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2018 Sex Differences in the Effects of Low- Dose Ketamine in Rats: A Behavioral, Pharmacokinetic and Pharmacodynamic Analysis Samantha K. Saland

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COLLEGE OF MEDICINE

SEX DIFFERENCES IN THE EFFECTS OF LOW-DOSE KETAMINE IN RATS:

A BEHAVIORAL, PHARMACOKINETIC AND PHARMACODYNAMIC ANALYSIS

By

SAMANTHA K. SALAND

A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy

2018

Samantha Saland defended this dissertation on April 18, 2018. The members of the supervisory committee were:

Mohamed Kabbaj Professor Directing Dissertation

Thomas Keller University Representative

James Olcese Committee Member

Branko Stefanovic Committee Member

Zuoxin Wang Committee Member

The Graduate School has verified and approved the above-named committee members, and certifies that the dissertation has been approved in accordance with university requirements.

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I dedicate this work to my loving husband and to my parents—without their endless encouragement and unconditional support throughout the years, this would not have been possible.

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ACKNOWLEDGEMENTS

I would like to acknowledge all those in the Department of Biomedical Sciences here at Florida State University who have made it possible for me to succeed, not only through financial and research support, but by providing an environment of opportunity, understanding and encouragement time and again during my time in the graduate program. I would also like to express my gratitude to my committee members for their continued guidance and support throughout all these years—thank you for challenging me and expanding my continuous search for knowledge. Most importantly, I am sincerely thankful to my advisor, Dr. Mohamed Kabbaj, whose mentorship has been transformative to my life as a scientist and as a person. I owe my continued pursuit of science and growth as a researcher to his unwavering guidance and support throughout the great moments, and most notably, the difficult ones. Thank you for teaching me the value of perseverance, and for fostering a setting in which asking challenging questions and thinking creatively are both encouraged and valued. Your mentorship has been invaluable.

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TABLE OF CONTENTS

List of Tables ...... vii List of Figures ...... viii Abstract ...... ix

1. INTRODUCTION...... 1

1.1 Primer on Ketamine and Sex Differences in Depression...... 1 1.2 Current state of knowledge on sex differences in effects of low-dose ketamine...... 2 1.2.1 Effects of ketamine under baseline conditions...... 2 1.2.2 Effects of ketamine under conditions of stress...... 3 1.2.2.1 Sex differences in response to chronic stress ...... 3 1.2.2.2 Sex differences in antidepressant response following chronic stress ...... 4 1.3 Pharmacokinetic considerations and relevance to clinical populations ...... 6 1.3.1 Preclinical sex differences in ketamine pharmacokinetics...... 7 1.3.2 Clinical sex differences in ketamine pharmacokinetics ...... 7 1.3.3 Possible explanations for sex- and species-specific pharmacokinetic differences ...8 1.4 Dissertation Plan ...... 9

2. HEDONIC SENSITIVITY TO LOW-DOSE KETAMINE IS MODULATED BY GONADAL HORMONES IN A SEX-DEPENDENT MANNER ...... 10

2.1 Introduction ...... 10 2.2 Materials and Methods...... 12 2.2.1 Animals ...... 12 2.2.2 Ovariectomy/Gonadectomy ...... 12 2.2.3 Cyclic hormone treatment regimen and testosterone supplementation...... 13 2.2.4 Estrous cycle monitoring...... 13 2.2.5 Continuous-access sucrose preference test ...... 13 2.2.6 Experimental design ...... 14 2.2.6.1 Experiment 1a: Effect of cyclic E2 and P4 treatment on hedonic response to low-dose ketamine in ovariectomized female rats...... 14 2.2.6.2 Experiment 1b: Effect of low-dose ketamine on hedonic behavior following cyclic E2 and P4 treatment in intact male rats ...... 14 2.2.6.3 Experiment 2a: Effect of chronic testosterone treatment on hedonic response to low-dose ketamine in intact female rats...... 15 2.2.6.4 Experiment 2b: Effect of low-dose ketamine on hedonic behavior following gonadectomy and testosterone supplementation in male rats ...15 2.2.7 Western blotting ...... 16 2.2.8 Statistical analysis ...... 16 2.3 Results ...... 18 2.3.1 Influence of cyclic E2 and P4 treatment on hedonic response to low-dose ketamine in ovariectomized female rats ...... 18 v

2.3.2 Effect of low-dose ketamine on hedonic behavior following cyclic E2 and P4 treatment in intact male rats ...... 18 2.3.3 Effect of chronic testosterone treatment on hedonic response to low-dose ketamine in intact female rats ...... 22 2.3.4 Effect of low-dose ketamine on hedonic behavior following gonadectomy and testosterone supplementation in male rats...... 23 2.3.5 Integrated analysis of ketamine’s effects across sex and hormonal status: Z-score normalization of sucrose preference ...... 25 2.3.6 Effect of cyclic E2 and P4 treatment on hippocampal BDNF protein levels and downstream signaling effectors in female and male rats ...... 27 2.4 Discussion ...... 30

3. SEX DIFFERENCES IN THE PHARMACOKINETICS OF LOW-DOSE KETAMINE IN PLASMA AND BRAIN OF MALE AND FEMALE RATS ...... 39

3.1 Introduction ...... 39 3.2 Materials and Methods...... 41 3.2.1 Animals ...... 41 3.2.2 Estrous cycle monitoring...... 41 3.2.3 Pharmacokinetics experimental procedures ...... 42 3.2.3.1 Ketamine treatment and sample collection ...... 42 3.2.3.2 Sample preparation...... 42 3.2.4 Quantification of ketamine and metabolites in biological matrices ...... 44 3.2.4.1 HPLC for plasma samples...... 44 3.2.4.2 HPLC for brain tissue samples...... 44 3.2.4.3 Mass spectrometry...... 44 3.2.5 Pharmacokinetics data analysis ...... 45 3.2.6 Statistical analysis ...... 47 3.3 Results ...... 47 3.3.1 Plasma concentrations of ketamine and metabolites in male and female rats ...... 47 3.3.2 Brain tissue concentrations of ketamine and metabolites in male and female rats .49 3.3.3 Sex differences in metabolism and brain distribution of ketamine and norketamine ...... 50 3.4 Discussion ...... 50

4. CONCLUSIONS AND FUTURE DIRECTIONS ...... 63

APPENDIX ...... 69

A. ACUC PROTOCOL APPROVAL ...... 69 B. SUPPLEMENTARY FIGURES ...... 70

REFERENCES ...... 73

BIOGRAPHICAL SKETCH ...... 83

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LIST OF TABLES

3.1 Pharmacokinetic parameters for ketamine and metabolites in plasma and brain tissue of male and female rats...... 52

3.2 Pharmacokinetic parameters for ketamine and metabolites in plasma and brain tissue of diestrus and proestrus female rats...... 53

3.3 Comparison of metabolite and brain-to-plasma ratios in male and female rats following low-dose ketamine administration...... 56

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LIST OF FIGURES

2.1 Timeline of procedures and cyclic hormone treatment regimen for Experiment 1...... 17

2.2 Estradiol and progesterone are required for rapid and sustained hedonic-like effects of low- dose ketamine in female rats ...... 19

2.3 Cyclic P4 treatment enhances the hedonic sensitivity of intact male rats to low-dose ketamine ...... 21

2.4 Chronic testosterone treatment blocks pro-hedonic like effects of ketamine in intact female rats via persistent disruption of estrous cyclicity ...... 24

2.5 Gonadal testosterone does not influence sensitivity to low-dose ketamine in male rats...... 26

2.6 Integrated analysis of ketamine’s effects across sex and hormonal status ...... 28

2.7 Protein levels of BDNF and downstream signaling effectors 24 h after ketamine in estradiol- and progesterone-treated female and male rats ...... 31

3.1 Pharmacokinetics experimental design and sample preparation workflow ...... 43

3.2 Calibration curves for ketamine and metabolite standards in blank plasma and brain matrices...... 46

3.3 Plasma concentration-time profiles of ketamine and its metabolites in male and cycling female rats ...... 48

3.4 Brain concentration-time profiles of ketamine and norketamine in male and cycling female rats ...... 51

3.5 Metabolite ratios and brain distribution of ketamine and norketamine in male and cycling female rats ...... 55

4.1 Conceptual framework for an individualized multi-domain analysis of antidepressant response ...... 67

B1 Phosphoproteomics experimental design and sample preparation workflow ...... 70

B2 Low-dose ketamine alters hippocampal protein phosphorylation in a sex- and estrous- dependent manner...... 71

B3 Functional enrichment of significantly altered phosphoproteins in male and female rats .....72 viii

ABSTRACT

As the global burden of depression continues to rise, development of more efficacious and faster-acting antidepressant treatments has remained stagnant over several decades. This has created a dire need for a newer generation of therapeutics aimed at helping a greater percentage of the patient population in a shorter period of time. Identifying which subpopulations of patients experience optimal responses to certain treatments has become of great interest, as a means of tailoring treatment strategies for more individualized clinical outcomes. In major depressive disorder, women exhibit a lifetime prevalence roughly twice that of men, and tend to display different profiles of symptomology and antidepressant response rates when compared to men, illustrating the importance of examining sex and related variables as individual differences in the pathophysiology of depression and therapeutic response. Indeed, in recent years, consideration of sex has gained interest in depression relevant preclinical research—particularly in light of the discovery that the N-methyl d-aspartate receptor (NMDAR) antagonist, ketamine, rapidly relieves depressive symptoms and suicidal ideation, even in those with treatment-resistant depression. Notably, recent work from our group and others have revealed a higher sensitivity of females to the antidepressant effects of the NMDAR antagonist ketamine. Combined with its fast-acting and relatively sustained properties, ketamine may be a particularly interesting therapeutic alternative for this sensitive population. However, a comparatively small proportion of preclinical and clinical ketamine studies have included females and/or included sex as a variable in analyses. Therefore, the aim of the current work sought to develop the current gap in understanding of how sex and hormones may contribute to the heightened sensitivity of female rats to the rapid antidepressant effects of ketamine by taking a multidisciplinary approach using behavioral, pharmacokinetic and pharmacodynamic analyses in male and female rats. We recently reported that ovarian-derived estradiol (E2) and progesterone (P4) are essential for the greater sensitivity of female rats to rapid antidepressant-like effects of ketamine compared to male rats. However, whether or not the duration of response to ketamine is modulated in a sex- and hormone-dependent manner remains unknown, in addition to the possible contribution of testosterone to such sex differences. Therefore, in the second chapter we explored this systematically by investigating the influence of testosterone, estradiol and ix progesterone on initiation and maintenance of hedonic response to low-dose ketamine in intact and gonadectomized male and female rats. Females, but not males, experienced a sustained increase in sucrose preference following low-dose ketamine, and did so in an E2P4-dependent manner. Whereas testosterone failed to alter male treatment response, hedonic response to low- dose ketamine was enhanced in intact males when P4 was administered concurrently with low- dose ketamine. Treatment responsiveness was associated with greater hippocampal BDNF levels in female, but not male rats 24h after ketamine administration, without activation of key downstream signaling effectors. This work provides novel evidence supporting activational roles for ovarian-, but not testicular-, derived hormones in mediating hedonic sensitivity to low-dose ketamine in female and male rats. The persistence of sex differences following gonadectomy and selective involvement of BDNF in treatment response may indicate a partial role for organizational differences in these effects. In the absence of any preclinical studies of pharmacokinetic sex differences using low- dose ketamine, it is unclear whether the effects reported in the second chapter may be the result of differences in ketamine metabolism between male and female rats, or whether functional differences in the brain are the predominant driver of behavioral sex differences. Therefore, the third chapter examined whether or not sex and hormonal status affect the metabolism of low- dose ketamine in male and female rats. Intact male rats and female rats in either diestrus (low E2, P4) or proestrus (high E2, P4) were administered low-dose ketamine, and their plasma and brains collected to analyze levels of ketamine and its metabolites, norketamine (NK) and dehydronorketamine (DHNK). Females exhibited greater concentrations of ketamine and NK over the first 30 minutes following treatment in both the brain and plasma, largely accounted for by slower clearance rates and longer half-lives. Interestingly, despite the impact of ovarian hormones on behavioral sensitivity to ketamine, no appreciable differences in pharmacokinetic parameters existed between proestrus and diestrus female rats. Together, this work suggests that while sex differences in metabolism may influence the amount of ketamine and NK reaching target areas in the brain, the impact of circulating hormone levels on behavioral sensitivity is more likely an effect of actions within the brain at the time of ketamine administration. As the mechanisms underlying this sex-dependent sensitivity to ketamine’s antidepressant-like effects remain elusive, ongoing phosphoproteomics work is underway to

x investigate the molecular mechanisms underlying this sex-dependent sensitivity to ketamine. Preliminary results revealed striking dissimilarities in the dHPC proteome and phosphoproteome of male and female rats both at baseline, and following low-dose ketamine treatment. Notably, these differences appear to be heavily influenced by hormonal status in female rats. While future work is needed to determine the functional significance of these findings, the collective data presented herein suggest that both biological sex and the hormonal milieu are critical modulators of ketamine’s rapid actions on drug metabolism and within the brain, and provide greater insight into potential physiological and post-translational processes underlying sex- and hormone- dependent modulation of ketamine’s therapeutic effects.

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CHAPTER 1

INTRODUCTION

Adapted from: Saland SK, Duclot F, Kabbaj M (2017). Integrative analysis of sex differences in the rapid antidepressant effects of ketamine in preclinical models for individualized clinical outcomes. Curr Opin Behav Sci. 14, 19-26.

1.1 Primer on Ketamine and Sex Differences in Depression

As the global burden of depression continues its rise as the leading cause of disability worldwide (Patel et al., 2016), the urgent need for more effective treatments is dire. A new wave of excitement, however, has been generated by recent discovery that the N-methyl d-aspartate receptor (NMDAR) antagonist, ketamine, rapidly relieves depressive symptoms and suicidal ideation, particularly amongst those with treatment-resistant depression (Abdallah et al., 2016). Since then, a significant amount of effort has gone into understanding the underlying mechanisms by both preclinical and clinical researchers alike, with the hope of developing novel rapid-acting treatments effective in a broader range of patients (Abdallah et al., 2016). In the era of personalized medicine, a greater focus on identifying biomarkers or predictors of rapid antidepressant response to ketamine has emerged (Zarate et al., 2013), but despite the well-established female preponderance in depressive disorders (Patel et al., 2016) and sex differences in antidepressant efficacy (Keers and Aitchison, 2010), sex has yet to be investigated as a potential moderating variable. Much like genetic and environmental factors, sex is a naturally- occurring disease and treatment modifier (Keers and Aitchison, 2010; Becker et al., 2016), such that factors either protecting against disease or enhancing treatment response in one sex may indicate prevention or treatment strategies in the other sex (de Vries and Forger, 2015). This introduction will highlight recent preclinical evidence demonstrating sex differences in the rapid antidepressant-like response to acute low-dose ketamine, and discuss how a variety of factors including stress, hormonal state, context, and the presence of baseline sex differences, significantly contribute to behavioral or molecular readout following ketamine treatment. This new evidence

1 encourages that sex be seen as an important factor influencing the individual’s response to antidepressant treatment rather than a phenotypic dichotomy.

1.2 Current state of knowledge on sex differences in effects of low-dose ketamine in rodents

1.2.1 Effects of ketamine under baseline conditions

Sex differences in the rapid antidepressant-like effects of ketamine were first reported only a few years ago by work from our lab revealing the heightened sensitivity of female rats to these effects compared to males. These conclusions were demonstrated by the lower dose (2.5 mg/kg) required to rapidly reduce immobility in the forced swim test (FST) and latency to feed in the novelty-suppressed feeding test (NSFT) in naturally-cycling female rats compared to their male counterparts (Carrier and Kabbaj, 2013). This finding, using FST measures as a behavioral readout, has since been replicated by our lab in rats (Sarkar and Kabbaj, 2016), and corroborated in mice (Franceschelli et al., 2015; Zanos et al., 2016). While studies conducted in mice to date have focused solely on intact females, our work in rats demonstrated that this heightened female sensitivity required cyclic fluctuations of both gonadal estradiol and progesterone in female rats (Carrier and Kabbaj, 2013). The higher sensitivity of females to low-dose ketamine interestingly does not simply translate to greater activation of the known molecular mediators in males, mammalian target of rapamycin (mTOR) in the medial prefrontal cortex (mPFC), and eukaryotic elongation factor 2 in the hippocampus (Carrier and Kabbaj, 2013), suggesting that behavioral sex differences in response to ketamine extend beyond differential sensitivity at the molecular level, but rather involve distinct mechanisms in a dose-dependent manner. Interestingly, daily injections of 10 mg/kg ketamine in mice for 21 days induce anti- or pro-depressant-like effects in males or females, respectively (Thelen et al. 2016), which, while still potentially linked to the females’ higher sensitivity to ketamine, highlights the importance of administration paradigm in preclinical studies and warrants further investigations into the interaction between the treatment regimen and ketamine’s sex-dependent behavioral outcome.

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1.2.2 Effects of ketamine under conditions of stress

Although substantial data on brain dysregulations in human depressed subjects are now available, preclinical studies have brought a detailed understanding of their underlying molecular mechanisms and response to therapeutic interventions. In this context, repeated exposures to stress triggers behavioral, molecular, and functional alterations resembling depressive symptoms observed in humans (Czéh et al., 2016). Notably, because several key mediators of the antidepressant response are sexually biased at baseline or following stress itself, it is critical to first investigate their regulation under chronic stress to better understand how males and females differ in response to antidepressants under pathological conditions.

1.2.2.1 Sex differences in response to chronic stress. Stress triggers a fast endocrine response characterized by the release of glucocorticoids, which, in the brain, directly control neurotransmission at multiple levels (McEwen et al., 2016). In addition to being highly dependent on the nature and intensity of the stressor, the consequences of this regulation are affected by both sex and hormonal fluctuations. In male rodents, prolonged exposure to stress or glucocorticoids impairs learning and memory, cognitive performances, and induces anxiety and depressive-like behaviors (Czéh et al., 2016; McEwen et al., 2016; Darcet et al., 2016), whereas in females, the effects of chronic stress differ in a stress-specific manner (Luine, 2016; Franceschelli et al., 2014). For instance, chronic social defeat, restraint, isolation, or unpredictable stress, provoke learning and memory impairments as well as anxiety and depressive-like behaviors in male rats and mice (Czéh et al., 2016), whereas chronic restraint stress induces memory deficits in male but not female rats (Dalla et al., 2011; Kitraki et al., 2004). Similarly, males generally appear more sensitive to the development of anhedonia following chronic mild or isolation stress (Sarkar and Kabbaj, 2016; Dalla et al., 2016; Mileva and Bielajew, 2015), whereas females are more sensitive to induction of depressive-like symptoms by subchronic variable stress (Hodes et al., 2015). These behavioral sex differences are paralleled by coherent adaptations in neuronal activity underlined by dendritic and spine plasticity in key structures such as the hippocampus and mPFC. In these structures, chronic stress generally results in spine loss in both rats and mice (Sarkar and Kabbaj, 2016; Qiao et al., 2016), with concomitant down-regulation of synaptic proteins including synapsin I, PSD-95, and GluR1 (Sarkar and Kabbaj, 2016; Li et al., 2010; 2011),

3 and reduced synaptic function and depressive-like behaviors, as observed in human depressed patients (Duman and Duman, 2015). These effects are specific to males, however, as females show greater spine density in hippocampal CA1 (McLaughlin et al., 2010; Conrad et al., 2012) and infralimbic neurons projecting to the basolateral amygdala (Shansky et al., 2010). Notably, we recently found that chronic isolation stress down-regulates spine density and synaptic proteins in the mPFC of both male and female rats (Sarkar and Kabbaj, 2016), indicating that sex differences in stress-induced spinogenesis in the mPFC are stress-specific. Furthermore, these sex differences are also species-specific as while observed in rats, both male and female mice exhibit hippocampal spine loss following chronic restraint stress (Qiao et al., 2016), requiring further consideration when investigating sex-differences in the effects of ketamine on hippocampal spinogenesis. Despite the lack of data in females, glutamatergic neurotransmission is critically involved in these events, as chronic stress-induced dendritic atrophy is prevented by NMDAR antagonists in males (Magariños and McEwen, 1995; Rubio-Casillas and Fernández-Guasti, 2016; Shors et al., 2004). Importantly, stress-induced spine alterations can recover following interruption of the stress (Radley et al., 2005), in addition to illustrating the highly dynamic nature of neuronal and synaptic plasticity, opens the way for novel therapeutic intervention, and warrants targeting the glutamatergic neurotransmission for antidepressant treatment.

1.2.2.2 Sex differences in antidepressant response following chronic stress. Since the original detailed description of ketamine’s antidepressant effect in a preclinical model (Li et al., 2010), the study of its underlying molecular mechanisms led to the development of a model whereby acute ketamine—through NMDAR inhibition on GABAergic interneurons—increases synaptic strength and subsequent activation of postsynaptic neuroplasticity-promoting pathways such as mTOR and brain-derived neurotrophic factor (BDNF), which, when coupled with extrasynaptic NMDAR inhibition, enhances synaptogenesis (Abdallah et al., 2016). This model, however, originates from studies conducted solely in males. In light of the aforementioned sex differences in synaptic plasticity following chronic stress, and the higher sensitivity of stress-naive females to ketamine’s antidepressant-like effects when compared to males, it is difficult to directly extrapolate these findings to both sexes.

