I CHEMOKINE MODULATION of MDPV-INDUCED BEHAVIOR AND

CHEMOKINE MODULATION OF MDPV-INDUCED BEHAVIOR AND NEUROPLASTICITY

A Dissertation Submitted to the Temple University Graduate Board

In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY

by Chicora F. Oliver May 2019

Examining Committee Members:

Dr. Scott M. Rawls, Advisory Chair, Lewis Katz School of Medicine at Temple University, Center for Substance Abuse Research Dr. Debra Bangasser, Temple University, Psychology Department Dr. Vinay Parikh, Temple University, Psychology Department Dr. Mathieu Wimmer, Temple University, Psychology Department Dr. Deborah Drabick, Temple University, Psychology Department Dr. Tania Giovannetti, Temple University, Psychology Department i

2018

ABSTRACT

Psychostimulant abuse is a major public health concern yet no FDA-approved medications exist. Synthetic cathinones (“bath salts”) are a class of psychostimulants that have emerged relatively recently worldwide. One synthetic cathinone, MDPV (3,4- methylenedioxypyrovalerone) is mechanistically similar to cocaine but is over ten times more potent, possesses high abuse potential, and is relatively understudied. Recent studies have revealed involvement of inflammatory proteins called chemokines in the rewarding effects of MDPV and the mechanistically similar drug, cocaine. We and others have shown that the chemokine-receptor ligand pair CXCL12-CXCR4 is recruited in the rewarding effects of cocaine and MDPV. Humans and animal models of cocaine addiction have dysregulated CXCL12 and the commercially-available CXCR4 antagonist, AMD3100, can reverse cocaine use and relapse in preclinical models of addiction. Specifically, AMD3100 reduces self-administration and reinstatement to cocaine-seeking with concomitant alterations in CXCL12 gene expression in the midbrain. Here, I employ several complementary methods to demonstrate that AMD3100 also reverses MDPV-elicited behaviors. I demonstrate that (i) AMD3100 reverses

MDPV-induced hyperlocomotion, conditioned place preference (preclinical model of drug reward), self-administration and reinstatement to MDPV-seeking behavior; (ii)

AMD3100 can rescue MDPV-induced deficits in measures of anxiety and recognition memory shortly after a binge; and (iii) repeated MDPV exposure upregulates CXCL12 gene expression in the nucleus accumbens with concomitant downregulation of dendrite

iii morphometrics and a related synapse scaffolding protein gene expression. These findings implicate CXCR4-CXCL12 signaling in the modulation of MDPV-elicited behaviors, suggesting that AMD3100 is a viable therapeutic option for the effects of this synthetic cathinone.

ACKNOWLEDGEMENTS

I would like to acknowledge all the people who contributed to the completion of my doctoral dissertation. Thank you, Scott Rawls for being a great PI. Thank you undergraduate research assistants for being excellent pupils. Thank you to my family for providing endless support. Thank you to all friends for being a varied group of wonderful and complex people with whom I’ve enjoyed many adventures. Thank you, podcasts audiobooks, and online streaming services for being welcome distractions and sources of knowledge I’ve consumed while writing and scoring videos. Thank you, Temple

University for being my home for over half a decade. Thank you, thank you, thank you!

TABLE OF CONTENTS

Page

ABSTRACT ...... iii

ACKNOWLEDGEMENTS ...... v

LIST OF FIGURES ...... ix

CHAPTER 1 ...... 1

GENERAL INTRODUCTION ...... 1

Psychostimulant Addiction ...... 1 Neural Substrates of Psychostimulant Addiction ...... 1 MDPV ...... 2 Neuroinflammatory Contributions to Psychostimulant Addiction ...... 5 Chemokines ...... 6 CXCR4 Modulation of Psychostimulant Addiction ...... 7 CXCL12-CXCR4 Signaling ...... 9 AMD3100 ...... 12 AMD3100 Reversal of Effects of Psychostimulants ...... 13 CHAPTER 2: AMD3100 EFFECTS ON MDPV REWARD ...... 14

Background ...... 14 Methods ...... 15 Animals ...... 15 Drugs...... 15 Conditioned place preference...... 16 Ultrasonic vocalizations...... 17 Intrajugular catheterization...... 18 Self-Administration...... 19 Results ...... 20 vi

Locomotor activity...... 20 Conditioned place preference...... 23 Ultrasonic vocalizations...... 25 Self-administration...... 27 Discussion ...... 29 CHAPTER 3: AMD3100 MODULATION OF THE COGNITIVE AND EMOTIONAL EFFECTS OF MDPV ...... 30

Background ...... 30 Methods ...... 32 Drug binge...... 32 Elevated zero maze...... 32 Locomotor Control Experiments...... 33 Results ...... 33 Elevated zero maze...... 33 Locomotor Control Experiment...... 39 Novel Object Recognition...... 39 Discussion ...... 42 CHAPTER 4: NEURAL SUBSTRATES OF THE EFFECTS OF MDPV ...... 43 Background ...... 43 Methods ...... 45 Drug Binge...... 45 CXCL12 gene expression...... 45 Golgi-Cox Staining...... 46 Statistical Analysis...... 47 Results ...... 47 Dendrite Morphology ...... 47 CXCL12 Gene Expression...... 47 PSD95 Gene Expression...... 47 Discussion ...... 53 vii

CHAPTER 5: CONCLUSIONS ...... 55

Limitations ...... 61 Directions for future research ...... 62 REFERENCES CITED ...... 64

viii

LIST OF FIGURES

Figure Page

Figure 1. AMD3100 reduced MDPV-induced hyper-locomotion...... 22

Figure 2. AMD3100 reduced MDPV Conditioned Place Preference...... 24

Figure 3. MDPV-induced ultrasonic vocalization modestly reduced by AMD3100...... 25

Figure 4. AMD3100 reduced MDPV self-administration...... 26

Figure 5. Cumulative infusions of MDPV and effects of AMD3100 in self- administration...... 28

Figure 6. 24 h Withdrawal from Binge MDPV is Anxiolytic, AMD3100 reverses effect...... 34

Figure 7. Effects of AMD3100 on MDPV-elicited Anxiogenesis 72 h after binge...... 35

Figure 8. 24 h Locomotor Control for MDPV EZM: Total Activity...... 36

Figure 9. 24 h Locomotor Control for MDPV EZM: Ambulation...... 37

Figure 10. 24 h Locomotor Control for MDPV EZM: Stereotypy...... 38

Figure 11. AMD3100 does not rescue expression of novel object recognition...... 40

Figure 12. The effects of AMD3100 on novel object recognition 24 h after MDPV binge...... 41

Figure 13. Dendrite Morphometrics in nucleus accumbens core following cocaine or MDPV binge...... 49

Figure 14. Nucleus accumbens core dendrites following cocaine or MDPV ...... 50

Figure 15. PSD95 is downregulated in the nucleus accumbens core following MDPV binge...... 51 ix

Figure 16. CXCL12 in nucleus accumbens 24 h withdrawal from MDPV Binge ...... 52

CHAPTER 1

GENERAL INTRODUCTION

Psychostimulant Addiction

Psychostimulant addiction is a major public health concern with an estimated 24.1 million dependent individuals reported in 2010 (Degenhardt et al., 2014), over 9,000 overdose deaths reported in 2014 (Vital Statistics Reports, 2016), and an economic burden estimated in the tens of billions of dollars (Bouchery, Harwood, & The Lewin

Group, 2001; Drug Intelligence Center, 2011). The addictive properties of psychostimulants such as cocaine are attributed to the drug’s ability to increase synaptic levels of neurotransmitters with reinforcing properties, such as dopamine and norepinephrine (Koob & Bloom, 1988; White & Kalivas, 1998) yet direct modulation of these neurotransmitters fails to prevent addiction or relapse to psychostimulant use. As a result, there is currently no FDA-approved medication for psychostimulant addiction and thus a better understanding of the neural correlates of psychostimulant use and relapse are needed.

Neural Substrates of Psychostimulant Addiction

Several decades of research have localized the addictive properties of psychostimulants such as cocaine to alterations in nucleus accumbens (NAC) dopamine activity (Cornish &

Kalivas, 2000a; Kelly, Seviour, & Iversen, 1975; Koob, Le, & Creese, 1987; Kourrich &

Thomas, 2009). For example, cocaine increases in extracellular dopamine in the NAC (

Hurd & Ungerstedt, 1989; Ritz, Lamb, Goldberg, & Kuhar, 1987) and blockade of this

1 increase attenuates cocaine self-administration (Maldonado, Robledo, Chover, Caine, &

Koob, 1993). Cocaine also increases NAC morphometrics, such as length, complexity, and spine density (Robinson & Kolb, 1999a; Robinson, Gorny, Mitton, & Kolb, 2001a) in a dose- and NAC-dopamine-dependent manner (Ferrario et al., 2005; Martin et al.,

2011; Norrholm et al., 2003). Cocaine-induced increases in NAC dendrite morphology are considered markers for psychostimulant addiction because they are long-lasting and coincide with behavioral sensitization and return to drug-seeking behavior (Ferrario et al.,

2005; Robinson & Kolb, 1997; Robinson & Kolb, 2004). In sum, cocaine-taking, return to taking, and NAC structural plasticity are mediated by drug-induced alterations in NAC dopamine tone. These aspects of cocaine addiction are well-established, however these effects are largely unexplored in many of the novel psychoactive substances that are currently being abused.

