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SEX DIFFERENCES IN THE BEHAVIORAL AND NEUROMOLECULAR EFFECTS OF

THE RAPID-ACTING ANTIDEPRESSANT DRUG KETAMINE IN MICE

Dissertation

Submitted to

The College of Arts and Sciences of the

UNIVERSITY OF DAYTON

In Partial Fulfillment of the Requirements for

The Degree of

Doctor of Philosophy in Biology

Connor Francis Thelen

UNIVERSITY OF DAYTON

Dayton, OH

December 2019

SEX DIFFERENCES IN THE BEHAVIORAL AND NEUROMOLECULAR EFFECTS OF

THE RAPID-ACTING ANTIDEPRESSANT DRUG KETAMINE IN MICE

Name: Thelen, Connor Francis

APPROVED BY:

______Pothitos M. Pitychoutis, Ph.D. Faculty Advisor

______Mark Nielsen, Ph.D. Committee member

______Madhuri Kango-Singh, Ph.D. Committee member

______Renu Sah, Ph.D. Committee member

______Amit Singh, Ph.D. Committee member

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ABSTRACT

SEX DIFFERENCES IN THE BEHAVIORAL AND NEUROMOLECULAR EFFECTS OF

THE RAPID-ACTING ANTIDEPRESSANT DRUG KETAMINE IN MICE

Name: Thelen, Connor Francis University of Dayton

Advisor: Dr. Pothitos M. Pitychoutis

Over 350 million people currently suffer from Major Depressive Disorder (MDD).

This debilitating neuropsychiatric disease is the greatest cause of disability worldwide, and available pharmacotherapeutic treatment options are largely ineffective in many depressed patients. Currently, conventional antidepressants (e.g., selective serotonin reuptake inhibitors; SSRIs and tricyclic antidepressants; TCAs) are only partially effective in managing depressive symptoms in MDD patients. Additionally, currently marketed antidepressant drugs require weeks to alleviate symptomology in depressed patients.

Notably, women are twice as likely to be diagnosed with MDD as compared to men, yet until recently most clinical and preclinical studies in this field were conducted in the male sex. Ketamine, a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist is the first rapid-acting antidepressant agent discovered that has the unique ability to relieve depressive symptoms within hours in both MDD patients and in animal models of depression. While earlier studies have determined that this drug has great promise for treating refractory depression, little is still known as to ketamine’s putative sex- differentiated effects and about its long-term safety.

In the context of the current dissertation we explored the sex-differentiated behavioral and neuromolecular effects of antidepressant-relevant doses of ketamine following acute and repeated drug treatment in male and female mice. Specifically, we showed that behavioral responsiveness to ketamine in female mice is not accompanied by the neurochemical and synaptogenic effects that are typically observed in the male brain, and we further exposed a brain region that may be implicated in the sex-differentiated response to this drug. Moreover, we provided the first evidence that repeated ketamine dosing induced beneficial antidepressant-like effects in male mice but was associated with adverse anxiety-like and depressive-like effects in females. Taken together, the research findings pertaining to the current dissertation provide novel insights into the sex-dependent effects of the rapid-acting antidepressant drug ketamine and also suggests that the heightened sensitivity of females to this drug may put them at risk for sex-specific adverse effects following chronic treatment.

Dedicated to my parents, lab-mates, and my mentor Dr. Pothitos Pitychoutis, all of whom

helped support and guide my research

ACKNOWLEDGEMENTS

My special thanks are in order to my mentor Dr. Pothitos “Takis” Pitychoutis, for providing the time, equipment, and funding necessary to complete the work contained herein, and for patiently providing guidance during the construction of this dissertation.

Takis has been the embodiment of what it means to be a graduate advisor. His ability to celebrate our successes and provide steadfast support during periods of difficulty was the driving force that pushed me to completing my research and schooling at the University of

Dayton. My appreciation also goes out to the other members of my graduate advisory committee who, alongside Dr. Pitychoutis, lent their knowledge and advice to help me become a better scientist and to conduct ethical and meaningful research that reaches beyond the bounds of our university; in this context I would like to thank, Dr. Amit Singh,

Dr. Madhuri Kango-Singh, Dr. Mark Nielsen, Dr. Renu Sah, and the late Dr. Panagiotis

Tsonis. Moreover, I would like to thank the current and the past Chairs of the Biology

Department, Dr. Karolyn Hansen and Dr. Mark Nielsen for their meaningful support throughout my Ph.D. candidacy.

I would also like to thank the undergraduate research assistants whose daily commitment to this research was invaluable. Special thanks goes to: Emily Flaherty, who helped with a variety of projects, including the quantification and analysis of dendritic spine densities as well as the in vivo microdialysis experiments; Joey Saurine for his help in Golgi staining; Jonathon Sens and Sara Mohamed for their help in western blotting and protein analysis, as well as Anthony Franceschelli, M.Sc., whose work I continued, for his support in the behavioral experiments. Moreover, I would like to thank all the vivarium

vii assistants and vivarium director, Dr. Jeffrey Kavanaugh, who maintained a clean and orderly space to conduct research.

I would also like to thank the other past and present members of the Pitychoutis lab for their help completing the long list of tasks needed to maintain a highly productive research environment. Graduate students Aikaterini Britzolaki and Benjamin Klocke, as well as all the undergraduate students I had the pleasure to mentor in the lab, including:

Patrick Flaherty, Claire Cronin, Madison Schulze, John Coffey, Sean Koeller, Joseph

Mauch, Jake Michalakes, Allison Noe, Jonathan Bode, Nicklaus Halloy, Sam Herchick,

Evan Birmingham, Katie Fasoli, Anna Schaffstein, Radhika Pandit and Eric Schneider as they all played integral roles in helping me complete the research presented in this dissertation. This research could not have been conducted without the financial support provided by the University of Dayton Graduate School, the University of Dayton honors program, and awarded grants and fellowships.

My sincere thanks also go to Cathy Wolfe, Janice Bertke, Rita McGinn, Casey

Hanley, Grover Allen, and Susan Trainum for all their help and support, as well as to the

Graduate Directors of the Biology Department, Dr. Madhuri Kango-Singh and Dr. Amit

Singh.

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TABLE OF CONTENTS

ABSTRACT…………………………………………………………………………...…iv

DEDICATION……………………………………………………………………………vi

ACKNOWLEDGEMENTS…………………………………………………………..….vii

LIST OF FIGURES………………………………………………………………..……xiii

LIST OF TABLES……………………………………………………………………….xv

LIST OF ABBREVIATIONS AND NOTATIONS……………..……………..…….…xvi

CHAPTER 1 ON THE SEX-DIFFERENTIATED EFFECTS OF THE

RAPID-ACTING ANTIDEPRESSANT DRUG KETAMINE: AN INTRODUCTION…1

1.1 A Short History of Antidepressant Drug Discovery: from Imipramine to

Ketamine…………………………………………………………………………..1

1.2 Antidepressant Effects of Ketamine: Clinical Evidence………………………4

1.3 Ketamine: The Prototype Rapid-acting Antidepressant Drug with a Novel

Synaptogenic Mechanism of Action………………………………………………7

1.4 Sex Differences in Depression and Response to Conventional

Monoaminergic Antidepressants…………………………………………….…..12

1.5 Sex Differences in the Antidepressant Effects of Ketamine:

Insights from the Clinic and Animal Models……………………………...……..15

1.6 Focus of the Dissertation…………………………………………………….18

1.7 Figures………………………………………………………………………..20

CHAPTER 2 SEX DIFFERENCES IN THE TEMPORAL NEUROMOLECULAR

AND SYNAPTOGENIC EFFECTS OF THE RAPID ACTING ANTIDEPRESSANT

DRUG KETAMINE IN THE MOUSE BRAIN…………………………………………22

2.1 Introduction…………………………………………………………………..22

2.2 Experimental Procedures…………………………………………………….26

2.2.1 Animals…………………………………………………………….26

2.2.2 Experimental design and drug treatments………………………….26

2.2.3 Experiment #1: sex differences in ketamine-induced glutamate

release in the mPFC of male and female mice…………………………...27

2.2.4 Experiment #2: sex differences in the synaptic molecular effects of

ketamine………………………………………………………………….28

2.2.5 Experiment #3: sex differences in the time course of spine

formation…………………………………………………………………28

2.2.6 In vivo brain microdialysis………………………………………....29

2.2.7 Glutamate analysis with high-performance liquid chromatography

(HPLC)…………………………………………………………………...30

2.2.8 Synaptoneurosome preparation and immunoblotting……………...30

2.2.9 Golgi staining and spine analysis…………………………………..31

2.2.10 Statistical analysis………………………………………………...32

2.3 Results………………………………………………………………………..33

2.3.1 Experiment #1……………………………………………………...33

2.3.2 Experiment #2……………………………………………………...33

2.3.3 Experiment #3……………………………………………………...35

2.4 Discussion……………………………………………………………………36

2.5 Figures………………………………………………………………………..45

CHAPTER 3 REPEATED KETAMINE TREATMENT INDUCES SEX-SPECIFIC

BEHAVIORAL AND NEUROCHEMICAL EFFECTS IN MICE……………………..51

3.1 Introduction…………………………………………………………………..51

3.2 Materials and Methods……………………………………………………….54

3.2.1 Animals…………………………………………………………….54

3.2.2 Experimental design and drug treatments………………………….55

3.2.3 Spontaneous locomotor activity in the open field test (OFT)…...…56

3.2.4 Forced swim test (FST)…………………………………………….56

3.2.5 Neurochemical analysis……………………………………………57

3.2.6 Synaptoneurosome preparation and western blotting……………...58

3.2.7 Statistical analysis………………………………………………….59

3.3 Results………………………………………………………………………..59

3.3.1 Repeated ketamine treatment is beneficial for males but induces

anxiety-like and depressive-like effects in females……………………...59

3.3.2 Repeated ketamine treatment affects hippocampal

neurochemistry in a sex-dependent manner………………………..…….61

3.3.3 Repeated ketamine treatment affects the expression of synaptic

proteins only in male mice……………………………………………….61

3.3.4 Neurochemical effects correlate with synaptic protein

alterations in the male HIPP………………………………………..……62

3.4 Discussion…………………………………………………………………....63

3.5 Conclusions…………………………………………………………………..68

3.6 Figures and Tables………………………………………...…………...…….69

BIBLIOGRAPHY………………………………………………………………………..75

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LIST OF FIGURES

Figure 1.1 Timeline of antidepressant drug discovery……………………………..……20

Figure 1.2 Ketamine’s mechanism of action…………………………………….………21

Figure 2.1 Sex differences in response to acute ketamine treatment in the forced swim test……………………………………………………………………….…45

Figure 2.2 Experimental design to understand the temporal neuromolecular alterations following ketamine treatment………………………………………...………46

Figure 2.3 Ketamine induced glutamate release within the mPFC of male and female mice…………………………………………………………………….……47

Figure 2.4 Ketamine induced sex-differentiated synaptic molecular effects in mouse prefrontocortical synaptoneurosomes……………………………………………………48

Figure 2.5 Ketamine-induced synaptic molecular effects in male and female hippocampal synaptoneursomes…………………………………..……………..49

Figure 2.6 Ketamine induces sex-dependent synaptogenic effects in stress-naïve mice...50

Figure 3.1 Experimental timeline of repeated ketamine treatment and sex-specific anxiogenic effects of ketamine in the open field………………………………………...71

Figure 3.2 Female-specific depressogenic effects of repeated ketamine treatment in the forced swim test……………………………………………………..….72

Figure 3.3 Sex-specific alterations in neurotransmission and synaptic protein concentration following repeated ketamine treatment…………………………………...73

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Figure 3.4 Correlative indices between synapsin-1 and glutamate as well as syntaxin and 5HIAA levels in the hippocampus following repeated ketamine treatment…………………………………………………………………………………74

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LIST OF TABLES

Table 3.1 Previous studies assessing the antidepressant-like behavioral effects of repeated ketamine treatment……………………………………………….….69

Table 3.2 Effects of repeated ketamine treatment on neurotransmitter concentration and synaptic protein density in the PFC………………………………………….……...70

LIST OF ABBREVIATIONS AND NOTATIONS

5-HIAA 5-hydroxy-indoleacetic acid

5-HT Serotonin (5-hydroxytryptamine) aCSF Artificial cerebrospinal fluid

ADRs Adverse drug reactions

AkT Protein kinase B

AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor

ANOVA Analysis of variance

BDNF Brain derived neurotrophic factor

CA Cornu ammonis

CBD Cannabidiol

CES-D Center for Epidemiological Studies Depression Scale

CMS Chronic mild stress

DG Dentate gyrus

DRN Dorsal raphe nucleus

EAA Excitatory amino acid

FDA Food and Drug Administration

FST Forced swim test

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GABA γ-aminobutyric acid

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

GluR1 Glutamate receptor 1

GluR2 Glutamate receptor 2

HIPP Hippocampus

HNK Hydroxynorketamine

HPLC High-performance liquid chromatography i.p. Intraperitoneally

IS Isolation stress mAchR muscarinic acetylcholine receptor

MDD Major depressive disorder mPFC Medial prefrontal cortex mTORC1 Mammalian target of rapamycin complex 1

NMDA N-methyl-d-aspartate

NMDAR N-methyl-D-aspartate receptor

NSFT Novelty-suppressed feeding test

OFT Open field test

PFC Prefrontal cortex

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RSC Retrosplenial cortex

SNARE Soluble N-ethylmaleimide-sensitive factor attachment protein receptor

SSI Scale for Suicidal Ideation

SSRI Selective serotonin reuptake inhibitor

SYX Syntaxin-I

TCA Tricyclic antidepressant

TRD Treatment-resistant depression

TrkB Tyrosine receptor kinase B

VEH Vehicle

WHO World Health Organization

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CHAPTER 1

ON THE SEX-DIFFERENTIATED EFFECTS OF THE RAPID-ACTING

ANTIDEPRESSANT DRUG KETAMINE: AN INTRODUCTION

1.1. A Short History of Antidepressant Drug Discovery: from Imipramine to Ketamine

Major depression is a devastating stress-related neuropsychiatric disease that presents with depressed mood, anhedonia, feelings of guilt or low self-esteem, disturbed sleep or appetite, cognitive deficits, and at its worst, can lead to suicide. According to the World

Health Organization (WHO), major depression affects 350 million people around the globe and by 2030 is expected to be the leading cause of disease burden worldwide (Collins et al., 2011; WHO, Global Burden, 2015). Major depression is also is a very costly brain disorder. In the United States (US) alone, the economic burden of this disorder has increased by 21.5% between 2005 and 2010 (from $173.2 to $210.5 billion inflation- adjusted dollars); approximately 45% of this cost is attributed to direct medical costs, 5% to suicide-related mortality costs and 50% to workplace indirect costs (Greenburg et al.,

2015). Not surprisingly, major depression is the leading cause of disability in Americans aged 15-44 years, resulting in approximately 400 million disability days annually

(Merikangas et al., 2007).