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Accordingly, while acute ketamine can reverse chronic stress-induced depressive- like behaviors in male rats and mice, its effects differ in females. For instance, although 10 mg/kg ketamine reduces chronic mild stress-induced behavioral despair in the FST 24 hours after acute treatment in both male and female mice, this effect is more pronounced in females but lasts longer in males (Franceschelli et al., 2015), suggesting that the mechanisms underlying maintenance of ketamine’s lasting antidepressant-like effects in chronically stressed animals may be sexually biased. Alternatively, this sex discrepancy could result from the activation of sexually-distinct molecular mechanisms. Accordingly, we recently analyzed the dose-dependent effects of acute ketamine on spine density on apical dendrites of prelimbic pyramidal neurons of the socially- isolated male and female rat mPFC (Sarkar and Kabbaj, 2016). Similar to unstressed rats, a single dose of ketamine at 5 but not 2.5 mg/kg reversed isolation-induced behavioral despair in males, whereas both doses were effective in females (Sarkar and Kabbaj, 2016)—findings coherent with the higher female sensitivity to ketamine’s antidepressant-like effects (Carrier and Kabbaj, 2013). Consistent with the current model for ketamine’s effects on spinogenesis, ketamine reversed the stress-induced spine loss in the male mPFC only at the 5 mg/kg dose, associated with increases in synapsin I, PSD-95, and GluR1. In females, however, neither dose affected synaptic proteins expression or spine density within the mPFC, despite behavioral antidepressant-like effects (Sarkar and Kabbaj, 2016). Although pharmacokinetic differences or stress-specificity cannot be ruled out, these findings indicate that the molecular mechanisms underlying the reversal of stress-induced depressive-like behaviors differ between sexes. We do possess several elements of interest for such alternative mechanisms, originating from the dependence of females’ higher sensitivity to ketamine on estrogen, progesterone, and hippocampal BDNF. First, hippocampal spinogenesis is markedly influenced by ovarian hormones, and as such varies across the estrous cycle (Kato et al., 2013). Second, BDNF is a critical regulator of the antidepressant and spinogenesis-enhancing effects of ketamine in the mPFC (Liu et al., 2012). Interestingly, although controversial, multiple studies report differential stress-induced hippocampal BDNF regulation between male and female rodents (Lin et al., 2009; Niknazar et al., 2016; Autry et al., 2009), which places hippocampal BDNF as a potential critical mediator of sex differences in sensitivity to both the induction of a depressive - like state, as well as response to ketamine.

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While behavioral responses to low-dose ketamine discussed thus far are generally consistent between rats and mice, they can vary between strains. In mice, for example, while ketamine’s antidepressant-like effects can be observed for up to 2 weeks (Maeng et al., 2008), they dissipate by 7 days post-treatment in the male CD-1 strain (Bechtholt-Gompf et al., 2011). Similarly, both male and female ICR mice exhibit an antidepressant-like response to acute 10 mg/kg ketamine, but not 5 mg/kg (Kara et al. 2016), a dose effective in C57BL6/J mice. Moreover, ketamine dose-dependently reduces immobility in the FST in Wistar-Kyoto female rats but not in their relative control Wistar strain (Tizabi et al., 2011). Although the non-response of Wistar rats could result from a flooring effect, these studies illustrate the need for an appropriate selection of strain based on study design. The Wistar-Kyoto strain, for instance, meets several criteria for modeling treatment-resistant depression (Willner and Belzung, 2015), and given the interest in ketamine’s efficacy in treatment-resistant patients (Zarate et al., 2006), represents an interesting choice for deciphering ketamine’s potential in this population.

1.3 Pharmacokinetic considerations and relevance to clinical populations

Once administered, ketamine is predominantly N-demethylated into norketamine (NK), which is further transformed into dehydronorketamine (DHNK) and six diastereomeric hydroxynorketamine (HNK) metabolites (Mion and Villevieille, 2013). As a highly lipophilic, weak base, ketamine is rapidly distributed to the brain where it readily penetrates the blood-brain barrier via passive diffusion, along with its pharmacologically active metabolites—albeit to a slightly lesser extent as a result of greater hydrophilicity (Zanos et al., 2016 ;Moaddel et al., 2016). As a prerequisite, ketamine’s therapeutic efficacy in a given individual is limited by the availability of unbound drug (and/or metabolites) present at its relevant site(s) of action within the brain, making pharmacokinetic processes fundamental in understanding the heterogeneity in treatment response within and between sexes. Importantly, sex is a variable that influences nearly all pharmacokinetic processes—absorption, distribution, metabolism, and elimination— which may or may not ultimately influence treatment response (de Vries and Forger, 2015).

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1.3.1 Preclinical sex differences in ketamine pharmacokinetics.

To this point, recent preclinical work has found that higher HNK, but not ketamine or NK, levels are observed in the brain of female mice following acute administration of 10 mg/kg ketamine (i.p.), in addition to greater female behavioral sensitivity to ketamine’s antidepressant- like effects when compared to males (Zanos et al., 2016). Excitingly, further experiments showed that systemically administered HNK is able to cross the blood-brain barrier and elicit antidepressant-like activity in mice without inducing ketamine-like side effects. However, sex differences were not examined in this case, so it is unclear whether behavioral sensitivity to HNK differs between males and females. Here, it should be noted that females, but not males, exhibited an antidepressant-like response to 3 mg/kg ketamine, whereas both sexes responded to the 10 mg/kg dose used for pharmacokinetic analysis. Therefore, a direct association between greater HNK levels and enhanced female antidepressant-like response to ketamine cannot be conclusively inferred. Interestingly, investigation into potential sex differences in ketamine metabolism in rats and species differences are remarkably absent. This leaves a significant gap between preclinical behavioral findings—specifically in rats—and clinical efficacy in humans, and highlights the need for pharmacokinetic analysis across multiple behaviorally-relevant doses across species in both sexes.

1.3.2 Clinical sex differences in ketamine pharmacokinetics.

Surprisingly, very little evidence exists regarding sex differences in ketamine pharmacokinetics in humans, primarily owing to a lack of studies investigating such effects. However, Zarate and colleagues (2012) recently identified modest sex differences in metabolism of low-dose ketamine in MDD and bipolar patients, where females displayed greater plasma levels of DHNK and HNK4a/c metabolites compared to males—however, these differences had no association with treatment response, and no sex differences in antidepressant response were observed. In fact, HNK was negatively associated with treatment response in bipolar depression patients (independent of sex) (Zarate et al., 2012), suggesting that pharmacokinetic sex differences may not actually impact treatment response in clinical depression. These observations are likely dose-dependent, however, as 20% greater ketamine and NK clearance and lower drug/metabolite concentrations are observed in women than men following ketamine infusion at a higher dose

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(Sigtermans et al., 2009). These sex differences were also reflected at the behavioral level in healthy male and female humans, with greater effects on cardiac output and heat pain related indices in men compared to women. Together, the limited clinical data available do not currently support sex differences in rapid antidepressant-response to acute low-dose ketamine. However, sex aside, inclusion of analysis controlling for hormone levels in both men and women is notably absent in currently available examinations of treatment response and pharmacokinetic correlates in patients treated clinically with low-dose ketamine. Given that functional activity within brain regions functionally-relevant to ketamine’s antidepressant actions—particularly prefrontal cortical and hippocampal areas—are modulated by circulating ovarian hormones across the menstrual cycle in adult women (Arelin et al., 2015), it is reasonable to suspect that the hormonal milieu may impact antidepressant effects of ketamine, especially in women. Indeed, preclinical evidence discussed herein may warrant further investigation through the use of proper experimental design and controls for within- and between-sex differences in hormone levels.

1.3.3 Possible explanations for sex- and species-specific pharmacokinetic differences.

While underlying factors responsible for these varying differences in ketamine metabolism observed between males and females remain unknown, sex differences in hepatic expression and activity of ketamine-metabolizing cytochrome P450 enzymes are well-known (Waxman and Holloway, 2009)—and subject to hormonal regulation by estrogen, progesterone and testosterone, which also happen to be substrates of several P450 enzymes responsible for ketamine metabolism (de Vries and Forger, 2015; Waxman and Holloway, 2009). As well, physiological differences influencing xenobiotic distribution, metabolism and clearance (i.e., body weight, adipose tissue levels and distribution) are present between males and females of a variety of species (de Vries and Forger, 2015). Ultimately, whether sex-dependent pharmacokinetic processes contribute to differences between males and females in ketamine’s antidepressant response is unclear, but the evidence strongly supports their consideration both preclinically and clinically. Likewise, non- negligible pharmacokinetic-related species differences have been highlighted herein, encouraging further examination to better translate findings between rodents and humans—an appreciable barrier currently hindering translational neuroscience.

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1.4 Dissertation Plan

The experiments outlined in this dissertation were designed to enhance our understanding of the mechanisms—both physiological and molecular—underlying the differential behavioral sensitivity of male and female rats to low-dose ketamine. We began our studies by exploring the magnitude and duration of impact of ovarian and testicular hormones, if any, on hedonic response to low-dose ketamine in male and female rats. Levels of hippocampal BDNF protein and downstream signaling effectors were also examined as potential predictors of treatment response under sex- and/or hormone-dependent conditions. As presented in Chapter Two, this work provided novel evidence supporting activational roles for ovarian-, but not testicular-, derived hormones in mediating hedonic sensitivity to low-dose ketamine in female and male rats, and was published in the journal Scientific Reports. We then investigated whether this enhanced behavioral sensitivity of female rats could be explained, in part, by pharmacokinetic sex differences in metabolism and distribution of ketamine and its metabolites in the plasma and brain of male rats and female rats with low and high hormonal status, as presented in Chapter Three. All work was conducted in the absence of stressors in order to gain fundamental insight into mechanisms influencing such sex differences at baseline, which will be essential for interpretation of future ketamine studies employing stress to model depressive-like states in rodents.

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CHAPTER 2

HEDONIC SENSITIVITY TO LOW-DOSE KETAMINE IS MODULATED BY GONADAL HORMONES IN A SEX-DEPENDENT MANNER

Adapted from: Saland SK, Schoepfer KJ, Kabbaj M (2016). Hedonic sensitivity to low-dose ketamine is modulated by gonadal hormones in a sex-dependent manner. Sci Rep 6, 21322; doi: 10.1038/srep21322.

2.1 Introduction

Beyond the well-established female preponderance in depressive disorders (Kessler, 2003; Seedat et al., 2009), sex differences have also been identified in antidepressant efficacy that suggest a role for gonadal hormones in moderating treatment response (Hammarström et al., 2009; Keers and Aitchison, 2010). Despite its clear importance, the precise nature of hormonal influence on antidepressant efficacy is equivocal and understudied. This issue is secondary to a more fundamental problem concerning the stagnant progress in development of more efficacious medications for the treatment of depression and other mental health disorders (Hyman, 2014; Insel and Landis, 2013). It is therefore understandable why significant excitement has been generated by the recent discovery that the N-methyl d-aspartate receptor (NMDAR) antagonist, ketamine, produces rapid relief of depressive symptoms in patients with treatment-resistant depression (Berman et al., 2000; Zarate CA et al., 2006). We recently reported new evidence of a greater sensitivity of female rats to the rapid antidepressant-like effects of low-dose ketamine (2.5 mg/kg) in the forced swim and novelty suppressed feeding tests when compared to male rats, and that ovarian-derived estrogen (E2) and progesterone (P4) are both required for this heightened response (Carrier and Kabbaj, 2013). This work has since been validated in mice (Franceschelli et al., 2015). However, sex differences in baseline responding in these acutely-stressful behavioral paradigms can interfere with the ability to tease apart hormone-dependent contributions to ketamine’s response profile from those consequent to sex differences in response to stress, environmental variables and other factors. In addition, these behavioral assays were developed to detect the efficacy of drugs with similar 10 mechanisms of action to first-generation prototype drugs (Hyman, 2014), and therefore yield little benefit for identification of novel therapeutic targets for newer drugs with distinct mechanisms of action. Because we still know so little about the proximal effects mediating ketamine’s rapid and sustained therapeutic efficacy, combined with the already complex actions of gonadal- and brain- derived hormones, a more fruitful approach may be achieved through use of more translationally- relevant behavioral components with well-known circuitry, where subjects can be repeatedly measured over time. Anhedonia, the decreased ability to experience pleasure from or desire for normally pleasurable activities, is one symptom common to several mental health illnesses that may serve as a tractable intermediate phenotype for drug discovery (Rømer Thomsen et al., 2015). There is a substantial knowledge of reward circuitry, and these circuits appear to be reasonably well- conserved between rodents and humans (Berridge, 1996; Hyman, 2014; Treadway and Zald, 2011). Clinically significant anhedonia is experienced by significant proportion of patients with major depressive disorder (Pelizza and Ferrari, 2009), and is a predictor of poorer treatment prognosis relative to their non-anhedonic counterparts (McMakin et al., 2012; Spijker et al., 2001; Uher et al., 2012). Excitingly, while currently available antidepressants are generally ineffective in alleviating anhedonia, ketamine has recently been shown to relieve anhedonic symptoms in both rodents (Carrier and Kabbaj, 2013; Garcia et al., 2009; Li et al., 2011) and human patients with depression (Lally et al., 2015, 2014). As such, preclinical tests assessing hedonic behavior are an ideal starting point for teasing apart the influence of hormonal status on the differential sensitivity of male and female rodents to the antidepressant-like actions of low-dose ketamine. This strategy falls in line with the recent National Institute of Mental Health Research Domain Criteria (RDoC) initiative, which proposes a dimensional approach to diagnostic criteria for mental health disorders that implicate specific emotional, behavioral and cognitive domains as the basis for understanding of such illnesses (Cuthbert and Insel, 2013). Therefore, in this work we used a continuous-access sucrose preference test to evaluate whether activational and/or organizational effects of gonadal hormones underlie their contribution to the differential sensitivity of male and female rats to low-dose ketamine. Here, the effect of ketamine on hedonic behavior was systematically investigated in intact and gonadectomized adult male and female rats treated identically with physiologically-relevant doses of estradiol (E2),

11 progesterone (P4), E2 + P4 or testosterone (T) (see Fig. 2.1 for experimental design). By collecting repeated measurements for each individual throughout the experiment, this mixed between- /within-subjects approach provides a highly sensitive measure of hedonic behavior on an individual basis, yielding high statistical power and simultaneous evaluation of hormone- and subject-specific predictors of treatment response. In addition, because the neurotrophic factor BDNF represents a major point of convergence in the hippocampus between known mechanisms of action of ketamine on antidepressant-like response (Autry and Monteggia, 2012; Monteggia and Zarate Jr, 2015) and gonadal hormones in affective behavior (Numakawa et al., 2014), we investigated whether alterations of this protein and its downstream signaling effectors were associated with hedonic-like response to low-dose ketamine in male and female rats with various hormonal profiles.

2.2 Materials and Methods

2.2.1 Animals

Adult male (250–270 g) and female (200–225 g) Sprague-Dawley rats (Charles River, Wilmington, MA) were pair-housed in 43 × 21.5 × 25.5 cm plastic cages and maintained on a 12 h:12 h light:dark cycle (lights on at 0700 h) in a temperature- and humidity-controlled room. Food and water were available ad libitum through- out the duration of the study, and all animal protocols were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Florida State University.

2.2.2 Ovariectomy/Gonadectomy

Ovariectomy, gonadectomy and sham surgeries were performed as previously described (Carrier and Kabbaj, 2013, 2012a, 2012b) with the exception that isoflurane (4% for induction, 1– 3% for maintenance) was used as an anesthetic (Butler Schein Animal Health, Dublin, OH). The non-steroidal anti-inflammatory drug meloxicam (1.0 mg/mL) was injected subcutaneously before and after surgery, and bupivacaine (0.25% solution; 0.4 mL/kg) was applied topically as an analgesic.

12

2.2.3 Cyclic hormone treatment regimen and testosterone supplementation

Chronic testosterone (0 or 25 mg/pellet; Innovative Research of America, Sarasota FL) supplementation and cyclic administration of 17-β-estradiol benzoate (0 or 2 μg in 100 μL sesame oil, s.c.) and progesterone (0 or 500 μg in 100 μL sesame oil, s.c.) (Sigma, St. Louis, MO) were performed as previously described (Carrier and Kabbaj, 2013, 2012a, 2012b). See Experimental Design for details.

2.2.4 Estrous cycle monitoring

Estrous cycles were monitored continuously in intact female rats (Experiment 4) via daily vaginal lavage as previously described (Stack et al., 2010). Cytological smears were used to assign stages as follows: diestrus 1 was characterized by the presence of leukocytes and clusters of cornified epithelial cells, diestrus 2 was characterized by a predominance of leukocytes in combination with larger rounded pavement cells, proestrus was characterized by moderate numbers nucleated epithelial cells, and estrus was defined by the predominance of cornified epithelial cells.

2.2.5 Continuous-access sucrose preference test

The sucrose preference test consisted of a two-bottle choice paradigm (Carrier and Kabbaj, 2013, 2012a, 2012b; Hollis et al., 2011) administered continuously throughout the experiment, except during the one-week surgery recovery period. After a 5-day habituation to two bottles of water, rats were given access to two pre-weighed bottles, one containing water and the other 0.25% sucrose. The position of the sucrose solution was alternated with water every 24 hours to account for possible location preference. The bottles were weighed at 0900 h and 1800 h daily and the preference for sucrose over water was used as a measure of hedonic behavior. The concentration of sucrose was chosen based on the similar preference scores it produced between male and female rats prior to hormonal manipulation, thus minimizing potential sex bias introduced by unequal baseline measures. In addition, the almost negligible contribution of a 0.25% sucrose solution to daily caloric intake minimized the dependency of preference levels on general consummatory behavior. Importantly, providing continuous free access to both sucrose and water solutions permitted a more reliable assessment of treatment-induced changes in hedonic behavior by

13 dissociating transient (or “state”) levels of sucrose preference from more stable underlying “trait” preference levels on an individual basis.

2.2.6 Experimental design

2.2.6.1 Experiment 1a: Effect of cyclic E2 and P4 treatment on hedonic response to low-dose ketamine in ovariectomized female rats. One week following arrival, pair-housed adult female rats began testing in the continuous-access sucrose preference test to determine baseline hedonic behavior. Rats were then ovariectomized (OVX) and allowed one week for recovery before resuming sucrose preference testing. Upon re-stabilization of sucrose preference levels, rats were matched for baseline sucrose preference and body weight and assigned to one of four groups. One group (OVX + E2P4; n = 12) was injected subcutaneously with 2 μg 17-β- estradiol benzoate (E2) in 100 μL sesame oil at 1100 h every fourth day and 500 μg progesterone (P4) in 100 μL sesame oil 24 h later; a second group (OVX + E2; n = 14) received E2 and sesame oil vehicle 24 h later; a third group (OVX + P4; n = 12) received sesame oil vehicle and P4 24 h later; the final group (OVX + OIL; n= 10) received sesame oil vehicle on both days. Every 2 days of injection were followed by 2 days without injection. Hormone doses were chosen based on previous work from our lab (Carrier and Kabbaj, 2013), and produce near-physiological levels of E2 (Asarian and Geary, 2002) and P4 (al-Dahan and Thalmann, 1996) observed throughout a typical 4-day estrous cycle in female rats (Yu et al., 2008). The experimental design and hormone treatment regimen are presented in Fig. 2.1. Hormone treatments began after habituation to subcutaneous oil injections, and were continued throughout the duration of the experiment. On Day 3 of the third hormone treatment cycle Fig. 2.1), rats were injected (i.p.) with saline vehicle (SAL), followed 4 d later by 2.5 mg/kg ketamine (KET). SAL and KET were administered 4 h after P4 or oil injections on Day 3 of the treatment cycle to ensure the presence of elevated hormone levels at the time of drug administration. Sucrose preference measurements continued for two additional hormone treatment cycles to examine the duration of KET effects on hedonic behavior.

2.2.6.2 Experiment 1b: Effect of low-dose ketamine on hedonic behavior following cyclic E2 and P4 treatment in intact male rats. Adult pair-housed male rats were tested in the continuous-access sucrose preference test after one week of habituation to the facility to obtain 14 baseline measures of hedonic behavior. Once stable baselines were achieved, rats were matched based on sucrose preference and body weight and assigned to one of four cyclic hor- mone treatment groups as described above: Intact+ E2P4 (n= 10), Intact+ E2 (n= 10), Intact+ P4 (n= 10), or Intact+ OIL (n= 8). Identical experimental procedures and doses of hormone/drug used in Experiment 1 were followed to determine whether cyclic administration of E2 and/or P4 alter behavioral sensitivity of gonad-intact male rats to low-dose ketamine in the sucrose preference test.

2.2.6.3 Experiment 2a: Effect of chronic testosterone treatment on hedonic response to low-dose ketamine in intact female rats. One week following arrival, pair-housed adult female rats began testing in the continuous-access sucrose preference test to determine baseline hedonic behavior. Once stable baselines were achieved, rats were matched by sucrose preference and assigned to one of two groups. One group (Intact + T; n= 10) received a subcutaneous testosterone pellet implant (25 mg/pellet), and a second group (Intact+ P; n= 10) received a placebo pellet as a control. Rats were allowed one week for recovery prior to resuming testing. Experimental procedures and drug doses identical to those outlined in Experiment 1 were followed to examine whether activational effects of chronic testosterone treatment influence the hedonic response of intact female rats to low-dose ketamine, except that all groups received oil injections on both days of the hormone treatment cycle instead of E2 and/or P4 to control for injection stress and account for potential effects of oil treatment alone.

2.2.6.4 Experiment 2b: Effect of low-dose ketamine on hedonic behavior following gonadectomy and testosterone supplementation in male rats. One week after habituation to the facility, adult pair-housed male rats began testing in the continuous-access sucrose preference test to determine baseline measures of hedonic behavior. Once stable baselines were achieved, male rats were matched by sucrose preference and body weight and assigned to one of three groups. Two groups were gonadectomized (GDX)—one group (GDX + T; n= 10) received a testosterone pellet implant (25 mg/pellet), and a second group (GDX + P; n = 10) received a placebo pellet implant. A third group (SHAM; n = 10) was sham-operated and implanted with a placebo pellet as a control. Recovery from surgery and all experimental procedures were identical to those

15 described for Experiment 2a. This experiment determined whether gonadal testosterone modulates the effect of low-dose (2.5 mg/kg) ketamine on hedonic behavior in male rats.