MDPV

3,4-methylenedioxypyrovalerone (MDPV), a type of ‘bath salt’, is a powerful psychostimulant with addictive effects similar to cocaine, amphetamine, and methamphetamine (Aarde, Huang, Dickerson, & Taffe, 2015; Harvey & Baker, 2016;

Watterson et al., 2014). MDPV has a mechanism most similar to that of cocaine, blocking activity of dopamine, norepinephrine and serotonin transporters thereby increasing levels of these neurotransmitters in the synapse (Baumann et al., 2013, 2017). However,

MDPV is at least ten times more potent than cocaine (Baumann et al., 2013; Schindler et al., 2016), frequently consumed unknowingly (Palamar, Salomone, Vincenti, & Cleland,

2016), and has a chemical structure similar to newer ‘bath salts’ (e.g., αPVP; Marusich et al., 2014). Like cocaine, the addictive effects of MDPV are mediated by dopamine alterations in the NAC. Decreasing cocaine-evoked dopamine in the NAC (Barr et al.,

2015) also attenuates MDPV reward behavior (Gregg & Rawls, 2015), providing further evidence that the reinforcing effects of MDPV are mediated by NAC dopamine. The clear involvement of dopamine in the addictive effects of psychostimulants necessitates treatment options that modulate this neurotransmitter. Indeed dopamine modulators have had some success in treating cocaine addiction in humans (Dackis, Kampman, Lynch,

Pettinati, & O’Brien, 2005; Somoza et al., 2004), however, no Federal Drug

Administration (FDA)-approved medication for psychostimulant addiction currently exists. Therefore, an exploration into alternative means of modulating psychostimulant- induced dopamine dynamics is needed.

Like other psychostimulants, the salient behavioral effects of MDPV are attributed to elevations of synaptic monoamines dopamine, norepinephrine and serotonin.

Some psychostimulants such as methamphetamine act as substrate for DAT, enabling the drug to enter the cell, trigger the release of dopamine from vesicular stores, and reverse the direction of the DAT from reuptake to a releaser of dopamine. Cocaine and MDPV, on the other hand are only transporter blockers and thus cause elevations in synaptic dopamine by merely blocking the ability of DAT to reuptake synaptic dopamine. It is therefore not surprising that MDPV (DAT blocker) protects against methamphetamine- induced neurotoxicity at dopamine nerve terminals (Anneken, Angoa-Pérez, & Kuhn,

2015). This is attributed to the DAT-blocking properties of MDPV preventing the entry 3 of methamphetamine into the presynaptic terminal. On the other hand, when methamphetamine is combined with synthetic cathinones that are also substrate releasers, such as methylone and mephedrone, methamphetamine-induced neurotoxicity at dopamine nerve terminals is enhanced due to synergistic dopamine enhancing properties amongst the drugs (Anneken et al., 2015). These findings highlight the importance of understanding drug combinations prior to consumption.

Unintentional synthetic cathinone consumption is a growing public health concern. There are several reported incidents involving accidental death from consuming fatal combinations of drugs that include synthetic cathinones. A recent review describes several incidents of persons consuming what they assume to be ecstasy, that turns out to be a much more potent synthetic cathinone (Oliver, Palamar, et al., 2018). In infamous case of this prompted to shut down of a popular electronic dance festival called Electric

Zoo (Hadlock et al., 2011). A concertgoer was found unconscious febrile, tachycardic, and exhibited sustained clonus (muscle rigidity). Despite maximal supportive care at a hospital (sedatives, fluid replacement, ventilation, and aggressive cooling), the patient expired within 48 hours of entering the hospital. Pills found on her person that were similar to the two ingested were revealed to contain the synthetic cathinones, methylone and butylone, with no traces of the MDMA the “ecstasy” was sold as. While methylone and butylone are 2-6x less potent than MDMA in terms of dopamine and serotonin transporter substrate and blocking-properties, the excessive quantity of drug found in each pill was the likely cause of the fatal overdose. Indeed the three drugs (MDMA, methylone, and butylone) are consumed at similar doses recreationally, likely due to 4 similar efficacy in increasing dopamine (Hall & Henry, 2006; López-Arnau, Martínez-

Clemente, Pubill, Escubedo, & Camarasa, 2012; Oliver, Palamar, et al., 2018; Warrick et al., 2012). However, each pill ingested in this case contained 422 mg of methylone and

53 mg of butylone, manifold greater quantities that what is considered pleasurable or safe.

Neuroinflammatory Contributions to Psychostimulant Addiction

Neuroinflammation is emerging as a major contributor to psychostimulant addiction. For example, cocaine addicts have repeatedly been shown to possess heightened levels of inflammatory proteins call cytokines (Araos, Pedraz, Serrano,

Lucena, Barrios, García-Marchena, et al., 2015a; Ersche et al., 2013; Ersche & Döffinger,

2017; Fox & Sinha, 2009). These elevations in inflammatory cytokines are also evident during periods of abstinence from cocaine in dependent patients (Araos, Pedraz, Serrano,

Lucena, Barrios, García-Marchena, et al., 2015a; Levandowski et al., 2014; Moreira et al., 2016; Pedraz et al., 2015) and non-dependent users (Van Dyke, Stesin, Jones,

Chuntharapai, & Seaman, 1986). Moreover, chronic inflammation in cocaine users is associated with chronic stress ( Fox & Sinha, 2009; Jong & Kloet, 2004; Sinha et al.,

2003) as well as stress- (Abrahams et al., 1996; Fox et al., 2005; Sinha, Garcia, Paliwal,

Kreek, & Rounsaville, 2006) and cue-induced (Sinha et al., 2003) reinstatement of cocaine use. The interaction between cocaine addiction and inflammatory cytokines is clear thus modulation of this interaction may be a viable therapeutic option for addiction to cocaine and perhaps other psychostimulants.

Chemokines

Chemokines are small (7-14 kDA) chemotactic neuroinflammatory cytokines

(Belmadani, Tran, Ren, & Miller, 2006; Mennicken, Maki, de Souza, & Quirion, 1999) that are currently being investigated as therapeutic targets for psychostimulant addiction

(Araos, Pedraz, Serrano, Lucena, Barrios, García‐Marchena, et al., 2015; Kim, Connelly,

Unterwald, & Rawls, 2016a; Trecki & Unterwald, 2009; Yoshie, 2013). Chemokines are classified and named based on the spacing of conserved cysteine residues in the N- terminus: C, CC, CXC, CX3C (X is any amino acid) followed by an R for receptors or an

L for ligands, ending with a number reflecting chronicity of discovery (Bacon et al.,

2002). Though some chemokine ligands and receptors are also known by monikers with historical significance. For example, CXCL12 is also known as stromal (connective tissue) cell-derived factor 1 alpha (SDF1α) because the protein was first observed in bone marrow stromal cells (Bleul, Fuhlbrigge, Casasnovas, Aiuti, & Springer, 1996).

Beyond, their role in inflammation, chemokines affect neural processes that make them promising targets for psychostimulant addiction therapy. Chemokines possess neuromodulatory properties in that they have little to no effect on basal conditions but can modulate neuronal excitability, neurotransmitter release, and can activate second messenger systems (Adler, Geller, Chen, & Rogers, 2006). Of particular relevance is the ability of chemokines to modulate dopamine in the brain as well as behaviors and structural plasticity associated with this neurotransmitter. For example, fractaline

(CX3CL1), a chemokine modulated by cocaine exposure (Araos, Pedraz, Serrano,

Lucena, Barrios, García‐Marchena, et al., 2015; Pedraz et al., 2015), has been shown to 6 modulate hippocampal dendrite length and complexity in mice (Xiao, Xu, & Jiang,

2015) in a way that mimics the effects of cocaine on hippocampal structural plasticity (H.

Yao, Bethel-Brown, & Buch, 2009). CX3CL1 can also reduce dopamine neuron loss

(Chien, Lee, Liou, Liou, & Fu, 2016; Nash et al., 2015) and the chemokine CCL2 can increase dopamine transmission (Guyon et al., 2009). In addition, CCR2 (CCL2 receptor) knockout mice exhibit reduced cocaine locomotor sensitization (Trocello et al., 2011).

Thus chemokines are regulators of dopamine and may therefore represent viable targets for treatment of psychostimulant addiction.

CXCR4 Modulation of Psychostimulant Addiction

The chemokine receptor-ligand pair CXCR4-CXCL12 (Oberlin et al., 1996) has been linked to psychostimulant-use. Plasma CXCL12 is positively correlated with cocaine use in humans and rodents (Araos, Pedraz, Serrano, Lucena, Barrios, García‐

Marchena, et al., 2015; Pedraz et al., 2015). Systemic and striatal administration of

CXCL12 potentiates cocaine-induced hyperlocomotion (Trecki & Unterwald, 2009).

Moreover, we recently found that CXCR4 antagonism attenuates cocaine-induced hyperlocomotion and development of conditioned place preference (Kim, Connelly, et al., 2016). CXCR4 activation by CXCL12 triggers dopamine (Apartis, Mélik-

Parsadaniantz, Guyon, Kitabgi, & Rostène, 2010; Guyon et al., 2008; Skrzydelski et al.,

2007) and glutamate release (Bezzi et al., 2001b; Calì & Bezzi, 2010a), neurotransmitters dysregulated by cocaine and MDPV (Cornish & Kalivas, 2000a; Kim, Rawls, & Walker,

2016; Pettit & Justice, 1989). In addition, repeated cocaine produced an increase in

CXCL12 mRNA in the ventral tegmental area (Kim, Rawls, et al., 2016), a brain region that releases dopamine in the nucleus accumbens with psychostimulant use (Wise &

Rompre, 1989). Thus, CXCR4-CXCL12 signaling is a powerful regulator of the neural substrates and behavioral effects of psychostimulants.

There is human and preclinical data showing that cocaine dysregulates CXCL12 and that CXCL12 can be used to stratify human users based on cocaine use symptom severity (Araos, Pedraz, Serrano, Lucena, Barrios, García-Marchena, et al., 2015a).

Human male intranasal cocaine users recruited from outpatient treatment facilities have lower plasma levels of CXCL12. Specifically, these CXCL12 deficits were exhibited in patients who were abstinent from cocaine for at least two weeks and the same study observed elevated CXCL12 after a single injection of cocaine in mice. These findings suggest that CXCL12 may be elevated during use but downregulated during withdrawal.

