATION OF FEAR EXTINCTI ON MEMORY AND REDUCES CONDITIONED FEAR RELAPSE IN A SEX DEPENDENT MANNER Running title: Exercise and fear extinction Courtney A. Bouchet 1 Brian A. Lloyd 1 Esteban C. Loetz 1 Caroline E. Farmer 1 Mykola Ostrovskyy 1 Natalie Haddad 1 Rebecca M. Foright 2 Be njamin N. Greenwood 1 1 University of Colorado, Denver. Department of Psychology 2 University of Colorado, Denver. School of Medicine, Anschutz Medical Campus. *Corresponding author Keywords: wheel running, fear conditioning, fear renewal, anxiety Accepted w ith revisions: Learning and Memory. 3/10/17 Abstract Fear extinction based exposure therapy is the most common behavioral therapy for anxiety and trauma related disorders, but fear extinction memories are labile and fear tends to return even after success ful extinction. The relapse of fear contributes to the poor long term efficacy of exposure therapy. A single session of voluntary exercise can enhance the acquisition and consolidation of fear extinction in male rats, but the effects of exercise on relapse of fear after extinction is not well understood. Here, we characterized the effects of 2 h of voluntary exercise during the consolidation phase of contextual or auditory fear extinction learning on long term fear extinction memory and renewal in adult, ma le and female, Long Evans rats. Results indicate that exercise enhances consolidation of fear extinction memory and reduces fear relapse after extinction in a sex dependent manner. These data suggest that brief bouts of exercise could be used as an augment ation strategy for exposure therapy, even in previously sedentary subjects. Fear memories of discrete cues, rather than of contextual ones, may be most susceptible to exercise augmented extinction, especially in males. Additionally, exercise seems to have the biggest impact on fear relapse phenomena, even if fear extinction memories themselves are only minimally enhanced.

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8 Introduction Fear conditioning is an associative learning phenomenon during which a previously innocuous cue (conditioned stimulus; CS) c omes to elicit an emotional fear response because of its association with an aversive stimulus (unconditioned stimulus; US). Mechanisms underlying fear conditioning are thought to go awry in anxiety and trauma related disorders (Elzinga & Bremner 2002) Fear extinction, the decay of a fear response to a CS following repeated presentation of the fear evoking CS in the absence of the aversive US (Pavlov 1927 ) forms the basis of exposure therapy, the behavioral therapy of choice for anxiety and trauma relate d disorders. Unfortunately, extinction memories are labile and susceptible to relapse phenomena such as renewal (Bouton 1988) spontaneous recovery (Pavlov 19 27 ) and reinstatement (Rescorla & Heth 1975) ; which together contribute to the poor long term efficacy of exposure therapy (Boschen et al 2009, Neumann & Kitlertsirivatana 2010) Identification of nove l means to render fear extinction memories impervious to relapse is of utmost importance to mental health. One factor that enhances many learning and memory processes and could potentially modulate fear extinction is physical activity. Despite the well es tablished benefits of chronic exercise on cognition and learning processes (Cassilhas et al 2015, Cotman & Berchtold 2002, Hillman et al 2014, Hillman et al 2003, Prakash et al 2015) the lingering changes in the b rain produced by habitual exercise are not sufficient to enhance fear extinction (Greenwood et al 2009) Moreover, maintenance of chronic exercise is essential for use in a clinical setting, yet maintaining regular exercise is a constant challenge and long term exercise adherence rates are low (Dishman 1982, Hogg et al 2012, Zuckoff 2012) even when initial motivation is high (Van Roie et al 2015) In contrast to weeks of repeated exercise, individuals may be more likely to adhere to a re commendation of a relatively few sessions of exercise. A few acute bouts of exercise could be more practical to implement in clinical settings than repeated exercise. Even so, prior research investigating the effects of a single, recent bout of exercise (a cute exercise) as a treatment for anxiety or trauma related disorders is limited. Investigating the effects of acute exercise within the context of fear extinction learning and memory in an animal model is an initial step toward utilization of acute exerci se in a clinical setting.

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9 Fear extinction learning incorporates two critical phases: the acquisition phase and the consolidation phase. During the acquisition phase, the association between the fear eliciting CS and the lack of the predicted aversive eve nt is initially encoded. Later, during the consolidation phase, molecular processes take place in the neural circuits responsible for forming the long term memory of fear extinction. Previous research investigating the effects of chronic exercise on subseq uent contextual (Greenwood et al 2009) or auditory (Dubreucq et al 2015) fear extinction report negative results, possibly due to the inability of prior chronic exercise to target a distinct extinction learning phase. Exercise initiates a plethora of physiological and neurobiological events; the timing of which in relation to a specific phase of fear extinction learning could be critical for the ability of exercise to modula te fear extinction. There is evidence that acute exercise, within a small temporal window in relation to fear extinction learning, can modulate later fear extinction memory and relapse. Siette et al. (2014) reported that previously sedentary male rats al lowed 3 h of voluntary exercise either immediately before or after contextual fear extinction displayed improved retention of fear extinction memory when tested the next day. However, if rats were allowed the same 3 h of acute exercise 6 h after fear extin ction, presumably after the consolidation phase, exercise had no effect on extinction memory retention (Siette et al 2014) A pilot study in humans revealed a similar effect. Powers and colleagues (Powers et al 2015) allowed mostly female patients suffering from post traumatic stress disorder (PTSD) to engage in moderate intensity treadmill exercise for 30 min immediately prior to each of 12 sessions of exposure therapy. Those patients who exercised prior to exposure therapy showed significant improvement of PTSD symptoms immediately following the final therapeutic session, relative to patients who had 90 min of exposure therapy without exerci se. Further, Mika et al. (2015) found that male rats allowed to run in wheels during the acquisition of auditory fear extinction, relative to rats extinguished in locked wheels, demonstrated reduced fear when re exposed to the CS one wk later in a novel co ntext. Although prior data suggest that acute exercise can enhance the acquisition or consolidation of fear extinction in such a way as to reduce fear relapse, critical questions remain unanswered. Siette et al. (2014) did not investigate the effects of a cute exercise on relapse of contextual fear conditioning following extinction, and the experimental design used in Mika et al. (2015) did not allow the differentiation of the

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10 effects of acute exercise on auditory fear extinction memory from its relapse. Fu rther, none of these previous experiments have investigated the effects of voluntary wheel running on fear extinction learning, memory and relapse in female rats. This is a critical oversight, as females can have impaired fear extinction depending on their estrus cycle phase (Milad et al 2009) and, relative to males, have a higher incidence of anxiety (McLean et al 2011) and are more likely to develop PTSD (Breslau et al 1997) The goal of the current studies was to further characterize effects of acute, voluntary exercise on consolidation and relapse of c ontextual and auditory conditioned fear in both males and cycling females. It is hypothesized that acute exercise during the consolidation phase of fear extinction learning will enhance fear extinction memory and reduce later fear relapse in both sexes. Ma terials and Methods Subjects Adult, male (N=71, p~54 on date of arrival) or female ( N=40 p~54 on date of arrival) Long Evans rats were used for all experiments. Animals were pair housed in ventilated rat cages (45 W x 25.2 D x 14.7 H cm) with ad libitum a ccess to food (standard Rat Chow) and water. The housing room was kept on a 12 12 h light dark cycle with lights on from 0700 to 1900 and temperature was maintained at 25 All procedures took place in the University of Colorado Denver Auraria campus ani mal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care Animals were allowed 1 wk to acclimate to their housing conditions before start of experimental manipulations. All rats were gently handled once daily du ring the last 4 days of this acclimation week. Care was taken to minimize pain and all protocols were approved by the University of Colorado Denver Institutional Animal Care and Use Committee. Estrus Cycle Monitoring After 1 wk of acclimation to the vivar ium, vaginal lavages were conducted every 24 h for 5 consecutive days in order to establish cycle and habituate rats to lavage. Briefly, a sterile, blunt tip eyedropper was used to flush the vagina with approximately 0.5 mL sterile filtered 0.2% PBS Brij s olution (Brij 35 Solution 30%; Sigma, B4184) using the ventral method (Becker et al 2005) to obtain vaginal epithelial cells. The fluid collected from lavages was transferred to a microscope slide and tissue was an alyzed under

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11 bright field at 400X (Olympus BX53). A single lavage was again collected ~ 6 h prior to fear extinction learning, immediately following the fear renewal test, and immediately following the long term extinction memory test to confirm phase of e strus cycle on these days. Estrus cycle phase was determined by the morphology of the cells collected during vaginal lavages as previously described (Cora et al 2015) Briefly, proestrus consists of small, round, o ften clumped mononucleated cells of relatively the same size (Figure 5F), the estrus phase is predominated by anucleated keratinized epithelial cells (Figure 5G), metestrus consists of anucleated keratinized epithelial cells interspersed with leukocytes (F igure 5H), and diestrus is predominated by leukocytes and nucleated epithelial cells with a decrease in anucleated keratinized epithelial cells (Figure 5I). Wheel R unning At the start of all experiments, rats were placed into pre assigned running wheels ( 1.1 m circumference; Lafayette Instruments, Lafayette, IN, USA) for the duration of the dark (active) cycle for 4 consecutive nights. On alternating nights, the wheels were rendered immobile so that, in total, rats had 2 nights of voluntary running in mobi le running wheels and 2 nights in immobile (locked) running wheels. The purpose of this familiarization procedure was two fold: (1) to ensure that both the mobile and locked wheel environments were equally familiar and (2) to increase running behavior afte r extinction, as in our experience rats lacking prior experience with a running wheel run minimally. All wheel running activity was recorded with Activity Wheel Monitor software (Lafayette Instruments; Lafayette, IN, USA). Behavioral Analyses Fear Conditi oning Between 0800 and 1100, rats were placed into custom, rectangular conditioning chambers (20"W x 10"D x 12"H; Context A) with a shock grid floor (Coulbourn Instruments, Allentown, PA) housed inside individual sound attenuating cabinets. Rats were tr ansported to Context A in their home cages. A fan located near the floor of the cabinets provided ventilation and background noise and bright white lights illuminated the chambers. For contextual fear conditioning (Experiment 1), rats were allowed 5 min to explore the context, after which 3 foot shock US (1s, 0.8mA) were delivered with a 1 min ITI. For auditory fear conditioning (Experiments 2 and 3), rats were allowed to explore the context for 3 min, followed by 4

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12 exposures to an auditory CS (10s, 80dB, 2 kH), each co terminating with a 1s, 0.08 mA foot shock US delivered on a 1 min ITI. Auditory stimuli and foot shocks were delivered through Coulbourn tone generators and shock scramblers controlled via Noldus EthoVision XT software (Tacoma, WA) through a c ustom interface. All rats remained in the conditioning chamber for 1 min after the last shock before being transported back to their home cages. Chambers were cleaned with water between rats. Freezing behavior, an innate fear response, was defined as the a bsence of all movement except that required for respiration (Fanselow 1 980) and was used as the measure of fear in all behavior tests. Fear Extinction Each fear extinction training session took place near the start of the active (dark) cycle, to maximize running behavior of rats in the Mobile group (Greenwood et al 2011) Rats exposed to contextual fear extinction (Experiment 1) were placed into context A for 15 min in the absence of the shock US. All transport, lighting, and cleaning conditions were identical to fear conditioning. Rats exposed to auditory fear extinction (Experiments 2 and 3) were placed into a novel Context B that was either a custom Plexiglas rectangular chamber (15" W x 15" D x 20" H) with a textured floor or a custom Plexiglas triangular chamber (15 sides x 20 H) with a smooth floor. Rectangular and triangular Context B chambers were counterbalanced so half the rats were exposed to fear extinction training in the rectangular chambers and the other half in the triangular chambers. Context B was housed in the sam e sound attenuating cabinets used for conditioning, but all other contextual features and discrete cues differed between contexts. Rats were transported to the sound attenuating chambers, which included vanilla scent, in their assigned Context B custom Ple xiglas chamber. The fan within the chamber was turned off and the room was dimly lit by a lamp outside of the sound attenuating chambers. Context B was cleaned with 10% ethanol between rats. After a 3 min exploration period, the auditory CS was administere d 30 times (1 min ITI) in the absence of the foot shock US. Similar auditory extinction parameters have been used previously in Long Evans rats (Maren 2014)