2.2.7 Western blotting

Total proteins were extracted from dorsal hippocampal tissue punches and processed as previously described (Carrier and Kabbaj, 2013, 2012b; Hollis et al., 2011). Immunoblots were blocked in 5% non-fat dry milk in TBS for 1 h at room temperature and incubated at 4 °C overnight with BDNF (Santa Cruz Biotechnology; 1:500), actin (Millipore; 1:5,000), phospho- p44/42T202/Y204 (Cell Signaling; 1:1,000), p44/42 (Cell Signaling; 1:1,000), phospho-AKTS453 (Cell Signaling; 1:1,000), AKT (Cell Signaling; 1:1,000), phospho-CaMKIIT286 (Cell Signaling; 1:1,000), or CaMKIIα (6G9) (Cell Signaling; 1:1,000) antibodies. Membranes were washed four times for 5 m each with TBST, then incubated 1 h at room temperature with donkey anti-rabbit IR Dye 680LT (Li-COR Biosciences; 1:10 000) and goat anti-mouse IR Dye 800CW (Li-COR; 1:20 000) fluorescent secondary antibodies. Following four 5-minute TBS washes, membranes were visualized using an Odyssey infrared imaging system (Li-COR Biosciences). Quantification was performed using NIH ImageJ (http://rsbweb.nih.gov/ij). Background-subtracted densities of proteins of interest were normalized to those of either the corresponding total protein for phosphorylated targets, or loading control (actin or GAPDH) for total protein. Normalized data are expressed as fold change relative to control, with control animals set at 1.0.

2.2.8 Statistical analysis

All data were first subjected to the Anderson-Darling Normality test, and followed a normal distribution. Raw data for sucrose preference, fluid consumption and caloric intake were analyzed by two-way repeated measures analysis of variance (ANOVA). Dunnett’s multiple comparisons tests were then per- formed where appropriate to determine simple effects of ketamine treatment across time within each hormone condition. Multiplicity-adjusted p-values are reported. Simple linear regression was conducted using SAL baseline sucrose preference as the predictor variable and post-treatment preference scores (expressed as percent change from baseline collapsed across 7 post-treatment days) as the response variable, in order to determine the degree to which baseline hedonic behavior could account for variability in the magnitude of response to

16

KET within each treatment group. Within-group Pearson correlations were used to identify possible associations between daily fluid and caloric intake levels and raw sucrose preference scores following KET treatment. Z-normalized sucrose preference scores and body weight data were analyzed by two-tailed Welsh’s unpaired t-tests or one-way ANOVA, followed by Dunnett’s multiple comparisons tests where appropriate. A detailed description of z-score calculations are presented in Supplementary Materials and Methods online. Comprehensive listings of results from all statistical analyses of behavioral data can be found in Supplementary Tables S1–4 online. Western blot data were analyzed by one-way ANOVA, followed by Dunnett’s tests where appropriate. Alpha was set to 0.05 for all statistical analyses.

Figure 2.1 Timeline of procedures and cyclic hormone treatment regimen for Experiment 1. Abbreviations: E2, 17β-estradiol benzoate; KET, ketamine (2.5 mg/kg); OIL, sesame oil; OVX, ovariectomy; P4, progesterone; SAL, saline.

17

2.3 Results

2.3.1 Influence of cyclic E2 and P4 treatment on hedonic response to low-dose ketamine in ovariectomized female rats

Following a single injection of low-dose of KET (2.5 mg/kg), OVX female rats treated with both E2 and P4 showed a robust increase in sucrose preference relative to their SAL-treated levels, persisting for 7 days post-treatment (Fig. 2.2a; Treatment/Day: F(8,352) = 5.134, p < 0.0001; Hormone: F(3,44) = 2.531, p= 0.0693; Interaction: F(24,352) = 1.839, p= 0.0103; p< 0.01; Supplementary Table S1 online). Conversely, sucrose preference was unaltered in OVX females treated with OIL, E2 or P4 alone (all p > 0.05), suggesting that both hormones are required to increase hedonic response to low-dose KET in this paradigm. Separate analyses of fluid and caloric intake confirmed that increased sucrose preference levels in E2P4-treated rats following ketamine were not secondary to changes in general consummatory behavior or metabolic needs (Supplementary Figure S1 online). No significant differences in baseline sucrose preference between groups were apparent (p> 0.05). Simple linear regression analyses revealed that SAL baseline sucrose preference scores only predicted response to ketamine in E2P4-treated rats (Fig. 2.2e; F(1,10) = 23.73, p = 0.0007, R2 = 0.7035), suggesting that the predictive ability of basal hedonic preference levels on treatment response in OVX females depend on hormonal status (Fig. 2.2b–d; OIL: F(1,8) = 0.7167, R2 = 0.08222; E2: F(1,12) = 0.3812, R2 = 0.03079; P4: F(1,10) = 0.2293, R2 = 0.02242; all p’s >0.05). Results from all statistical analyses are reported in Supplementary Table S1 online.

2.3.2 Effect of low-dose ketamine on hedonic behavior following cyclic E2 and P4 treatment in intact male rats

When intact male rats were administered cyclic hormone treatment identical to that used in OVX females, KET induced a long-lasting increase in sucrose preference of P4-treated rats (all p < 0.05; Supplementary Table S2 online), but was without effect (p > 0.05) in any other treatment group (Fig. 2.3a; Treatment/Day: F(8,272) = 3.939, p= 0.0002; Hormone: F(3,34) = 8.386, p= 0.0003; Interaction: F(24,272) = 2.195, p= 0.0014). Further analysis of general consummatory

18

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Figure 2.2 Estradiol and progesterone are required for rapid and sustained hedonic-like effects of low-dose ketamine in female rats. (a) Ketamine (KET, 2.5 mg/kg, i.p.) induced a… 19

Figure 2.2 – continued. …significant and protracted increase in sucrose preference in ovariectomized (OVX) female rats treated with E2P4 (****p < 0.0001, ***p < 0.001, **p < 0.01 vs. SAL), but not OIL, E2 or P4 alone (Main Effects: Treatment/Day, ****p < 0.0001; Hormone: † p = 0.0693). Data are expressed as mean± SEM (n= 48). (b–e) Saline (SAL) baseline sucrose preference levels predicted magnitude of positive response to KET in E2P4-treated OVX females only (r2 = 0.7035, p = 0.0007). (f) Significantly reduced overall body weight gain of E2-treated OVX female rats compared to OVX + OIL females (***p < 0.001) confirmed efficacy of hormone treatment. Data are expressed as mean± SEM (n= 48).

behavior (Supplementary Figure S2 online) revealed that the pro-hedonic response to ketamine observed in P4-treated males was not simply due to changes in fluid or caloric intake. Overall weight gained throughout the study by E2- and E2P4-administered intact male rats was substantially lower than that of their OIL-treated counterparts (Fig. 2.3f; E2, E2P4: p < 0.0001; F(3,34) = 28.86, p < 0.0001), which was associated with comparatively lower sucrose preference (E2: q(34) = 2.591, p = 0.0363 vs. OIL; E2P4: q(34) = 3.172, p= 0.0086 vs. OIL) and caloric intake, and higher levels of water consumed (Supplementary Figure S2 online), before and after KET treatment. However, lower baseline preference levels and calories consumed do not likely account for the lack of hedonic response to KET in these animals, supported by the lack of positive correlation between sucrose preference and consummatory fluctuations for all treatment groups across the post-treatment period (see Supplementary Figure S2 online). Interestingly, SAL baseline preference levels were highly predictive of magnitude of response to KET in OIL-, E2- and P4-treated, but not E2P4-treated, intact male rats (Fig. 2.3b–e; OIL: F(1,6) = 60.51, p= 0.0002 R2 = 0.9098; E2: F(1,8) = 15.13, p = 0.0046, R2 = 0.6541; P4: F(1,8) = 251.7, p < 0.0001, R2 = 0.9692; E2P4: F(1,8) = 1.272, p= 0.2921, R2 = 0.1372). However, regression scatter plots show roughly equivalent numbers of data points falling above and below the 100% baseline indicator in OIL- (Fig. 2.3b) and E2-treatment (Fig. 2.3c) groups. Here, rats with higher baseline sucrose preferences (>70%) showed reduced hedonic response to KET, whereas increased responses were observed in those with lower (<70%) preferences. Therefore, baseline sucrose preference predicts both positive and negative treatment response in OIL- and E2-treated male rats, whereas the magnitude of positive response is predicted in P4-treated rats. Results from all statistical analyses are presented in Supplementary Table S2.

20

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Figure 2.3 Cyclic P4 treatment enhances the hedonic sensitivity of intact male rats to low- dose ketamine. (a) Cyclic P4 treatment increased sensitivity of male rats to low-dose ketamine… 21

Figure 2.3 – continued. (KET) treatment (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 vs. SAL) for up to 6 days following drug administration (Main Effects: Treatment/Day, *** p < 0.001; Hormone: ***p < 0.001). Data are expressed as mean± SEM (n = 40). (b–e) Lower saline (SAL) baseline sucrose preference levels predicted a higher magnitude of positive response to KET in treatment-responsive P4-treated intact males (r2 = 0.9692, p < 0.0001). (f) Cyclic treatment with E2 alone or in combination with P4 significantly reduced overall body weight gain of intact male rats compared to Intact + OIL males (***p < 0.001), confirming effective hormone treatment. Data are expressed as mean± SEM (n = 38).

2.3.3 Effect of chronic testosterone treatment on hedonic response to low-dose ketamine in intact female rats

To determine whether activational effects of testosterone might reduce sensitivity to low- dose KET, we first administered testosterone or cholesterol placebo chronically to intact adult female rats. After receiving a low-dose injection of KET, placebo-treated female rats displayed heightened sucrose preference levels that fluctuated throughout the 7 days following treatment (Fig. 2.4a; p < 0.05, Supplementary Table S3 online). In stark contrast, KET had no effect in female rats with testosterone pellet implants (Fig. 2.4a; Treatment/Day: F(8,144) = 3.390, p = 0.0014; Hormone: F(1,18) = 6.689, p = 0.0172; Interaction: F(8,144) = 1.289, p = 0.2539). As sucrose intake was similar between groups, this discrepancy was largely attributed to the greater volumes of water consumed by testosterone-administered female rats, both prior to and following KET treatment, relative to those receiving placebo (Supplementary Figure S3 online). Unlike findings reported above, KET-induced reductions in water and caloric intake were either absent (Intact+ P) or mild (Intact+ T) in both groups, and neither fluid nor caloric intake positively correlated with preference scores across the post-treatment period, discounting the influence of altered consummatory behavior or energy requirements in determining hedonic response to KET (see Supplementary Figure S3 online). Both baseline preference (F(1,8) = 44.05, p = 0.0002, R2 = 0.8463) and sucrose intake levels (Supplementary Figure S3 online; F(1,8) = 14.74, p= 0.0050, R2 = 0.6482) were strongly associated with the magnitude of hedonic response to KET in placebo-treated females, but not those receiving testosterone pellets (Fig. 2.4b,c; p> 0.05). Here, a greater positive response to KET was observed in animals consuming lower quantities of sucrose solution prior to treatment. Neither

22 water consumption, caloric intake nor body weight correlated with treatment response in either group (p> 0.05). Given the co-requirement of E2 and P4 in female rats for pro-hedonic effects of low-dose KET in this behavioral paradigm (Experiment 1), estrous cycles were continuously monitored by vaginal lavage to account for potential hormone- or injection-stress induced disruptions. As portrayed in Fig. 2.4e, chronic testosterone treatment led to sustained disruption of estrous cyclicity in intact female rats. Persistent diestrus smears were evident from 10–14 d post-surgery throughout experiment’s entirety, shown by the continuous presence of sparsely-packed leukocytes and, in some females, cornified epithelial cells. Disruption by testosterone, rather than injection stress, is supported by the maintenance of normal 4–5 d cycles in placebo-treated rats. Notable physiological alterations observed in Intact +T females, including greater caloric intake and body weight gain (Fig. 2.4d; Welch’s t-test: t(17.08) = 3.869, p= 0.0012 vs. Intact + P), are consistent with absent or abnormal E2/P4 fluctuations. Results from all statistical analyses are presented in Supplementary Table S3 online.

2.3.4 Effect of low-dose ketamine on hedonic behavior following gonadectomy and testosterone supplementation in male rats

Despite having enduring effects on general consummatory behavior (Supplementary Figure S4 online), depletion of gonadal testosterone in adult male rats had no effect on sensitivity to hedonic actions of KET. As expected, low-dose KET failed to elicit any effect on sucrose preference in males above and beyond normal daily fluctuations, regardless of hormonal status (Fig. 2.5a; Treatment/Day: F(8,216) = 3.577, p= 0.0006; Hormone: F(2,27) = 6.995, p= 0.0036; Interaction: F(16,216) = 1.093, p= 0.3627). A gradual reduction in water intake in placebo-treated GDX rats reached significance + 6d post-treatment, and modest increases in sucrose consumed on the day of KET treatment were exhibited by both SHAM and GDX + T male rats (Supplementary Figure S4 online); however, the brevity of this effect along with concurrent caloric intake elevations suggest a general rise in consummatory behavior, rather than pro-hedonic actions of KET, per se (Supplementary Figure S4 online).

23

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Figure 2.4 Chronic testosterone treatment blocks pro-hedonic like effects of ketamine in intact female rats via persistent disruption of estrous cyclicity. (a) Chronic testosterone treatment in intact female rats induced anhedonic behavior (**p < 0.005) and blocked response to ketamine (2.5 mg/kg, i.p.) (p > 0.05). Acute ketamine treatment led to a modest increase in sucrose preferences of placebo-treated intact female rats (**p < 0.01, *p < 0.05 vs. SAL) that persisted for 5 days (Main Effects: Treatment/Day, **p < 0.01; Hormone: *p < 0.05). Data are expressed as mean± SEM (n= 18). (b,c) Lower SAL baseline sucrose preference levels predicted a higher magnitude of positive response to KET in treatment-responsive placebo-treated intact females (r2 = 0.8463, p = 0.0002), but not those receiving testosterone pellets (p > 0.05).

24

Figure 2.4 – continued. (d) Chronic treatment with testosterone significantly increased overall body weight gain of intact female rats relative to intact females receiving placebo pellets (**p < 0.01). Data are expressed as mean± SEM (n= 18). (e) Chronic testosterone treatment resulted in persistent disruption of estrous cyclicity in intact female rats.

Independent of treatment with KET, GDX and testosterone replacement induced robust effects on baseline measures for all parameters considered. Gonadal hormone depletion significantly reduced sucrose preference (Fig. 2.5a; p < 0.05 vs. SHAM, GDX + T; see Supplementary Table S4 online), consumption (p < 0.01 vs. SHAM, GDX + T) and caloric intake (p < 0.0005 vs. SHAM, GDX + T) at baseline and throughout the post-treatment period (Supplementary Figure S4 online). Testosterone supplementation at the time of castration protected against development of these decrements, confirming robust efficacy of the chronic treatment regimen used herein. Despite the large behavioral distinctions between male rats deprived or not of peripheral testosterone supplies, levels of hedonic responding following KET administration were unrelated to baseline preference levels (Fig. 2.5b–d). A comprehensive list of results from all statistical analyses can be found in Supplementary Table S4 online.

2.3.5 Integrated analysis of ketamine’s effects across sex and hormonal status: Z-score normalization of sucrose preference

Post-treatment sucrose preference measures were summarized across experiments to compare the hedonic response to low-dose KET under different hormonal conditions within each sex (Fig. 2.6). Individual sucrose preference scores were rescaled by expressing values as percent change from baseline, allowing within-sex comparison of treatment response in groups from independent experiments along the same scale. Data depicted in Fig. 2.6a,b represent group means collapsed across days of the post-treatment period. When compared in this manner, the same effects of KET are observed for both sexes. When compared to OVX + OIL controls, E2P4-treated OVX females (p = 0.0103) and intact females receiving placebo (p = 0.0222) display significant increases in sucrose preference relative to their SAL baseline levels (Fig. 2.6a; F(5,60) = 4.958, p = 0.0007). Conversely, only P4-treated intact males (p = 0.0143) show a positive hedonic response relative to their OIL-treated counterparts (Fig. 2.6b; F(6,61) = 5.256, p = 0.0002).

25

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Figure 2.5 Gonadal testosterone does not influence sensitivity to low-dose ketamine in male rats. (a) Gonadectomized (GDX) male rats displayed a significantly lower sucrose preference when compared to SHAM & testosterone-supplemented male rats (**p < 0.01). Ketamine (KET; 2.5 mg/kg, i.p.) was without effect in male rats, regardless of hormonal status (Main Effects: Treatment/Day, ***p < 0.001; Hormone: **p < 0.01). Data are expressed as mean± SEM (n= 30). (b–d) Saline (SAL) baseline preference levels of all males were not associated with magnitude of response to KET (p > 0.05). (e) Gonadal testosterone depletion resulted in significantly less body weight gain throughout the experiment relative to SHAM-operated males (*p < 0.05). Chronic testosterone supplementation at the time of gonadectomy was sufficient to block this effect (p > 0.05). Data are expressed as mean± SEM (n= 30).

26

In order to identify the relative magnitude of response to KET between same sex-groups, data in Fig. 2.6a,b were standardized via z-score normalization within each sex, eliminating behavioral “noise” from repeated measures data by accounting for non-uniformity of variances between experimental cohorts. Z-score values are presented in Fig. 2.6c,d as the number of standard deviations of each group from their respective OIL-treated control means. In females, only cyclic E2P4 treatment in OVX rats (p = 0.01) restored the behavioral response to KET to levels similar to those observed for normally cycling females (Fig. 2.6c, Intact+ P: p = 0.0222; F(5,60) = 4.958, p = 0.0007), validating the treatment regimen used and emphasizing the requirement of both hormones in female rats for a pro-hedonic to low-dose KET. Interestingly, this regimen is ineffective in intact males; whereas treatment with only P4 (p = 0.0143) enhanced the sensitivity of males to this dose of KET, significantly increasing sucrose preference relative to OIL-treated males (Fig. 2.6d; F(6,61) = 5.256, p = 0.0002). By correcting for sex differences in basal preference levels (Fig. 2.6e), we found that the magnitude of the P4-mediated response to KET in males (p = 0.0301) was similar to that of intact (p = 0.0057) and E2P4-treated OVX (p = 0.0018) females F(12,121) = 4.551, p < 0.0001).

2.3.6 Effect of cyclic E2 and P4 treatment on hippocampal BDNF protein levels and downstream signaling effectors in female and male rats

Protein levels of BDNF were substantially increased in the dorsal hippocampus of E2P4- treated OVX female rats 24 h following an acute low dose of KET relative to OIL-treated controls (p = 0.0345), but were unaltered (p > 0.05) in those receiving cyclic E2 or P4 alone (Fig. 2.7a; F(3,20) = 1.574, p = 0.0004). Conversely, BDNF levels were unaffected by ketamine in treatment- responsive intact male rats treated with cyclic P4 (p > 0.05), and showed decreases in E2- (p = 0.0006) and E2P4-treated (p = 0.0034) male rats when compared to OIL-treated controls (Fig. 2.7b; F(3,20) = 1.385, p< 0.0001). A significant positive correlation was observed between BDNF levels and sucrose preference (average percent change from baseline across the post-treatment period) for E2P4-treated OVX females (data not shown; p = 0.0036, R2 = 0.9034), but not for any other group of OVX females or intact males (p > 0.05).

27

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% ( O IL E 2 P 4 E 2 P 4 P T O IL E 2 P 4 E 2 P 4 S H A M P T -1 .5 OVX Intact Intact GDX Figure 2.6 Integrated analysis of ketamine’s effects across sex and hormonal status. (a) Comparison of all groups of ovariectomized (OVX) and intact female rats demonstrating that…

28

Figure 2.6 – continued. …normal cyclic fluctuation of both estradiol (E2) and progesterone (P4) levels are essential for pro-hedonic like response to low-dose ketamine (KET; 2.5 mg/kg) (*p < 0.05 vs. OVX + OIL). Data are expressed as mean± SEM (n = 66). (b) Comparison of all groups of gonadectomized (GDX) and intact male rats reiterate the negligible effect of circulating testosterone (T) levels on male sensitivity to low-dose KET (p > 0.05), but identify effective enhancement of KET sensitivity in intact male rats by P4 treatment (*p < 0.05 vs. Intact + OIL). Data in (a,b) are presented as the percent change in sucrose preference following KET administration relative to saline (SAL) baseline levels, averaged across all days of the post- treatment period. Data are expressed as mean± SEM (n = 68). (c,d) Standardization of female (n = 66) and male (n = 68) sucrose preference scores presented in (a,b), respectively, via Z-score transformation relative to OIL-treated groups of each sex (**p < 0.01, *p < 0.05). Data are expressed as mean± SEM. (e) Percent change in sucrose preference levels from baseline following KET administration compared across sex and all hormone treatments via Z-score normalization of each group’s scores to OVX + OIL female rats. The magnitude of pro-hedonic like effects of KET was found to be similar in OVX + E2P4 (**p < 0.01) and Intact + P (*p < 0.05) female and P4- treated intact males (*p < 0.05) when controlling for unequal variances between all experimental cohorts. Data are expressed as mean± SEM (n = 134).

Phosphorylation of key proteins within three primary downstream BDNF-TrkB signaling pathways was next evaluated to identify potential mechanisms contributing to, or resulting from, sex-dependent involvement of hippocampal BDNF protein in treatment response. Regardless of hormone regimen, levels of total (Females: F(3,20) = 1.154, p = 0.3517; Males: F(3,20) = 0.2424, p = 0.8657) and phosphorylated (Females: F(3,20) = 1.122, p = 0.3640; Males: F(3,20) = 2.440, p = 0.0943) hippocampal AKT protein were similar (p > 0.05) between ketamine-responsive and non-responsive groups of male and female rats 24 h following treatment (Fig. 2.7c,d). An effect of hormone treatment on ERK phosphorylation was observed in females (Fig. 2.7e), with greater levels of p-ERK detected in OVX rats receiving E2 alone (p = 0.0047), but not those receiving P4 or E2P4 (p > 0.05), when compared to OIL-treated controls (F(3,20) = 4.503, p = 0.0143). No differences in total ERK abundance were found between treatment groups in female rats (F(3,20) = 1.516, p = 0.2409). Conversely, neither total levels of ERK1/2 (F(3,20) = 2.571, p = 0.0829) nor its phosphorylation status (Fig. 2.7f; F(3,20) = 0.7044, p = 0.5605) were affected by hormone treatment in male rats (Fig. 2.7f). Examination of hippocampal CaMKIIα protein expression by western blot revealed distinct sex-dependent patterns of regulation by hormone treatment in female and male rats (Fig. 2.7g,h). While similar levels of both phosphorylated and total CaMKIIα protein were observed between E2P4- and OIL-treated female rats (p > 0.05), non-treatment responsive OVX females 29 receiving either E2 (p = 0.0033) or P4 (p < 0.0001) alone exhibited reduced levels of total, but not phosphorylated, CaMKIIα relative to their OIL-treated counterparts (Fig. 2.7g; total: F(3,20) = 16.45, p< 0.0001, phospho: F(3,20) = 1.936, p= 0.1563). Interestingly, levels of both phosphorylated and total CaMKIIα were detected in E2- (total: p = 0.0196; phospho: p < 0.0001) and E2P4- (total: p = 0.0017; phospho: p < 0.0001) treated, but not P4-treated, male rats compared to OIL-treated controls (Fig. 2.7h; total: F(3,20) = 7.282, p= 0.0017, phospho: F(3,20) = 18.51, p< 0.0001).