Cocaine users with high symptom severity (9 to 11 DSM-IV criteria) have increased prevalence for comorbid psychiatric disorders. When 85 total cocaine users were assessed, 30 (36.6%) experienced mood disorders (e.g., depression, dysthymia, bipolar disorder) but when stratified by symptom severity, those with high symptom severity were significantly more likely to experience a mood disorder compared (n = 20 or 54.1%) to the low-severity group (n = 10 or 22.2%). A similar pattern was seen with comorbid anxiety disorders, with 17 (20.7%) of the entire cocaine group exhibiting anxiety disorders but with a lower prevalence of anxiety disorders in the low symptom severity group 5 (11.1%) compared to 12 (32.4%) in the high severity group. The strongest association was found with personality disorders with 27 (32.9%) of all cocaine 8 users exhibiting comorbidity and amongst these prevalence was most significantly lower in the low symptom-severity group (n = 5; 11.1%) compared to the high severity group (n

= 22; 59.5%).

CXCL12-CXCR4 Signaling

CXCL12 is a C-X-C motif chemokine that was the 12th to be discovered.

CXCL12 is one of the few chemokines found in the brain (Kim, Connelly, Unterwald, &

Rawls, 2017a; Oliver, Simmons, et al., 2018; Trojan et al., 2017) and activates two receptors, CXCR4 (C-X-C chemokine receptor 4) and CXCR7 (C-X-C chemokine receptor 7). CXCR4 is the major brain receptor though CXCL12 does bind sparsely with

CXCR7 in the central nervous system (Bleul et al., 1996; Jordan Trecki, Brailoiu, &

Unterwald, 2010).

The neural substrates of addiction involve neurotransmitters and brain regions that are strongly modulated by CXCL12-CXCR4 signaling. CXCL12 can increase GABA and glutamate synaptic activity through a number of presynaptic actions that vary from structure to structure. CXCL12 elicits glutamate increases to indirectly increase the frequency of post-synaptic events in GABAergic cells in the cerebellum and in serotonergic cells in the dorsal raphe (Heinisch & Kirby, 2010a; Limatola et al., 2000).

However, CXCL12 directly increases dopamine cell activity in the substantia nigra

(Guyon et al., 2006b). Moreover, glutamate release is tetrodotoxin (TTX)-dependent in the lateral hypothalamus (Guyon et al., 2005) and in the raphe (Heinisch & Kirby, 2010b) but is tetrodotoxin-independent in the substantia nigra (Guyon et al., 2006b), with the 9

TTX-dependent mechanism indicating involvement of glutamate release from postsynaptic terminals. CXCL12 also acts postsynaptically in ways that differ across brain structures. For example, CXCL12 elicits an increase in GABA release in the lateral hypothalamus to evoke tonic GABA type A currents in melanin-concentrating hormone hypothalamic neurons (Guyon et al., 2005). However, CXCL12 elicits phasic dopaminergic currents in the substantia nigra through GABA type B receptor activity

(Guyon et al., 2006b). In addition, CXCL12 exerts biphasic effects on high-voltage calcium currents of dopaminergic neurons: a potentiation at low currents (0.1-10 nM) and a depression at higher concentrations (50-100 nM) with the depression likely due to

GABA overflow (Guyon et al., 2008; Rostène, Kitabgi, & Parsadaniantz, 2007). These and other studies provide clear evidence that CXCL12 activation of CXCR4 can exert powerful effects on synaptic neurotransmitter levels that are relevant to addiction.

One of the most important studies linking CXCL12-CXCR4 signaling to psychostimulant addiction was conducted by Araos and colleagues (2014) on both human subjects and mouse models. This study examine the cytokines and chemokines TNFα,

CCL2, CX3CL1 and CXCL12 in the plasma of cocaine addicts seeking outpatient treatment in Spain. All volunteers had a lifetime pathological use of cocaine and 89% of individuals had been diagnosed with cocaine abuse or dependence disorder using criteria present in both the DSM-IV-TR and more recent DSM-V and all volunteers were confirmed to be clean from cocaine for at least two weeks prior to sample collection. All cocaine users had lower levels of TNFα, CCL2, and CXCL12 assayed, suggesting an abstinence-related downregulation of these inflammatory proteins. Moreover, the group 10 was able to stratify subjects by symptom severity using sample means and the number of criteria met for abuse or dependence (i.e. low severity and high-severity groups).

Interestingly, only CXCL12 levels predicted both symptom severity as well history of pathological use of cocaine (Araos, Pedraz, Serrano, Lucena, Barrios, García-Marchena, et al., 2015b). These findings demonstrate the role of CXCL12 in particular as a putative mediator of cocaine abuse and dependence.

Araos and colleagues (2014) also examined CXCL12 in mice at various timepoints following acute (one dose) or chronic (once daily for seven days) cocaine exposure. Both acute and chronically-exposed animals increased plasma CXCL12, relative to baseline. The elevations were greater in the chronic group with the most significant upregulation of CXCL12 seen at 120 and 240 min after the last injection of cocaine. This is curious given that the cocaine itself has been almost completely metabolized at these time points thus increases in CXCL12 are secondary, rather than resultant from cocaine exposure (Mets, Diaz, Soo, & Jamdar, 1999). Such a comprehensive assessment of CXCL12 in humans or rodents following MDPV exposure has yet to be completed.

Several preclinical studies implicate CXCR4-CXCL12 signaling in the effects of cocaine. Systemic and striatal administration of CXCL12 potentiates cocaine-induced hyperlocomotion (Trecki & Unterwald, 2009). We recently found that CXCR4 antagonism attenuates cocaine-induced hyperlocomotion and development of conditioned place preference (Kim, Connelly, et al., 2016). CXCR4 activation by CXCL12 triggers dopamine (Apartis et al., 2010; Guyon et al., 2008; Skrzydelski et al., 2007) and 11 glutamate release (Bezzi et al., 2001b; Calì & Bezzi, 2010a), neurotransmitters dysregulated by both cocaine and MDPV (Cornish & Kalivas, 2000a; Kim, Rawls, et al.,

2016; Pettit & Justice, 1989). In addition, repeated cocaine produces an increase in

CXCL12 mRNA in the ventral tegmental area (Kim, Rawls, et al., 2016), a brain region that releases dopamine in the nucleus accumbens with psychostimulant use (Wise &

Rompre, 1989). Thus, CXCR4-CXCL12 signaling is a powerful regulator of the neural substrates and behavioral effects of cocaine, a psychostimulant that is mechanistically similar to MDPV.

AMD3100

Importantly, CXCR4 is one of the few chemokines with a commercially-available receptor antagonist (AMD3100), which has proven useful to the investigation of addiction-related mechanisms. AMD3100, also known as plerixafor or Mozobil, displays high selectivity for CXCR4 receptors as demonstrated by calcium flux assays that reveal no interaction of AMD3100 with the chemokine receptors CXCR1, CXCR2, CXCR3, or

CCR1 through CCR9 (Hatse, Princen, Bridger, De Clercq, & Schols, 2002; Matthys et al., 2001). Currently, AMD3100 is approved by the FDA as an immunostimulant that mobilizes stem cells in cancer patients (Bilgin & de Greef, 2016; Khan, Greenman, &

Archibald, 2007). While currently used as a cancer treatment, AMD3100 has recently been effective in the amelioration of several preclinical aspects of psychostimulant addiction.

AMD3100 Reversal of Effects of Psychostimulants

Antagonizing CXCR4 with AMD3100 has been effective in reversing the effects of CXCR4-CXCL12 signaling that are relevant to psychostimulant addiction. AMD3100, a CXCR4 antagonist (Hatse et al., 2002), attenuates the reinforcing strength of cocaine by reducing the number of times an animal will lever-press for an infusion of the drug (Kim,

Rawls, et al., 2016). AMD3100 also prevents reinstatement to cocaine-seeking when a cue or a drug with a cue is used to prime relapse to cocaine use (Kim, Rawls, et al.,

2016). CXCR4 inhibition with AMD3100 has also been shown to reverse CXCL12- induced increases in dendrite morphology (Muzio et al., 2016; Pitcher et al., 2014) which is especially relevant given the well-established, long-lasting increases in dendrite morphometrics that coincide with return to psychostimulant use following long periods of abstinence (Robinson & Kolb, 1999; Robinson, Gorny, Mitton, & Kolb, 2001a). Given the mechanistic similarities of MDPV to cocaine, the results of these studies suggest that antagonism of CXCR4 with AMD3100 can attenuate the effects of MDPV-taking behavior as well as the effects of MDPV on structural plasticity.

CHAPTER 2: AMD3100 EFFECTS ON MDPV REWARD

Background

Addiction to psychostimulants such as cocaine and MDPV is attributed to their locomotor-activating, rewarding and reinforcing properties in humans and animals. Both psychostimulants exert these effects through blockade of DAT, NET, and SERT in brain regions such as the nucleus accumbens, with DAT and NET-blocking properties especially associated with effects on the brain and behavior (Baumann, Partilla, Lehner, et al., 2013b). While mechanistically-similar, the synthetic cathinone, MDPV is ten times more potent at NET, ten times less potent at SERT, and fifty times more potent at DAT

(Baumann, Partilla, & Lehner, 2013; Baumann et al., 2013). Despite these known properties, direct modulation of neurotransmitters is not an effective treatment for these and other psychostimulant abuse disorders.

In recent years, psychostimulant users and animal models have been shown to exhibit dysregulated levels of inflammatory proteins called chemokines. Cocaine addicts at least two weeks into withdrawal have significantly lower levels of the chemokine

CXCL12 (Araos, Pedraz, Serrano, Lucena, Barrios, García‐Marchena, et al., 2015). In mice, acute and chronic cocaine upregulate CXCL12 up to four hours after the last drug exposure. We have shown similar effects in rats wherein repeated cocaine increases

CXCL12 gene expression in the VTA, a brain region that sends dopamine projections to the nucleus accumbens with psychostimulant exposure (Kim et al., 2017a). The chemokine CXCL12 is unique in that it exerts powerful effects on dopamine activity.