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13 Fear Renewal Test Between 0800 1100, rats exposed to auditory fear conditioning and extinction were re exposed to the auditory CS in either the same context used for extinction training (Context B; Same) or a novel Context C (Different). Rats were assigned to Same or Different contexts based on freezing levels during fear extinction, such that freezing levels during extinction were balanced between groups. Rats assigned to the Di fferent group were transported to Context C in a novel inner Plexiglass chamber, so that rats extinguished in the square Plexiglass chamber were now placed into the triangle, and vise versa. Context C consisted of a raspberry scent, red box lights, a fan n ear the top of the behavior cabinets was turned on, and chambers were cleaned with 1% acetic acid between tests. After a 3 min exploration period, the auditory CS was presented 4 times (1 min ITI) in the absence of the foot shock US. Because fear renewal i s the return of fear in contexts different from where fear extinction was learned (Bouton & Ricker 1994a) the difference in freezing between the Same and Different contexts was considered fear renewal. Post Extinc tion Wheel Running Immediately following contextual or auditory fear extinction, rats were transferred to their familiar running wheels that were either rendered immobile (Locked) or freely mobile (Mobile). Rats were assigned to Locked or Mobile conditions based on freezing levels during fear conditioning, such that freezing levels during conditioning were balanced between wheel assignments. Rats were returned to their home cages following 2 h of exposure to the locked or mobile wheels. Rats in the "No Exti nction" group were placed in their assigned Locked or Mobile wheels for 2 h / night, but were not exposed to fear extinction. Procedures E xperiment 1: Effects of Acute Exercise on E xtinction o f Contextual Fear C onditioning A timeline for the experiment is shown in Figure 1A. Prior to fear conditioning, rats (N=32 males) were exposed to the running wheel familiarization procedure, so each rat spent 2 active cycles in mobile,

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14 Figure 1 Experimental design. (A) All rats were placed into unlocked and locked wheels on alternating nights for 4 nights to equally familiarize rats with mobile and locked running wheels. Three days following the last running opportunity, rats were exposed to contextual fear conditioning in Context A by admi nistering an un signaled 1 s, 0.8 mA foot shock unconditioned stimulus (US) 3 times with a 1 min inter trial interval (ITI). The following evening, all rats were placed back into the familiar conditioning Context A for 15 min immediately followed by 2 h in either locked or mobile running wheels. Contextual fear extinction followed by 2 h in locked or mobile wheels was repeated the following 2 evenings. One week following the last extinction trial, rats were placed into the conditioning Context A to assess l ong term fear extinction memory. (B) All rats were placed into mobile and locked wheels on alternating nights for 4 nights to equally familiarize rats with mobile and locked running wheels. Three days following the last running opportunity, rats were condi tioned to fear a tone in Context A by co terminating a 10 s, 80 dB tone conditioned stimulus (CS) with a 1 s, 0.8 mA foot shock US four times with a 1 min inter trial interval. The following evening, all rats were placed into a novel extinction Context B a nd exposed to the auditory CS 30 times in the absence of the foothsock US (1 min ITI). Immediately following fear extinction, rats were placed into either mobile or locked running wheels for 2 h. The fear extinction and running procedure was repeated the f ollowing evening, for a total of 2 nights of fear extinction following by either mobile or locked conditions. The morning after the second fear extinction trial, half of the rats were placed into the familiar, extinction Context B and the other half were p laced into a novel Context C to test for fear renewal. (C). Experiment 2 was repeated with female rats with extinction consisting of 20 CS presentations instead of the 30 CS presentations as in experiment 2. One week following the fear renewal test, rats w ere placed back into Context B to assess long term fear extinction memory. voluntary running wheels alternating with 2 active cycles in the same locked running wheel. Three days following the second running opportunity, rats underwent contextual fear cond itioning in Context A. During the following 3 evenings, immediately prior to the dark (active) cycle, rats were re exposed to Context A for 15 min in the absence of shock, in order to extinguish contextual fear. Each of the three, 15 min extinction traini ng sessions was immediately followed by 2 h in familiar Locked (n =11) or Mobile (n = 11) running wheels, so that rats in the Mobile group ran in wheels during the consolidation phase of contextual

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15 fear extinction. Rats were again exposed to Context A 1 wk following the third contextual fear extinction training session in order to assess the effects of post extinction wheel running on long term extinction memory / spontaneous recovery of fear. An additional cohort of rats were conditioned and placed into th eir assigned mobile or locked running wheels at the same time as the other rats, but were not exposed to fear extinction training. This subset of Locked (n=5) or Mobile (n=5) rats was placed into Context A on the day of the long term extinction memory / sp ontaneous recovery test to assess potential effects of running in the absence of fear extinction training on subsequent freezing behavior. Experiment 2: Effects of Acute Exercise on E xtinction a nd Renewal of Auditory Conditioned Fear in M ales A timeline fo r the experiment is shown in Figure 1B. Three days following the second familiarization running opportunity, rats (N=39 males) underwent auditory fear conditioning in Context A. The following 2 evenings, immediately prior to the dark (active) cycle, rats w ere exposed to auditory fear extinction training in Context B immediately followed by placement for 2 h into their familiar Locked (n=20) or Mobile (n=19) running wheels, so that rats assigned to the Mobile condition had the opportunity to run during the f ear extinction consolidation period. The morning after the second fear extinction training session, rats were again exposed to the CS either in the extinction Context B (Locked Same n = 10; Mobile Same n = 10) or in a novel Context C (Locked Different n = 10; Mobile Different n = 9) to assess fear renewal. Experiment 3: Effects of Acute Exercise on Extinction and Renewal of Auditory Fear Conditioning in F emales A timeline for the experiment is shown in Figure 1C. Experiment 2 was repeated except cycling f emales (n = 40 females) were used instead of males. Since males and females have been reported to similarly acquire auditory fear conditioning (Maren et al 1994) female rats in experiment 3 were administered the same number of CS US pairings during conditioning as male rats in experiment 2. Pilot studies revealed that females displayed less freezing behavior during fear extinction memory tests than males. Therefore, to help avoid floor effects on freezing, females were exposed to 20 CS presentations during fear extinction training. A long term fear extinction memory / spontaneous recovery test was added 1 wk after the fear renewal test.

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16 All behavioral tests were recorded with overhead cameras and freezing was scor ed both by multiple experimenters blind to the experimental conditions of the animals and by automated behavioral analyses software (Noldus Ethovision XT) during both the CS and the ITIs. Because regularly scheduled ITIs can become part of the CS, and anal yses indicated a lack of differential effects of exercise on freezing during ITIs and CS, freezing during each CS and subsequent ITI were combined and expressed as freezing during a trial, as in prior work (Fitzgera ld et al 2015, Goode et al 2015, Mika et al 2015) Statistical Analysis Running distances were calculated using the Lafayette Instruments Activity Wheel Monitor data management add on for Microsoft Excel, then total running distances were analyzed using A NOVA. Percent time spent freezing was calculated by averaging freezing data from individual experimenters blind to treatment condition of the animals with immobility times obtained from Noldus Ethovision XT. Pre shock freezing for each test was averaged an d group differences were analyzed with ANOVA. Average freezing across minutes during conditioning (Experiment 1) or across trials during conditioning and extinction (Experiments 2 and 3) were analyzed using repeated measures ANOVA. Freezing scores within e ach extinction session in Experiment 1 were averaged and group differences compared using ANOVA. For renewal, freezing across trails was averaged and compared using 2 way ANOVA with Exercise and Context conditions as factors. For long term memory / spontan eous recovery tests, overall group means were analyzed using ANOVA. Estrus phase was added as a factor in Experiment 3; therefore, 2 way repeated measures ANOVAs (Exercise x Estrus Phase) were used to analyze freezing during extinction days, and a 3 way AN OVA (Exercise x Context x Estrus Phase) was used to compare group means during the renewal test. Simple regressions were run to determine whether running distance during the running familiarization phase or after fear extinction predicted freezing levels d uring subsequent behavioral tests. Fisher's least significant differences post hoc analyses were performed when appropriate. Group differences were considered different when p 0.05.

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17 Results E xperiment 1: Exercise During Consolidation of Contextual Fear E xtinction Improves Long Term Memory of Fear Extinction in M ales Rats used in experiment 1 ran equal amounts during the running familiarization period regardless of subsequent exposure to fear extinction (Figure 2A). Rats exhibited low levels of freezing p rior to the first US during contextual fear conditioning (pre; Figure 3A). Freezing levels increased throughout US presentations (F(2,56) = 62.25, p < 0.0001) and did not differ between rats subsequently assigned to Locked or Mobile conditions, nor between No Extinction (n = 10) and Extinction ( Figure 3A). There was indication of a trend toward a positive correlation between average distance run during the wheel running familiarization period and freezing during fear conditioning (R = 0.299, F(1,29) = 2.86; p = 0.1; Figure 6A); an observation that would be consistent with prior reports indicating that wheel running enhances contextual fear conditioning (Burghardt et al 2006, Greenwood et al 2009, Kohman et al 2012, Va n Hoomissen et al 2004) Figure 2 Running distances. Female rats in experiment 3 (Exp 3) ran more than male rats used in experiment 1 (Exp 1) and experiment 2 (Exp 2), both (A) during the running familiarization phase and (B) d uring the 2 h running bouts after fear extinction training. Data displayed represent mean SEM. *** Exp 3 different from all other groups ( p < 0.0001); Exp 3 day 2 different from Exp 3 day 1 (p < 0.01); # Exp 2 day 2 different from Exp 2 day 1 (p < 0.01 ); Exp 1 Extinction day 2 different from Exp 1 Extinction day 1 (p < 0.01). Freezing levels during the first fear extinction training session did not differ between rats subsequently placed into Locked or Mobile wheels (Figure 3B), and distance run du ring the running familiarization period did not predict the amount of freezing during the first extinction training session. Running distances during the 2 h following the first and subsequent fear extinction training sessions are shown in Figure 2B. Exerc ise immediately following contextual fear extinction training had no effect on fear extinction

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18 memory. Rats in Locked and Mobile conditions displayed similar levels of freezing and within session extinction (not shown) during the second (Figure 3C) and thi rd (Figure 3D) fear extinction training sessions, which also served as fear extinction memory tests. Distance run during the consolidation of the first and second contextual fear extinction sessions was; however, negatively correlated to freezing levels du ring the second and third fear extinction memory tests (R = 0.60; F(1,9) = 5.07, p = 0.05; Figure 6C). Rats were again re exposed to Context A 1 wk after the third fear extinction training session in order to determine if post extinction wheel running fac ilitated long term memory of contextual fear extinction. Because freezing levels a week after fear extinction training are modulated both by the strength of the extinction memory and the spontaneous recovery of fear (Bouton 1993, Ponnusamy et al 2016) this memory test at day 10 represented both a long term extinction memory test and a test of spontaneous recovery. However, freezing levels displayed by all rats exposed to fear extinction training remained at low lev els during the extinction memory test at day 10 (Figure 3E), indicating minimal spontaneous recovery. The lack of spontaneous recovery could be due to rats receiving 3 sessions of fear extinction training, thus producing a strong fear extinction memory. De spite this, rats that ran after fear extinction displayed significantly less fear during the long term extinction memory test than rats placed into locked wheels after fear extinction ( p < 0.05); further, this effect of exercise was dependent on exercise b eing contingent with fear extinction (interaction between Exercise and Extinction: F(1,28) = 6.47, p = 0.01; Figure 3E). Rats that ran similar amounts (Figure 2) but were not exposed to fear extinction (Mobile No Ext), displayed levels of freezing greater than that expressed by rats exposed to extinction (p < 0.05), and no different from Locked rats not exposed to fear extinction (Locked No Ext, Figure 3E). These data indicate that exercise during the consolidation of contextual fear extinction facilitates retrieval of fear extinction memory at long term time points.

