Role of Oxytocin and GABA in the Prefrontal Cortex in Mediating Anxiety Behavior

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Sara Sabihi

Graduate Program in Psychology

The Ohio State University

2017

Dissertation Committee:

Dr. Benedetta Leuner, Advisor

Dr. Laurence Coutellier,

Dr. Baldwin Way

Copyrighted by

Sara Sabihi

2017

Abstract

This dissertation consists of two parts both of which aim to understand the neurobiological mechanisms underlying the anxiolytic actions of oxytocin (OT), focusing specifically on its interactions with the inhibitory neurotransmitter GABA in the medial prefrontal cortex (mPFC). Anxiety disorders affect about 40 million adults and are devastating in the personal, societal, and financial costs, yet, current treatment strategies are not completely effective which is likely related to the our limited understanding of the neural underpinnings of anxiety disorders. Both OT and GABA are well-known regulators of anxiety-like behavior. Furthermore, the mPFC has been shown to play a role in the modulation of anxiety behavior and contains OT-sensitive neurons as well as many

GABAergic interneurons, some of which express OT receptors (OTR). Together, it seems likely that an OT-GABA interaction in the mPFC may play an important role in regulating anxiety but this hypothesis has not been tested.

In Part 1, we examine the anxiolytic effects of exogenous OT in the mPFC and its interactions with GABA. After an introduction in Chapter 1, we assess the regional and receptor specificity of OT’s anxiolytic actions within the mPFC in Chapter 2. We confirm and extend previous work by demonstrating that infusion of OT into the prelimbic (PL) region of the mPFC, but not other regions, decreased anxiety-like behavior and show that the attenuation in anxiety-like behavior following OT administration into the PL mPFC is

ii abolished with pretreatment with a selective OTR antagonist (OTR-A). Further, although

OT has been implicated in the attenuation of the HPA axis response to stress, our results show that OT acting within the mPFC does not diminish the stress-induced glucocorticoid levels. In Chapter 3, we assess OT’s mechanism of action and demonstrate that the attenuation in anxiety-like behavior following OT administration into the PL mPFC is abolished by pretreatment with a GABAA receptor antagonist.

Furthermore, OT in the mPFC increased activation of GABA neurons and was associated with altered neuronal activation in the amygdala following exposure to the enxiogenic environment of the elevated plus maze.

The postpartum period is a time that is often accompanied by a natural reduction in anxiety and an upregulation of the OT and GABA systems. However, anywhere from

3-43% of women experience postpartum anxiety within the first year postpartum. Both

OT and GABA are known regulators of postpartum anxiety, and just as in anxiety in the non-postpartum state, the mPFC has been shown to regulate aspects of maternal care behaviors and postpartum anxiety. Thus, in Part 2 of this dissertation we examine the anxiolytic effects of endogenous OT and GABA in the PL mPFC of postpartum females.

Chapter 4 provides an overview of maternal anxiety and in Chapter 5, we show that OT acting within the PL mPFC modulates anxiety-like behavior during the postpartum period. Chapter 6 expands our investigation of postpartum anxiety to GABA by showing that blockade of GABAAR in the PL mPFC prevents the normal reduction in postpartum anxiety. Further, we demonstrate that mother-pup separation, which increases anxiety, was accompanied by decreased activation of GABAergic neurons in the PL mPFC and

iii that reduced maternal anxiety could be restored by activation of the GABAAR in the PL mPFC.

Overall, the findings from this dissertation suggest that OT and GABA act within the PL mPFC to reduce anxiety-like behavior likely in an interactive manner. In doing so, this work provides new insights into the neural circuitry and the mechanisms that underlie anxiety-like behavior.

Dedication

This dissertation is dedicated to….

....my parents for providing me with the support, resources, and opportunities to pursue

my dream without asking for anything in return.

….my husband, Josh. For going through this process with me. For supporting me every

step of the way. For pushing me forward when I didn’t think I could do it anymore. For

putting up with my moments of high anxiety and stress. And mostly, for helping me

understand (to the best of my ability) statistics.

….Dr. Benedetta Leuner, my advisor, mentor, and a kick-ass female scientist. For taking

a chance on me. For holding me to high expectations and making sure I achieved every one of them. For making sure I never gave up. For being, literally, the best advisor ever.

For mentoring me in science, writing, teaching, and life. For teaching me how to be a

kick-ass female scientist.

….my two dogs, Oscar and Ziggy. Because there’s no way I could have made it through

this without their cuddles.

….for the Baha’is in Iran, who are denied the right to higher education, yet they persist.

Acknowledgments

I would like to express my utmost gratitude to Dr. Benedetta Leuner for all of the knowledge and support she has given me over the last six years. This work would not be

possible without her guidance and support.

I would like to also thank my colleagues who have provided additional support during my

graduate career, including Chris Albin-Brooks, Peter Fredericks, Achik Haim, and

Dominic Julian.

Thank you to the undergraduates who volunteered so much of their time to assist with many of these projects, including Shirley Dong, Nicole Durosko, Caitlin Post and Skyler

Maurer.

To Josh, thank you, again, for all of you statistical help. And thank you for supporting me

always and pushing me to keep going.

Finally, I’d like to thank my parents, for their love and support and understanding as to

why I couldn’t visit all the time. You can stop asking me when I’ll be done with school

now….

Vita

June 2007 ...... Howland High School

2011...... B.S. Psychobiology, The Ohio State

University

2013...... M.A. Psychology, The Ohio State

University

2015 to present ...... Doctoral Candidate, Department of

Psychology, focus in Behavioral

Neuroscience, The Ohio State University

Publications

Sabihi, S., Dong, S.M., Maurer, S.D., Post, C.M., Leuner, B., 2017. Oxytocin in the medial prefrontal cortex attenuates anxiety: regional and receptor specificity and mechanism of action. Neuropharmacology, under review.

Albin-Brooks, C.C., Nealer, C.A., Sabihi, S., Haim, A., Leuner, B., 2017. The influence of parity, pups, and oxytocin on cognitive flexibility during the postpartum period. Hormones and Behavior, 89, 130-136.

vii

Leuner, B., Sabihi, S., 2016. The birth of new neurons in the maternal brain: hormonal regulation and functional implications. Frontiers in Neuroendocrinology, 41, 99-113.

Sabihi, S., Dong, S.M., Durosko, N.E., Leuner, B., 2014. Oxytocin in the medial prefrontal cortex regulates maternal care, maternal aggression and anxiety during the postpartum period. Frontiers in Behavioral Neuroscience, 8, 258.

Sabihi, S., Durosko, N.E., Dong, S.M., Leuner, B., 2014. Oxytocin in the prelimbic medial prefrontal cortex reduced anxiety-like behavior in female and male rats. Psychoneuroendocrinology 45, 31-42.

Fields of Study

Major Field: Psychology

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Table of Contents

Abstract ...... ii

Dedication ...... v

Acknowledgments ...... vi

Vita ...... vii

Publications ...... vii

Fields of Study ...... viii

Table of Contents ...... ix

List of Figures ...... xi

PART 1 ...... 1

Chapter 1: Introduction ...... 2

Chapter 2: Oxytocin in the medial prefrontal cortex attenuates anxiety: regional and receptor specificity ...... 41

Chapter 3: Oxytocin in the medial prefrontal cortex attenuates anxiety: mechanism of action ...... 61

PART 2 ...... 80

Chapter 4: Introduction ...... 81 ix

Chapter 5: Oxytocin in the medial prefrontal cortex regulates maternal anxiolysis during the postpartum period ...... 94

Chapter 6: Can the postpartum reduction in anxiety also be affected by GABA manipulations in the mPFC? ...... 109

Chapter 7: Conclusions ...... 133

References ...... 136

List of Figures

Figure 1: Few new pharmacotherapies for the treatment of anxiety have been developed since the 1940s. The cumulative FDA approvals of medications with an indication for anxiety (blue line) are compared to those for medications with an indication for hypertension (dark blue line), a more thoroughly understood condition. In addition to the comparatively slow rate of overall approvals of anxiolytics, a lesser number of mechanistically distinct targets have been identified for the treatment of anxiety than hypertension during the past 75 years (inset). The relative paucity of pharmacological strategies for the treatment of anxiety disorders and the imperfect efficacy of these drugs betrays a need for a more thorough understanding of the neural substrates of anxiety. Figure adapted from “Resolving the neural circuits of anxiety,” by G. G. Calhoon and K.M. Tye, 2015, Nature Neuroscience, volume 18, No. 10. Copyright 2015 by Nature Publishing Group. Adapted with permission...... 5 Figure 2: Schematic representation of mPFC regions (blue = Cg1, purple = PL, green = IL; adapted from Paxinos and Watson 1998)...... 12 Figure 3: Prefrontal control of anxiety and fear expression and extinction. Anxiety and fear excitation involves PL projections back to basolateral amygdala (BA), whereas anxiety and fear inhibition involves IL projections to amygdala-intercalated cells (ITC). In turn, BA excites neurons in the medial division of the central nucleus of the amygdala (CeM) to produce anxiety and fear responses, while ITCs inhibit these amygdala output neurons thereby inhibiting anxiety and fear responses. Thus, the same stimulus can signal either high anxiety and fear (red) or low anxiety and fear (green) states in the appropriate circumstances. Figure adapted from “Prefrontal control of fear: more than just extinction,” by R. Sotres-Bayon and G.J. Quirk, 2010, Current Opinion in Neurobiology, volume 20, No. 2. Copyright 2010 by Elsevier. Adapted with permission...... 14

Figure 4: Schematic illustration of the GABAAR and its associated binding sites. The receptor is pentameric, being composed of two α, two β, and one γ subunit. GABAA receptors contain recognition sites for a variety of clinically relevant drugs. The binding of GABA in two GABA binding sites at the interface between α and β subunits open the receptor-associated chloride (Cl−) channel. The benzodiazepine binding site is located at the interface between α and γ2 subunits. Barbiturates, ethanol, and neurosteroids bind to sites in the membrane-spanning transmembrane regions of the subunits. Figure adapted from “Regulation of GABAA receptor subunit expression by pharmacological agents,” by xi

M. Uusi-Oukari and E.R. Korpi, 2010, Pharmocological Reviews, volume 62, No. 1. Copyright 2010 by The American Society for Pharmacology and Experimental Therapeutics...... 36 Figure 5: OT in the PL mPFC decreases anxiety-like behavior. (a) Schematic representation of mPFC regions in which bilateral cannulae were implanted (blue = Cg1, red = PL, black = IL; adapted from Paxinos and Watson 1998). In the EPM, (b) the number of open arms entries and (c) the percentage of time spent in the open arms was greatest in the group that received 1.0 µg OT in the PL region of the mPFC. (d) Locomotor activity, as measured by the number of closed arm entries, was not altered by infusion type or region of infusion. (e) In the SI test, the group infused with the 1.0 µg dose of OT in the PL mPFC spent a greater amount of time interacting with a novel conspecific compared to all other groups which did not differ from each other. * Indicates a significant interaction (p < 0.05) followed by post-hoc analyses, ** Indicates a trend (p = 0.07) for an interaction. Bars represent mean + SEM...... 52 Figure 6: OT acting on the OTR in the PL mPFC decreases anxiety-like behavior in the SI test. The AVPR-A+OT and S+OT groups spent more time interacting with an unknown conspecific than the group treated with OTR-A+OT indicating that the anxiolytic effect of OT in the PL mPFC was blocked by pretreatment with an OTR-A, but not an AVPR-A. The S+S, OTR-A+OT, OTR-A+S and AVPR-A+S groups all showed similar levels of anxiety-like behavior in the SI test. Bars represent mean + SEM; *p < 0.05...... 53 Figure 7: OT infusion to the PL mPFC prior to an acute stressor does not affect plasma CORT levels. Males that were stressed, regardless of whether they received saline (a) or OT (b), displayed significantly higher levels of CORT compared to unstressed males immediately and 30 min after the stressor but by 60 min there were no differences among the groups. Bars represent mean + SEM; *p < 0.05...... 54 Figure 8: OT interacts with GABA in the PL mPFC to decrease anxiety-like behavior. In the EPM, the group that received an infusion of OT in the PL mPFC (a) made more open arm entries and (b) spent a greater percentage of time in the open arms unless they also received an mPFC infusion of the GABAA antagonist, bicuculline methiodide (BIC) which by itself did not affect anxiety-like behavior in the EPM. (c) Locomotor activity (closed arm entries) was not altered by infusion type. (d) In the SI test, OT increased time spent interacting with an unknown stimulus rat, but BIC blocked this anxiolytic effect. Bars represent mean + SEM; *p < 0.05...... 71 Figure 9: Males infused with OT in the PL mPFC display decreased anxiety-like behavior and increased activation of GABAergic cells in the PL mPFC. Males infused with OT to the PL mPFC spent more time in the open arms (a) and had more open arm entries (b) in the EPM when compared to males infused with saline. Locomotor activity was not affected by drug infusion (c). OT infusion to the PL mPFC followed by EPM exposure resulted in a greater number of c-Fos (d) but not GAD67 (e) expressing cells in the PL mPFC. Further, infusion of OT yielded a significant increase in the number (f) and percentage (g) of GAD67+ neurons expressing c-Fos in the PL mPFC. (h) xii

Confocal images of a cell positive for c-Fos (green, top), GAD67 (red, middle), and GAD67 colabeled with c-Fos (bottom). Bars represent mean + SEM.* p < 0.05...... 73 Figure 10: OT in the PL mPFC reduces anxiety-like behavior and alters neuronal activation in the amygdala. In the EPM, OT increased both (a) the number of open arm entries and (b) the percentage of time spent in the open arms without altering (c) the number of closed arm entries. (d) Schematic representation of regions throughout the BLA and CEA in which c-Fos counts were taken. Grey shaded regions = CEA; black shaded regions = BLA (adapted from Paxinos and Watson, 1998). Following exposure to the EPM, c-Fos expression in the BLA was reduced in rats that received an infusion of OT in the PL mPFC (e). In contrast, EPM exposure increased c-Fos expression in the CEA of rats that received OT in the PL mPFC (f). Scale bars = 100µm. (h). Bars represent mean +SEM, *p< 0.05...... 75 Figure 11: Schematic representation of mPFC cannula placements. Cannula tip placements were in the prelimbic region (PL) of the mPFC (AP: +3.2 mm, ML: ±0.5 mm, DV: -3.2 mm). Each dot indicates an individual subject. Infusions were bilateral but are represented unilaterally. Cannula placements for virgin females receiving an infusion of 0.1µg/1µl OTR-A, 0.5µg/1µl OTR-A, or saline (a). Cannula placements for postpartum females receiving an infusion of 0.1µg/1µl OTR-A, 0.5µg/1µl OTR-A, or saline (b). Animals with missed cannula placements in IL or the ventricle were excluded from analysis. Adapted from Paxinos and Watson, 1998...... 102 Figure 12: Blocking OTR in the PL mPFC enhances postpartum anxiety, but has no effect on anxiety in virgin females. Postpartum females infused with saline or the lower dose of the OTR-A in the mPFC spent a greater percentage of time in the open arms (a) and made more open arm entries (b) as compared to virgins. In contrast, postpartum females receiving the higher 0.5 µg/µl dose of OTR-A displayed a decrease in the percentage of time spent in the open arms (a) and made fewer open arm entries (b) as compared to saline or low dose infusion dams. Locomotor activity, as measured by the number of closed arm entries (c), was not altered. None of the virgin groups differed significantly from one another (a,b,c). Bars represent mean + SEM; ** P < 0.01 postpartum saline and postpartum 0.1 µg/µl OTR-A vs all other groups...... 105 Figure 13: Schematic representation of mPFC cannula placements for Experiment 1 and 2. Cannula tip placements were in the prelimbic region (PL) of the mPFC (AP: +3.2 mm, ML: ±0.5 mm, DV: -3.2 mm). Each dot indicates an individual subject. Infusions were bilateral but are represented unilaterally. Cannula placements for virgin and postpartum females receiving an infusion of 2.5 ng BIC, or saline (left). Cannula placements for postpartum females receiving an infusion of 0.5 ug muscimol, or saline and either having no separation from their pups or 4h of separation (right). Animals with missed cannula placements in Cg1 or the ventricle (indicated by a red X) were excluded from analysis. Adapted from Paxinos and Watson, 1998...... 117

Figure 14: Blocking GABAAR in the mPFC prevents the postpartum reduction in anxiety, but has no effect on anxiety in virgin females. Postpartum females infused with saline in the mPFC spent a greater percentage of time in the open arms (a) and made xiii more open arm entries (b) as compared to virgins and BIC infused dams indicating reduced anxiety-like behavior. In contrast, postpartum females receiving BIC (2.5 ng) in the mPFC displayed a decrease in the percentage of time spent in the open arms (a) and made fewer open arm entries (b) as compared to saline infused dams and did not differ from virgin groups. Locomotor activity, as measured by the number of closed arm entries (c), was not altered. Neither of the virgin groups differed significantly from one another on any measure (a,b,c). Bars represent mean + SEM; * p < 0.05 vs all other groups. ... 122 Figure 15: Attenuated anxiety during the postpartum period is prevented by mother-pup separation and restored by agonizing GABAAR in the mPFC. In the EPM, postpartum females that were separated from their pups for a period of 4 h spent less time in the open arms (a) and made fewer open arm entries (b) as compared to all other groups indicating increased anxiety-like behavior. Muscimol (0.5ug/µl) into the PL mPFC reversed the anxiogenic effect of pup separation as evidenced by an increase in the percentage of time spent in the open arms (a) and more open arm entries (b). Locomotor activity, as measured by the number of closed arm entries, was not altered by pup separation or drug administration (c). Bars represent mean + SEM; *p < 0.05...... 124 Figure 16: Postpartum females separated from their pups show increased anxiety in the EPM and reduced activation of GABAergic neurons in the PL mPFC. Postpartum females separated from their pups spent less time in the open arms (a) and made fewer open arm entries (b) compared to mothers that were allowed continual contact with their pups. Locomotor activity, as measured by the number of closed arm entries (c), was not altered. Pup separation followed by EPM exposure did not alter the number of c-Fos (d) or GAD67 (e) expressing cells within the PL mPFC. However, in mothers with high levels of anxiety due to pup separation the number (f) and percentage (g) of GAD67+ neurons expressing c-Fos in the PL mPFC was reduced. (h) Confocal images of a cell positive for c-Fos (green, top), GAD67 (red, middle), and GAD67 colabeled with c-Fos (bottom). Bars represent mean + SEM; *p < 0.05...... 126 Figure 17: Pup manipulation followed by EPM exposure did not alter activation of PV neurons in the PL mPFC. Pup separation followed by EPM exposure did not alter the number of c-Fos (a) or PV (b) expressing cells within the PL mPFC. In addition, the number (c) and percentage (d) of PV+ neurons expressing c-Fos in the PL mPFC was unaffected by pup separation. (e) Confocal images of a cell positive for c-Fos (green, top), PV (red, middle), and PV colabeled with c-Fos (bottom). Bars represent mean + SEM...... 127

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PART 1

Chapter 1: Introduction

1.1 Anxiety Disorders in Humans

According to the National Institute of Mental Health (NIMH), anxiety disorders are among the most prevalent categories of mental illness experienced by Americans affecting about 40 million American adults, or about 18% in a given year (Kessler et al.

2005). The median age of onset is 11 years of age with a lifetime prevalence rate of

28.8% (Kessler et al. 2005). Additionally, women are 60% more likely than men to experience an anxiety disorder over their lifetime (Kessler et al. 2005). When left untreated, anxiety symptoms can persist and are associated with significant impairments in functioning, poor quality of life, and a huge economic burden estimated to be more than $42 billion a year in the United States (Greenberg et al. 1999). As such, anxiety disorders are devastating in their personal, societal, and financial costs.

There are several types of anxiety disorders; among them are panic disorders, social anxiety disorder, and phobias. The Diagnostic and Statistical Manual of Mental

Disorders, Fifth Edition (DSM-5) classifies anxiety disorders into three high-level categories: anxiety disorders, obsessive-compulsive and related disorders, and trauma- and stressor-related disorders (American Psychiatric Association Association 2013). All three categories of anxiety disorders are marked by a frequent negative emotional state

2 characterized by feelings of worry and apprehension accompanied by specific somatic, cognitive, and behavioral manifestations. Anxiety is also common during the postpartum period (Pawluski et al. 2017) but this will be considered in PART 2.

It is important to note that anxiety disorders are distinguishable from fear in that there is a nonspecific state of heightened awareness and apprehension, whereas fear is directed at a specific identified threat (Nuss 2015). Additionally, fear is typically defined as a phasic and abrupt flight-or-flight response while anxiety is defined by a more prolonged state of tension, worry, and apprehension (Barlow 2000). Anxiety behaviors are part of the normal behavioral repertoire and are of value as a defense mechanism.

However, when anxiety becomes excessively severe or frequent and exists in inappropriate contexts, it can interfere with normal daily functioning and is thus considered pathological. The DSM-5 diagnostic criteria state that anxiety should be considered pathological when “the anxiety, worry, or physical symptoms cause clinically significant distress or impairment in social, occupational, or other important areas of functioning” (American Psychiatric Association 2013).

Exposure-based behavioral interventions currently represent the “gold standard” for treating anxiety disorders (Hofmann 2007). However, this is not effective in all patients, and pharmacotherapies originally designed for depression or anxiety, such as antidepressants and benzodiazepines, can also be used—either alone or in addition to behavioral therapies. A major problem with both treatment options is that many patients achieve only partial remission of symptoms or show a high rate of relapse (Blanco et al.

2002; Blanco et al. 2013). Therefore, development of novel pharmacotherapies is required.

A review by Calhoon and Tye (2015) points out that since the 1940s there has been very little progress in the advancement of new pharmacotherapies for the treatment of anxiety disorders. This is due at least in part to our limited understanding of the neural mechanisms underlying anxiety disorders. As such, the number of FDA approvals of medications and the number of mechanistic targets for the treatment of anxiety has remained scarce (Fig. 1). Thus, it is imperative that we continue to strive for a better understanding of the specific neural mechanisms underlying anxiety disorders and the mechanisms of action by which effective treatments reduce anxiety symptomology.

1.2 The Prefrontal Cortex and Anxiety

Many brain regions appear to be involved in the recognition and regulation of negative emotional stimuli and in the generation of cognitive, behavioral, or somatic responses to these stimuli. Indeed, anxiety disorders arise from disruptions in highly interconnected circuits that interpret and evaluate emotional stimuli from our environment (Calhoon and Tye 2015). Perturbations in this distributed circuit result in a misinterpretation of external stimuli as threatening, resulting in an inappropriate

5 emotional and behavioral response. Human imaging studies suggest that brain regions including the striatum, amygdala, nucleus accumbens, hippocampus, and insula play a large role in identifying anxiogenic stimuli and generating a behavioral response. These areas often have structural abnormalities and are hyperactive in anxiety patients compared to controls (Duval et al. 2015; Etkin and Wager 2007; Myers-Schulz and

Koenigs 2012; Price and Drevets 2012; Ressler and Mayberg 2007; Shin and Liberzon

2010). However, a particular set of corticolimbic structures appears to be most critical for the regulation of anxiety.

In particular, the amygdala has been greatly implicated in the regulation of anxiety. For example, in humans, bilateral lesions of the amygdala have been associated with deficits in recognizing negative emotions whereas electrical stimulation of this region leads to feelings of fear and anxiety (Adolphs et al. 1999; Lanteaume et al. 2007;

Nuss 2015). Additionally, human brain imaging studies consistently find that patients with anxiety disorders appear to activate the amygdala in response to a given stimulus more than non-anxious controls. Indeed, several neuroimaging studies have shown that patients with a variety of anxiety disorders exhibit heightened amygdala hyperactivity in response to negative emotional stimuli (Evans et al. 2008; Goldin et al. 2009; Phan et al.

2006; Rauch et al. 2003). Conversely, treatment of the disorder with selective-serotonin reuptake inhibitors, benzodiazepines, and cognitive behavioral therapy leads to extinction of this hyperactivation (Etkin and Wager 2007; Furmark et al. 2002; Harmer et al. 2006;

Paulus et al. 2005; Straube et al. 2006). The neural network modulating anxiety involves not only the amygdala but also several areas of the prefrontal cortex (PFC) and more

6 recent studies have begun to focus on connectivity between these brain regions in anxiety states. Generally, results suggest that there is decreased connectivity between emotion processing areas including the amygdala and emotion modulation regions such as the

PFC and anterior cingulate regions in anxiety disorders including social and generalized anxiety disorders, and post-traumatic stress disorder (Dodhia et al. 2014; Duval et al.

2015; Jovanovic and Norrholm 2011; Tromp et al. 2012). By contrast, generalized anxiety has further been associated with increased connectivity between the amygdala and insular cortex in response to emotional cues or an emotional task (Makovac et al.

2016; Mochcovitch et al. 2014).