Physicians attempting to treat this mental disorder are largely prescribing partially effective antidepressant drugs whose therapeutic mechanism of action typically depends on gradual changes in the brain’s monoaminergic systems (i.e., serotonergic, noradrenergic and dopaminergic). The 1950’s were marked by the serendipitous discovery of imipramine,

1 the first tricyclic antidepressant (TCA), which was introduced in the clinic shortly afterwards, while the first selective serotonin reuptake inhibitor (SSRI) drug, fluoxetine

(i.e., Prozac), was marketed in the United States back in 1987 (Fig. 1.1). Fluoxetine was effective in managing depressive symptoms, was better tolerated by depressed patients, and lacked many of the aversive TCA side-effects. Following its release fluoxetine rapidly became a “blockbuster” drug; Prozac was the most widely prescribed drug in North

America by 1990, and the second best-selling drug in the world (Lopez-Munoz and Alamo,

2009; Pereira and Hiroaki-Sato, 2018). Besides fluoxetine, four additional SSRIs were also released in the market; citalopram, fluvoxamine, paroxetine and sertraline. Widespread and systematic clinical use of TCAs and SSRIs during the 1990’s indicated that only 60% of depressed patients will initially respond to the first wave of antidepressant drug therapy and from this percentage only half will eventually achieve remission (Al-Harabi, 2012).

When an antidepressant drug proves ineffective in managing depressive symptoms, doctors suggest switching or adding another SSRI drug to the therapeutic regimen. After going through different combination therapies there are still 30% of depressed patients who will not respond to the available antidepressant treatments and are classified as treatment- resistant depressed (TRD) patients. To make matters worse, these “conventional” antidepressant drugs require time to induce their therapeutic effect as it may take weeks or months to improve depressive symptoms. Not surprisingly, this time-lag between the onset of antidepressant drug therapy and alleviation of depressive symptoms is associated with increased “drop-out” rates in the clinic.

Following the release of SSRIs, several monoaminergic antidepressants were developed in the 1990s, including: mirtazapine (α2 adrenergic receptor antagonist),

2 venlafaxine (selective noradrenaline and serotonin inhibitor) and reboxetine (selective noradrenaline reuptake inhibitor). These drugs were not considered to be a huge advancement in the field as they acted on the same monoaminergic systems targeted by

TCAs and SSRIs and did not offer a significantly improved efficacy (Millan et al., 2006;

Pereira and Hiroaki-Sato, 2018).

The latest advancement in the field of antidepressant drug discovery came about in

2000 with the intriguing clinical finding that ketamine, an anesthetic already on the market, may induce rapid and long-lasting antidepressant effects. First discovered in 1962, ketamine was approved for use in humans in 1970 as a dissociative anesthetic. As an induction agent, ketamine was widely used for general sedation due to its analgesic properties and relative safety, as it has a high overdose threshold and does not depress the circulatory or respiratory systems (Adams, 1997; Wong, 2014). Unfortunately, use of ketamine for pain management and surgical procedures became less common for humans in the following years as the “psychedelic” effects induced by high doses of ketamine (i.e., dysphoria, hallucinations, and general alterations in sensory systems) were associated with an elevated abuse potential, and it became common for individuals to use the drug recreationally. While still used in clinical procedures, ketamine was designated a class III substance in 1999 under the US controlled substances act and therefore became much more common for use in veterinary medicine (Hartsfield S.M., 1992). In more recent years, focus on ketamine has shifted from its use as an anesthetic to its potential use as a prototype rapid-acting antidepressant agent mostly following the clinical study by Berman et al.,

(2000) showing that ketamine induced a rapid antidepressant response in TRD patients.

What’s more, due to ketamine’s rapid alleviation of depressive symptomology great

3 attention has been given to ketamine for its potential in treating patients with ongoing suicidal ideation (DiazGranados, 2010; Price, 2009).

1.2. Antidepressant Effects of Ketamine: Clinical Evidence

The earliest indication for ketamine’s putative antidepressant-like actions in humans was reported by Khorramzadeh and Lotfy (1973). The authors showed that intravenous

(i.v.) infusion of ketamine at the subanesthetic doses of 0.2–1.0 mg/kg (i.v. bolus) in a cohort of 100 psychiatric inpatients resulted in facilitation of psychotherapy and emotional discharge, yet the specific depression symptoms that were alleviated were not properly addressed (Zanos et al., 2018). Moreover, around the same time, ketamine was found to exert behavioral effects similar to those observed following administration of conventional antidepressant drugs (e.g., TCAs) in preclinical studies conducted in rodents (Sofia and

Harakal, 1975). Excitingly, a recent clinical study in 2000 revolutionized the field of antidepressant drug discovery. In a seminal placebo-controlled study, Berman et al. (2000) showed that a single 40 min intravenous infusion of 0.5mg/kg of ketamine hydrochloride significantly reduced the mean depression scores of 7 depressed individuals as early as 3 hours post-injection; the antidepressant effect lasted up to 72 hours, as measured using the clinician-administered Hamilton Depression Rating Scale (HDRS). This first study was further replicated by two larger double-blind placebo controlled clinical studies by Zarate et al. (2006) and Murrough et al. (2013). Zarate et al., (2006) treated 18 patients (12 women,

6 men) suffering from TRD, and had thus failed to respond to at least two prior classical antidepressant drugs, using a 0.5mg/kg dose of ketamine hydrochloride. Remarkably, 71%

4 of individuals saw a positive treatment response manifested as early as 2 hours and lasting as long as 7 days (Zarate et al., 2006). In a much larger study, Murrough et al. (2013) treated 72 TRD patients (37 women, 35 men) using the same 0.5mg/kg dose of ketamine; in that study ketamine significantly reduced depression scores from 24 hours up to 7 days, as assessed using the clinician-administered Montgomery-Asberg Depression Rating Scale

(MADRS). Notably, in that study Murrough et al., (2013) also used the drug midazolam as a psychoactive placebo and reported a higher response rate for TRD patients treated with ketamine, as compared to those treated with midazolam (64% versus 28%). Of note, ketamine also appears to exert robust anti-suicidal effects. Depressed patients treated with a single i.v. dose of 0.5mg/kg ketamine showed a rapid resolution in suicidal ideation as early as 40 minutes post-injection as measured using the Scale for Suicide Ideation (SSI),

MADRS, and HDRS (DiazGranados, 2010; Price, 2009). These effects were sustained for at least 24 hours and were found to last up to 12 days with repeated dosing (i.e., every 2-3 days) (Price 2009). Overall, these clinical findings strongly indicate that the non- competitive N-methyl-D-aspartate receptor (NMDAR) antagonist ketamine exerts rapid and long-lasting antidepressant effects in patients previously resistant to conventional antidepressant treatments.

Ketamine is a racemic mixture consisting of equal portions of its two enantiomers (S)- and (R)-ketamine that was introduced in the clinic as an anesthetic agent in 1970. A randomized controlled clinical trial showed that intranasally-administered (S)-ketamine

(i.e., 28–84 mg, twice a week for a total of 2 weeks) induced dose-dependent antidepressant effects in TRD patients ) as adjuvant to oral antidepressant drug therapy (Daly et al., 2017).

Moreover, intranasal (S)-ketamine administration was also found to decrease suicidal

5 ideation in depressed patients (Canuso et al., 2018). Concerted efforts by basic and clinical scientists working in the depression and ketamine fields led to the US Food and Drug

Administration (FDA) granting “fast-track” and “breakthrough therapy” designations to this drug in order to take advantage of its rapid-acting antidepressant and antisuicidal actions. Notably, by March 5th, 2019 the intranasally administered drug Esketamine

(SpravatoTM) was released by Janssen Pharmaceuticals, Inc. to the U.S. market for the treatment of TRD in adults along with an antidepressant taken by mouth. Esketamine is synthesized using the (S)-ketamine enantiomer, which has been shown to have greater binding affinity to NMDAR, as compared to the (R)-ketamine enantiomer and produces significantly fewer adverse effects (i.e., psychomimetic effects, drowsiness, and cognitive impairment) (Canuso et al., 2018). Previously, ketamine has almost exclusively been administered intravenously due to its low bioavailability when administered orally. To create a drug that could be safely self-administered outside the clinic yet still retain its potency, Esketamine was developed for intranasal administration, which has been shown to have significantly greater bioavailability as compared to Esketamine taken orally

(Covvey et al., 2012; Malhi et al., 2016; Thomas et al., 2016). As of June 2019, 32 clinical trials using Esketamine have either concluded or are still in progress (Bahr et al., 2019).

The long-term phase III clinical trials have shown that Esketamine successfully decreases suicidal risk and symptoms of depression as evidenced by reduced MADRS depression scores (Morrison et al., 2018; Popova et al., 2018; Ochs-Ross et al., 2018; Daly et al., 2018;

Wais et al., 2018; Starr et al., 2018). Despite these promising findings, treatment efficacy and possible long-term adverse side effects after one year have yet to be determined.

1.3. Ketamine: The Prototype Rapid-acting Antidepressant Drug with a Novel Synaptogenic Mechanism of Action

Clinical and preclinical studies in patients suffering from major depression and in rodent models of depression, have consistently reported that brain derived neurotrophic factor (BDNF) levels are significantly decreased in the prefrontal cortex (PFC), hippocampus (HIPP), and in the blood (Bocchio-Chiavetto, 2010; Dwivedi, 2009).

Moreover, post-mortem studies have found that the mammalian target of rapamycin complex 1 (mTORC1) expression and activity are reduced in the PFC of depressed individuals, and that in the same brain region the stress response protein REDD1, a negative regulator of mTORC1 is increased (Jernigan et al., 2011; Ota et al., 2014). Studies conducted in rodents have shown that chronic stress also decreases mTORC1 signaling and that overexpression of REDD1 leads to a loss of synapses within the PFC (Ota et al., 2014;

Li et al., 2011). Based on this information it can be theorized that a selective increase in

BDNF or mTORC1 activation within the PFC or HIPP may lessen the negative effects of stress. Indeed, classic antidepressants such as SSRIs have been shown to alleviate stress- induced decreases in BDNF levels after chronic treatment, although there are also other studies which have shown no impact on BDNF following antidepressant treatment (Zhou et al., 2017; Arumugam et al., 2017). Electroconvulsive therapy (ECT) has also been shown to increase BDNF expression in the PFC and HIPP which can induce antidepressant effects in both depressed patients and in animal models of depression (Dukart et al., 2014; Abbott et al., 2014). To date, ECT is widely considered the best way to treat patients suffering from severe depression who show poor response to SSRIs and TCAs as it enhances synaptic connectivity by alternate means. Unfortunately, ECT also has severe side effects

7 such as retrograde amnesia due to the broad depolarization of large areas of the brain

(Semkovska and McLoughlin 2010). The finding that the recently spotlighted drug ketamine swiftly reduces depressive symptoms and simultaneously increases BDNF and mTORC1 signaling within selective areas of the brain has given hope for the development of the first relatively safe rapid-acting antidepressant that acts without the need for slow changes in monoaminergic neurotransmission.

Since 2000, neuroscientists have strived to decode ketamine’s rapid-acting mechanism of antidepressant action in order to develop novel molecules that would lack ketamine’s adverse psychedelic effects. The antidepressant properties of ketamine have been shown to rely heavily on post-synaptic release of BDNF and downstream activation of the mTORC1 pathway (Li 2010, Miller 2014, Paul 2014, Zhou 2014). Specifically, it is thought that the secondary effects of ketamine’s excitatory influence on the mTORC1 are responsible for the drug’s long-lasting antidepressant effects. Brief, but robust activation of the mTORC1 pathway and the translation of synaptic proteins’ mRNAs leads to an increase in dendritic spine formation and connectivity in brain regions implicated in the pathophysiology of major depression and in the neurobiology of antidepressant response, with most prominent being the mPFC and the HIPP (Li 2011, Gass 2014). This rapid induction of synaptogenesis and the amelioration of depression and/or stress-induced neuronal atrophy is thought to be the unifying characteristic of all new rapid-acting antidepressant pharmacotherapies.

According to the “disinhibition hypothesis” that has gained ground, ketamine, a non- competitive NMDAR antagonist, binds to γ-aminobutyric acid (GABA)-ergic interneurons that regulate localized release of glutamate in the mPFC (Li et al., 2010). Disinhibition of

8 glutamatergic neurotransmission within the mPFC leads to a “glutamate burst” and a subsequent activation of post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), as shown in Fig. 1.2 (Maeng et al., 2008; Li et al., 2010).

Depolarization then activates voltage dependent Ca2+ channels leading to a release of

BDNF, which then binds to tyrosine kinase B (TrkB) receptor and initiates the phosphorylation of mTORC1 by protein kinase B (AkT) (Li et al., 2010; Paul et al., 2014;

Zhou et al., 2014). Phosphorylated mTORC1 stimulates p70S6K which begins the synthesis of synaptic proteins and additionally represses phosphorylation of eukaryotic elongation factor 2 (eEF2), a protein synthesis inhibitor (Monteggia et al., 2013). This activation of the mTORC1 signaling pathway by ketamine happens rapidly (within 30 minutes) and is transient, with elevated phosphorylation of mTOR returning to basal levels within hours, well before ketamine’s antidepressant effects subside (Li et al., 2010). It has been shown that the synaptic proteins GluR1, GluR2, postsynaptic density protein-95

(PSD95), and synapsin 1, among others, are increased and sustained following just a single dose of ketamine (Li et al., 2010; Duman et al., 2012). It is thought that the increased translation of these synaptic growth and structural proteins leads to the strengthening and the formation of new dendritic spines which persist for a week or longer in rodent animal models (Abdallah et al., 2015; Zanos and Gould, 2018). Again, this increase in the density of dendritic spines within the mPFC is most-likely responsible for the sustained antidepressant action of ketamine as it has been shown that degradation of newly formed spines coincides with a relapse in antidepressant response (Duman, 2014). In addition to advancing the release of BDNF and initiating the local translation of synaptic proteins within the mPFC, it has also been shown that a single dose of ketamine increases BDNF

9 concentration and decreases in the phosphorylation of eEF2 within the HIPP as well (Takei et al., 2004; Zhou et al., 2014; Garcia et al., 2008).