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19 Figure 3 Running after contextual fear extinction, but not running alone, enhances long term extinction memory. (A) Freezing levels increased throughout US presentati ons during contextual fear conditioning (3, 1 s, 0.8mA foot shocks, 1 min ITI), regardless of subsequent group assignments. (B) There was no significant difference in freezing during the first fear extinction training session 1 day after conditioning betwe en rats subsequently placed in either locked (Locked) or mobile (Mobile) running wheels. (C D) Wheel running during the consolidation of contextual fear extinction had no impact on fear extinction memory during memory tests on days 2 and 3. (E) Rats that r an after extinction exhibited significantly less fear than those placed into locked wheels after extinction. Both Locked and Mobile rats that went through fear extinction (Ext) froze significantly less than those that were not exposed to fear extinction (N o Ext). Data displayed represent mean SEM; p < 0.01. *** p < 0.0001 Experiment 2: Exercise During Consolidation of Auditory Fear Extinction Improves F ear Extinction M emor y and Reduces Fear Renewal in M ales Rats exhibited low levels of freezing prior to the first CS US pairing (pre; Figure 4A). Freezing levels increased throughout conditioning (F(3,111) = 58.20, p < 0.0001) and did not differ between rats subsequently assigned to Locked or Mobile conditions. Rats in Experiment 2 ran distances similar to rats in Experiment 1 during the running familiarization period (Figure 2A). Interestingly, the average running distance during the wheel running familiarization period negatively correlated with freezing levels during fear conditioning (R = 0.48; F(1, 36) = 10.699; p < 0.01; Figure 6A). These data suggest that unlike chronic wheel running which has been reported to have no effect on the acquisition of auditory fear conditioning (Baruch et al 2004) brief access to running wheels could interfere with auditory fear conditioning lear ning. One wheel did not record running distance during the wheel running familiarization period, so this rat was omitted from this correlational analysis. All rats exhibited negligible amounts of freezing prior to the first CS during day 1 of auditory fea r

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20 extinction training in Context B, indicating that minimal fear transferred from the conditioning Context A to the fear extinction Context B (not shown). Freezing levels decreased across trials (F(5,185) = 147.12, p 0.0001) and did not differ between rats subsequently placed into Locked or Mobile wheels (Figure 4B). The average running distance during the familiarization period negatively correlated with freezing levels during the first 4 trials of the first fear ex tinction training session (R = 0.34; F(1,36) = 4.60; p < 0.05; Figure 6B), consistent with the negative relationship between running distance and the initial acquisition of auditory fear conditioning (Figure 6A). Running distances during the 2 h followin g the auditory fear extinction training sessions are shown in Figure 2B. Post extinction wheel running facilitated fear extinction memory. When re exposed to the extinction Context B the evening following post fear extinction running, Mobile rats displayed significantly less freezing than locked rats across the duration of the fear extinction training session, which also served as a fear extinction memory test (F(1,37) = 6.10, p = 0.01; Figure 4C). Locked and Mobile rats displayed equivalent within session extinction, as freezing levels decreased over time in both groups (F(5,185) = 43.67; p < 0.0001; Figure 4C). Distance run after the first auditory fear extinction training session did not predict levels of freezing during the second fear extinction trainin g session. Rats were again placed into their locked or mobile wheels for 2 h following the second fear extinction training session. Then, to determine the effect of wheel running during consolidation of auditory fear extinction on renewal of auditory con ditioned fear, rats were re exposed to the extinguished CS in either Context B (Same) or a novel Context C (Different) the following day. Locked rats displayed typical fear renewal, as Locked rats re exposed to the extinguished CS in a context different fr om where fear extinction was learned exhibited more fear than Locked rats re exposed to the CS in the same extinction context (p < 0.05). Rats allowed to exercise during the consolidation of auditory fear extinction; however, were protected from fear renew al (interaction between Exercise and Context: F(1,35) = 5.11, p < 0.05). Distance run after the second fear extinction training session did not predict the levels of freezing in either context during the renewal test on day 3.

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21 Figure 4 Exercise enhances auditory fear extinction memory and blocks renewal in males. (A) Fear increased throughout conditioning trials (4 pairings of 10s, 80dB, 2 KHz auditory CS co terminating with 1s, 0.8mA foot shock US), and was not different between rats subsequently assigned to locked (Locked) or mobile (Mobile) wheel conditions. (B) Rats were placed into a novel extinction context and exposed to 30 CS presentations (I min ITI) in the absence of the US. Freezing decreased throughout the extinction t rials and did not differ between rats subsequently assigned to locked or mobile conditions. (C) When re exposed to the CS in the extinction context, rats in the Mobile group displayed significantly less fear behavior than those in the Locked group. (D) Loc ked rats placed into a context different from that in which extinction was learned (Different) displayed typical fear renewal. Rats placed into Mobile wheels after fear extinction were protected from fear renewal. Data displayed represent mean SEM; p < 0.01 Experiment 3: Exercise during consolidation of auditory fear extinction fails to improve fear extinction memory or reduce fear renewal in females Analyses of the freezing data during the renewal test on day 3 was consistent with prior work (Lebron Milad & Milad 2012) suggesting that estrus phase during fear extinction learning modulates later retrieval of fear extinction. Specifically, levels of freezing expressed during the renewal test by rats that were in metestrus and diestrus (Met&Di; n = 17) during initial fear extinction learning on extinction day 1 differed from rats that were in proestrus and estrus (Pro&E; n = 23) on extinction day 1. In contrast, estrus phase during conditioning or renewal had no impact on freezing levels during any behavioral test. Therefore, females were separated into groups according to phase of the estrus cycle on the first day of fear extinction training (Extinction Day 1). Photomicrographs of vaginal cytology defining the e strus phases are shown in Figure 5F I. Overall distance run on the second wheel running familiarization day increased significantly from

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22 the first wheel familiarization opportunity (main effect of time: F(1,105) = 54.297; p < 0.0001), with f emale rats ru nning significantly further than male rats (main effect of experiment: F(3,105) = 17.456; p < 0.0001; Figure 2A). Unlike the males, the distance run during the wheel running familiarization period did not predict levels of freezing during fear conditioning in female rats. Rats exhibited low levels of fear prior to the first CS US pairing during conditioning (pre; Figure 5A). Freezing levels increased throughout CS US pairings (F(3,108)= 45.63, p < 0.0001; Figure 5A) for all rats regardless of subsequent est rus cycle phase or subsequent wheel assignment. Freezing levels in all groups were negligible prior to the first CS during day 1 of auditory fear extinction training in Context B (data not shown). Freezing levels decreased across trials (F(4,144)= 76.28, p < 0.0001; Figure 5B) and did not differ between estrus phases or between rats subsequently placed into Locked or Mobile wheels. The average running distance during wheel familiarization did not predict levels of freezing during the first fear extinction training session. Rats were placed into either locked or mobile wheels immediately following fear extinction. Post fear extinction running distances did not differ between rats at different stages of the estrus cycle, so running distances were averaged a cross all females (Figure 2B). Female rats ran significantly more than male rats during the 2 h running opportunities following fear extinction (F(3,48) = 29.09; p < 0.0001). Additionally, female rats running distance escalated across nights (p < 0.05), wh ereas the males maintained running distances across subsequent post extinction running opportunities (Figure 2B). Despite the greater distances run by females, post extinction wheel running did not facilitate auditory fear extinction memory in females. Whe n re exposed to the auditory CS in the extinction Context B the evening following post fear extinction running, freezing levels were similar between groups and all rats demonstrated similar within session extinction (F(4, 144)= 8.91, p < 0.0001; Figure 5C) Freezing within the first few trials of the second fear extinction training session is considered extinction memory retrieval (Bukalo et al 2015, Do Monte et al 2015) Thus, to verify that post extinction running did not impact fear extinction memory, freezing during the first 4 trials of extinction day 2 was analyzed separately. No differences between groups were observed during the first 4 trials (data not shown). Distance run after the first auditory fear

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23 extin ction training session did not predict levels of freezing during the second fear extinction training session. Rats in the Mobile group were again allowed to run for 2 h following the second fear extinction training session. Then, to determine the effect of wheel running during consolidation of auditory fear extinction on renewal of auditory conditioned fear, rats were re exposed to the extinguished CS in either Context B (Sam) or a novel Context C (Different) the following day. Results are shown in Figure 5D. Rats placed into the Different context the next day displayed more freezing behavior than rats placed into the Same context (F(1,32) = 14.40, p < 0.001), indicating renewal of fear in the Different context. Rats in the Met&Di group displayed significa ntly higher freezing levels compared to Pro&E rats (F(1,32) = 6.54, p = 0.01), regardless of context. Moreover, ANOVA revealed an interaction between context and estrus phase (F(1,32) = 5.22, p < 0.05), indicating that rats in the Met&Di group displayed si gnificantly more fear renewal than rats in the Pro&E group. Unlike males, wheel running during the consolidation of auditory fear extinction had no effect on freezing levels during the renewal test in females. Interestingly; though, the average running dis tance after both extinction training sessions negatively correlated with freezing levels in the Same context, but this association just missed significance (R = 0.58, F(1,8) = 4.14, p = 0.07; Figure 6D). No other significant correlations between freezing and running distance were found. Rats were again re exposed to Context B 1 wk after the fear renewal test to determine if post extinction wheel running facilitated long term memory of auditory fear extinction. Neither context on day 3 of testing (Same vs Different), phase of estrus cycle during day 1 of fear extinction training (Met&Di vs Pro&E), nor wheel running (Locked vs Mobile) impacted freezing levels when re exposed to the CS 1 week later (Figure 5E).

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24 Figure 5 Phase of e strus cycle, but not exercise, modulates fear renewal in female rats. Rats were grouped by phase of estrus cycle during fear extinction learning (fear extinction day 1). (A) Fear increased throughout conditioning trials (4 pairings of 10s, 80dB, 2 KHz aud itory CS co terminating with 1s, 0.8mA foot shock US), and was not different between rats subsequently assigned to locked (Locked) or mobile (Mobile) wheel conditions. (B) Rats were placed into a novel extinction context and exposed to 20 CS presentations (1 min ITI) in the absence of the US. Freezing decreased throughout the extinction trials and did not differ between rats in metestrus and diestrus (Met&Di) or proestrus and estrus (Pro&E) phases of the estrus cycle, or subsequently assigned to Locked or M obile conditions. (C) Freezing levels were similar between all groups and all rats demonstrated similar within session extinction regardless of estrus phase or exercise condition. (D) Rats that were in Met&Di during fear extinction Day 1 displayed typical fear renewal when re exposed to the CS in a context different from where extinction was learned (Different). Rats that were in Pro&E during fear extinction Day 1 were protected against the renewal of fear. (E) No differences between groups were observed wh en rats were re exposed to the CS in Context B 1 wk after the fear renewal test. (F) Photomicrograph of vaginal cytology showing keratinized epithelial cells (white arrow) and leukocytes (black arrow) characteristic of metestrus. (G) Photomicrograph of vag inal cytology showing predominately leukocytes (white arrow) and nucleated epithelial cells (black arrow) with a decrease in anucleated keratinized epithelial cells. (H) Photomicrograph of vaginal cytology showing small, round, often clumped mononucleated cells (arrow) of relatively the same size. (I) Photomicrograph of vaginal cytology showing predominately anucleated keratinized epithelial cells. Data displayed represent mean SEM; ** p < 0.001

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25 Figure 6 Correlations between ru nning and freezing levels. (A) Average running distance during running familiarization did not predict freezing during contextual fear conditioning in experiment 1 (Exp 1), but negatively correlated with freezing during auditory fear conditioning in experi ment 2 (Exp 2). (B) The average running distance during running familiarization negatively correlated with freezing during the first 4 tones of fear extinction day 1 in Exp 2. (C) There was a trend for the average running distance following the auditory fe ar extinction sessions to negatively correlate with freezing in the Same context during the fear renewal test in females in experiment 3 (Exp 3). (D) Average running distance after the first and second fear extinction training sessions negatively correlat ed with average freezing levels during the second and third fear extinction training sessions (Exp 1). Discussion Here we report the novel findings that a brief increase in physical activity during the consolidation phase of fear extinction learning can enhance fear extinction and render fear extinction memory resistant to relapse. In males, wheel running during consolidation of contextual fear extinction improved long term memory of fear extinction, recall of which is influenced by spontaneous recovery. Similarly, wheel running during the consolidation of auditory fear extinction improved fear extinction memory and prevented the renewal of fear. In both cases, rats that ran the greatest distance during the consolidation phase of fear extinction learning also tended to have the strongest extinction memory. Exercise, however, only improved fear extinction in males. In females, wheel running had no impact on either fear extinction consolidation or renewal of fear. However, females that ran the greatest dista nce during auditory fear extinction