Methods

Animals. Adult male Sprague-Dawley rats (275-300 g) from Taconic Biosciences

(Hudson, NY) were used. Rats were pair-housed in a temperature and humidity- controlled room on a 12 h light/dark cycle (lights on at 7:00 am) and had ad libitum access to food and water in the home cage. All procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory

Animals and approved by the Temple University Institutional Animal Care and Use

Committee.

Drugs. (±)-MDPV was synthesized in accordance with previously published methods

(Abiedalla, Abdel-Hay, DeRuiter, & Clark, 2012; Kolanos et al., 2015), by Dr. Allen

Reitz. AMD3100 was purchased from AstaTech (Bristol, PA). MDPV and AMD3100 were dissolved in sterile water. Intraperitoneal (i.p.) drugs were injected at a volume of 1 ml/kg. Doses were based on AMD3100 (1–10 mg/kg) reducing cocaine’s rewarding effects (Kim et al., 2017) and MDPV (2 mg/kg) causing conditioned place preference

(CPP), locomotor activation, and 50-kHz USV calls (Gregg et al., 2016; Simmons et al.,

2017). Each rat was used once for an experiment (either locomotor, CPP or USV) and then euthanized immediately following behavioral experimentation (Oliver, Simmons, et al., 2018).

Locomotor activity. Ambulation and stereotypy were assessed using a Digiscan DMicro system (Hicks et al., 2018; Oliver, Simmons, et al., 2018). The chambers were made of plastic (45 cm x 20 cm x 20 cm) and were set inside metal frames equipped with 16

15 infrared light emitters and detectors (Lisek et al., 2012). Following a 60-min habituation period, rats were pretreated with 5 mg/kg AMD3100 or vehicle (sterile water) 30 min prior to MDPV (2 mg/kg) or vehicle administration, and activity was measured for 90 min. This experiment was also repeated with the AMD3100 doses 1, 2.5, and 10 mg/kg

(Oliver, Simmons, et al., 2018).

Conditioned place preference. CPP was assessed by manual scoring of time spent in various chambers as previously described in detail (Hicks et al., 2018; Lisek et al., 2012).

CPP was conducted in a Plexiglas container (45 cm x 20 cm x 20 cm) which was constructed in-house. One chamber had black walls with a sandpaper-like textured floor.

The other compartment had white walls with black vertical stripes and a smooth, non- textured floor. A counterbalanced, biased design was employed wherein each rat’s innate preference for one side of the chamber was assessed during preconditioning. MDPV was always paired with the non-preferred side. During preconditioning, rats were allowed access to the entire apparatus for 30 min and time spent each chamber was later assessed by observing a video recording of the session. A rat was considered in distinct compartments if its forelimbs were inside the compartment. The CPP conditioning phase began 24 h later. At this time rats were given MDPV (2.5, 5, and 10 mg/kg) via i.p. injection and placed in their non-preferred side for 30 min. Rats were then returned to the homecage for 4 h. Rats were then injected with vehicle and placed in the preferred compartment for 30 min. CPP conditioning phases were completed once a day, for 4 consecutive days. CPP testing occurred 24 h after the final CPP conditioning phase. For

CPP testing, rats were allowed to freely explore both sides of the chamber in a drug-free state for 30 min. Time spent in each chamber was later assessed by observing a video recording of the session (Oliver, Simmons, et al., 2018).

Ultrasonic vocalizations. 50 kHz USVs were assessed because they are thought to reflect positive affective states produced by cocaine or MDPV exposure (Avvisati et al., 2016;

Barker, Simmons, et al., 2014; Simmons et al., 2016). USVs were recorded in 90 min sessions with 10 mg/kg AMD3100 or vehicle given at minute 0 and 2 mg/kg MDPV or vehicle given at minute 30. Recordings took place in self-administration chambers (Med-

Associates) as described below. Importantly, this was merely the setting for recordings and self-administration experiments were conducted on separate sets of animals.

Ultrasonic condenser microphones (Avisoft Bioacoustics; Dodotronic) were positioned atop Plexiglas chambers which were themselves contained within larger, sound- attenuating wooden chambers (Simmons et al., 2016). USVs were manually detected and quantified using a Matlab program (Barker, Herrera, & West, 2014; Simmons et al.,

2016). Anticipation for MDPV was operationalized as USV calls made during minutes 0-

30. MDPV was given at minute 30. Calls made during minutes 30-90 were considered

MDPV-evoked USVs. Baseline USVs were measured on Day 1 and vehicle was given at minute 0 and minute 30. On Days 2-5, rats were given AMD3100 at minute 0 and MDPV was administered at minute 30. USVs were recorded during the baseline session on Day 1 and during the last session on Day 5. Putative 50 kHz USVs were detected using an

17 automated scoring program and were manually confirmed using spectrograms, as previously described (Barker, Simmons, et al., 2014; Oliver, Simmons, et al., 2018).

Intrajugular catheterization. Self-administration was conducted on rats implanted with an intrajugular catheter, as previously described (Simmons et al., 2016). Rats were anesthetized with isofluorane gas (5% induction, 2-3% maintenance, in oxygen, 2 liters per min) in preparation for surgery. The mid-scapular region of the dorsal surface of the rat and right neck region of the ventral surface of the rat were wiped with 70% ethanol and betadine prior to being shaved. Rats were then given 1 mg/ml of meloxicam, subcutaneously (s.c.), as a means of post-operative analgesia. The same dose of meloxicam was administered once a day, for 2 days following surgery. Following the initial dose of meloxicam, a small incision was made on both dorsal and ventral surfaces.

Connective tissue was cleared from the ventral surface incision and the right jugular vein was isolated. The intrajugular catheter itself consisted of a backmount made of mesh which was secured to a 22-gauge stainless steel vascular port, connected to a catheter made of plastic (PlasticsOne, Roanoake, VA). The catheter was passed subcutaneously through the rat’s dorsal incision through the ventral incision. The catheter was then placed in the right jugular vein and suture thread was used to secure this position.

Catheters were then flushed with 0.1 ml of 0.5 mg/ml baytril saline. A dust cap was then placed over the catheter port. All wounds were secured with 9-mm surgical staples. After surgery, rats were individually-housed and allowed to recover for one week prior to self- administration procedures. In the first three days following surgery, catheters were

18 flushed once a day with 0.1 ml of baytril saline. In the last four days of the week-long recovery period, catheters were flushed with 0.1 ml of 100 IU/ml of heparinized saline combined with 0.5 mg/ml of baytril saline, once a day to maintain patency.

Self-Administration. Self-administration sessions were conducted during the dark cycle, in standard operant self-administration chambers (Med Associates, St. Albans, VT) equipped with two retractable levers designated as the active (right lever) or the inactive

(left lever), as described previously (Hicks et al., 2018). Thirty minutes prior to each session, rats were pretreated with vehicle or AMD3100 (2.5, 5 or 10 mg/kg, i.p.). Rats were trained to self-administer 0.056 mg/kg/infusion of MDPV in 2 hour daily session, for 7 days, on an FR-1 schedule of reinforcement wherein a 20 second intertrial interval was employed. Active lever responses produced a 50 µl intravenous infusion of MDPV delivered over 3 seconds via an infusion pump connected to a metal spring tether, which was connected to the catheter port of each rat. Infusion of drug was paired with a tone

(4.5 kHz, 78 dB tone) and illumination of a cue light positioned 50 mm above the active lever and deactivation of the house-light (Hicks et al., 2018).

Statistical analysis. GraphPad Prism 6 software was used for all statistical analyses.

Locomotor data were analyzed by a two-way or one-way ANOVA and a Bonferroni posttest was employed. CPP data were analyzed by a one-way ANOVA and a Dunnett’s posttest. USV data were normalized using change-scores [(B-A/(A+B)]. USV change- scores ranged from -1 to +1 and was used to determine the change in 50 kHz USVs from treatment, against the within-subjects baseline score. T-tests compared USV change-

19 scores for each group during anticipation and post-MDPV time-points against 0, with zero indicating no change from baseline (Oliver, Simmons, et al., 2018). Self- administration data were analyzed by two-way ANOVA followed by a Bonferroni posttest, which were each performed on the total number of infusions on each day of self- administration.

Results

Locomotor activity. AMD3100 Reduced MDPV-evoked locomotor activity. For the time course data, a two-way ANOVA showed effects of MDPV treatment [F(3,684) = 423.84, p < 0.0001] and time (F(18,684) = 13.25, p < 0.0001] and there was a significant interaction (F(54,684) = 5.35, p < 0.0001] as shown in Figure 1A. In MDPV-treated rats,

5 mg/kg AMD3100 (AMD-MDPV) pretreatment reduced hyperlocomotion compared to vehicle pretreated rats (VEH-MDPV). For the cumulative data shown in Figure 1A inset, a two-way ANOVA revealed an effect of MDPV treatment [F(1,36) = 59.94, p < 0.0001] but not an effect of pretreatment [F(1,36) = 3.18, p > 0.05] or significant interaction

[F(1,36) = 2.82, p > 0.05]. MDPV-treated rats (VEH-MDPV) displayed greater hyperlocomotion than those treated with vehicle-alone (VEH-VEH; p < 0.001). MDPV- treated rats that were pretreated with AMD3100 (AMD-MDPV), however, displayed a significant reduction in locomotor activity compared to those pretreated with vehicle

(VEH-MDPV; p < 0.01). Figure 1B displays dose-effect results for AMD3100. For these data, a one-way ANOVA revealed a main effect of AMD3100 doses [F(4,47) = 11.55,

20 p < 0.0001]. Each dose of AMD3100 reduced locomotor activation produced by MDPV

(1 mg/kg, p < 0.01; 2.5 mg/kg, p < 0.001; 5 mg/kg, p < 0.05; 10 mg/kg, p < 0.001).