From task-based studies of anxiety, the mPFC in humans can be roughly divided into two subregions relative to the genu of the corpus callosum- the dorsal mPFC

(dmPFC) and the ventral mPFC (vmPFC; Kim et al. 2011a). The dmPFC typically includes the supragenual anterior cingulate and the medial frontal gyrus, whereas the vmPFC includes the subgenual anterior cingulate and parts of the medial orbitofrontal cortex (Kim et al. 2011b). Consistent findings across many studies suggest that higher levels of anxiety are associated with both attenuated vmPFC activity and exaggerated dmPFC activity (Kim et al. 2011b; Schienle et al. 2011; Simmons et al. 2008; Straube et al. 2009). Early human lesion studies support the idea that the dmPFC is involved in human emotional processes as lesions were shown to produce apathy and emotional instability (reviewed in Heilbronner and Hayden 2016; Paus 2001), whereas electrical stimulation or hyperactivity of the dmPFC are associated with increased states of fear and anxiety (Bruhl et al. 2011; Caseras et al. 2010; Lueken et al. 2011; Patel et al. 2012;

Wager et al. 2009). Treatment with anxiolytics or cognitive behavioral therapy has been found to decrease hyperactivity in the dmPFC in response to anxiety-provoking situations

(Hoehn-Saric et al. 2004; Lipka et al. 2014; Nitschke et al. 2009). The vmPFC is also a key regulator of mood and anxiety (Myers-Schulz and Koenigs 2012; Price and Drevets

2012). The vmPFC serves to regulate negative affect via top-down inhibition of regions such as the amygdala and pathologically elevated levels of anxiety disorders result from deficient vmPFC-mediated inhibition of amygdala activity (Kim et al. 2011a; Kim and

Whalen 2009; Rauch et al. 2006). Furthermore, lesions of the vmPFC result in social disinhibition and have been associated with reduced likelihood of developing posttraumatic stress disorder and depression (Barrash et al. 2000; Koenigs et al. 2008).

Together, it is clear that although the dmPFC and the vmPFC have opposing influences, both play an important role in regulating anxiety.

1.3 Animal Models of Anxiety

Historically, animal models have provided insight into the neurotransmitter systems and brain circuitry underlying psychiatric illness, enabled the screening of potential therapeutics for efficacy, and guided the search for new pharmacotherapies. As such, research performed in rodents has been central to efforts to understand the neural mechanisms regulating anxiety. While there are differences in brain anatomy between humans and rodents, it is clear that many brain structures involved in limbic regulation of emotion are evolutionarily conserved from human to rodent (Davis 2004; Richardson-

Jones et al. 2010). Additionally, there are many fundamental physiological and

8 behavioral responses that are evolutionarily conserved between species. Thus, we can employ rodents to understand human behavior by studying their responses in order to elucidate the neural circuits underlying human conditions.

Rather than attempting to model a psychiatric disorder, neuroscientists focus on individual aspects of a disorder and use physical manifestations and measurable behaviors (Lapiz-Bluhm et al. 2008; Richardson-Jones et al. 2010; Walf and Frye 2007).

For rodent research, scientists focus on “state” anxiety, or, the behavioral state of enhanced arousal and vigilance in response to an anxiety-provoking stimulus. This is in contrast to “trait” anxiety which is often studied in humans wherein anxiety is a persistent state of heightened arousal in response to unspecific stimuli. Despite the contrast in state versus trait anxiety, the neural circuitry underlying both forms of anxiety in humans and rodents is fairly consistent (Adhikari 2014; Calhoon and Tye 2015; Etkin et al. 2011;

Likhtik et al. 2005; Tye et al. 2011). State anxiety can be studied in rodents because of their innate desire to avoid open areas and brightness, likely because this allows them to be more vulnerable to predators. Additionally, rodents are a naturally foraging, exploratory species, and exploration-based tasks exploit the conflicting tendencies to approach versus avoid a potentially dangerous area. As a measure of anxiety-like behavior, this pattern of avoidance has face validity, given that many human anxiety disorders are typified by a pervasive avoidance of an object or situation (American

Psychiatric Association 2013).

Approach-avoidance tasks that use the innate desire to avoid open areas and brightness are employed to measure anxiety-like behavior and take different forms in

9 different tests and include the open field, elevated plus maze, social interaction test, light- dark box, and elevated zero maze (Cryan and Holmes 2005; Lapiz-Bluhm et al. 2008;

Richardson-Jones et al. 2010; Treit 1985; Walf and Frye 2007). In tests such as the elevated plus maze, rats confined to an open arm of the maze have elevated plasma corticosterone levels compared to controls, confirming the aversive quality of the open arms (Pellow et al. 1985), suggesting high construct validity. Additional evidence of construct validity comes from pharmacological validation. For example, drugs that decrease anxiety in humans, such as benzodiazepines, decrease the innate aversion rodents have towards open spaces (Lapiz-Bluhm et al. 2008; Pellow et al. 1985; Walf and

Frye 2007). Finally, the elevated plus maze has predictive validity, in that a dependent measure such as open arm activity can predict behavior on a related measure such as time spent in the center of a brightly lit open field (File 1980; Walf and Frye 2007). Thus, with these paradigms, the anxiolytic and anxiogenic effects of pharmacological agents can be investigated with appropriate face, construct, and predictive validity.

1.4 The Rodent mPFC and Anxiety

For a mechanistic understanding of the neural underpinnings of anxiety, the ability to manipulate specific circuit components is required. Rodents are well suited for this purpose and through methods such as lesion, inactivation, and targeted manipulations via optogenetics, prior studies have confirmed that regions that play a role in human anxiety behavior also regulate anxiety-like behavior in rodents. Key loci mediating anxiety in rodents include the amygdala, bed nucleus of the stria terminalis, and ventral

10 hippocampus (Adhikari 2014; Calhoon and Tye 2015; Papez 1995; Price and Drevets

2012; Shin and Liberzon 2010). Thus, it is clear that just as in humans, anxiety-like behavior in rodents involves a broad interconnected neural circuit. This distributed circuit regulating anxiety also includes the medial prefrontal cortex (mPFC) which is a critical structure for evaluating threatening situations that can provoke anxiety and furthermore, regulate downstream limbic structures (Calhoon and Tye 2015). Although there remains some debate about whether rodents have a PFC with homology to that of primates (Heidbreder and Groenewegen 2003), there is evidence suggesting that the primate PFC and the rodent mPFC share similar anatomical as well functional characteristics.

The rodent mPFC contains two major types of neurons: pyramidal projection neurons and a diverse array of interneurons (Calhoon and Tye 2015; Markram et al.

2004). The projection neurons are excitatory and use glutamate as their primary neurotransmitter whereas the interneurons are inhibitory and use GABA as their primary neurotransmitter (Amaral 1991). The interneurons constitute approximately 20-25% of all neurons in the cortex and exist in all cortical layers (Amaral 1991).

The rat mPFC can be parsed into three distinct subregions on the basis of their cytoarchitectures, which are consistent with divergent functions among these regions

(Fig. 2; Van De Werd and Uylings 2014). The primary subdivisions of the mPFC in rodents include the anterior cingulate (Cg1, homologous to the human dmPFC region), prelimbic (PL; homologous to the human more dorsal regions of the dmPFC), and

11 infralimbic (IL; homologous to ventral regions of the human dmPFC and vmPFC) cortices.

Figure 2: Schematic representation of mPFC regions (blue = Cg1, purple = PL, green = IL; adapted from Paxinos and Watson 1998).

These areas receive inputs from midline thalamic nuclei, the basolateral amygdala

(BLA), and the hippocampus, and send reciprocal projections to BLA, central amygdala, as well as efferent projections to the striatum (Calhoon and Tye 2015; Vertes 2004). It is important to consider that the various subregions of the mPFC show different patterns of connectivity with subcortical and cortical structures and thus often differentially contribute to a variety of physiological and behavioral processes, including fear and anxiety (Hamani et al. 2010; Hoover and Vertes 2007; Pereira and Morrell 2011; Radley et al. 2006; Sierra-Mercado et al. 2011; Vertes 2004). For example, activation, lesion,

12 inactivation, and molecular approaches have shown that the PL subregion of the mPFC plays a role in regulating anxiety-like behavior, and specifically that increased activity of this region is positively correlated with anxiety-like behavior. This idea has been assessed and confirmed in a variety of rodent behavioral paradigms including the elevated plus maze, open field, and social interaction test (Gonzalez et al. 2000; Lacroix et al. 2000;

Maaswinkel et al. 1996; Resstel et al. 2008; Saitoh et al. 2014; Shah et al. 2004; Shah and

Treit 2003; Stack et al. 2010; Stern et al. 2010; Sullivan and Gratton 2002; Suzuki et al.

2016). The mechanism by which the PL region acts to increase anxiety-like behavior is likely due to its ability to modulate amygdalar responses via its excitatory projections to the BLA and the central nucleus of the amygdala (Hoover and Vertes 2007; Vertes 2004).

Thus, increased activation of the PL region results in greater activation of the amygdala, and thus increased anxiety-like and fear behavior (see Fig. 3).

Conversely, increased activity in the IL subregion inhibits downstream activity in the amygdala and inhibits fear and anxiety responses (Calhoon and Tye 2015;

Maaswinkel et al. 1996; Shah and Treit 2003; Vidal-Gonzalez et al. 2006). Increased activity in the IL cortex is associated with fear extinction and electrical stimulation of this region reduces freezing in test animals (Maroun 2013; Maroun et al. 2012; Quirk et al.

2000; Vidal-Gonzalez et al. 2006). The mechanism by which the IL region modulates fear, and in some cases anxiety-like behavior, is likely due to its ability to modulate amygdalar responses via its projections to a group of inhibitory cells called the intercalated cells (ITC) which are situated between the basolateral amygdala and the central nucleus of the amygdala (Hoover and Vertes 2007; Vertes 2004). Thus, activation

13 of the IL cortex results in inhibition of amygdalar output, and therefore reduced fear and anxiety-like behavior.

Figure 3: Prefrontal control of anxiety and fear expression and extinction. Anxiety and fear excitation involves PL projections back to basolateral amygdala (BA), whereas anxiety and fear inhibition involves IL projections to amygdala- intercalated cells (ITC). In turn, BA excites neurons in the medial division of the central nucleus of the amygdala (CeM) to produce anxiety and fear responses, while ITCs inhibit these amygdala output neurons thereby inhibiting anxiety and fear responses. Thus, the same stimulus can signal either high anxiety and fear (red) or low anxiety and fear (green) states in the appropriate circumstances. Figure adapted from “Prefrontal control of fear: more than just extinction,” by R. Sotres-Bayon and G.J. Quirk, 2010, Current Opinion in Neurobiology, volume 20, No. 2. Copyright 2010 by Elsevier. Adapted with permission.

In contrast to the IL and PL subregions of the mPFC, only a few studies have investigated the role of the rodent Cg1 subregion in these disorders. The Cg1 subregion also extensively connects with limbic structures (Etkin et al. 2011; Milad et al. 2006;

Price and Drevets 2012) and has been shown to play a role in the regulation of emotional behavior and modulation of negative mood states (Etkin et al. 2011; Myers-Schulz and

Koenigs 2012). For example, inactivation of the Cg1 is capable of reducing fear expression and neurons in this region are activated by fearful stimuli (Bissiere et al.

2008). Conversely, others have shown that stimulation of this region produces no discernible effects on fear (Vidal-Gonzalez et al. 2006). However, investigations into the role of the Cg1 subregion on depression seem to be consistent in their findings which suggest that this region does mediate depressive-like behavior (Bissiere et al. 2006; Kim et al. 2011c). Still, a definitive role of this region on anxiety-like behavior has yet to be established as some studies indicate that the Cg1 does mediate anxiety-like behavior

(Kim et al. 2011c) while others indicate that this region does not (Bissiere et al. 2006).

Nonetheless, it is clear that regional differences within the mPFC in mediating anxiety- like behavior exist (Bi et al. 2013; Bissiere et al. 2008; Jinks and McGregor 1997; Kim et al. 2011c; Sullivan and Gratton 2002).

1.5 Oxytocin

Storage, Release, and Signaling

Oxytocin (OT) is a nonapeptide hormone most widely known for its role in lactation and parturition. The word “oxytocin” was coined from the Greek words meaning “quick birth” after its uterine-contracting properties were discovered (Dale

1906) and shortly thereafter, the milk ejection property of OT was described (Ott 1910).

OT is present not only in the hypothalamic neurohypophyseal system as a hormone, but also in other areas of the central nervous system (CNS) where it exerts widespread

15 neuromodulatory effects on behavior (de Wied et al. 1993; McCarthy and Altemus 1997;

Wojciak et al. 2012).

OT was the first peptide hormone to have its structure determined and the first to be chemically synthesized in biologically active form (Du Vigneaud et al. 1953). OT is composed of nine amino acids (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-GlyNH2) with a sulfur bridge between the two cysteines. The structure of OT is very similar to another nonapeptide, arginine vasopressin (AVP), which differs from OT by only two amino acids. In the vertebrate brain, OT and AVP are synthesized in separate neuronal populations in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. As such, expression of AVP and OT in the PVN and SON occurs in strictly separate neuronal populations (Gainer 2012).

OT is stored in secretory vesicles called large dense-core vesicles (LDCV) along with its respective carrier proteins called neurophysins. Neurophysins are synthesized in the cell body as part of a precursor protein that also contains the OT sequence. Processing of this prohormone takes place in the LDCV which contains the enzymes for post- translational processing during its transport to the axon terminal (Stoop 2012). Thus, OT is synthesized as a non-glycosylated protein which undergoes endoproteolytic cleavage by aminopeptidases. The product of this cleavage is converted to the final nonapeptide,

OT (Burbach et al. 1995; Ebstein et al. 2012). As a result, release at the nerve endings includes the hormone, the carrier proteins, and residual bits of precursor (Stoop 2012).

The LDCV containing OT are distributed in the nerve endings and also in the soma, dendrites, and axonal varicosities, and are released by calcium (Ca2+)-dependent

16 exocytosis in vivo and in vitro by physiological and pharmacological stimuli (Benarroch

2013). OT can also undergo somatodendritic release; this requires high-frequency stimulation and involves both Ca2+ influx and mobilization of intracellular Ca2+ stores

(Tobin et al. 2011).

Neuroanatomical and immunocytochemical studies have shown that in the hypothalamus, the PVN and the SON contain cell bodies of two kinds of oxytonergic neurons: magnocellular neurons and parvocellular neurons. The magnocellular neurons synthesizing OT send their projections to the posterior pituitary. Here, the peptide is released into the blood circulation where OT acts as a hormone and plays a major role in parturition and lactation (Benarroch 2013; Poulain and Wakerley 1982). In the PVN and

SON, magnocellular OT neurons release OT from the soma and dendrites, thus acting as an autocrine signal which controls activity of the magnocellular neurons themselves.

Conversely, the axons of the parvocellular neurons are widely distributed throughout the

CNS including the amygdala, septum, hippocampus, ventral tegmental area, medial preoptic area, bed nucleus of the stria terminalis, frontal cortex, brainstem, pons, medulla, and spinal cord (Gimpl and Fahrenholz 2001). At the axonal level, OT from parvocellular neurons may be released at presynaptic terminals and at nonterminal axonal varicosities and can thus act in synaptic manner or in a paracrine manner and diffuse to targets via the extracellular fluid. It is important to keep in mind that in the CNS, OT acts as a neuromodulator. As such, OT is co-released with other classical neurotransmitters such as glutamate and GABA at higher frequencies of stimulation (Ludwig and Leng 2006).

Microdialysis studies have shown that social stimuli, including sexual behavior and stress, trigger OT release via diffusion or targeted release from magnocellular OT neurons to the CNS (Landgraf and Neumann 2004). Local release from dendrites and subsequent diffusion of OT have been proposed to be an important route of action, however it is unclear how OT spreads after release, to where it diffuses, how quickly, and at what concentrations. Alternatively, because axonal fibers containing OT have been found in a large number of brain areas (Buijs et al. 1978; Knobloch et al. 2012;

Sofroniew 1983; Tobin et al. 2011), it is possible that OT from the hypothalamus arrives at various brain regions by axonal release from OT containing fibers specifically targeting brain areas expressing OT receptors. Thus, centrally, OT participates in classical synaptic transmission via long-range axonal projections as well as volume transmission by diffusion to nearby or remote receptors via the extracellular fluid (de

Vries et al. 2012; Neumann and Landgraf 2012; Stoop 2012). This allows for OT to communicate with neurons and modulate different brain structures in a multimodal manner, both through a “wired,” axonal , fast, and focal manner as well as an “unwired,” diffusive, slow, and global fashion (Landgraf and Neumann 2004).

OT has a short half-life of about 20 min in cerebrospinal fluid (CSF) (Ludwig and

Leng 2006) and much shorter in blood (only 1-2 min) (Gimpl and Fahrenholz 2001). OT concentrations in the extracellular fluid of the SON are calculated to be >100-1,000 fold higher than the basal OT concentration in plasma. Plasma OT does not readily cross the blood-brain barrier, however, if pharmacological doses are administered peripherally, the exogenous neuropeptide may reach the brain parenchyma in minute, but functionally

18 significant amounts (Landgraf and Neumann 2004). Centrally released OT is degraded by aminopeptidases within brain tissue and then enters the CSF, where it is cleared into the circulation. Interestingly, the aminopeptidases, can produce shorter peptides from OT that in some cases may have biological effects (Ludwig and Leng 2006). These shorter peptides have been shown to facilitate avoidance behavior of rats at concentrations 1000x smaller than AVP, although their direct efficiency as neuromodulators is much smaller than AVP (Burbach et al. 1995). Moreover, these shorter OT fragments could cross the blood-brain barrier more easily.

Oxytocin Receptors

OT receptors (OTRs) are present in the smooth muscle of the uterus and myoepithelial cells of the mammary gland. OTRs are also abundant in the central nervous system (CNS) on both neurons and astrocytes (Zingg and Laporte 2003). The distribution of OTR expression within the CNS of numerous species has been examined using in situ hybridization (Ostrowski 1998; Yoshimura et al. 1993), transgenic mouse models (Gould and Zingg 2003), and receptor autoradiography and these have shown widespread distribution of OTR throughout the brain. In rodents, OTRs are prominent in the olfactory bulb, central (CEA) and lateral (LA) amygdala, CA1 region of the hippocampus, frontal cortex, ventromedial hypothalamus, BNST, nucleus accumbens, VTA, autonomic nuclei of the brainstem, and dorsal horn (Benarroch 2013; Gimpl and Fahrenholz 2001; Insel et al. 1991). In the adult rat, there are no major differences in receptor distribution between male and female brains (Gimpl and Fahrenholz 2001). It is important to note that the

19 distribution of OTRs in the human brain differs slightly from that of rodents (Tribollet et al. 1997) and have been detected in the basal nucleus of Meynert, vertical limb of the diagonal band of Broca, ventral part of the lateral septal nucleus, preoptic/anterior hypothalamic area, posterior hypothalamic area, globus pallidus, and ventral pallidum

(Tribollet et al. 1997). Interestingly, the distribution pattern of OT binding sites is markedly different from that of binding sites for AVP; whenever both are present in the same area, binding sites for OT and AVP are located in different regions of that particular area (Gimpl and Fahrenholz 2001). The OTR is relatively unselective with only about a

10-fold higher affinity of the receptor for OT than for AVP (Smeltzer et al. 2006;

Tribollet et al. 1997). Thus, AVP can act as a partial agonist on the OTR. However, to elicit the same response as induced by OT, ~100-fold higher concentrations of AVP would be necessary (Gimpl and Fahrenholz 2001).

OT is currently known to have only one receptor isoform. This receptor belongs to the rhodopsin-type (class I) G protein- coupled receptor (GPCR) family. These GPCRs are coupled to various intracellular signaling cascades and activation of these cascades brings about biochemical and transcriptional changes that account for immediate and long-term neuromodulatory effects. Specifically, the OTR is coupled to three different G proteins, Gq/11, Gi/o, and Gs (Stoop 2012; van den Burg and Neumann 2011) . Signaling via

Gq (most common) leads to activation of the phospholipase-Cβ (PLC β) cascade which stimulates Ca2+ release from intracellular stores via inositol triphosphate production (IP3) and then activates diacyl glycerol (DAG). Gq also stimulates the activity of two mitogen- activated protein kinase (MAPK) cascades, leading to phosphorylation of extracellular

20 signal-regulated kinase 1/2 (ERK 1/2; Zhong et al. 2003), or the related MAPK p38

(Devost et al. 2008). This pathway underlies uterine smooth muscle cell contraction

(Alberi et al. 1997), and in neurons can inhibit inward rectifying currents (Gravati et al.

2010). In neurons, OT can also activate inward rectifying currents through Gi/o protein, thus modulating neuronal excitability (Gravati et al. 2010). Further, Gi signaling mobilizes intracellular Ca2+ stores independent of IP3, and only activates p38 (Hoare et al. 1999). In addition to Gq and Gi signaling, OT can activate adenylate cyclase via Gs and activate cyclic adenosine monophosphate production (Campbell and Macqueen 2004;

Stoop 2012). It is possible that these various signaling pathways are differentially expressed in neuronal versus peripheral tissues. Importantly, endogenous OT is able to bind and activate any OTR, regardless of what G protein it is coupled to (Stoop, 2012).

Via these mechanisms, OT may indirectly affect excitatory or inhibitory synaptic transmission.

When receptors are persistently stimulated with agonists, they desensitize.

Desensitization is a two-step process which consists of phosphorylation and subsequent arrestin binding. First, the receptor uncouples from G proteins and undergoes endocytosis, internalization, or sequestration. OTRs are phosphorylated by G protein coupled receptor kinase-2, then bind beta-arrestin and are endocytosed via clathrin-coated vesicles. After internalization, they do not recycle back to the cell surface (Gimpl and

Fahrenholz 2001). This internalization is thought to underlie the rapid desensitization that may occur upon OTR activation (Smith et al. 2006). Interestingly, whereas endogenous

OT can activate OTRs regardless to which G protein they are coupled, specific agonists

21 and antagonists may exhibit differential affinity to OTRs, depending on the specific G protein to which they are coupled and therefore not cause such internalization (Stoop

2012).

Sex Differences in the Oxytocin System

Although OT was initially characterized as a “female reproductive hormone,” it is now clear that it is synthesized, distributed, and binds fairly equally in both males and females. Overall, there seems to be few sex differences in the central OT system, and when differences exist, they are particular to specific brain regions and species. For example, some work in voles and rats has found sex differences in OT-immunoreactive neurons within hypothalamic neurons suggesting that a few species of female rodents, have more OT neurons in regions including the PVN and SON (Carter 2007; Smeltzer et al. 2006; Xu et al. 2010; Zingg and Laporte 2003). However, numerous studies in several rodent species, non-human primates, and humans report an absence of sex differences in

OT mRNA expression or immunoreactivity in brain regions including the PVN, SON, and BNST (Caffe et al. 1989; Dumais et al. 2013; Ishunina and Swaab 1999; Wang et al.

1997).

In regards to sex differences in brain OT fibers, a quantitative comparison between sexes among rodent species is very limited. The work that has been completed indicates that in some species of mice and mandarin voles, females have more OT- immunoractive fibers compared to males in the lateral septum and BNST (Haussler et al.

1990; Qiao et al. 2014). However, evidence from prairie voles and macaques indicate no

22 sex differences in OT fibers in regions including the amygdala, solitary tract, nucleus accumbens, and spinal cord (Caffe et al. 1989; Lim et al. 2004). A very recent and thorough study in rats is one of the first to quantitatively analyze age and sex differences in AVP and OT immunoreactivity in the “social behavior neural network-” a circuit of interconnected brain regions including the amygdala, BNST, lateral septum, medial preoptic area, and hypothalamus (DiBenedictis et al. 2017). In contrast to AVP, there were no observed age or sex differences in OT-ir fibers or cell bodies in any of the 22 brain regions examined, confirming some prior work.

Despite the lack of sex differences in OT synthesis in the brain, in humans, females show higher concentrations of OT in cerebral spinal fluid compared to men suggesting that brain OT release may be higher in females (Altemus et al. 1999; Ishunina and Swaab 1999). Still, it is unclear as to whether sex differences in brain OT synthesis reflect differences in OT release in the periphery. In humans, studies examining sex differences of OT plasma concentrations have been inconsistent with many reporting no differences or inconclusive results (Grewen et al. 2005; Weisman et al. 2012; Zhong et al.

2012). Overall, there seems to be a lack of sex difference in OT synthesis in the brain in most species that are studied (see Dumais and Veenema 2016 for review).

The distribution of OTR is a critical aspect of the OT system as the pattern of receptor expression indicates which brain regions are sensitive to the local release of OT.

Similar to OT, sex differences in OTR expression and binding are brain region and species specific, although consistent differences have not been found (Dumais et al. 2013;

Dumais and Veenema 2016; Insel et al. 1991; Tribollet et al. 1990). Studies in rodents

23 have generally found that in males, OTR density is usually higher in forebrain regions compared to females (Dumais et al. 2013). In other regions, such as the central amygdala, females have been shown to have increased OTR density compared to males (Bales et al.