2.4 Discussion

The present study is the first of its kind to systematically investigate the nature of gonadal hormone influence on the differential sensitivity of male and female rats to low-dose ketamine in the context of hedonic behavior, as well as the therapeutic potential of these hormones as adjuncts to enhance the effectiveness of ketamine at suboptimal doses. As expected, a single low dose of ketamine (2.5 mg/kg) selectively enhanced sucrose preference of female rats in an E2P4-dependent manner, with no effect in males, confirming selective enhancement of female responsivity to this dose reported in our previous work (Carrier and Kabbaj, 2013). In extension, this hormone- mediated effect was protracted, lasting up to 7 days. Of note was the finding that cyclic treatment with P4 alone, but not E2P4, significantly enhanced hedonic response of intact male rats to the same low dose of ketamine. Furthermore, positive treatment response was associated with increased BDNF protein levels in the dorsal hippocampus of female rats only, suggesting that hedonic response to ketamine and its modulation by sex steroids in male and female rats are mediated via distinct mechanisms. Collectively, these findings provide novel evidence supporting both activational and therapeutic roles for ovarian-, but not testicular-, derived hormones in mediating hedonic sensitivity to ketamine in both sexes, and suggest potential therapeutic implications for progesterone or progesterone-like compounds as adjunctive treatments in males. In extension of our previous work (Carrier and Kabbaj, 2013) and supporting evidence recently demonstrated in mice (Franceschelli et al., 2015), we first sought to confirm the E2P4- dependent enhancement of female rats to low-dose ketamine, using hedonic behavior as the dependent variable of interest. A continuous-access sucrose preference paradigm was used in order

30

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Figure 2.7 Protein levels of BDNF and downstream signaling effectors 24 h after ketamine in estradiol- and progesterone-treated female and male rats. (a) Hippocampal BDNF levels… 31

Figure 2.7– continued. …were significantly increased 24 h after low-dose ketamine (2.5 mg/kg, i.p.) only in ovariectomized female rats receiving cyclic treatment with both estradiol and progesterone (E2P4) relative to OIL-treated controls (*p = 0.0345; all other p > 0.05). (b) BDNF in the hippocampus of intact male rats was decreased 24 h after ketamine in estradiol (E2; ***p = 0.0006) and E2P4-treated (**p = 0.0034) male rats relative to OIL-treated controls, but was unaffected in those receiving progesterone (P4) alone (p > 0.05). (c,d) Neither total nor phosphorylated levels of hippocampal AKT were altered 24 h post- ketamine ketamine in male and female rats regardless of hormone treatment. (e,f) Phosphorylated ERK1/2 levels were increased following ketamine in E2-treated females (**p = 0.0047), but were otherwise unaffected (p > 0.05) in all other treatment conditions. Total ERK abundance was similar between groups, except in P4-treated males which displayed decreased ERK relative to OIL-treated controls (*p = 0.0338). (g,h) While CaMKIIα phosphorylation was not associated with treatment-response in either sex, lower levels were apparent in E2- (****p < 0.0001) and E2P4-treated (****p < 0.0001) male rats 24 h following ketamine. Parallel decreases in total CaMKIIα levels were observed in the same males relative to OIL-treated counterparts (E2: *p = 0.0196; E2P4: **p = 0.0017). Lower CaMKIIα abundance was also observed at this timepoint in E2- (**p = 0.0033) and P4-treated (****p < 0.0001)—but not E2P4-treated—female rats when compared to same-sex controls. (i) Representative western blots for proteins depicted in (c,h) across all treatment groups. Vertical lines indicate juxtaposition of non-adjacent regions within the same membrane for each phosphorylated and total protein assayed. All data expressed as mean± SEM (n = 24 female/24 male). to investigate both the magnitude and duration of response induced by ketamine. In agreement with our original report (Carrier and Kabbaj, 2013), a single low dose of ketamine significantly enhanced sucrose preference above saline-treated levels in E2P4-treated OVX female rats, without effect in OIL-, E2- or P4-treated OVX rats. These effects were not secondary to changes in fluid or caloric intake, confirming that low-dose ketamine selectively enhanced hedonic valence in female rats in the presence of both E2 and P4. Interestingly, baseline sucrose preference levels only predicted the magnitude of response to ketamine in E2P4-treated OVX female rats, suggesting that the predictive ability of baseline hedonic valence on the magnitude of response to ketamine in OVX females is dependent on hormonal status. Here, a greater enhancement of hedonic response was observed in animals with lower baseline preference levels. These findings suggest that the influence of E2 and P4 on enhanced sensitivity to ketamine in females are, at least in part, activational in nature. Therefore, we administered identical cyclic hormone treatment regimens to intact male rats in order to determine whether these hormones might increase their sensitivity to low-dose ketamine. Interestingly, cyclic treatment with P4 alone was sufficient to enhance ketamine’s efficacy in intact males, significantly increasing sucrose

32 preference levels for up to one week. Neither fluid nor caloric intake could explain the increased preference levels across the post-treatment period. Conversely, ketamine was without effect in OIL-, E2-, and E2P4-treated male rats. While both E2- and E2P4-treated male rats both gained significantly less weight throughout the course of the experiment, their sucrose preference levels following ketamine treatment were unaffected by overall fluid or caloric intake. In contrast to observations in OVX female rats, saline baseline preference levels were highly predictive of magnitude of response to ketamine in OIL-, E2- and P4-treated, but not E2P4-treated, intact male rats. However, the magnitude of positive response was predicted by baseline sucrose preference only in treatment-responsive groups of female (OVX + E2P4) and male (Intact + P4) rats, whereas baselines of non-responsive OIL-, E2- and E2P4-treated intact males were predictive of response in both directions, as reflected by the similar number of points falling above and below baseline in the regression scatterplots. An alternative hypothesis for the sex-dependent sensitivity to ketamine is that testosterone may reduce responsivity in males. To address this possibility, hedonic effects of low-dose ketamine were investigated in sham-operated (SHAM) and gonadectomized (GDX) adult male rats receiving either placebo (GDX + P) or testosterone pellet (GDX + T) supplementation. While gonadectomy induced an anhedonic-like state in male rats, ketamine failed to alter sucrose preference levels in all males, regardless of baseline preference or circulating testosterone levels. As well, negligible effects of this drug were observed on fluid or caloric intake throughout the post-treatment period. The lack of effect of ketamine in these animals across all parameters measured strongly supports than an organizational sex difference is involved in the differential sensitivity of male and female rats to ketamine. To confirm this hypothesis, we examined whether chronic supplementation of the same dose of testosterone altered ketamine’s efficacy in intact female rats. Confirming our earlier findings, intact female rats exhibited a protracted enhancement of hedonic behavior following a single injection of ketamine. It is worth noting that this effect was not as robust as those observed in E2P4-treated OVX female rats, likely due to differences in estrous cycle stage between subjects at the time ketamine was administered. As observed in OVX + E2P4 rats, lower baseline sucrose preference levels in cycling female rats predicted a greater increase in hedonic response to keta- mine. Interestingly, chronic testosterone treatment significantly reduced sucrose preference levels

33 in female rats prior to treatment, relative to their own baseline preference levels and to that of normally cycling female rats, and completely prevented the pro-hedonic actions of ketamine observed in female rats. Of note, estrous cycles were persistently disrupted in all testosterone - treated females prior to and throughout the testing period. It is therefore likely that abnormal or absent fluctuations in ovarian hormone levels prevented treatment response, rather than a direct consequence of testosterone itself. When comparing the effective hormone treatments and/or treatment-responsive conditions in both intact and gonadectomized male and female rats, the present data support the original hypothesis that activational effects of ovarian, rather than testicular, hormones primarily mediate the enhanced sensitivity of female rats to low-dose ketamine; however, organizational differences may, in part, account for the persistence of sex differences following gonadectomy in male rats. Specifically, the requirement of P4 for pro-hedonic response to low-dose ketamine in both sexes suggests a primary activational effect of this hormone. It seems likely that the co-requirement of E2 in females reflects an organizational difference between male and female rats, where P4- mediated events act through substrates available only in the context of a preceding E2 surge that prime the physiological environment necessary for it to act. Together, these findings provide the first evidence of robust and protracted enhancement of hedonic responsivity to low-dose ketamine by adjunctive hormone treatment. The neurotrophic factor BDNF represents a major point of convergence in the hippocampus between known mechanisms of action of ketamine on antidepressant-like response (Autry and Monteggia, 2012; Monteggia and Zarate Jr, 2015) and gonadal hormones in affective behavior (Numakawa et al., 2014). As such, we examined levels of BDNF as well as phosphorylation of key proteins within three primary downstream BDNF-TrkB signaling pathways to identify potential mechanisms mediating the enhancement of hedonic-like response to low-dose ketamine in E2P4-treated female and P4-treated male rats. Interestingly, results demonstrated a sex- and hormone-dependent effect of ketamine on BDNF protein levels in the dorsal hippocampus. Here, a significant increase in BDNF was observed in the hippocampus 24 h following ketamine treatment in E2P4-treated OVX female rats, but not in male rats receiving P4, relative to their OIL-treated counterparts. This selective increase of hippocampal BDNF in treatment-responsive female rats was not associated with alterations in phosphorylation status of

34 either ERK1/2, AKT or CaMKIIα at this timepoint, suggesting that these signaling effectors downstream of BDNF-TrkB activation may have contributed to, rather than resulted from, increased BDNF secretion and/or translation. These findings are consistent with those of Duman and colleagues (2011), demonstrating a transient increase in ERK and AKT phosphorylation which returned to baseline within 2 hours of acute ketamine administration (Li et al., 2011). It is interesting to note that hippocampal BDNF protein levels were reduced in E2- and E2P4-treated male rats when compared to OIL-treated males, accompanied by corresponding reductions in total CaMKII abundance. These results parallel the significantly lower sucrose preference scores observed in E2- and E2P4-treated male rats prior to and following ketamine treatment. Specifically, OIL- and P4-treated rats show similar raw sucrose preference scores and levels of BDNF and CaMKII both before and after ketamine treatment, compared with the substantially lower sucrose preference and protein levels displayed by E2- and E2P4-treated males at the same timepoints. Based on these observations, hippocampal BDNF and CaMKII levels in E2/P4-treated male rats may reflect changes associated with hormonal modulation of baseline hedonic behavior, independent of treatment response. Nonetheless, it appears that the role of BDNF translation and/or release in mediating the heightened sensitivity of female rats to the pro- hedonic effects of ketamine is sex-dependent and may reflect underlying organizational differences in both ketamine’s mechanisms of action, as well as in the activational effects of estradiol and progesterone within the hippocampus. This is supported by the significant correlation between hippocampal BDNF levels and change in sucrose preference following ketamine in treatment-responsive E2P4-treated OVX animals only—a relationship absent in males and non- responsive females. It should not be discounted, however, that ovarian hormone-treated females used in this study were in a gonadal hormone-deprived anhedonic state, whereas males receiving the same treatment were not—it is therefore possible that a ceiling effect may account, in part, for the lack of effect of ketamine on BDNF protein levels in treatment-responsive P4-treated male rats. Interactions between sex, hormones and environment generate significant complexity that makes it difficult to isolate independent contributions of any of these factors to behavioral outcomes, either at baseline or in response to a drug—this is particularly true under conditions of stress (Joel and Yankelevitch-Yahav, 2014). Therefore, we sought to reduce as many sources of

35 this complexity as possible in order to first understand how gonadal hormones influence response to ketamine under non-stressful conditions in a sex-specific manner. Our choice of sucrose preference as a behavioral readout was guided by the extensive knowledge of circuitries mediating reward-related behavior—which is reasonably well-conserved across species (Hyman, 2014)— and the ability of hedonic behavior to be easily modeled in rodents (Berridge and Kringelbach, 2008). Reward can be further subdivided into several measurable components that include consummatory “liking” (hedonic impact), “wanting” (motivation/anticipation for reward), and “learning” (reward representation and prediction) (Berridge, 1996; Berridge and Kringelbach, 2008). The continuous-access sucrose preference paradigm employed in the present experiments reflect hedonic “liking”—a fundamental experience of pleasure reflecting the hedonic valence of a stimulus (Berridge and Kringelbach, 2008)—and lends several advantages to the interpretation and impact of our findings. Most importantly, continuous measurement in the homecage environment permitted assessment of stable baseline, or trait, hedonic behavior for each animal over time in a non-stressful environment, devoid of confounds introduced by reactivity to novel testing environments. By collecting continuous measures for each individual, “state” changes in hedonic behavior following ketamine administration in the present results can be directly attributed to treatment effects (relative to vehicle), rather than artifacts of variability over time. The within- subjects component of this design yielded more sensitive outcome measures by allowing each animal to serve as their own control, and by the ability to account for normal intra-individual variability over time. Substantial statistical power is generated by this type of repeated measures analysis, generating more sensitive data with fewer animals needed per group. In addition, confounding influences on the interpretation of outcome measures was reduced by utilizing the within-subject comparison of treatment response and simultaneous measurements of fluid and food intake as predictors and covariates, respectively, rather than restricting them to independent analysis. Prediction of treatment response as a factor of baseline hedonic behavior and hormonal status has relevant translational implications. Despite the aforementioned advantages, some limitations presented by this approach relevant to the interpretation and generalizability of the present findings should be acknowledged. As the present work was conducted entirely under non-stressful conditions, it is possible that

36 potential behavioral and/or molecular responses to ketamine in non-treatment responsive animals may have been masked by ceiling effects in either domain. High baseline sucrose preference levels in intact rats, for example, could preclude any further increase in hedonic behavior following low- dose ketamine treatment. While this possibility cannot be excluded, that we still observed a pro- hedonic response to ketamine in intact female rats and P4-treated intact male rats not exhibiting anhedonic-like behavior supports a degree of sensitivity in our paradigm high enough to detect subtle treatment-induced changes regardless of stress exposure. Indeed, treatment-response occurred in both intact and gonadectomized animals, despite persistent reductions in sucrose preference—reflecting an anhedonic state— induced by gonadal hormone depletion in the latter. It is also well-established that sex, gonadal hormones and environmental stress exert independent and interacting influences on depressive-like behaviors and antidepressant response. Sex differences in baseline FST behaviors, for example, have been consistently reported (albeit in conflicting directions) (Kokras et al., 2015; Kokras and Dalla, 2014). While estrous cycle effects in this behavior are generally small (Andrade et al., 2010; Craft et al., 2010; Flores-Serrano et al., 2013), their impact on antidepressant response is considerably larger (Allen et al., 2012; Flores- Serrano et al., 2013). Additionally, work from our lab has demonstrated on numerous occasions the significant impact of gonadectomy and hormone replacement on depressive-like behavior and antidepressant response in the sucrose preference test, FST and NSFT (Carrier et al., 2015; Carrier and Kabbaj, 2013, 2012a, 2012b). Sex and hormone effects become further complicated when animals are first exposed to varying social and environmental stressors (Carrier and Kabbaj, 2012a; Franceschelli et al., 2015), which may result in similar or distinct (even opposite) effects on behavior and within the brain (Kokras and Dalla, 2014). With this in mind, it is unclear at this time whether the present findings may extend to other depression-relevant behaviors and brain regions in rodents under conditions of stress—particularly concerning the efficacy of P4 treatment on enhancement of response to low-dose ketamine. Given the complex roles within the nucleus accumbens (NAc) and ventral tegmental area (VTA) that BDNF plays in susceptibility and resiliency to stress-induced anhedonia (Duclot and Kabbaj, 2015; Taliaz et al., 2010), as well as antidepressant response (Duclot and Kabbaj, 2015), investigation of these brain regions would be a worthwhile avenue for further exploration of this work.

37

Collectively, the findings presented herein support a primary and essential role of E2 and P4 in mediating the enhanced sensitivity of female rats to ketamine. Among the most exciting of the present findings was the P4-mediated enhancement of ketamine’s actions in intact male rats, providing the first evidence of robust and protracted enhancement of hedonic response to low-dose ketamine by adjunctive hormone treatment. This sex-specific hormonal response profile and persistence of sex differences following gonadectomy in male rats also suggest a strong influence of organizational differences in the behavioral effects of ketamine, as well its underlying mechanisms—as supported by the selective increase in hippocampal BDNF in treatment- responsive female rats. With strong efforts currently dedicated to finding safer ways to maintain antidepressant response, this novel evidence has great implications for the use of ketamine as an antidepressant treatment in both men and women. In particular, these findings may be of high relevance for the development of effective antidepressants in women suffering from postmenopausal depression and other forms of hormone-related depressive states, in light of the critical roles ovarian-derived hormones serve in the enhanced sensitivity of female rats to low- dose ketamine. On a more fundamental level, the systematic identification of hormonal influence across sexes on “trait” hedonic behavior and “state” hedonic response to low-dose ketamine provide a good foundation for future antidepressant research development across a wide range of behavioral domains.

38

CHAPTER 3

SEX DIFFERENCES IN THE PHARMACOKINETICS OF LOW-DOSE KETAMINE IN PLASMA AND BRAIN OF MALE AND FEMALE RATS

3.1 Introduction

The pressing need for more effective and faster-acting treatments for depression is underscored by the increasing global burden of depression as a leading cause of disability worldwide (Patel et al., 2016), owed in part to ignorance of complex disease mechanisms and stagnant progress in novel pharmacotherapeutic development. In light of this urgency, renewed hope was recently generated by the discovery that the noncompetitive N-methyl d-aspartate receptor (NMDAR) antagonist, ketamine, can rapidly relieve depressive symptoms and suicidal ideation in many patients—notably amongst those with treatment-resistant depression (Abdallah et al., 2016). These findings have since been corroborated numerous times in clinical and preclinical settings, spurring significant efforts into understanding ketamine’s underlying mechanisms with the goal of identifying new targets for rapid-acting treatments with sustained efficacy in a broader range of patients (Abdallah et al., 2016). In the era of personalized medicine, greater emphasis on identifying biomarkers or predictors of rapid antidepressant response to ketamine has emerged in an attempt to address the large heterogeneity observed in patient response to currently-available treatments (Zarate et al., 2013). Yet despite a well-established female preponderance in depressive disorders (Patel et al., 2016) and variable sex differences in antidepressant response (Keers and Aitchison, 2010a), sex is a variable yet to be thoroughly investigated as a potential moderator of response to ketamine. Akin to genetic and environmental factors, sex is a naturally-occurring disease and treatment modifier of particular interest (Becker et al., 2016; Keers and Aitchison, 2010b), in that protective or treatment-enhancing factors in one sex may reveal prevention or treatment strategies in the other (de Vries and Forger, 2015). Importantly, sex is a variable that influences nearly all pharmacokinetic processes— absorption, distribution, metabolism, and elimination—which might ultimately influence treatment response (de Vries and Forger, 2015). As a weak and highly lipophilic base, ketamine 39 rapidly distributes to the brain upon administration primarily via passive diffusion across the blood-brain barrier (BBB). This parent drug is predominantly N-demethylated into norketamine (NK), and further transformed into dehydronorketamine (DHNK) and six diastereomeric hydroxynorketamine (HNK) metabolites (Mion and Villevieille, 2013)—all of which are BBB- penetrant and pharmacologically active within the brain. On a basic level, the therapeutic efficacy and tolerability of ketamine is limited by the availability of the drug and/or its active metabolites in unbound form at relevant target sites within the brain and periphery, making pharmacokinetic processes fundamental to the understanding of variability in treatment response across individuals both within and between sexes (Saland et al., 2016a). Unfortunately, the lack of studies investigating sex differences in ketamine pharmacokinetics has left very little evidence regarding such effects in humans. The few that do exist suggest that observations of sex effects in ketamine metabolism and clearance are likely dose-dependent. As drug/metabolite levels available at the site(s) of action within the brain do not necessarily correlate with those in the periphery, preclinical studies can provide essential information not able to be directly obtained in humans. Indeed, recent work found HNK, but not ketamine or NK, levels to be greater in the brain of female mice following acute systemic administration of 10 mg/kg ketamine (i.p.), in addition to greater female behavioral sensitivity to ketamine’s antidepressant-like effects when compared to males (Zanos et al., 2016). While systemically administered HNK was able to cross the blood-brain barrier and elicit antidepressant- like activity in mice without inducing ketamine-like side effects, sex differences were not examined in this case, so it is unclear whether behavioral sensitivity to HNK truly differs between males and females. It should also be noted that females, but not males, exhibited an antidepressant- like response to 3 mg/kg ketamine, whereas both sexes responded to the 10 mg/kg dose used for pharmacokinetic analysis. Therefore, a direct association between greater HNK levels and enhanced female antidepressant-like response to ketamine cannot be conclusively inferred. As in humans, hormone levels were not controlled for in this study. Clearly, species- and dose-specific differences impede a clear understanding of how sex may influence ketamine pharmacokinetics and treatment response at low doses used in clinic. Notably absent are related studies in rats, which may help bridge this gap in knowledge to paint a clearer picture of whether and how sex differences observed preclinically may apply to the human

40 condition. As well, there is an absence of any information regarding how fluctuating hormone levels may influence ketamine pharmacokinetics, as they are known to affect antidepressant- like behavioral responses to this drug at the preclinical level (Carrier and Kabbaj, 2013; Saland et al., 2016a,b). Therefore, herein we investigated ketamine, NK and DHNK exposure levels in the plasma and brain of proestrus (high hormone) and diestrus (low hormone) female versus male rats following 2.5 mg/kg ketamine—a dose behaviorally effective in females but not in males. In addition, potential regional differences in the medial prefrontal cortex (mPFC) and hippocampus, depression- and ketamine-relevant brain regions, were examined independently.