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26 consolidation did tend to have the strongest fear extinction memory. Interestingly, females that learned fear extinction during proestrus and estrus phases of the estrus cycle were protected against fear renewal. Along w ith prior data (Mika et al 2015, Powers et al 2015, Siette et al 2014) these results suggest that brief exercise bouts could be used as an augmentation strategy for exposure therapy, even in previously sedentary s ubjects. Fear memories of discrete cues, rather than of contextual ones, may be most susceptible to exercise augmented extinction, especially in males. Moreover, exercise seems to have the biggest impact on fear relapse phenomena, even if fear extinction m emories themselves are only minimally enhanced, as is the case with contextual fear extinction. Further research is warranted to determine the effectiveness of exercise augmentation of fear extinction in a clinical setting. In experiment 1, post extincti on exercise only enhanced consolidation of contextual fear extinction when memory was assessed 1 wk after the final extinction training session. This observation differs from that of Siette et al. (2014), who observed that Wistar rats allowed to run during consolidation of contextual fear extinction had improved recall of fear extinction the next day. One difference between our experiment and the Siette et al. (2014) study is that rats in the current experiment had access to running wheels for 2 active cycl es prior to fear conditioning, whereas the rats in the Siette (2014) study were na•ve to running wheels. This prior brief access to running wheels may have made the contextual fear memory more difficult to extinguish. Indeed, only 2 days of wheel running i ncreases BDNF mRNA in the hippocampus (Neeper et al 1995, Neeper et al 1996) and it is well established that wheel running prior to conditioning enhances contextual fear conditioning (Burghardt et al 2006, Greenwood et al 2009, Kohman et al 2012, Van Hoomissen et al 2004) and makes it more difficult to extinguish (Greenwood et al 2009) Nevertheless, we did observe a negative correla tion between the distance run after extinction training sessions 1 and 2 and freezing during the extinction memory tests on days 2 and 3, as well as a significant improvement in contextual fear extinction recall a week later. These observations suggest tha t exercise can enhance the consolidation of contextual fear extinction memory, even if the effect was not large enough to be significantly different from locked controls during the initial memory tests. That exercise enhances recall of contextual fear exti nction memory a week after the third fear extinction training session is especially interesting, as fear expression at long term timepoints can be influenced by a number of factors in addition

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27 to the strength of the fear extinction memory, including genera lization of the original fear memory (Wiltgen & Silva 2007) and the spontaneous recovery of fear (Bouton 1993) Thus, our data suggest that ex ercise during consolidation of contextual fear extinction may render fear extinction memory resistant to processes that tend to increase conditioned fear responding over time, even in the absence of improving short term extinction memory, per se. This effe ct of exercise may be especially relevant to the long term efficacy of extinction based exposure therapy. Chronic wheel running begun after contextual fear conditioning has been reported to reduce conditioned fear responding, even in the absence of fear e xtinction (Akers et al 2014, Ishikawa et al 2016) It was therefore important to determine whether the reduction in freezing observed on day 10 in rats that ran after contextual fear extinction was dependent on whe el running being contingent with the consolidation of contextual fear extinction. Mobile rats not exposed to fear extinction, but that ran an equivalent time and distance to Mobile rats exposed to fear extinction, displayed freezing behavior equal to Locke d rats not exposed to fear extinction (Figure 3E), indicating that brief exercise sessions did not themselves alter the later expression of conditioned freezing. The reduction in freezing displayed by the Mobile rats on Day 10 is therefore dependent on exe rcise being contingent with the consolidation of contextual fear extinction. We have previously reported that wheel running during the acquisition of auditory fear extinction reduces freezing to the CS in a novel context one wk later (Mika et al 2015) The design that was used, however, rendered fear extinction memory indistinguishable from renewal and spontaneous recovery. Experiment 2 thus investigated whether exercise during the consolidation of auditory fear e xtinction could both enhance fear extinction memory and reduce fear renewal. We found that exercise during the consolidation phase of auditory fear extinction both enhances fear extinction memory and blocks fear renewal one day following fear extinction. R ats that ran in the absence of extinction were excluded from this experiment because Experiment 1 revealed that brief running bouts in the absence of fear extinction learning have no effect on freezing (Figure 4). Interestingly, running distance during th e wheel familiarization period prior to conditioning was negatively correlated to freezing during the acquisition of auditory conditioned fear (Figure 6A). Con-

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28 sistent with this observation, distance run during the wheel familiarization period was also nega tively correlated to the strength of the fear conditioning memory assessed by freezing during the first 4 trials of the first fear extinction training session (Figure 6B). While short term exercise has profound effects on the hippocampus (Molteni et al 2002, Neeper et al 1995, Neeper et al 1996) a structure important for contextual fear conditioning (Chang & Liang 2016, Matus Amat et al 2004, Rudy et al 2002) less is known regarding exercise effects on the amygdala, a region critical for the CS US association formed during auditory fear conditioning (Bergstrom 2016, Huff & Rudy 2004) Moreover, the effects of exercise on auditory fe ar conditioning are not as well characterized as contextual fear conditioning (Baruch et al 2004, Falls et al 2010) To our knowledge, no prior research has investigated effects of only a few days of wheel running on auditory fear conditioning. Investigation of this question is warranted given the current results. Rats that ran after the first day of auditory fear extinction displayed significantly less fear during re exposure to the auditory CS the following day. This is the first time, to our knowledge, that brief exercise has been reported to improve the consolidation of auditory fear extinction. Interestingly, the effect of exercise observed on extinction training day 2 was not observed again the next day in the same context. Mobile rats did not freeze any less than Locked rats when re exposed to the CS in the extinction Context B (Same) during the fear renewal test on day 3. This could be because all rats had, by this time, been exposed to 2 prior fear extincti on training sessions, thus enabling Locked rats ample opportunity to learn fear extinction in Context B and reducing the difference between Locked and Mobile groups. Nevertheless, the fear extinction memory strengthened by exercise was resistant to renewal When re exposed to the CS in an environment different from the extinction context, fear renewed in the rats placed into locked, but not mobile, wheels after extinction (Figure 4D). The mechanisms by which brief exercise could enhance the consolidation of fear extinction are unknown, but could involve several factors sensitive to brief exercise sessions, including increases in glucocorticoids, endocannabinoids, glutamatergic, noradrenergic, and / or dopaminergic signaling. Indeed, acute increases in each of these factors can enhance fear extinction (for reviews see (Fitzgerald et al 2014, Myskiw et al 2014, Singewald et al 2015) ), although in some cases the effects on fear relapse is not well established. In our p revious study (Mika et al 2015) we observed that the recall of relapse resistant fear

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29 extinction in rats that ran during fear extinction acquisition was associated with potentiated activity of direct pathway neuro ns in the striatum. This activity was above that which would be expected by locomotor activity or fear memory recall, suggesting that it was the recall of the fear extinction memory and not the reduction in freezing or fear response itself that was driving activation of striatal direct pathway neurons. These data suggest that striatal direct pathway neurons could be involved in the mechanisms by which brief exercise facilities fear extinction learning and reduces relapse. Although striatal direct pathway n eurons are typically considered in the context of locomotor activity, recent technical advances have revealed roles for these neurons in emotional behavior (Kravitz & Kreitzer 2012, Lenz & Lobo 2013) Further resea rch is required to determine how these neurons could interact with other exercise signals and the canonical fear circuitry. Because exercise had a more robust effect on auditory fear extinction compared to contextual fear extinction, we used auditory fea r extinction to determine the effects of brief exercise on fear extinction and relapse in females. Consistent with the literature (Kandasamy et al 2016, Venezia et al 2016) female rats ran significantly more than male rats during both the familiarization and post extinction phases (Figure 2). Despite this vigorous wheel running, exercise neither enhanced the consolidation of auditory fear extinction nor prevented fear renewal in females. These data suggest that exe rcise might be a more useful strategy for augmentation of exposure therapy in males than in females. It is well established that fear extinction learning and memory is dependent on the phase of the estrus / menstrual cycle in which female rats and humans initially learn extinction (Daviu et al 2014, Milad et al 2009) Consistent with these prior data, we observed that female rats that initially learned fear extinction in proestrus and estrus, phases of the estrus c ycle during which levels of estradiol are highest, were protected against fear renewal (Figure 5D). Relatively high levels of estradiol during proestrus and estrus phases could have contributed to this effect. Indeed, women with naturally low or experiment ally reduced estrogen have impaired fear extinction (Graham & Milad 2013, Zeidan et al 2011) and memory deficits present when rats learn fear extinction during phases of low estradiol can be prevented by pre extin ction estrogen receptor agonist, or rescued by post extinction estradiol (Chang et al 2009, Zeidan et al 2011) Activation of estrogen receptor § in the hippocampus 24 h prior to passive avoidance learning can incr ease

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30 the generalization of the memory of the context in which passive avoidance was learned to a neutral context (Lynch et al 2014, Lynch et al 2016) It is possible that high estrogen levels during fear extinction learning could similarly promote later generalization of the extinction memory, therefore reducing the contextual dependency of fear extinction. Collectively, the current data suggest that brief exercise sessions could be a useful strategy to augment tr eatment for anxiety and trauma related disorders to help prevent relapse. This set of experiments further emphasizes the importance of investigating manipulations in females in addition to males, as we found that exercise differentially impacts auditory f ear extinction learning and memory in male and female rats. These experiments expand our knowledge of factors able to modulate fear extinction learning and memory and provide building blocks upon which to further characterize the mechanisms by which exerci se modulates fear extinction.

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31 CHAPTER II I ACTIVATION OF NIGROSTRIATAL DOPAMINE NEURONS DURING FEAR EXTINCTION PREVENTS THE RENEWAL OF FEAR Abbreviated title: Nigrostriatal dopamine facilitates fear extinction A uthor list: Courtney A. Bouchet, Megan Mine r, Esteban C. Loetz Holly S Ha ke, Adam J. Rosberg, Caroline Farmer Mykola Ostrovskyy Nathan Gray Benjamin N. Greenwood U niversity of Colorado, Denver. Department of Psychology. Abstract Manipulations that increase dopamine (DA) signaling have been rep orted to enhance fear extinction, but the circuits involved remain unknown. DA neurons originating in the substantia pars compacta (SNc) projecting to the dorsal striatum (DS) are traditionally viewed in the context of motor behav ior, but growing data impl icate this nigrostriatal circuit in emotion. Here we investigated the role of nigrostriatal DA in fear extinction. Activation of SNc DA neurons with designer receptors exclusively activated by designer drugs (DREADD) during fear extinction had no effect on fear extinction acquisition, but enhanced fear extinction memory and blocked the renewal of fear in a novel context D1 receptors in the DS are a likely target mediating the effect s of SNc DA activation. Indeed, D1 expressing neurons in the medial DS (DMS ) were recruited during fear extinction, and DREADD induced DA potentiated activity of D1 expressing neurons in both the DMS and the lateral DS R ats whose SNc DA neurons were activated by DREADD during prior fear extinction displayed potentiated renewal i nduced cFos expression in the CA1 of the hippocampus, a region important for contextual processing. Pharmacological activation of D1 rece ptors in the DS did not impact fear extinction acquisition or memory, but blocked fear renewal T hese data suggest that activation of SNc DA neurons and DS D1 receptors could alter contextual processing during fear extinction, thus reducing the context specificity of the memory and preventing fear renewal. Nigrostriatal DA thus represents a novel target to enhance long ter m efficacy of extinction based therapies for anxiety and trauma related disorders.