2007). However, many studies have found no difference in OTR density between males and females (Duchemin et al. 2017; Mitre et al. 2016; Tribollet et al. 1990). Indeed, two very recent studies demonstrated that within the forebrain and mPFC regions, there are no differences in OTR density between male and female prairie voles or mice (Duchemin et al. 2017; Mitre et al. 2016).

It is important to note that the OTR is regulated by gonadal hormones which may account for some of the sex differences observed in OTR density. Indeed, estrogens upregulate OTR expression, OT release from the hypothalamic neurons, and OT binding in the amygdala (Young et al. 1998). Female rats treated neonatally with testosterone

(converted to estrogen) show higher levels of OTR binding in the ventromedial hypothalamus, BNST, and medial amygdala compared to control female Sprague-Dawley rats (Uhl-Bronner et al. 2005) and gonadectomy of adult rats decreases OTR binding in areas such as the BNST and caudate putamen in males and females (Tribollet et al. 1990).

Additionally, in female rats, OTR expression increases in a number of brain areas just prior to parturition (Meddle et al. 2007) accompanied by an increase in gonadal hormones, particularly estrogen (Rosenblatt et al. 1988). However, sex differences have been found in OTR density in regions that may not be sensitive to gonadal steroids such as the caudate putamen and hippocampal CA1 regions (Dumais et al. 2013), suggesting

24 that hormonal influences on OTR do not explain all of the sex differences that have been observed.

Functions of Oxytocin

OT was initially known for its endocrine effects and the role it plays in parturition and lactation (Pedersen et al. 2006) but it is now clear that OT regulates a variety of functions including stress, social behavior, and anxiety (Heinrichs et al. 2009; Neumann

2008; Neumann and Landgraf 2012; Uvnas-Moberg and Petersson 2005).

Stress. In both rodents and humans, OT is commonly regarded as a stress hormone as it is released in response to a variety of stressful conditions (Heinrichs et al.

2001; Neumann et al. 2000b; Onaka et al. 2012). However, OT also buffers stress hormone effects on the brain and body (Neumann 2002). Specifically, OT reduces blood pressure (Petersson et al. 1996; Petersson et al. 1999b), attenuates the hypothalamic- pituitary-adrenal (HPA) axis response to stress including the release of stress hormones such as ACTH and glucortcortiods (Ditzen et al. 2009; Heinrichs et al. 2003; Smith et al.

2016; Smith and Wang 2013; Windle et al. 1997), and dampens stress-induced neuronal activation (Windle et al. 2004). Adult male and female rats show elevated levels of plasma OT following exposure to a variety of stressors, including restraint, immobilization, footshock, and forced swim (Hashimoto et al. 1989; Laguna-Abreu et al.

2005; Wotjak et al. 1998; Yoshida et al. 2009), however, sex differences in rodents have been noted in stress-induced OT responses in adults, typically with females showing greater responses than males (Carter and Lightman 1986; 1987; Williams et al. 1985).

Similarly, lactating females exhibit a reduction in the HPA response to stress which has been attributed to suckling-induced increases in OT release (Febo et al. 2009; Heinrichs et al. 2001; Lonstein 2005b; Neumann et al. 2000a). In humans, many studies have suggested an inhibitory influence of OT on stress-responsiveness of the HPA axis (Ditzen et al. 2009; Heinrichs et al. 2003; Heinrichs et al. 2001; Uvnas-Moberg 1998; Uvnas-

Moberg and Petersson 2005). One in particular, demonstrated that men who receive intranasal OT and are then exposed to a social stress test exhibit lower salivary cortisol concentrations than those receiving saline (Heinrichs et al. 2003). Moreover, in lactating women, a suppression of the endocrine stress response has been observed if breastfeeding starts 30-60 min prior to stress exposure (Heinrichs et al. 2001). However, it is important to note that few studies in this area have investigated mixed sex samples, and the few that have tested both men and women have found no robust sex differences

(Ditzen et al. 2009; Linnen et al. 2012; Simeon et al. 2011). These findings suggest that

OT may be playing different roles in male versus female responses to stressful interactions, although there are many mixed findings and results are difficult to interpret.

Social behavior. Behaviorally, OT plays a prominent role in a wide range of social functions that, in rodents, includes maternal care (Bosch and Neumann 2012;

Pedersen et al. 2006), sexual behavior (Bale et al. 2001), and pair boding (Lee et al. 2009;

Norman et al. 2010). In both males and females, OT also plays a role in social memory

(Engelmann et al. 1998), social preference (Lukas et al. 2011), social cognition (Popik and van Ree 1991), social play (Bredewold et al. 2014), and social anxiety (Sabihi et al.

2014b). Classic studies have investigated OT-mediated sex differences in pair bonding

26 and parental behavior in voles. In female prairie voles, parenting behavior and pair bonding is more dependent on OT while male parenting behavior and bonding in this species relies more on OT’s counterpart, AVP (Bales and Carter 2003; Bales et al. 2004).

Outside of voles, there are few comparative studies that have investigated the behavioral function of this peptide in both males and females. ICV administration of OT reverses social defeat-induced social avoidance and improves social recognition in male but not female rats (Engelmann et al. 1998; Lukas and Neumann 2014; Lukas et al. 2011).

However, ICV administration of an OTR-A impairs social recognition in both male and female rats (Engelmann et al. 1998). Another study examining the effects of ICV OT in males and females demonstrated that OT infusion is accompanied by increased aggression in females, but not in males (Bales and Carter 2003).

Similarly, a large number of human studies have also demonstrated the pro-social effects of OT including trust, social support, social cognition, and emotion recognition

(Bethlehem et al. 2013; Heinrichs and Domes 2008; Meyer-Lindenberg et al. 2011).

However, just as in rodents, there is increasing evidence that OT can produce opposite effects in men and women. Behavioral studies have reported that intranasally applied OT facilitates positive social judgements, social approach, kinship, and altruism in females

(Fischer-Shofty et al. 2013; Hoge et al. 2014; Preckel et al. 2014; Scheele et al. 2014).

Contrastingly in males, intranasally applied OT can facilitate negative social judgements, social avoidance, competition, and selfishness (Fischer-Shofty et al. 2013; Hoge et al.

2014; Scheele et al. 2012; Scheele et al. 2014). Together, these results suggest that OT

27 may be playing different roles in the modulation of social information processing between males and females.

Anxiety. In both rodents and humans, OT has been further implicated in the regulation of anxiety (Neumann and Landgraf 2012; Neumann et al. 2000a; Neumann et al. 2000b; Sabihi et al. 2014a; Sabihi et al. 2014b; Slattery and Neumann 2010; Waldherr and Neumann 2007) which is the focus of this dissertation. Numerous studies in rodents have shown that OT reduces anxiety. OT knockouts present with an anxious phenotype indicating an involvement of endogenous OT (Mantella et al. 2003). Endogenous OT is also directly involved in anxiolysis during the postpartum period (Bosch and Neumann

2012) as well as in males after mating (Waldherr and Neumann 2007). Peripheral or central administration of OT to male and female rats reduces anxiety-related behavior in the elevated plus maze (EPM), open field (OF), and other related tests measuring inherent anxiety suggesting that both sexes are equally sensitive to the anxiolytic effects of exogenous OT in this region (Ayers et al. 2011; Bale et al. 2001; Blume et al. 2008;

Figueira et al. 2008; Mak et al. 2012; McCarthy et al. 1996; Sabihi et al. 2014b; Uvnas-

Moberg et al. 1994; Windle et al. 1997; Yoshida et al. 2009), although some sex-specific effects have been reported in rodents after chronic infusion of OT (Slattery and Neumann

2010). These anxiolytic effects of OT have been localized to actions within the PVN of males and central amygdala of females (Bale et al. 2001; Knobloch et al. 2012; Smith et al. 2016) as well as the PL mPFC of both males and females (Sabihi et al. 2014b).

Likewise, intranasal administration of OT to humans has been shown to suppress anxiety responses in healthy and clinical populations (de Oliveira et al. 2012; Guastella et

28 al. 2010; Heinrichs et al. 2003). While human studies have consistently indicated an anxiolytic effect of OT (Heinrichs et al. 2003; Kirsch et al. 2005; Meyer-Lindenberg et al. 2011; Striepens et al. 2012), many of these have only investigated the effects in males.

Neuroimaging studies suggest OT’s anxiolytic effects in humans may be mediated by the sexually dimorphic actions of OT within the anxiety circuitry (Domes et al. 2007; Domes et al. 2010). For example, compared to placebo, OT increased reactivity in the amygdala to fearful facial expressions in a healthy sample of women (Bertsch et al. 2013; Domes et al. 2010; Lischke et al. 2012), although studies investigating the effects of intranasal OT in postpartum women have reported dampened amygdala activity (Riem et al. 2011;

Riem et al. 2012; Rupp et al. 2014) suggesting differential effects based on reproductive status. The increased amygdala reactivity under OT found in women contrasts with studies in men which consistently report attenuated amygdala responses following OT treatment (Heinrichs et al. 2003; Kirsch et al. 2005; Petrovic et al. 2008) indicating sex-specific modulation of the salience and processing of social cues. The reasons for such differences may be manifold as no fMRI study so far has systematically addressed the question of differential effects of OT on brain activity in men and women within a single experimental protocol.

In the rodent literature, OT has also emerged as a neuropeptide that promotes social approach behavior and helps to overcome social anxiety (Bales and Carter 2003;

Lukas et al. 2011; Mitre et al. 2016; Veenema and Neumann 2008; Witt et al. 1992). As such, behavioral tests in rodents for anxiety that measure social approach behavior, such as the social interaction (SI) test (Lapiz-Bluhm et al. 2008), have shown that ICV OT

29 administration (Lukas et al. 2011; Norman et al. 2010) and site-specific OT administration to the PL mPFC (Sabihi et al. 2014b) reduces anxiety in social situations and thus, increases the amount of time spent in social interaction with a conspecific.

Similarly, in humans, OT is recognized as a social neuropeptide and exogenous intranasal

OT administration mediates social cognition and increases trust, perhaps secondary to effects on anxiety (Baumgartner et al. 2008; Heinrichs and Domes 2008; Meyer-

Lindenberg et al. 2011).

It is important to note that the closely related neuropeptide AVP is also a well- known regulator of anxiety but its effects are often opposite to that of OT and thus act to increase anxiety (Neumann and Landgraf 2012). Specifically, peripheral or central (ICV) administration of AVP antagonists have confirmed the anxiogenic effects of endogenous

AVP (Landgraf 2006; Mak et al. 2012; Pitkow et al. 2001; Ring 2005). The only brain sites that have been investigated for AVP’s effects on anxiety are the lateral septum and the PL mPFC. While administration of exogenous AVP to the both regions was not able to affect anxiety-like behavior in male or female rats (Liebsch et al. 1996; Sabihi et al.

2014b), males infused with a V1a antagonist into the lateral septum displayed a significant reduction in anxiety compared to those infused with saline (Liebsch et al.

1996).

Together, these findings suggest that OT plays a major role in the attenuation of anxiety as well as anxiety that presents in a social context. However, the neural basis and the mechanism of action of OT remain unclear. One possible mechanism that OT may use to reduce anxiety may include modulation of GABA signaling.

1.6 GABA

GABA: Structure, and Synthesis within the Prefrontal Cortex

GABA (γ-aminobutyric acid) is the principal inhibitory neurotransmitter in the brain. Its primary role is to reduce neuronal excitability throughout the nervous system and as such has been implicated in the modulation of many cognitive and behavioral processes. GABA is synthesized in the brain by GABAergic neurons only and does not penetrate the blood-brain barrier. It is synthesized from glutamate using the enzyme glutamate decarboxylase (GAD) and pyridoxal phosphate as a cofactor. GAD from mammalian brain occurs in two molecular forms, GAD65 and GAD67 (referring to subunit relative molecular weight in kilodaltons). These different forms of GAD are the product of different genes, differing in immunoreactivity, nucleotide sequence, and subcellular localization (Tillakaratne et al. 1995). Together, GAD and pyridoxal phosphate convert the principal excitatory neurotransmitter in the brain, glutamate, into the principal inhibitory neurotransmitter (Petroff 2002). Once synthesized, the vesicular GABA transporter (VGAT) transports GABA into vesicles. Upon release, GABA is removed from the cleft by three different transporters; GAT1 and GAT2 are expressed in neurons and astrocytes whereas GAT3 is only expressed in astrocytes. Inside the cell, GABA is metabolized to glutamate and succinate by GABA-transaminase (GABA-T) (Olsen and

DeLorey 1999).

GABA can be found in all areas of the brain, although here, we will focus on the

GABAergic system within the PFC. In fact, 10-40% of nerve terminals in the cerebral

31 cortex use GABA as their neurotransmitter (Paulsen and Fonnum 1987). Many major

GABAergic pathways project to and arise from the cortex and play a central role in controlling the excitability of local circuits. A heterogeneous population of GABA- producing interneurons exist within the PFC that shape excitatory output in the central nervous system, these include parvalbumin (PV) expressing basket type interneurons, vasoactive intestinal peptide (VIP) containing interneurons, and somatostatin (SOM) containing interneurons (Gaykema et al. 2014). The PV interneurons are tightly coupled to pyramidal cells in the PFC, synchronizing their output and regulating shifts in the excitation-inhibition balance (Somogyi et al. 1983). VIP containing interneurons mainly exist as radially projecting bipolar neurons which extend processes between cortical layers to innervate other groups of inhibitory interneurons (David et al. 2007). As such,

VIP interneurons promote a disinhibitory mechanism in the PFC. Finally, SOM expressing interneurons target pyramidal cells in the cortex and inhibit them when activated (Kvitsiani et al. 2013). These three inhibitory neuronal cell types within the

PFC comprise up to 84% of all cortical interneurons (Gaykema et al. 2014).

GABA Receptors

GABA acts at inhibitory synapses in the brain by binding to specific transmembrane receptors in the plasma membrane of both pre- and postsynaptic neuronal processes. Centrally, GABA can bind to one of three receptors, GABAA, GABAB, or

GABAC receptors. GABAA receptors are fast acting receptors associated with chloride

2+ + ion channels while metabotropic GABAB receptors are coupled to Ca or K channels as

32 second messenger systems to produce slow and prolonged inhibitory responses

(Watanabe et al. 2002). The third class of GABA binding sites, GABAC receptors appear to be simple ligand-gated Cl- channels with a distinct pharmacology in that they are not blocked by bicuculline, a GABA receptor antagonist (Enz and Cutting 1998; Watanabe et al. 2002). Here, we will be focusing on the GABAA receptor within the mPFC as it has been greatly implicated in the regulation of anxiety-like behavior and since it is the primary binding site for many anxiolytic drugs.

Distribution, Structure, and Function of GABAA Receptors

GABAA receptors (GABAAR) occur throughout the entire central nervous system in all organisms and are fast-acting ionotropic receptors. (Nutt 2006). The binding of

GABA causes the opening of the ion channel allowing the flow of either negatively charged chloride ions (Cl-) into the cell or positively charged potassium ions (K+) out of the cell. Binding of GABA to its receptor results in a negative change in the transmembrane potential, thus, causing hyperpolarization.

The GABAAR is a pentameric transmembrane receptor that consists of five subunits arranged around a central pore (Johnston 1996). Three or four different kinds of subunits may be found within a particular GABAAR complex. The subunits are designated α, β, γ, and δ and can combine in different ways (up to 20 isoforms) to form

GABAA channels (Rudolph et al. 2001). Each isoform can determine the receptor’s agonist affinity, chance of opening, conductance and other properties. About 40% of the

GABAAR in the brain are pentamers comprised of two α subunits, two β subunits, and

33 one γ subunit ((α1)2(β2)2(γ)), however other subunits including the δ, π, θ, and ϵ exist but are much less common (Rudolph et al. 2001).

GABAAR contain at least five different binding sites (Fig. 4), the primary site being for GABA. GABA is the endogenous ligand that causes the GABAAR to open.

Upon activation by GABA, the receptor undergoes a conformational change in the protein subunits leading to the transient creation of a pore along the axis of the pentameric cylinder through which chloride ions can flow from one side of the membrane to the other resulting in hyperpolarization of the neuron (Horenstein et al. 2001). This causes an inhibitory effect on neurotransmission by reducing the likelihood of a successful action potential occurring. In addition to being the binding site for GABA, the active site of the GABAAR is also a binding site for several drugs that act as orthosteric ligands such as muscimol (GABAAR agonist) and bicuculline (GABAAR antagonist). The

GABAAR also contains a number of allosteric binding sites which allow other drugs to modulate the activity of the receptor indirectly (Johnston 1996; Uusi-Oukari and Korpi

2010). For example, a second binding site on the GABAAR is for the binding of benzodiazepines, a class of drugs commonly used as anxiolytics. The third site binds with barbiturates and the fourth with various steroids. Benzodiazepines and barbiturates require the binding of GABA to its site and they act to potentiate the action of GABAAR activity by increasing the potency of GABA to the receptor and by allowing the channel to open more easily and more often. Finally, the fifth site on the GABAAR binds with picrotoxin, a poison and GABAAR antagonist found in an East Indian Shrub. In addition, alcohol binds with as as-yet unknown site on the GABAAR. Typically, ligands that

34 activate the GABAAR or are positive allosteric modulators of the receptor, such as muscimol or benzodiazepines respectively, have anxiolytic, anticonvulsant, and relaxing effects (Rudolph et al. 2001). Conversely, ligands that decrease receptor activation or are negative allosteric modulators of the GABAAR usually have opposite effects including anxiogenesis and convulsion (Rudolph et al. 2001). Together, it is not surprising that much effort has been devoted to studying the effects of modulating this receptor’s activity in connection with anxiety behaviors.

GABA, the mPFC, and Anxiety

A number of studies in humans have provided evidence for an important role of

GABAergic neurotransmission in the medial prefrontal network in the modulation of anxiety-like responses. For example, greater and abnormal activation in cortical regions including the dmPFC has been attributed to reduced inhibition and hypofunction in the

GABAergic system (Goddard et al. 2001; Ham et al. 2007; Long et al. 2013; Simpson et

36 al. 2012). In addition, hypofunction in the cortical GABAergic system has been correlated with increased anxiety behavior such that patients with anxiety disorders demonstrate lower brain levels of GABA, specifically in the cortex (dmPFC/mPFC areas), than healthy controls (Goddard et al. 2001; Ham et al. 2007; Long et al. 2013;

Simpson et al. 2012). Furthermore, patients with anxiety disorders display reduced benzodiazepine binding in various brain regions including the dmPFC in comparison with controls (Bremner et al. 2000; Hasler et al. 2008). Low cortical GABA levels in patients with anxiety may be due to dysfunctional GABA synthesis enzymes or due to dysfunction in enzymes involved in glutamate–glutamine cycling, which accounts for

10–15% of the neuronal pool of GABA (Kugler 1993). Contrastingly, administration of the benzodiazepine, alprazolam, attenuates the increased activation of the dmPFC in the presence of negative stimuli. This drug also increases functional connectivity between this area and other anxiety-related brain regions including the amygdala (Leicht et al.

2013).

Animal studies further support the idea of increased neuronal activity in the mPFC in anxiety. Stress and anxiety-inducing stimuli consistently activate the mPFC in rats (Singewald et al. 2003) as demonstrated by increased expression of Fos, an indicator of neuronal activation, in the mPFC after exposure to a variety of anxiety-provoking challenges (Duncan et al. 1996). This increase in activation can be blocked by increased

GABAAR activity via benzodiazepine administration (Morrow et al. 2000). Additional studies have demonstrated that lesions and pharmacological inactivation of the mPFC by local administration of muscimol, a direct GABAaR agonist, or an indirect GABAAR

37 agonist, midazolam, induce anxiolysis (Shah et al. 2004; Shah and Treit 2003; Solati et al. 2013). Conversely, infusion of the direct GABAAR antagonist, bicuculline, to the mPFC has been shown to result in increased anxiety-like behavior (Bi et al. 2013; Solati et al. 2013). Together, it is clear that increased activity of the prefrontal regions in both humans and rodents is associated with increased anxiety-like behavior and that hypofunction of the GABAergic system plays a key role in this phenomenon.

GABA’s Link to Oxytocin and Anxiety

Recent studies have begun to explore the neural basis and mechanism of action of

OT’s anxiolytic effect. One possible mechanism that OT may use to reduce anxiety in the mPFC may include modulation of GABA signaling in this region and the effect this may have on regions downstream, including the BLA and CEA (Mitchell et al. 2015). It has recently been found that regions such as the mPFC, amygdala, hypothalamus, and hippocampus express OTR on PV and SOM expressing GABAergic interneurons (Marlin et al. 2015; Nakajima et al. 2014; Smeltzer et al. 2006; Smith et al. 2016). Activation of these interneurons by OT has generally been found to balance inhibition and improve the signal to noise ratio in order to mediate behaviors such as anxiety, social interaction, and maternal care behaviors (Marlin et al. 2015; Nakajima et al. 2014; Owen et al. 2013;

Smith et al. 2016). For instance, in the hippocampus, OT enhances cortical information transfer by increasing spike probability in GABAergic interneuron firing while simultaneously lowering background activity, thus improving the signal to noise ratio

(Owen et al. 2013). In the mPFC, OT has been shown to suppress glutamatergic

38 neurotransmission and increase basal levels of extracellular GABA (Ninan 2011; Qi et al.

2012; Qi et al. 2009). Together these data point to interactions between the OT and

GABA systems.

The behavioral implications of increased GABAergic neurotransmission by OT have just begun to be investigated. A recent study demonstrated that OT administration to the PVN increases GABAergic neuronal activity and GABA release in this region (Smith et al. 2016). The anxiolytic effects of OT administration to the PVN were inhibited by concomitant treatment of a GABAAR antagonist, indicating that within the PVN the anxiolytic effects of OT are dependent upon enhanced GABAergic neurotransmission

(Smith et al. 2016). Another study that specifically examined the mPFC reported the discovery of a specific population of SOM expressing interneurons in this region that express OTR. Silencing of these interneurons in female mice resulted in the loss of social interest in female mice during the sexually receptive phase (Nakajima et al. 2014). In a follow-up study, the group found that optogenetic activation of OTR expressing interneurons in the mPFC of male mice had a strong anxiolytic effect and no impact on social interaction, whereas activation of these neurons in female mice resulted in increased sociality and no change in anxiety-related behaviors. This effect was mediated by corticotropin-releasing-hormone-binding protein (CRHBP), an antagonist of the stress hormone CRH, which is specifically expressed in OT interneurons. Furthermore, activation of these interneurons in both males and females resulted in increased inhibition of pyramidal cells in that region (Li et al. 2016). These data indicate that OT likely acts through OT expressing interneurons in the mPFC in order to mediate anxiety-like

39 behavior, although may do so in a sex-specific manner. Together, OT-GABA interactions in the mPFC may play an important role in regulating anxiety.

1.7 Experiments

The aim of the first part of this dissertation was to examine the anxiolytic actions of exogenous OT and GABA in the mPFC of virgin rats. This chapter builds upon our previously published work (Sabihi et al. 2014b) which showed that the PL mPFC is a site in which exogenous OT acts to mediate anxiety-like behavior in male and female rats.

Here we examined the regional and receptor specificity of OT in the mPFC as well as its mechanism of action by exploring OT’s interactions with GABAergic system.

Chapter 2: Oxytocin in the medial prefrontal cortex attenuates anxiety: regional and

receptor specificity

2.1 Introduction

In addition to its well-known role in various social behaviors, (Bale et al. 2001;

Bosch and Neumann 2012; Calcagnoli et al. 2015; Caldwell 2012; Engelmann et al.

1998; Lim and Young 2006; Meyer-Lindenberg et al. 2011) the neuropeptide oxytocin

(OT) has been implicated in the regulation of anxiety (Benarroch 2013; Macdonald and

Feifel 2014; Neumann and Landgraf 2012; Veenema and Neumann 2008). In rats and mice, exogenous OT has repeatedly been shown to attenuate anxiety-like behavior when administered peripherally or centrally (Ayers et al. 2011; Bale et al. 2001; Blume et al.

2008; Mak et al. 2012; McCarthy et al. 1996; Ring et al. 2006; Sabihi et al. 2014b;

Slattery and Neumann 2010; Uvnas-Moberg et al. 1994; Windle et al. 1997). The anxiolytic effect of OT in rodents translates to humans with several studies demonstrating that intranasal administration of OT suppresses anxiety responses in healthy individuals as well as patients with anxiety disorders (de Oliveira et al. 2012; Feifel et al. 2011;

Guastella et al. 2009; Heinrichs et al. 2003; Macdonald and Feifel 2014).

Numerous brain regions have been identified as sites of action for the anxiolytic effect of OT, including the hypothalamic paraventricular nucleus (Blume et al. 2008;

Smith et al. 2016), amygdala (Bale et al. 2001; Neumann 2002), raphe nucleus (Yoshida et al. 2009), and most recently, the prelimbic (PL) region of the medial prefrontal cortex

(mPFC) (Sabihi et al. 2014a; Sabihi et al. 2014b). In addition to the PL region, the mPFC of the rodent brain also includes the infralimbic (IL) and anterior cingulate (Cg1) cortices. The various subregions of the mPFC show different patterns of connectivity with subcortical and cortical structures which are known to regulate the expression of anxiety-like behavior (Calhoon and Tye 2015; Hoover and Vertes 2007; Likhtik et al.