3.2 Materials and Methods

3.2.1 Animals

Adult male (250-270g) and female (200-225g) Sprague-Dawley rats (Charles River, Wilmington, MA) were pair-housed in 43x21.5x25.5cm plastic cages. Animals were maintained on a 12h:12h light:dark cycle (lights on at 0700 hours) in a temperature- and humidity-controlled room, and food and water were available ad libitum throughout the duration of the study. All animal protocols were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Florida State University.

3.2.2 Estrous cycle monitoring

Animals were habituated to handling procedures for one week upon arrival. Following habituation, daily estrous cycle monitoring and stage assignment of intact female rats was performed via vaginal lavage and characterization of cytological smears as previously detailed (Hollis et al., 2011; Saland et al., 2016). Only rats exhibiting at least 2 consecutive 4-day cycles were used in the present work. During this time, male rats received a similar brief daily handling treatment to minimize potential for stress and handling confounds between sexes.

41

3.2.3 Pharmacokinetics experimental procedures

3.2.3.1 Ketamine treatment and sample collection. The complete experimental design and sample preparation workflow are illustrated in Figure 3.1. Separate adult male rats (n=4/time point) and female rats in either diestrus (low E2P4, n=4/time point) or proestrus (high E2P4, n=4/time point) received a single intraperitoneal (i.p.) injection of ketamine hydrochloride (Butler Schein Animal Health, Inc.) at 2.5 mg/kg, and were sacrificed under non-stressful conditions after 5, 10, 30, 60, 90 or 180 min. Brains were immediately removed, snap-frozen in 2-methylbutane and stored at -80°C, along with plasma separated from trunk blood, until further processing. Drug was administered at a volume of 1 mL/kg. 3.2.3.2 Sample preparation. 260 uL of plasma was transferred to a pre-chilled 1.5 mL microcentrifuge tube and centrifuged for 15 min at 2000xg (4°C) to pellet any contaminants. Supernatant was transferred to a clean tube and an appropriate volume of ketamine-d4 (Cerilliant, Round Rock, TX) was added as an internal standard (IS) at 100 ng/mL for a 300 uL final volume. Samples were pulse-vortexed for 1 min then mixed on a shaker plate for 10 min (RT) to equilibrate the IS. 250 uL spiked plasma was then transferred to a clean, pre-chilled tube, diluted 1:1 with acidified water (0.4N HCl), vortexed and stored at 4°C overnight to disrupt plasma protein binding and precipitate proteins. The following day, samples were thawed on ice, vortexed and centrifuged for 20 min at 8000xg (RT) to clarify prior to solid phase extraction (SPE). For brain tissue samples, ketamine-d4 was added at 300 ng/g to frozen tissue punches (1.0mm) collected from 200μm sections of the dorsal hippocampus (HPC) and medial prefrontal cortex (mPFC). Spiked tissue samples were homogenized via sonication in 200 uL MilliQ water. 800 uL 100% methanol (MeOH) was immediately added and samples were vortexed for 1 min to mix. Following homogenization, samples were sonicated in an ice bath for 15 min and stored at 4°C overnight. The following day, samples were briefly vortexed centrifuged for 10 min at 3000 x g (4°C). Supernatant was transferred to a clean tube, dried via SpeedVac and resuspended in 500 uL 0.2N HCl.

42

Figure 3.1 Pharmacokinetics experimental design and sample preparation workflow. Abbreviations: AUC, area under the curve; DHNK, dehydronorketamine; HCl, hydrochloric acid; HPC, hippocampus; K, ketamine; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MeOH, methanol; mPFC, medial prefrontal cortex; NK, norketamine; PK, pharmacokinetic.

43

For SPE, acidified samples (250 uL for plasma, 500 uL for brain) were loaded onto Oasis MCX (1cc/30mgm) cartridges (Water, Milford, MA) preconditioned with 1mL MeOH and 1 mL MilliQ water, and allowed to elute via gravity. Columns were washed with 1 mL 0.1N HCl, followed by 1 mL MeOH. K, NK and DHNK analytes were then eluted twice with 500 uL 5% NH4OH in MeOH via gravity, and dried via SpeedVac. Pellets were stored at -20°C until further processing.

3.2.4 Quantification of ketamine and metabolites in biological matrices

3.2.4.1 HPLC for plasma samples. Dried plasma extracts after SPE were re-dissolved in 0.5% acetonitrile aqueous solution with 0.1% formic acid (1:2 v:v plasma:solvent). Two uL of the above solutions were loaded to the NanoAquity nanoLC system (Waters, Milford, MA) for liquid chromatography (LC) separations with a single pump trapping fluidic configuration. A Symmetry C18 5um 180 um x 20 mm trap column was followed by a HSS T3 1.8 um 75 um x 150 mm analytical column (both by Waters, Milford, MA) in a vented configuration. Buffer A was aqueous solution with 0.1% formic acid; buffer B was acetonitrile with 0.1% formic acid. The LC gradient profile was as follows with a flow rate of 400 nL/min: 1% B at 0 minute, 85% B at 15-20 minutes and 1% B at 25-35 minutes.

3.2.4.2 HPLC for brain tissue samples. Dried brain extracts after SPE were re-dissolved in 2% acetonitrile aqueous solution with 0.1% formic acid (1:12 mg:uL tissue:solvent). Two uL of the above solutions were loaded to the NanoAquity nanoLC system (Waters, Milford, MA) for LC separations with a single pump trapping fluidic configuration. A Symmetry C18 5um 180 um x 20 mm trap column was followed by a HSS T3 1.8 um 150 um x 100 mm analytical column (both by Waters, Milford, MA) in a vented configuration. Buffer A was aqueous solution with 0.1% formic acid; buffer B was acetonitrile with 0.1% formic acid. The LC gradient profile was as follows with a flow rate of 2 uL/min: 1% B at 0 minute, 85% B at 5-5.5 minutes and 1% B at 6-7 minutes.

3.2.4.3 Mass spectrometry. LC eluents were ionized in positive ion mode by nano electrospray ionization (nESI). Thus, generated analyte ions were detected on-line with a Xevo TQ-S Triple Quadrupole Mass Spectrometer (Waters, Milford, MA). We optimized the conditions

44 for nanoelectrospray source as follows: +3.3 kV capillary voltage, 43 V cone voltage, 50 V source offset, 100°C source temperature, and 0.20 bar spray gas. Standard solutions were directly infused (without LC separation) to optimize the multiple reaction monitoring (MRM) transitions for quantification: ketamine (m/z 238 → 125), norketamine (m/z 224 → 125), dehydronorketamine (m/z 222 → 142) and ketamine-d4 (internal standard, IS, m/z 242 → 129) with 3 ms dwell time and 25 V collision energy. For plasma samples, seven calibration standards were prepared by spiking standards to “blank” plasma followed by SPE extraction. Concentration of those spiked standards were as follows: ketamine-d4 (IS) 100 ng/mL; ketamine, norketamine and dehydronorketamine 1, 5, 25, 100, 500, 800 & 1000 ng/mL. The calibration linearity was observed for 1-500 ng/mL range. For brain tissue samples, five calibration standards were prepared by spiking standards to “blank” tissues followed by SPE extraction. Concentration of those spiked standards were as follows: ketamine-d4 (IS) 300 ng/g; ketamine, norketamine and dehydronorketamine 5, 25, 100, 500, & 1000 ng/g. The calibration linearity was observed for 5- 1000 ng/g range. Standard curves are presented in Fig. 3.2 for plasma (Fig. 3.2a) and brain tissue (Fig. 3.2b). All samples and calibrants LC-MS/MS experiments were run in triplicates. TargetLynx software (Waters, Milford, MA) was used to automatically quantify ketamine, norketamine and dehydronorketamine in plasma and brain tissues. TargetLynx extracted the area under curve (AUC) of those targets of interest in the MRM chromatograms with a restraint of retention time. TargetLynx then normalized the response by comparing the AUC to that of IS. Calibrants response were used to generate calibration curves which were used to calculate the concentration in the unknown samples.

3.2.5 Pharmacokinetics data analysis

Pharmacokinetic parameters following i.p. ketamnine administration in male and female rats were determined from mean plasma concentration-time data via noncompartmental analysis (NCA) using PKSolver 2.0 (Zhang et al., 2010). The maximum plasma and tissue concentrations (Cmax) and time to reach Cmax (Tmax) were directly observed from data. The area under the concentration-time curves from 0-180 min after ketamine administration (AUC0-t) and extrapolated to infinity (AUC0-∞) were calculated for K, NK and DHNK using the linear-up/log-

45 down trapezoidal method. As well, the mean residence time (MRT), or the amount of time a molecule remains in the body, was calculated for ketamine and both metabolites by dividing AUC by the area under the first moment concentration-time curve (AUMC). For mean K and NK concentration-time profiles, NCA was used to calculate terminal elimination half-life (t1/2) and

a P la s m a 6

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0 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 C o n c e n tr a tio n (n g /m L )

Figure 3.2 Calibration curves for ketamine and metabolite standards in blank plasma and brain matrices. Linearity (R2 > 0.9995) was observed for ketamine (K), norketamine (NK) and dehydronorketamine (DHNK) calibrants (Cal) within a range of 1-500 ng/mL for plasma, and 5- 1000 ng/g for brain. Response depicted as the ratio of the peak area (AUC) of each analyte to that of the internal standard (IS). Data expressed as mean of 3 technical replicates ± SEM.

46 rate constant (kel) values in both plasma and brain tissue, as well as apparent volume of distribution at the terminal phase (Vz/F) and the total systemic clearance (CL/F) for plasma samples. The terminal phase of DHNK for female rats was unidentifiable due to the unsuitability of our timeframe in accommodating for the later peak observed in this sex; therefore, only Cmax, Tmax, and AUC0-t were calculated.

3.2.6 Statistical analysis

All data were first subjected to the Anderson-Darling Normality test, and determined to follow a normal distribution. Plasma and brain concentrations of ketamine and metabolites across time were analyzed by two-way analysis of variance (ANOVA), with sex/estrous cycle and time as independent factors. Due to the sparse sampling protocol required herein for parallel analysis of plasma and brain tissue, repeated-measures analysis across timepoints collected was not possible. Where appropriate, significant main or interaction effects were followed by Bonferroni’s multiple comparisons test to determine between-group differences across time. Multiplicity- adjusted p-values are reported. GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA) was used for all statistical analyses and production of graphs. Alpha was set to 0.05 for all statistical analyses.

3.3 Results

3.3.1 Plasma concentrations of ketamine and metabolites in male and female rats

Following systemic administration of the same low dose of ketamine to male and female rats, plasma concentrations of the parent drug were significantly higher in female rats 5 (p=0.0367) and 10 (p=0.0385) min post-treatment as compared to male rats (Fig. 3.3a; Sex: F(1,59)=18.72, p<0.0001; Time: F(5,59)=48.53, p<0.0001; Interaction: F(5,59)=1.337, p=0.2615). Accordingly, greater NK levels in females versus males were observed at subsequent 30- (p=0.0013) and 90- minute (p=0.0111) time points (Fig. 3.3c; Sex: F(1,57)=26.20, p<0.0001; Time: F(5,57)=24.41, p<0.0001; Interaction: F(5,57)=1.708, p=0.1474). That DHNK levels were greater in male than female rats 10 (p=0.0328) and 30 (p=0.0031) minutes after ketamine administration suggests greater rate of transformation of DHNK from NK in males (Fig. 3.3e; Sex: F(1,60)=3.108, p=0.0830; Time: F(5,60)=3.744, p=0.0051; Interaction: F(5,60)=4.784, p=0.0010). 47

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Figure 3.3 Plasma concentration-time profiles of ketamine and its metabolites in male and cycling female rats. (a,b) Females displayed greater levels of ketamine (K) rapidly upon i.p. administration at 5 (p=0.0367) and 10 (p=0.0385) min post-dose, relative to their male counterparts, regardless of hormonal status. (c,d) Higher concentrations of the norketamine (NK) metabolite were also subsequently observed in female rats at 30- (p=0.0013) and 90-min (p=0.0111) timepoints, when compared to males, in an estrous-cycle independent manner. (e) However, dehydronorketamine (DHNK) was significantly elevated in male compared to female rats 10 (p=0.0328) and 30 (p=0.0031) min after ketamine administration. (f) Estrous cycle had minimal influence on DHNK levels in females. (g-j) Superimposed K, NK and DHNK concentrations reveal greater metabolite-to-parent concentrations in males than in females through

48

Figure 3.3 – continued. …the first 30 min post-treatment, despite their overall lower levels of K and NK during this time. *p < 0.05 vs. Male, **p < 0.01 vs. Male.

Interestingly, no meaningful differences were observed between diestrus and proestrus females in concentrations of ketamine (Fig. 3.3b; Estrous Cycle: F(1,36)=0.003740, p=0.9516; Time: F(5,36)=38.61, p<0.0001; Interaction: F(5,36)=0.8209, p=0.5430), norketamine (Fig. 3.3d; Estrous Cycle: F(1,36)=0.08903, p=0.7672; Time: F(5,36)=18.65, p<0.0001; Interaction: F(5,36)=0.5766, p=0.7175), or dehydronorketamine (Fig. 3.3f; Estrous Cycle: F(1,36)=0.2833, p=0.5978; Time: F(5,36)=5.370, p=0.0009; Interaction: F(5,36)=1.806, p=0.1365), suggesting that ovarian hormones did not significantly influence ketamine pharmacokinetics at this low dose and route of administration during these cycle stages (see Table 1 for pharmacokinetic parameter values).

3.3.2 Brain tissue concentrations of ketamine and metabolites in male and female rats

We next sought to measure the active-site concentrations of ketamine and norketamine within key brain regions relevant to ketamine’s antidepressant-like action. Ketamine levels in both the HPC (Fig. 3.4a) and mPFC (Fig. 3.4e) paralleled sex differences seen in plasma, with significantly greater concentrations of the parent drug in female rats 5 and 10 min post-treatment (p’s < 0.0001) relative to male rats (HPC, Sex: F(1,59)=134.0, p<0.0001; Time: F(5,59)=572.4, p<0.0001; Interaction: F(5,59)=78.74, p<0.0001; mPFC, Fig. 4E: Sex: F(1,58)=6415, p<0.0001; Time: F(5,58)=22557, p<0.0001; Interaction: F(5,58)=3690, p<0.0001). Females also exhibited higher norketamine levels than males in both brain regions (Fig. 3.4c,g) rapidly beginning at 5 min up to 60 min after administration (p’s < 0.0001). (HPC, Sex: F(1,58)=179.4, p<0.0001; Time: F(5,58)=112.1, p<0.0001; Interaction: F(5,58)=12.94, p<0.0001; mPFC, Fig. 4E: Sex: F(1,59)=567.4, p<0.0001; Time: F(5,59)=251.8, p<0.0001; Interaction: F(5,59)=42.59, p<0.0001). DHNK was detected below the lower limit of quantitation (LLOQ) at all timepoints in both brain regions, and was therefore omitted from analysis. As in plasma, while HPC NK was slightly greater in diestrus versus proestrus female rats, measured concentrations of K and NK were markedly similar between these groups in the HPC (K, Fig. 3.4b; Estrous Cycle: F(1,35)=0.004494, p=0.9469; Time: F(5,35)=468.2, p<0.0001; Interaction: F(5,35)=0.1070, p=0.9900; NK, Fig. 3.4d: Estrous Cycle: F(1,34)=5.179, p=0.0293;

49

Time: F(5,34)=113.6, p<0.0001; Interaction: F(5,34)=0.9861, p=0.4406) and mPFC (K, Fig. 3.4f; Estrous Cycle: F(1,36)=0.07046, p=0.7992; Time: F(5,36)=25491, p<0.0001; Interaction: F(5,36)=1.326, p=0.2754; NK, Fig. 3.4h: Estrous Cycle: F(1,36)=1.179, p=0.2848; Time: F(5,36)=277.4, p<0.0001; Interaction: F(5,36)=0.2875, p=0.9168). The only exception was a modestly greater initial NK level in diestrus females 5 min (p=0.0413) post-dose restricted to the HPC (Fig. 3.4d) relative to that of proestrus female rats (see Table 2 for pharmacokinetic parameter values).

3.3.3 Sex differences in metabolism and brain distribution of ketamine and norketamine

Concentration-time relationships between ketamine and its metabolites in plasma and brain tissues were examined to identify whether sex and/or hormonal milieu influence metabolism and CNS distribution of low-dose ketamine in behaviorally-relevant brain regions. As DHNK was not reliably detected at or above quantifiable levels in brain tissue, primary emphasis was on ketamine and norketamine in the present analyses. In order to directly compare ketamine and metabolite exposure levels, their plasma (ng/mL) and brain tissue (ng/g) concentrations—assuming a specific mass of 1 g/mL—were first converted into micromolar (µM) to correct for molecular weight differences between molecules which contribute to overall AUC (exposure) values. The results of these comparisons are depicted graphically in Figure 5 for visual comparison of exposure levels between sexes across time—numerical values presented therein (Fig. 5) are provided in Table 3, along with female-to-male ratios for comparison at each parameter.

3.4 Discussion

Comparative evaluation of pharmacokinetic profiles and brain tissue distribution of low-dose ketamine and its metabolites in male and female rats is necessary for improved understanding of their differential behavioral sensitivity to the drug observed preclinically, and translation of such information across species. In this work, we provide the first characterization of ketamine pharmacokinetics across sex and hormonal status in rats following a low-dose administration of 2.5 mg/kg—a dose selectively effective in female but not male rats in eliciting antidepressant- like and pro-hedonic behaviors. Here, females exhibited greater peak ketamine and norketamine (NK), but not dehydronorketamine (DHNK), levels rapidly upon systemic administration in both plasma

50

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Figure 3.4 Brain concentration-time profiles of ketamine and norketamine in male and cycling female rats. (a,e) Concentrations of ketamine (K) were ~2-fold greater in female compared to male rats 5-10 min (p < 0.0001) following administration in the hippocampus (HPC) and medial prefrontal cortex (mPFC), regardless of estrous cycle stage (b,f). (c,g) These differences were more pronounced for its metabolite norketamine (NK), whose levels in females were significantly higher 5-60 min post-treatment in both regions (p < 0.0001) when compared to those in males. (d) While NK levels in the HPC were higher in diestrus than in proestrus females 5-min after ketamine treatment (p = 0.0413), hormonal status had minimal effect on distribution of the parent drug and its metabolite within the HPC and mPFC. Data are expressed as mean ± SEM (n=3-4/group/timepoint). **p < 0.0001 vs. Male, *p < 0.05 vs. Proestrus. 51

Table 3.1. Pharmacokinetic parameters for ketamine and metabolites in plasma and brain tissue of male and female rats.

MRT0- Parameter kel t1/2 Tmax Cmax AUC0-t AUC0-∞ AUMC0-t AUMC0-∞ MRT0-t Vz/F Cl/F ∞ Unit 1/min min min ng/ml ng•min/L ng•min/L ng•min2/L ng•min2/L min min L/kg L/min/kg Plasma Ketamine Male 0.0156 44.42 5 100.52 2.86 3.10 127.52 186.09 44.60 60.05 51.69 0.807 Female 0.0116 59.99 5 135.83 5.48 6.07 276.24 434.03 50.45 71.53 35.66 0.412 Norketamine Male 0.0124 55.76 30 190.68 14.54 16.85 808.15 1409.02 55.58 83.63 11.94 0.148 Female 0.0095 72.72 30 311.80 28.04 34.41 1937.45 3751.16 69.10 109.03 7.62 0.073 Dehydronorketamine Male – – 30 9.88 0.61 – 38.48 – 62.67 – – – Female – – 90 5.49 0.66 – 62.28 – 94.02 – – – mPFC Ketamine Male 0.0471 14.72 5 198.47 9.09 – 288.26 – 31.72 – – – Female 0.0504 13.74 10 402.87 10.92 – 274.57 – 25.15 – – – Norketamine Male 0.0223 31.08 10 170.64 9.78 – 405.59 – 41.45 – – – Female 0.0170 40.66 10 413.26 26.07 – 1233.84 – 47.33 – – – HPC Ketamine Male 0.0487 14.24 10 199.21 9.12 – 289.12 – 31.70 – – – Female 0.0503 13.78 10 375.79 10.72 – 277.72 – 25.91 – – – Norketamine Male 0.0239 29.01 10 170.25 9.10 – 355.52 – 39.06 – – – Female 0.0163 42.42 10 374.46 21.69 – 1043.47 – 48.12 – – –

52

Table 3.2. Pharmacokinetic parameters for ketamine and metabolites in plasma and brain tissue of diestrus and proestrus female rats.

