The Role of Vesicular Zinc in Instrumental Conditioning and Drug-Evoked Plasticity

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2020-09-03 The Role of Vesicular Zinc in Instrumental Conditioning and Drug-Evoked Plasticity

Thackray, Sarah Elizabeth

Thackray, S. E. (2020). The Role of Vesicular Zinc in Instrumental Conditioning and Drug-Evoked Plasticity (Unpublished doctoral thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/112494 doctoral thesis

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The Role of Vesicular Zinc in Instrumental Conditioning and Drug-Evoked

Plasticity

Sarah Elizabeth Thackray

A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

GRADUATE PROGRAM IN PSYCHOLOGY

CALGARY, ALBERTA

SEPTEMBER, 2020

ABSTRACT

Zinc is critical for the functioning of all cells. A subset of the zinc in the brain

(vesicular zinc) acts as a neurotransmitter and is capable of modulating a variety of receptors. Not all areas of the brain contain vesicular zinc; however, there are high amounts found in the striatum, neocortex, and limbic regions. Some regions have received more attention than others concerning the function of vesicular zinc. Those that have been studied have found that vesicular zinc is important for synaptic plasticity. Less studied regions include areas involved in instrumental conditioning, motivation and reward. A commonly used model to study the role of vesicular zinc is the zinc transporter 3 (ZnT3) knockout (KO) mouse which lack the protein solely responsible for loading zinc into vesicles and thus shows a complete absence of vesicular zinc. The purpose of this thesis was to examine the behaviour of ZnT3 KO mice (compared to wildtype mice) on instrumental conditioning tasks as well as on their response to cocaine. Drugs of abuse, including cocaine, can be used to probe the functioning of the reward pathways.

Results found no difference in instrumental conditioning in ZnT3 KO mice. There were, however, differences in response to cocaine which, for the most part, were restricted to one sex or the other. In general, ZnT3 KO mice had reduced locomotor response to cocaine, particularly at higher doses and in females. They also showed differences in “memory” of cocaine experience, with male KO mice more affected. Overall, findings suggest that vesicular zinc is involved in both acute response to cocaine and in the long-term memory of drug-associated cues. iii

PREFACE

At the time of writing, none of the manuscripts included in this thesis have been submitted for publication. iv

ACKNOWLEDGEMENTS

First, I would like to thank Richard Dyck for guiding and supporting me through my PhD journey. I’d also like to thank the other “basement dwellers” that have provided both friendship and mentorship to me over the years including

Drs. Veronika Kiryanova, Brendan McAllister, Vicki Smith, Jhen Shankara, and

Simon Spanswick. I especially need to thank Sarah Bryden and Selena Fu for their extraordinary help with carrying out my research; it would have been much less organized and colourful without you two. I would also like to thank the other members of the Dyck Lab who I’ve worked with over the last five years including

Katy Sandoval, Nicoline Bihelek, Angela Pochakom, Nicole Niewinski, Mariya

Markovina, and Alex Debusschere. Finally, I’d like to thank my family for their support over the years. They keep putting up with me talking about a lot of things that go over their heads, but they listen none the less. v