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32 Significance Statement High relapse rates impede the efficacy of extinction based therapies for anxiety and trauma related disorders, thus identification of novel strategi es to reduce fear relapse after extinction is of utmost importance to mental health. We found that activation of nigrostriatal dopamine during extinction of conditioned fear prevents the return of fear in a context different from where fear extinction was learned, a form of relapse termed renewal. These data add fear extinction to the purview of nigrostriatal DA functions and suggest that nigrostriatal DA could be a novel target for increasing the long term efficacy of extinction based therapies. Introducti on Extinction of traumatic memories is a major goal of therapeutic strategies for anxiety and trauma related disorders, but extinction memories are labile and fear associated with traumatic memories tends to resurface even following successful extinction. Identification of novel means to enhance fear extinction to prevent the relapse of fear after extinction is of utmost importance to mental health. Dopamine (DA) is a memory modulator implicated in fear extinction. Recent theories proffer that fear extincti on could involve learning a new association between the conditioned stimulus (CS) and a positive affective state stemming from the relief that the predicted unconditioned stimulus (US) no longer follows the CS (prediction error; (Huh et al 2009) ; an association that could be supported by DA. Indeed, high frequency (phasic) DA release in the striat um encodes both reward value (Flagel et al 2011, Howe et al 2013) and prediction error (Schultz 2016) These functions of DA are traditionally thought to involve the mesolimbic DA pathway, originating in the ventral tegmental area (VTA) and terminating in the nucleus accumbens (NAc), in the ventral striatum. Cons istent with this view, phasic DA release in the NAc increases during fear extinction (Badrinarayan et al 2012) and D2 receptor signaling in the NAc has been reported to be necessary for fear extinction (Holtzman Assif et al 2010b) Emerging data indicate that functions of the nigrostriatal DA pathway, originating in the substantia nigra pars compacta (SNc) and terminating in the dorsal striatum (DS), overlap with those of mesolim bic DA (Kravitz & Kreitzer 2012, Wise 2009) Indeed, although nigrostriatal DA is traditionally viewed in the context of motor behavior, recent data reveal a role for the DS, particularly DS D1 expressing neurons p referentially responsive to

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33 phasic DA release (Dreyer et al 2010, Schultz 2007) in reinforcement (Kravitz et al 2012) and emotional behavior (Lenz & Lobo 2013) Despite potential involvement of multiple DA pathways in fear extinction, nigrostriatal DA has not before been considered in fear extinction research. The goal of the current set of experiments was to begin to elucidate the role of nigrostriatal DA in fear extinction. A combined, viral mediated expression of designer receptors exclusively activated by d esigner drugs (DREADD) and pharmacological approach was used to test the hypothesis that activation of SNc DA neurons and subsequent DS D1 receptor signaling can enhance fear extinction learning. Results indicate that activation of SNc DA neurons during fe ar extinction can facilitate fear extinction memory in a manner that resists the return of fear in a novel context (renewal; (Bouton 1988) Consistent with D1 receptors in the DS being a target of G q DREADD induced DA, D1 expressing neurons in the DS are recruited during fear extinction, and their activity is augmented by G q DREADD induced DA. Pharmacological activation of DS D1 receptors during extinction prevents the renewal of fear without impacting fear extinction memory tested in the fear extinction context. These data suggest that nigrostriatal DA and DS D1 receptors c ould be a novel target for the prevention of fear relapse after extinction. Materials and Methods Animals A total of 43 adult, male Tg(TH Cre)3.1Deis rats on a Long Evans background (TH Cre; (Witten et al 2011) wer e supplied by Karl Deisseroth via the NIH Rat Resource and Research Center (Columbia, MO). A total of 88 adult, male wildtype (WT) Long Evans rats were purchased from Charles River. Rats were pair housed in ventilated Nalgene Plexiglass cages (45.5 W x 24 D x 21 H cm) with ad libitum access to food (Standard Lab Chow) and water. All animals were well handled by experimenters once daily for at least 5 days prior to start of behavioral tests. Animals were kept on a 12 12 h light dark cycle with lights on from 0700 to 1900 in a temperature (22 ) and humidity controlled facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care located on the University of Colorado Denver Auraria campus. Care was taken to minimize discomfort in all procedures and all protoc ols were approved by the University of Colorado Denver Institutional Animal Care and Use Committee.

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34 Surgeries All surgical procedures were performed under Ketamine (75.0 mg / kg i.p.) and Medetomidine (0.5 mg / kg i.p.) anesthesia. Carprofen (5 mg / kg s.c .) was administered for pain management at induction and then every 24 h for 72 h post surgery. Atipamezole (0.5 mg/kg i.p.) was used to reverse the effects of Metetomidine to speed the recovery from anesthesia. Penicillin G (22,000 IU/rat, s.c.) was given every 24 h for 72 h following surgery to avoid infection. Viral Transfection AAV5/hSyn DIO hm3D(G q ) mCherry (G q DREADD) viral vector was provided by Dr. Bryan Roth via the University of North Carolina viral core (Gene Therapy Center Vector Core; Chapel H ill, NC; (Zhu & Roth 2014) The virus was kept on wet ice during surgery and loaded into 10l Hamilton syringes back filled with silicon oil immediately before microinjection. After drilling holes into the skull, 1 #L of undiluted virus was injected bilaterall y into the SNc (from Bregma: 5.4mm anterior, 2.3mm lateral, 8.8mm ventral from top of the skull) at a rate of 0.1 #L/min using a Micro4 Microsyringe Pump Controller (World Precision Instruments, Inc. Sarasota, FL, USA). The bevel on the tip of the Hami lton was oriented laterally to minimize spread of virus medially toward the VTA. The syringe was left in place for 10 min after the injection to avoid spreading of virus. The incision was closed with Woundclips. Behavior tests were conducted at least 1 mon th after viral injection to allow ample time for viral expression. Location of viral transfection in the midbrain was verified in all rats by inspection of mCherry expression under 200X magnification on an Olympus BX53. Relative expression of mCherry in va rious projection regions of midbrain DA neurons was quantified with densitometry as previously described (Lloyd et al. 2016). Images through the SNc, dorsal medial striatum (DMS), dorsal lateral striatum (DLS), nucleus accumbens (NAc), medial prefrontal co rtex (mPFC), dentate gyrus region of the hippocampus (DG) and basolateral amygdala (BLA) were captured at 200X magnification from at least 4 sections per brain region per animal, and intensity of signal above background (densitometry) was calculated with C ell Sense software (Olympus).

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35 Cannulae implantation Bilateral 26 gauge guide cannula (4.6mm below pedestal; Plastics One) were implanted into the DS (from Bregma: +5.0mm anterior, +3.0mm lateral, 4.5mm ventral from top of the skull) following our publi shed protocols (Greenwood et al 2012, Strong et al 2011) The guide cannulae were held in place by flowing dental acrylic around the cannulae and 4 anchoring screws. Dummy cannulae (Plastics One) were inserted into the guide cannulae to avoid clogging of the cannula. Rats were allowed at least 1 week of recovery before behavior testing. Microinjections Rats were held gently and microinjectors that extended 1 mm past the tip of the guide cannulae (PlasticsOne) were i nserted through the guide cannulae into the DS. SKF38393 (1 g / hemisphere; (Agnoli et al 2013) SKF 81297 (0.1 g / hemisphere; (Larkin et al 2016, Pezze et al 2015) or sali ne (1 l) were injected at a rate of 0.5 l/min bilaterally through the microinjector controlled by a Micro4 pump. The injector was left in place for 2 min following injection to allow for diffusion and avoid backflow up the guide cannulae. D1 agonists or s aline were injected 10 min prior to fear extinction training trials. Drugs Clozapine N Oxide (CNO; provided by National Institute of Mental Health Chemical Synthesis and Drug Supply Program, Bethesda, MD) was stored at 20 and CNO solution was prepared i mmediately before use. CNO was dissolved in sterile saline with 0.6% DMSO and administered i.p. at a dose of 1 mg/kg (Boekhoudt et al 20 16) Vehicle treated rats received equal volume 0.6% DMSO in saline (i.p.). Vehicle or CNO was administered 30 min before each of 2 fear extinction training sessions. This timing of CNO administration relative to behavior has been used previously with suc cess (MacLaren et al 2016) Two different D1 like (D1 and D5, referred to here as D1 receptors) receptor agonists were used for this study: SKF38393 ( 2,3,4,5, tetrahydro 7,8 dihydroxy 1 phenyl 1 H 3 benzazepine hydrochloride) and SKF81297 ( 6 chloro 7,8 dihydroxy 1 phenyl 2,3,4,5 tetrahydro 1 H 3 benzazepine hydrobromide) Both SKF38393 and SKF81297 have very high binding affinities for D1 receptors in the striatum (Mannour y la Cour et al 2007) SKF3839 (Sigma Aldrich) was dissolved in sterile saline to a final concentration of

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36 0.5 g/ l which is the most effective dose to activate D1 receptors (Agnoli et al 2013, Pezze et al 2007) SKF81297 ( Tocris Biosciences) was dissolved in sterile saline to a final concentration of 0.1 g/ l. This dose has been recently reported to impair appetitive learning when injected into the PFC (Pezze et al 2015) and systemic administ ration of SKF81297 can facilitate contextual fear extinction (Abraham et al 2016) Behavioral Analyses Auditory Fear Conditioning Rats were placed into custom, rectangular conditioning chambers (20 W x 10 D x 12 H cm; Context A) with a shock grid floor (Coulbourn Instruments, Allentown, PA) housed inside individual sound attenuating cabinets. Rats were transported to Context A in their home cages. A fan located near the floor of the cabinets provided ventilation and background noise and bright white lights illuminated the chambers. Chambers were cleaned with water between rats. Rats were allowed 3 min to explore the context, after which rats were exposed to 4 auditory CS (10s, 80dB, 2kH), each co terminating with a foot shock US (1s, 0.8mA; 1 min ITI). The rats were left in the conditioning chamber for 30 s after the last shock before being returned to their home cages. Auditory Fear Extinction Approximately 24 h after fear conditioning, rats were placed into a no vel Context B that was either a custom Plexiglas rectangular chamber (15"W x 15"D x 20"H) with a smooth floor or a custom Plexiglas triangular chamber (15"D sides x 20"H) with a textured floor for fear extinction training. Rectangular and triangular Contex t B chambers were counterbalanced so half the rats were exposed to fear extinction training in the rectangular chambers and the other half in the triangular chambers. Context B was housed in the same sound attenuating cabinets used for conditioning, but al l other contextual features and discrete cues differed between contexts. Rats were transported to the sound attenuating chambers, which included vanilla scent, in their respective Context B custom Plexiglas chambers. The fan within the chamber was turned o ff and the room was dimly lit by a lamp outside of the sound attenuating chambers. Context B was cleaned with 10% ethanol between rats. After a 3 min exploration period, the tone CS was administered 20

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37 times (1 min ITI) in the absence of the foot shock US. One min after the last tone CS, rats were transported to the housing room in their extinction chambers and placed into their home cage. Fear renewal test Approximately 24 h following the second fear extinction training session, rats were exposed to the a uditory CS (1 min ITI) in either the same context used for extinction training (Context B; same ) or a novel Context C ( different ). Rats assigned to the different group were transported to Context C in a novel inner chamber, so that rats extinguished in the square were now placed into the triangle, and vise versa. Context C consisted of a raspberry scent, red box lights, a fan near the top of the behavior cabinets was turned on, and chambers were cleaned with 1% acetic acid between tests. After a 3 min explo ration period, the tone CS was presented 4 times (1 min ITI) in the absence of the foot shock US. Because fear renewal is the return of fear in contexts different from where fear extinction was learned (Bouton and Ricker 1994), the difference in freezing b etween same and different contexts was considered fear renewal. All behavioral tests were recorded with overhead cameras and freezing was scored both by multiple experimenters who were blind to the experimental conditions of the animals and by automated b ehavioral analyses software (Noldus Ethovision XT) during both the CS and the ITIs. Because regularly scheduled ITIs can become a part of the CS, and analyses indicated a lack of differential effects of exercise on freezing during ITIs and CS, freezing dur ing each CS and subsequent ITI were combined and expressed as freezing during a trial, as in prior work (Fitzgerald et al 2015, Goode et al 2015, Mika et al 2015) Each trial consists of a 10 s tone and a 60 s ITI. Double label fluorescent in situ hybridization Two days after the renewal test, a subset of TH Cre rats were injected i.p. with either CNO (1 mg/kg) or vehicle and, 30 min later, either exposed to fear extinction or left in their home cages (no extinctio n). Immediately after the extinction session or equivalent time in home cages, rats were rapidly decapitated and brains were removed, flash frozen with isopentane maintained between 20 and 30 ¡ C, then stored at 80 ¡ C Brains were prepared for in situ hy bridization per our previously published protocols (Herrera et al 2016, Mika et al 2015) Brains were sectioned using RNase free techniques at 10 m onto