MRT0- Parameter kel t1/2 Tmax Cmax AUC0-t AUC0-∞ AUMC0-t AUMC0-∞ MRT0-t Vz/F Cl/F ∞ Unit 1/min min min ng/ml ng•min/L ng•min/L ng•min2/L ng•min2/L min min L/kg L/min/kg Plasma Ketamine Diestrus 0.0122 56.70 5 123.93 5.56 6.06 270.64 400.96 48.64 66.15 33.74 0.412 Proestrus 0.0110 63.15 5 147.73 5.38 6.07 281.36 468.94 52.33 77.27 37.53 0.412 Norketamine Diestrus 0.0108 64.21 30 333.05 27.39 31.99 1817.55 3070.51 66.36 96.00 7.24 0.078 Proestrus 0.0085 81.22 30 290.54 28.63 37.03 2050.42 4548.97 71.62 122.83 7.91 0.068 Dehydronorketamine Diestrus – – 90 7.30 0.73 – 72.73 – 99.40 – – – Proestrus – – 60 4.87 0.59 – 51.59 – 87.50 – – – mPFC Ketamine Diestrus 0.0505 13.74 10 404.82 10.91 – 274.31 – 25.14 – – – Proestrus 0.0504 13.74 10 400.93 10.92 – 274.83 – 25.17 – – – Norketamine Diestrus 0.0167 41.57 10 415.86 26.49 – 1255.24 – 47.39 – – – Proestrus 0.0175 39.67 10 410.66 25.63 – 1210.16 – 47.21 – – – HPC Ketamine Diestrus 0.0500 13.87 5 373.87 10.69 – 276.91 – 25.90 – – – Proestrus 0.0504 13.76 10 380.44 10.74 – 278.37 – 25.92 – – – Norketamine Diestrus 0.0163 42.47 10 383.14 22.99 – 1115.64 – 48.53 – – – Proestrus 0.0164 42.38 10 362.88 20.39 – 971.90 – 47.67 – – –

53 and two depression-relevant brain regions, the medial prefrontal cortex (mPFC) and hippocampus (HPC). Longer half-lives and slower clearance rates in females contributed to their greater exposure levels of ketamine and its primary metabolite over the three-hour time course. Notably, while our previous work demonstrated an important role for ovarian hormones in the enhanced female behavioral sensitivity to low-dose ketamine (Carrier and Kabbaj, 2013; Saland et al., 2016), proestrus and diestrus female rats exhibited remarkably similar pharmacokinetic profiles, suggesting a more prominent influence of sex hormones on pharmacodynamic rather than pharmacokinetic systems in conferring sex-dependent behavioral sensitivity to ketamine. Absent from preclinical work are pharmacokinetic analyses of ketamine and metabolite concentrations across a broad range of doses within which behaviorally-effective doses of ketamine reside. Therefore, it was of particular interest here to determine concentrations of ketamine and its metabolites for a low dose of ketamine that exerts antidepressant-like effects in female rats, but not in males, in order to obtain results that directly parallel behavioral and molecular findings presented in our previous studies utilizing the same low dose (Carrier and Kabbaj, 2013; Saland et al., 2016; Sarkar and Kabbaj, 2016). Following systemic administration of 2.5 mg/kg ketamine, ketamine concentrations peaked rapidly 5 minutes after dosing the plasma of male and female rats. Notably, peak concentrations of ketamine in female rats were significantly greater than those in males for the first 10 minutes after the drug was administered. Concentrations of its primary metabolite NK peaked slightly later at 30 minutes in both sexes as ketamine declined, exhibiting greater levels in female rats from its peak time through 90 minutes following treatment when compared to males. Interestingly, DHNK (derived from NK) showed an opposite trend, in which males displayed greater and earlier peak concentrations at 30 minutes than females, whose levels peaked much later around 90 minutes. Over the time points examined, AUC values indicated that total exposure levels to both ketamine and NK in females were nearly double those observed in males, whereas DHNK exposure was similar between sexes. Longer half-lives and slower clearance rates of both ketamine and NK in females contributed to their overall greater exposure levels compared to males. Given that the same trends in these parameters were observed for both ketamine and its primary metabolite, NK, greater peak concentrations of ketamine in the plasma of females cannot be explained by more extensive or rapid metabolism to NK in males.

54

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T im e (m in ) Figure 3.5 Metabolite ratios and brain distribution of ketamine and norketamine in male and cycling female rats. (a) Ratio of norketamine to ketamine area under the concentration-time curve (AUC) values in plasma and brain of male (n=3-4/timepoint) and female (n=7-8/timepoint) rats (brain regions were averaged due to overall similarity). (b,c) Cumulative brain-to-plasma AUC ratios for ketamine and norketamine, respectively, over time in male and female rats. Data expressed as ratios of AUC0-t values calculated using pooled group data from biological replicate measurements at each timepoint—plasma (ng/mL) and brain tissue (ng/g) concentrations were converted into micromolar (µM) units beforehand for direct comparison between analytes. AUC0- 180 Ratio: total analyte exposure across all timepoints measured for each analysis, depicted by gray vertical bar. N.Q.: not quantifiable, ketamine detected in brain samples < LLOQ 90-180 min; here, AUC0-60 represents total exposure up to this timepoint, indicated by a dashed vertical bar for comparison. 55

Table 3.3. Comparison of metabolite and brain-to-plasma ratios in male and female rats following low-dose ketamine administration.

Metabolite-to-Parent Ratios Brain-to-Plasma Ratios

Parameter AUCNK / AUCK AUCDHNK / AUCK AUCNK + DHNK / AUCK AUCbrain / AUCplasma AUC Units (umol•min/L) (umol•min/L) (umol•min/L) (umol•min/L) Ketamine Norketamine Minutes Male Female F:M Male Female F:M Male Female F:M Male Female F:M Male Female F:M Plasma 5 1.88 1.54 -1.22 0.01 0.01 -1.20 1.91 1.55 -1.23 – – – – – – 10 2.27 1.82 -1.24 0.04 0.01 -3.47 2.31 1.84 -1.26 – – – – – – 30 3.68 3.02 -1.22 0.14 0.03 -5.37 3.82 3.05 -1.25 – – – – – – 60 4.86 4.09 -1.19 0.19 0.05 -3.93 5.06 4.14 -1.22 – – – – – – 90 5.16 4.65 -1.11 0.21 0.08 -2.67 5.37 4.73 -1.13 – – – – – – 180 5.40 5.44 -0.99 0.23 0.13 -1.78 5.64 5.57 -1.01 – – – – – – mPFC 5 0.85 1.09 1.28 – – – – – – 1.97 2.93 1.48 0.89 2.07 2.32 10 0.88 1.09 1.24 – – – – – – 2.27 3.28 1.45 0.88 1.96 2.23 30 0.94 1.49 1.59 – – – – – – 3.15 2.97 -1.06 0.80 1.47 1.82 60 0.95 2.00 2.09 – – – – – – 4.05 2.75 -1.47 0.79 1.34 1.69 HPC 5 0.80 0.91 1.15 – – – – – – 1.97 2.73 1.38 0.83 1.62 1.94 10 0.83 0.96 1.16 – – – – – – 2.35 3.06 1.30 0.86 1.62 1.87 30 0.93 1.32 1.42 – – – – – – 3.17 2.84 -1.11 0.80 1.24 1.55 60 0.96 1.68 1.74 – – – – – – 3.94 2.69 -1.46 0.78 1.10 1.41 Brain (Avg) 5 0.82 1.01 1.22 – – – – – – 1.97 2.83 1.43 0.86 1.84 2.14 10 0.87 1.03 1.19 – – – – – – 2.31 3.17 1.37 0.88 1.79 2.03 30 0.95 1.41 1.49 – – – – – – 3.16 2.91 -1.09 0.81 1.36 1.67 60 0.97 1.84 1.91 – – – – – – 3.99 2.72 -1.47 0.79 1.22 1.54

56

Interestingly, male rats did exhibit a larger apparent volume of distribution than females for ketamine, and to a lesser extent NK, according to values obtained via non-compartmental analysis. It is therefore possible that this drug was more extensively distributed to other tissue and fluid compartments in male rats, which could help to explain its lower levels observed in plasma compared to females across the time period measured. To this end, we examined ketamine and metabolite concentrations within the brain in two regions relevant to depressive-like behaviors and ketamine’s related mechanism of action in rodents—the mPFC and HPC. Ketamine and NK were detected rapidly in the brains of male and female rats, both peaking 5-10 minutes after systemic administration of the parent compound. These findings agree with previous reports in mice and rats performed using similar routes of administration, and confirm what is already known regarding distribution of ketamine afte r systemic and intravenous administration of the drug across species (Mion and Villevieille, 2013). As in plasma, ketamine levels were significantly higher in female versus male rats 5-10 minutes after injection. Norketamine peaked sooner in the brain than in plasma in both sexes, but was also detected at much greater levels in the mPFC and HPC of females compared to males over the first 90 minutes after administration. Unfortunately, DHNK levels detected were below the limit of quantification in both brain regions, and were therefore not reported. Failure to reliably detect DHNK in the brain at a dose this low is unsurprising based on similar reports in rats administered low-dose ketamine systemically. Given that females displayed greater ketamine and NK concentrations than males in both the brain and plasma, greater distribution of ketamine to brain tissue in males cannot explain lower levels of ketamine and NK in males. Determination of drug levels in other tissues and compartments are needed to help explain the greater apparent volume of distribution observed in males. In order to better understand the observed sex differences in ketamine concentrations over time, direct comparisons of pharmacokinetic parameters and concentration-time curves between the plasma and brain were made within each sex, then plotted against each other for further insight. First, to determine whether differences metabolism of ketamine to downstream metabolites could help explain the greater female exposure to ketamine in the plasma and brain, cumulative ratios of total exposure levels (depicted by AUC values) for norketamine to ketamine were plotted over time at each measurement taken in the plasma and brain. Interestingly, despite greater

57 concentrations of NK in the plasma of females compared to males, the NK:ketamine ratios were remarkably similar over the 180-minute time period examined, with roughly 5 times greater norketamine exposure compared to ketamine in both sexes. This similarity was true at each timepoint examined, suggesting that metabolism of ketamine into NK does not contribute to differing plasma concentrations between male and female rats. In the brain, however, NK exposure was roughly twice that of ketamine in females, whereas males displayed an NK:ketamine ratio in equilibrium. Given that ketamine is not known to undergo local metabolism within the brain, this suggests that either distribution or permeability of NK into the brain is greater in females than in males independent from the rate or extent of hepatic metabolism of the parent drug. To examine this further, AUC values of ketamine and NK were compared between the brain and plasma in each sex, then plotted against each other for evaluation. In agreement with previous studies, ketamine levels were roughly 3-4 times greater in the brain than in plasma of both males and females; however, the rate of brain penetration of the parent compound differed between sexes. Over the first 10 minutes following ketamine administration, females displayed brain:plasma ketamine ratios of ~3 compared to ~2 in males. This trend shifted to favor males by 60 minutes, who displayed nearly 4 times greater ketamine concentrations in the brain than in plasma, compared to ~3 times greater levels in females at this timepoint. Despite greater brain concentrations of ketamine in females, it is possible that males exhibit either slower elimination or greater retention of ketamine within the brain than females. Indeed, the mean residence time (MRT) of ketamine was nearly 20% greater in males than in females. Differences in the rate of ketamine’s elimination from the brain over time could have a significant impact on the time course of molecular consequences of ketamine action within the brain, and therefore behavior, at the low dose used in the present work. Because NK is also known to be a pharmacologically active metabolite, sex differences in its concentrations within the brain also have the potential to influence ketamine’s rapid behavioral effects. Owing to its reduced lipophilicity compared to ketamine, brain penetrance of NK was reduced relative to ketamine in both sexes. Norketamine brain:plasma ratios were near 1 in males, suggesting near equilibrium between the 2 compartments. This ratio was roughly doubled in females for the first 10 minutes following ketamine administration, but lowered to equilibrium by 60-90 minutes post-dose. These data suggest similar elimination from and penetrance of NK in the

58 brain, with slight differences in elimination rates for ketamine, between male and female rats, suggesting a low probability that these parameters contribute to the sex-dependent behavioral sensitivity to low-dose ketamine in rats. However, it is important to note that ketamine and NK displayed regional differences in concentrations in females, where concentrations of both were significantly greater in the mPFC than the HPC for the first 10 minutes after ketamine was administered. No difference was apparent in males, however, whose concentrations were similar in both regions examined. This sex discrepancy could help to explain the molecular differences between male and female rats shown to occur in both the mPFC and HPC rapidly following low- dose ketamine administration (Carrier and Kabbaj, 2013; Saland et al., 2016; Sarkar and Kabbaj, 2016). As this is the first report of regional differences in ketamine distribution within the brain between males and females, it is unknown the extent to which these differences occur throughout other regions of the brain which may have behaviorally-relevant implications. It is an exciting finding, nonetheless, and warrants further investigation. While the pharmacokinetic differences between male and female rats are certainly of interest, the lack of difference between proestrus and diestrus females are equally important to highlight when considering potential relevance of pharmacokinetic differences to those reported in depression-relevant behavioral assays. Despite differences in peripheral and central levels of estradiol and progesterone in proestrus and diestrus stages of the estrous cycle, levels of ketamine and NK in the plasma and brain tissue of these females were remarkably similar. Alone, this is not necessarily surprising, as no studies prior to the present work have investigated hormonal influence on pharmacokinetic parameters of ketamine in either rodents or humans. However, these findings hold great import when considered in the context of behavioral data reporting hormone-dependent effects of ketamine on depressive-like behavior in female rats. Whereas circulating estradiol and progesterone appear essential to the heightened behavioral sensitivity of female rats to low-dose ketamine, differences in their circulating levels do not appear to significantly affect metabolism or brain distribution of the same behaviorally-relevant dose of ketamine in intact females (at least in the regions investigated herein). Therefore, it is more plausible that estrous cycle-dependent behavioral effects of a single low-dose of ketamine in rats are a consequence of pharmacodynamic, rather than pharmacokinetic, differences that occur with cyclic hormonal fluctuations. It should be noted that secondary metabolites, including HNK, were not examined in the present work. As

59 such, estrous cycle-dependent differences in metabolism of NK to HNK and its distribution are unknown, and warrant further investigation. Given the single dose administration used in this study, these findings should not be generalized to repeated administration regimens. It is possible that estrous cycle could influence pharmacokinetic parameters of ketamine over repeated administration of a low dose. For example, estrous cycle appears to influence both maintenance of intravenous ketamine self-administration, as well as reinstatement to ketamine-paired cues in rats (Wright et al., 2017). While pharmacokinetic differences between proestrus and diestrus rats are not apparent after a single systemic injection of low-dose ketamine, one cannot exclude the possibility that cycle-dependent reinforcing properties of ketamine following repeated administration (intravenous or otherwise) are influenced by pharmacokinetics. Future pharmacokinetic analyses of ketamine using different routes of administrations, doses and treatment regimens (single vs. repeated) in females across the estrous cycle and males would greatly benefit comprehension of sex and cycle-dependent behavioral and molecular differences reported across studies. While this is the first investigation to examine and report pharmacokinetic sex differences of ketamine in brain and plasma of rats, conflicting findings have recently been reported in mice (Zanos et al., 2016). Here, Zanos and colleagues found that higher HNK, but not ketamine or NK, levels are observed in the brain of female mice following acute administration of 10 mg/kg ketamine (i.p.), in addition to greater female behavioral sensitivity to ketamine’s antidepressant- like effects when compared to males (2016). Further experiments showed that systemically administered HNK is able to cross the blood-brain barrier and elicit antidepressant-like activity in mice without inducing ketamine-like side effects. However, sex differences were either not examined or not reported in this case, so it is unclear whether behavioral sensitivity to HNK differs between males and females. Here, it should be noted that females, but not males, exhibited an antidepressant-like response to 3 mg/kg ketamine, whereas both sexes responded to the 10 mg/kg dose used for pharmacokinetic analysis. Therefore, a direct association between greater HNK levels and enhanced female antidepressant-like response to ketamine cannot be conclusively inferred. In contrast, the present findings demonstrate greater ketamine and NK exposure in the plasma and brain of cycling female versus male rats following 2.5 mg/kg ketamine—a dose behaviorally effective in females but not males. In addition, the regional differences observed

60 herein when the mPFC and hippocampus were examined independently may not be directly comparable to the whole brain analysis performed by Zanos et al. (2016). These findings further suggest species differences in not only behavioral, but also pharmacokinetic parameters following low-dose ketamine exposure, and highlight the need for pharmacokinetic analysis across multiple behaviorally-relevant doses across species in both sexes. Unfortunately, the lack of studies investigating sex differences in ketamine pharmacokinetics has left very little evidence regarding such effects in humans. The few that do exist suggest that observations of sex effects in ketamine metabolism and clearance are likely dose- dependent. For example, 20% greater ketamine and NK clearance and lower drug/metabolite concentrations have been observed in healthy women when compared to men following ketamine infusion at a higher dose >10 mg/kg, i.v. (Sigtermans et al., 2009). These sex differences were reflected at the behavioral level, with greater effects on cardiac output and heat pain-related indices in men than in women (Sigtermans et al., 2010). Conversely, Zarate and colleagues (2012) identified smaller sex differences in metabolism of low-dose ketamine in MDD and bipolar patients, where females displayed greater plasma levels of DHNK and HNK4a/c metabolites compared to males. Importantly, however, no sex differences in antidepressant response were apparent, and the differences in metabolite levels—notably that of HNK4a—had no significant association with treatment response. In fact, independent of sex, HNK5 was negatively associated with treatment response in bipolar depression patients (Zarate et al., 2012), suggesting that pharmacokinetic sex differences may not actually impact treatment response in clinical depression. Of note, hormone levels were not controlled for in these studies, which may have obscured potential differences in clinical response between sexes—particularly given the overlap in brain regions whose activity is modulated by both circulating ovarian hormone levels in women and ketamine itself (Arelin et al., 2015). While underlying factors responsible for these varying differences in ketamine metabolism observed between males and females remain unknown, sex differences in hepatic expression and activity of ketamine-metabolizing cytochrome P450 enzymes are well-known (Waxman and Holloway, 2009)—and subject to hormonal regulation by estrogen, progesterone and testosterone, which also happen to be substrates of several P450 enzymes responsible for ketamine metabolism (de Vries and Forger, 2015; Waxman and Holloway, 2009). As well, physiological differences

61 influencing xenobiotic distribution, metabolism and clearance (i.e., body weight, adipose tissue levels and distribution) are present between males and females of a variety of species (de Vries and Forger, 2015). Ultimately, whether sex-dependent pharmacokinetic processes contribute to differences between males and females in ketamine’s antidepressant response is unclear, but the evidence strongly supports their consideration both preclinically and clinically. Likewise, non- negligible pharmacokinetic-related species differences have been highlighted herein, encouraging further examination to better translate findings between rodents and humans.

62

CHAPTER 4

CONCLUSIONS AND FUTURE DIRECTIONS

The overall goal of the work presented herein was to gain a better understanding of the role sex and hormonal milieu play in the differential female sensitivity of rats to low-dose ketamine under baseline conditions, so as to build a better foundation upon which to design future studies and interpret findings regarding mechanisms by which female rats respond with greater sensitivity to ketamine’s rapid antidepressant actions. Behavioral, pharmacokinetic and pharmacodynamic approaches were taken to develop a more comprehensive picture of processes mediating the heightened sensitivity of one sex over another to low-dose ketamine. We began by demonstrating in Chapter Two that females exhibited a sustained increase in sucrose preference following low-dose ketamine in an E2P4-dependent manner. In contrast, whereas testosterone failed to alter male treatment response, hedonic response to low-dose ketamine could be enhanced in intact males with concurrent P4 administration. This treatment responsiveness was associated with greater hippocampal BDNF levels only in female rats 24h after ketamine administration, without activation of key downstream signaling effectors at this timepoint. Based on these findings, it is more likely that rapid activation of signaling networks by ketamine is critical for initiation of more delayed plasticity-related changes (e.g. increased BDNF translation and/or release) associated with treatment response as seen in this study, rather than alteration of the general activity of such cascades long-term. Overall, this work suggests activational roles for ovarian-, but not testicular-, derived hormones in mediating hedonic sensitivity to low-dose ketamine in female and male rats. The sex- and hormone-dependent effect of ketamine on BDNF protein levels in the dorsal hippocampus and persistence of behavioral sex differences following gonadectomy may reflect underlying organizational differences in both ketamine’s mechanisms of action, as well as in the activational effects of estradiol and progesterone within the hippocampus. This makes it difficult to disentangle the weight of either sex or hormonal status on behavioral sensitivity to and molecular mediators of ketamine’s antidepressant-like properties; nonetheless, it supports the notion that both factors should be considered and/or controlled for experimentally when examining sex differences in ketamine’s effects in preclinical and clinical studies. 63

To this end, the very few clinical studies that have included or reported on sex as a variable in analyses of ketamine’s antidepressant effects have reported no effect of sex with respect to clinical symptomology, biomarkers of treatment response or ketamine metabolism. Notably, no studies to date have examined hormonal status in women receiving ketamine treatment; as such, this is an important gap in current literature worth exploring. Given the known effects of sex and hormones on xenobiotic metabolism, in Chapter Three we determined the relative contributions of sex and hormonal milieu on distribution and metabolism of low-dose ketamine in the brain and plasma of rats. Here, we demonstrated appreciable sex differences in ketamine pharmacokinetics, where females displayed higher levels of ketamine and NK rapidly following treatment in both plasma and depression-relevant brain regions. Interestingly, hormonal status in female rats had no significant impact on either metabolism or distribution of ketamine and its primary metabolites to the brain. Together, this work suggests that while sex differences in metabolism may influence the amount of ketamine and NK reaching target areas in the brain, the impact of circulating hormones on behavioral sensitivity is more likely an effect of actions within the brain at the time of ketamine administration. Despite recent reports in mice and rats confirming our lab’s original findings of enhanced behavioral sensitivity to the antidepressant-like effects of low-dose ketamine, no significant progress has been made in identifying molecular processes underlying the enhanced effects of this drug in female rodents. What is known to date is primarily that the functionally-relevant actions of ketamine in the brain of females appears to differ from those reported at behaviorally-relevant doses in males. To fill this gap, ongoing phosphoproteomic analyses are underway as a high- throughput, unbiased approach to uncovering potential signaling pathways rapidly altered by low- dose ketamine in male and female rats, with the goal of uncovering novel targets that may permit enhanced sensitivity to the beneficial effects ketamine at low doses. In order to compare phosphoproteomics findings with drug concentrations in the brain presented in Chapter 3, an experimental design identical to that used for the pharmacokinetics study was employed (Figure B1). Analysis of ketamine-induced signaling pathway activation and baseline sex differences in the dorsal hippocampal (dHPC) proteome and phosphoproteome is underway. Preliminary data show that a low dose of ketamine behaviorally effective in females, but not males, rapidly induces distinct protein phosphorylation events in male and female rats, with a much greater number of

64 upregulated phosphoproteins observed in females (Figure B2). Among female rats, estrous cycle stage also influenced the number, but not direction of phosphorylation events in female rats, where those in diestrus (low E2P4) displayed upregulation of phosphorylation of many distinct proteins when compared to proestrus (high E2P4) rats (Figure B2). Functional enrichment analyses of significantly altered phosphoproteins following ketamine treatment in male and female rats revealed that processes selectively affected by ketamine in female rats are primarily those involved in synaptic transmission, calcium signaling and energy/metabolism within cells (Figure B3). While preliminary at this point, a deeper exploration of how these selectively altered processes in female rats work in concert with each other after ketamine treatment may provide several yet unexplored functionally-relevant targets for further study. While future work is needed to determine the functional significance of these findings, the collective data presented herein suggest that both biological sex and the hormonal milieu are critical modulators of ketamine’s rapid actions on drug metabolism and effects within the brain, and provide greater insight into potential physiological and molecular processes underlying sex- and hormone-dependent modulation of ketamine’s therapeutic effects. Circulating hormone levels in female rats appear to be essential for their enhanced behavioral sensitivity to low-dose ketamine when compared to males, having a large influence on activation of large networks of phosphoproteins within depression-relevant brain regions (here, the hippocampus) not affected in males, without influencing metabolism or distribution of ketamine to targets within the brain. At the same time, the female sex—but not hormonal milieu—appears to predict greater concentrations of ketamine and NK within the brain versus males at early timepoints post-treatment, during which ketamine initiates activation of a network of signaling cascades important for rapid and sustained behavioral effects. Future work should build upon this work to include animal models under conditions of stress for comparison and further development of more specified therapeutic targets, with the goal of identifying strategies to enhance the effectiveness of ketamine at lower doses, or exploring alternative approaches that may yield similar effects to ketamine without the negative side effects. In view of the bigger picture, while the increasing inclusion of females in animal models of depression and antidepressant response in recent years have revealed sex-specificities in response to low-dose ketamine, these observations are somewhat inconsistent. Through the current

65 work and other studies, it has become clear that observed sex differences (or not) depend on a variety of factors, baseline sex differences and species differences among the most important. These factors, among others, are likely to interfere with behavioral and molecular readouts following the administration of ketamine in a variety of experimental contexts. The studies presented in this dissertation were designed to address the “Baseline” study level depicted in Figure 4.1, as a small component of and contribution to the large amount of work that is yet to be done. It is important to investigate the multiple factors influencing behavior together, revealing the advantage of an individualized approach over a group-based strategy (Figure 4.1). In addition to other factors, the inclusion of sex as a variable will help to more closely align preclinical experiments with the considerable heterogeneity in clinical populations, improving the translatability of findings from preclinical to clinical, and further our understanding of sex and individual differences in antidepressant response (Saland et al., 2017).