While there is evidence that ketamine has a greater affinity for NMDAR on GABAergic interneurons, which explains the aforementioned “disinhibition hypothesis,” new studies have shown that direct activation of NMDARs and AMPARs by ketamine and its metabolites respectively may also be implicated in antidepressant response (Zanos et al.,

2016). In a groundbreaking study by Zanos et al., (2016), an active metabolite of ketamine,

(2R,6R)-hydroxynorketamine (HNK) was shown to induce its rapid antidepressant effects by directly activating post-synaptic AMPAR leading to the stimulation of the same synaptic molecular growth pathways as the parent molecule. It is thought that this metabolite is at least partially responsible for the cumulative activation of the mTORC1 pathway and therefore antidepressant action (Zanos et al. 2016).

Following the revelation that ketamine could rapidly improve depressive symptomology at a time where current medications required weeks to induce their effect, a scramble to find other drugs that similarly targeted glutamatergic systems as well as synaptogenic growth pathways took place. To date, GluN2B antagonists, (2R,6R)-HNK,

GABAA negative allosteric modulators, Glyx-13 (Rapastinel), scopolamine, cannabidiol, and psychedelics (i.e. LSD, DMT, MDMA, DOI) have all been shown to induce antidepressant responses similar to those seen with ketamine. Selective GluN2B subunit antagonists, such as ifenprodil, have been shown to enhance the antidepressant efficacy of

TCAs and SSRIs (Poleszak, 2014; Raybuck, 2017). These drugs, like ketamine, inhibit

NMDARs; however, the antidepressant efficacy of GluN2B subunit antagonists are yet to be elucidated. While some studies have shown robust antidepressant effects in rodents, a

2014 study found that GluN2A antagonists may be better suited for treating refractory depression (Lima-Ojeda, 2013; Jiménez-Sánchez et al., 2014). (2R,6R)-HNK is a ketamine metabolite that does not inhibit NMDAR directly but also leads to a rapid-antidepressant effect in animal models (Morris et al., 2017; Suzuki et al., 2017; Zanos et al., 2016). It has been shown that AMPAR activation is required for the rapid induction of antidepressant response from all “rapid-acting antidepressants” as NBQX, an AMPAR inhibitor, blocks the therapeutic effects of all the aforementioned treatments (Zanos et al., 2016, Maeng et al., 2008; Burgdorf et al., 2013; Voleti et al., 2013; Zanos et al., 2017; Koike et al., 2014;

Zhou et al., 2014; Martin et al., 2017; Karasawa et al., 2005). Studies showing an increase in glutamatergic AMPAR activity following (2R,6R)-HNK administration provide support for the theory that BDNF release and synaptogenesis are triggered by direct AMPAR stimulation by this metabolite (Zanos et al., 2016). Negative allosteric modulators of the

α5 subunit of GABAA receptors reduce GABA-ergic regulation of glutamate transmission.

These drugs can cause a “glutamate burst” just like ketamine in the mPFC and HIPP that leads to antidepressant-like behavioral effects in rodents (Sur et al., 1999; Fischell et al.,

2015). Glyx-13 (Rapastinel), a drug in phase III clinical trials, is a partial NMDAR agonist acting at the glycine-binding site. By activating post synaptic NMDAR in the mPFC, eEF2 phosphorylation by eEF2K is reduced. Consequently, translation of BDNF is increased, activation of the TrkB receptor and the mTORC1 pathway takes place, and finally synaptogenesis is initiated (Opal et al, 2014; Kato et al., 2017; Liu et al., 2017).

Interestingly, it appears that AMPAR activation is still necessary for Glyx-13 initiated antidepressant-like effects (Burgdorf et al., 2013). Scopolamine is a muscarinic acetylcholine receptor (mAchR) antagonist that when bound to type 1 or type 2 mAchRs

11 on GABA-ergic interneurons leads to a similar disinhibition of glutamate release in mPFC pyramidal neurons as ketamine (Voleti et al., 2013). Silencing of the GABAergic regulatory system and the release of glutamate by scopolamine leads to stimulation of the mTORC1 pathway, the release of BDNF, the induction of synaptogenesis in the mPFC, and a rapid antidepressant-like effect (Navarria et al., 2015). While less is known about how cannabidiol (CBD) exerts its antidepressant effects, it has been shown that intraperitoneal (i.p.) administration of this drug leads to a rapid antidepressant response in rodents (30 min), which is followed by increased BDNF expression and synaptogenesis in the mPFC (Sales et al., 2018; Shbiro et al., 2018; Shoval et al., 2016); interestingly, blocking the activity of the mTOR pathway eliminates the rapid antidepressant actions of

CBD, suggesting a similar mechanism of action to that of ketamine (Sales et al., 2018).

1.4. Sex Differences in Depression and Response to Conventional Monoaminergic Antidepressants

The advancement of personalized medicine has highlighted sex, genetics, hormonal activity, age, body composition, metabolic activity, and a myriad of other factors that should be taken into consideration when developing and prescribing drugs for the greatest therapeutic effect. The treatment of major depressive disorder has been one of the focuses during this medical revolution. While women are diagnosed with major depression at over twice the rate of men, up until recently preclinical research has almost exclusively used male subjects (Beery and Zucker 2011). Up until 1993 women were not allowed to participate in clinical trials, thus much of our knowledge on disease processes or the mechanisms of action of the different drugs depend on studies conducted in the male sex

(Lee, 2018). Remarkably, the National Institutes of Health (NIH) recently introduced a policy on the consideration of sex as a biological variable in NIH-funded research. Thus, all NIH-funded researchers are expected to study both vertebrate animals and humans of both sexes, where possible, in order to improve our understanding of physiological and pathophysiological processes in both men and women

(https://orwh.od.nih.gov/resources/pdf/NOT-OD-15-102_Guidance.pdf) (Clayton, 2014).

Interestingly, treatment and diagnosis of major depressive symptoms is universal, despite evidence suggesting that males and females respond differently to current medications (i.e. men tend to respond more favorably to TCA’s, whereas women report greater treatment success taking SSRI’s) (Kornstein et al. 2000, Khan et al. 2005). While little is known as to the exact reason why males and females present with different rates and symptoms of depression, it is thought that hormonal activity and genetic variability play the largest role. Other than the innate physiological differences between the sexes, it is worth noting that males and females respond differently to stressful life experiences, which may partially attribute to the disparity in major depression diagnosis (Kendler et al.,

2001). Women, who on average are part of larger social circles than men, are also more negatively impacted by discord within these proximal friend groups (Kendler et al., 2001).

Also, while men report higher rates of stress attributed to job loss and work problems, women seem to have higher exposure rates to more common stressors such as housing problems and social crisis within their social network (Kendler et al., 2001). These findings are further supported in preclinical rodent studies where researchers have found that females have a greater sensitivity to stress, show an exaggerated response to stressors, and have longer recovery times following stress exposure (Bale T.L. 2006).

On a deeper level, the sexually dimorphic onset of major depression seems to be closely tied with levels of female sex hormones. The first emergence of sex differences in stress induced alterations of synaptic connectivity occur only after the onset of puberty (Eiland

2012). Additionally, the prevalence of developing symptoms of major depression drops dramatically in women after menopause, following the accompanying decrease in estrogen levels (Freeman et al. 2014). Interestingly, post-partum depression is also tied to periods of rapid alterations in estradiol and progesterone, although current studies still have not fully elucidated the role of sex hormones in influencing susceptibility to major depression

(Schiller et al. 2015). Despite the exact causal relationship between reproductive hormones and major depression remaining uncharacterized, many studies have shown that estrogens greatly influence neurotransmission, synaptic plasticity, and overall cognitive function

(Fugger et al., 2000, Packard et al., 1997; Wei et al., 2014; Luine, 2014). Specifically, elevated estrogen levels during menstruation increase synaptic plasticity, BDNF levels, and spine density within the HIPP and PFC (Woolley et al., 1992; Hao et al., 2006; Shansky et al., 2009; Cavus et al., 2003; Kiss et al., 2012). These short-term neural enhancements subside following the end of the menstrual cycle and may lead to a depressive-like state

(Epperson et al., 2012). Interestingly, estrogen administration in rodent models and some clinical studies have produced antidepressant and neuroprotective effects, which may provide support for the suggestion that deficiencies in estrogen levels may be partially responsible for depressive-like states (Rubinow et al., 2011; Hughes et al., 2009). It has been theorized that the benefits of increased estrogen may be due to its influence on some of the same neural growth pathways that are also impacted by the new rapid-acting antidepressants. Of note, several studies have shown that estrogen acts upon upstream

14 regulators of the mTORC1 such as the PI3K-Akt and MAPK-ERK signaling pathways

(Hughes et al., 2009; Nilsson et al., 2011; Spencer et al., 2008).

1.5. Sex Differences in the Antidepressant Effects of Ketamine: Insights from the Clinic and Animal Models

The sex-differentiated effects of ketamine have been documented since its early use in both clinical and preclinical settings. Ketamine has been used as an induction agent since the 1970s and initial studies of its anesthetic properties have suggested a sex-differentiated response. It has been reported that following an anesthetic dose, middle-aged women were more likely than middle-aged men to experience the psychoactive effects of ketamine such as delirium and unpleasant dreams (Knox et al., 1970). In fact, women experienced unpleasant dreams at twice the rate as men; 14:6 when pre-medicated with atropine and

24:12 when pre-medicated with droperidol fentanyl (Knox et al., 1970; Bovill et al. 1971).

The notion of sex-differentiated responses to anesthetic levels of ketamine was further supported by preclinical data in Sprague-Dawley rats following administration of two different doses of ketamine. In this early study, female rats slept nearly twice as long as their male counterparts; following a 60 mg/kg dose, females slept for 32 minutes while males slept for 15 minutes and when given a 90 mg/kg dose females slept for 57 minutes and males for 30 minutes (Douglas et al., 1975). Notably, in preclinical studies examining the molecular effects of anesthetic doses of NMDAr antagonists, researchers found that females were more susceptible to their neurotoxic effects that result in neuronal degradation (Auer, 1996). For instance, it has been reported that Sprague-Dawley rats exposed to different anesthetic doses of ketamine exhibited sex-specific neurotoxic

15 reactions within the retrosplenial cortex (RSC) of the brain (Jevtovic-Todorovic et al.,

2001). Overall, female rats were more sensitive to ketamine’s neurotoxic effects at all doses administered (Jevtovic-Todorovic et al, 2001; Auer, 1996). While there are no studies tracking the long-term treatment efficacy of ketamine and the possible sex differentiated response or side effects, it has been shown that female, but not male, chronic ketamine abusers show increased depression scores compared to healthy individuals as measured using the Center for Epidemiological Studies Depression Scale (CES-D) (Li et al., 2017).

This study enforces the need for continuous monitoring of female-specific neurotoxic responses to ketamine when prescribed for repeated use.

Notably, newer studies examining low antidepressant-relevant doses of ketamine have also begun to uncover differences in how males and females respond to the drug. Clinical studies assessing ketamine’s antidepressant efficacy have often consisted of small sample sizes, and as such did not stratify their results by sex. From what information is available, men and women seem to respond similarly to ketamine, at least early on, and show equal treatment response rates (Freeman et al., 2019; Coyle and Laws, 2015). Interestingly, a meta-analysis from 2015 reported that males and females treated with 0.5mg/kg ketamine

(i.v.) had similar treatment outcomes at 4 hours and 24 hours, but that males had a prolonged antidepressant response by 7 days post injection (Coyle and Laws, 2015).

Interestingly, preclinical research data from our group first hinted a sex-differentiated effect on the kinetics of antidepressant response following administration of a low-dose of ketamine in chronically stressed mice. Specifically, we reported that male, but not female, mice exposed to the chronic mild stress (CMS) model of depression and treated with

10mg/kg ketamine (i.p.) displayed reduced immobility scores in the FST at 7 days post-

16 treatment (Franceschelli et al., 2015). Men and women seem to have similar rates of adverse effects following ketamine treatment, but women do report greater frequency of headaches and nausea and in one small study, only females reported feeling delirious following ketamine administration (Freeman et al., 2019; Soetens, 1969). Despite the evidence for a more persistent antidepressant response in males, in our recent study we found that CMS-exposed female mice were more sensitive to the early effects of ketamine as they responded to lower doses of the drug at 30 minutes and 24 hours post injection

(Frenceschelli et al., 2015). The greater reactivity of female rodents to low dose ketamine treatment was first identified when female rats treated with 2.5mg/kg ketamine displayed reduced immobility in the FST and reduced latency to feed in the novelty-suppressed feeding test (NSFT); this same dose had no effect on their male counterparts (Carrier and

Kabbaj, 2013). Female rodent sensitivity to antidepressant-relevant doses of ketamine was later corroborated by a third study in which female mice presented reduced immobility scores at 24 hours post-ketamine injection (3mg/kg) in the FST (Zanos et al., 2016). Follow up studies focusing on this sex-specific response to the antidepressant drug ketamine have found that the underlying differences may be due to hormonal activity, sex-dependent neurochemical, molecular and synaptogenic pathways, differences in pharmacokinetics, or some combination thereof (Franceschelli et al, 2015; Saland et al., 2017; Thelen et al.,

2019; Zanos et al., 2016). In a recent study, male and freely cycling female C57BL/6J mice showed similar antidepressant behavioral responses to the drug at 30 minutes post- administration of a 3mg/kg dose of ketamine (Dossat et al., 2018); this dose that was not found to induce an antidepressant effect in male C57BL/6J mice in earlier studies

(Franceschelli et al., 2015). However, females in proestrus characterized by high

17 circulating estrogen levels, also responded to a lower 1.5mg/kg dose of ketamine suggesting that this female-specific sensitivity to this drug is dependent on sex hormones

(Dossat et al., 2018). This sex-dependent response to ketamine has also been shown in female Sprague-Dawley rats exposed to social isolation (IS) stress. Male and female SI- stressed rats responded to 5mg/kg ketamine treatment; females, however, also responded to a lower 2.5 mg/kg dose of ketamine that did not affect their male counterparts (Sarkar and Kabbaj, 2016). Interestingly, while IS decreased synaptic spine density within the mPFC of both male and female rats, administration of 5mg/kg ketamine rescued spine atrophy only in male rats (Sarkar and Kabbaj, 2016). This result runs contrary to the behavioral effects seen at the same dose. What’s more, stressed-induced declines in synaptic proteins (i.e., Synapsin-1, PSD-95, GluR1) were only rescued upon ketamine treatment in males, showing a female antidepressant-like behavioral response independent of synaptogenesis or increases of synaptic protein concentrations in the mPFC (Sarkar and

Kabbaj, 2016).

1.6. Focus of the Dissertation

The purpose of the current dissertation is to better understand how ketamine exerts its therapeutic effects and to provide insights on how this drug may act in a sex-specific manner. The discovery of new fast-acting antidepressant agents has revolutionized the way clinicians treat patients and accelerated a previously stagnant field of academic and pharmaceutical research.