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38 Superfrost Plus slides (FisherBrand) then stored at 80 ¡ C unt il in situ hybridization. Riboprobes complimentary to D1, D2 or cfos were filtered through a G50 50 Saphadex column and enzymatically stabilized with 2M dithiothreitol (DTT). Mounted tissue from 1.7 to 0.8 mm Bregma were moved directly from 80 ¡ C into 4% paraformaldehyde (PFA) for 1 h. Following a wash in 2X saline sodium citrate (SSC), slides were placed in 0.01% triethanolamine solution (triethanolamine in H 2 O pH to 8.0, 0.25% acetic anhydride) for 20 min then dehydrated with EtOH. The labeled probe was added to hybridization buffer (formamide, dextran sulfate, sterile water, 20X SSC, 50X Denhart's solution, tRNA, 0.5 M sodium phosphate) and applied to the slide via coverslip. Slides were kept in humidified chambers overnight at 55¡C to promote hybridiza tion. The following day, cover slips were removed in 2X SSC. Slides were then incubated in RNase digestion solution (Tris HCl, NaCl in H2O pH to 8.0, RNase A) for 1 h to degrade remaining single stranded RNA. Slides were then rinsed with graded SSC washes followed by 0.1X SSC at 65 ¡ C for 60 min, after which they were transferred to a 0.05 M phosphate buffered saline (PBS) solution. Slides were incubated in 2% peroxide solution (30% H 2 O 2 in 0.05 M PBS) for 30 min. Slides were then washed in 1X Tris buffered saline with 0.05% Tween 20 (TBS T) and then incubated in blocking buffer (blocking reagent in 1X TBS; Perkin Elmer) for 1 h. Digoxigen and fluorescein flurophores (Perkin Elmer kit; reconstituted in DMSO) were added under dim light as follows. All solutio ns were applied to tissue with coverslips and incubated in humidified chambers. Slides were incubated with anti digoxigen horseradish peroxidase for 30 min (anti digoxigen in blocking buffer). Cover slips were removed in TBS T then slides were washed in TB S T. Slides were incubated with Cyanine 3 Amplification Reagent for 45 min (1:100 dilution in 1X Amplification diluent) followed by TBS T then TBS washes. Slides were then incubated with anti Fluorescein horseradish peroxidase for 2 h (1:100 anti fluoresce in HRP in blocking buffer). Slides were washed in TBS T then incubated with fluorescein fluorophore tyramide for 1 h (1:100 tyramide fluorophore in 1X amplification diluent). Slides were washed in TBS T then TBS, then cover slipped with ProLong Gold antifa de reagent with DAPI (Life Technologies) and stored in the dark to dry. FISH image capture and analysis Images for the DMS, DLS, NAcC and NAcS were captured at 200x magnification using a confocal fluorescent microscope (Axio Observer Z1; Zeiss Microscopy, Jena, Germany). Images were captured

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39 from 4 separate hemispheres from 1.7 to 0.8 mm from Bregma. Single cfos D1, D2 and double labeled cells were counted using Zeiss Zen software by multiple experimenter's blind to treatment conditions of the animals. m Cherry expression was quantified in alternate brain sections not used for FISH. Immunohistochemistry Ninety min after the fear renewal test, TH Cre and WT rats not used for FISH were perfused transcardially and brains prepared for immunohistochemistry as previously described (Herrera et al 2016, Lloyd et al 2017) Thirty five m coronal slices through the PFC (3.2 to 1.7 mm Bregma), AMG / hippocampus ( 1.8 to 4.3 mm Bregma) or midbrain ( 4.5 to 7.0 mm Bregma) wer e sliced on a cryostat at 20 C and stored in cryoprotectant (glycerol, ethylene glycol, 0.1M PB at pH 7.4) at 20 ¡ C until use. Sections were incubated in rabbit anti cFos (1:3000; Santa Cruz sc 52) or mouse anti TH (1:1000; Immuno Star cat #22941) at 4 ¡ C for 48 h. cFos primary binding was visualized using biotinylated secondary (1:200 ; Jackson ImmunoResearch), avidin bioten complex (Vector Laboratories) and nickel enhanced 3,3' diaminobenzidine. TH primary was bound with fluorescent Donkey anti Mouse IgG (H&L) DyLight 650 Conjugate (1:2000; Immunoreagents, Inc) Immunohistochemistry quantification For cFos quantification, images of serial sections spaced 200 # m apart were captured at 200X magnification on an Olympus BX53. Cells expressing cFos protein wer e counted within a counting frame ( 1.48 x 10 5 m ) by multiple experimenter's blind to treatment conditions of animals. Regions counted included the infralimbic (IL) and prelimbic (PL) regions of the prefrontal cortex, the basolateral (BLA) and central (CeA ) nuclei of the amygdala, and the dentate gyrus (DG), CA1, CA2, and CA3 regions of the hippocampus. At least 6 hemispheres were counted per region per animal, but the number varied due to tissue damage incurred during slicing or staining procedures (range 6 10). Cells containing dark brown or black nuclei were considered cFos positive and lightly stained cells were not counted. Laser scanning confocal microscopy software (Axio Observer Z1; Zeiss Microscopy) was used to image TH (green; A488) and mCherry ( red; A568) positive cells in the SNc at 200x. Both hemispheres of five representative rats were visualized, 3 sections each. Numbers of single TH+, mCherry+, or double labeled cells were quantified.

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40 Statistical Analyses Percent time spent freezing was calc ulated by averaging freezing data from individual experimenter's blind to treatment condition of the animals with immobility times obtained from Noldus Ethovision XT. Pre shock freezing for each test was averaged and group differences were analyzed with AN OVA. Average freezing across trials during conditioning and extinction were analyzed using repeated measures ANOVA with subsequent Viral Expression Site or Drug as factors. For renewal, freezing across trails was averaged and compared using 2 ( same vs diff erent ) x 3 (Veh vs SN vs Off Target OR Veh vs SKF38393 vs SKF81297). Group differences in cFos protein expression were analyzed with 2 x 3 ANOVA. Group differences in the total number of cfos or D1 mRNA expressing neurons and % D1 expressing neurons contai ning cfos mRNA were also analyzed with a 2 x 2 ANOVA. Differences in mCherry density between brain regions was analyzed with ANOVA, Fisher's protected least significant differences post hoc analyses were performed when appropriate. Group differences were c onsidered different when p 0.05. Results Activation of SNc DA neurons enhances fear extinction memory and blocks fear renewal Viral transfection was confirmed in all rats. Figure 1A depicts bilateral mCherry expression in the SNc. Rats with mCherry ex pression restricted to the SNc were assigned after the experiment to the SNc group. Of the SNc rats injected with CNO, 6 were observed to have bilateral SNc mCherry expression and 2 had unilateral SNc mCherry; for a total of 8 rats in the SNc CNO group. An other 6 SNc rats received vehicle. Rats with mCherry extending into the VTA were considered "off target" and were assigned to the off target group. These rats tended to have limited expression in the SNc. Twelve off target rats were injected with CNO and 5 were injected with vehicle. No significant differences were found between SNc and off target rats injected with vehicle, so these rats were combined into the "Vehicle" group for all analyses. mCherry expression in brain regions involved in fear extinction and known to receive DA innervation from midbrain DA neurons was quantified with densitometry in SNc rats. The highest expression of mCherry was observed in the SN and DLS, moderate expression observed in the DMS and NAc, and minor expression in the PL, IL, DG and AMG (Figure 1B). A photomicrograph depicting terminal expression

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41 of mCherry in the DS is shown in Figure 1C. Four rats had no visible mCherry and were excluded from all analyses. TH immunohistochemistry revealed that 92% of the mCherry was conta ined within TH expressing neurons (Figure 1D F), indicating the majority of the viral transfection was restricted to DA neurons. Figure 1 G q DREADD selectively targeted the SNc and was selective to TH expressing neuro ns. (A) Representative picture of bilateral SNc G q DREADD injection; (B) mCherry intensity in SNc and potential terminal regions; (C) Representative picture of mCherry in the DS; (D) TH labeled neurons; (E) mCherry autofluorescence; (F) Merge of TH and mC herry. # denotes significantly different from BLA (0.002), DG (p = 0.005), IL (p = 0.01), PL (p = 0.01); *** denotes p < 0.0001 from all other groups. To determine the effects of G q DREADD induced SNc DA activation during fear extinction on subsequent fear extinction memory and fear renewal, rats were injected with either vehicle or CNO during 2 subsequent days of fear extinction training. Fear conditioning and fear renewal were conducted drug free (see Figure 2A for experimental timeline). Freezing levels were negligible prior to the first CS US presentation during conditioning (Figure 2B; pre). All rats acquired fear conditioning equally (F(3, 105) = 16.421, p <0.0001); Figure 2B), regardless of subsequent group assignment. The following day, all groups di splayed equivalent within session fear extinction in the presence of vehicle or CNO (F(4,140) = 55.004, p < 0.0001; Figure 2C). Given that SNc DA neurons are closely associated with movement, cage crossings were counted during the first 3 min of the extinc tion training sessions, before the first CS, as a measure of locomotor activity. No differences in overall locomotion were observed between groups on extinction day 1 ( Vehicle 9.29 1.04; Off Target CNO 8.08 1.33; SN CNO 11.00 3.32; p = 0.52) or extin ction day 2

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42 ( Vehicle 9.23 0.93; Off Target CNO 10.50 2.20; 11.20 1.24; p = 0.74). When tested for fear extinction memory the following day, all rats displayed within session fear extinction (F(4,140) = 26.45, p < 0.0001; Figure 2D), but the SNc CNO g roup displayed significantly less freezing than rats in other groups (F (2,35) = 3.22, p = 0.05; Figure 2D), indicating enhanced fear extinction memory recall in rats whose SNc DA neurons were activated during fear extinction. The interaction between vira l expression and time just missed significance (F (8,140) = 1.89, p = 0.06). Rats were again exposed to the CS the next day in either the same or a different context for assessment of fear renewal. Consistent with fear renewal, within the vehicle group, ra ts exposed to the CS in the different context froze more than rats exposed to the CS in the same context in which extinction was learned (main effect of context; F(1,32) = 9.20, p = 0.005; Figure 2E). However, activation of SNc DA neurons during extinction blocked fear renewal. Indeed, post hoc analysis revealed no difference between the SN CNO same and different groups The reduction in freezing displayed by the SNc CNO group during fear extinction day 2 was not observed in the same context again during th e renewal test (Figure 2E). This is likely because by this point all groups had received sufficient extinction training in this context; thus, levels of freezing were at a floor. It is interesting that activation of G q DREADD expressing DA neurons with CNO had no impact on fear extinction or renewal in rats in which viral transfection was observed in the VTA (off target; Figure 2D and 2E). This observation could be due to the simple fact that fewer SNc DA neurons were transfected in off target rats, or it c ould reflect a lack of effect of VTA DA neurons on fear extinction.