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Figure 4.1 Conceptual framework for an individualized multi-domain analysis of antidepressant response (applied to sex differences in anti-anhedonic effects of ketamine, as studied and discussed herein). In this diagram, key findings on the current state of knowledge of sex differences in ketamine’s effects on hedonic behaviors across three primary domains of investigation (“Behavioral correlates”, “Molecular correlates”, and “Pharmacokinetics”) are depicted at three main study levels (“Baseline”, “Preclinical”, and “Clinical”). Furthermore, each level x domain combination is summarized with (black background) and without (white background) ketamine treatment, thereby accounting for baseline sex differences when analyzing the drug’s effect(s). Note that all changes in the “Ketamine” category refer to comparisons with vehicle-treated controls (“Vehicle” category of the same “level”). Despite a previous sparsity in

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Figure 4.1 – continued. …experimental evidence, growing preclinical research further characterizes the similarities and inconsistencies in sex differences in the effects of ketamine between rodents and humans across the three study levels defined (baseline, preclinical, and clinical). In this example, for instance, a parallel comparison reveals a consistent sex difference in ketamine-induced spinogenesis in rats between baseline and a preclinical model, whereas such a comparison can be limited either by the presence of a sex difference in the preclinical model itself (as seen for the development of anhedonia in males but not females, “Behavioral correlates” domain), or by the absence of data on eventual sex differences (as seen in the “Pharmacokinetics” domain). Moreover, this example also illustrates the missing consideration of sex differences in clinical populations—both in vehicle- and drug-treated groups—that further limits the translation from the preclinical to clinical level and thus represents a significant barrier to progress in individualized treatment approaches. Cl/F: oral clearance, DHNK: dehydronorketamine, HNK: hydroxynorketamine, HPC: hippocampus, K: ketamine, MDD: major depressive disorder, mPFC: medial prefrontal cortex, NK: norketamine. Adapted from Saland et al. (2017), Curr Opin Behav Sci.

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APPENDIX A

ACUC PROTOCOL APPROVAL

69

APPENDIX B

SUPPLEMENTARY FIGURES

Figure B1 Phosphoproteomics experimental design and sample preparation workflow. Dorsal hippocampi in male and female rats were collected 30 minutes following saline or an acute low dose (2.5 mg/kg) of ketamine, after which total and TiO2-enriched phosphopeptide lysates were prepared and processed via high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the LTQ Orbitrap Velos. Collected LC-MS/MS data were processed and quantified (by label-free approach), and proteins identified with Progenesis QI software and MASCOT Search Engine, respectively. Pathway and gene ontology analyses were then performed. Abbreviations: TiO2, titanium dioxide; SDS, sodium dodecyl sulfate; LC-MS/MS, liquid chromatography-tandem mass spectrometry.

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Figure B2 Low-dose ketamine alters hippocampal protein phosphorylation in a sex- and estrous-dependent manner. (a) Ketamine rapidly induces distinct protein phosphorylation events in male and female rats, with a much greater number of upregulated phosphoproteins seen in females. A majority of phosphoproteins altered by ketamine in males were downregulated; whereas those altered in females were upregulated by ketamine. Among those proteins affected by ketamine in both sexes, phosphorylation of all were increased in females, but decreased in males. (b) Estrous cycle stage also influenced the number, but not direction of phosphorylation events in female rats, where those in diestrus displayed upregulation of phosphorylation of many distinct proteins not altered in proestrus female rats. (c) Heat map depicting fold change values of phosphoproteins significantly altered by ketamine in male and intact female rats. Notable differences in the number of upregulated phosphoproteins were observed between proestrus and diestrus female rats.

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Figure B3 Functional enrichment of significantly altered phosphoproteins in male and female rats. Significantly enriched pathways (KEGG) and gene ontologies for phosphoproteins differentially expressed following ketamine in male (a) and female (b) rats.

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REFERENCES

Abdallah CG, Adams TG, Kelmendi B, Esterlis I, Sanacora G, Krystal JH (2016). Ketamine’s mechanism of action: a path to rapid-acting antidepressants. Depress Anxiety doi:10.1002/da.22501.

Allen PJ, D’Anci KE, Kanarek RB, Renshaw PF (2012). Sex-specific antidepressant effects of dietary creatine with and without sub-acute fluoxetine in rats. Pharmacol Biochem Behav 101: 588–601.

Andrade S, Silveira SL, Arbo BD, Batista B a. M, Gomez R, Barros HMT, et al (2010). Sex- dependent antidepressant effects of lower doses of progesterone in rats. Physiol Behav 99: 687–690.

Arélin K, Mueller K, Barth C, Rekkas PV, Kratzsch J, Burmann I, et al (2015). Progesterone mediates brain functional connectivity changes during the menstrual cycle—a pilot resting state MRI study. Front Neurosci 9: 44.

Asarian L, Geary N (2002). Cyclic Estradiol Treatment Normalizes Body Weight and Restores Physiological Patterns of Spontaneous Feeding and Sexual Receptivity in Ovariectomized Rats. Hormones and Behavior 42: 461–471.

Asarian L, Geary N (2006). Modulation of appetite by gonadal steroid hormones. Philos Trans R Soc Lond, B, Biol Sci 361: 1251–1263.

Autry AE, Adachi M, Cheng P, Monteggia LM (2009). Gender-specific impact of brain-derived neurotrophic factor signaling on stress-induced depression-like behavior. Biol Psychiatry 66: 84–90.

Autry AE, Adachi M, Nosyreva E, Na ES, Los MF, Cheng P, et al (2011). NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses. Nature 475: 91–95.

Autry AE, Monteggia LM (2012). Brain-Derived Neurotrophic Factor and Neuropsychiatric Disorders. Pharmacol Rev 64: 238–258.

Bechtholt-Gompf AJ, Smith KL, John CS, Kang HH, Carlezon WA Jr, Cohen BM, et al (2011). CD-1 and Balb/cJ mice do not show enduring antidepressant-like effects of ketamine in tests of acute antidepressant efficacy. Psychopharmacology 215: 689–695.

Becker JB, Prendergast BJ, Liang JW (2016). Female rats are not more variable than male rats: a meta-analysis of neuroscience studies. Biol Sex Differ 7.

Berman RM, Cappiello A, Anand A, Oren DA, Heninger GR, Charney DS, et al (2000). Antidepressant effects of ketamine in depressed patients. Biol Psychiatry 47: 351–354.

73

Berridge KC (1996). Food reward: Brain substrates of wanting and liking. Neuroscience & Biobehavioral Reviews 20: 1–25.

Berridge KC, Kringelbach ML (2008). Affective neuroscience of pleasure: reward in humans and animals. Psychopharmacology (Berl) 199: 457–480.

Carrier N, Kabbaj M (2012a). Extracellular Signal-Regulated Kinase 2 Signaling in the Hippocampal Dentate Gyrus Mediates the Antidepressant Effects of Testosterone. Biological Psychiatry 71: 642–651.

Carrier N, Kabbaj M (2012b). Testosterone and imipramine have antidepressant effects in socially isolated male but not female rats. Hormones and Behavior 61: 678–685.

Carrier N, Kabbaj M (2013a). Sex differences in the antidepressant-like effects of ketamine. Neuropharmacology 70: 27–34.

Carrier N, Kabbaj M (2013b). Sex differences in the antidepressant-like effects of ketamine. Neuropharmacology 70: 27–34.

Carrier N, Saland SK, Duclot F, He H, Mercer R, Kabbaj M (2015). The Anxiolytic and Antidepressant-like Effects of Testosterone and Estrogen in Gonadectomized Male Rats. Biol Psychiatry 78: 259–269.

Chen Y-W, Rada PV, Bützler BP, Leibowitz SF, Hoebel BG (2012). Corticotropin-releasing factor in the nucleus accumbens shell induces swim depression, anxiety, and anhedonia along with changes in local dopamine/acetylcholine balance. Neuroscience 206: 155–166.

Conrad CD, McLaughlin KJ, Huynh TN, El-Ashmawy M, Sparks M (2012). Chronic stress and a cyclic regimen of estradiol administration separately facilitate spatial memory: relationship with hippocampal CA1 spine density and dendritic complexity. Behav Neurosci 126: 142– 156.

Craft RM, Kostick ML, Rogers JA, White CL, Tsutsui KT (2010). Forced swim test behavior in postpartum rats. Pharmacol Biochem Behav 96: 402–412.

Cuthbert BN (2014a). The RDoC framework: facilitating transition from ICD/DSM to dimensional approaches that integrate neuroscience and psychopathology. World Psychiatry 13: 28–35.

Cuthbert BN (2014b). Translating intermediate phenotypes to psychopathology: The NIMH Research Domain Criteria. Psychophysiol 51: 1205–1206.

Cuthbert BN, Insel TR (2013). Toward the future of psychiatric diagnosis: the seven pillars of RDoC. BMC Medicine 11: 126.

74

Czéh B, Fuchs E, Wiborg O, Simon M (2016). Animal models of major depression and their clinical implications. Prog Neuropsychopharmacol Biol Psychiatry 64: 293–310.

Dahan MI al-, Thalmann RH (1996). Progesterone regulates gamma-aminobutyric acid B (GABAB) receptors in the neocortex of female rats. Brain Res 727: 40–48.

Dalla C, Pitychoutis PM, Kokras N, Papadopoulou-Daifoti Z (2011). Sex differences in response to stress and expression of depressive-like behaviours in the rat. Curr Top Behav Neurosci 8: 97–118.

Darcet F, Gardier AM, Gaillard R, David DJ, Guilloux J-P (2016). Cognitive Dysfunction in Major Depressive Disorder. A Translational Review in Animal Models of the Disease. Pharmaceuticals 9.

Donaldson ZR, Hen R (2015). From Psychiatric Disorders to Animal Models: A Bidirectional and Dimensional Approach. Biological Psychiatry 77: 15–21.

Drossopoulou G, Antoniou K, Kitraki E, Papathanasiou G, Papalexi E, Dalla C, et al (2004a). Sex differences in behavioral, neurochemical and neuroendocrine effects induced by the forced swim test in rats. Neuroscience 126: 849–857.

Drossopoulou G, Antoniou K, Kitraki E, Papathanasiou G, Papalexi E, Dalla C, et al (2004b). Sex differences in behavioral, neurochemical and neuroendocrine effects induced by the forced swim test in rats. Neuroscience 126: 849–857.

Duclot F, Kabbaj M (2015). Epigenetic mechanisms underlying the role of brain-derived neurotrophic factor in depression and response to antidepressants. J Exp Biol 218: 21–31.

Duman CH, Duman RS (2015). Spine synapse remodeling in the pathophysiology and treatment of depression. Neurosci Lett 601: 20–29.

Flores-Serrano AG, Vila-Luna ML, Álvarez-Cervera FJ, Heredia-López FJ, Góngora-Alfaro JL, Pineda JC (2013). Clinical doses of citalopram or reboxetine differentially modulate passive and active behaviors of female Wistar rats with high or low immobility time in the forced swimming test. Pharmacol Biochem Behav 110: 89–97.

Franceschelli A, Herchick S, Thelen C, Papadopoulou-Daifoti Z, Pitychoutis PM (2014). Sex differences in the chronic mild stress model of depression. Behav Pharmacol 25: 372–383.

Franceschelli A, Sens J, Herchick S, Thelen C, Pitychoutis PM (2015a). Sex differences in the rapid and the sustained antidepressant-like effects of ketamine in stress-naïve and “depressed” mice exposed to chronic mild stress. Neuroscience doi:10.1016/j.neuroscience.2015.01.008.

75

Franceschelli A, Sens J, Herchick S, Thelen C, Pitychoutis PM (2015b). Sex differences in the rapid and the sustained antidepressant-like effects of ketamine in stress-naïve and “depressed” mice exposed to chronic mild stress. Neuroscience 290: 49–60.

Fuller GB, Hobson WC, Reyes FI, Winter JS, Faiman C (1984). Influence of restraint and ketamine anesthesia on adrenal steroids, progesterone, and gonadotropins in rhesus monkeys. Proc Soc Exp Biol Med 175: 487–490.

Garcia LSB, Comim CM, Valvassori SS, Réus GZ, Stertz L, Kapczinski F, et al (2009). Ketamine treatment reverses behavioral and physiological alterations induced by chronic mild stress in rats. Progress in Neuro-Psychopharmacology and Biological Psychiatry 33: 450–455.

Guilloux J-P, Seney M, Edgar N, Sibille E (2011). Integrated behavioral z-scoring increases the sensitivity and reliability of behavioral phenotyping in mice: Relevance to emotionality and sex. Journal of Neuroscience Methods 197: 21–31.

Gutiérrez-Lobos K, Scherer M, Anderer P, Katschnig H (2002). The influence of age on the female/male ratio of treated incidence rates in depression. BMC Psychiatry 2: 3.

Hammarström A, Lehti A, Danielsson U, Bengs C, Johansson EE (2009). Gender-related explanatory models of depression: A critical evaluation of medical articles. Public Health 123: 689–693.

Hodes GE, Pfau ML, Purushothaman I, Ahn HF, Golden SA, Christoffel DJ, et al (2015). Sex Differences in Nucleus Accumbens Transcriptome Profiles Associated with Susceptibility versus Resilience to Subchronic Variable Stress. J Neurosci 35: 16362–16376.

Hollis F, Duclot F, Gunjan A, Kabbaj M (2011). Individual differences in the effect of social defeat on anhedonia and histone acetylation in the rat hippocampus. Horm Behav 59: 331–337.

Hyman SE (2014). Revitalizing Psychiatric Therapeutics. Neuropsychopharmacology 39: 220– 229.

Insel T, Cuthbert B, Garvey M, Heinssen R, Pine DS, Quinn K, et al (2010). Research domain criteria (RDoC): toward a new classification framework for research on mental disorders. Am J Psychiatry 167: 748–751.

Insel TR, Landis SC (2013). Twenty-five Years of Progress: The View from NIMH and NINDS. Neuron 80.

Joel D, Yankelevitch-Yahav R (2014). Reconceptualizing sex, brain and psychopathology: interaction, interaction, interaction. Br J Pharmacol 171: 4620–4635.

76

Kara NZ, Agam G, Anderson GW, Zitron N, Einat H (2016). Lack of effect of chronic ketamine administration on depression-like behavior or frontal cortex autophagy in female and male ICR mice. Behav Brain Res doi:10.1016/j.bbr.2016.09.056.

Kato A, Hojo Y, Higo S, Komatsuzaki Y, Murakami G, Yoshino H, et al (2013). Female hippocampal estrogens have a significant correlation with cyclic fluctuation of hippocampal spines. Front Neural Circuits 7: 149.

Keers R, Aitchison KJ (2010a). Gender differences in antidepressant drug response. Int Rev Psychiatry 22: 485–500.

Keers R, Aitchison KJ (2010b). Gender differences in antidepressant drug response. Int Rev Psychiatry 22: 485–500.

Kentner AC, McLeod SA, Field EF, Pittman QJ (2010). Sex-dependent effects of neonatal inflammation on adult inflammatory markers and behavior. Endocrinology 151: 2689– 2699.

Kessler RC (2003). Epidemiology of women and depression. J Affect Disord 74: 5–13.

Kitraki E, Kremmyda O, Youlatos D, Alexis MN, Kittas C (2004). Gender-dependent alterations in corticosteroid receptor status and spatial performance following 21 days of restraint stress. Neuroscience 125: 47–55.

Kokras N, Antoniou K, Mikail HG, Kafetzopoulos V, Papadopoulou-Daifoti Z, Dalla C (2015). Forced swim test: What about females? Neuropharmacology 99: 408–421.

Kokras N, Dalla C (2014). Sex differences in animal models of psychiatric disorders. Br J Pharmacol 171: 4595–4619.

Krishnan V, Han M-H, Graham DL, Berton O, Renthal W, Russo SJ, et al (2007). Molecular Adaptations Underlying Susceptibility and Resistance to Social Defeat in Brain Reward Regions. Cell 131: 391–404.

Lally N, Nugent AC, Luckenbaugh DA, Ameli R, Roiser JP, Zarate CA (2014). Anti-anhedonic effect of ketamine and its neural correlates in treatment-resistant bipolar depression. Transl Psychiatry 4: e469.

Lally N, Nugent AC, Luckenbaugh DA, Niciu MJ, Roiser JP, Zarate CA (2015). Neural correlates of change in major depressive disorder anhedonia following open-label ketamine. J Psychopharmacol 269881114568041doi:10.1177/0269881114568041.

Lee CJ, Do BR, Kim JK, Yoon YD (2000). Pentobarbital and ketamine suppress serum concentrations of sex hormones in the female rat. J Anesth 14: 187–190.

77

Li N, Lee B, Liu R-J, Banasr M, Dwyer JM, Iwata M, et al (2010). mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science 329: 959–964.

Li N, Liu R-J, Dwyer JM, Banasr M, Lee B, Son H, et al (2011a). Glutamate N-methyl-D-aspartate Receptor Antagonists Rapidly Reverse Behavioral and Synaptic Deficits Caused by Chronic Stress Exposure. Biological Psychiatry 69: 754–761.

Li N, Liu R-J, Dwyer JM, Banasr M, Lee B, Son H, et al (2011b). Glutamate N-methyl-D-aspartate receptor antagonists rapidly reverse behavioral and synaptic deficits caused by chronic stress exposure. Biol Psychiatry 69: 754–761.

Lin Y, Ter Horst GJ, Wichmann R, Bakker P, Liu A, Li X, et al (2009). Sex differences in the effects of acute and chronic stress and recovery after long-term stress on stress-related brain regions of rats. Cereb Cortex 19: 1978–1989.

Liu R-J, Lee FS, Li X-Y, Bambico F, Duman RS, Aghajanian GK (2012). Brain-derived neurotrophic factor Val66Met allele impairs basal and ketamine-stimulated synaptogenesis in prefrontal cortex. Biol Psychiatry 71: 996–1005.

Luine V (2016). Estradiol: Mediator of memories, spine density and cognitive resilience to stress in female rodents. J Steroid Biochem Mol Biol 160: 189–195.

Maeng S, Zarate CA Jr, Du J, Schloesser RJ, McCammon J, Chen G, et al (2008). Cellular mechanisms underlying the antidepressant effects of ketamine: role of alpha-amino-3- hydroxy-5-methylisoxazole-4-propionic acid receptors. Biol Psychiatry 63: 349–352.

Magariños AM, McEwen BS (1995). Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: involvement of glucocorticoid secretion and excitatory amino acid receptors. Neuroscience 69: 89–98.

McEwen BS, Nasca C, Gray JD (2016). Stress Effects on Neuronal Structure: Hippocampus, Amygdala, and Prefrontal Cortex. Neuropsychopharmacology 41: 3–23.

McLaughlin KJ, Wilson JO, Harman J, Wright RL, Wieczorek L, Gomez J, et al (2010). Chronic 17beta-estradiol or cholesterol prevents stress-induced hippocampal CA3 dendritic retraction in ovariectomized female rats: possible correspondence between CA1 spine properties and spatial acquisition. Hippocampus 20: 768–786.

McMakin DL, Olino TM, Porta G, Dietz LJ, Emslie G, Clarke G, et al (2012). Anhedonia Predicts Poorer Recovery Among Youth With Selective Serotonin Reuptake Inhibitor Treatment– Resistant Depression. Journal of the American Academy of Child & Adolescent Psychiatry 51: 404–411.