DEDICATION

To my parents, Janet and Michael vi

TABLE OF CONTENTS

ABSTRACT ...... ii

PREFACE...... iii

ACKNOWLEDGEMENTS ...... iv

DEDICATION ...... v

TABLE OF CONTENTS ...... vi

LIST OF TABLES ...... xi

LIST OF FIGURES ...... xii

LIST OF SYMBOLS, ABBREVIATIONS, NOMENCLATURE ...... xviii

1 CHAPTER 1: ...... 1

1.1 INTRODUCTION ...... 1

1.2 ZINC ...... 2

1.2.1 Zinc in the Body ...... 2

1.2.2 ZnT3 and Vesicular Zinc ...... 3

1.2.3 Zincergic Anatomy ...... 5

1.2.4 Zinc Signaling in the Brain ...... 10

1.2.4.1 Vesicular zinc as a neuromodulator ...... 11

1.2.4.2 Vesicular zinc in synaptic plasticity ...... 16

1.2.4.2.1 In behaviour ...... 18

1.2.4.2.2 In LTP and LTD ...... 20

1.2.4.2.3 Zinc in the postsynaptic density ...... 21

1.2.5 Genetic Manipulations and ZnT3 Knockout Mice ...... 22

1.3 STRIATUM ...... 24

1.3.1 Striatal Anatomy ...... 24

1.3.1.1 Macrocircuitry ...... 25 vii

1.3.1.2 Microcircuitry ...... 28

1.3.1.2.1 Striatal synapses ...... 29

1.3.2 Striatal Plasticity ...... 32

1.3.2.1 LTP and LTD ...... 32

1.3.3 Striatal Function ...... 33

1.4 DRUG-EVOKED BEHAVIOUR AND PLASTICITY ...... 35

1.4.1 Behavioural Plasticity ...... 35

1.4.2 Circuits and Neurotransmitters ...... 38

1.4.3 Cellular Mechanisms of Plasticity ...... 39

1.4.3.1 Cocaine-specific mechanisms ...... 40

1.4.4 Sex Differences ...... 44

1.5 rationale and overview of experiments ...... 44

2 CHAPTER 2: ...... 49

2.1 INTRODUCTION ...... 49

2.2 METHOD ...... 50

2.2.1 Animals ...... 50

2.2.2 Training ...... 51

2.2.2.1 Food restriction ...... 51

2.2.2.2 Conditioning boxes ...... 51

2.2.2.3 Training procedure ...... 52

2.2.3 Differential Reinforcement of Low Rate (DRL) Schedule ...... 53

2.2.4 Progressive Ratio (PR) ...... 55

2.2.5 Statistical Analyses ...... 55

2.3 RESULTS ...... 55

2.3.1 Training ...... 55 viii

2.3.2 DRL Schedule ...... 59

2.3.3 PR Schedule ...... 64

2.4 DISCUSSION ...... 66

2.5 Acknowledgements ...... 70

3 CHAPTER 3: ...... 71

3.1 INTRODUCTION ...... 71

3.2 METHOD ...... 73

3.2.1 Animals ...... 73

3.2.2 Drugs ...... 73

3.2.3 Behaviour ...... 74

3.2.3.1 Habituation ...... 74

3.2.3.2 Dose response ...... 74

3.2.3.3 Locomotor sensitization ...... 77

3.2.3.4 Stereotyped behaviours ...... 77

3.2.4 Fast Scan Cyclic Voltammetry ...... 78

3.2.4.1 Electrode fabrication ...... 78

3.2.4.2 Electrode calibration ...... 79

3.2.4.3 Surgeries ...... 79

3.2.4.4 Analysis ...... 81

3.2.5 Morphology ...... 82

3.2.5.1 Procedure ...... 82

3.2.5.2 Quantification ...... 83

3.2.6 Vaginal Lavage and Cytology ...... 84

3.2.7 Statistical Analyses ...... 85

3.3 RESULTS ...... 85 ix

3.3.1 Behaviour ...... 85

3.3.1.1 Dose response ...... 85

3.3.1.2 Sensitization ...... 89

3.3.1.3 Focused stereotypy ...... 89

3.3.2 Fast Scan Cyclic Voltammetry ...... 92

3.3.3 Morphology ...... 96

3.3.4 Estrus Cycle Effects ...... 96

3.4 DISCUSSION ...... 98

3.5 Acknowledgements ...... 104

4 CHAPTER 4: ...... 105

4.1 INTRODUCTION ...... 105

4.2 METHOD ...... 107

4.2.1 Animals ...... 107

4.2.2 Drugs ...... 107

4.2.3 Conditioned Place Preference ...... 108

4.2.4 Statistical Analyses ...... 109

4.3 RESULTS ...... 112

4.3.1 Distance Travelled under Influence of Cocaine ...... 112

4.3.2 Conditioned Place Preference ...... 115

4.4 DISCUSSION ...... 119

4.5 Acknowledgements ...... 123

5 CHAPTER 5: ...... 124

5.1 INTRODUCTION ...... 124

5.2 METHOD ...... 125

5.2.1 Animals ...... 125 x

5.2.2 Drugs ...... 126

5.2.3 Behaviour ...... 127

5.2.4 Brain Processing ...... 130

5.2.5 Statistical Analyses ...... 130

5.3 RESULTS ...... 131

5.4 DISCUSSION ...... 137

5.5 Acknowledgements ...... 141

6 CHAPTER 6: ...... 142

6.1 Instrumental conditioning ...... 145

6.2 Differences in short-term response ...... 145

6.3 Differences in long-term effects ...... 149

6.4 Sex differences ...... 150

6.5 Future directions ...... 153

6.6 Conclusions ...... 154

References ...... 156

Appendix ...... 185

Figures ...... 186

LIST OF TABLES

Table 4.1 P values for cocaine value compared to the saline value (i.e. do they spend significantly more time in the cocaine compartment than the saline compartment?). * represents p < .05, ** represents p < .01; a represents medium effect size, aa represents large effect size...... 118