2005; Myers-Schulz and Koenigs 2012; Vertes 2004) and as such have been shown in some studies to differentially contribute to anxiety (Albrechet-Souza et al. 2009; Bi et al.

2013; Gonzalez et al. 2000; Jinks and McGregor 1997; Maaswinkel et al. 1996; Resstel et al. 2008; Saitoh et al. 2014; Shah et al. 2004; Stern et al. 2010; Suzuki et al. 2016). Thus, it is possible that the effect of exogenous OT within the mPFC on anxiety-like behavior may be subregion specific.

Oxytocin receptors (OTR) are expressed in the mPFC (Gould and Zingg 2003;

Insel and Shapiro 1992; Liu et al. 2005; Mitre et al. 2016; Smeltzer et al. 2006) and so it is reasonable to assume that OT in the PL mPFC reduces anxiety by activating the OTR.

However, receptors for the structurally similar neuropeptide, vasopressin (AVP), are also found in the mPFC (Kozorovitskiy et al. 2006; Smeltzer et al. 2006). Cross-reactivity at the receptor level has been described (Postina et al. 1998) due to OT’s moderate to strong affinity for the V1a subtype of the AVP receptor (Chini et al. 1996; Hicks et al. 2012) and there are studies showing that some behavioral effects of OT involve the V1a receptor (Hicks et al. 2012; Ramos et al. 2013; Sala et al. 2011). As such, it remains to be

42 determined whether exogenous OT may be acting as a partial agonist at the AVP V1a receptor to reduce anxiety.

In addition to its anxiolytic effects, OT is also commonly regarded as a stress hormone as it is released in response to a variety of stressful conditions which activate the hypothalamic-pituitary –adrenal (HPA) axis (Heinrichs et al. 2001; Neumann et al.

2000b; Onaka et al. 2012). Here, OT acts as a negative feedback mechanism to attenuate the HPA axis response to stress including dampening the release of stress hormones such as ACTH, corticosterone, and glucocorticoids (Smith et al. 2016; Smith and Wang 2013;

Windle et al. 2004; Windle et al. 1997). The mPFC is a well-known regulator of the HPA axis because it contains a high density of glucocorticoid receptors, it is prone to stress- induced increases in immediate early gene expression, and it has various ascending and descending projections to limbic structures involved in HPA regulation (Carmichael and

Price 1995; Diorio et al. 1993; Figueiredo et al. 2003; Meaney et al. 1985; Radley 2012;

Radley et al. 2008). However, it is unknown whether OT acting within the mPFC can attenuate the HPA axis response to stress.

In the present study, we assessed whether the regulation of anxiety by OT within the mPFC is restricted to the PL subregion and evaluated whether OTR activation is required for OT to have an anxiolytic effect. In addition, we examined whether OT infusion into the PL mPFC can attenuate the HPA axis response to stress.

2.2 Materials and Methods

Animals

Adult (9-12 weeks of age) male (300-350g) Sprague-Dawley rats (Taconic;

Germantown, NY) were housed individually in a temperature and humidity controlled room and maintained on a 12h/12h light/dark cycle (lights on at 0600 hr) with access to food and water ad libitum. All procedures were conducted in accordance with The Guide for the Care and Use of Laboratory Animals published by the National Institutes of

Health and approved by The Ohio State University Institutional Animal Care and Use

Committee.

Surgical procedures

After at least 7 d of acclimation to the colony, rats were anesthetized with a 2-4% isoflurane gas/air mixture and aligned on a stereotaxic apparatus (Kopf Instruments,

Tujunga, CA). Body temperature was maintained throughout the surgery with a warming pad. Bilateral cannula guides (pedestal mounted 22-gauge stainless steel tubes with 1.5 mm separation and cut either 1.4 mm (Cg1), 3.5 mm (PL), or 4.6 mm (IL) below the pedestal; Plastics One, Roanoke, VA) were secured in a stereotaxic holder and lowered into one of the three mPFC subregions (Cg1: AP: + 2.7 mm, ML: ± 0.5 mm, DV: -1.4 mm; PL: AP: + 3.2 mm, ML: ± 0.5 mm, DV: -3.5 mm; IL: AP: + 3.2 mm, ML: ± 0.5 mm, DV: -4.6 mm) (Paxinos and Watson 1998). All cannulae were secured by stainless steel screws and dental cement. A bilateral stainless steel obturator (0.35 mm diameter;

Plastics One) extending 0.2 mm beyond the tip of the guide cannula was placed into the

44 guide cannula after surgeries. The scalp was closed around the protruding portion of the cannula with sutures. Rats were allowed to recover 7 d before behavioral testing.

Central infusions

On days 3 and 5 post-surgery, rats were habituated to the handling and infusion procedures. During habituation, rats were removed from their home cage and handled for

3 min while being lightly restrained in a terrycloth towel. The obturators were then removed and a 28-gauge bilateral injection cannula extending 0.2 mm beyond the tip of the guide cannula was inserted into the guide. The injection cannula was left in place for

3 min then removed and the obturator replaced. On the day of testing, rats underwent the same procedure as described above except that an injection cannula attached to a 1 µl

Hamilton Syringe via PE-10 tubing was inserted into the guide cannula. Infusions were made using a Harvard Apparatus Pico Plus Elite infusion pump (Holliston, MA) which delivered a 0.5 µl volume over 1.5 min. The injector was left in place for an additional 1 min before withdrawal. Testing for anxiety-related behavior was done 15 min after the

OT or saline infusion which is consistent with other studies examining the behavioral effects of OT (Bale et al. 2001; Lukas et al. 2011; Sabihi et al. 2014b). Because of the short half-life of brain OT (approximately 30 min), all behavioral testing was completed within 30 min of infusion (Gimpl and Fahrenholz 2001).

Anxiety-like behavior

Anxiety-like behavior was evaluated one week post-surgery using the elevated plus maze (EPM) and/or social interaction (SI) test (Lapiz-Bluhm et al. 2008; Rotzinger et al. 2010). When both the EPM and SI test were done in the same animal, the order of the two tests was counterbalanced and were done 5 min apart.

The EPM consisted of a cross-shaped platform (height: 50 cm) with four arms

(width: 10 cm, length: 50 cm), two of which were enclosed by walls 50 cm in height.

Rats were placed in the center of the platform (10 x 10 cm), facing a junction between an open and closed arm and allowed to explore for 5 min. The number of entries into the open arms and the percentage of time spent in the open arms (time in open arms/time in open and closed arms x 100) were used as measures of anxiety-like behavior (Pellow et al. 1985; Cruz et al. 1994; Lapiz-Bluhm et al. 2008). An increase in the percentage of time spent in the open arms and a greater number of open arm entries are indicative of reduced anxiety. Closed arm entries were used as a measure of locomotion independent of anxiety (Cruz et al. 1994; Lapiz-Bluhm et al. 2008; Pellow et al. 1985).

In the SI test, an age and weight (+/- 10 g) matched novel male conspecific was placed in the corner of a 60 x 60 cm Plexiglas arena with walls 40 cm high opposite from the corner in which the test rat was placed. Conspecifics were used a maximum of two times and were never used twice in the same day. The assignment of a conspecific to a test rat was random and not restricted to a particular drug or dose of drug. During a 5 min test, the time spent in active social behavior (i.e. communal grooming, sniffing, approaching, following, climbing on or under the stimulus rat) initiated by the test rat was

46 scored. The time the experimental rat spent interacting with the novel conspecific was used as a measure of anxiety-like behavior. Greater social interaction time is indicative of reduced anxiety (File 1980).

All behavioral tests were performed under normal fluorescent overhead ambient lighting of ~550 lux which generated high levels of anxiety in the EPM. Tests were performed within the same time range each day (approximately 0900-1300h), which is sufficiently separated from light-dark transitions (lights on at 0600h, lights off at 1800h) to avoid any potential diurnal variations in exploratory behavior (Lapiz-Bluhm et al.

2008). Tests were digitally recorded and later scored blind by a trained observer using

BEST Collection and BEST Analysis software (Education Consulting Inc., Hobe Sound,

FL).

Experimental design

Experiment 1 investigated the extent to which the anxiolytic effect of OT in the mPFC is subregion specific. Separate groups of male rats received infusions of OT (cat# O6379;

Sigma, St. Louis, MO) into one of the three regions of the mPFC (Fig. 5). OT was dissolved in 0.5 µl saline at a dose of 0.1 µg (Cg1: n = 6; PL: n = 6; IL: n = 9) or 1.0 µg

(Cg1: n = 6; PL: n = 6; IL n = 10). Doses were selected based on prior studies of anxiety- like behavior using site specific administration of OT (Ayers et al. 2011; Bale et al. 2001;

Lee et al. 2005; Sabihi et al. 2014b). Control rats received a 0.5 µl infusion of saline

(Cg1: n = 7; PL: n = 6; IL: n = 8). All rats were tested for anxiety-like behavior on both the EPM and SI test.

Experiment 2 evaluated whether the anxiolytic effect of OT in the PL mPFC is dependent on OTR activation. Separate groups of male rats received an infusion of saline, OTR antagonist (OTR-A; 0.1 µg) or AVPR antagonist (AVPR-A; 0.1 µg) followed 10 min later (Sala et al. 2011; Yosten and Samson 2010) by an infusion of saline or OT (1 µg) resulting in the following six groups: saline+saline (n = 7), saline+OT (n = 7), OTR-

A+OT (n = 7), OTR-A+saline (n = 5), AVPR-A+OT (n = 7), AVPR-A+saline (n = 5).

Testing for anxiety-related behavior was done using only the SI test since both anxiety tests yielded similar results in experiment 1. All drugs were dissolved in 0.5 µl saline.

2 4 The OTR-A (desGly-NH2-d(CH2)5[D-Tyr ,Thr ]OVT, courtesy of Dr. Maurice Manning,

University of Toledo) is highly specific, being 95 times more selective for the OTR over the V1aR (Manning et al. 2008). The AVPR-A (d[Tyr(Me)2, Dab5]AVP, courtesy of Dr.

Manning) is highly specific to the V1a receptor and is devoid of any anti-OT activity in vivo, thus allowing for the discrimination between V1a and OT receptors (Manning et al.

2012). Both antagonists have been used in rodent behavioral studies at the dose used here

(Lukas et al. 2011; Sabihi et al. 2014a). This dose was lower than that of the peptide

(Bales et al. 2004; Cho et al. 1999) since OTR and AVPR antagonists have been shown to be approximately 10-100 times more effective in receptor binding than the natural ligands (Barberis and Tribollet 1996).

Experiment 3 investigated whether OT infusion into the PL mPFC can attenuate the HPA axis response to stress. 1 week after surgery, separate groups of male rats received either

48 one bilateral infusion of OT (cat# O6379; Sigma, St. Louis, MO) at a dose of 1.0 µg / 0.5

µl saline or one bilateral infusion of 0.5 µl saline into the PL mPFC. After infusion, males were split into one of two conditions, either no stress or stress. Males in the no stress condition had their cages placed into an adjacent room for 15 min while males in the stress condition habituated to a stress room for 15 min and were then exposed to an acute footshock stress. Footshock stress consisted of five 1mA electric footshocks at 10 s intervals for 2 s each (Almaguer-Melian et al. 2012). After stress (or no stress) animals were killed by decapitation 0, 30, or 60 min later. The groups were as follows:

0 min 30 min 60 min Saline / No Stress n = 7 n = 6 n = 6 Saline / Stress n = 6 n = 6 n = 6 OT / No Stress n = 6 n = 7 n = 6 OT / Stress n = 6 n = 7 n = 7

Tissue Collection and Processing

For Experiments 1 and 2, rats were deeply anesthetized and transcardially perfused with 4% paraformaldehyde. Brains were postfixed for 24 h at 4°C, transferred to

0.1M PBS, and a Vibratome used to obtain 40 µm thick coronal sections throughout the area of the cannula implant. Sections were stained with 0.2% cresyl violet for verification of correct placement. Examination under high magnification (100X) revealed limited to no damage at the tip of the cannula in any of the animals. Those animals with cannula placements outside of the intended region of the mPFC were excluded from analyses. For Experiment 1, this included a total of 5 animals: 1 in the PL 0.1 µg OT infusion group, 2 in the PL 1.0 µg infusion group, 1 in the IL saline group, and 1 in the IL

0.1 µg OT group. For Experiment 2, only 1 animal in the S+OT group was removed due 49 to missed cannula placement. For Experiment 3, rats were rapidly decapitated and truck blood samples obtained.

Plasma corticosterone measurement

Blood samples were centrifuged at 3000 g for 15 min, plasma collected and stored at -80 °C. Samples were thawed at the time of assay. Corticosterone was assayed, in duplicates, using a commercially available corticosterone ELISA kit (Enzo Life Sciences,

Ann Arbor, MI). ELISA procedures were conducted following the manufacturer’s instructions.

Statistical analysis

Statistical analyses for Experiment 1 and 2 were performed using GraphPad Prism software version 5.01 (La Jolla, CA). For Experiment 1 examining anatomical specificity, data from the EPM (percent time in open arms, number of open arm entries, number of closed arm entries) and SI test (time interacting) were analyzed using a two- way Analysis of Variance (ANOVA) with region (Cg1, PL or IL) and infusion type

(saline, 0.1 µg OT or 1.0 µg OT) as factors. SI test data from the Experiment 2 was analyzed using one-way ANOVA. When applicable, post-hoc analyses were conducted using Tukey’s HSD post-hoc comparison test. For CORT data in experiment 3, a three- way ANOVA with drug, stress, and timepoint as factors was performed in R (Version

3.3.2) and followed by all possible multiple comparisons using Tukey’s HSD post-hoc comparison test. Data are expressed as the mean + SEM. Significance was set at p < 0.05.

2.3 Results

OT in the PL, but not IL or AC, mPFC reduces anxiety-like behavior

In the EPM, OT had a subregion specific effect on anxiety-like behavior (Fig. 5).

For the number of open arm entries (Fig. 5b) there was a significant main effect of infusion type (F2,55 = 4.48, p < 0.05) and brain region (F2,55 = 6.98, p < 0.05) as well as an infusion type X brain region interaction (F4,55 = 3.51, p < 0.05). Post-hoc analysis revealed that the number of open arm entries was greatest in the group that received 1.0

µg OT in the PL region of the mPFC as compared to all other groups (p’s < 0.05) which did not differ from one another (p’s > 0.05). For the percentage of time spent in the open arms (Fig. 5c), there were also significant main effects of infusion type (F2,55 = 4.56, p <

0.05) and brain region (F2,55 = 7.55, p < 0.05) and a trend for an infusion type X brain region interaction (F4,55 = 2.24, p = 0.07) likely driven by the high percentage in the group that received 1.0 µg OT in the PL mPFC. Locomotor activity as measured by the number of closed arm entries (Fig. 5d) was not altered by infusion type (F2,55 = 2.21, p >

0.05) or region of infusion (F2,55 = 0.08, p > 0.05) and there was no significant infusion type X brain region interaction (F4,55 = 0.36, p > 0.05).

In the SI test (Fig. 5e), there was a significant main effect of infusion type (F2,55 =

3.47, p < 0.05) and brain region (F2,55 = 8.54, p < 0.05) and a significant infusion type X brain region interaction (F4,55 = 4.33, p < 0.05) on the amount of time spent interacting with an unknown stimulus rat. Post-hoc analysis showed that the group given the 1.0 µg dose of OT in the PL mPFC spent a greater amount of time interacting with a novel

51 conspecific when compared to all other groups (p’s < 0.05) which did not differ from each other (p’s > 0.05).

Figure 5: OT in the PL mPFC decreases anxiety-like behavior. (a) Schematic representation of mPFC regions in which bilateral cannulae were implanted (blue = Cg1, red = PL, black = IL; adapted from Paxinos and Watson 1998). In the EPM, (b) the number of open arms entries and (c) the percentage of time spent in the open arms was greatest in the group that received 1.0 µg OT in the PL region of the mPFC. (d) Locomotor activity, as measured by the number of closed arm entries, was not altered by infusion type or region of infusion. (e) In the SI test, the group infused with the 1.0 µg dose of OT in the PL mPFC spent a greater amount of time interacting with a novel conspecific compared to all other groups which did not differ from each other. * Indicates a significant interaction (p < 0.05) followed by post-hoc analyses, ** Indicates a trend (p = 0.07) for an interaction. Bars represent mean + SEM.

The anxiolytic effect of OT in the PL mPFC is dependent on OTR activation The anxiolytic effect of OT in the PL mPFC was blocked by pretreatment with an OTR-A, but not an AVPR-A (Fig. 6). There was a significant effect of treatment in the SI test (F5,41 = 9.14, p < 0.0001) with post-hoc analysis revealing that the group infused with S+OT and AVPR-A+OT spent more time interacting with an unknown conspecific than the group treated with OTR-A+OT (p’s < 0.05) which did not differ from the S+S group (p > 0.05). Neither antagonist alone affected anxiety-like behavior as social interaction time did not differ among the S+S, OTR-A+S or AVPR-A+S groups (p’s > 0.05).

Figure 6: OT acting on the OTR in the PL mPFC decreases anxiety-like behavior in the SI test. The AVPR-A+OT and S+OT groups spent more time interacting with an unknown conspecific than the group treated with OTR-A+OT indicating that the anxiolytic effect of OT in the PL mPFC was blocked by pretreatment with an OTR-A, but not an AVPR-A. The S+S, OTR-A+OT, OTR-A+S and AVPR-A+S groups all showed similar levels of anxiety-like behavior in the SI test. Bars represent mean + SEM; *p < 0.05.

OT infusion to the PL mPFC prior to an acute stressor does not affect plasma CORT levels Infusion of OT prior to an acute footshock stress did not affect plasma levels of

CORT following stressor exposure (Fig. 7). While there was no main effect of drug (F1,64

= 0.68, p > 0.05), there was a significant main effect of stress (F1,64 = 17.89, p < 0.05) , a main effect of timepoint (F2,64 = 18.05, p < 0.05), and a significant stress by timepoint interaction (F2,64 = 4.92, p < 0.05). All other interactions were not significant (p’s > 0.05). Posthoc analysis revealed that stressed males had higher CORT levels than unstressed males immediately and 30 min (p’s < 0.05) after the stressor but by 60 min there were no differences among the groups (p > 0.05).

Figure 7: OT infusion to the PL mPFC prior to an acute stressor does not affect plasma CORT levels. Males that were stressed, regardless of whether they received saline (a) or OT (b), displayed significantly higher levels of CORT compared to unstressed males immediately and 30 min after the stressor but by 60 min there were no differences among the groups. Bars represent mean + SEM; *p < 0.05.

2.4 Discussion

Numerous studies in animals and humans have established that OT reduces anxiety

(Ayers et al. 2011; Bale et al. 2001; Blume et al. 2008; de Oliveira et al. 2012; Feifel et al. 2011; Guastella et al. 2009; Heinrichs et al. 2003; Macdonald and Feifel 2014; Mak et al. 2012; McCarthy et al. 1996; Ring et al. 2006; Sabihi et al. 2014b; Slattery and

Neumann 2010; Uvnas-Moberg et al. 1994; Windle et al. 1997). In rodents, the mPFC is among the brain regions implicated in the anxiolytic actions of OT (Sabihi et al. 2014a; 54

Sabihi et al. 2014b). Here we assessed whether the regulation of anxiety by OT within the mPFC is restricted to the PL subregion and evaluated whether OTR activation is required for OT to have an anxiolytic effect. We confirm and extend previous work by demonstrating that infusion of OT into the PL region of the mPFC, but not the IL or Cg1 regions, decreased anxiety-like behavior. Further, we show that the attenuation in anxiety-like behavior following OT administration into the PL mPFC is abolished when the binding of OT to the OTR was impeded by pretreatment with a selective OTR-A.

Taken together, these results demonstrate that OT, acting on OTR in the PL mPFC attenuates anxiety-related.

Here we used two well-validated models that assess different types of anxiety-like behavior – the EPM and the SI test. While the EPM features exploration and relies on the rat's innate fear of open spaces, the SI test places emphasis on social behavioral responses and is based on the premise that anxiety is incompatible with social behavior (Bethlehem et al. 2014; Lapiz-Bluhm et al. 2008; Rotzinger et al. 2010). The anxiolytic effect of OT in the PL mPFC was evident in both tests suggesting that anxiety in non-social and social contexts is sensitive to PL OT although for the SI test, it is difficult to attribute the increased social interaction time to diminished anxiety alone since OT administration is also widely known to promote prosocial behavior (Benarroch 2013; Lukas et al. 2011;

Meyer-Lindenberg et al. 2011; Neumann and Slattery 2015).

The EPM testing conditions in the current study produced high levels of anxiety in the control groups making it necessary that a large threshold be breached for an anxiolytic effect of OT to be observed. Our results show that a 1 µg, but not a 0.1 µg,

55 dose of OT in the PL mPFC was able to overcome this threshold and reduce anxiety.

Although 1 µg OT is a relatively high dose, this dose was also shown to be anxiolytic in the amygdala (Bale et al. 2001). However, in the Cg1 and IL subregions of the mPFC, neither the 1 µg nor the 0.1 µg dose of OT was effective, even though both regions have also been implicated in fear and anxiety regulation (Cg1: Bissiere et al. 2008; Albrechet-

Souza et al. 2009; but see Bissiere et al. 2006; IL: Vidal-Gonzalez et al. 2006; Sierra-

Mercado et al. 2011; Bi et al. 2013). Importantly, OT’s influence was likely limited to each subregion of the mPFC targeted by the microinjection as a previous study found that when a larger volume of radiolabeled OT was injected into the CeA, which has a slightly smaller area than subregions of the mPFC, there was no spread beyond this area (Lee et al. 2005). As such, it appears that anxiety-like behavior is differentially sensitive to exogenous OT depending on the mPFC subregion although it could be the case that the

Cg1 and IL subregions may require a different OT dose than tested here for an effect on anxiety to be revealed. As nonlinear effects of OT have been reported (Figueira et al.

2008; Leuner et al. 2012), it is possible that a dose lower than 0.1 µg could yield a different outcome. Consistent with this possibility are findings showing that 0.01 µg OT in the IL mPFC promotes fear extinction (Lahoud and Maroun 2013), although these results could also suggest that learned fear versus innate anxiety, and the different paradigms used to assess these processes, may not be equally sensitive to mPFC OT. It is also important to consider emerging evidence that OT is not solely involved in reducing fear and anxiety but can also have the opposite action and can increase fear and anxiety

(Bartz et al. 2011; Grillon et al. 2012; Guzman et al. 2013; Lahoud and Maroun 2013;

Toth et al. 2012). However, as noted above, anxiety levels in the EPM were already fairly high which could have precluded the ability to detect an anxiogenic effect of OT. In this regard, it is worth noting that baseline anxiety levels in the SI test were not high and yet, like the EPM, OT was anxiolytic when administered in the PL mPFC at a dose of 1 µg.

Even so, it is possible that different EPM testing conditions would yield anxiolytic effects of OT at lower doses in the PL mPFC and/or in the other mPFC subregions. It is also possible that different testing conditions could possibly reveal increased anxiety after OT administration.

OTR are located in the mPFC (Duchemin et al. 2017; Gould and Zingg 2003;

Insel and Shapiro 1992; Liu et al. 2005; Mak et al. 2012; Mitre et al. 2016; Nakajima et al. 2014; Smeltzer et al. 2006) although little is known about subregional differences in

OTR density which may be a factor contributing to the observed behavioral variations across mPFC subregions. Nonetheless, it is reasonable to assume that within the PL mPFC, OT reduces anxiety by acting on the OTR. However, receptors for the structurally similar neuropeptide, vasopressin (AVP), are also found in the mPFC (Kozorovitskiy et al. 2006; Smeltzer et al. 2006). Cross-reactivity at the receptor level has been described

(Chini et al. 1996; Hicks et al. 2012; Postina et al. 1998) due to OT’s moderate to strong affinity for the V1a subtype of the AVP receptor and there are studies showing that some behavioral effects of OT, particularly social behaviors, involve the V1a receptor (Hicks et al. 2012; Mak et al. 2012; Ramos et al. 2013; Sala et al. 2011). Thus, central OT at high concentrations could possibly act as a partial agonist at the AVP V1a receptor to alter anxiety. Although this is unlikely because when it comes to anxiety-like behavior

57 activation of the V1a receptor has been linked to anxiogenic effects (Neumann and

Landgraf, 2012), we addressed this issue using a co-administration design in which OT was administered along with highly specific antagonists for either the OTR or the V1aR

(Manning et al. 2012). Our results demonstrate that the anxiolytic effect of OT in the PL region of the mPFC was blocked by the OTR-A, but not the AVPR-A, and thus confirm the anxiolytic actions of OT through its binding to the OTR. In doing so, these data eliminate the possibility of OT’s anxiety reducing actions through potential effects on the

AVPR and indicate that activation of the OTR is required to have behavioral significance.