Figure 1. AMD3100 reduced MDPV-induced hyper-locomotion. (A) Total activity across time, inset is cumulative activity. (B) Cumulative total activity AMD3100 dose response.

Conditioned place preference. AMD3100 reduced MDPV-induced CPP. A One-way

ANOVA revealed a main effect of difference scores (F(5,47) = 3.490, p = 0.01) as shown in Figure 2. Rats treated with MDPV (VEH-MDPV) had greater difference scores than did vehicle-treated control rats (VEH-VEH; p < 0.01). In rats administered MDPV, pretreatment with 5 mg/kg AMD3100 (5-AMD-MDPV) or 10 mg/kg AMD3100 (10-

AMD-MDPV) had reduced difference scores relative to MDPV-treated rats that did not receive AMD3100 (VEH-MDPV; p < 0.01).

800 ##

600

400 200 * * 0

-200 VEH VEH 1-AMD 5-AMD 10-AMD

Preference Score + S.E.M. Preference -400 MDPV MDPV MDPV MDPV

Figure 2. AMD3100 reduced MDPV Conditioned Place Preference.

Ultrasonic vocalizations. AMD3100 modestly suppressed 50-kHz USVs during the anticipation phase as demonstrated by within-subjects change-score data from the fourth day of injection, relative to baseline USVs (t(7) = 1.81, p = 0.057; H1: μ < 0). As hypothesized, MDPV enhanced 50 kHz USVs above baseline in that MDPV injection elicited significantly greater 50 kHz USVs in vehicle pretreated rats (t(7) = 2.33, p = 0.026; H1: μ > 0). In contrast, MDPV did not significantly elevate 50 kHz USVs in

AMD3100-pretreated rats (t(7) = 0.75, n.s.). There were no significant between-subjects differences observed during the anticipation (t(14) = 0.84, n.s.) or post-MDPV

(t(14) = 0.93, n.s.) time points (Oliver, Simmons, et al., 2018).

Figure 3. MDPV-induced ultrasonic vocalization modestly reduced by AMD3100.

Figure 4. AMD3100 reduced MDPV self-administration.

Self-administration. A two-way ANOVA revealed a significant interaction of number infusions across days and doses of AMD3100 (F(21, 126) = 3.963, p < 0.0001). A

Bonferonni post test revealed an effect of AMD3100 on Day 6 (5 mg/kg AMD3100, p <

0.05; 10 mg/kg AMD3100 p < 0.01) and Day 7 (5 mg/kg AMD3100, p < 0.05; 10 mg/kg

AMD3100 p < 0.001) of self-administration. Cumulative infusions of MDPV were also reduced (F(3, 30) = 3.581, p = 0.052) following 5 mg/kg (p < 0.05) and 10 mg/kg

AMD3100 (p < 0.05).

Cumulative Infusions 300

# 200 *

100

Number of Infusions of Number 0 VEH 2.5 AMD 5 AMD 10 AMD

Figure 5. CumulativeF(3, 30) infusions = 3.581, of pMDPV = 0.0252 and effects of AMD3100 in self- Bonferroni Posttest administration#VEH. vs. 5 AMD (p < 0.05) *VEH vs. 10 AMD (p < 0.05)

Discussion

The CXCR4 antagonist, AMD3100, reduced MDPV-evoked hyperlocomotion and conditioned place preference. This suggests that tonically active CXCR4 receptors mediate the stimulant and rewarding properties of MDPV. AMD3100 also suppressed

MDPV-elicited 50 kHz USV calls, relative to within-subject baseline rates and AMD- pretreated animals elicited fewer USVs prior to the fourth MDPV injection, during the anticipation phase. These results are in line with previous studies with a mechanistically similar psychostimulant, cocaine. As with MDPV, 5 mg/kg AMD3100 inhibited cocaine- induced hyperlocomotion and CPP (Kim et al., 2017a). Moreover, the suppression of

MDPV-induced hyperlocomotion is not likely due to generalized behavioral suppression as 10 mg/kg AMD3100 (the highest dose used here) did not affect basal locomotor behavior in the Kim et al. (2017) study. The USV results are also in agreement with previous studies in that MDPV has been shown to elicit 50 kHz USVs following injection or self-administration of MDPV and an MDPV-paired context also consistently elicits 50 kHz USVs (Simmons et al., 2016, 2017).

CHAPTER 3: AMD3100 MODULATION OF THE COGNITIVE AND EMOTIONAL

EFFECTS OF MDPV

Background

Psychostimulants such as amphetamine and cocaine are known to increase anxiety during withdrawal (Blanchard & Blanchard, 1999; Jeri, Sanchez, del Pozo, Fernandez, &

Carbajal, 1978; Sareen, Chartier, Paulus, & Stein, 2006; Vorspan, Mehtelli, Dupuy,

Bloch, & Lépine, 2015; Wang et al., 2013; Washton & Gold, 1984) and this withdrawal state is thought to drive drug craving and relapse (Erb, 2010; Wang et al., 2013).

Cognition is also modulated by synthetic cathinone use and CXCR4-CXCL12 signaling.

Synthetic cathinones mephedrone and methylone produce deficits in clinical and preclinical models of working memory, verbal recall, and object recognition (animals,

Motbey et al., 2012, den Hollander et al., 2013, Shortall et al., 2013, Lopez-Arnau et al.,

2014, Lopez-Arnau et al., 2015 humans, (Freeman et al., 2012, de Sousa Fernandes Perna et al., 2016, Jones et al., 2016). Recently, MDPV was shown to produce deficits in novel object recognition with concomitant neurodegeneration seen in related parahippocampal structures (Sewalia et al., 2018).

Synthetic cathinones also increase anxiety (Erb, 2010; Gregg & Rawls, 2014;

Ross, Reisfield, Watson, Chronister, & Goldberger, 2012). The synthetic cathinone,

MDPV was recently shown to increase anxiety 72 h after repeated drug exposure

(Philogene-Khalid, Hicks, Reitz, Liu-Chen, & Rawls, 2017a). In this study, the elevated plus maze (EPM) was employed to assess anxiety-like behavior in rodents 72 h after a

10-day binge (3 times daily, 1 hour intervals). The EPM exploits rodents’ innate aversion 30 to exposed, well-lit spaces, presumably as a means to avoid predation (Ellenbroek &

Charles Marsden, 2010) and accordingly, MDPV-exposed rats spent significantly more time in the closed arms of the EPM than controls. The effects of MDPV on anxiety-like behavior at earlier withdrawal time points is not known.

The early phase of withdrawal from psychostimulant use is associated with increased anxiety and deficits in cognition. In humans, anxiety is observed in the first 1-2 days of withdrawal from cocaine abuse (Aronson & Craig, 1986; McDougle, 1994). In rodents, cocaine also produces anxiety 24 h after binge-like exposure (Lisieski & Perrine,

2017; Sarnyai et al., 1995). Deficits in cognition are also observed at the 24 h withdrawal time point following exposure to cocaine, specifically in a test of novel object recognition

(NOR; Briand, Gross, & Robinson, 2008).

CXCL12 has been linked to novel object deficits. A recent study of gene transcripts associated with high altitude stress in mice found that CXCL12 is amongst a small handful of genes upregulated in the amygdala and hippocampus as shown by RNA sequencing (Cramer et al., 2019). While the authors ascribe this to changes in blood brain barrier permeability and angiogenesis, these findings demonstrate a possible role for

CXCL12 in novel object recognition task performance.

The CXCL12-CXCR4 system has been shown to modulate anxiety that results from inflammation (Yang et al., 2016). LPS injection in mice produced anxiety-like behavior that was reversed by AMD3100 or CXCR4 shRNA administration. Patch clamp experiments showed that CXCL12 increased glutamate transmission in the basolateral amygdala, a brain region that mediates anxiety-like behavior. A separate study found that 31

CXCL12 was increased in those with anxiety and personality disorders (Ogłodek, Szota,

Just, Moś, & Araszkiewicz, 2015; Ogłodek, Szota, Moś, Araszkiewicz, & Szromek,

2015). These studies suggest that MDPV-induced deficits in and changes in anxiety may be reversed with the CXCR4 antagonist, AMD3100.

Methods

Drug binge. A four-day escalating binge paradigm was employed to mimic the human tendency to both binge and escalate MDPV use. Injections of MDPV took place once every 3 hours, on each day. The first daily injection of MDPV was followed by a second injection of AMD3100 or vehicle. For the second and third daily injections, MDPV was given alone. On Days 1-3 of the binge, animals were administered 1 mg/kg MDPV. On

Day 4, animals were given 2 mg/kg MDPV. Elevated zero maze took place 24 hours or

72 hours after the last injection of MDPV.

Elevated zero maze. The elevated zero maze apparatus consisted of equal-sized runways

(19 in x 4 in) elevated 20 in from the ground, in the shape of a plus sign. Two of the arms had walls 13 in high on three sides (closed arm) and the open arm had no walls. The open arm was illuminated at 200 lux and the closed arm at 160 lux, as previously described

(Philogene-Khalid, Hicks, Reitz, Liu-Chen, & Rawls, 2017b). An animal was considered in an arm when the head, shoulders, and front limbs were present in the arm. The EZM was cleaned with 70% ethanol and allowed to dry between sessions. For each rat, the session began by placing the rat in the center of the maze, with the snout facing open and closed arms equally. Rats were allowed to explore arms freely for 10 min. Each session 32 was video recorded and time spent in each arm type was later scored by an experimenter blind to conditions. Amount of time spent in open arms is reported as a percentage of the entire 10 min session (Philogene-Khalid et al., 2017b).

Locomotor Control Experiments. To assess whether the effects of MDPV withdrawal on anxiety-like behavior was due to confounding locomotor abnormalities, locomotive behavior was assessed at time points that matched the EPM experiments. Rats underwent the 4-day escalating binge paradigm described above and locomotor control experiments were conducted at the 24 hour withdrawal time point.