In the current dissertation we built on our previous findings on the sex-differentiated sensitivity of female mice to the prototype rapid-acting antidepressant drug ketamine

(Francescelli et al., 2015) and explored the sex-differentiated behavioral and neuromolecular effects following acute and repeated ketamine dosing in male and female mice. Specifically, we showed that behavioral responsiveness to ketamine in female mice is not accompanied by the neurochemical and synaptogenic effects that are typically observed in the male mPFC (Thelen et al., 2019; Chapter 2); moreover, we found that ketamine administration induced a robust synaptogenic response in the female HIPP, suggesting that this brain region may play a greater role in mediating the female antidepressant response to ketamine (Thelen et al., 2019; Chapter 2). Notably, ketamine’s antidepressant-like actions are transient and can only be sustained by repeated drug infusions. In an additional study we provided first evidence that repeated ketamine treatment induced beneficial antidepressant-like effects in male mice but induced both anxiety-like and depressive-like effects in female mice (Thelen et al., 2016; Chapter 3).

Taken together, findings from the current dissertation exposed novel sex differences in response to the rapid-acting antidepressant drug ketamine and have far-reaching implications for the sex-oriented use of this drug in both preclinical and clinical settings.

1.7 Figures

Figure 1.1: A timeline for Antidepressant Drug Development. See text for details.

Figure 1.2: Ketamine mechanism of antidepressant action within the PFC. “Disinhibition hypothesis”

CHAPTER 2

SEX DIFFERENCES IN THE TEMPORAL NEUROMOLECULAR AND SYNAPTOGENIC EFFECTS OF THE RAPID ACTING ANTIDEPRESSANT DRUG KETAMINE IN THE MOUSE BRAIN*

* This dissertation chapter is an adapted version of our peer-reviewed publication in the journal Neuroscience (Elsevier). Permission to reuse published material (text and figures) in the current Ph.D. thesis has been granted by Elsevier. (Citation: Thelen C, Flaherty E,

Saurine J, Sens J, Mohamed S, Pitychoutis PM, Sex Differences in the Temporal

Neuromolecular and Synaptogenic Effects of the Rapid-acting Antidepressant Drug

Ketamine in the Mouse Brain. Neuroscience, 398: 182-192)

2.1. Introduction

The World Health Organization (WHO) has predicted that by 2030 major depressive disorder (MDD) will be the top contributor to the global burden of disease

(Collins et al., 2011, WHO, 2011). Unfortunately, currently marketed antidepressant drugs

(e.g., tricyclic antidepressants; TCAs and selective serotonin reuptake inhibitors; SSRIs) are partially effective, and have been strongly associated with increased drop-out rates due to the time-lag between the onset of antidepressant drug therapy and the alleviation of depressive symptoms (i.e., from 3 weeks to several months) (Lapidus et al., 2013). Indeed,

22 patients suffering from treatment-resistant depression (TRD) are in need of novel and rapid-acting antidepressant medications. Notably, women suffer from MDD at roughly twice the rate of men (Holden, 2005, Marcus et al., 2005, Grigoriadis and Robinson, 2007), yet most novel insights on the neurobiological processes underlying the pathophysiology and the treatment of MDD depend on studies conducted in males.

Remarkably, infusion of a single sub-anesthetic dose of the non-competitive N- methyl-D-aspartate receptor (NMDAR) antagonist ketamine has been reported to induce rapid (i.e., within 1h) and sustained (i.e., for 7-10 days) antidepressant effects in both depressed patients (Berman et al., 2000, Zarate et al., 2006) and in rodents (Li et al., 2010,

Zanos and Gould, 2018). Ketamine’s fast-acting therapeutic potential has been attributed to the rapid induction of spine formation (i.e., synaptogenesis) in brain regions implicated in MDD, such as the medial prefrontal cortex (mPFC) and the hippocampus (HIPP)

(Skolnick et al., 2009, Abdallah et al., 2015, Zanos and Gould, 2018). It is currently believed that the sustained synaptogenic actions of ketamine in the male brain are rapidly induced, possibly as early as 2h post-administration, and also that these new synapses become unstable and are lost after around 7-10 days, which coincides with relapse in MDD patients (Duman, 2014). Intriguingly, the actions of ketamine appear to be unique, as its antidepressant potential emerges as a reaction to its acute effects (Abdallah et al., 2015). It is currently believed that acute blockade of the NMDAR in the brain mobilizes a neurobiological molecular cascade that involves brain region-dependent alterations in the activity of excitatory glutamatergic systems, and downstream alterations in synaptic protein synthesis that ultimately lead to spine formation (Abdallah et al., 2015, Gerhard and Duman, 2018). The mPFC is considered to be the most critical brain region involved

23 in ketamine’s antidepressant actions (Gerhard and Duman, 2018). Indeed, a battery of studies conducted in male rodents have shown that low-dose ketamine administration induces a pre-synaptic disinhibition of glutamatergic neurons in the mPFC, which in turn leads to rapid glutamate release (i.e., “glutamate burst”) that triggers post-synaptic neuroplasticity-related molecular cascades that involve the mammalian target of rapamycin complex 1 (mTORC1) pathway; activation of this pathway orchestrates the rapid synthesis of synaptic proteins, spine formation and subsequent activity-dependent enhancement of synaptic strength (Li et al., 2010, Zanos and Gould, 2018). Of note, elegant preclinical studies have shown that glutamate activation of α-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid receptors (AMPAR) is necessary for both the behavioral and the synaptogenic actions of ketamine in rodent models (Li et al., 2010). However, the complex neurobiological mechanisms implicated in ketamine’s antidepressant actions appear to be dose- and brain region-dependent and remain to be elucidated (Autry et al., 2011, Abdallah et al., 2015).

Notably, preclinical research regarding the behavioral and neurobiological actions of the rapid-acting antidepressant drug ketamine has focused on the male sex. Recent data from our group and others provide evidence that female mice respond differently to both acute and repeated ketamine administration. Indeed, stress-naïve female rodents are more sensitive, and/or reactive to both the rapid and the sustained antidepressant-like effects of ketamine, as they respond to lower antidepressant-relevant doses of this drug in antidepressant-predictive behavioral tests (e.g., forced swim test; FST) (Carrier and

Kabbaj, 2013, Franceschelli et al., 2015). Moreover, we recently showed that repeated (i.e.,

21 days) daily treatment with ketamine appears to induce beneficial antidepressant-like

24 effects in male mice, but induced anxiety- and depressive-like effects in their female counterparts (Thelen et al., 2016). Most importantly, this sex-differentiated responsiveness to ketamine has also been observed in mice subjected to the chronic mild stress (CMS) model of depression; data from our lab provided first evidence that CMS-exposed females were behaviorally more reactive to the earlier sustained effects of a 10 mg/kg dose of ketamine (i.e., at 24 h post-administration), but the antidepressant actions of the drug lasted longer in males (i.e., sustained up to 7 days post-administration) (Franceschelli et al.,

2015).

Based on our previous data, we hypothesized that the sex-dependent reactivity and antidepressant time-course of ketamine could reflect a sex-dependent time course of induction and maintenance of spine formation in the mPFC and the HIPP. It is noteworthy that most studies in the field typically assess only a single endpoint following ketamine treatment and essentially provide only a “snapshot” of the neurobiological alterations that occur in the brain upon ketamine administration. Thus, it is possible that important sex- dependent neurobiological events that occurred before this time-point or even later could be missed. In the current study we performed a deep temporal sampling of ketamine’s synaptogenic program, in order to understand how a single ketamine injection may induce its antidepressant-like effects in the male and the female brain. Herein, we addressed this matter by assessing the kinetics of prefrontocortical glutamate release and downstream activation of synaptic plasticity processes in behaviorally-naïve mice administered a single dose of ketamine (10mg/kg) that we have previously reported to reliably induce both rapid

(i.e., at 30 min) and sustained (i.e., at 24h) antidepressant like-effects in both male and female mice subjected to FST (Franceschelli et al., 2015; Fig. 2.1.).

2.2. Experimental Procedures

2.2.1. Animals

Adult male and female C57BL/6J mice (N=156) used in this study were bred at the

Vivarium of the University of Dayton from a mouse colony originally obtained from the

Jackson Laboratory (ME, USA). Experimental procedures and animal husbandry were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23; revised 1996) and approved by the

University of Dayton Animal Care and Use Committee. Mice were kept on a 12 h/12 h light/dark cycle, in groups of four per cage, under standard laboratory conditions and were given access to food and water ad libitum. All efforts were made to minimize the number of animals used and their suffering.

2.2.2. Experimental design and drug treatments

In the current study we investigated the temporal effects of ketamine on prefrontocortical glutamate release (Experiment #1; Fig. 2.2a), activation of synaptic plasticity pathways (Experiment #2; Fig. 2.2b), and spine formation in the mPFC and the

HIPP (Experiment #3; Fig. 2.2c) of behaviorally-naïve C57BL/6J mice of both sexes. Mice received a single injection of ketamine hydrochloride (10 mg/kg; Henry-Schein; NY; USA) or vehicle (VEH; 0.9% NaCl). Drugs were injected intraperitoneally (i.p.) in a volume of

10 mL/kg of body weight. The 10 mg/kg sub-anesthetic dose of ketamine was implemented herein based on our previous studies showing that it reliably induces both rapid and

26 sustained antidepressant-like effects in the FST in both male and female mice

(Franceschelli et al., 2015). In the current study, we performed a deep temporal sampling of the synaptogenic program following acute ketamine treatment in the mPFC and HIPP of male and female mice. Our study was designed to assess whether there is a basic sex difference in the temporal neurobiological effects of ketamine in the mouse brain. As the first stage of any sex-difference study should be a comparison of gonadally-intact adult male and free-cycling female rodents, the mouse reproductive cycle status was not taken into account (Greenspan et al., 2007, McCarthy et al., 2012, Miller et al., 2017). Therefore, a two-group design (males and females) was implemented for testing for sex-differentiated traits, as in previous studies from our laboratory and others (Pitychoutis et al., 2012, Carrier and Kabbaj, 2013, Franceschelli et al., 2015, Thelen et al., 2016).

2.2.3. Experiment #1: Sex differences in ketamine-induced glutamate release in the mPFC of male and female mice

Male (VEH: N=6; Ketamine: N=8) and female mice (VEH: N=8; Ketamine: N=8) stereotaxically implanted with a microdialysis probe received a single injection of ketamine

(10 mg/kg; i.p.) or VEH. Ketamine-induced release of glutamate was evaluated in extracellular dialysates obtained every 10 min for 60 min, by means of high performance liquid chromatography (HPLC) with a coulometric detector, following precolumn derivatization with o-phthaldialdehyde, according to previous studies from our laboratory

(Marazioti et al., 2008, Franceschelli et al., 2015, Thelen et al., 2016) (Fig. 2.2a).

2.2.4. Experiment #2: Sex differences in the synaptic molecular effects of ketamine.

Male and female mice (N=4-6/time-point), received a single injection of ketamine

(10 mg/kg; i.p.) and were sacrificed immediately (i.e., baseline; B), or after 0.5h, 4h, 24h,

3days (3d), and 7days (7d) following ketamine administration (Fig. 2.2b). Protein concentrations of selected synaptic molecular targets (i.e., mTORC1 phosphorylation,

GluR1 and GluR2) were assessed with western blotting in preparations of enriched prefrontocortical and hippocampal synaptoneurosomes, based on previous studies from our laboratory and others (Li et al., 2010, Thelen et al., 2016).

2.2.5. Experiment #3: Sex differences in the time course of spine formation.

In order to assess the temporal synaptogenic actions of ketamine, male and female mice were administered a single injection of ketamine (10 mg/kg; i.p.) and were euthanized immediately (i.e., baseline; B), or after 2h, 24h, 3d, and 7d following ketamine administration (Fig. 2.2c). Following sacrifice, the brain was isolated and processed for analysis of spines within the mPFC (N=4-5 per group) and the HIPP (N=5-6 per group), using the FD Rapid GolgiStain™ Kit (FD NeuroTechnologies, Inc.).

2.2.6. In vivo brain microdialysis

Male and female mice were anaesthetized with sodium pentobarbital (80 mg/kg; i.p.). After securing the mouse in the stereotaxic frame, a concentric microdialysis probe

(Eicom, membrane length 2mm) was implanted unilaterally in the mPFC (membrane length: 2 mm; coordinates: AP: + 1.98, ML: + 3.0, DV: − 2.75), according to the mouse brain atlas of Paxinos and Franklin (2012) (Paxinos, 2001), based on standard protocols

(Zapata et al., 2009) . Following stereotaxic surgery mice were placed in a dedicated microdialysis cage and kept warm while recovering from the anesthesia. The probe was connected to a microdialysis pump (Eicom ESP-101) through a liquid swivel system

(Eicom TC52-23) for freely moving animals and was perfused with a sterile artificial cerebrospinal fluid (aCSF) solution (i.e., 147 mM NaCl, 3 mM KCl, 1.3 mM CaCl2·2H2O,

1 mM MgCl2·6H2O, 1mM Na3-PO4·12H2O, pH=7.4) at a low flow rate while the animal was recovering overnight. The next day, the flow rate was increased to 1.5 μL/min, and samples were collected into microcentrifuge tubes at 4oC every 10 min. After a 2h stabilization period three samples were collected that were used to assess basal glutamate release. Following collection of the third baseline sample animals received either ketamine

(10 mg/kg; i.p.) or VEH injection and the subsequent measurements were expressed as percent of basal release. Samples were collected every 10 min for 60 min. Immediately after collection, the samples were stored at −80◦C for subsequent neurochemical analysis of glutamate with HPLC. The proper implantation of the probes in the mPFC was verified histologically at the end of each experiment.

2.2.7. Glutamate analysis with high performance liquid chromatography (HPLC)

Glutamate analysis was performed by HPLC with a coulometric detector, as previously described (Franceschelli et al., 2015, Thelen et al., 2016). Briefly, microdialysis samples were injected into an HPLC system that consisted of a pump, a reverse-phase column (Accucore C18 2.6μm, 3.0x100mm) and coulometric detector (Dionex Ulitmate

3000, Fisher Scientific, PA, USA) with a 6011RS ultra analytical cell to quantify extracellular levels of the excitatory amino acid glutamate following pre-column derivatization with OPA/βME, as previously described (Franceschelli et al., 2015). The

v v mobile phase consisted of 100mM Na2HPO4·2H2O, 20% ( /v) methanol and 3.5% ( /v) acetone, pH 6.7. The first electrode was set at +150 mV and the second electrode at +550 mV, column temperature was set at 40oC and the flow rate at 0.5 mL/min. Assay sensitivity was tested for each series of samples using external standards. The signal of the second electrode was used to quantify glutamate levels in microdialysates by comparing of the area under the curve (AUC) with the AUC of reference standard peaks, using a commercial

HPLC software (Chromeleon 7; Fisher Scientific, PA, USA).