In contrast to exogenous OT, endogenous OT in the PL mPFC does not appear to be directly involved in regulating anxiety-like behavior in the SI test since administration of an OTR-A alone was without effect. This finding is consistent with numerous studies using anxiety tests which lack a social component (i.e. EPM, OF, light-dark box) that have shown an anxiolytic effect of endogenous brain OT only during periods of high OT system activity such as the postpartum period (Lukas et al. 2011; Neumann 2002;

Neumann et al. 2000a; Sabihi et al. 2014a; Slattery and Neumann 2010). That we did not find an anxiolytic effect of endogenous PL OT on social anxiety may be somewhat surprising given the prosocial effects of endogenous brain OT (Dumais and Veenema

2016) and emerging studies implicating the PFC OT in various social behaviors (Marlin et al. 2015; Nakajima et al. 2014; Sabihi et al. 2014a; Sabihi et al. 2014b; Young et al.

2014). Methodological aspects of the SI test versus other tests of social behavior (i.e. social preference, social memory) may explain this discrepancy although the possibility remains that anxiety in a social context is meditated by components of the endogenous

58 brain OT system other than the PL mPFC (Neumann and Slattery 2015). It should also be noted that like the OTR-A, administration of the AVP V1aR antagonist into the PL mPFC had no effect on anxiety-like behavior in the SI test. Thus, although AVP also modulates anxiety-like behavior and some aspects of social behavior (Dumais and

Veenema 2016; Neumann and Landgraf 2012; Veenema and Neumann 2008), our data suggest that endogenous action of AVP at the V1aR in the PL mPFC is not essential.

Pharmacological studies have shown that exogenous treatment of OT prevents stress-induced activity, ultimately dampening HPA axis function (Blume et al. 2008;

Heinrichs et al. 2003; Petersson et al. 1999a; Smith et al. 2016; Windle et al. 2004;

Windle et al. 1997). Conversely, targeted inhibition of OT in regions including the PVN and central amygdala potentiates the behavioral and physiological response to stress

(Bosch et al. 2004; Ebner et al. 2005; Smith and Wang 2013). Because the mPFC has been implicated in negative feedback regulation of the HPA axis response to stress

(Diorio et al. 1993; Meaney et al. 1985; Radley 2012; Radley et al. 2008), we examined whether OT infusion to the PL mPFC would blunt the plasma CORT response to stress.

However, we did not find a calming effect of OT on the CORT response to an acute stressor. These results suggest that the anxiolytic properties of OT in the PL mPFC may be independent of any effects on stress hormones. However, it is important to note that the lack of stress-attenuating effects by OT can be attributed to a number of factors. For example, the intensity of the stressor may have been too much for OT to overcome and if a more mild stress or a different dose of OT was used, a calming effect of OT may have been revealed. Alternatively, a recent meta-analysis found that the attenuating effect of

OT on the cortisol response in humans is larger in response to challenging laboratory tasks that produce a robust stimulation of the HPA axis and the effect was even greater in clinical populations (Cardoso et al. 2014). These findings indicate that perhaps a more stressful experience that activates the HPA axis even more may uncover the attenuating effects of OT on the CORT response. Additionally, although many studies have confirmed the role of the PL mPFC in mediating the HPA axis (Diorio et al. 1993;

Figueiredo et al. 2003; Radley et al. 2006) it may be that another subregion of the mPFC, including Cg1 or IL may play a more prominent role in mediating OT’s effects on the

HPA axis (Diorio et al. 1993; Radley et al. 2006; Tavares et al. 2009).

In conclusion, these results demonstrate that OT acting on OTR in the PL mPFC attenuates anxiety, but has no effect on acute stress-induced elevations of CORT. In doing so, our results provide novel insights into OT’s anxiolytic actions for which little is currently known despite its potential therapeutic uses.

Chapter 3: Oxytocin in the medial prefrontal cortex attenuates anxiety: mechanism of

action

3.1 Introduction

In addition to open questions about anatomical and receptor specificity addressed in Chapter, 2, OT’s mechanism of action within the PL mPFC remains unclear. Several lines of evidence suggest that OT may be interacting with GABA, the main inhibitory neurotransmitter in the brain, to reduce anxiety (Nuss 2015). For example, recent work has shown that within the cortex, OTR are located on GABAergic interneurons (Marlin et al. 2015; Nakajima et al. 2014). In addition, OT has been found to increase cortical

GABA levels (Qi et al. 2012). When combined with the observation that activation of

GABAA receptors in the mPFC produces an anxiolytic-like response (Solati et al. 2013), it could be postulated that OT in the PL mPFC attenuates anxiety by enhancing local

GABA activity. The resulting increase in inhibition might in turn inhibit glutamatergic projections from the PL mPFC to attenuate activity of downstream limbic areas that generate anxiety-like behavior. Of particular interest are the basolateral (BLA) and central (CEA) amygdala, two components of the neural network regulating anxiety that receive direct and indirect input from the mPFC (Adhikari 2014; Berretta et al. 2005;

Brinley-Reed et al. 1995; Gabbott et al. 2005; Leuner and Shors 2013; McDonald et al.

1996; Vertes 2004).

In the present study, we examined whether OT interacts with the GABA system in the PL mPFC to reduce anxiety and investigated the extent to which OT in the PL mPFC affects neuronal activation of the PL mPFC as well as the BLA and CEA.

3.2 Materials and Methods

Animals

Adult (9-12 weeks of age) male (300-350g) Sprague-Dawley rats (Taconic;

Health and approved by The Ohio State University Institutional Animal Care and Use

Committee.

Surgical procedures

After at least 7 d of acclimation to the colony, rats were anesthetized with a 2-4% isoflurane gas/air mixture and aligned on a stereotaxic apparatus (Kopf Instruments,

62 the pedestal; Plastics One, Roanoke, VA) were secured in a stereotaxic holder and lowered into the PL mPFC (AP: + 3.2 mm, ML: ± 0.5 mm, DV: -3.5 mm) (Paxinos and

Watson 1998). All cannulae were secured by stainless steel screws and dental cement. A bilateral stainless steel obturator (0.35 mm diameter; Plastics One) extending 0.2 mm beyond the tip of the guide cannula was placed into the guide cannula after surgeries.

Obturators for animals in Experiment 2 extended 1.0 mm beyond the tip of the guide cannula in order to minimize damage to the PL region by guide cannulae. The scalp was closed around the protruding portion of the cannula with sutures. Rats were allowed to recover 7 d before behavioral testing.

Central infusions

Infusions for all three experiments followed the same procedures as described in

Chapter 2 with the exception that the injection cannula used in Experiment 2 extended

1.0 mm beyond the tip of the guide cannula

Anxiety-like behavior

Anxiety-like behavior was evaluated one week post-surgery using the elevated plus maze (EPM) and/or social interaction (SI) test (Lapiz-Bluhm et al. 2008; Rotzinger et al. 2010) as described in Chapter 2. When both the EPM and SI test were done in the same animal, the order of the two tests was counterbalanced and were done 5 min apart.

Experimental design

Experiment 1 evaluated whether OT in the PL mPFC exerts an anxiolytic effect by recruiting GABA neurons. Separate groups of male rats received one 0.5 µl infusion of saline followed by an infusion of 1.0 µg OT (S+OT; n = 6), one infusion of bicuculline methiodide (BIC), a specific GABAA receptor antagonist at a dose of 2.5 ng followed by an infusion of 1.0 µg OT (BIC+OT; n = 7), or one infusion of BIC at a dose of 2.5 ng followed by an infusion of saline (BIC+S; n=7). OT and BIC were both dissolved in 0.5

µl saline. An additional group of rats received two 0.5 µl infusions of saline (S+S; n = 6).

In each case, the second infusion occurred 5 min after the first. Anxiety-like behavior was evaluated in the EPM and SI test.

Experiment 2 examined how OT in the PL mPFC affects neuronal activation in this region following exposure to the EPM. Separate groups of male rats received infusions of

OT dissolved in 0.5 µl saline at a dose of 1.0 µg (n = 8) (Ayers et al. 2011; Bale et al.

2001; Blume et al. 2008; Ring et al. 2006; Sabihi et al. 2014b; Toth et al. 2012) or 0.5 µl saline (n = 8) and were tested for anxiety-like behavior in the EPM. Rats were euthanized and brains removed ~70 min after anxiety testing consistent with previous studies and peak c-Fos expression (Bossert et al. 2011; Knapska and Maren 2009; Rey et al. 2014).

Experiment 3 examined how OT in the PL mPFC affects neuronal activation in the BLA and CeA following exposure to the EPM. Separate groups of male rats received infusions of OT dissolved in 0.5 µl saline at a dose of 1.0 µg (n = 6) (Ayers et al. 2011; Bale et al.

2001; Blume et al. 2008; Ring et al. 2006; Sabihi et al. 2014b; Toth et al. 2012) or 0.5 µl saline (n = 5) and were tested for anxiety-like behavior in the EPM. Rats were euthanized and brains removed ~70 min after anxiety testing consistent with previous studies and peak c-Fos expression (Bossert et al. 2011; Knapska and Maren 2009; Rey et al. 2014).

The integrity of brain tissue slices from one animal receiving OT was compromised during tissue collection and slicing and was consequently removed from c-Fos analysis.

Tissue Collection and Processing

Rats were deeply anesthetized and transcardially perfused with 4% paraformaldehyde. For experiment 1, brains were postfixed for 24 h at 4°C, transferred to

0.1M PBS, and a Vibratome used to obtain 40 µm thick coronal sections throughout the area of the cannula implant. Sections were stained with 0.2% cresyl violet for verification of correct placement. Those animals with cannula placements outside of the intended region of the mPFC were excluded from analyses. Examination under high magnification (100X) revealed limited to no damage at the tip of the cannula in any of the animals. For experiment 2, brains were postfixed for overnight at 4 °C. On the following day, brains were transferred to 30% sucrose in 0.1M PBS until sectioning. 50 µm sections extending through the mPFC (AP: 4.2 – 2.0mm) were obtained with a cryostat and stored in a sucrose-based cryoprotectant at -20ºC until immunohistochemical processing on free-floating sections. Four sections (1:6) were processed for double-labeling for GAD67 and c-Fos as well as PV and c-Fos. Briefly, sections were washed in 0.1M PBS (3x5min) and then incubated with 0.1% Tween in PBS for 10 min. Next, sections were blocked in

10% normal goat serum (NGS) and 0.3% Triton X in PBS for 60 min followed by incubation in rabbit anti-Fos primary antibody (1:300; Santa Cruz Biotechnology, Santa

Cruz, CA) in PBS with 1% Triton X and 3% NGS for 72 hr at 4°C. After a PBS rinse, sections were incubated for 2.5 hr at room temperature in goat anti-rabbit secondary antibody with Alexa 488 (1:500; Vector Laboratories, Burlingame, CA) and rinsed in

PBS. sections were then incubated in mouse anti-GAD67 primary antibody (1:2000;

Millipore, Billerica, MA) in PBS with 0.5% tween overnight at 4°C. After a PBS rinse, sections were incubated for 1 hr at room temperature in DyLight 549 horse anti-mouse secondary antibody (1:500; Vector Laboratories, Burlingame, CA). After a final rinse, all sections were mounted on SupraFrost Plus microscope slides, and coverslipped with

DABCO and kept in the dark at 4°C until imaging. As the 200 micron microinjection needle creates mechanical tissue disturbance in some of the PL mPFC, we preferentially selected sections with minimal or no tissue damage for quantification (Smith et al. 2016).

For experiment 3, brains were kept in 4% paraformaldehyde for 4h at 4°C then switched to a solution of 0.1M PBS with 0.01% NaN3 until coronal sections (1:6 series) extending through the amygdala (AP: -1.6 - -2.8mm) were obtained with a Vibratome.

For c-Fos immunohistochemistry, sections were washed in 0.1M PBS (3 x 10min), quenched for 30 min with 3% H202 in 50% EtOH, and rinsed with PBS. Sections were then blocked for 60 min in 3% normal goat serum in 0.6% Triton-X followed by incubation in rabbit anti-Fos primary antibody (1:2000; Santa Cruz Biotechnology, Santa

Cruz, CA) for 48 h at 4°C. After a PBS rinse, sections were incubated for 2 h in biotinylated anti-rabbit secondary antibody (1:200; Vector Laboratories, Burlingame,

CA), rinsed in PBS, and then incubated for 1 hr in avidin-biotin complex (ABC

Vectastain kit, Vector Laboratories). After another rinse in PBS, sections were reacted in diaminobenzadine (Vector Laboratories) for ~7 min. After a final rinse, sections were mounted on SupraFrost Plus microscope slides, dehydrated through an alcohol series, cleared with xylene, and coverslipped with Permount.

For Experiment 2 and 3, separate sections throughout the area of the cannula implant were stained with 0.2% cresyl violet for verification of correct placement. Those animals with cannula placements outside of the intended region of the mPFC were excluded from analyses; this included one animal from Experiment 1 in the S+OT group.

Examination under high magnification (100X) revealed limited to no damage at the tip of the cannula in any of the animals.

c-Fos Analysis

For confocal analysis in Experiment 2, a Nikon 90i microscope was used to obtain

20x image stacks of the mPFC (~100 steps in 0.3 µm intervals along the z-plane). Image stacks were then projected using NIS Elements software and total number of c-Fos+,

GAD67+, and GAD67+/c-Fos+ double-labeled cells in the PL mPFC were counted by a researcher blind to experimental conditions. Identification of this region was conducted with reference to illustrations from a standard stereotaxic rat brain atlas (Paxinos and

Watson, 1998) and landmarks such as the location of the corpus callosum. For analysis of

GAD67+/c-Fos+ cells, 4 unilateral images of the PL mPFC per animal were taken at 20x.

On each image, c-Fos+, GAD67+, and GAD67+/c-Fos+ double-labeled cells were

67 quantified in a set ROI (250000 µm2). Data are expressed as total number of positive cells within an ROI (250000 µm2). Percentages of GAD67+ cells expressing c-Fos were calculated by dividing the number of these cells expressing c-Fos by the total number of single-labeled cells expressing GAD67. Counts and percentages were averaged across sections within an animal and the group mean determined from these values.

For Experiment 3, densities of c-Fos positive cells were quantified in the BLA and CEA (Fig. 10). Identification of these regions was conducted with reference to illustrations from a standard stereotaxic rat brain atlas (Paxinos and Watson 1998) and landmarks such as the location of white matter tracts (e.g., fornix, optic tracts) and ventricles. These regions corresponded to anterior-posterior (AP) coordinates -1.6 to -2.8 mm from bregma in the rat brain atlas. Images of from four bilateral sections per rat were taken at 10× magnification with a camera (Zeiss AxioCam and software) affixed to a microscope (Zeiss Axio Imager M2). On these images, c-Fos positive cells (i.e. round or oval-shaped nuclei with brown-black immunostaining darker than background) were counted manually by a researcher blind to experimental conditions in 4-6 regions of interest (ROIs) using StereoInvestigator (Williston, VT). For each ROI, counts were divided by the area of the ROI (averaging 0.112 mm2 for the BLA and 0.097 mm2 for the

CEA) then averaged for each animal and the group mean determined from these values.

Data are expressed as the number of c-Fos immunoreactive cells per 1 mm2.

Statistical analysis

All statistical analyses were performed using GraphPad Prism software version

5.01 (La Jolla, CA). EPM and SI test data from the Experiment 1 was analyzed using one-way ANOVA, while c-Fos counts and EPM data from experiment 2 and 3 were analyzed using a Student’s one-tailed t-test. When applicable, post-hoc analyses were conducted using Tukey’s HSD post-hoc comparison test. Data are expressed as the mean

+ SEM. Significance was set at p < 0.05.

3.3 Results

The anxiolytic effect of OT in the PL mPFC relies on GABAergic neurotransmission

The anxiolytic effect of OT in the PL mPFC was blocked by pretreatment with the

GABAA receptor antagonist, BIC (Fig. 8). In the EPM, there was a significant effect of treatment for the number of open arm entries (Fig. 8a; F3,25 = 7.16, p < 0.05) and percentage of time spent in the open arms (Fig. 8b; F3,25 = 6.68, p < 0.05). Post-hoc analysis revealed that the S+OT group made a greater number of entries into the open arms and spent more time in the open arms as compared to all other groups (p’s < 0.05), which did not differ from each other (p’s > 0.05) This anxiolytic effect of OT in the PL mPFC was prevented by pretreatment with BIC as the number of open arm entries and the percentage of time spent in the open arms did not differ between the BIC+OT and

S+S groups (p > 0.05). In addition, the BIC+S group did not differ from the S+S group on either measure of anxiety-like behavior indicating the BIC itself was without effect

(p’s > 0.05). Locomotor activity, as measured by the number of closed arm entries (Fig.

8c), was not altered by treatment (F3,25 = 0.24, p > 0.05).

In the SI test (Fig. 8d), there was also a significant effect of treatment (F3,25 =

319.43, p < 0.05) on the amount of time spent interacting with an unknown stimulus rat.

Post-hoc analysis showed that the S+OT group spent a greater amount of time interacting with a novel conspecific when compared to all other groups (p’s < 0.05) which did not differ from each other (p’s > 0.05). Again, this anxiolytic effect of OT in the PL mPFC was prevented by pretreatment with BIC as indicated by a similar amount of time spent interacting with an unknown conspecific (Fig. 8d) in the BIC+OT and S+S groups (p >

0.05). BIC itself did not affect anxiety-like behavior in the SI test as the BIC+S group did not differ from the S+S group (p > 0.05).

Figure 8: OT interacts with GABA in the PL mPFC to decrease anxiety-like behavior. In the EPM, the group that received an infusion of OT in the PL mPFC (a) made more open arm entries and (b) spent a greater percentage of time in the open arms unless they also received an mPFC infusion of the GABAA antagonist, bicuculline methiodide (BIC) which by itself did not affect anxiety-like behavior in the EPM. (c) Locomotor activity (closed arm entries) was not altered by infusion type. (d) In the SI test, OT increased time spent interacting with an unknown stimulus rat, but BIC blocked this anxiolytic effect. Bars represent mean + SEM; *p < 0.05.

OT in the PL mPFC increases activation of GABAergic neurons As we have previously shown, OT in the the PL mPFC reduced anxiety-like behavior in the EPM, without affecting locomotor activity (Fig. 9). As compared to saline-infusion, males infused with OT displayed an increase in the percentage of time spent in the open arms (Fig. 9a; t(15) = 3.99, p < 0.05) and an increase in the number of open arm entries (Fig. 9b; t(15) = 3.50, p < 0.05) but no change in the number of closed arm entries (Fig. 9c; t(15) = 0.26, p > 0.05). Furthermore, OT infusions into the PL mPFC resulted in more c-Fos+ cells after EPM exposure (Fig. 9d; t(15) = 2.58, p < 0.05) 71 without affecting the number of GAD67+ cells (Fig. 9e; t(15) = 0.35, p > 0.05) in the PL mPFC. OT in the PL mPFC also yielded a significant increase in the number (Fig. 9f; t(15) = 2.35, p < 0.05) and percentage (Fig. 9g; t(15) = 2.46, p < 0.05) of GAD67+ cells co-expressing c-Fos in the PL mPFC indicating greater activation of GABA neurons in the mPFC after OT treatment.

Figure 9: Males infused with OT in the PL mPFC display decreased anxiety-like behavior and increased activation of GABAergic cells in the PL mPFC. Males infused with OT to the PL mPFC spent more time in the open arms (a) and had more open arm entries (b) in the EPM when compared to males infused with saline. Locomotor activity was not affected by drug infusion (c). OT infusion to the PL mPFC followed by EPM exposure resulted in a greater number of c-Fos (d) but not GAD67 (e) expressing cells in the PL mPFC. Further, infusion of OT yielded a significant increase in the number (f) and percentage (g) of GAD67+ neurons expressing c-Fos in the PL mPFC. (h) Confocal images of a cell positive for c-Fos (green, top), GAD67 (red, middle), and GAD67 colabeled with c-Fos (bottom). Bars represent mean + SEM.* p < 0.05.

OT in the PL mPFC alters neuronal activation in the amygdala

OT in the PL mPFC reduced anxiety-like behavior and altered neuronal activation in the amygdala (Fig. 10). Once again, OT increased both the number of open arm entries

(Fig. 10a; t(9) = 2.51, p < 0.05) and the percentage of time spent in the open arms (Fig.

10b; t(9) = 2.08, p < 0.05) without altering the number of closed arm entries (Fig. 10c; t(9) = 0.91, p > 0.05). Following exposure to the EPM, c-Fos expression in the BLA was reduced in rats that received an infusion of OT in the PL mPFC relative to saline infused rats (Fig. 10d, e; t(8) = 2.38, p < 0.05). In contrast, EPM exposure increased c-Fos expression in the CEA of rats that received OT in the PL mPFC (Fig. 10e,f; t(8) = 2.94, p

< 0.05).

Figure 10: OT in the PL mPFC reduces anxiety-like behavior and alters neuronal activation in the amygdala. In the EPM, OT increased both (a) the number of open arm entries and (b) the percentage of time spent in the open arms without altering (c) the number of closed arm entries. (d) Schematic representation of regions throughout the BLA and CEA in which c-Fos counts were taken. Grey shaded regions = CEA; black shaded regions = BLA (adapted from Paxinos and Watson, 1998). Following exposure to the EPM, c-Fos expression in the BLA was reduced in rats that received an infusion of OT in the PL mPFC (e). In contrast, EPM exposure increased c-Fos expression in the CEA of rats that received OT in the PL mPFC (f). Scale bars = 100µm. (h). Bars represent mean +SEM, *p< 0.05.

3.4 Discussion

Numerous studies in animals and humans have established that OT reduces anxiety

(Ayers et al. 2011; Bale et al. 2001; Blume et al. 2008; de Oliveira et al. 2012; Feifel et 75 al. 2011; Guastella et al. 2009; Heinrichs et al. 2003; Macdonald and Feifel 2014; Mak et al. 2012; McCarthy et al. 1996; Ring et al. 2006; Sabihi et al. 2014b; Slattery and

Neumann 2010; Uvnas-Moberg et al. 1994; Windle et al. 1997). In rodents, the mPFC is among the brain regions implicated in the anxiolytic actions of OT (Sabihi et al. 2014a;

Sabihi et al. 2014b) but the mechanism by which exogenous OT in the mPFC regulates anxiety is unknown. Here we examined whether OT interacts with the GABA system in the PL mPFC to reduce anxiety and investigated the extent to which OT in the mPFC affects neuronal activation of BLA and CeA, two primary targets of the mPFC which are components of the neural network regulating anxiety (Adhikari 2014; Calhoon and Tye

2015). We show that the attenuation in anxiety-like behavior following OT administration into the PL mPFC was impeded by pretreatment with a GABAA receptor antagonist. Further, the reduction in anxiety-like behavior following OT in the PL mPFC was accompanied by a significant increase in the number and percentage of GAD67+ neurons expressing c-Fos in the PL mPFC suggesting greater activation of GABA neurons. Lastly, administration of OT to the PL region was accompanied by differential effects on neuronal activation in the BLA and CeA following EPM exposure. Taken together, these results demonstrate that OT in the PL mPFC attenuates anxiety-related behavior and may do so by engaging GABAergic neurons which ultimately modulate downstream brain regions implicated in anxiety-like behavior.

While the mPFC is often regarded as having an inhibitory influence on fear and anxiety, numerous studies suggest that it can also stimulate fear and anxiety (Calhoon and

Tye 2015; Quirk and Beer 2006). This bidirectional regulation of anxiety by the mPFC

76 can be attributable to differences among the subregions with the more dorsally located PL region having an excitatory influence through its glutamatergic projections to downstream limbic regions including the amygdala (Adhikari 2014; Berretta et al. 2005;

Brinley-Reed et al. 1995; Gabbott et al. 2005; Leuner and Shors 2013; McDonald et al.

1996; Vertes 2004). Because these glutamatergic projection neurons within the PL mPFC are modulated by local GABA interneurons, which have been shown to express OTR

(Marlin et al. 2015; Nakajima et al. 2014) one way OT could attenuate anxiety would be by engaging local GABAergic inhibition. This possibility is supported by evidence that

OT increases cortical GABA release in the mPFC (Qi et al. 2012), OT balances cortical inhibition (Marlin et al. 2015), as well the the demonstration that activation of mPFC

GABAA receptors is anxiolytic (Solati et al. 2013). Our results provide further support for an OT-GABA interaction in the PL mPFC. First, we show that the anxiolytic effect of

OT in the PL mPFC was inhibited by concomitant treatment with the GABAA receptor antagonist BIC. Notably, higher concentrations of BIC infusions in the mPFC (˃ 0.25 µg) have been reported to increase anxiety (Solati et al., 2013) while the current study used a lower concentration (2.5 ng) of BIC which did not. Second, we found that the decrease in anxiety-like behavior following OT delivery to the PL mPFC was accompanied by an increase in the number and percentage of activated GAD67 neurons in this region. These findings suggest that OT in the PL mPFC may act to reduce anxiety-like behavior by acting on receptors located on GABAergic interneurons, thus increasing local inhibition and altering GABAergic output. Interestingly, GABA has also been previously shown to mediate the anxiolytic action of OT within the amygdala (Huber et al. 2005; Knobloch et

77 al. 2012) and PVN (Smith et al. 2016) suggesting a similar mechanism of across these brain regions. Although it also possible that OT may act directly on glutamatergic neurotransmission in the mPFC to affect anxiety (Ninan 2011; Qi et al. 2012; Qi et al.