Results

Elevated zero maze. AMD3100 attenuates MDPV-induced anxiolysis 24 hours ([F(5, 40)

= 3.908, p = 0.0056], Tukey 10-AMD/MDPV, p < 0.05) and 72 hours (not shown [F(2,

21) = 7.098, p = 0.0044], Bonferroni 10-AMD/MDPV, p < 0.05) following 4 days of repeated MDPV (1 mg/kg days 1-3; 2 mg/kg day 4), n = 7-8.

50 * ** 40

% Open Arm% Time+SEM 0 VEH VEH (5)AMD (5)AMD (10)AMD(10)AMD MDPV VEH MDPV VEH MDPV

Figure 6. 24 h Withdrawal from Binge MDPV is Anxiolytic, AMD3100 reverses effect.

80 ** 60

% Open Arm %Time+SEM Arm Open 0 VEH VEH (10)AMD MDPV MDPV

Figure 7. Effects of AMD3100 on MDPV-elicited Anxiogenesis 72 h after binge.

Figure 8. 24 h Locomotor Control for MDPV EZM: Total Activity.

Figure 9. 24 h Locomotor Control for MDPV EZM: Ambulation.

Figure 10. 24 h Locomotor Control for MDPV EZM: Stereotypy.

Locomotor Control Experiment. To determine whether the anxiolytic effects of MDPV observed at 24 h are due to changes in general locomotion, the open field test was performed on a separate set of animals, at the same time EPM would have been performed.

For Total activity, there was a main effect of time (F(23, 138) = 16.84, p < 0.0001) but no interaction (F(23, 138) = 1.209, p = 0.2475) or difference in cumulative total activity (t(6)

= 1.598, p = 0.1612). For ambulation, there was a main effect of time (F(23, 138) = 17.36, p<0.0001), but no interaction (F(23, 138) = 1.183, p = 0.2703), or cumulative difference

(t(6) = 1.562, p = 0.1692). For stereotypy, there was a main effect of time (F(23,

138)=1.195, p<0.0001) but no interaction (F(23, 138) = 1.209, p = 0.2593) or cumulative differences (t(6) = 1.483, p = 0.1886).

Novel Object Recognition. Repeat MDPV (10 days, 1 mg/kg, 24 hour withdrawal) impairs novel object recognition (F(3, 28) = 7.732, p = 0.0006, Bonferroni VEH vs.

VEH-MDPV, p < 0.05). A single dose of AMD3100 fails to attenuate this effect (p >

0.05). When AMD3100 is administered during MDPV binge, the antagonist does rescue

MDPV-induced deficits in NOR (F(5,38) = 3.864, p = 0.0117; Bonferroni VEH vs. VEH-

MDPV, p < 0.05, VEH-MDPV vs. 5AMD-MDPV, p < 0.01)

Effects of AMD on 24 h MDPV Binge WD Novel Object Recognition 0.8

0.6 * 0.4

0.2

Discrimination Index + SEM Index Discrimination 0.0 VEH VEH 5-AMD 5-AMD MDPV VEH MDPV

Figure 11. AMD3100 does not rescue expression of novel object recognition.

10 day binge 24 h withdrawal single dose of AMD prior to NOR

Effects of AMD on 24 h MDPV Binge WD Novel Object Recognition

0.8 ** 0.6 * 0.4

0.2

Discrimination Index + SEM + DiscriminationIndex 0.0 VEH VEH (5)AMD (5)AMD (10)AMD (10)AMD MDPV VEH MDPV VEH MDPV

Figure 12. The effects of AMD3100 on novel object recognition 24 h after MDPV binge.

Discussion

We found that withdrawal from MDPV paradoxically reduces anxiety-like behavior and that AMD3100 can ameliorate these effects, without affecting locomotor activity. The 24 hour withdrawal time point reflects expectation of a subsequent injection of MDPV (Vuong, Oliver, Scholl, Oliver, & Forster, 2010). Indeed, in Chapter 2, we showed that animals previously and repeatedly given MDPV elicit ‘anticipatory’ 50 kHz calls, which are thought to reflect a positive affective state experienced during anticipation for MDPV (Barker, Simmons, et al., 2014; Simmons et al., 2018).

It is possible that animals are experiencing a positive affective state more associated with anticipation for MDPV, rather than the anxiety state more often associated with withdrawal from psychostimulants in general. It would be interesting to record USVs during EPM to test this hypothesis. The EPM capitalizes on the conflict between natural avoidance and the exploratory drive of rodents (Ellenbroek & Charles

Marsden, 2010). Thus the reduced anxiety observed at the 24 h withdrawal time point may reflect an increase in exploratory drive produced by MDPV. The significance in this interpretation lies in the possibility that acute withdrawal from MDPV may be a time of increased risk-taking behavior. Indeed risky and sometimes fatal behaviors are observed in the days following an MDPV binge (Oliver, Palamar, et al., 2018).

CHAPTER 4: NEURAL SUBSTRATES OF THE EFFECTS OF MDPV

Background

Psychostimulants such as cocaine and MDPV produce changes in brain reward regions that are thought to drive addiction. The reinforcing effects of both psychostimulants are attributed to increases in extracellular dopamine in the nucleus accumbens (Baumann et al., 2012; Baumann, Partilla, Lehner, et al., 2013a; Koob &

Bloom, 1988; White & Kalivas, 1998). Binge-like intake is common in users of both drugs and after repeated use, neuroadaptations to high levels of dopamine in the NAC occur.

Post-synaptic density-95 (PSD95) is a synaptic scaffolding protein in the postsynaptic density of dendritic spines. PSD95 anchors glutamate receptors to the postsynaptic density and thus expression of PSD95 fluctuates with both glutamate receptor expression as well as with dendrite morphometrics as a whole (Ghasemzadeh,

Vasudevan, Mueller, Seubert, & Mantsch, 2009; Ghasemzadeh, Mueller, & Vasudevan,

2009; Yao et al., 2004). For example, nucleus accumbens dendrite length, complexity, spine density, and AMPA (glutamate) receptor density decreases in the weeks following repeated cocaine exposure. This reduction in dendrite morphometrics as well as sites of excitatory input is thought to drive the behavioral sensitization and relapse to cocaine seeking behavior known to occur at these protracted withdrawal time points (Grimm,

Hope, Wise, & Shaham, 2001; Wolf & Ferrario, 2010a). PSD95 in the nucleus accumbens core is especially linked to the reemergence of cocaine-seeking behavior in

43 the weeks following cessation of cocaine administration (Ghasemzadeh, Vasudevan,

Mueller, Seubert, & Mantsch, 2009; Ghasemzadeh, Mueller, & Vasudevan, 2009).

The NAC core in particular is involved in impulsivity and motor programs related to reward-seeking behavior. Impulsive choice is exemplified by choosing a small or poor reward that is available immediately instead of a larger but delayed reward (Cardinal et al., 2001). The NAC core is also involved in the acquisition of appetitive instrumental learning (e.g. lever-pressing for food) and this process is dependent on coincident glutamate and dopamine receptor activation (Smith-Roe & Kelley, 2000).

Dendrites get narrower as they extend further away from the cell soma

(Athabasca, n.d.). Overall synaptic efficacy is proportional to the surface area of dendrite branches (Komendantov & Ascoli, 2009). Indeed dendrites and spines themselves are thought to be efficient means of increasing the receptive surface area of a neuron in the most spatially conservative way. A similar evolutionary advantage is ascribed to brain sulci, cortical folds that expand the function of the cortex without increasing the size of the skull and head (Cusack, 2005).

The first few days of abstinence from repeated cocaine exposure is distinct from more protracted cocaine withdrawal time points. NAC neurons exhibit a decrease in firing capacity as demonstrated by alterations in sodium, calcium, and potassium conductance (Cooper, 2002; Dong et al., 2006; Hu, Basu, & White, 2004; Kourrich &

Thomas, 2009; Zhang, Hu, & White, 1998).

One approach to establishing the neural substrates of psychostimulant addiction is to identify shared adaptations between different drugs that produce a common behavioral and neurochemical output (Kourrich & Thomas, 2009). Thus we tested whether cocaine and the mechanistically similar psychostimulant, MDPV, produce similar effects on nucleus accumbens dendrite morphometrics.

Methods

Drug Binge. Animals were given i.p. injections of 1 mg/kg MDPV three times a day, at one hour intervals, for 10 days. Twenty-four hours after the last injection, animals were euthanized for gene expression and Golgi-Cox staining.

CXCL12 gene expression. CXCL12 mRNA was assessed in animals that underwent the

10-day binge MDPV procedure. Brains were harvested 24 h after the last MDPV injection. Animals were briefly anesthetized with 5% isofluorane gas and rapidly decapitated with a guillotine. Brains were harvested by dissection and alternating hemispheres were flash frozen in 2-methylbutane and stored in a -80 degree freezer.

Hemispheres were mounted on a cryostat and sections were made until the anterior commissure was visible. Tissue punches (1 mm) were made centered around the anterior commissure to ensure capture of the nucleus accumbens. RNA was isolated using a

Quick-RNA Miniprep kit (Zymo Research, Irvine, CA). cDNA was synthesized using a

High Capacity cDNA Reverse Transcription kit (Applied Biosciences, Foster City, CA).

Quantitative real-time PCR was employed with the TaqMan Fast Advanced Master Mix and the TaqMan Fast Advanced Master Mix and the TaqMan Gene Expression Assays

45 for CXCL12 (Rn00573260_m1) and the internal control gene 18S rRNA

(Hs99999901_s1) using the StepOnePlus Real-Time PCR System (Applied Biosystems).

Relative gene expression was measured according to the 2-ΔΔCT method, as described previously (Kim et al., 2017a).