2.2.8. Synaptoneurosome preparation and immunoblotting

Crude synaptoneurosome fractions were purified as previously described (Li et al.,

2010, Thelen et al., 2016). Synaptoneurosomal protein concentrations were determined by the bicinchoninic (BCA) protein assay. For immunoblotting equal amount of proteins (30-

40 μg/lane) for each sample was loaded into 10-12% SDS PAGE gel for electrophoresis.

Upon protein transfer onto nitrocellulose membranes, the membranes were probed with primary antibodies overnight at 4ºC. The following primary antibodies were used: pmTOR

(1:1000; Cell Signaling Technology), mTOR (1:1000; Cell Signaling Technology),

Glutamate Receptor 1 (GluR1; 1:1000; Cell Signaling Technology), Glutamate Receptor 2

(GluR2; 1:1000; Cell Signaling Technology); glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) was used as loading control. Total immunoreactivity for each protein band was conducted using the NIH Image J software.

2.2.9. Golgi staining and spine analysis

A Golgi impregnation technique was used to assess the synaptogenic effects of ketamine, based on a modified version of the FD Rapid GolgiStain™ Kit (FD

NeuroTechnologies, Inc). Mouse brains were immersed in an impregnation solution (A/B

Solution). Following immersion into fresh Solution A/B after 24h, the brains remained in the dark at room temperature for two weeks. Following the two-week incubation period the brains were transferred into Solution C for three days in the dark and Solution C was replaced after 24 hours. Coronal sections (100 µm) of the entire cerebrum were cut on a

Vibratome (Lafayette Instruments; IN, USA). Prior to developing, all sections were mounted onto positively-charged Super-frost slides using Solution C. After absorption of excess solution, sections were dried for 2-3 days in the dark and were then processed according to the manufacturer’s instructions. Images of dendrites within mPFC, cornu ammonis 1 (CA1), CA3 and dentate gyrus (DG) of the HIPP, were captured using a 100× objective of a confocal Olympus BX51 microscope. Specifically, secondary branches from

31 layer V neurons within the mPFC and secondary branches from neurons in the CA1, CA3 and DG hippocampal subfields measuring at least 50 µm long were used to calculate average dendritic spine density per 10 µm in each region. For spine number measurements, only spines that emerged perpendicular to the dendritic shaft were blindly counted. For each mouse a total of nine neurons were quantified (3 neurons per coronal section and 3 coronal sections per mouse). The averages for each region were obtained and individual averages for each mouse were combined for an overall average per region.

2.2.10. Statistical analysis

All data are presented as means±SEM. Alterations in the expression of molecular synaptic targets and spine density in the different brain regions of male and female mice were analyzed with one-way ANOVA. Microdialysis data were analyzed with repeated measures ANOVA. Homogeneity of covariance was assessed with the Mauchly’s test of sphericity. In cases where sphericity was violated, the ANOVA F was modiﬁed according to the Greenhouse-Geisser correction to make it more conservative (i.e., less likely to reject the null hypothesis). Approximate normal distribution of our data was assessed using the

Shapiro-Wilk’s test for each level of the dependent variable; in cases where the Shapiro-

Wilk’s test proved to reject the null hypothesis for normal distribution, a non-parametric

Mann-Whitney test was used. Western blotting data are depicted as box-whisker plots in which the box represents the interquartile (IQ) range which contains the middle 50% of the records and the line across the box indicates the median; the whiskers are lines that extend from the upper and lower edge of the box to the highest and lowest values which are no

N1.5 times the IQ range; outliers are depicted as cases with values N 1.5 times the IQ range

(Sens et al., 2017). The level of statistical significance was set at 0.05.

2.3. Results

2.3.1. Experiment #1

Ketamine administration induces a sex-specific “glutamate burst” in the mPFC of male but not female mice. Male and female C57BL/6J mice displayed differential sensitivity to the rapid neurochemical effects of a single dose of ketamine (10 mg/kg), as assessed by in vivo brain microdialysis. A repeated measures ANOVA in male mice revealed sex-specific significant effects for both time and treatment [F(2.686, 32.234) = 4.069, p = .018 and F(1,12) =

7.635, p = .017, respectively]. Further analysis revealed that acute administration of ketamine induced a rapid release of glutamate in the male mPFC at 10 min following ketamine administration [F(1,13) = 5.951, p = .031; Shapiro-Wilk: p>0.05 for both ketamine and VEH-treated groups] (Fig. 2.3a). Ketamine did not induce any apparent statistically significant alterations in glutamate release in the mPFC of female mice (Fig. 2.3b).

2.3.2. Experiment #2

Ketamine activates the mTORC1 pathway and induces rapid and sustained synthesis of AMPAR subunits in male but not female PFC. Ketamine induced a rapid

33 activation of the mTORC1 pathway in prefrontocortical synaptoneurosomes derived from male mice at 0.5h post-administration, as compared to baseline (B) [Shapiro-Wilk test: B: p = .174; 0.5h: p = 0.02; Mann-Whitney test: p = .008] that was sustained up to 24h [F(1,9)

= 6.540, p = .034; Shapiro-Wilk: p>0.05; Fig. 2.4a]. This rapid activation of mTORC1 pathway was accompanied by a rapid increase (i.e., at 4h) of synaptic protein levels of

GluR1 [F(1,9) = 5.573, p = .046; Shapiro-Wilk: p>0.05; Fig. 2.4b], and GluR2 [F(1,9) =

10.680, p = .010; Shapiro-Wilk: p>0.05; Fig. 2.4c] in male mice. Despite the fact that ketamine-induced alterations in synaptic GluR1 levels appeared to be short-lived, GluR2 levels remained elevated up to 24h post-administration [F(1,9) = 9.361, p = .016]. On the other hand, ketamine administration did not activate the mTORC1 pathway in the female prefrontal cortex; in fact, a sex-specific decrease of mTOR phosphorylation was evidenced at 4h post-ketamine administration in female synaptoneurosomes [F(1,11) = 6.826, p = .026;

Shapiro-Wilk: p>0.05; Fig. 2.4a]. No alterations in synaptoneurosomal GluR1 and GluR2 levels were evidenced in the female PFC.

Ketamine enhances GluR1 protein levels in male hippocampal synaptoneurosomes in a male-specific manner. In our experimental setup, ketamine administration did not activate the mTORC1 pathway in the HIPP of either sex (Fig. 2.5a). However, at 4h post- ketamine administration a small sex-specific increase was noted in synaptic concentrations of GluR1 [F(1,9) = 6.100, p = .039; Shapiro-Wilk: p>0.05; Fig. 2.5b], in male hippocampal synaptoneurosomes. Ketamine administration did not affect the protein expression of the synaptic molecular targets assessed in female hippocampal synaptoneurosomes. Moreover, an intriguing sex difference was noted for baseline synaptoneurosomal GluR2 protein

34 levels in the HIPP [F(1,9) = .097, p = .02; Fig. 2.5c] and the PFC [F(1,9) = 5.704, p = .064; statistical trend; Fig. 2.4c], that is in accordance with a recent report that the GluR2 protein content is higher in the hippocampal post-synaptic density (PSD)-fraction of female rats

(Monfort et al., 2015).

2.3.3. Experiment #3

Ketamine induces marked synaptogenic effects in the male mPFC. Shapiro-Wilk analysis showed that all data involved in the between-group comparisons presented below were approximately normally distributed (i.e., p > .05). Ketamine administration induced a long-lasting enhancement of dendritic spine formation in the mPFC of male mice that was already evident at 24h [F(1,7) = 6.337, p = .045] and lasted all the way up to 7d post- drug administration [3d: F(1,7) = 64.837, p < .001; 7d: F(1,8) = 12.549, p = .009; Fig. 2.6a].

On the other hand, ketamine administration was not accompanied by synaptogenesis in the female mPFC.

Ketamine induces sex-dependent temporal synaptogenic effects in the HIPP of male and female mice. Shapiro-Wilk analysis showed that all data involved in the between- group comparisons presented below were approximately normally distributed (i.e.,

Shapiro-Wilk test: p > .05). Ketamine enhanced dendritic spine formation in the CA1 and

CA3 subfields in male mice at 24h [CA1: F(1,10) = 9.824, p = .012; CA3: F(1,10) = 9.797, p

= .012; Fig. 2.6a]; this increase in spine density returned to baseline by 3 days post-drug administration . On the other hand, an increase in dendritic spine density was evidenced at

24h in the CA1 and CA3 regions of the female HIPP [CA1: F(1,11) = 7.895, p = .018; CA3:

F(1,11) = 3.852, p = .078; statistical trend], and was sustained all the way up to 3d post- ketamine treatment [CA1: F(1,11) = 10.300, p = .009; CA3: F(1,10) = 4.859, p = .05] (Fig.

2.6b). Moreover, ketamine was shown to induce a more delayed, yet non-significant, synaptogenic effect in the female DG [F(1,10) = 3.681, p = .087; statistical trend].

Interestingly, at 7 days post-ketamine administration spine density had returned to baseline in the CA1 of both sexes, but spine density in the male CA3 [F(1,10) = .628, p = .022], and

DG was found further decreased in a statistically significant manner [F(1,10) = 14.1015; p =

.005; Fig. 2.6a].

2.4. Discussion

Preclinical studies have provided strong evidence that ketamine induces its antidepressant effects by disinhibition of presynaptic glutamatergic neurons in the mPFC, which in turn leads to a rapid release of glutamate (i.e., a “glutamate burst”) that ultimately activates post-synaptic neuroplasticity-related molecular pathways that involve the mTORC1 (Li et al., 2010, Autry et al., 2011, Abdallah et al., 2015). Indeed, lower sub- anesthetic doses of ketamine (i.e., 5-10 mg/kg) have long been known to rapidly increase extracellular glutamate levels in the mPFC of male rats, assessed with in vivo microdialysis

(Moghaddam et al., 1997, Lorrain et al., 2003). Herein, we report that administration of a single dose of ketamine (10mg/kg) previously shown to induce both rapid and sustained antidepressant-like effects in mice of both sexes (Franceschelli et al., 2015), induced a rapid “glutamate burst” in the mPFC of male but not female mice, in a sex-specific manner

(Fig. 2.3). Glutamate release in the male mPFC was increased during the first 10 min following acute ketamine administration, recapitulating previous microdialysis data in male rats (Voleti et al., 2013). However, ketamine is not completely devoid of neurochemical effects in the female PFC, as we have previously shown that administration of the same dose of ketamine in female mice induces a rapid increase in aspartate prefrontocortical tissue levels at 30 min post-administration (Franceschelli et al., 2015).

Overall, the lack of a “glutamate burst” in the female mPFC suggests that ketamine mediates its rapid and sustained antidepressant-like effects in female mice through a different mechanism of action, or that the mPFC is not as important in mediating ketamine’s effects in female mice.

Compelling preclinical research evidence suggests that the ketamine-induced

“glutamate burst” in the mPFC initiates post-synaptic neuroplasticity-related molecular cascades that involve the activation of the mTORC1 pathway, and the AMPAR (Li et al.,

2010, Abdallah et al., 2015). As noted in recent excellent reviews, a battery of studies conducted mainly in male rodents suggests that the mTORC1 is a key downstream point of convergence accounting for the rapid-acting antidepressant actions of ketamine (Zanos and Gould, 2018, Zanos et al., 2018). The mTORC1 is ubiquitously expressed in the cytoplasm of dendrites and, once activated, it orchestrates the localized translation of synaptic proteins that enhance synaptic strength and promote synaptogenesis, including

AMPAR subunits. Indeed, mTOR phosphorylation has been functionally implicated to the localized synthesis of synaptic proteins required for the birth, maturation, and function of new spines (Hoeffer and Klann, 2010). Li et al., (2010) first showed that a single sub- anesthetic dose of ketamine in male rats induces a transient increase in mTORC1

37 phosphorylation that occurs as early as 30 min post-administration, followed by enhancement of GluR1 protein levels in PFC synaptoneurosomes (Li et al., 2010). Herein, we also assessed the levels of glutamatergic postsynaptic molecular targets involved in synaptic plasticity, namely the GluR1 and GluR2 AMPAR subunits. GluR1 are enriched in the postsynaptic membrane on dendritic spines, and their number is dynamically regulated in an activity-dependent manner (see review by (Chater and Goda, 2014), while synaptoneurosomal levels of GluR2 are highly predictive of synaptic strength (Heynen et al., 2000). Interestingly, decreases in synaptic levels of GluR1 in the PFC have been reported in male rats exposed to chronic stress (Li et al., 2010). Importantly, a number of studies have shown that synaptoneurosomal GluR1 and GluR2 levels are enhanced in the mPFC and the HIPP of rats and mice upon acute ketamine administration (Li et al., 2010,

Zanos et al., 2016, Zanos and Gould, 2018). In our experimental setup, ketamine administration (10mg/kg; i.p.) resulted in a male-specific rapid activation of the mTORC1 pathway in prefrontocortical synaptoneurosomes that was sustained up to 24h (Fig. 2.4a).

Importantly, mTOR activation in males was accompanied by a rapid and sustained increase of synaptoneurosomal protein levels of GluR1 and GluR2 (i.e., 4h-24h; Fig. 2.4bc). On the other hand, ketamine treatment did not increase, but rather decreased, mTOR phosphorylation in females at 4h post-administration (Fig. 2.4a). In accordance with the lack of apparent mTOR activation in the female mPFC, ketamine did not affect GluR1 and

GluR2 levels (Fig. 2.3bc). mTOR phosphorylation was not evidenced in the HIPP of either sex (Fig. 2.5a). However, in males, ketamine induced a small transient increase of synaptic

GluR1 protein levels (i.e., 4h) (Fig. 2.5b). Taken together, our data suggest that administration of a dose of ketamine that is behaviorally active in both sexes induces

38 activation of the mTOR pathway and synaptic AMPAR protein synthesis only in the male mPFC. However, it should be mentioned that the list of synaptic plasticity markers assessed herein was not exhaustive; therefore, ketamine-induced alterations in the levels of other neuroplasticity-related targets in the male and the female brain cannot be ruled out.