Table 5.1 Times to reach the maximum response to cocaine and time taken to return to baseline in mice in the cocaine groups over the 7 days of testing. Data are presented as M ± SD (Median)...... 136

xii

LIST OF FIGURES

Figure 1.1 Zincergic Anatomy. Brown regions indicate presence of vesicular zinc, with darker regions having higher concentrations. AMY: amygdala, DSTR: dorsal striatum, HPC: hippocampus, NAc: nucleus accumbens, PFC: prefrontal cortex,

SN: substantia nigra, THAL: thalamus, VTA: ventral tegmental area...... 9

Figure 1.2 Striatal Circuitry. An overview of the macrocircuitry of the striatum including afferent projections and direct vs indirect efferent projections...... 27

Figure 1.3 Striatal Synapse. A model synapse in the striatum showing the various cell types and receptors that are present...... 31

Figure 1.4 Overview of cocaine changes at a striatal synapse. Cocaine acts by blocking the dopamine reuptake transporter (DAT) leading to prolongment of dopamine in the synapse. Acute or chronic cocaine has been shown to increase activation of D1 dopamine receptors, increase insertion of AMPA receptors into the postsynaptic membrane, increase BDNF, and increase activation of the ERK signaling pathway. Additionally, it has been shown to alter the functioning of the

NMDA receptor...... 43

Figure 1.5 Circuits Under Investigation. A sagittal section of a mouse brain stained for vesicular zinc showing the circuits under investigation in this thesis.

Solid white arrows indicate the pathway affected by drugs of abuse. Dashed white arrows are the pathway involved in instrumental conditioning (and somewhat with addiction). The yellow dashed lines are other brain regions involved in modulating those circuits. AMY: amygdala, DSTR: dorsal striatum, xiii

HPC: hippocampus, NAc: nucleus accumbens, PFC: prefrontal cortex, SN: substantia nigra, THAL: thalamus, VTA: ventral tegmental area...... 46

6 sec delay and advanced by 6 sec every 4 days. Following 24 days on the DRL schedule, mice were kept on a PR schedule for 5 days...... 54

Figure 2.2 Mice Meeting Inclusion Criteria. Mice were given a minimum of 7 days to meet the inclusion criteria. Other studies have found that ~75% meet the criteria within 10 days. Mice were allowed up to 17 days of training then excluded from the study if the criteria were not met by this time. This figure shows the breakdown per group of when and if mice met the inclusion criteria...... 57

Figure 2.3 . Weight Change. Mice were started on food restriction regimen 2 days prior to commencement of training and remained restricted for the duration of the training phase. Weight change was calculated by subtracting weight on the first day of training from initial weight before restriction began. (A) Weight change for all mice in each group. (B) Weight change for mice that met inclusion criteria.

Figure 2.4 . DRL Results. Average success rate (A & B), number of pellets earned (C & D), nose pokes in the active hole (E & F), and active/inactive nose poke ratio (G & H) for male (A, C, E, G) and female (B, D, F, H) mice on each xiv stage of DRL reinforcement. Error bars represent ± SEM. *: p < .05; ***: p <.001.

...... 62

Figure 2.5 Inter-Response Time. Average frequency of IRTs less than 3 ses (A

& B) and IRTs less than DRL time (C & D) for male (A, C) and female (B, D) mice. Panels E-I show a histogram of IRTs on the last day of DRL-36 for each group. Error bars represent ± SEM. *: p < .05; ***: p <.001...... 63

Figure 2.6 Progressive Ratio Results. Breakpoint for each day on the PR schedule for male (A) and female (B) mice. Error bars represent ± SEM. *: p <

.05; ***: p < .001...... 65

Figure 3.1 Dose Response and Sensitization Schedule. Mice were habituated to the test arena and received I.P. saline injections for 3 days to determine baseline locomotor activity (BL1-BL3). Mice were then assigned to either a saline control group or cocaine group. Mice in the cocaine group received escalating doses of cocaine (5-30 mg/kg) over 10 days. Following a two-week incubation period, all mice were given 10 mg/kg cocaine...... 76