Golgi-Cox Staining. Alternating hemispheres from animals were placed in Golgi-Cox solution (Glaser & Van der Loos, 1981) and stored in the dark for 12 days, as previously described (Holliday et al., 2016). Golgi-Cox solution was refreshed after 2 days. At the end of the 12-day immersion period, brains were placed in 30% sucrose until vibratome sectioning. Brains were sectioned in 200 µm coronal sections and mounted on a gel- subbed slide. Sections were then developed according to an established method (Gibb &

Kolb, 1998) wherein sections were be alkalinized in ammonium hydroxide and fixed and developed in Kodak rapid fixer prior to dehydration in increasing concentrations of ethanol baths and then cleared in a 1:1:1 mixture of xylene, 100% ethanol, and chloroform. Medium spiny neurons (MSNs) neurons from the nucleus accumbens were traced at 60X magnification using Neurolucida software (MBF Bioscience, Williston,

VT, USA) on a Nikon Optiphot-2 bright-filed microscope (Holliday et al., 2016). MSNs were defined as having distinct individual dendrites and dense groupings of at least four adjacent spines distributed along the length of dendritic segments. Only well-stained

MSNs with dendrites unobstructed by neighboring cells or blood vessels that can be traced without interruption were analyzed. Interneurons, with distinct individual dendrites, but without dense spines, and astrocytes, with numerous projections in a radial distribution, were not traced (Kobrin et al., 2016; Tepper, Tecuapetla, Koós, & Ibáñez- 46

Sandoval, 2010). Dendrite length and complexity (arborizations and branching; Ehlinger,

Bergstrom, McDonald, & Smith, 2012) were quantified with a Sholl analysis as previously described (Sholl, 1953).

Statistical Analysis. Gene expression data were analyzed with a Student’s t-test. Each dendrite morphometric was assessed using a two-way ANOVA followed by Bonferroni posttests.

Results

Dendrite Morphology. Nucleus accumbens core Golgi-Cox-stained neurons 24 h after withdrawal from 10 days drug binge. Intersections (dendrite complexity) reduced by

MDPV (F(2,21)=4.419, p=0.0250; Saline vs MDPV p<0.05). Dendrite length reduced by cocaine and MDPV (F(2,21)=7.248, p=0.0040; Saline vs Cocaine, p<0.05; Saline vs

MDPV, p<0.01). Surface area reduced by MDPV (F(2,21)=4.498, p=0.0237; Saline vs

MDPV, p<0.05). Volume reduced by cocaine (F(2,21)=1.733, p=0.2010; Saline vs

Cocaine, p<0.05). Diameter reduced by MDPV (F(2,21)=2.121, p=0.1449; Saline vs

MDPV, p<0.05). Endings reduced by MDPV (F(2,21)=3.826, p=0.0383; Saline vs

MDPV, p<0.05). No significant changes in dendrite morphology in nucleus accumbens shell.

CXCL12 Gene Expression. CXCL12 gene expression increased in the nucleus accumbens core 24 h after a 10-day drug binge.

PSD95 Gene Expression. There was a trend toward a decrease in PSD95 gene expression at the 24 h withdrawal time point, following 10 days of drug binge. The cocaine-induced

47 decreases in PSD95 were not significantly different from saline-treated control animals

(t(10) = 1.374, p = 0.0997). The MDPV-induced decrease was also not significant (t(11)

= 1.790, p = 0.0505). While these are only a non-significant trends, the effect sizes are robust in the MDPV group (d = 1.08).

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Figure 13. Dendrite Morphometrics in nucleus accumbens core following cocaine or

MDPV binge.

Saline

Cocaine

MDPV

Figure 14. Nucleus accumbens core dendrites following cocaine or MDPV

Figure 15. PSD95 is downregulated in the nucleus accumbens core following MDPV binge.

NAc CXCL12 2.5 * 2.0

1.5

1.0

0.5

0.0

Fold Change CXCL12 + CXCL12 SEM Change Fold

MDPV Vehicle Treatment

Figure 16. CXCL12 in nucleus accumbens 24 h withdrawal from MDPV Binge

Discussion

We showed that binge-like consumption of MDPV increases CXCL12 gene expression in the nucleus accumbens. This finding is in line with the central hypothesis that MDPV upregulates CXCL12 in brain reward region and that CXCR4 activation by

CXCL12 is a critical component of the behavioral effects of MDPV. Moreover, CXCR4 receptors are found in the nucleus accumbens (Banisadr et al., 2002; Jordan Trecki et al.,

2010) and CXCL12 in the nucleus accumbens potentiates the locomotor activating effects of cocaine (Trecki & Unterwald, 2009). The functional relevance of CXCL12 upregulation in the NAC core lies in the ability of this chemokine to modulate neuronal activity.

Interestingly, NAC CXCL12 gene expression was not changed by cocaine exposure (Kim et al., 2017a) and this may reflect mechanistic disparities between the two psychostimulants. The dose of MDPV employed was 10 times lower than that of cocaine, however, MDPV is 50 times more effective than cocaine at blocking dopamine transporters and 10 times less effective than cocaine at blocking serotonin transporters

(Baumann, Partilla, Lehner, et al., 2013b). These mechanistic distinctions are the likely cause of behavioral and neurochemical differences between MDPV and cocaine.

While many reports highlight increases in nucleus accumbens dendrite morphometrics and excitability, this position reflects protracted withdrawal time points, namely the weeks to months following repeated drug exposure. This is an important aspect of addiction as it provides a neural substrate for relapse, a hallmark of psychostimulant, and drug addiction in general. A less oft considered aspect of addiction 53 involves early time points in particular the first few days of withdrawal from repeated drug exposure. The first reports of alterations in the excitability of nucleus accumbens neurons following repeat cocaine exposure exhibit decreased excitability 16 h to 3 days into withdrawal (Henry & White, 1991; Zhang et al., 1998) which switches to an increase in excitability 30 days into withdrawal (Henry & White, 1991). Moreover, 24 h after return to cocaine exposure following a binge and 10-14 days of withdrawal, a similar decrease in NAC excitability is observed (Thomas, Beurrier, Bonci, & Malenka, 2001).

In sum, the NAC is less excitable during early withdrawal and more excitable during late withdrawal from repeated cocaine exposure.

CHAPTER 5: CONCLUSIONS

These studies provide compelling evidence that CXCL12-CXCR4 signaling drives the effects of MDPV on the brain and behavior. MDPV is a psychostimulant that is mechanistically similar, but far more potent than cocaine and many of the effects seen here reflect this. Like cocaine, MDPV exposure increased CXCL12 in a brain reward region (Chapter 4), produced hyperlocomotive, rewarding, and reinforcing effects

(Chapter 2) and altered effects on measures of cognition and anxiety (Chapter 3).

Important differences emerged in many of these measures that likely reflect mechanistic differences between these drugs.

The efficacy of AMD3100 against MDPV-evoked reward behaviors is in line with previous findings with cocaine. Cocaine, a less potent but mechanistically similar drug produces hyperlocomotive, rewarding, and reinforcing effects in the open field,

CPP, and self-administration tests, respectively. Similar effects are seen with MDPV

(Chapter 1) and importantly, CXCR4 inhibition with AMD3100 rescued these effects with each both psychostimulants (Kim, Connelly, Unterwald, & Rawls, 2017; Oliver,

Simmons, et al., 2018). These findings implicate CXCR4-CXCL12 signaling in the rewarding effects of each drug. A putative mechanism involved AMD3100 blockade of

CXCR4-CXCL12 signaling that occurs with psychostimulant exposure. Both cocaine and

MDPV increase CXCL12 in brain reward regions (Kim et al., 2017b; Kim, Rawls, et al.,

2016; Chapter 4) and CXCL12 has been shown to modulate neuronal activity in these brain regions. For example, midbrain CXCL12 increases dopamine in the striatum

(Skrzydelski et al., 2007). Thus psychostimulant-induced increases in CXCL12 may elicit 55 increases in dopamine in the striatum which is known to drive drug-related behaviors such as hyperlocomotion, CPP, and self-administration. In agreement, CXCL12 potentiates cocaine-induced hyperlocomotion and AMD3100 blocks this effect

(Skrzydelski et al., 2007; Trecki & Unterwald, 2009).

We have shown that AMD3100 decreases active lever responding during cue and drug plus cue-primed reinstatement (Kim, Rawls, et al., 2016). Further, dopamine receptor activation also drives drug-primed reinstatement to psychostimulant-seeking behavior (Kalivas & McFarland, 2003; Kalivas & Volkow, 2005; See, Kruzich, &

Grimm, 2001) and CXCL12 has been shown to modulate dopamine release

(Bhattacharyya et al., 2008; Guyon et al., 2006; Heinisch & Kirby, 2010; Miller,

Banisadr, & Bhattacharyya, 2008). Changes in reinstatement suggest alterations in glutamate as this neurotransmitter drives this relapse-like process (Knackstedt & Kalivas,

2009; Knackstedt, Melendez, & Kalivas, 2010a).

PSD95 is a synaptic scaffolding protein that is found in abundance in the post- snaptic density of dendritic spines (Chen et al., 2011). Specifically, PSD95 anchors glutamate receptors (e.g., AMPA, NMDA, mGluR1) and other synapse scaffolding proteins to the cell membrane and thus expression of PSD95 fluctuates with glutamate receptor expression and dendrite morphometrics as a whole (Ghasemzadeh, Vasudevan,

Mueller, Seubert, & Mantsch, 2009; Ghasemzadeh, Mueller, & Vasudevan, 2009; Yao et al., 2004). Decreased PSD95 likely reflects a decrease in glutamate receptor and synaptic scaffolding protein content in the reduced dendrites present in the NAC core following cocaine and MDPV exposure (Chapter 4). These findings are in line with previous studies 56 showing reduced PSD95 and AMPA receptor expression 24 h after binge-like cocaine exposure (Wolf & Ferrario, 2010b; Yao et al., 2004). PSD95 in the nucleus accumbens core is especially linked to the reemergence of cocaine-seeking behavior in the weeks following cessation of cocaine administration (Ghasemzadeh, Vasudevan, Mueller,

Seubert, & Mantsch, 2009; Ghasemzadeh, Mueller, & Vasudevan, 2009). The implications of these findings lies in the function of glutamate receptor expression at different points of psychostimulant withdrawal.