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43 Figure 2 Gq DREADD activation of SNc DA neurons, but not off target DA neurons, enhances fear extinction and blocks fear renewal. (A) Experimental de sign. (B) All rats conditioned equally, take note that rats had not yet received CNO or vehicle. (C) 30 min after injection of either vehicle or CNO, all groups extinguished equally. (D) Rats received Vehicle or CNO 30 min prior to a second fear extinction session. When placed back into the extinction context 24 hours, CNO rats whose mCherry was selective to the SN exhibited significantly less freezing behavior, indicative of enhanced fear extinction memory. Main effect of SN CNO p = 0.05. (E) When tested in a context different form the fear extinction context, rats in the Vehicle and Off target CNO groups had a significant increase in freezing behavior. Rats in the SN CNO had significantly lower freezing than both different groups; fear did not return in a different context. Vehicle and Off target CNO different groups are significantly different from SN CNO different group. denotes p = 0.05; ** denotes p < 0.01. Gq DREADD activates target D1 expressing neurons in the DS We used double D1/ cfos FISH to det ermine if the G q DREADD approach successfully activates target D1 expressing neurons in the DS Similar to prior reports (Bertran Gonzalez et al 2008, Bertran Gonzalez et al 2010) virtually no co localization of D1 and D2 mRNAs were observed in the DS (Figure 3 A ) This is in contrast to the NAc, in which D1 and D2 receptors have been observed to be colocalized (Bertran Gonzalez et al 2008, Shetreat et al 1996) Since almost all neurons in the DS express either D1 or

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44 D2 mRNA, cells expressing single cfos mRNA can be assumed to be putative D2 expressing neurons (Gerfen et al 1990) Figure 3 Double fluorescent in situ hybridization labeling cfos and D1 mRNA during fear extinction. (A) Representative picture from DS showing D1 and D2 receptor mRNA with low levels of co localization. White bar represents 20 m. (B) Representative picture from the DS fr om a Vehicle No Extinction rat showing very little D1 cfos coexpression; insert is a coronal section modified from the Paxinos and Watson rat brain atlas (Paxinos & Watson 1998) (C) Representative picture from the DS CNO extinction rat showing high levels of D1 cfos coexpression. (D ) Activa tion of D1 expressing neurons in the DS. Fear extinction alone recruits D1 expressing neurons in the DMS, while CNO injection activates D1 expressin g neurons in the DMS and DLS. (E ) Activation of D1 expressing neurons in the NAc is not increased with fear extinction nor peripheral CNO injection. denotes Extinction is p < 0.05 from No Extinction; # denotes CNO p < 0.05 from Vehicle. Representative photomicrographs showing D1 and cfos mRNAs in the DMS of rats exposed to no extinction or extinction are depic ted in Figure 3B and 3C. Extinction learning alone increased the % of D1 expressing neurons containing cfos mRNA in the DMS (Main effect of extinction: F(1,14) = 5.548, p = 0.03; Figure 3 D ) and the number of single cfos mRNA expressing cells in the DLS (Ma in effect of extinction: F(1,14) = 4.750, p = 0.04, Table 1). Fear extinction learning did not alter the number of single cfos mRNA containing cells nor the % of D1 expressing neurons containing cfos mRNA in the NAcC or NAcS (Table 1), but extinction learn ing increased the number of neurons expressing D1 mRNA in the NAcC (F(1,14) = 4.634, p = 0.04; Table 1). Activation of G q DREADD with CNO increased both the number of cells expressing cfos mRNA (F(1,14) = 4.474, p = 0.05; Table 1) and the % of D1 expressin g neurons containing cfos mRNA in the DMS (F(1,14) = 13.492, p = 0.003; Figure 3 D ) and the number of double labeled D1/ cfos mRNA expressing neurons in the DLS (F(1,16) = 5.564, p = 0.0 3; Figure 3 D ), but had no effect in the NAc C or NAcS ( Table 1 and Figure 3 E ). CNO did not impact cfos or D1 mRNAs in any other

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45 region. These data suggest that fear extinction recruits D1 expressing neurons in the DMS more than in other regions of the striatum. Further, the G q DREADD approach successfully activates D1 expressin g neurons in the DS. Table 1 *Main effect of extinction p < 0.05 Gq DREADD induced S Nc DA activation during fear extinction alters brain activation patterns during renewal To probe which brain regions in the fear extinction circuitry might be impacted by SNc DA activation, rats were sacrificed after the fear renewal test and cFos was quan tified in the hippocampus, mPFC, and amygdala; regions known to be important for fear extinction and renewal (Bergstrom 2016, Giustino & Maren 2015, Hirsch et al 2015, Huff & Rudy 2004, Jin & Maren 2015, Knapska et al 2012, Knapska & Maren 2009, Maren 2014, Thompson et al 2010) See Figure 4A for a graphic depiction of where cFos was Brain Region Veh No Ext Mean (SEM) Veh Ext Mean (SEM) CNO No Ext Mean (SEM) CNO Ext Mean (SEM) # of single D1 mRN A positive cells DMS 33.59 (2.57) 34.77 (6.84) 22.81 (4.81) 34.70 (2.42) DLS 28.50 (4.67) 27. 90 (3.82) 23.67 (4.04) 29.66 (1.34) NAcC* 29.78 (2.75) 38.08 (6.51) 25.49 (2.32) 39.79 (3.77) NAcS 35.46 (3.96) 45.62 (7.17) 35.17 (7.59) 37.87 (2.19) # of single cfos mRN A positive cells DMS 3.28 (0.65) 4.2 (0.86) 4.57 (1.32) 7 .03 (1.12) DLS* 4.02 (0.59) 6.875 (1.367) 4.25 (1.32) 8.31 (2.50) NAcC 4.65 (1.19) 6.24 (1.66) 4.36 (2.45) 6.04 (1.86) NAcS 4.18 (1.02) 6.39 (1.39) 4.86 (1.84) 7.37 (0.72)

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46 quantified. No differences in cFos were noted in the PL (Figure 4 B ). Similar to a prior report (Knapska & Maren 2009) rats re exposed to the extinguished CS in the same context in which fear extinction was learned demonstrated slightly hi gher cFos in the IL than rats re exposed to the extinguished CS in a different context, although the main effect of context just missed significance (F(1,22) = 3.876, p = 0.06; Figure 4 B ). The increase in cFos in the IL noted in the same context could refl ect this region's role in fear extinction recall (Chang & Maren 2011, Thompson et al 2010) SNc DA activation had no significant impact on cFos expression in the IL, suggesting that the IL is unlikely to be the sit e of action mediating the effects of SNc DA activation. Both the PL region of the PFC and the CeA are thought to drive the fear response in response to conditioned cues (Burgos Robles et al 2009, Corcoran & Quirk 20 07, Fendt & Fanselow 1999, LeDoux et al 1988) No group differences in cFos expression we re observed in the PL (Figure 4 B ). However, consistent with the fear renewal observed in the different context, cFos expression in the CeA was significantly higher in rats placed into the different context than those placed into the same context (F(1,22) = 5.784, p = 0.02). Interestingly, though, cFos expression in the CeA of rats whose SNc DA neurons were activated during fear extinction was ident ical between contex ts (Figure 4B ); a pattern of data that parallels the freezing behavior No significant differences were observed in the BLA, CA3, or CA2. In the DG; however, cFos expression was higher in rats re exposed to the extinguished CS in the same context compared to the different context (F(1, 21) = 4.131, p = 0.05; Figure 4 C ) This observation in the DG is similar to a prior report (Knapska & Maren 2009) and could reflect the recognition of the familiar extinction context by the hippocampus. Interesting data observed in CA1 where greater cFos expression was observed in the CA1 of rats whose SNc DA neurons were activat ed during fear extinction compared to rats in other groups, regardless of the context in which rats were re exposed to the extinguished CS (F(2,21) = 3.566, p = 0.04; Figure 4 C ). Representative photomicrographs depicting cFos immunoreactivity in the CA1 a r e shown in Figures 4D and 4 E.

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47 Figure 4 Immunohistochemistry cFos protein expression within regions implicated in fear extinction during fear renewal. (A) Representative images of regions quantified; including regions of the hippocampus, mPFC, and amygdala modified from the Paxinos and Watson rat brain atlas (Paxinos & Watson 1998) (B) Regions within the mPFC and amygdala. cFos protein expression in the CeA mirrors the fear expression observed in the renewal behavioral test, with significantly higher ac tivation in the different vs same groups for the Vehicle and Off Target CNO groups but no significant difference between the same and different SN CNO groups. (C) Regions within the hippocampus; testing in the same context increases cFos protein in the DG. Significantly higher levels of cFos protein were observed in the CA1 region of SN CNO rats. No differences were observed in the CA3 or CA2 regions of the hippocampus. (D) Representative picture of the CA1 region with low cFos protein expression; (E) Repre sentative picture of the CA1 region with high cFos protein expression. Scale bar represents 50 m. represents p < 0.05. Activation of DS D1 receptors during fear extinction blocks fear renewal without enhancing fear extinction memory To determine the cont ribution of D1 receptors in the DS to the observed effects of G q DREADD induced activation of SNc DA neurons, we microinjected D1 agonists into the DS prior to fear extinction learning on 2 subsequent days, and fear renewal was assessed drug free 24 h late r (see Figure 5A for experimental timeline). Given a recent report that systemic administration of SKF81297 and SKF38393 have

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48 different effects on fear extinction; whereby SKF81297, but not SKF38393, administered prior to fear extinction enhances fear exti nction memory (Abraham et al 2016, Borowski & Kokkinidis 1998) but see (Mannoury la Cour et al 2007) we microinjected both drugs into the DS in different cohorts of rats prior to fear extinction. Fifty two out of 54 rats had successful bilateral DS cannulae and the locations of the can nulae tips are shown in Figure 5 B. Exclusion of missed injections resulted in the following group sizes: Saline same = 12, Saline different = 11, SKF38393 same = 8, SKF38393 different = 9, SKF81297 same = 6, SKF81297 different = 6. Figure 5

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49 Figure 5 D1 agonists injected into the DS during fear extinction block fear renewal without enhancing fear extinc tion memory. (A) Experimental design. (B) Cannula placement verification for C F. Coronal sections modified from the Paxinos and Watson rat brain atlas (Paxinos & Watson 1998) (C) All groups were equally conditioned; (D) Saline, SKF38393, or SKF 81297 were injected 10 minutes prior to fear extinction training. Presence of a D1 agonist had no impact on fear extinction learning. (E) Saline, SKF38393, or SKF81297 was injected 10 minutes prior to the second day of fear extinction training. No difference between groups was observed. (F) Saline ra ts placed into a different context exhibited significantly higher freezing than those placed into the same context; however, no significant differences were observed between same and different groups if rats were injected with D1 agonists prior to fear ext inction training. (G) Experimental design for drug free extinction memory test. (H) Cannula placements for drug free extinction experiment. (I) All rats were equally conditioned to fear the tone; (J) Rats received microinjections of Vehicle or SKF38393 pri or to fear extinction learning. No difference in fear extinction learning was observed. (K) When tested drug free, no differences between groups were observed. denotes p < 0.05 from same group. Rats acquired auditory fear conditioning equally (Main effec t of time: F(3,147) = 65.679, p < 0.0001; Figure 5 C), regardless of subsequent group assignment. Neither agonist impacted freezing during the first fear extinction training session. All rats displayed within session extinction (Main effect of time: F(4,196 ) = 56.950, p < 0.0001) that did not differ between groups (Figure 5 D). Similar effects were observed during the second fear extinction training session the next day, during which all rats similarly acquired within session extinction (Main effect of time: F(4,196) = 46.846, p < 0.0001; Figure 6E). Freezing to the first few CS during a fear extinction session represents memory of previously acquired fear extinction (Bukalo et al 2015, Do Monte et al 2015) thus freez ing during the first 4 trials were analyzed separately. Intra DMS D1 agonist had no impact on fear extinction memory during the first few trials of the second fear extinction training session. Similarly, all rats displayed equivalent freezing in the same c ontext when tested drug free the next day, although freezing levels in the same context were approaching a floor. Despite the fact that D1 activation had no effect on fear extinction learning or memory in the extinction context, rats that received D1 agoni st prior to fear extinction were protected against fear renewal tested in a novel environment 24 h after the second fear extinction training session (drug by context interaction; F(2,46) = 3.1, p < 0. 0001; Figure 5 F). Post hoc analysis revealed that the sa line different rats froze significantly more than all other groups (saline same p = 0.006, SKF38393 different p = 0.008, SKF38393 same p = 0.02, SKF81297 different p = 0.009, SKF81297 same p = 0.03), and neither the SKF38393 different or SKF81297 different groups different from their same counterparts (p > 0.05).