78

Micevych P, Sinchak K (2008). Estradiol regulation of progesterone synthesis in the brain. Molecular and Cellular Endocrinology 290: 44–50.

Mileva GR, Bielajew C (2015). Environmental manipulation affects depressive-like behaviours in female Wistar-Kyoto rats. Behav Brain Res 293: 208–216.

Mion G, Villevieille T (2013). Ketamine pharmacology: an update (pharmacodynamics and molecular aspects, recent findings). CNS Neurosci Ther 19: 370–380.

Moaddel R, Sanghvi M, Ramamoorthy A, Jozwiak K, Singh N, Green C, et al (2016). Subchronic administration of (R,S)-ketamine induces ketamine ring hydroxylation in Wistar rats. J Pharm Biomed Anal 127: 3–8.

Monteggia LM, Zarate Jr C (2015). Antidepressant actions of ketamine: from molecular mechanisms to clinical practice. Current Opinion in Neurobiology 30: 139–143.

Niknazar S, Nahavandi A, Peyvandi AA, Peyvandi H, Akhtari AS, Karimi M (2016). Comparison of the Adulthood Chronic Stress Effect on Hippocampal BDNF Signaling in Male and Female Rats. Mol Neurobiol 53: 4026–4033.

Numakawa T, Richards M, Nakajima S, Adachi N, Furuta M, Odaka H, et al (2014). The role of brain-derived neurotrophic factor in comorbid depression: possible linkage with steroid hormones, cytokines, and nutrition. Front Psychiatry 5: 136.

Patel V, Vikram P, Dan C, Rachana P, Charlson FJ, Louisa D, et al (2016). Addressing the burden of mental, neurological, and substance use disorders: key messages from Disease Control Priorities, 3rd edition. Lancet 387: 1672–1685.

Pelizza L, Ferrari A (2009). Anhedonia in schizophrenia and major depression: state or trait? Ann Gen Psychiatry 8: 22.

Qiao H, Li M-X, Xu C, Chen H-B, An S-C, Ma X-M (2016). Dendritic Spines in Depression: What We Learned from Animal Models. Neural Plast 2016: 8056370.

Radley JJ, Rocher AB, Janssen WGM, Hof PR, McEwen BS, Morrison JH (2005). Reversibility of apical dendritic retraction in the rat medial prefrontal cortex following repeated stress. Exp Neurol 196: 199–203.

Reddy DS, Zeng Y-C (2007a). Differential anesthetic activity of ketamine and the GABAergic neurosteroid allopregnanolone in mice lacking progesterone receptor A and B subtypes. Methods Find Exp Clin Pharmacol 29: 659–664.

Reddy DS, Zeng Y-C (2007b). Differential anesthetic activity of ketamine and the GABAergic neurosteroid allopregnanolone in mice lacking progesterone receptor A and B subtypes. Methods Find Exp Clin Pharmacol 29: 659–664.

79

Rømer Thomsen K, Whybrow PC, Kringelbach ML (2015). Reconceptualizing anhedonia: novel perspectives on balancing the pleasure networks in the human brain. Front Behav Neurosci 9: .

Rubio-Casillas A, Fernández-Guasti A (2016). The dose makes the poison: from glutamate- mediated neurogenesis to neuronal atrophy and depression. Rev Neurosci doi:10.1515/revneuro-2015-0066.

Saland SK, Schoepfer KJ, Kabbaj M (2016). Hedonic sensitivity to low-dose ketamine is modulated by gonadal hormones in a sex-dependent manner. Sci Rep 6: 21322.

Saland SK, Duclot F, Kabbaj M (2017). Integrative analysis of sex differences in the rapid antidepressant effects of ketamine in preclinical models for individualized clinical outcomes. Curr Opin Behav Sci 14: 19-26.

Sarkar A, Kabbaj M (2016). Sex Differences in Effects of Ketamine on Behavior, Spine Density, and Synaptic Proteins in Socially Isolated Rats. Biol Psychiatry 80: 448–456.

Seedat S, Scott KM, Angermeyer MC, Berglund P, Bromet EJ, Brugha TS, et al (2009). Cross- national associations between gender and mental disorders in the World Health Organization World Mental Health Surveys. Arch Gen Psychiatry 66: 785–795.

Shansky RM, Hamo C, Hof PR, Lou W, McEwen BS, Morrison JH (2010). Estrogen promotes stress sensitivity in a prefrontal cortex-amygdala pathway. Cereb Cortex 20: 2560–2567.

Shors TJ, Falduto J, Leuner B (2004). The opposite effects of stress on dendritic spines in male vs. female rats are NMDA receptor-dependent. Eur J Neurosci 19: 145–150.

Sigtermans M, Dahan A, Mooren R, Bauer M, Kest B, Sarton E, et al (2009). S(+)-ketamine effect on experimental pain and cardiac output: a population pharmacokinetic-pharmacodynamic modeling study in healthy volunteers. Anesthesiology 111: 892–903.

Sigtermans M, Marnix S, Ingeborg N, Elise S, Martin B, René M, et al (2010). An observational study on the effect of S( )-ketamine on chronic pain versus experimental acute pain in Complex Regional Pain Syndrome type 1 patients. Eur J Pain 14: 302–307.

Simmons WK, Drevets WC (2012). A “Taste” of What is to Come: Reward Sensitivity as a Potential Endophenotype for Major Depressive Disorder. Biological Psychiatry 72: 526– 527.

Spijker J, Bijl RV, De Graaf R, Nolen WA (2001). Determinants of poor 1-year outcome of DSM- III-R major depression in the general population: results of the Netherlands Mental Health Survey and Incidence Study (NEMESIS). Acta Psychiatrica Scandinavica 103: 122–130.

80

Stack A, Carrier N, Dietz D, Hollis F, Sorenson J, Kabbaj M (2010). Sex differences in social interaction in rats: role of the immediate-early gene zif268. Neuropsychopharmacology 35: 570–580.

Taliaz D, Loya A, Gersner R, Haramati S, Chen A, Zangen A (2011). Resilience to Chronic Stress Is Mediated by Hippocampal Brain-Derived Neurotrophic Factor. J Neurosci 31: 4475– 4483.

Taliaz D, Nagaraj V, Haramati S, Chen A, Zangen A (2013). Altered brain-derived neurotrophic factor expression in the ventral tegmental area, but not in the hippocampus, is essential for antidepressant-like effects of electroconvulsive therapy. Biol Psychiatry 74: 305–312.

Taliaz D, Stall N, Dar DE, Zangen A (2010). Knockdown of brain-derived neurotrophic factor in specific brain sites precipitates behaviors associated with depression and reduces neurogenesis. Mol Psychiatry 15: 80–92.

Thelen C, Sens J, Mauch J, Pandit R, Pitychoutis PM (2016). Repeated ketamine treatment induces sex-specific behavioral and neurochemical effects in mice. Behav Brain Res 312: 305–312.

Tizabi Y, Bhatti BH, Manaye KF, Das JR, Akinfiresoye L (2012). Antidepressant-like effects of low ketamine dose is associated with increased hippocampal AMPA/NMDA receptor density ratio in female Wistar-Kyoto rats. Neuroscience 213: 72–80.

Treadway MT, Zald DH (2011). Reconsidering anhedonia in depression: Lessons from translational neuroscience. Neuroscience & Biobehavioral Reviews 35: 537–555.

Treadway MT, Zald DH (2013). Parsing Anhedonia: Translational Models of Reward-Processing Deficits in Psychopathology. Curr Dir Psychol Sci 22: 244–249.

Uher R, Perlis RH, Henigsberg N, Zobel A, Rietschel M, Mors O, et al (2012). Depression symptom dimensions as predictors of antidepressant treatment outcome: replicable evidence for interest-activity symptoms. Psychological Medicine 42: 967–980.

Vries GJ de, Forger NG (2015). Sex differences in the brain: a whole body perspective. Biol Sex Differ 6.

Waxman DJ, Holloway MG (2009). Sex differences in the expression of hepatic drug metabolizing enzymes. Mol Pharmacol 76: 215–228.

Willner P, Belzung C (2015). Treatment-resistant depression: are animal models of depression fit for purpose? Psychopharmacology 232: 3473–3495.

Yu Z, Geary N, Corwin RL (2008). Ovarian hormones inhibit fat intake under binge-type conditions in ovariectomized rats. Physiol Behav 95: 501–507.

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Zanos P, Moaddel R, Morris PJ, Georgiou P, Fischell J, Elmer GI, et al (2016). NMDAR inhibition-independent antidepressant actions of ketamine metabolites. Nature 533: 481– 486.

Zarate CA Jr, Singh JB, Carlson PJ, Brutsche NE, Ameli R, Luckenbaugh DA, et al (2006a). A randomized trial of an N-methyl-D-aspartate antagonist in treatment-resistant major depression. Arch Gen Psychiatry 63: 856–864.

Zarate CA, Mathews DC, Furey ML (2013). Human Biomarkers of Rapid Antidepressant Effects. Biol Psychiatry 73: 1142–1155.

Zarate CA, Nancy B, Gonzalo L, Luckenbaugh DA, Vattem Venkata SL, Anuradha R, et al (2012). Relationship of Ketamine’s Plasma Metabolites with Response, Diagnosis, and Side Effects in Major Depression. Biol Psychiatry 72: 331–338.

Zarate CA, Singh JB, Quiroz JA, De Jesus G, Denicoff KK, Luckenbaugh DA, et al (2006b). A double-blind, placebo-controlled study of memantine in the treatment of major depression. Am J Psychiatry 163: 153–155.

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BIOGRAPHICAL SKETCH

EDUCATION

Ph.D. Candidate, Doctor of Philosophy in Neuroscience, expected May 2018 Florida State University, College of Medicine, Tallahassee, FL Advisor: Dr. Mohamed Kabbaj

M.Sc. Psychobiology, May 2012 Florida State University, Tallahassee, FL Advisor: Dr. Joshua Rodefer

B.S. Psychology, May 2009 University of Iowa, Iowa City, IA Minor: Spanish

PROFESSIONAL MEMBERSHIPS

2014-present Organization for the Study of Sex Differences 2008-present Society for Neuroscience

TEACHING EXPERIENCE

2010-2012 Teaching Assistant, Conditioning and Learning Laboratory Course, Graduate, Florida State University, Tallahassee, FL

OUTREACH EXPERIENCE

2013-2014 Graduate Student Mentor, SSTRIDE Summer Institute, College of Medicine, Florida State University, Tallahassee, Florida 2009-2012 Outreach and Education Sub-Committee (OES), Florida State University, Tallahassee, Florida

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AWARDS

2012 First Place Grant, Bryan W. Robinson Endowment, Tallahassee Memorial Foundation 2011 Ermine M. Owenby Jr. Travel Award, Florida State University

PUBLICATIONS

Saland SK, Lam T, Wilzcak K, Singh R, Mercer R, Kabbaj M. Phosphoproteomic analysis of sex- dependent changes in signaling pathway activation and protein levels in the hippocampus by low- dose ketamine. In preparation.

Saland SK, He H, Mercer R, Kabbaj M (2016). Sex differences in the pharmacokinetics of low- dose ketamine in plasma and brain of male and female rats. In preparation.

Schoepfer KJ, Strong CE, Saland SK, Wright KN, Kabbaj M (2017). Sex- and dose-dependent abuse liability of repeated subanesthetic ketamine in rats. Physiol Behav doi:10.1016/j.physbeh.2017.10.021.

Strong CE, Schoepfer KJ, Dossat AM, Saland SK, Wright KN, Kabbaj M (2017). Locomotor sensitization to intermittent ketamine administration is associated with nucleus accumbens plasticity in male and female rats. Neuropharmacology 121: 195–203.

Saland SK, Duclot F, Kabbaj M (2017). Integrative analysis of sex differences in the rapid antidepressant effects of ketamine in preclinical models for individualized clinical outcomes. Curr Opin Behav Sci 14: 19–26.

Saland SK, Schoepfer KJ, Kabbaj M (2016). Hedonic sensitivity to low-dose ketamine is modulated by gonadal hormones in a sex-dependent manner. Sci Rep 6: 21322.

Carrier N*, Saland SK*, Duclot F, He H, Mercer R, Kabbaj M (2015). The Anxiolytic and Antidepressant-like Effects of Testosterone and Estrogen in Gonadectomized Male Rats. Biol Psychiatry 78: 259–269. (* denotes co-first authorship).

Rodefer JS, Saland SK, Eckrich SJ (2012). Selective phosphodiesterase inhibitors improve performance on the ED/ID cognitive task in rats. Neuropharmacology 62: 1182–1190.

Saland SK, Rodefer JS (2011). Environmental enrichment ameliorates phencyclidine-induced cognitive deficits. Pharmacol Biochem Behav 98: 455–461.

Droste SM, Saland SK, Schlitter EK, Rodefer JS (2010). AM 251 differentially effects food- maintained responding depending on food palatability. Pharmacol Biochem Behav 95: 443–448.

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ORAL COMMUNICATIONS

Saland SK, Schoepfer KJ, Kabbaj M (2014). Influence of gonadal hormones on behavioral sensitivity to low-dose ketamine. Natural Sciences Graduate Research Day Symposium, Florida State University, Tallahassee, FL.

Eckrich SJ, Saland SK, Rodefer JS (2012). The impact of monoamine transporters in the rat gambling task. Society for Neuroscience Conference, New Orleans, LA.

Saland SK, Eckrich SJ, Rodefer JS (2011). Contributions of social interaction and environmental complexity on cognitive development in male rats. Society for Neuroscience Conference, Washington, D.C.

POSTERS AND ABSTRACTS

Saland SK, Singh R, Mercer R, Lam TT, Wilczak K, Kabbaj M (2015). Rapid sex- and hormone- dependent changes in signaling pathway activation and protein levels in the hippocampus following low-dose ketamine administration: A phosphoproteomics approach. Society for Neuroscience Conference, Chicago, IL.

Schoepfer KJ, Strong CE, Saland SK, Dossat AM, Johnson F, Kabbaj M (2015). Sex differences in the abuse potential of low-dose ketamine. Society for Neuroscience Conference, Chicago, IL.

Kabbaj M, Schoepfer KJ, Saland SK (2014). Influence of gonadal hormones on behavioral sensitivity to low-dose ketamine. American College of Neuropsychopharmacology Annual Meeting, Phoenix, AZ.

Saland SK, Schoepfer KJ, Kabbaj M (2014). Influence of gonadal hormones on behavioral sensitivity to low-dose ketamine. Society for Neuroscience Conference, Washington, D.C.

Saland SK, Carrier N, Duclot F, Kabbaj MK (2013). Effects of testosterone on depressive-like behavior and hippocampal gene expression in male rats. Society for Neuroscience Conference, San Diego, CA.

McAllister SM, Saland SK, Lieberwirth C, Smith AS, Stathopoulos AC, Biggs L (2012). Neuroscience graduate students at the Florida State University share their enthusiasm about the brain with the public. Society for Neuroscience Conference, New Orleans, LA.

Eckrich SJ, Saland SK, Rodefer JS (2011). Evaluation of risky decision making using the rat gambling task and attentional set-shifting. Society for Neuroscience Conference, Washington, D.C.

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Berkley KJ, Saland SK, McGinty KM, Rodefer JS (2011). Endometriosis (ENDO) in the rat: a pilot study of ENDO’s influence on anxiety-like behaviors as assessed by the elevated plus maze and open field tests. Society for Neuroscience Conference, Washington, D.C.

Smith AS, Lieberwirth C, McAllister SM, Saland SK (2011). Celebrating Brain Awareness Week throughout the entire academic school year: a strategy for growth. Society for Neuroscience Conference, Washington, D.C.

Saland SK, Rodefer JS (2010). Dissociable pro-cognitive contributions of social interaction and environmental enrichment on cognitive flexibility in developing male rats. Society for Neuroscience Conference, San Diego, CA.

Moschak TM, Saland SK, Delgado MG, Hafling JM, Rodefer JS (2010). Modulation of cognitive flexibility by CB1 ligands. Society for Neuroscience Conference, San Diego, CA.

Lieberwirth C, Maffeo M, McAllister SM, Saland SK, Smith AS (2010). Neuroscience educational outreach by the Florida State University. Society for Neuroscience Conference, San Diego, CA.

Saland SK, Rodefer JS (2009). Evaluation of dopamine involvement in PCP-induced cognitive deficits in the rodent attentional set-shifting task. Society for Neuroscience Conference, Chicago, IL.

RESEARCH EXPERIENCE

Graduate Work, Rodefer Lab: 2009-2012

 Evaluation of dopamine involvement in phencyclidine-induced deficits in cognitive flexibility. Dopaminergic involvement in prefrontal cortex-mediated cognitive deficits induced by subchronic administration of the NMDA receptor antagonist, phencyclidine (PCP), was investigated by evaluating the effects of acute administration of dopamine D1 and D2 receptor agonists and antagonists on behavioral performance in the attentional set-shifting task. Phosphodiesterase (PDE) inhibitors known to modulate dopaminergic activity, including papaverine (PDE10A inhibitor), were also evaluated for their ability to attenuate PCP-induced deficits in cognitive flexibility. This work was published in Neuropharmacology.  Contributions of social interaction and environmental complexity on cognitive flexibility and non-spatial memory in developing male rats. Systematic manipulations of the social and physical environment in post-weanling male rats throughout development were performed

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to identify the impact of dissociable early-life experience factors on execute function and non- spatial memory in adulthood, as well as on cognitive dysfunction pharmacologically induced in adulthood via subchronic PCP administration. Cognitive flexibility and non-spatial memory were assessed during adulthood in the attentional set-shifting and novel object recognition tasks, respectively. In addition, cognitive impulsivity and delay-based reward processing were also assessed in these animals in a delay discounting operant task. This work led to a first author manuscript published in Pharmacology, Biochemistry & Behavior, as well as a Master of Science degree in Psychobiology.  Influence of gonadal hormones on cognitive flexibility and non-spatial memory in female rats. Using the attentional set-shifting and novel object recognition behavioral tasks described above, cognitive flexibility and non-spatial learning were assessed in adult ovariectomized female rats supplemented or not with estradiol. Estradiol or sesame oil were administered at different points throughout the training and testing procedures in both tasks to determine the stage of learning and performance, if any, influenced by gonadal estradiol at the time of testing. This work provided a foundational knowledge that facilitated the transition to my work in Dr. Kabbaj’s lab focused on sex differences in behavior relevant to psychiatric disorders.

Graduate Work, Kabbaj Lab: 2012-present

 Effects of testosterone and estrogen on anxiolytic and antidepressant-like behavior and hippocampal gene expression in gonadectomized male rats. Testosterone has been shown to have protective effects against anxiety and depressive(-like) behavior in both humans and rodents, which may be due to either its androgenic or estrogenic metabolites, or both. To this end, we compared the effects of testosterone (T) and its aromatase-derived metabolite, estradiol (E), both systemically and locally within the dentate gyrus of gonadectomized (GDX) male rats. Microarray analysis of mRNA within the dorsal hippocampus (dHPC) of T- and E- supplemented GDX male rats revealed substantial overlap in regulation of the expression of MAPK pathway and synaptic plasticity-related genes, supporting the high similarity of protective effects of both hormones against the development of depressive-like behavior in GDX male rats. To determine whether aromatized E within this brain region was functionally relevant to its antidepressant-like properties, we blocked local conversion of T to E within the

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dHPC of GDX male rats supplemented or not with T by continuous infusion of the aromatase inhibitor, fadrozole, via osmotic minipumps attached by catheter to bilateral cannula stereotaxically implanted into the dentate gyrus. Anxiety- and depressive-like behavior was then assessed. This work led to a co-first author manuscript published in Biological Psychiatry.  Modulation of hedonic sensitivity to low-dose ketamine by gonadal hormones and sex in male and female rats. We recently reported a greater sensitivity of female rats to the rapid antidepressant- like effects of ketamine (KET) when compared to males, and that ovarian- derived estradiol (E2) and progesterone (P4) were essential for this response. However, it remained unclear to what extent testosterone (T) may also contribute to this sex difference, and whether the duration of response to ketamine was also modulated in a sex- and hormone- dependent manner. A mixed between-/within- subjects approach was implemented to tease apart hormonal contributions to ketamine’s sex- dependent response profile in rats. Using a continuous-access sucrose preference paradigm, we systematically investigated the influence of T, E2 and P4 on initiation and maintenance of hedonic response to low-dose ketamine in gonadectomized and intact male and female rats receiving identical physiologically-relevant hormone treatments. Hippocampal BDNF levels and activation of downstream signaling effectors were subsequently investigated. A first-author manuscript covering this work was recently published in Scientific Reports.  Phosphoproteomic analysis of rapid sex- and hormone-dependent changes in signaling pathway activation and protein levels in the hippocampus following low-dose ketamine administration in rats. Given the profound role of gonadal hormones in mediating the greater antidepressant- and pro-hedonic-like effects of low-dose ketamine in female, compared to male rats, a phosphoproteomics approach was used to identify ketamine-induced changes in signaling pathway activation and protein abundance within the hippocampus of intact male rats and female rats in either diestrus (low E2P4) or proestrus (high E2P4) stages of their estrous cycles. Tissue was collected 30 minutes following an acute low dose of ketamine that is behaviorally ineffective in male rats, after which dHPC total and phosphopeptide-enriched protein lysate were extracted and submitted for high- resolution mass spectrometric analysis. Data were analyzed at the Keck Institute at Yale University. Downstream ontology and pathway enrichment analyses were performed to provide greater insight into potential

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translational and post-translational processes in the HPC contributing to sex- and hormone- dependent modulation of ketamine’s therapeutic effects. This work is complete and in preparation for submission.  Sex differences in the pharmacokinetics of low-dose ketamine in plasma and brain of male and female rats. In addition, sex differences in the pharmacokinetics of ketamine may also contribute to the differential behavioral sensitivity of male and female rats to low doses of this drug. To address this possibility, concentrations of ketamine and its metabolites extracted from the plasma and brain (mPFC, HPC) of male, diestrus (low E2P4) and proestrus (high E2P4) female rats 5-180 minutes following a single injection of low-dose of ketamine were determined via mass spectrometric analysis. This work is complete and in preparation for submission.

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