During protracted psychostimulant withdrawal, AMPA receptor and dendrite morphometrics in the nucleus accumbens are known to increase and these changes coincide with incubation of drug-seeking behavior. For example, NAC dendrite morphometrics, PSD95 expression, and AMPA receptor density increase in the weeks following cessation of repeated cocaine exposure (Ferrario, Goussakov, Stutzmann, &

Wolf, 2012; Wolf & Ferrario, 2010b). Early withdrawal time points, however are associated with opposite effects.

Glutamate homeostasis is critical to neural function and over 90% of brain glutamate is cleared by the glutamate transporter, GLT-1 (Tanaka et al., 1997). Repeated cocaine reduces GLT-1 expression and GLT-1 activators rescue this effect and can reverse the rewarding effects of cocaine (Cornish & Kalivas, 2000b; Knackstedt,

Melendez, & Kalivas, 2010b; Rasmussen, Baron, Kim, Unterwald, & Rawls, 2011).

Indeed, the GLT-1 activator, ceftriaxone, is currently being investigated as a potential cocaine therapeutic in ongoing clinical trials (Reissner et al., 2015).

ICV CXCL12 increases glutamate in the nucleus accumbens as shown by microdialysis (Kim, Rawls, et al., 2016). This likely occurs through activation of CXCR on astrocytes which has been shown to induce TNFα and then glutamate release (Calì &

Bezzi, 2010a; Callewaere et al., 2006). This process can occur on a millisecond timescale and thus may be detectable by electrophysiological measures of neural activity.

Importantly, cocaine-induced increases in NAC glutamate are blocked by AMD3100

(Kim, Rawls, et al., 2016).

The effects of cocaine on CXCL12 in the nucleus accumbens take hours to manifest. We have shown that both acute and chronic cocaine increase in CXCL12 in the

NAC 60-240 min after systemic cocaine administration. In this microdialysis study, the increases in CXCL12 were greater in chronic cocaine-exposed animals. AMD3100 blockade of reinstatement likely reflects a reduction in glutamate and CXCL12 can increase astrocytic release of glutamate on a millisecond time scale (Bezzi et al., 2001a).

Repeated cocaine exposure decreases GLT-1 expression in nucleus accumbens, but not in the prefrontal cortex. AMD3100 given prior to self-administration sessions reversed this effect (Kim, Rawls, et al., 2016).

The psychostimulants cocaine and MDPV produce deficits in cognition

(Mahoney, 2018; Sewalia et al., 2018; Spronk, van Wel, Ramaekers, & Verkes, 2013).

MDPV was shown to produce deficits in novel object recognition with concomitant neurodegeneration seen in related parahippocampal structures (Sewalia et al., 2018).

Cognitive deficits resulting from psychostimulant exposure is associated with poorer

58 treatment outcomes (Mahoney, 2018; Spronk et al., 2013), which expands the importance of assessing these deficits in novel psychostimulants, such as MDPV.

Reductions in NAC core dendrite morphometrics may be related to the NOR deficits observed at the same time point as depletion of dopamine in the NAC core impairs NOR performance (Coccurello, Adriani, Oliverio, & Mele, 2000; Nelson, Thur,

Marsden, & Cassaday, 2010). AMD3100 reversal of MDPV-induced deficits in NOR suggest CXCR4-CXCL12 signaling involvement in this process. Few studies have examined chemokine involvement in NOR and only one study has linked CXCL12 to

NOR performance. A recent study of gene transcripts associated with high altitude stress in mice found that CXCL12 is amongst a small handful of genes upregulated in the amygdala and hippocampus as shown by RNA sequencing (Cramer et al., 2019). While the authors ascribe this to changes in blood brain barrier permeability and angiogenesis, these findings demonstrate a possible role for CXCL12 in NOR and supports the finding that AMD3100 rescues MDPV-induced NOR deficits.

It is not surprising that MDPV alters anxiety and cognition at the 24 hour withdrawal time point. What is surprising is that MDPV produced decreases in anxiety at this time point. Cocaine, the mechanistically similar psychostimulant has repeatedly been shown to increase anxiety within the first day of withdrawal from repeated exposure

(Aronson & Craig, 1986; Lisieski & Perrine, 2017; McDougle, 1994; Sarnyai et al.,

1995) and MDPV also increases anxiety at the 72 hour withdrawal time point (Sewalia et al., 2018). We have shown increases in CXCL12 at early withdrawal time points and a trend toward a decrease in CXCL12 at more protracted withdrawal time points (Kim et 59 al., 2017b; Kim, Rawls, et al., 2016). For example, at 24 hour withdrawal from MDPV and cocaine there is an increase in CXCL12 gene expression in the NAC and VTA and there is a trend toward a decrease in CXCL12 30 days after repeated cocaine administration. Humans more than 2 weeks abstinent from cocaine exhibit lower levels of

CXCL12 (Araos, Pedraz, Serrano, Lucena, Barrios, García‐Marchena, et al., 2015). Thus it appear that CXCL12 is higher early in withdrawal and lower later in withdrawal.

However, parallel changes in CXCL12 with cocaine and MDPV do not explain differences in anxiety-like behavior observed throughout withdrawal.

Mechanistic differences between cocaine and MDPV may explain why only

MDPV is anxiolytic early in withdrawal but anxiogenic later in withdrawal. Both cocaine and MDPV increase dopamine, norepinephrine, and serotonin by blocking the transporters (i.e. DAT, NET, SERT) for these neurotransmitters (Baumann, Partilla,

Lehner, et al., 2013a). MDPV, however, blocks NET at 10 times the potency and DAT at

50 times the potency, and SERT at 10 times lower potency than cocaine. These differences in potency is why MDPV is administered at doses 10 times lower than cocaine doses employed and indeed parallel behavioral results are obtained using these dose equivalencies (e.g., Gregg et al., 2016; Oliver, Simmons, et al., 2018; Simmons et al., 2018). Indeed serotonin deficiencies after binge-like cocaine cessation is thought to drive the anxiogenic effects of cocaine (Parsons, Koob, & Weiss, 1995). These deficiencies in serotonin would be absent in MDPV users. Moreover, MDPV users exhibit manic, irrational behavior days after initial drug consumption (Oliver, Palamar, et

60 al., 2018). Thus the early withdrawal syndrome produced by MDPV may be unique to this synthetic cathinone and certainly warrants further investigation.

AMD3100 blockade of the effects of MDPV on anxiety are limited to the early withdrawal anxiolytic effect. This may be due to still heightened levels of CXCL12 present at the 24 hour (Chapter 4) but not at more protracted withdrawal time points with the mechanistically similar drug, cocaine (Araos, Pedraz, Serrano, Lucena, Barrios,

García-Marchena, et al., 2015a; Kim, Connelly, Unterwald, & Rawls, 2017c; Kim,

Rawls, et al., 2016). Higher levels of CXCL12 in the striatum (Chapter 4; Guyon et al.,

2006c) suggest heightened neuronal activity in this brain region (Calì & Bezzi, 2010b,

2010a; Guyon et al., 2006c). The NAC projects to the basal ganglia to produce reward and other motivated behaviors (Yin, Ostlund, & Balleine, 2008). Indeed deep brain stimulation of the NAC has been shown to ameliorate symptoms of anxiety disorders including obsessive compulsive disorder (Sturm et al., 2007).

Limitations

There are several limitations of the current studies. These and previous studies strongly implicate CXCR4-CXCL12 involvement in the effects of psychostimulants, however, the CXCR4 antagonist AMD3100 may not be a viable therapeutic option for psychostimulant addicted patients. AMD3100 requires parenteral administration (Hendrix et al., 2000; Stone et al., 2007) which could be triggering for addicts. This is especially problematic for intravenous psychostimulant users exposed to drug paraphernalia such as the needles used to administer the drug (Dudish-Poulsen & Hatsukami, 1997; Reid et al.,

2003; Robbins, Ehrman, Childress, & O’Brien, 1999). Moreover, the highest dose of

AMD3100 used here, 10 mg/kg, produces nonsignificant but noticeable hypolocomotive effects. This can be seen in the open field results here (Chapter 2) and previously published (Kim, Connelly, et al., 2016). Importantly, lower doses of AMD3100 (e.g. 5 mg/kg) do not significantly or noticeably affect locomotion and future studies may explore means of modulating CXCR4-CXCL12 signaling without the use of a drug that may trigger relapse in intravenous drug users.

Another important limitation of the current studies is that AMD3100 may only be effective if administered before or during psychostimulant abuse. This prediction is based on the finding that AMD3100 was only effective against NOR when coadministered with

MDPV (Chapter 4). Efficacy of AMD3100 on MDPV-induced anxiety may not require coadministration but all other behavioral tasks did (locomotion, CPP, USV, and self- administration.

Directions for future research

This exciting body of work has laid the foundation for numerous future investigations of chemokine modulation of psychostimulant addiction. The paradoxical anxiolytic effect of MDPV can be further explored using other models of anxiety or impulsivity, such as the light-dark box or a delayed reinforcer task, respectively (Cardinal et al., 2001; Takao & Miyakawa, 2006).

Future studies will examine the efficacy of AMD3100 in rescuing cocaine and

MDPV-induced decreases in dendrite morphometrics. AMD3100 is expected to reverse

62 morphological alterations in both drug groups. This prediction is based on the finding that acute and protracted withdrawal from cocaine as well as CXCR4 activation by CXCL12 are associated with increases in dendrite morphometrics (Muzio et al., 2016) and

AMD3100 has been shown to reverse these CXCR4-mediated effects (Pitcher et al.,

2014). These results would suggest that CXCR4 signaling can modulate MDPV-induced neuroadaptations in a region of the brain that is critical to drug reward.

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