Previous molecular studies conducted in rats and mice have used different ketamine dose- ranges that included doses that either induced only rapid antidepressant-like effects in the

FST in both sexes (rats: 5mg/kg; mice: 3mg/kg) or only in females (rats: 2.5mg/kg; mice:

1.5mg/kg) (Carrier and Kabbaj, 2013, Dossat et al., 2017). These studies have reported that mTOR phosphorylation is rapidly increased in PFC synaptoneurosomes of male and freely- cycling behaviorally-naïve female rats at 30 min post-ketamine administration (5mg/kg)

(Carrier and Kabbaj, 2013). Moreover, mTOR phosphorylation was also noted in the mPFC and HIPP synaptoneurosomes derived from male mice and female mice on PE but not DE-

1 suggesting that those alterations are estrous cycle-dependent; these studies were conducted in stressed (i.e., FST-exposed) mice following administration of a dose of ketamine (3 mg/kg) that induced rapid antidepressant-like effects in both sexes (Dossat et al., 2017). Thus, discrepancies between present data and previous studies can be attributed to the different methodology implemented (e.g., behaviorally-naïve versus FST-exposed mice), tissue samples (i.e., whole tissue versus synaptoneurosomes), estrous cyclicity (i.e., freely-cycling versus DE-1/PE categorization), species used (i.e., rats and mice) and ketamine dosing (i.e., dose that induces rapid antidepressant effects versus dose that induces rapid and sustained antidepressant effects).

Most importantly, our data revealed that the temporal effects of ketamine on spine formation are sex-differentiated and brain region-dependent. Specifically, ketamine

39 treatment induced a robust and long-lasting synaptogenic response in the mPFC of male mice in a sex-specific manner (Fig. 2.6a; males). On the other hand, ketamine did not affect spine density in the female mPFC at any time-point examined (Fig. 2.6b; females). Indeed, ketamine administration (2.5 and 5.0 mg/kg) in stress-naïve paired housed rats has not been shown to affect spine formation in the mPFC at 3h post-drug administration (Sarkar and

Kabbaj, 2016). Interestingly, acute ketamine treatment (5mg/kg) has been shown to rapidly rescue chronic isolation stress (IS)-induced decreases of synaptoneurosomal proteins and spine density in the mPFC of male rats at 3h post-administration (Sarkar and Kabbaj,

2016). Conversely, those neuroplastic deficits were not rapidly reversed by ketamine in female rats despite the fact this dose was shown to induce an antidepressant-like effect in both sexes (Sarkar and Kabbaj, 2016). Overall, our data revealed that the kinetics of ketamine-induced spine formation in the mouse mPFC is sex-differentiated, and further point to the notion that ketamine may be devoid of robust synaptogenic effects in the mPFC of stress-naïve female mice. The lack of a synaptogenic response in the female mPFC suggests that ketamine may not induce its rapid and sustained antidepressant-like effects in females by enhancing spine formation, at least in this brain region.

Moreover, in the current study we provide first evidence that ketamine affects the kinetics of spine formation in the HIPP in a sex-dependent fashion. Specifically, in our experimental setup ketamine induced a transient increase of spine density in the CA1 and

CA3 of the male HIPP at 24h post-administration (Fig. 2.6a). In female mice, the synaptogenic response in female mice was first evidenced in the CA1 and the CA3

(marginal trend) hippocampal subfields at 24h, and became more widespread at 3days post-administration to also include the DG (marginal trend) (Fig. 2.6b). Intriguingly, this

40 female-specific increase of hippocampal spine density was not accompanied by relevant synaptic alterations in mTOR activation or AMPAR subunit protein synthesis. These findings can be interpreted at both the conceptual and the technical levels. Our data suggest that synaptogenesis in the female HIPP may be independent of mTOR-activation and downstream activation of AMPA receptors, and possibly that sex- and/or estrous cycle- dependent molecular pathways are activated in the HIPP leading to increased spine formation in male and female mice. For instance, behavioral sensitivity of female mice in proestrus (PE) to the rapid effects of ketamine was specifically shown to be accompanied by increased activation of Akt and CaMKIIα in the HIPP (Dossat et al., 2017). It should also be mentioned that whole hippocampi were used for synaptoneurosomal immunoblotting assessments, whereas Golgi-Cox spine density microscopic assessments focused on individual neurons of selected hippocampal subfields (i.e., CA1, CA3 and DG); given the different level of analysis between these two techniques it is possible that small, yet biologically significant alterations in the levels of synaptic proteins could be missed.

Ketamine was also shown to decrease spine density in the CA3 and the DG of male mice beyond baseline at 7 days post-administration; this finding could be secondary to the ketamine-driven transient increase in hippocampal spine number and warrants further investigation. Overall, our data exposed a sharp sex difference in the synaptogenic response to ketamine, and further suggest that the mPFC may play a more important role in mediating the antidepressant effects of ketamine in males, while the HIPP may be more important for females. It should be borne in mind that stress exposure may largely alter responsiveness of male and female rats and mice to the rapid and the sustained antidepressant-like effects of ketamine. Specifically, we recently reported that CMS-

41 exposed male mice may benefit to a greater extent from acute ketamine treatment (10 mg/kg; i.p.); the antidepressant-like effects of the drug last longer in males, as compared to their female stressed counterparts (Franceschelli et al., 2015). Interestingly, our data on stressed mice were strongly supported by a recent preliminary meta-analysis of clinical studies on ketamine treatment in MDD patients, that identified the male sex as a predictor of a sustained antidepressant response to ketamine at 7 days post-infusion (Coyle and

Laws, 2015). Thus, further research in male and female mice and rats exposed to different animal models of depression, and in different phases of the estrous cycle and ketamine treatment regimens (e.g., repeated administration 2-3 times per week) is considered imperative in order to better understand the neurobiological mechanisms underlying the sex-differentiated responsiveness to this rapid-acting antidepressant drug.

Preclinical studies in rats and mice support the notion that the enhanced behavioral responsiveness of the female sex to ketamine is dependent on the hormonal status and the sex-differentiated pharmacokinetic disposition of ketamine’s metabolite (2S,6S;2R,6R)- hydroxynorketamine (HNK). Indeed, the sex-differentiated responsiveness of rats and mice to ketamine has been typically attributed to the sex-specific levels of sex hormones

(i.e., estrogens and progesterone), as it was reported that the female-specific rapid antidepressant effects of a low acute dose of ketamine were abolished in ovariectomized

(OVX) rats exposed to FST at 30 min post-administration, but restored when supplemented with physiological levels of progesterone and estrogen (Carrier and Kabbaj, 2013).

Interestingly, ovarian hormones may also impact the expression and/or the sensitivity of glutamatergic receptors (Gazzaley et al., 1996, Woolley et al., 1997), which in turn may affect responsiveness to the effects of ketamine (Saland et al., 2017). Following the

42 demonstration by our group and others that female rodents are more reactive to the rapid and the sustained antidepressant-like effects of ketamine (Carrier and Kabbaj, 2013,

Franceschelli et al., 2015), Zanos et al. (2016) reported that sex differences in response to a single dose of ketamine may be due to the sex-differentiated metabolism of ketamine to

(2S,6S;2R,6R)-HNK (Zanos et al., 2016). In that study the authors showed that while levels of ketamine and its main metabolite norketamine were similar between the two sexes, the levels of (2S,6S;2R,6R)-HNK metabolite were approximately three-fold higher in the female mouse brain following acute ketamine treatment (Zanos et al., 2016). Indeed, it is hypothesized that increased exposure of the female brain to the (2S,6S;2R,6R)-HNK metabolite may explain the enhanced female response to ketamine (Gerhard and Duman,

2018). Given that it is also likely that sex-differentiated, estrous cycle-dependent, and/or brain region-dependent molecular pathways may be mobilized by ketamine and/or

(2S,6S;2R,6R)-HNK in the brain of the two sexes, further investigation into this important field is warranted.

It is noteworthy that the intricate neurobiological mechanisms underlying ketamine’s actions appear to be brain region- and dose-dependent and remain to be fully elucidated. As highlighted in excellent recent reviews, current hypotheses on ketamine’s antidepressant mechanism of action focus on the mPFC (Abdallah et al., 2015, 2018).

Despite the well characterized ketamine-induced “glutamate burst” in the mPFC, attenuation of glutamatergic activity has been rather proposed to underlie its rapid antidepressant actions in the HIPP (Autry et al., 2011). Recently, acute systemic administration of S-ketamine (15 mg/kg, i.p.) was reported to decrease evoked glutamate release in the subiculum of the HIPP in male mice, assessed with a highly sensitive

43 microelectrode array fast analytical sensing technology (FAST) (Stan et al., 2014). As implementation of this high throughput technique was not possible herein, the effects of ketamine in regulating hippocampal glutamate release in male and female mice will be assessed in future studies.

In the present study we report that ketamine induces sex-specific neurochemical, synaptic molecular, and synaptogenic effects in the mPFC and the HIPP of male and female stress-naïve C57BL/6J mice. Specifically, present data suggest that ketamine induces a

“glutamate burst” followed by downstream activation of the mTOR pathway, synaptic protein synthesis and spine formation in the mPFC of stress-naïve male mice. Conversely, in our experimental setup ketamine was devoid of robust neuromolecular and synaptogenic effects in the female mPFC; this finding further supports the notion that ketamine may induce its antidepressant effects by either mobilizing a female-specific mechanism in the mPFC, or by acting preferentially in a different brain circuit. This hypothesis warrants further investigation and will be dissected in future studies. Notably, the fact that ketamine was able to enhance spine formation in the female HIPP further shows that ketamine is not devoid of synaptogenic effects in the female brain, but rather in the mPFC. Excitingly, the female-specific sustained synaptogenic response evidenced in the HIPP further implicates this brain region in the sex-differentiated response to this rapid-acting antidepressant drug.

2.5 Figures

Figure 2.1: Male and female stress-naïve C57BL/6J mice were administered increasing doses of ketamine (3, 5, or 10 mg/kg; i.p.) or vehicle (VEH) and were subjected to the forced swim test (FST) at (A) 30 min, or (B) at 24 h to assess the rapid and the sustained antidepressant-like effects of acute ketamine administration. The 10 mg/kg ketamine dose was found to induce rapid and sustained antidepressant effects in both male and female mice. Bars represent immobility means ± SEM (N=6–8/group). *p < .05; *** p < .001 as compared to VEH-treated mice of the same sex. The figure was adapted from Neuroscience, 290, Francescelli A, Sens J, Herchick S, Thelen C, Pitychoutis PM, Sex differences in the rapid and the sustained antidepressant-like effects of ketamine in stress- naïve and “depressed” mice exposed to chronic mild stress, 49-60, Copyright (2015), with permission from Elsevier.

Figure 2.2: Experimental Design: A) Separate cohorts of male and female mice were implanted with microdialysis probes in the mPFC, and upon recovery were administered a single dose of ketamine (10 mg/kg; i.p.) or VEH; the effects of ketamine on glutamate release in the mPFC were assessed by means of HPLC in samples collected every 10 min for 60 min; B) Separate cohorts of male and female mice were administered ketamine (10 mg/kg; i.p.) and were sacrificed at critical time points; protein concentrations of selected synaptic molecular targets were assessed with western blotting in preparations of PFC and HIPP synaptoneurosomes (i.e., P2 fraction) enriched in synaptic proteins (e.g., Synapsin I); C) the effects of ketamine (10 mg/kg; i.p.) on the time-course of spine formation were assessed with Golgi-Cox staining in the mPFC and the CA1, CA3 and DG subfields of the HIPP in male and female mice.

Figure 2.3: Ketamine induced a sex-specific rapid “glutamate burst” in the mPFC of male mice. Glutamate release was assessed with in vivo brain microdialysis in the mPFC of A) male (VEH: N=6; Ketamine: N=8) and B) female mice (VEH: N=8; Ketamine: N=8), following ketamine (10mg/kg; i.p.) or VEH administration. *p<0.05 statistically significant difference from VEH-treated mice.

Figure 2.4: Ketamine induced sex-differentiated synaptic molecular effects in mouse prefrontocortical synaptoneurosomes. Protein expression levels are shown for A) pmTOR/mTOR (males: N=4-6/time-point; females: 5-6/time-point), B) GluR1 and C) GluR2 AMPAR subunits (males: N=5-6/time-point; females: 5/time-point) in the PFC of male and female mice. *p<0.05, **p<0.01; statistically significant difference from baseline (B) group of the same sex.

Figure 2.5: Ketamine-induced synaptic molecular effects in male and female hippocampal synaptoneurosomes. Protein expression levels of a) pmTOR/mTOR (males: N=5/time-point; females: 6/time-point), or b) GluR1 and c) GluR2 AMPAR subunits in the HIPP of male and female mice (N=4-6/time-point). *p<0.05; statistically significant difference from baseline (B) group.

Figure 2.6: Ketamine induces sex-dependent synaptogenic effects in stress-naïve mice. Ketamine affects spine formation in the mPFC (N=4-5/time-point) and the HIPP (N=5- 6/time-point) of a) male and b) female mice in a sex-specific manner. Representative images of high magnification Z-stacks of secondary dendrite branches are shown (red scale bar: 10 μm). *p<0.05, **p<0.01, ***p<0.001; statistically significant difference from baseline (B) group; +statistical trend.

CHAPTER 3

REPEATED KETAMINE TREATMENT INDUCES SEX-SPECIFIC BEHAVIORAL

AND NEUROCHEMICAL EFFECTS IN MICE*

* This dissertation chapter is an adapted version of our peer-reviewed publication in the journal Behavioral Brain Research. Permission to reuse published material (text and figures) in the current Ph.D. thesis has been granted by Elsevier. (Citation: Thelen C, Sens

J, Mauch J, Pandit R, Pitychoutis PM, (2016) Repeated ketamine treatment induces sex- specific behavioural and neurochemical effects in mice. Behavioural Brain Research,

312:305-312).

3.1. Introduction

During the past decade, one of the most striking discoveries in the treatment of major depressive disorder (MDD) was the finding that infusion of a single sub-anesthetic dose of the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine induces rapid and sustained antidepressant effects in treatment-resistant MDD patients and in rodents subjected to various antidepressant-predictive animal models, such as the forced swim test

(FST) and the chronic mild stress (CMS) model of depression (Abdallah et al., 2015).

The intricate behavioral and neurobiological mechanisms underlying ketamine’s antidepressant actions appear to be brain region- and dose-dependent and have not yet been fully elucidated (Abdallah et al., 2015). Compelling preclinical evidence indicates that

51 ketamine’s antidepressant potential lies on its rapid synaptogenic effects in the medial prefrontal cortex (mPFC) and the hippocampus (HIPP), two brain regions that have been strongly implicated in the pathophysiology of MDD (Autry et al., 2011; Li et al., 2010).