2009), this seems unlikely as all studies to date have only found OTR on GABAergic interneurons (Nakajima et al., 2014; Marlin et al., 2015).

Increased activation of GABAergic interneurons in the mPFC could act to decrease activation of downstream limbic regions, such as the amygdala. Experiment 3 set out to examine this possibility. Our results also show that OT in the PL mPFC was associated with changes in the activity of the amygdala such that c-Fos expression was reduced in the BLA but increased in the CeA. The BLA is the main target of glutamatergic projections from the PL mPFC (Gabbott et al. 2005; Vertes 2004) and increased activation of the BLA is associated with heightened anxiety (Hale et al. 2006;

Tye et al. 2011; Wang et al. 2011). Thus, the reduction in activation of the BLA after OT administration and the accompanying attenuation of anxiety-like behavior is consistent with diminished excitatory drive to BLA neurons and corroborates other work showing reduced amygdala activation following peripheral OT administration (Sobota et al. 2015).

The reason for the reciprocal pattern of c-Fos expression in the CeA, which receives robust direct projections from the BLA (Tye et al. 2011), isn’t entirely clear but may be explained by the fact that there are also intercalated cells (ITCs) interposed between the

BLA and CeA (Cassell et al. 1999; Millhouse 1986) which generate feedforward inhibition in CeA (Pare et al. 2003; Royer et al. 1999). Thus, the c-Fos responses detected in the CeA might in part be attributable to changes in BLA efferent activity via the ITC.

Although, without examining the phenotype of the activated cells or differentiating among the different subregions within the CeA (medial vs lateral CeA), as they can have opposing influences on anxiety (Calhoon and Tye 2015; Ressler 2010), it is difficult to make any further conclusions.

In conclusion, these results demonstrate that OT in the PL mPFC attenuates anxiety-related behavior via a GABAergic mechanism which may ultimately calm the downstream excitatory circuitry that would otherwise produce an anxious state. In doing so, our results provide novel mechanistic insights into OT’s anxiolytic actions for which little is currently known despite its potential therapeutic uses.

PART 2

Chapter 4: Introduction

4.1 Postpartum Anxiety in Humans

The postpartum period is commonly accompanied by emotional changes, which for many new mothers includes a reduction in anxiety (Hard and Hansen 1985; Lonstein

2005a; 2007; Lonstein et al. 2014). Attenuated postpartum anxiety is critical for normal mother-infant interactions and consequently infant development. Physical contact with the offspring mediates the reduction in maternal anxiety. In lactating women, either suckling or non-suckling contact with infants can produce an anxiolytic effect in mothers

(Feijo et al. 2006; Heinrichs et al. 2001; Lonstein 2007).

Because childbirth is a major life event, it represents a time of enhanced risk for the development of anxiety, and as such, a significant percentage of women experience elevated anxiety levels following parturition (Paul et al. 2013). In fact, the incidence rate of postpartum anxiety ranges from 3-43% in the first year postpartum with evidence that it may occur independently and at a higher rate than postpartum depression (Fallon et al.

2016; Paul et al. 2013). There are a few reasons for such a large range in incidence rate.

First, postpartum anxiety is less well known and less scientifically studied than postpartum depression and accurately detecting and diagnosing anxiety in the postpartum period is very low (Pawluski et al. 2017). Second, there is no clear diagnostic criteria or

81 specific tool for postpartum anxiety that currently exists, and it is often overshadowed or grouped in with postpartum depression as depression supersedes anxiety diagnostically

(Fallon et al. 2016; Goodman et al. 2016; Miller et al. 2006; Misri et al. 2015; Pawluski et al. 2017). In fact, more postpartum women are likely affected by high anxiety than they are by depression (Britton 2008). In addition, there is no clear understanding of postpartum anxiety or the neural mechanisms involved. Our understanding relies on a few clinical studies that point to several brain regions affected by postpartum depression, not anxiety, in response to infant or non-infant cues (Gingnell et al. 2015; Moses-Kolko et al. 2014; Wang et al. 2011). Unfortunately, imaging data have not been collected from women with postpartum anxiety alone (Pawluski et al. 2017).

Risk factors that are associated with anxiety during the postpartum period include sociodemographic factors such as age, ethnicity, marital status, and income, biological factors including psychiatric history of the mother and family, pregnancy and obstetric complications, life stressors, social support, and maternal adjustment (for review see

Dennis et al. 2016). Symptoms of postpartum anxiety in mothers include low self- efficacy in the parenting role, higher levels of fatigue, diminished maternal reactivity and sensitivity, decreased coping capability, and a higher risk for postpartum depression

(Fallon et al. 2016; Porter and Hsu 2003; Reck et al. 2013; Taylor and Johnson 2013).

Further, mothers that experience postpartum anxiety touch their infants less often and less affectionately compared to unaffected mothers (Ferber et al. 2008; Herrera et al. 2004).

This reduced level of contact between mother and infant, combined with a reduced

82 likelihood of breast-feeding in anxious mothers, in turn, can exacerbate postpartum anxiety (Dias and Figueiredo 2015; Ystrom 2012).

Heightened maternal anxiety is not only detrimental for maternal well-being, but can also interfere with effective caregiving and therefore negatively impact the development of the offspring. In infants, increased anxiety experienced by the mother has been linked to insecure attachment behaviors, disturbed sleep patterns, excessive crying, distress to novelty, delayed motor and cognitive development, negative temperament, and low social engagement (Fallon et al. 2016; Feldman et al. 2009; Galler et al. 2000; Keim et al. 2011; Manassis et al. 1994; Petzoldt et al. 2014; Pinheiro et al. 2014; Reck et al.

2013; Warren et al. 2006).

The most common treatment for postpartum anxiety is the use of selective serotonin reuptake inhibitors (SSRIs; Charlton et al. 2015; Pawluski et al. 2017) even though it is clear that dysfunction of many neurotransmitters and neuropeptides contribute to postpartum anxiety, not just serotonin (Lonstein et al. 2014). Further, questions remain about their risks for use in breastfeeding women because SSRIs can cross the placenta and are often evident in breastmilk (Berle and Spigset 2011; Lanza di

Scalea and Wisner 2009). As such, non-pharmacological interventions have been considered including cognitive behavioral therapy (CBT) (Challacombe and Salkovskis

2011; Green et al. 2015), exercise, dietary supplements, and physical contact with one’s infant (i.e. “kangaroo care”), all of which show some degree of effectiveness in alleviating the symptoms of postpartum anxiety (Ferber et al. 2008; Pawluski et al. 2017;

Sockol 2015; Ystrom 2012). OT has also been proposed as a possible treatment in

83 postpartum mental illness (Kim et al. 2014) but it remains to be determined how OT might alter the neurobiology of the maternal brain and improve maternal anxiety

(Pawluski et al., 2017).

Animal Models of Postpartum Anxiety

Given the detrimental effects of postpartum anxiety on the mother and her offspring, understanding the underlying neurobiology in an animal model is greatly beneficial. Just as in humans, postpartum rodents also undergo complex behavioral changes during this time period. Among these changes is a reduction in anxiety-related behavior which may be a prerequisite for a dams’ heightened maternal responsiveness to neonates and aggression towards intruders (Fleming and Luebke 1981; Hard and Hansen

1985; Macbeth and Luine 2010). These adaptations complement direct pup-caring to ensure the survival of the mother as well as her offspring.

The temporal progression of anxiety-like behavior in reproductive female rats is characterized by an increase during some points of pregnancy followed by a postpartum decrease to levels that fall below those seen in cycling virgins (Lonstein et al. 2014) and this anxiolytic effect of the maternal state in rodents lasts through the first postpartum week (Lonstein 2005a). Reduced anxiety-like behavior in postpartum females compared to virgin females has been observed in many behavioral paradigms including the light- dark box, elevated plus maze, and open field (Lonstein 2007; Miller et al. 2011;

Neumann 2003; Neumann et al. 2000a; Sabihi et al. 2014a). Furthermore, just as in humans, the degree of anxiety expressed by a mother rat can influence her capacity to

84 display maternal behavior which can have dramatic effects on the later physiology and behavior of the offspring (Caldji et al. 1998; Cameron et al. 2005; Champagne and

Meaney 2001).

While the hormones of pregnancy and parturition are necessary for the onset of a suite of behavioral changes in female rats, they are maintained thereafter by physical interactions with the offspring. Likewise, the postpartum reduction in anxiety is probably established by the endocrine profile of the peripartum period because dams' anxiety is already low within hours after parturition (Lonstein, 2005 and Picazo and Fernández-

Guasti, 1993). Thereafter, low maternal anxiety does not require peripheral endocrine systems and is instead maintained by neurochemicals released intracerebrally when mothers physically interact with the litter. This is evidenced by the fact that removing the ovaries, adrenals or pituitary gland does not affect the anxiety-related behavior of postpartum rats (Figueira et al. 2008; Lonstein 2005a; Miller et al. 2011; Neumann 2003;

Smith and Lonstein 2008) while separation of a mother from her litter for a period of 2-

24h prior to anxiety-behavior testing increases dams’ anxiety levels to those found in virgin females (Lonstein 2005a; Miller et al. 2011; Neumann 2003; Smith and Lonstein

2008). Based on the similarities to humans, postpartum rats can provide great insight into the neurobiology of reduced postpartum anxiety as well as insight into what can go awry in cases of elevated postpartum anxiety.

4.2 Brain Regions Regulating Postpartum Anxiety

Investigation into specific brain sites regulating postpartum anxiety has been limited. As mentioned above, there have been no studies in humans that employ neuroimaging strategies in mothers with only postpartum anxiety. However, studies examining mothers with postpartum depression (who also have elevated anxiety) have shown that these women have increased activity in the insula and decreased connectivity among the amygdala, anterior cingulate cortex, and dorsal lateral prefrontal cortex

(Gingnell et al. 2015; Moses-Kolko et al. 2010; Wang et al. 2011). Of course, there is not one particular brain region that is responsible for postpartum anxiety, and just as in non- postpartum subjects, there is likely a distributed neural circuitry which regulates anxiety during the postpartum period.

It is important to keep in mind that even though anxiety-related brain regions in both postpartum and non-postpartum subjects are likely the same, this circuitry is uniquely modified by a variety of hormones and neurotransmitters that are present and ever-changing during the postpartum period, contributing to a distinctive neurobiological profile during the postpartum period (Neumann 2001; 2003). For example, subjects with generalized anxiety disorder (GAD) show hyperactivity in the amygdala and insula and increased connectivity between these two regions in response to emotional cues (Etkin and Wager 2007; Makovac et al. 2016) whereas higher anxiety in postpartum subjects is associated with lower amygdala response to emotional cues and decreased connectivity between the amygdala and insula (Gingnell et al. 2015; Moses-Kolko et al. 2010; Wang et al. 2011). Additionally, many of the symptom characteristics of postpartum anxiety are

86 centered on the infant and family, while this is not the case in a disorder such as GAD.

This mother-infant interaction introduces an initially exogenous factor that is not present in GAD and, under normal circumstances, can help lower a mother’s anxiety (Lonstein

2007; Numan and Insel 2003; Smith and Lonstein 2008) as described above.

Much of our understanding of the neurobiology of postpartum anxiety has come from rodent studies. In rodents, the periaqueductal gray (PAG; Figueira et al. 2008), PVN

(Jurek et al. 2012), BNST (Smith et al. 2013), and amygdala (Bosch et al. 2005) have been implicated in the regulation of anxiety in postpartum females. All of these regions are interconnected with the mPFC (Numan and Woodside 2010; Peters et al. 2009; Vertes

2004), and we have recently shown that the PL region of the mPFC in postpartum females also plays a role in mediating postpartum anxiolysis (Sabihi et al. 2014a). Thus, the brain regions regulating postpartum anxiety in both rodents and humans are largely the same but the neural mechanisms within these regions that regulate postpartum anxiety require further investigation in order to fully understand this disorder and assist in prevention and treatment strategies for women.

4.3 Postpartum Anxiety and Oxytocin

Numerous hormones, neuropeptides, and neurotransmitters have been implicated in the regulation of postpartum mood disorders and are thought to do so by acting on the brain sites discussed above. These include norepinephrine, serotonin, glutamate, prolactin, CRF, and a variety of other steroid and peptide hormones (Hillerer et al. 2011;

Lonstein et al. 2014; Marchesi et al. 2016; Moses-Kolko et al. 2008; Pawluski et al. 2017;

Woodside 2016). Importantly many of these neurochemicals have been associated with postpartum anxiety in both rodents and women, supporting the face validity of rodent models (Pawuluski et al. 2017). It is likely that the various neurochemical factors do not only function in parallel and with redundancies, but also sequentially and interactively

(Lonstein, 2007). Part 2 of this dissertation will focus on OT in the mPFC and its possible synergistic role with GABA to alter maternal anxiety.

As described above, OT is critical for parturition, lactation, and maternal care behaviors (Bosch and Neumann 2012; Febo et al. 2009; Heinrichs et al. 2001; Lonstein

2005b; Neumann et al. 2000a; Pedersen et al. 2006). The only assessments of the OT system in human mothers come from OT measured in the blood, CSF, or saliva. OT levels rise in the plasma and CSF during labor (Takeda et al. 1985) and remain high postpartum to aid in milk ejection (Bellmann 1976). Salivary OT has been typically used to characterize changes in OT secretion patterns in mothers across the breastfeeding cycle. A number of studies have concluded that OT increases in anticipation of breastfeeding and remains high until at least 30 min after feeding (Grewen et al. 2010;

Niwayama et al. 2017; White-Traut et al. 2009). A few studies have demonstrated a negative association between plasma and salivary OT and aspects of anxiety in peripartum women (Nissen et al. 1998; Niwayama et al. 2017; Uvnas-Moberg et al. 1990) and this may be attributed to breastfeeding and physical contact with the infant

(Heinrichs et al. 2001). Likewise, higher OT during pregnancy or postpartum has been linked to less depressive-like behavior (Bell et al. 2015; de Moura et al. 2016; Skrundz et al. 2011) and presumably less anxiety since these two conditions present with high co-

88 morbidity (Falah-Hassani et al. 2016; Grigoriadis et al. 2011; Reck et al. 2008). However, there are studies which have failed to find a relationship between OT levels and postpartum anxiety/depression. These inconsistencies are likely due to difficulty of measuring OT in the blood and CSF because of this peptide’s short half-life in the periphery. Additionally, measurements in the periphery do not correlate well with measures of the central system because only 1-5% of many peptides circulating in blood plasma cross the blood brain barrier (Ermisch et al. 1993; Ermisch et al. 1985).

Compared to humans, of which we know little about changes in central OT signaling during pregnancy and/or the postpartum period, rodent studies show an upregulation of the central OT system with motherhood. For example, local release of OT is elevated in many brain regions during the postpartum period (Kendrick et al. 1992;

Moos et al. 1984; Neumann et al. 1993). In addition to OT release, OTR mRNA and OTR binding in regions including the lateral septum, amygdala, medial preoptic area, and cortex are also elevated during pregnancy and the postpartum period (Meddle et al. 2007;

Mitre et al. 2016; Young et al. 1997). Alterations in circulating ovarian steroids including progesterone and estrogens likely underlie the changes in OT and OTR expression observed (Windle et al. 2006).

The reduction in postpartum anxiety has been attributed to OT acting within brain regions including the PAG (Figueira et al. 2008), PVN (Jurek et al. 2012), and amygdala

(Bosch and Neumann 2008). Thus, blockade of OTR-mediated effects by either site- specific or ICV infusion of an OTR antagonist has anxiogenic effects in pregnant and lactating rats but not in virgin females (Bosch et al. 2004; Figueira et al. 2008; Sabihi et

89 al. 2014a). Furthermore, just as in humans, offspring contact stimulates central release of

OT (Lonstein et al. 2014; Neumann et al. 2000a) contributing to the reduction in anxiety that is typically observed during this time. Conversely, in mothers that have not had recent physical contact with their pups and display heightened anxiety-like behavior (as described above) an infusion of OT into the PAG results in restored low anxiety-like behavior (Figueira et al. 2008).

The mPFC is interconnected with each of these regions (Vertes, 2004; Peters et al., 2009; Numan and Woodside, 2010) and has been shown to regulate aspects of maternal care behaviors and postpartum anxiety (Afonso et al. 2007; Febo et al. 2010;

Gonzalez et al. 2000; Maaswinkel et al. 1996; Sabihi et al. 2014b; Stack et al. 2010). For example, the mPFC of postpartum females has been shown to be activated by offspring contact (Febo, 2012). Furthermore, inactivation or lesions of the mPFC result in pup retrieval deficits (Afonso et al. 2007; Febo et al. 2010). In regards to OT, OTR blockade in the postpartum mPFC has been shown to impair maternal care behaviors and enhance maternal aggression (Sabihi et al. 2014a). However, it’s important to note that reduced anxiety-like behavior during the postpartum period likely isn’t just mediated by elevated

OT. Similar consequences on postpartum anxiety have been reported in cases of elevated

GABA (described below), suggesting potential interactions between these systems in order to ensure optimal maternal care behaviors.

4.4 Postpartum GABA and its Link to OT and the Maternal mPFC A discussed in Part 1, GABA is the main inhibitory neurotransmitter in the brain and there is a vast literature linking GABA to anxiety (Goddard et al. 2001; Ham et al.

2007; Long et al. 2013; Morrow et al. 2000; Shah et al. 2004; Shah and Treit 2003;

Simpson et al. 2012). GABA has also been related to changes in anxiety during the postpartum period. In laboratory rats, CSF concentrations of GABA are high in lactating rats that interact with pups, but are almost nondetectable in dams whose pups have been removed (Qureshi et al. 1987). Furthermore, administration of GABA agonists bring emotional responding in cycling female rats to a level similar to that found in postpartum females (Ferreira et al. 1989). Treating postpartum females with GABAAR antagonists peripherally or centrally (PAG) prevents the normal anxiolytic phenotype (Hansen et al.

1985; Miller et al. 2011; Miller et al. 2010). However, in regions including the ventromedial hypothalamus and amygdala, treatment with GABAAR antagonists does not seem to have any effect on postpartum anxiety-like behavior (Hansen and Ferreira 1986).

In the mPFC, postpartum rats display elevated basal GABA release and turnover

(Arriaga-Avila et al. 2014; Kornblatt and Grattan 2001) as well as higher expression of

GAD65 and the vesicular GABA transporter (Ahmed and Lonstein 2012) compared to virgin females, suggesting greater potential for cortical GABA synthesis and release in mothers. There is also significant increase in the affinity of total forebrain GABAAR for

GABA in mid to late pregnant rats compared to cycling females (Majewska et al. 1989).

While research performed in rodents paints a fairly clear picture of how the GABAergic system changes across pregnancy and the postpartum period and may contribute to

91 reduced anxiety-like behavior, research performed in humans is much more limited.

Little is known about changes in the central GABA system in postpartum women, however, one study has shown that women’s CSF levels of GABA drop during late pregnancy and increase during labor in the occipital cortex (Altemus et al. 2004;

Sethuraman et al. 2006).

Recent work has shown that just as in the non-maternal brain, OTR are found on somatostatin and parvalbumin positive GABAergic interneurons in the maternal cortex, suggesting that OT is important for mediating cortical inhibition during the postpartum period (Marlin et al. 2015). Activation of these receptors was shown to enable pup retrieval behavior by enhancing auditory cortical pup call responses through increasing the signal-to-noise ratio in maternal mice (Marlin et al. 2015). In addition, OT increases

GABA release and GABAAR mediated inhibition in the mPFC (Qi et al. 2012). Given these links, the mPFC may represent an important region where OT and GABA interact to regulate anxiety during the postpartum period.

4.5 Experiments

The postpartum period, which is accompanied by a natural reduction in anxiety, serves as an opportune time in which to study the mechanism by which increased levels of endogenous mPFC OT act in order to reduce anxiety-like behavior. Thus, in part 2 of this dissertation we examined the natural reduction in anxiety that occurs postpartum and determined whether endogenous OT within the postpartum mPFC mediates this effect.

To do so, we blocked OTR in the maternal mPFC using a highly specific OTR antagonist

92 and evaluated anxiety-like behavior in the EPM. In addition, because it seems likely that

OT and GABA interact and have synergistic effects during the postpartum period, we investigated the extent to which high levels of endogenous GABA in the maternal mPFC attenuate anxiety. This was accomplished by blocking GABAAR in the PL mPFC and evaluating anxiety-like behavior in the EPM. Furthermore, we investigated the anxiogenic effects of mother-pup separation on postpartum anxiety-like behavior and its effects on activation of GABAergic neurons in the PL mPFC. Finally, we examined whether the natural reduction in anxiety-like behavior that occurs postpartum can be restored by activation of the GABAAR in the PL mPFC.

Chapter 5: Oxytocin in the medial prefrontal cortex regulates maternal anxiolysis during

the postpartum period

This chapter contains information that has been published in the August, 2014 Frontiers in Behavioral Neuroscience journal under the title “Oxytocin in the medial prefrontal cortex regulates maternal care, maternal aggression and anxiety during the postpartum period” by Sabihi et al., 2014.

5.1 Introduction

The postpartum period is accompanied by dramatic behavioral changes in all mammalian species. In rats, females that were previously unresponsive or infanticidal towards pups will engage in an elaborate repertoire of caregiving activities after parturition that serve to nurture and protect the young thus promote their development and survival (Erskine et al. 1978; Numan and Woodside 2010; Rosenblatt

1967). In addition, postpartum females also show changes in their emotional state characterized by attenuated levels of anxiety-like behavior (Figueira et al. 2008; Fleming and Luebke 1981; Hard and Hansen 1985; Jurek et al. 2012; Lonstein 2005a; 2007;

Macbeth and Luine 2010; Neumann et al. 2000a). The co-occurrence of reduced anxiety along with heightened maternal responsiveness during the postpartum period suggests a link between emotionality and proper parenting (Fleming and Luebke

1981; Lonstein 2005a).

Each of the behavioral changes that emerge postpartum is mediated by a vast array of neurochemicals, including oxytocin (OT). In postpartum rats, suckling simultaneously stimulates the release of OT from the pituitary into the bloodstream as well as into the CNS (Bosch and Neumann 2012; Landgraf et al. 1992; Neumann et al.

1993). In the periphery, OT enhances smooth muscle contractility for milk ejection while the effects of OT on maternal care, maternal aggression, and postpartum anxiety are mediated by CNS OT. However, both the central and peripheral actions of OT are transduced by a single isoform of the oxytocin receptor (OTR) (Gimpl and Fahrenholz

2001). During the peripartum period, expression of the OTR increases not only on mammary contractile cells but also within various regions of the brain including the lateral septum, medial preoptic area (MPOA), central nucleus of the amygdala (CEA), ventromedial nucleus of the hypothalamus, nucleus accumbens, olfactory bulb (OB),

PVN, bed nucleus of the stria terminalis (BNST), and ventral tegmental area (VTA)

(Bosch and Neumann 2012; Bosch et al. 2010; Caughey et al. 2011; Francis et al. 2000;

Insel 1990; Pedersen et al. 1994). Not surprisingly, many of these brain regions have been implicated as sites mediating the behavioral effects of OT during the postpartum period. Although less studied, the postpartum-associated reduction in anxiety has been attributed to OT acting within the midbrain periaqueductal gray (PAG) (Figueira et al.

2008), paraventricular nucleus (PVN) (Jurek et al. 2012), and CEA (Bosch et al. 2005).

Thus, OT acts on a widespread network of brain regions to influence postpartum behaviors.

Several lines of evidence suggest that another component of this network may include the medial prefrontal cortex (mPFC). First, in addition to expressing OTR

(Gould and Zingg 2003; Insel and Shapiro 1992; Liu et al. 2005; Smeltzer et al. 2006), which are upregulated postpartum (Mitre et al. 2016), the mPFC contains OT-sensitive neurons (Ninan 2011) and receives long-range axonal projections from OT producing neurons in the hypothalamus (Knobloch et al. 2012; Sofroniew 1983). Second, the mPFC of postpartum rats becomes activated by suckling or OT administration (Febo 2012; Febo et al. 2005). Third, lesion and inactivation studies have implicated the mPFC in the regulation of anxiety (Shah and Treit 2003; Stern et al. 2010). Lastly, OT regulates social

(Young et al. 2014) and anxiety (Sabihi et al., 2014b) behaviors in female rodents at least in part through its actions in the PL region of the mPFC. Although these findings collectively suggest that the mPFC may be a common target underlying the behavioral effects of OT during the postpartum period, this possibility has not been previously explored. Thus, in the present study, we examined anxiety-like behavior in postpartum rats following administration of a highly specific OTR-A into the PL mPFC.