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50 The observation that G q DREADD induced DA release both enhances fear extinction memory and reduces renewal, whereas selective activation of DS D1 with D1 agonists reduce fear renewal without enhan cing fear extinction, is an interesting finding. To rule out the possibility that the presence of D1 agonist drug during the extinction memory test (extinction day 2) obscured a potential effect of prior D1 agonist on extinction memory, SKF38393 was inject ed into the DS prior to the first fear extinction training session in a different cohort of rats, and fear extinction memory was tested the next day drug free ( s ee f igure 5G for experimental design ). Location of can nula tips are shown in figure 5H All rat s acquired fear conditioning throughout CS US pairings (Main effect of time: F(3,42) = 25.201, p < 0.0001) regardless of subsequ ent group assignments (Figure 5I ). The following day, all rats acquired within session fear extinction (Main effect of time: F(5 ,70) = 72.155, p < 0.0001; Figure 5J ) and there was no difference between rats that received intra DS injections of vehicle (n = 8) or SKF38393 (n = 8). When tested the following day drug free, we again observed that D1 receptor activation during extinctio n failed to enhance fear extinction memory (Main effect of time: F(5,70) = 15.102, p < 0.0001; Figure 5K ). Therefore, although activation of SNc DA neurons and DS D1 receptors during fear extinction both prevent fear renewal, they seem to have different ef fects on fear extinction memory when tested in the extinction context. Discussion Data presented here reveal a novel role for nigrostriatal DA in fear extinction. Activation of SNc DA neurons with G q DREADD during fear extinction had no effect on fear ext inction acquisition, but enhanced fear extinction memory and blocked the renewal of fear; a pattern of data paralleled by cFos expression in the CeA. D1 expressing neurons in the DS are a likely target mediating at least part of the effect of SNc DA activa tion. Indeed, expression of terminal mCherry, indicating presence of G q DREADD, was highest in the DS, D1 expressing neurons in the DMS were observed to be recruited during fear extinction, and G q DREADD induced DA potentiated activity of D1 expressing neu rons in both the DMS and the DLS. Interestingly, rats whose SNc DA neurons were activated by G q DREADD during prior fear extinction displayed potentiated cFos expression in the CA1 of the hippocampus, a region importan t for contextual processing (Mat us Amat et al 2004) Pharmacological activation of D1 receptors in the DS had no impact on fear extinction acquisition or memory, but blocked fear renewal in a novel context. Together,

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51 these data suggest that activation of SNc DA neurons and DS D1 recepto rs could alter the contextual processing of the fear extinction memory, thus reducing the context specificity of the memory and preventing fear renewal. Prior studies investigating the roles of specific DA circuits reveal inconsistent results. A recent st udy suggests that VTA DA neurons projecting to the mPFC can impair fear extinction (Hitora Imamura et al 2015) However, other studies implicate roles for D1, D2, & D4 receptors in the mPFC in fear extinction (Hikind & Maroun 2008, Mueller et al 2010, Pfeiffer & Fendt 2006) VTA DA neurons also provide DA innervation to other limbic structures including the BLA, hippocampus and NAc. In the BLA, DA manipulations have no effect on fear extinc tion (Fiorenza et al 2012, Hikind & Maroun 2008) In contrast, D1 activation in the hippocampus can enhance short term fear extinction memory (Fiorenza et al 2012) while i nhibition of D2 in the NAc has been reported to impair fear extinction (Holtzman Assif et al 2010a) Notably, relapse was not investigated in these studies; therefore, the effects of these manipulation on fear rene wal is unknown. No studies to our knowledge have investigated the involvement of SNc DA neurons and their primary target, the DS, in fear extinction. Here we used a G q DREADD approach in TH Cre rats to selectively increase phasic activity of SNc DA neurons (Urban & Roth 2015) This approach was selective at targeting DA neurons in the SNc (Figure 1) and produced a functional increase in activity of target neurons downstream of SNc DA neurons. G q DREADD induced activation of SNc DA neurons resulted in an increase in cfos mRNA in D1 mRNA expressing neurons in the DMS and DLS (Figure 4A), but not the NAc. This is not surprising, since the DS is the primary recipient of DA terminals originating from the SNc (Figure 1B). The effects of SNc DA activation was not due to a non specific effect DA on locomotor activity. Indeed, the 1 mg/kg dose of CNO did not alter general locomotor activity measured by spontaneous cage crossings in Context B prior to the first CS during either fear extinction training session, nor did CNO reduce freezing during the first extinction training day. These observations suggest that the reduced freezing displayed by the SNc CNO group during extinction training day 2 was not due to an ef fect of CNO on the expression of freezing per se but rather to prior SNc DA facilitating the later recall of fear extinction memory. Since CNO was injected prior to fear extinction and has a 3h half life, the effect of SNc DA activation on later extinctio n

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52 memory recall could have been due to enhancements of either extinction acquisition or consolidation. The observation that CNO had no impact on fear extinction acquisition on fear extinction day 1 argues for an effect on consolidation, but the current exp eriment was not designed to directly address the role of DA in acquisition versus consolidation. A critical question is where DA is acting to produce the observed effects of SNc DA activation. Although the SNc projects to regions other than just the DS, mi nimal terminal mCherry expression was observed in regions traditionally implicated in fear extinction, such as the mPFC, hippocampus, and AMG. Therefore, it is unlikely that the observed effects of SNc DA activation was mediated by DA in these regions, alt hough a role for DA in these regions cannot be completely ruled out. Double FISH revealed that D1 expressing neurons in the DMS are recruited during fear extinction learning (Figure 3 D) DMS cortical circuits are involved in guiding goal directed behavior (Shiflett et al 2010, Yin et al 2005) Goal directed strategies may guide behavior as the animal acquires extinction and begins to evaluate the environment. We have similarly observed potentiated activity of DMS D1 expressing neurons during recall of relapse resistant fear extinction in rats that ran in running wheels during fear extinction acquisition (Mika et al 2015) Together, these data suggest that DMS D1 expressing ne urons could represent a previously unidentified component of fear extinction, recruitment of which could be involved in the learning or recall of fear extinction. Additionally, although DLS D1 expressing neurons were not recruited during fear extinction, G q DREADD induced SNc DA activity resulted in increased cfos mRNA in these DLS neurons (Figure 3A). The DLS is involved in guiding inflexible behavioral strategies that can occur at the expense of goal directed or spatial strategies (i.e. "habit"; (Lovinger 2010, Schwabe et al 2008, Yin & Knowlton 2006) These data therefore raise the possibility that DA in the DLS elicited by G q DREADD could have altered the learning strategy used during fear extinction acquisition to one involving the DLS. This is an intriguing possibility, as learning strategies involving the DLS may be less susceptible to the disruptive effects of contingency changes, such as context, during recall (Dias Fe rreira et al 2009, Schwabe et al 2008) More selective targeting of DMS vs. DLS DA circuits will be required to determine the role of DA in these regions in fear extinction. Similarly, how these DS regions communicate with canonical fear circuitry is curr ently unknown. The observation of altered cFos expression patterns during fear extinction

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53 memory recall in the CeA and hippocampus of rats whose SNc DA neurons were activated during fear extinction ( Figure 4B and 4C ) implicates these regions in the mechani sms by which nigrostriatal DA enhances fear extinction. Modulation of neural activity in the hippocampus, in particular, could reflect altered contextual processing by nigrostriatal DA; a prospect that is supported by the complete blockade of fear renewal elicited by both SNc DA activation (Figure 2E) and pharmacological activation of DS D1 receptors ( Figure 5F ). The current data are consistent with prior work implicating the nigrostriatal pathway in learning processes that are independent of the hippocampu s & thus contextual modulation (Da Cunha et al 2003, Faure et al 2005) D1 agonists injected into the DS prior to fear extinction prevented fear renewal without impacting extinction memory, even when extinction me mory was assessed drug free (Figure 5 K ); a pattern of data differing from that of SNc DA activation. This discrepancy could be explained by phasic DA elicited by G q DREADD interacting with DA receptors differently than specific receptor agonists. For examp le, phasic DA activates both D1 and D2 expressing neurons (table 1), whereas the partial D1 agonist SKF38393 is selective to D1 (Conroy et al 2015) SKF81297; however, agonizes both D1 and D2 receptors (Rashid et al 2007a) as well as D1/D2 heteromers (Rashid et al 2007b) Another possibility is that DA in the DS could render extinction memories resistant to relapse (e.g. renewal), whereas DA in a different region could be responsible for en hancing extinction memory in the same context. Since we didn't target D1 agonists to the DMS or DLS selectively (Figure 6B), the effects of pharmacological D1 activation cannot shed light on where within the DS G q DREADD induced DA could have been acting. However, the pharmacological data does support the idea that nigrostriatal DA frees extinction memory from its contextual modulation through a mechanism involving D1 receptors in the DS. The current data implicate DS D1 expressing neurons in fear extinc tion and demonstrate for the first time that SNc DA neurons can facilitation fear extinction memory. Importantly, fear extinction supported by nigrostriatal DA is resistant to fear renewal. As relapse phenomena, including renewal, are a major barrier to th e long term success of current treatment strategies for anxiety and trauma related disorders, the nigrostriatal DA pathway provides a promising target for the development of more effective therapeutic strategies.

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54 Acknowledgements We would like to thank Dr Michael Baratta and Dr. Erik Oleson for technical advice on the use of DREADDs.

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55 CHAPTER IV CONCLUSION S Data presented in this thesis increase our understanding of fear extinction learning and memory, specifically techniques to modify fear extinction le arning so as to r educe the renewal of fear. Exercise is a safe, healthy, inexpensive behavioral modification that can easily be integrated into exposure therapy sessions (Powers et al 2015) We found that voluntary exercise enhances auditory fear extinction memory and blocks the renewal of fear, as well as enhancing long term contextual fear extinction memory in male Long Evans rats. Interestingly, voluntary exercise integrated into fear extinction had no effect on fear extinction memory or fear renewal in female L ong Evans rat s. This is an important finding, particularly because of established differences in anxiety and trauma disorders between males and females (Maeng & Milad 2015) E merging sex differences in animal models can reveal pertinent know ledge gaps and change the way we address future experiments and future treatm ents. Further experiments are needed to further understand our observed sex differences in exercise manipulation of fear extinction learning and memory manipulated. The finding that exercise can modulate fear extinction learning and memory could h ave inte resting applications for the clinical setting; however, the underlying mechanisms are unknown Exercise impacts many central and peripheral systems, one of which is the DA system. The DA system provides an interesting intersection between reward and moveme nt, which makes it an excellent target for the neurobiological basis of the effects of exercise on fear extinction learning and memory. We targeted the nigrostriatal DA system using G q DREADD and pharmacological manipulations We found that G q DREADD induc ed phasic DA release enhances fear extinction memory and creates a memory that is resistant to relapse ; however, this effect was only observed if G q DREADD activation was selective to the SNc. This observation mimicked the effects of exercise during fear e xtinction consolidation Interestingly pharmacological activation of the D1 receptor in the DS, the main terminal region of SNc DA projections according to our mCherry data, blocked the renewal of fear without effecting fear extinction memory. These data s uggest that different memory systems modulate fear exti nction memory and fear renewal Further, to the best of our knowledge, these data are the first to implicate the dorsal striatum in fear extinction learning

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56 and memory. Data presented in this thesis pr ovide evidence that increased activation of the D1 receptor in the DS during fear extinction learning, either through DREADD activation of SNc DA neurons or pharmacological activation of the D1 receptor in the DS, reduces fear renewal. Further studies inve stigating the neural circuitry connecting the DS to the canonical fear circuitry are needed. Given the similar effects of nigrostriatal DA during fear extinction and exercise during fear extinction consolidation, it is likely that nigrostriatal DA plays a role in the observed exercise effect from chapter 1. Results from this thesis show that nigrostriatal DA activation is sufficient to produce the effects of exercise during fear extinction consolidation; however, further experiments are needed to assess wh ether nigrostriatal DA is necessary for these effects. Inhibitory DREADD d uring exercise after extinction would be an interesting means to test this question. If this manipulation interferes with the ability of exercise to enhance fear extinction memory an d block renewal, then it could be concluded that an increase in nigrostriatal DA is both necessary and sufficient for the effects. It is possible; however, that inhibitory DREADD during exercise could have an adverse effect on movement and impair the rat's ability to exercise. Data presented in this thesis identify a behavioral manipulation, voluntary exercise, that can enhance fear extinction learning and memory and bloc k the renewal of fear. Further we show that activation of SNc DA neurons, resulting in phasic DA release, during fear extinction produces the same behavioral outcome enhanced fear extinction and lack o f fear renewal. Fear extinction is the basis of exposure therapy, a common behavioral therapy used for the treatment of PTSD and anxiety r elated disorders. Enhancing our understanding of the neural circuitry underlying fear extinction, and factors that can modulate fear extinction learning and memory can have useful clinical implications for use in improving th e efficacy of exposure therapy REFERENCES Abraham AD, Cunningham CL, Lattal KM. 2012. Methylphenidate enhances extinction of contextual fear. Learn Mem 19: 67 72 Abraham AD, Neve KA, Lattal KM. 2014a. Dopamine and extinction: a convergence of theory with fear and r eward circuitry. Neurobiol Learn Mem 108: 65 77

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