Acute ketamine-induced NMDA antagonism in the brain accounts for the first step in the neurobiological cascade of events leading to regional alterations in the activity of brain’s neurotransmitter systems and synaptic protein synthesis, eventually resulting in synaptogenesis (Abdallah et al., 2015). Specifically, it is currently believed that ketamine- induced alterations of glutamatergic tone in the HIPP and the PFC trigger neuroplasticity- related molecular cascades that in turn regulate the synthesis of synaptic proteins involved in synaptogenesis (e.g. synapsin-I) and in the pre-synaptic release machinery (e.g. syntaxin-

I;SYX) (Abdallah et al., 2015; Li et al., 2010; Muller et al., 2013). Indeed, acute low-dose ketamine administration has been shown to rapidly increase synapsin-I protein levels and to decrease the accumulation of SYX-consisting SNARE (soluble N-ethylmaleimide- sensitive factor attachment protein receptor) complexes in synaptoneurosomal preparations from the HIPP and/or the PFC (Li et al., 2010; Muller et al., 2013). Moreover, it has been reported that enhancement of central serotonergic activity also underlies the antidepressant-like effects of ketamine (Li et al., 2010). Specifically, activation of the dorsal raphe nucleus (DRN) and subsequent serotonin (5-hydroxytryptamine; 5-HT) release in the PFC and the HIPP (Nishitani et al., 2014), is possibly implicated in the sustained antidepressant-like effects of ketamine in the FST (Gigliucci et al., 2013).

A battery of evidence indicates that behaviorally females are more sensitive to

NMDA receptor antagonists such as dizocilpine (MK-801), phencyclidine and ketamine than males (Auer et al., 1996; Honack et al., 1993; Shors et al., 2004). For instance, female

52 rats have been reported to be more responsive to the motor-enhancing properties of MK-

801 and to respond to lower doses of the drug that are not effective in their male counterparts (Honack et al., 1993). Notably, in an early study it was also shown that female rats tend to sleep longer than males following administration of anesthetic doses of ketamine (Douglas et al., 1975). Interestingly, ketamine’s (40-80 mg/kg; s.c.) neurotoxic effects in the retrosplenial cortex have been found to be more severe in female rats

(Jevtovic-Todorovic et al., 2001). Moreover, women have also been reported to experience more psychotropic effects (i.e. emergence hallucinations) than men upon ketamine anesthesia (Knox et al., 1970; Bovill et al., 1971). Despite the fact that women experience

MDD at roughly twice the rate of men (Marcus et al., 2005; Grigoriadis et al., 2007; Holden et al., 2005), research regarding the neurobiological antidepressant-relevant effects of ketamine has focused almost exclusively on the male sex. Recent preclinical data from our group and others show that female rodents are more sensitive and/or reactive to the rapid and the sustained antidepressant-like effects of acute ketamine treatment, as assessed in antidepressant-predictive behavioral tasks, such as the FST and the CMS model of depression (Carrier and Kabbaj., 2013; Franceschelli et al., 2015). Most importantly, we recently reported that acute ketamine administration affects the levels of excitatory amino acid neurotransmitters (EAAs; glutamate and aspartate) and serotonergic activity in the

HIPP and the PFC of stress-naïve mice in a sex- and brain region-dependent manner

(Franceschelli et al., 2015). Indeed, both these brain regions have been strongly implicated in sex-related neurobehavioral responses to stress and antidepressant treatments

(Groenewegen and Uylings, 2000; Celada et al., 2004; Drevets, 2007; Carballedo et al.,

2016; Anderson et al., 2016; Pitychoutis et al., 2012; Pitychoutis et al., 2011).

Strikingly, the clinical antidepressant-like effects of ketamine are transient and can only be sustained by repeated drug treatment (Browne and Lucki, 2013). However, our knowledge regarding the frequency and the dose on which repeated ketamine administration stops being beneficial and becomes harmful is limited (Abdallah et al.,

2015). Following our recent demonstration that female mice are more sensitive to the antidepressant-like effects of a single dose of ketamine in the FST (Franceschelli et al.,

2015), we hypothesized that the female sex may also be at greater risk for developing adverse drug reactions (ADRs) upon repeated ketamine treatment.

3.2. Materials and Methods

3.2.1. Animals

C57BL/6J mice used in the present study were bred at the University of Dayton from a mouse colony originally obtained from the Jackson Laboratory (ME, USA). Adult

(8-12 week-old) mice of both sexes were maintained on a 12 light:12 dark schedule (lights on at 8:00). Experimental procedures and animal husbandry were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals

(NIH Publications No. 80-23; revised 1996) and approved by the University of Dayton

Animal Care and Use Committee (IACUC; No: 015-01).

3.2.2. Experimental design and drug treatment

Mice (N=6-8 per group) were administered a single intraperitoneal injection of 3, 5 or 10 mg/kg of ketamine hydrochloride (Henry-Schein; NY; USA) or vehicle (VEH; 0.9%

NaCl), once daily for 21 days. The sub-anesthetic doses of ketamine implemented and route of administration selected are commonly used to screen for the antidepressant-like effects of ketamine in preclinical rodent models (Franceschelli et al., 2015; Browne and Lucki,

2013). Earlier studies have implemented similar drug regimens to investigate the antidepressant-like effects of repeated ketamine treatment in rodents (Tizabi et al., 2012;

Garcia et al., 2008; Garcia et al., 2009; Owolabi et al., 2014; Akinfiresoye et al., 2013;

Parise et al., 2013) [27-32] (Table 3.1). In an initial dose-dependent study we wondered whether male and female mice would display differential responsiveness to increasing doses of repeated ketamine treatment in the open field test (OFT) and the FST (Fig. 3.1a).

Based on this behavioral experiment, the 10 mg/kg ketamine dose was further selected for neuromolecular assessments, as it induced behavioral effects in both sexes.

Neuromolecular analyses were conducted in a different mouse cohort of behaviorally-naïve mice treated repeatedly with ketamine (10 mg/kg) or VEH for 21 days and sacrificed on day 22 (i.e. at 24h after last injection). Following sacrifice the HIPP and the PFC were rapidly isolated on ice, split into right and left parts, snap frozen and stored at –80oC until processing for neurochemical analysis or western blotting, respectively.

Given our aim to determine whether there is a basic sex difference in the way males and females respond to repeated ketamine treatment, the female reproductive state was not taken into account (McCarthy et al., 2012). Since it is generally agreed that the first stage

55 of any sex-difference study should be a comparison of gonadally intact adult females and males (Greenspan et al., 2007), a two-group design (males and females) was implemented for testing for sex-differentiated traits, as in previous studies from our laboratory

(Franceschelli et al., 2015; Pitychoutis et al., 2012; Pitychoutis et al., 2011).

3.2.3. Spontaneous locomotor activity in the open field test (OFT)

In order to assess the effects of ketamine administration on spontaneous locomotor activity and anxiety, mice were placed in the center of an OFT arena (45x45x45cm), as previously described (Franceschelli et al., 2015). Horizontal locomotor activity was recorded for 30 min and the time spent in the center was evaluated for the first 15 min of the test, using a video tracking system (SMART v3.0; PanLab; Barcelona, Spain).

3.2.4. Forced swim test (FST)

FST was conducted, as previously described (Franceschelli et al., 2015). Briefly, mice were individually placed in a glass 4 L beaker filled with 3 L of water (23-25oC) and the duration of immobility (i.e. the time mice spent making only the required movements to keep their head above water) was measured during the last 4 min of a 6-min trial, as before (Can et al., 2012).

3.2.5. Neurochemical analysis

Neurochemical analysis for glutamate and aspartate, and for 5-HT and its metabolite (5- hydroxy-indoleacetic acid; 5-HIAA) was performed using high-performance liquid chromatography (HPLC) with coulometric detection, as previously described

(Franceschelli et al., 2015). Following decapitation, the brain was carefully removed from the skull and the brain regions of interest were rapidly isolated on ice. After weighing, the tissue was homogenized and deproteinized in 0.2 N perchloric acid solution. The homogenate was centrifuged at 15,000 rpm for 30 min at 4oC and the supernatant was stored at −80 °C until analysis. Samples were injected into an HPLC system that consisted of a pump (Dionex Ultimate 3000, Fisher Scientific, PA, USA), a reverse-phase column

(Acclaim Polar Advantage II, 2.2μm, 3.0x100 mm or Accucore C18 2.6μm, 3.0x100mm) and coulometric detector (Dionex Ulitmate 3000, Fisher Scientific, PA, USA) with a

6011RS ultra analytical cell to quantify tissue levels of 5-HT and its metabolite, 5-HIAA.

The composition of the monoamine mobile phase was 75 mM Na2HPO4·H2O, 1.7 mM 1-

v octanesulfonic acid sodium salt, 100μL/L triethylamine, 25μM EDTA and 10% ( /v) acetonitrile (pH 3.0). The first electrode was set at +50 mV and the second electrode at

+350 mV, column temperature was set at 32oC and the flow rate was constant at 0.5 mL/min. The EAAs glutamate and aspartate were measured as their OPA/βME derivatives

(Donzanti et al., 1988). The composition of the amino acid mobile phase was 100mM

v v Na2HPO4·2H2O, 20% ( /v) methanol and 3.5% ( /v) acetonitrile, pH 6.7. The first electrode was set at +150 mV and the second electrode at +550 mV, column temperature was set at

40oC and the flow rate at 0.6 mL/min. The sensitivity of the assay was tested for each series of samples using external standards. The signal of the second electrode was used to

57 quantify all compounds by comparison of the area under the peaks with the area of reference standards with specific HPLC software (Chromeleon 7; Fisher Scientific, PA,

USA). The 5-HIAA/5-HT ratio was calculated as an indicator of 5-HT turnover

(Franceschelli et al., 2015).

3.2.6. Synaptoneurosome preparation and western blotting

Purification of a crude synaptoneurosome fraction was conducted as previously described

(Li et al., 2010) and protein concentration was determined by the BCA protein assay. For western blotting, equal amount of proteins (30-40 μg/lane) for each sample were loaded into 10-12% SDS PAGE gel for electrophoresis. Following protein transfer, nitrocellulose membranes were incubated with primary antibodies overnight at 4ºC. The following primary antibodies were used: Synapsin-I (1:200; Santa-Cruz), SYX (1:5,000; Sigma-

Aldrich) and GAPDH (loading control; 1:200; Santa-Cruz). For the detection of SDS- resistant SNARE complexes, western blotting was performed on samples of electrophoresed synaproneurosomes (non-boiled before gel loading), incubating blotted membranes with the antibody for SYX, as previously described (Muller et al., 2013).

Densitometric analysis of total immunoreactivity for each protein was conducted using

NIH Image J software.

3.2.7. Statistical analysis

Statistical analyses were performed with the SPSS 16.0 statistical software (SPSS Inc.,

Chicago, IL). Differences between VEH and ketamine-treated mice were evaluated by two- way analysis of variance (ANOVA) with two between-subjects factors sex (males vs females) and treatment, followed by Dunnett’s post-hoc testing, when appropriate, in order to elucidate specific differences between groups. Locomotor activity data were initially analyzed with repeated measures ANOVA with two between-subjects factors sex (males vs females) and treatment and one within-subjects factor of time; subsequent repeated measures ANOVAs were used to elucidate the specific effects of ketamine treatment in each sex. Homogeneity of covariance was assessed with the Mauchly’s test of sphericity; when data failed the sphericity test, the ANOVA F was modified according to Greenhouse-

Geisser correction to make it more conservative (less likely to reject the null hypothesis).

Correlation analysis was undertaken by estimating Pearson’s correlation coefficients for multiple comparisons. Statistical significance was defined as p≤.05.

3.3. Results

3.3.1. Repeated ketamine treatment is beneficial for males but induces anxiety-like and depressive-like effects in females

Ketamine administration did not affect spontaneous locomotor activity in either sex (Fig.

3.1b,c). A repeated measures ANOVA regarding the time male and female mice spent in

59 the center of the OFT arena revealed a significant effect of sex [F(1,51)=8.699; p=.005] that was further investigated with follow-up repeated measures ANOVAs for each sex.

This analysis in turn revealed that ketamine treatment affected the total time female mice spent exploring the center of the OFT arena, as indicated by a significant sex-specific effect of treatment only in females [F(3,24)=3.902; p=.021]. Post-hoc analysis revealed that the two higher doses of ketamine (i.e. 5 and 10 mg/kg) significantly reduced the time that female mice spent exploring the center of the arena (p<.05 for both doses). Separate one- way ANOVAs for each time-point (i.e. 5, 10 and 15 min) revealed that ketamine treatment affected the time female mice spent exploring the center of the OFT arena during the first 5 min of the OFT only [F(3,27)=3.405; p=.034]. Subsequent post-hoc analysis for the 5 min time-point revealed that the 10 mg/kg ketamine dose decreased the time female mice spent exploring the center of the OFT arena (p=.028; Fig. 3.1e).

Importantly, male and female mice displayed differential responsiveness to the sustained antidepressant effects of ketamine, as assessed in the FST at 24h after the last drug injection. In particular, a two-way ANOVA revealed a significant sex x treatment interaction [F(3,56)=6.248, p<.001]. Further analysis revealed that the 10mg/kg dose of ketamine decreased immobility duration in male mice (p=.042), but increased it in their female counterparts (p=.012; Fig. 3.2).

3.3.2. Repeated ketamine treatment affects hippocampal neurochemistry in a sex-

dependent manner

Ketamine treatment (10mg/kg) induced sex- and brain region-specific

neurochemical alterations in male and female mice. Two-way ANOVAs revealed a

significant sex x treatment interaction for glutamate [F(1,27)=4.086; p=.005], and a main

treatment effect for aspartate levels [F(1,27)=4.086; p=.005]. Further analysis revealed that

at 24 h following the last injection, ketamine decreased glutamate (p=.010; Fig. 3.3a) and

aspartate tissue concentrations (p=.014, Fig. 3.3b) in the HIPP of female, but not male

mice. Moreover, two-way ANOVAs for 5-HIAA levels and for the 5-HIAA/5-HT ratio

yielded a significant effect of sex [F(1,27)=5.364; p=.030] and a sex x treatment interaction

[F(1,27)=6.195; p=.020], respectively, due to the fact that that ketamine treatment enhanced

both 5-HIAA levels, and the 5-HIAA/5-HT ratio in the male HIPP (p<.05; Fig. 3.3c-e).

Ketamine did not affect neurochemical responses in the PFC (data not shown).

3.3.3. Repeated ketamine treatment affects the expression of synaptic proteins only in male mice

Protein assessments were conducted in the crude synaptoneurosomal fraction (P2)

enriched in synaptic proteins (Fig. 3.3f). Two-way ANOVAs for Synapsin I and SNARE-

100kDa hippocampal levels revealed significant sex x treatment interactions

[F(1,25)=4.846, p=.039 and F(1,24)=4.322, p=.05, respectively]. Further analysis revealed

that ketamine enhanced Synapsin I expression (p=.017; Fig. 3.3g) and SNARE (100kDa)

complex accumulation in hippocampal synaptoneurosomes (p=.029; Fig. 3.3i) derived

from male, but not female mice. SYX monomer levels in the HIPP were not affected by

ketamine in either sex (35kDa; Fig. 3.3h), but were decreased in the female PFC (p<.05;

data not shown).