5.2 Materials and Methods

Animals

Age matched adult (9-12 weeks of age) virgin (225-250 g) and timed pregnant

[gestation day (GD) 14] female Sprague-Dawley rats from Taconic (Germantown, NY) were used. All rats were housed individually in a temperature and humidity controlled room and maintained on a 12/12 light/dark cycle (lights on at 0600 hr) with access to

96 food and water ad libitum. All procedures were conducted in accordance with The Guide for the Care and Use of Laboratory Animals published by the National Institutes of

Health and approved by The Ohio State University Institutional Animal Care and Use

Committee.

For postpartum females, the day of birth was designated as postpartum day 0

(PD0) and on PD1 each litter was culled to 5 male and 5 female pups. In virgin females, stages of estrous were monitored through daily vaginal swabs which were taken at least 2 hr prior to testing. Samples of cells were obtained with a sterile cotton swab saturated in

0.9% saline and applied to a glass slide. After drying, slides were stained with 1% aqueous Toluidine Blue and cell types characterized under 10X magnification (Everett

1989). Only those virgin females that had normal 4-5 d estrous cycles were used.

Surgical procedures

On GD16-17, rats were anesthetized with a 2-4% isoflurane gas/air mixture and aligned on a stereotaxic apparatus (Kopf Instruments, Tujunga, CA). This timepoint for surgery is consistent with prior studies assessing behavioral changes during the postpartum period following drug administration via cannulation (Neumann et al., 2000a;

Lubin et al., 2003; Figueira et al., 2008). Body temperature was maintained throughout the surgery with a warming pad. Bilateral cannula guides (pedestal mounted 22-gauge stainless steel tubes with 1.5 mm separation and cut 3.5 mm below the pedestal; Plastics

One, Roanoke, VA) were secured in a stereotaxic holder and lowered into the prelimbic region (PL) of the mPFC (AP: + 3.2 mm, ML: ± 0.75 mm, DV: -3.2mm; Paxinos and

Watson 1998). The PL mPFC was targeted because it has been most consistently linked to maternal anxiety (Febo 2012; Febo et al. 2010; Nephew et al. 2009; Pereira and

Morrell 2011). The cannula were secured by stainless steel screws and dental cement. A bilateral stainless steel obturator (0.35 mm diameter; Plastics One) extending 0.2 mm beyond the tip of the guide cannula was placed into the guide cannula after surgeries.

The scalp was closed around the protruding portion of the cannula with sutures.

Following surgery, rats were allowed to recover for at least 7 d before behavioral testing.

Central infusions

On days 2 and 4 post-surgery, all rats were habituated to the handling and infusion procedures. During habituation, rats were removed from their home cage and handled for approximately 3 min while being lightly restrained in a terrycloth towel. The obturators were then removed and a 28-gauge bilateral injection cannula extending 0.2 mm beyond the tip of the guide cannula into the PL cortex was inserted into the guide.

The injection cannula were left in place for 3 min then removed and the obturator replaced. On testing days (during diestrus for virgin females and PD5 for postpartum rats), rats underwent the same procedure as described above except that an injection cannula attached to two 1 µl Hamilton Syringes via PE-10 tubing was inserted into the guide cannula. Bilateral infusions were made using a Harvard Apparatus Pico Plus Elite infusion pump (Holliston, MA) which delivered a 1.0 µl volume into each hemisphere over 3 min. The injector was left in place for an additional 1 min before withdrawal.

Experimental design

To investigate the effects of OTR blockade in the mPFC, virgin and postpartum female rats received bilateral infusions of the highly specific OTR-A (Manning et al.

2 4 2012), desGly-NH2-d(CH2)5[D-Tyr ,Thr ]OVT (courtesy of Dr. Maurice Manning,

University of Toledo) into the PL mPFC at a dose of either 0.1µg/1µl (n = 7 postpartum; n = 7 virgin) or 0.5µg/1µl (n = 8 postpartum; n = 8 virgin) into each hemisphere (Bosch et al. 2005; Figueira et al. 2008; Lubin et al. 2003). Additional groups of control rats received a 1µl infusion of physiological saline (n = 8 postpartum; n = 8 virgin). Anxiety- like behavior was assessed on PD5, when postpartum females exhibit low levels of anxiety-like behavior (Figueira et al. 2008; Lonstein 2005a). Anxiety-like behavior in virgin females was done during diestrus in order to control for fluctuations in anxiety across the estrous cycle (Marcondes et al. 2001; Mora et al. 1996; Walf and Frye 2007).

Studies which have examined factors regulating anxiety-like behavior in virgin females commonly test during diestrus since this is the stage when anxiety is relatively stable (De

Almeida et al. 1998; Figueira et al. 2008; Marcondes et al. 2001). In all cases, behavioral testing was done between 0900-1200 hr 20 min after infusions (Nyuyki et al. 2011; Ring et al. 2006).

Anxiety-like behavior

Both postpartum females (PD5) and virgin females (diestrus) were tested for anxiety-like behavior using two well-validated models- the elevated plus maze (EPM) and the open field (OF) tests (Lapiz-Bluhm et al. 2008; Prut and Belzung 2003; Rotzinger

99 et al. 2010). Virgin females were included in this experiment in order to examine whether the OTR-A would prevent the reduction in anxiety typically observed postpartum as well as to confirm prior reports showing that the behavioral effects of OTR blockade are specific to the postpartum period (Figueira et al. 2008; Neumann et al.

2000a; Sabihi et al. 2014b).

All females were brought into the infusion room in their home cage. Following a

20 min habituation period, litters were removed from postpartum females and placed in a separate cage on a heating pad. Dams were then removed from their home cage and infused with either OTR-A or saline and returned to their home cage which was then placed in an adjacent room for testing. 20 min after infusion, anxiety testing began. The two anxiety tests were done 5 min apart on the same day and the order of these tests was counterbalanced among rats.

The EPM is as described above (Chapter 2) (Cruz et al. 1994; Lapiz-Bluhm et al.

2008; Pellow et al. 1985). For the OF test, a 60 x 60 Plexiglass arena with walls 40 cm high was used. The floor of the arena was covered with gridlines which allowed for measurement of locomtion. The gridlines were spaced 10 cm apart yielding a total of 36,

10 x 10 cm squares. The innear area was considered the central 16 squares which covered a 40 x 40 cm area. Rats were placed in the center of the OF and during a 5 min test, the percentage of gridlines crossed in the center of the arena (number of center gridliens crossed/total number of gridlines crossed x 100) were used as measuures of anxiety-like behavior. An increase in the percentage of center gridlines crossed correlate with lower anxiety. Locomotor activity was assessed using total number of gridlines crossed (Prut

100 and Belzung 2003). Testing occurred in bright light conditions (550 lux inner zone, 150 lux outer zone).

Histology

Rats were overdosed with Euthasol and transcardially perfused with 4% paraformaldehyde 24 - 48 hr after the completion of behavioral testing. Brains were removed, postfixed for 24 hr and then sectioned on a Vibratome. 40-µm thick coronal sections were collected throughout the area of the cannula implant and stained with 0.2% cresyl violet for verification of correct placement (Fig. 11). Those animals with cannula placements outside of the PL region of the mPFC (2 postpartum females, one in the saline control group and one in the 0.1µg/1µl OTR-A group) were excluded from the study.

Examination under high magnification (100X) revealed limited to no damage at the tip of the cannula in any of the animals.

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Figure 11: Schematic representation of mPFC cannula placements. Cannula tip placements were in the prelimbic region (PL) of the mPFC (AP: +3.2 mm, ML: ±0.5 mm, DV: -3.2 mm). Each dot indicates an individual subject. Infusions were bilateral but are represented unilaterally. Cannula placements for virgin females receiving an infusion of 0.1µg/1µl OTR-A, 0.5µg/1µl OTR-A, or saline (a). Cannula placements for postpartum females receiving an infusion of 0.1µg/1µl OTR-A, 0.5µg/1µl OTR-A, or saline (b). Animals with missed cannula placements in IL or the ventricle were excluded from analysis. Adapted from Paxinos and Watson, 1998.

Statistical analysis

All statistical analyses were performed using Graphpad Prism software version

5.01 (La Jolla, CA). Anxiety-like behavior was analyzed using a 2 x 3 Analysis of

Variance (ANOVA) with reproductive state (postpartum or virgin) and infusion type

(saline, 0.1µg/1µl OTR-A, or 0.5µg/1µl OTR-A) as factors. Statistical significance for main effects and interactions were indicated by p values < 0.05 and when significance was found were followed by Tukey’s HSD post hoc comparison test.

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5.3 Results

Attenuated anxiety during the postpartum period is prevented by blocking OTR in the mPFC

Reproductive state and OTR-A infusion into the PL mPFC had significant effects on anxiety-like behavior in the EPM. For the percentage of time spent in the open arms of the EPM (Fig. 12a), there was a main effect of reproductive state such that postpartum females spent more time in the open arms than virgins (F1, 40 = 15.07, p < 0.0005) indicating lower anxiety in postpartum females. There was also a significant main effect of infusion type (F2, 40 = 6.97, p < 0.005) and a significant reproductive state by infusion type interaction (F2, 40 = 4.5, p < 0.05). Post hoc analysis revealed that in postpartum rats, the group receiving the higher 0.5µg/µl dose of the OTR-A spent less time in the open arms compared to both the saline and 0.1µg/µl OTR-A groups (p’s < 0.01), which did not differ. Postpartum females given the higher 0.5µg/µl dose of the OTR-A did not differ from virgins in the percentage of time spent in the open arms (p’s > 0.05). None of the virgin groups significantly differed from each other.

The number of entries made into the open arms of the EPM (Fig. 12b) also showed a significant main effect of reproductive state such that postpartum females made more open arm entries than virgins again indicating reduced anxiety in postpartum females (F1, 40 = 15.21, p < 0.0005). There was also a significant main effect of infusion type (F2, 40 = 6.08, p < 0.005) and a marginally significant reproductive state by infusion type interaction (F2, 40 = 3.11, p = 0.06). Post hoc analysis revealed that in postpartum rats, the group receiving the higher 0.5µg/µl dose of the OTR-A made fewer open arm

103 entries compared to both the saline and the lower 0.1µg/µl OTR-A groups (p’s < 0.01), which did not differ. Postpartum females given the higher 0.5µg/µl dose of the OTR-A did not differ from virgins in the number of open arm entries (p’s > 0.05). None of the virgin groups significantly differed from each other.

For the number of closed arm entries in the EPM (Fig. 12c), there was no main effect of reproductive state (F1, 40 = 1.96, p > 0.05) or infusion type (F2, 40 = 1.17, p >

0.05) and no significant reproductive state by infusion type interaction (F2, 40 = 0.47, p >

0.05) indicating that locomotor activity was unaltered by reproductive state or infusion type. In contrast to the EPM, there were no significant main effects or interactions for any behaviors measured in the open field (p’s > 0.05; data not shown).

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Figure 12: Blocking OTR in the PL mPFC enhances postpartum anxiety, but has no effect on anxiety in virgin females. Postpartum females infused with saline or the lower dose of the OTR-A in the mPFC spent a greater percentage of time in the open arms (a) and made more open arm entries (b) as compared to virgins. In contrast, postpartum females receiving the higher 0.5 µg/µl dose of OTR-A displayed a decrease in the percentage of time spent in the open arms (a) and made fewer open arm entries (b) as compared to saline or low dose infusion dams. Locomotor activity, as measured by the number of closed arm entries (c), was not altered. None of the virgin groups differed significantly from one another (a,b,c). Bars represent mean + SEM; ** P < 0.01 postpartum saline and postpartum 0.1 µg/µl OTR-A vs all other groups.

5.4 Discussion

The present work shows that OT receptor activity within the PL region of the mPFC modulates anxiety-like behavior during the early/mid postpartum period. We observed that infusion of a highly specific OTR-A into mPFC of postpartum females prevents the reduction in anxiety typically observed during the postpartum period. Given that OTR blockade in the mPFC can also disrupt some aspects of maternal care (Sabihi et al. 2014a), these findings identify the mPFC as a common brain site for the regulation of postpartum-related behaviors by OT.

The postpartum period has repeatedly been shown to be a time that is associated with anxiolysis (Figueira et al. 2008; Fleming and Luebke 1981; Hard and Hansen 1985;

Jurek et al. 2012; Lonstein 2005a; 2007; Macbeth and Luine 2010; Neumann et al. 105

2000a). We again support these findings here by showing that postpartum rats spend a greater percentage of time in the open arms of an EPM and make more entries into the open arms as compared to diestrus virgins. OT has frequently been examined for its role in modulating anxiety (Bale et al. 2001; McCarthy et al. 1996; Neumann and Landgraf

2012; Neumann et al. 2000b; Waldherr and Neumann 2007; Windle et al. 1997) and our previous work found that OT in the mPFC of virgin male and female rats is anxiolytic

(Sabihi et al., 2014b). However, investigation into specific sites in the brain where OT acts to reduce anxiety-like behaviors in postpartum females has been limited. The PAG

(Figueira et al. 2008), PVN (Jurek et al. 2012), and amygdala (Bosch et al. 2005) are the only regions that have been implicated in the OT-mediated regulation of anxiety in postpartum females. All of these regions are interconnected with the mPFC (Numan and

Woodside 2010; Peters et al. 2009; Vertes 2004) and thus our findings suggest that the mPFC may be part of a complex network that modulates postpartum anxiety-like behavior.

In contrast to postpartum females, OTR-A infused into the mPFC of diestrus virgins did not affect the percentage of time or number of entries into the open arms of the EPM. These results are in line with previous findings demonstrating that OTR blockade decreases the percentage of time spent in open-arms by postpartum, but not virgin female rats (Figueira et al. 2008; Neumann et al. 2000a; Neumann et al. 2000b;

Sabihi et al. 2014b). The differential effects of OTR antagonism in lactating and virgin females likely reflects reproductive differences in OT release and OTR expression in many brain regions (Bosch and Neumann 2012). While postpartum females exhibit

106 elevated OTR expression and/or peptide release, virgin females do not (Bosch et al. 2010;

Caughey et al. 2011; Francis et al. 2000; Insel 1990; Landgraf et al. 1992; Neumann et al.

1993; Pedersen et al. 1994). Therefore, it is reasonable that OT manipulations within the mPFC only impacted anxiety-like behavior in postpartum females.

In the OF, the anxiogenic actions of OTR-A were undetectable in both lactating and virgin females. Although the EPM and OF both have an exploratory component, the

EPM is considered a more sensitive test of anxiety (Hilakivi and Lister 1990) and behavior in one test does not always predict behavior in the other (Bale et al. 2001;

Bhatnagar et al. 2004; Sabihi et al. 2014b). It is also possible that the inconsistencies in the OF may be related to variations in the testing conditions known to influence OF behavior (Lapiz-Bluhm et al., 2008) or differential sensitivity of the OF to the OTR-A which may require different doses than those used here for an anxiogenic effect to be revealed.

As noted in Part 1, the mPFC of the rodent brain consists of three subregions - the infralimbic cortex (IL), PL cortex, and anterior cingulate cortex (Cg1) (Heidbreder and

Groenewegen 2003). Here we targeted only the PL mPFC because of our findings showing a specific effect of exogenous OT in the PL mPFC (Sabihi et al., 2014b). Given the injected volume and expected diffusion of OTR-A, we believe that physiological inactivation induced by OTR-A microinjection was limited to the PL mPFC. Together with the anatomical accuracy of cannula tip placement, we believe that OTR-A diffused into the majority of the PL region without diffusing into neighboring structures.

Nonetheless, without experimentally measuring diffusion of the OTR-A or the behavioral

107 outcomes of blocking OTR in other regions of the mPFC, we cannot eliminate the possibility that leakage to nearby structures may mediate some of the observed effects.

A large body of work over many years has identified an extensive network of brain sites underlying the behavioral effects of OT during the postpartum period (Bosch and Neumann 2012; Gammie 2005; Lonstein and Gammie 2002; Numan and Woodside

2010). Our results may be the first to reveal that OT in the maternal mPFC modulates postpartum anxiety, although further investigation examining the mechanism by which

OT in the postpartum mPFC is required. Nonetheless, these data provide new insights into the neural circuitry of the OT-mediated anxiolysis during the postpartum period.

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Chapter 6: Can the postpartum reduction in anxiety also be affected by GABA

manipulations in the mPFC?

6.1 Introduction

As mentioned in the general introduction and previous chapters, the postpartum period is commonly accompanied by a reduction in anxiety (Feijo et al. 2006; Figueira et al. 2008; Fleming and Luebke 1981; Hard and Hansen 1985; Heinrichs et al. 2001;

Lonstein 2005a; 2007; Neumann et al. 2000a; Sabihi et al. 2014a). This reduction in anxiety is thought to be mediated, at least in part, by physical interaction with the offspring in both rodent and human mothers which is accompanied by increases in intracerebral oxytocin (OT) release (Ferber et al. 2008; Lonstein 2005a; Lonstein et al.

2014; Miller et al. 2011; Neumann et al. 2000a; Pawluski et al. 2017; Ystrom 2012).

Brain regions including the periaqueductal gray (PAG), paraventricular nucleus (PVN), amygdala, and mPFC (Blume et al. 2008; Figueira et al. 2008; Jurek et al. 2012; Sabihi et al. 2014a) are sites of action for the anxiolytic effect of OT during the postpartum period.

Within the mPFC, it is likely that GABA may contribute to the OT mediated reduction in postpartum anxiety as prior work has indicated that GABA and OT likely have an interactive, if not synergistic relationship (Lonstein et al. 2014). For example, recent work has shown that within the cortex, OT receptors (OTR) are located on

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GABAergic interneurons (Nakajima et al., 2014; Marlin et al., 2015) and OT has been found to increase cortical GABA levels (Qi et al., 2012). Furthermore, in the mPFC, postpartum rats display elevated basal GABA release and turnover (Arriaga-Avila et al.

2014; Kornblatt and Grattan 2001) as well as higher expression of GAD65 and the vesicular GABA transporter (Ahmed and Lonstein 2012) compared to virgin females, suggesting greater potential for cortical GABA synthesis and release in mothers.

Together, it could be postulated that OT in the mPFC attenuates anxiety during the postpartum period by enhancing local GABA activity.

6.2 Materials and Methods

Animals

Virgin female (200–250 g) and male (300–350 g) Sprague Dawley rats from

Taconic (Germantown, NY) arrived at our facility and were individually housed for 1 week of acclimation. All rats were housed in a temperature and humidity controlled room and maintained on a 12/12 light/dark cycle (lights on at 0600 hr) with access to food and water ad libitum. All procedures were conducted in accordance with The Guide for the

Care and Use of Laboratory Animals published by the National Institutes of Health and approved by The Ohio State University Institutional Animal Care and Use Committee.

For breeding, one male was placed with one virgin female in their home cage.

Pregnancies were verified through daily vaginal swabs and microscopic identification of sperm. Upon positive determination of pregnancy, designated as gestation day 0 (GD0),

110 female rats were individually housed. The day of birth was designated as postpartum day

(PD) 0, and on PD1, litters were culled to 8 pups (4 males, 4 females).

In virgin females, stages of estrous were monitored through daily vaginal swabs which were taken at least 2 hr prior to testing. Samples of cells were obtained with a sterile cotton swab saturated in 0.9% saline and applied to a glass slide. After drying, slides were stained with 1% aqueous Toluidine Blue and cell types characterized under

10X magnification (Everett 1989). Only those virgin females that had normal 4-5 d estrous cycles were used. All animals were weighed daily.

Surgical procedures

Virgin or pregnant (gestation day 16-18) rats were anesthetized with a 2-4% isoflurane gas/air mixture and aligned on a stereotaxic apparatus (Kopf Instruments,

Tujunga, CA). This timepoint for surgery is consistent with prior studies assessing behavioral changes during the postpartum period following drug administration via cannulation (Neumann et al., 2000a; Lubin et al., 2003; Figueira et al., 2008). Body temperature was maintained throughout the surgery with a warming pad. Bilateral cannula guides (pedestal mounted 22-gauge stainless steel tubes with 1.5 mm separation and cut 3.5 mm below the pedestal; Plastics One, Roanoke, VA) were secured in a stereotaxic holder and lowered into the prelimbic region (PL) of the mPFC (AP: + 3.2 mm, ML: ± 0.75 mm, DV: -3.2mm; Paxinos and Watson 1998). The PL mPFC was targeted because it has been most consistently linked to maternal anxiety (Febo 2012;

Febo et al. 2010; Nephew et al. 2009; Pereira and Morrell 2011). The cannula were

111 secured by stainless steel screws and dental cement. A bilateral stainless steel obturator

(0.35 mm diameter; Plastics One) extending 0.2 mm beyond the tip of the guide cannula was placed into the guide cannula after surgeries. The scalp was closed around the protruding portion of the cannula with sutures. On day 2 postpartum, dams underwent a

30 min maternal behavior test to ensure that cannulation surgery did not interfere with maternal behavior. Only females who retrieved all of their pups to a common nest site and nursed and groomed the pups within the 30 min test, and whose pups gained weight during the postpartum period were retained in the study.

Central infusions

On day 3 post-surgery all females were habituated to the handling and infusion procedures. Postpartum rats were again habituated to the handling and infusion process on PD6 while virgin females were habituated a second time at least one week after surgery on a day of estrus, with the assumption being that they would be in diestrus the next day, which was confirmed after behavioral testing via vaginal swabbing. During habituation, rats were removed from their home cage and handled for approximately 3 min while being lightly restrained in a terrycloth towel. The obturators were then removed and a 28-gauge bilateral injection cannula extending 0.2 mm beyond the tip of the guide cannula into the PL cortex was inserted into the guide. The injection cannula were left in place for 3 min then removed and the obturator replaced. On testing days, all females were brought into the infusion room in their home cage. Followigng a 20 min habituation period, rats underwent the same procedure as described above except that an

112 injection cannula attached to two 1 µl Hamilton Syringes via PE-10 tubing was inserted into the guide cannula. Bilateral infusions were made using a Harvard Apparatus Pico

Plus Elite infusion pump (Holliston, MA) which delivered a 0.5 µl volume into each hemisphere over 3 min. The injector was left in place for an additional 1 min before withdrawal. Rats were then returned to their home cage which was then placed in an adjacent room for testing. 10 min after infusion, rats were tested on the elevated plus maze (EPM).

Experimental design

Experiment 1. To determine whether blockade of GABAA receptors in the PL mPFC can increase postpartum anxiety, virgin and postpartum female rats received bilateral infusions of 0.5 µl saline (n = 5 postpartum; n = 7 virgin) or 2.5 ng bicuculline methiodide (BIC; n = 7 postpartum; n = 6 virgin) dissolved in 0.5 µl of saline into the PL mPFC. These doses were based on previously published work (Miller et al. 2010). In all cases, behavioral testing was done between 0900-1200 hr. Anxiety-like behavior was assessed on PD7 in postpartum females and during diestrus in virgin females in order to control for fluctuations in anxiety across the estrous cycle (Marcondes et al. 2001; Mora et al. 1996; Walf and Frye 2007). Studies which have examined factors regulating anxiety-like behavior in virgin females commonly test during diestrus since this is the stage in which anxiety-like behavior is relatively stable (De Almeida et al. 1998; Figueira et al. 2008; Marcondes et al. 2001).

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Experiment 2. In experiment 2, we examined whether activation of GABAA receptors in the PL mPFC of dams that have been separated from their pups can restore the reduced levels of anxiety-like behavior that are typically observed during this time. A two-factor design was used with litter presence before testing as one factor (no separation or 4h separation) and subsequent drug infusion type as the other factor (saline or muscimol), resulting in a total of 4 groups: no separation/saline, 4 h separation/saline, no separation/muscimol, 4 h separation/muscimol. On PD7 approximately half of the postpartum females had their pups removed and placed in an incubator set at 34 °C (nest temperature) 4 h before testing. This separation time has been previously found to increase postpartum female rats' anxiety-related behavior in an EPM (Figueira et al.

2008; Lonstein 2005a; Miller et al. 2011; Smith and Lonstein 2008). The other half of dams had the experimenter’s hand placed in their home cage, the pups picked up, and immediately replaced to control for perturbation caused by longer-term litter removal in females from the other condition. Otherwise, postpartum females from the no separation group were left in their home cages and allowed continual contact with their pups until the time of infusion. Dam and litter manipulation occurred between 0600-0700 h on PD7.

4 h after pup separation or 4 h after the morning litter manipulation, postpartum females from both groups (with pups and pups removed) received either 0.5 µl saline (n = 6 no separation; n = 5 4h separation) or 0.5 µg muscimol (n = 6 no separation; n = 6 4h separation), a GABAA receptor agonist, dissolved in 0.5 µl of saline into each hemisphere of the PL mPFC. These doses are based on previously published work (Chan et al. 2011;

Maeng and Shors 2013; Shah et al. 2004; Solati et al. 2013).