3.3.4. Neurochemical effects correlate with synaptic protein alterations in the male

HIPP

Pearson’s correlation analyses revealed that synapsin-I protein levels correlated positively with glutamate tissue concentrations in the HIPP of male, but not female mice irrespectively of drug treatment (r=0.699, p=0.011, n=12 and r=0.305, p=0.312, n=13, respectively; Fig. 3.4a). The same analysis revealed that SYX protein levels correlated positively with hippocampal 5-HIAA levels and 5-HT turnover (5-HIAA/5-HT ratio) in the HIPP of male, but not female mice (5-HIAA: r=0.635, p=0.026, n=12; and r=0.126, p=0.697, n=12, respectively; Fig. 3.4b and 5-HIAA/5-HT ratio: r=0.571, p=0.05, n=12 and r=-0.167, p=0.604, n=12, respectively). Importantly, the positive correlations observed in male mice can be attributed to the effects of repeated ketamine treatment since they were also observed in drug-treated male mice (Glu-Synapsin: r=0.829, p=0.041, n=6; 5-

HIAA/5-HT-SYX: r=0.935, p=0.006, n=6), but not in their VEH-treated counterparts.

3.4 Discussion

Herein, we report that repeated treatment with a low dose of ketamine (i.e. 10 mg/kg, once daily for 21 days) induced opposite behavioral effects in male and female mice. Ketamine treatment induced sustained antidepressant-like effects in male mice, as evidenced by the decreased immobility duration in the FST at 24 h post-administration

(Fig. 3.2). However, the same treatment regimen induced sex-specific anxiety-like and depressive-like effects in female mice, as evidenced by the reduced time ketamine-treated females spent exploring the center of the OFT arena (Fig. 3.1e), and by the increase in FST immobility durations, respectively (Fig. 3.2).

The beneficial effects of repeated ketamine treatment observed in male mice are consistent with earlier studies that have investigated the antidepressant-like effects of various repeated ketamine treatment regimens in rodents (Tizabi et al., 2012; Garcia et al.,

2008; Garcia et al., 2009; Owolabi et al., 2014; Akinfiresoye et al., 2013; Parise et al.,

2013). Of note, the adverse effects of repeated ketamine treatment observed in females fit nicely with our recent finding that a single dose of ketamine (10 mg/kg) may induce rapid sex-specific anxiety-like effects in female mice subjected to the OFT (Franceschelli et al.,

2015). Notably, in a most recent clinical study Singh et al. (2016) reported that twice- weekly and thrice-weekly administration of ketamine (0.5 mg/kg; i.v.) for up to four weeks maintained antidepressant efficacy over 15 days in 67 treatment-resistant depressed patients (67% women) (Singh et al., 2016). Even though the data of that study were not stratified by sex, it is clear that ketamine is effective in both sexes. However, it was also reported that the most common treatment-emergent ADRs associated with repeated

63 ketamine dosing were anxiety, headache, nausea, dissociation and dizziness (Singh et al.,

2016). Given the greater representation of women in that study, it is tempting to speculate that the emergence of these ADRs could be associated with our finding that repeated ketamine administration may induce sex-specific anxiety-like effects in the female sex.

Ketamine’s sex-differentiated behavioral effects were accompanied by pronounced sustained neurochemical and synaptic molecular alterations in the male HIPP. Remarkably, repeated ketamine treatment enhanced hippocampal 5-HT turnover (Fig. 3.3e) and synapsin-I protein levels (Fig. 3.3g), and also increased the accumulation of SNARE

100kDa complexes in the male HIPP (Fig. 3.3i). These neurochemical alterations are consistent with the expression of antidepressant phenotypes. Indeed, both the enhancement of serotonergic activity and synapsin-I levels are thought to underlie the antidepressant- like effects of ketamine (Li et al., 2010). Numerous studies have shown that the SNARE complex mediates the fusion of synaptic vesicles with the presynaptic membrane, and thus increase in SNARE complex accumulation is consistent with increased neurotransmitter release (Musazzi et al., 2011). Herein, this association between SNARE complex accumulation and serotonergic neurotransmission is further underlined by the positive correlation observed between hippocampal 5-HIAA levels and 5-HT turnover and SYX levels in ketamine-treated male mice (Fig. 3.4b). Notably, administration of a single sub- anesthetic dose of ketamine (15 mg/kg; i.p.) in male Sprague Dawley rats has been shown to induce a rapid decrease in the accumulation of SNARE complexes at 1-4 h post- administration that is suggestive of a rapid reduction of neurotransmitter release in the

HIPP (Muller et al., 2013). Indeed, Kavalali and Monteggia (2012) have suggested that rapid decrease in hippocampal glutamatergic activity may underlie the rapid antidepressant

64 actions of acute ketamine treatment (Kavalali and Monteggia, 2012). Thus, we hypothesize that the sex-specific increase in SNARE complex accumulation observed in the male HIPP upon repeated ketamine administration, and at 24h after the last injection, could be attributed to long-lasting adaptations of hippocampal serotonergic neurotransmission associated with repeated ketamine administration. Conversely, the same drug treatment regimen induced a sex-specific reduction of hippocampal EAA levels in female mice, as well as a decrease in SYX levels in the PFC (Fig. 3.3a,b). Of note, it could be suggested that the baseline sex difference observed in hippocampal 5-HIAA levels could have masked ketamine’s ability to further boost 5-HT turnover and induce synaptic molecular effects in females. Overall, our data suggest that male mice are more responsive to the antidepressant-like behavioral and molecular effects of repeated ketamine treatment, whereas females appear to be resistant.

Intriguingly, in our experimental setup repeated ketamine treatment induced sex- specific antidepressant-like effects in male mice that were accompanied by major brain region-specific neurochemical and synaptic molecular alterations (i.e. enhancement of 5-

HT turnover, synapsin I levels and SNARE accumulation) in the HIPP, but not in the PFC.

On the other hand, in females the same drug regimen induced sustained decrease in hippocampal EAA levels and in SYX levels in the PFC that could be associated with the lack of antidepressant-like effects in these mice. Notably, a battery of compelling preclinical evidence indicates that the antidepressant actions of acute ketamine treatment lie on its rapid and sustained synaptogenic effects in the PFC and the HIPP (Autry et al.,

2011; Li et al., 2010). Therefore, the fact that herein relevant sustained neuromolecular effects were observed only in the male HIPP possibly highlights the importance of this

65 brain region in mediating the protracted antidepressant actions of ketamine following repeated administration.

Even though the estrous cycle was not assessed in the current study, it has been proposed that the antidepressant effects of acute ketamine administration are estrous cycle dependent (Carrier and Kabbaj, 2013; Sarkar and Kabbaj, 2016). Indeed, sex differences in response to ketamine in rodents are most likely dependent on the sex-specific levels of estrogen and progesterone, as it was recently reported that the female-specific rapid antidepressant effects of a low acute dose of ketamine were completely abolished in ovariectomized rats subjected to FST, but emerged again upon restoration of physiological levels of estrogens and progesterone (Carrier and Kabbaj, 2013). Notably, sex differences in response to antidepressant treatments have been largely attributed to the sex- differentiated pharmacokinetic disposition of psychotropic agents (Kokras et al., 2011;

Hodes et al., 2010). Following the demonstration by our lab and others that female mice and rats are more sensitive to the antidepressant effects of ketamine, Zanos et al. (2016) most recently reported that sex differences in response to acute administration of this drug may be partly due to the sex-differentiated metabolism of ketamine to (2S,6S;2R,6R)- hydroxynorketamine (HNK) (Zanos et al., 2016). Indeed, (2S,6S;2R,6R)-HNK metabolite levels were shown to be approximately three-fold higher in the female mouse brain following ketamine administration (Zanos et al., 2016). Notably, administration of the

(2R,6R)-HNK enantiomer in mice was shown to induce the desired antidepressant effects of ketamine, while lacking its adverse psychotomimetic effects (Zanos et al., 2016).

Overall, we hypothesize that the sex-dependent responsiveness of male and female mice to repeated ketamine administration observed herein could be attributed to the interaction

66 between the hormonal milieu and the sex-differentiated pharmacokinetic disposition of ketamine and its metabolites.

It is noteworthy that exposure to stress may affect the responsiveness of male and female rodents to the antidepressant-like effects of ketamine. Indeed, we recently reported that administration of a single dose of ketamine (10 mg/kg; i.p.), that was effective in both male and female stress-naïve mice in the FST, induced marked sex-differentiated effects in mice exposed to the CMS model of depression (Franceschelli et al., 2015). Excitingly, our behavioral data in the CMS paradigm suggested that male mice benefit to a greater extent from acute ketamine treatment since the antidepressant-like effects of the drug wear- off sooner in their female counterparts (Franceschelli et al., 2015). Of note, our findings in

CMS-exposed mice are strongly supported by a most recent preliminary meta-analysis of clinical studies on ketamine treatment in MDD patients, that identified the male sex as a predictor of a longer-lasting response to ketamine at 7 days post-infusion (Coyle and Laws,

2015). More recently, Sarkar and Kabbaj (2016) also reported that acute ketamine administration elicits sex-dependent effects in socially isolated rats (Sarkar and Kabbaj,

2016). Thus, further research in stressed rodents subjected to various animal models of depression and other repeated ketamine treatment regimens (e.g. 2-3 times per week) is imperative in order to further dissect the behavioral and neurobiological mechanisms underlying the sex differences in response to repeated ketamine treatment exposed herein.

3.5 Conclusions

Taken together, our findings indicate that repeated ketamine treatment induces opposite behavioral effects in male and female mice. Notably, our data suggest that sex- differentiated responsiveness to ketamine appears to become problematic when females are treated repeatedly with higher antidepressant-relevant doses of this drug. Thus, present findings have far-reaching implications for the use of ketamine in both experimental and clinical research settings.

3.6 Figures and Tables

Table 3.1: Earlier studies that have investigated the antidepressant-like behavioral effects of various repeated ketamine treatment regimens in rodents. WKY: Wistar Kyoto, S.D.: Sprague Dawley; bid: twice a day.

Strain/Sp Treatment Reference ecies Sex Regimen Behavioral Output

Garcia et al., WKY 5, 10 & 15 ↓ FST immobility; 60 min Male 2008 rats mg/kg; 14d post-injection Garcia et al., Wistar Male 15 mg/kg; 7d ↓ anhedonic behavior 2009 rats Tizabi et al., WKY 0.5 & 2.5 ↓ FST immobility; 22 h Female 2012 rats mg/kg; 10d post-injection Parise et al., 20 mg/kg bid; ↓ FST immobility; 2 months S.D. rats Male 2013 20d post-injection Akinfiresoye et WKY ↓ FST immobility; 22 h Male 0.5 mg/kg; 10d al., 2013 rats post-injection Reversed fluoxetine- Owolabi et al., Albino Male 15 mg/kg; 21d induced ↓FST immobility; 2014 mice (50%) 24 h post-injection

Table 3.2: Effects of repeated ketamine treatment on glutamate, aspartate, serotonin (5- HT), 5-hydroxy-indolacetic acid (5-HIAA) tissue concentrations and 5-HT turnover (5- HIAA/5-HT ratio) in the prefrontal cortex (PFC) of male and female mice. Effects of repeated ketamine treatment on protein expression levels of synapsin-I, syntaxin-I (SYX) and the SNARE 100 kDa complex accumulation in PFC synaptoneurosomes. Data are expressed as means±SEM (N=6-8group). *p<.05; as compared to VEH-treated mice of the same sex.

MALES VEH Ketamine 10 mg/kg Glutamate 2404.02 ± 230.62 2686.04 ± 265.42 Aspartate 407.48 ± 66.05 442.71 ± 61.63 5-HT 297.61 ± 31.31 355.94 ± 29.03 5-HIAA 126.95 ± 15.25 134.01 ± 17.58 5-HIAA/5-HT 0.44 ± 0.06 0.38 ± 0.04 Synapsin-I 0.85 ± 0.15 0.80 ± 0.07 Syntaxin-I 1.65 ± 0.16 1.56 ± 0.17 SNARE- 100kDa 0.07 ± 0.02 0.06 ± 0.02

FEMALES VEH Ketamine 10 mg/kg Glutamate 2884.53 ± 373.46 3361.01 ± 497.97 Aspartate 499.83 ± 86.31 586.15 ± 97.01 5-HT 291.70 ± 26.58 351.35 ± 49.59 5-HIAA 136.43 ± 9.16 148.91 ± 13.96 5-HIAA/5-HT 0.50 ± 0.06 0.46 ± 0.06 Synapsin-I 0.93 ± 0.19 0.92 ± 0.10 Syntaxin-I 1.47 ± 0.084 1.17 ± 0.09 * SNARE- 100kDa 0.04 ± 0.008 0.04 ± 0.004

Figure 3.1: a) Experimental timeline for behavioral studies in the current study: male and female mice were treated repeatedly with increasing doses of ketamine (i.e. VEH, 3, 5 and 10 mg/kg) for 21 days, once daily. On day 15 (at 24 h post-injection) spontaneous locomotor activity was tested in the OFT and on day 22 (at 24 h post-treatment) mice were subjected to FST in order to assess the sustained antidepressant-like effects of ketamine; b-c) effects of repeated ketamine treatment on the horizontal distance travelled and d-e) time spent in the center of the OFT arena. Data are expressed as means±SEM (N=7-8 per group). *p<.05, as compared to VEH-treated mice of the same sex.

Figure 3.2: Sustained antidepressant-like effects of repeated ketamine treatment in the FST at 24 h post-administration. Data are expressed as means±SEM (N=7-8 per group). *p<.05; **p<.01, as compared to VEH-treated mice of the same sex.

Figure 3.3: Effects of repeated ketamine treatment on a) glutamate, b) aspartate, c) serotonin (5-HT), d) 5-hydroxy-indolacetic acid (5-HIAA) tissue concentrations and e) 5- HT turnover (5-HIAA/5-HT ratio) in the hippocampus (HIPP) of male and female mice. f) The synaptoneurosomal fraction (P2) was enriched in synaptic proteins (i.e. synapsin-I). Effects of repeated ketamine treatment on protein expression levels of g) synapsin-I, h) syntaxin-I (SYX) and i) the SNARE 100kDa complex accumulation in hippocampal synaptoneurosomes. Data are expressed as means±SEM (N=6-8group). *p<.05; **p<.01, as compared to VEH-treated mice of the same sex; #p<.05; ###p<.001, differences between VEH-treated male and female mice.

Figure 3.4: Hippocampal neurochemical indices correlate with synaptic molecular alterations in male, but not female mice; positive correlations were observed between a) glutamate and synapsin-I tissue concentrations and between b) 5-HIAA and SYX tissue concentrations in the HIPP of male, but not female mice.

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