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Experiment 3. In experiment 3, we examined the effects of pup separation on activation of GABAergic neurons in the PL mPFC following exposure to the EPM. On PD7, one group of postpartum females (n = 11) had their pups removed and placed in an incubator set at 34 °C (nest temperature) 4 h before testing. The other group of dams had the experimenter’s hand placed in their home cage, the pups picked up, and immediately replaced to control for perturbation caused by longer-term litter removal in females from the other condition (n = 12). Otherwise, dams from the no separation group were left in their home cages and allowed continual contact with their pups until the time of infusion.

Dam and litter manipulation occurred between 0600-0700 h on PD7. 4h after pup separation or after the morning litter manipulation, postpartum females from both groups were habituated to the testing room for 10 min prior to EPM testing. After testing, dams were returned to their home cage (which contained pups or not) which was carried back to the colony. 60 min after behavioral testing all females were euthanized and brains removed for immunohistochemical analysis as described below.

Anxiety-like behavior

The EPM was used to test for anxiety-like behavior as described earlier (Lapiz-

Bluhm et al. 2008; Prut and Belzung 2003; Rotzinger et al. 2010). All females were brought into the infusion room in their home cage. Following a 20 min habituation period, females were removed from their home cage and infused with either BIC,

115 muscimol, or saline and returned to their home cage which was then placed in an adjacent room for testing. 10 min after infusion, anxiety testing began.

Histology

For experiments 1 and 2, rats were overdosed with Euthasol and transcardially perfused with 4% paraformaldehyde 24- 48h after the completion of behavioral testing.

Brains were removed, postfixed for 24h and then sectioned on a Vibratome. 40-µm thick coronal sections were collected throughout the area of the cannula implant and stained with 0.2% cresyl violet for verification of correct placement (Fig. 13). Those animals with cannula placements outside of the PL region of the mPFC (2 virgin females from experiment 1, 1 from the saline group and one from the BIC group; and 1 postpartum female from experiment 2 in the no separation muscimol group) were excluded from the study. Examination under high magnification (100X) revealed limited to no damage at the tip of the cannula in any of the animals.

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Figure 13: Schematic representation of mPFC cannula placements for Experiment 1 and 2. Cannula tip placements were in the prelimbic region (PL) of the mPFC (AP: +3.2 mm, ML: ±0.5 mm, DV: -3.2 mm). Each dot indicates an individual subject. Infusions were bilateral but are represented unilaterally. Cannula placements for virgin and postpartum females receiving an infusion of 2.5 ng BIC, or saline (left). Cannula placements for postpartum females receiving an infusion of 0.5 ug muscimol, or saline and either having no separation from their pups or 4h of separation (right). Animals with missed cannula placements in Cg1 or the ventricle (indicated by a red X) were excluded from analysis. Adapted from Paxinos and Watson, 1998.

For experiment 3, subjects were overdosed with Euthasol and transcardially perfused with 4% paraformaldehyde 1 hr after EPM testing. This time point was based on the maximal Fos protein expression in the rat brain following exposure to the EPM

(Duncan et al., 1996). Brains were postfixed overnight at 4 °C. On the following day, brains were transferred to 30% sucrose in 0.1M PBS until sectioning. 50 µm sections extending through the mPFC (AP: 4.2 – 2.0mm) were obtained with a cryostat and stored 117 in a sucrose-based cryoprotectant at -20ºC until immunohistochemical processing on free-floating sections. Four sections (1:6) were processed for double-labeling for GAD67 and c-Fos as well as PV and c-Fos. Briefly, sections were washed in 0.1M PBS (3x5min) and then incubated with 0.1% Tween in PBS for 10 min. Next, sections were blocked in

10% normal goat serum (NGS) and 0.3% Triton X in PBS for 60 min followed by incubation in rabbit anti-Fos primary antibody (1:300; Santa Cruz Biotechnology, Santa

PBS.

For co-labeling with GAD67, sections were then incubated in mouse anti-GAD67 primary antibody (1:2000; Millipore, Billerica, MA) in PBS with 0.5% tween overnight at 4°C. After a PBS rinse, sections were incubated for 1 hr at room temperature in

DyLight 549 horse anti-mouse secondary antibody (1:500; Vector Laboratories,

Burlingame, CA). For co-labeling with PV, sections were blocked in 0.3% Triton X and

10% NHS for 60 min at room temperature before being incubated overnight at 4°C in mouse anti-PV primary antibody (1:100; Millipore, Billerica, MA) diluted in PBS with 1% Triton X and 3% normal horse serum. After a PBS rinse, sections were incubated for 2.5 hr at room temperature in DyLight 549 horse anti-mouse secondary antibody (1:500; Vector Laboratories, Burlingame, CA). After a final rinse in PBS, all sections were mounted on SupraFrost Plus microscope slides, coverslipped with

DABCO, and kept in the dark at 4°C until imaging.

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Quantification

For confocal analysis, a Nikon 90i microscope was used to obtain 10x (PV+/c-

Fos+) and 20x (GAD67+/c-Fos+) image stacks of the mPFC from PD7 mother rats (~100 steps in 0.3 µm intervals along the z-plane). Image stacks were then projected using NIS

Elements software and total number of c-Fos+, GAD67+, and PV+ cells as well as

GAD67+/c-Fos+ and PV+/c-Fos+ double-labeled cells in the PL mPFC were counted by an researcher blind to experimental conditions. Identification of this region was conducted with reference to illustrations from a standard stereotaxic rat brain atlas

(Paxinos and Watson, 1998) and landmarks such as the location of the corpus callosum.

For the GAD67 analysis, 4 unilateral images of the PL mPFC were taken at 20x and 4 counts were performed unilaterally in a 250000 µm2 ROI. For the PV analysis, 6-8 unilateral images of the PL mPFC were taken at 10x and 6-8 cell counts were performed from these sections in a 160000 µm2 ROI. Percentages of GAD67+ and PV+ cells expressing c-Fos were calculated by dividing the number of these cells expressing c-Fos by the total number of single-labeled cells for the respective neurochemical marker. For each ROI, counts and percentages were divided by the area of the ROI then averaged for each animal and the group mean determined from these values. Data are expressed as the number of immunoreactive cells per 1 mm2. Two brains from the 4h separation group were excluded due to an insufficient number of sections for analysis.

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Statistical analysis

All statistical analyses were performed using Graphpad Prism software version

5.01 (La Jolla, CA). Anxiety-like behavior for Experiments 1 and 2 was analyzed using a two-way Analysis of Variance (ANOVA) with reproductive state (postpartum or virgin) or pup interaction (no separation or 4h separation) and infusion type (saline, BIC, or muscimol) as factors. Anxiety-like behavior and immunohistochemical data from

Experiment 3 was analyzed using a two-tailed Student’s t-test. Statistical significance for main effects and interactions were indicated by p values < 0.05 and when significance was found were followed by Tukey’s HSD post hoc comparison test.

6.3 Results

Attenuated anxiety during the postpartum period is prevented by blocking

GABAAR in the mPFC

For the percentage of time spent in the open arms of the EPM (Fig. 14a), there was a main effect of reproductive state such that postpartum females spent more time in the open arms than virgins (F1, 21 = 6.31, p < 0.05) indicating lower anxiety in postpartum females. There was also a significant main effect of infusion type (F1, 21 = 6.76, p < 0.05) and a significant reproductive state by infusion type interaction (F1, 21 = 7.23, p < 0.05).

Post hoc analysis revealed that in postpartum rats, the group receiving BIC spent less time in the open arms compared to the saline infused postpartum group (p < 0.05).

Postpartum females given BIC did not differ from virgins in the percentage of time spent

120 in the open arms (p’s > 0.05). None of the virgin groups significantly differed from each other (p’s > 0.05).

The number of entries made into the open arms of the EPM (Fig. 14b) also showed a significant main effect of reproductive state such that postpartum females made more open arm entries than virgins again indicating reduced anxiety in postpartum females (F1, 21 = 14.90, p < 0.05). There was also a significant main effect of infusion type (F1, 21 = 12.90, p < 0.05) and a significant reproductive state by infusion type interaction (F1, 21 = 10.75, p < 0.05). Post hoc analysis revealed that in postpartum rats, the group receiving BIC had fewer open arm entries compared to the saline infused postpartum group (p’s < 0.05). Postpartum females given BIC did not differ from virgins in the percentage of time spent in the open arms (p’s > 0.05). None of the virgin groups significantly differed from each other (p’s > 0.05).

For the number of closed arm entries in the EPM (Fig. 14c), there was no main effect of reproductive state (F1, 21 = 2.02, p > 0.05) or infusion type (F1, 21 = 0.12, p >

0.05) and no significant reproductive state by infusion type interaction (F1, 21 = 0.05, p >

0.05) indicating that locomotor activity was unaltered by reproductive state or infusion type.

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Attenuated anxiety during the postpartum period is prevented by mother-pup separation and restored by agonizing GABAAR in the mPFC

While there was no main effect of pup separation (F1, 19 = 2.30, p > 0.05), there was a main effect of infusion type (F1, 19 = 8.12, p < 0.05) and a significant interaction between pup separation and infusion type (F1, 19 = 7.48, p < 0.05) for the percentage of time spent in the open arms of the EPM (Fig. 15a). Post hoc analysis revealed that in the

4h separation group, postpartum rats receiving saline spent less time in the open arms compared to the muscimol infused postpartum group (p < 0.05), indicating increased anxiety-like behavior. Postpartum females that were separated from their pups and then given muscimol did not differ from postpartum females that remained in contact with

122 their pups in the percentage of time spent in the open arms (p’s > 0.05). Dams in the no separation group did not significantly differ from each other (p > 0.05).

For the number of open arm entries in the EPM (Fig. 15b), there was no main effect of pup separation (F1, 19 = 1.61, p > 0.05). However, there was a main effect of infusion type (F1, 19 = 4.96, p < 0.05) and a pup separation by infusion type interaction

(F1, 19 = 4.96, p < 0.05). Post hoc analysis revealed that in the 4h separation group, postpartum rats receiving saline had fewer open arm entries compared to the muscimol infused postpartum group (p < 0.05). Postpartum females that were separated from their pups and then given muscimol did not differ from postpartum females that remained in contact with their pups in the number of open arm entries (p’s > 0.05). Dams in the no separation group did not significantly differ from each other (p > 0.05).

For the number of closed arm entries in the EPM (Fig. 15c), there was no main effect of pup interaction (F1, 19 = 0.31, p > 0.05) or infusion type (F1, 19 = 0.08, p > 0.05) and no significant pup interaction by infusion type interaction (F1, 19 = 1.24, p > 0.05) indicating that locomotor activity was unaltered by pup interaction or infusion type.

123

Figure 15: Attenuated anxiety during the postpartum period is prevented by mother-pup separation and restored by agonizing GABAAR in the mPFC. In the EPM, postpartum females that were separated from their pups for a period of 4 h spent less time in the open arms (a) and made fewer open arm entries (b) as compared to all other groups indicating increased anxiety-like behavior. Muscimol (0.5ug/µl) into the PL mPFC reversed the anxiogenic effect of pup separation as evidenced by an increase in the percentage of time spent in the open arms (a) and more open arm entries (b). Locomotor activity, as measured by the number of closed arm entries, was not altered by pup separation or drug administration (c). Bars represent mean + SEM; *p < 0.05.

Postpartum females that display low anxiety have a greater number of activated

GABAergic neurons in the PL mPFC

Mother-pup separation increased anxiety-like behavior and altered GABAergic neuronal activation in the PL mPFC (Fig. 16). In the EPM, mothers that were separated from their pups for a period of 4h spent less time in the open arms (Fig.16a; t(21) = 2.43, p < 0.05) and had fewer open arm entries (Fig. 16b; t(21) = 3.39, p < 0.05) than mothers that had continual contact with their pups. Locomotor activity, as measured by the number of closed arm entries (Fig. 16c), was not altered by pup separation (t(21) = 1.56, p > 0.05).

While mother-pup separation increased anxiety-like behavior, pup separation followed by EPM exposure did not alter the number of c-Fos (Fig. 16d; t(19) = 1.55, p >

0.05) or GAD67 (Fig. 16e; t(19) = 0.49, p > 0.05) expressing cells within the PL mPFC. 124

However, in mothers that displayed high anxiety levels due to pup separation, the number

(Fig. 16f; t(19) = 2.79, p > 0.05) and percentage (Fig. 16g; t(19) = 3.52, p < 0.05) of

GAD67+ neurons expressing c-Fos in the PL mPFC was reduced.

125

Figure 16: Postpartum females separated from their pups show increased anxiety in the EPM and reduced activation of GABAergic neurons in the PL mPFC. Postpartum females separated from their pups spent less time in the open arms (a) and made fewer open arm entries (b) compared to mothers that were allowed continual contact with their pups. Locomotor activity, as measured by the number of closed arm entries (c), was not altered. Pup separation followed by EPM exposure did not alter the number of c-Fos (d) or GAD67 (e) expressing cells within the PL mPFC. However, in mothers with high levels of anxiety due to pup separation the number (f) and percentage (g) of GAD67+ neurons expressing c-Fos in the PL mPFC was reduced. (h) Confocal images of a cell positive for c-Fos (green, top), GAD67 (red, middle), and GAD67 colabeled with c-Fos (bottom). Bars represent mean + SEM; *p < 0.05.

As OTR have been reported on PV-expressing GABA neurons in the mPFC, we examined whether pup manipulation followed by EPM exposure specifically altered activation of PV neurons in the PL mPFC. Our results show that pup manipulation

126 followed by EPM exposure did not alter the number of c-Fos (Fig. 17a; t(19) = 0.50, p >

0.05), PV (Fig. 17b; t(19) = 0.19, p > 0.05), or double-labeled (c-Fos and PV; Fig. 17c; t(19) = 0.25, p > 0.05) expressing cells within a in the PL mPFC. Additionally, pup separation did not affect the percentage (Fig. 17d; t(19) = 0.21, p > 0.05) of PV+ neurons expressing c-Fos in the PL mPFC.

Figure 17: Pup manipulation followed by EPM exposure did not alter activation of PV neurons in the PL mPFC. Pup separation followed by EPM exposure did not alter the number of c-Fos (a) or PV (b) expressing cells within the PL mPFC. In addition, the number (c) and percentage (d) of PV+ neurons expressing c-Fos in the PL mPFC was unaffected by pup separation. (e) Confocal images of a cell positive for c-Fos (green, top), PV (red, middle), and PV colabeled with c-Fos (bottom). Bars represent mean + SEM.

127

6.4 Discussion

The present work links the attenuation of anxiety-like behaviors in postpartum females to GABA signaling in the mPFC. Specifically, we show that blocking GABAAR in the PL mPFC prevents the normal reduction in postpartum anxiety without affecting anxiety in virgin females. Furthermore, we found that increased anxiety following mother-pup separation was accompanied by decreased activation of GABAergic neurons in the PL mPFC and that reduced maternal anxiety could be restored by activation of the

GABAAR in the PL mPFC.

GABA has been related to changes in anxiety during the postpartum period

(Lonstein et al. 2014). In laboratory rats, CSF concentrations of GABA are high in lactating rats that interact with pups, but are almost nondetectable in dams whose pups have been removed (Qureshi et al. 1987). Furthermore, administration of GABA agonists bring emotional responding in cycling female rats to a level similar to that found in postpartum females (Ferreira et al. 1989). Treating postpartum females with GABAA receptor antagonists peripherally or centrally (PAG) prevents the normal anxiolytic phenotype (Hansen et al. 1985; Miller et al. 2010). However, in regions including the ventromedial hypothalamus and amygdala, treatment with GABAAR antagonists does not seem to have any effect on postpartum anxiety-like behavior (Hansen and Ferreira 1986).

Within the rat maternal mPFC, elevated GABAergic neurotransmission has been reported (Lonstein et al. 2014; Smolen et al. 1993). For example, microdialysis studies have demonstrated that lactating rats display elevated basal GABA release and turnover

128 in the mPFC compared to virgin females (Arriaga-Avila et al. 2014). Additional data indicate that the mPFC of postpartum rats has higher expression of both the 65-kDa molecular weight isoform of GAD (GAD65) and the vesicular GABA transporter compared to diestrus virgins, suggesting greater potential for cortical GABA synthesis and release in mothers (Ahmed and Lonstein 2012; Arriaga-Avila et al. 2014; Kornblatt and Grattan 2001). While these data collectively suggest that GABAergic neurotransmission in the PL mPFC may play a role in the modulation of anxiety during the postpartum period, this had not been explicitly tested.

Here, we show that infusion of BIC, a GABAA receptor antagonist, into the PL mPFC of postpartum females prevents the typical reduction in anxiety and brings emotional responding to a level that is similar to that of cycling virgin females. In contrast to postpartum females, BIC infused into the PL mPFC of diestrus virgins did not affect the percentage of time or number of open arm entries in the EPM, a finding that is in agreement with previous studies (Miller et al. 2010). The difference in sensitivity to BIC is likely not due to differences in the density of GABAAR in the mPFC as a study performed by Miller and Lonstein (2012) demonstrated that, within the mPFC, there are similar levels of GABA receptor binding in postpartum and virgin rats. However, it could be explained as a result of reproductive differences in GABA release and receptor expression between virgin and postpartum females due to endogenous changes in ovarian hormones which result in increased inhibitory neurotransmission at the GABAAR in postpartum females (Concas et al. 1999; Frye 2009; Perez et al. 1988; Toufexis et al.

2006). Thus, administration of BIC to virgin females would not increase the already high

129 anxiety-like behavior because endogenous ligand levels are relatively low and antagonizing the GABAAR would have little effect. It may also reflect a floor effect such that the low duration of time diestrous virgin females spent in the open arms could not be decreased further.

Mother-pup interactions are known to be critical for reducing postpartum anxiety

(Figueira et al. 2008; Lonstein 2005a; Miller et al. 2011; Smith and Lonstein 2008). We replicate this by demonstrating that 4h mother-pup separation induces increased anxiety- like behavior in postpartum females, an effect which was accompanied by decreased activation of GABA neurons in the mPFC. Further we found that reduced anxiety was restored in pup-separated mothers by administration of the GABAAR agonist, muscimol, to the PL mPFC. It is important to note that although we only examined the GABAAR, two other subtypes of GABA receptors exist including GABAB and GABAC receptors.

Most prior research has implicated activity at the GABAAR as a mediator of postpartum anxiolysis (Hansen and Ferreira 1986; Hansen et al. 1985; Miller et al. 2011; Miller et al.

2010). Although the role of GABAB and GABAC receptors in postpartum anxiety has not yet been examined, some studies in non-maternal rats suggest an important role for the

GABAB receptor in modulating anxiety-like behavior (Cryan and Kaupmann 2005;

Kumar et al. 2013). Thus, the potential exists that the GABAB receptor may also modulate postpartum anxiolysis.

As noted above, pup separation led to increased anxiety-like behavior concomitant with decreased activation of GABA neurons labeled with GAD67. However, a heterogeneous population of GABA-producing interneurons exists within the mPFC that

130 shape excitatory output in the central nervous system, these include PV expressing basket type interneurons, vasoactive intestinal peptide (VIP) containing interneurons, and somatostatin (SOM) containing interneurons (Gaykema et al. 2014). The PV subtype is a prominent GABAergic interneuron subtype in the mPFC, has been previously implicated in the control of maternal behavior, and has been shown to express the OTR (Gaykema et al. 2014; Marlin et al. 2015). Thus, we examined activation of this subtype of GABA interneuron specifically but found no effect of EPM exposure after pup separation.

Although previous studies have implicated PV neurons in modulating anxiety-like behavior (Canetta et al. 2016), the possibility remains that pup-separation is affecting the

SOM and VIP expressing interneurons subtypes in the mPFC as the SOM subtype also express OTR (Marlin et al. 2015; Nakajima et al. 2014) and both have been implicated in the regulation of anxiety (Albrecht et al. 2013; Han 2013; Ivanova et al. 2014; Li et al.

2016; Nakajima et al. 2014).

Postpartum anxiety is not well understood and despite the fact that more postpartum women are affected by high anxiety than they are postpartum depression, the disorder is less scientifically studied (Britton 2008; Fairbrother et al. 2016; Pawluski et al. 2017). The current results suggest that GABA signaling within the mPFC is important for regulating maternal anxiety that is mediated by pup interaction and we propose that

GABA may contribute to the OT mediated reduction in postpartum anxiety within the mPFC although this hypothesis has yet to be directly tested. Nonetheless our findings collectively provide a better understanding of the anxiolytic effects of GABA and its

131 relationship with OT which may be relevant to understanding the potential therapeutic role of OT as an agent for the management of postpartum anxiety disorders.

132

Chapter 7: Conclusions

Anxiety disorders affect about 40 million American adults, or about 18% in a given year and have a lifetime prevalence rate of 28.8% (Kessler et al. 2005).

Additionally, women are 60% more likely than men to experience an anxiety disorder over their lifetime (Kessler et al. 2005) with anxiety being particularly prevalent during the postpartum period (Pawluski et al., 2017). Behavioral interventions, including psychotherapy, either alone or in addition to pharmacotherapies currently represent the

“gold standard” for treating anxiety disorders (Hofmann 2007). However, this is not effective in all patients, and a major problem with both treatment options is that many patients achieve only partial remission of symptoms or show a high rate of relapse

(Blanco et al. 2002; Blanco et al. 2013). Therefore, development of novel pharmacotherapies is required. Although anxiety disorders have been extensively studied, the literature examining the underlying neural mechanisms remains scarce, with little evidence identifying specific deficits. Thus, it is imperative that we continue to strive for a better understanding of the specific neural mechanisms underlying anxiety disorders and the mechanisms of action by which effective treatments reduce anxiety symptomology.

133

OT has been shown to reduce anxiety, however, the brain regions where OT acts to reduce anxiety and its mechanism of action are issues that have received limited attention. The mPFC in rodents (dmPFC in humans) has been consistently implicated as a region that modulates anxiety (Albrechet-Souza et al. 2009; Bi et al. 2013; Gonzalez et al. 2000; Heilbronner and Hayden 2016; Jinks and McGregor 1997; Maaswinkel et al.

1996; Paus 2001; Resstel et al. 2008; Saitoh et al. 2014; Shah et al. 2004; Stern et al.

2010; Suzuki et al. 2016; Wager et al. 2009) and we previously implicated the mPFC as a target where OT acts to reduce anxiety (Sabihi et al. 2014b). We extended this work here

(Chapters 2, 3) in a series of experiments in which we determined that OT, acting on

OTR in the PL mPFC, attenuates anxiety-related behavior and may do so by engaging

GABAergic neurons which ultimately modulate downstream brain regions (i.e. the BLA and CEA) implicated in anxiety-like behavior.

The postpartum period is a time that is accompanied by a natural reduction in anxiety and an upregulation of the central OT and GABAergic systems. In addition, it has been suggested that OT and GABA have an interactive and synergistic relationship during this time (Lonstein et al. 2014). Thus, the postpartum period serves as an opportune time in which to study the mechanism by which endogenous OT acts in order to reduce anxiety-like behavior. Just as in generalized anxiety, postpartum anxiety is also mediated by the mPFC (Ahmed and Lonstein 2012; Arriaga-Avila et al. 2014; Febo

2012; Kornblatt and Grattan 2001; Sabihi et al. 2014a). In a series of experiments

(Chapter 5, 6) we determined that endogenous OT in the mPFC regulates postpartum anxiety. Furthermore, we confirmed that postpartum females display attenuated anxiety

134 in comparison to virgin females and observed that blocking GABAAR in the PL mPFC enhances postpartum anxiety. Mother-pup separation decreased activation of GABAergic neurons in the PL mPFC and prevented the natural reduction in anxiety-like behavior that occurs postpartum. However, the reduction in anxiety was restored by activation of the

GABAAR in the PL mPFC.

Overall, these findings suggest that in virgin animals, OT and GABA interact together within the PL mPFC to reduce anxiety (Chapter 2 & 3). In postpartum females, naturally elevated levels of endogenous OT in the PL mPFC act to reduce anxiety

(Chapter 5) and may do so via interactions with GABA in this region (Chapter 6). These results provide new insights into the neural circuitry and the mechanisms that underlie anxiety in both virgin and postpartum animals. These observations also provide a better understanding of the anxiolytic effects of OT and its relationship with GABA which may be relevant to understanding the potential therapeutic role of OT as an agent for the management of anxiety disorders (Lin 2012; Mitchell et al. 2015). Because our findings and those of others demonstrate that elevated OT and GABA levels can each reduce symptoms of anxiety in both rodents and humans, it is possible that treatment with OT could potentially augment the effects of current anti-anxiety drugs, leading to greater therapeutic efficacy. Still, further work is required in order to better understand the anxiolytic actions of OT and GABA in the mPFC as stand-alone or adjunct therapies.

Filling these gaps will enhance the translational value of animal research and broaden our perspective on which systems would be best targeted to improve the mental health of those suffering from anxiety.

135

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