Figure 3.2 . Behavioural Results. Male (A) and female (B) ZnT3 WT and KO mice received either saline or escalating doses of cocaine and were tracked by overhead camera and computer software for 15 min prior to injection and 45 min after injection. Distance travelled was determined. Male (C) and female (G) ZnT3

WT and KO mice in the cocaine group were compared on distance travelled the first time they received 10 mg/kg cocaine and on the challenge day – the second time they received 10 mg/kg cocaine. Panels D & H show oral stereotypic behaviours; Panels E & I show repetitive stereotypic behaviours; and Panels F & xv

J show sniffing stereotypic behavior. Error bars represent ± standard error of the mean (SEM). *: p < .05; ***: p <.001; a: medium effect size; aa: large effect size.

...... 88

Figure 3.3 Fast-scan Cyclic Voltammetry Results. Male (Panels A-C) and female

(Panels D-F) ZnT3 WT and KO mice in saline and cocaine groups were injected

(I.P.) first with saline (0.9%) then with 10 mg/kg cocaine. The VTA was stimulated every 5 min and dopamine concentration was determined...... 95

Figure 3.4 . Anatomical Results. Brains of mice were processed for Golgi-Cox staining. Medium spiny neurons (n = 4/mouse) from the dorsolateral striatum were analyzed. Panel A shows a sagittal section of a Golgi-stained mouse brain.

Panel B shows a specific medium spiny neuron with the tracing of that cell in

Panel C. Dendritic length as measured by Sholl analysis is shown for males (D) and females (F). Spine density (number of spines/length of dendrite) is shown in

Panels E and G for males and females respectively. Error bars represent ± standard error of the mean (SEM). aa: large effect size...... 97

Figure 4.1 . Experimental Setup. Panel A shows a mock-up of the conditioned place preference boxes used; the door connecting the two chambers is absent during conditioning days and present during test days. Panel B shows the timeline for the experiment. Mice were counterbalanced in terms of whether they received the saline or cocaine pairing first...... 111

Figure 4.2 Distance Travelled During Conditioning Sessions. Panel A (male) and

Panel B (female) show distance traveled over the 30 min pairing sessions under xvi the influence of saline or cocaine. Error bars represent ± SEM. * represents p <

.05; *** represents p < .001...... 114

Figure 4.3 Conditioned Place Preference. Panels A (male) and B (female) show the time spent in the cocaine-paired chamber during the testing sessions. Time spent in each of the chambers for each test is shown below for males (Panels C-

F) and females (Panels G-J). The solid (Panels A & B) and dotted (Panels C-J) lines represent equal time spent in the two chambers. Error bars represent

±SEM. * represents p < .05; ** represents p <.01; *** represents p < .001...... 117

Figure 5.1 Experimental Overview. Male and female ZnT3 WT and KO mice were handled for 3 days (H1-3) prior to testing. Mice were assigned to receive either saline or cocaine (20 mg/kg) for 7 days (D1-7) and placed in an open field for 15 min habituation, given an I.P. injection then replaced for 90 min. On day 8, brains were removed for further analyses...... 129

Figure 5.2 Behavioural Results. Panels A & B show the response across the 7 days of testing in male and female mice, respectively. Panels C-I (male) and J-P

(female) show the response over time within each session across the 7 days as a ratio compared to the baseline response prior to injection. The dotted line represents baseline. Error bars represent ± SEM. *: p < .05; a: medium effect size; aa: large effect size...... 135

Figure 6.1 Striatal Synapse. A model synapse in the striatum showing the various receptors that zinc has binding sites on and is able to modulate, as well as the direction of modulation (potentiation vs inhibition)...... 144 xvii

Figure A.1 Within Trial Data for Males. Distance travelled in 15 min intervals for each dose of cocaine. Data are presented as mean ± SEM…………………….186

Figure A.2 Within Trial Data for Females. Distance travelled in 15 min intervals for each dose of cocaine. Data are presented as mean ± SEM…...……………187

xviii

LIST OF SYMBOLS, ABBREVIATIONS, NOMENCLATURE

AC: adenylyl cyclase Ach: acetylcholine AChE: acetylcholinesterase aCSF: artificial cerebrospinal fluid AEA: anandamide ALK: anaplastic lymphoma kinase AMPA: α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AMY: amygdala ANOVA: analysis of variance AP-3: adaptor protein 3 AUC: area under the curve BDNF: brain derived neurotrophic factor BLA: basolateral amygdala BNST: bed nucleus of the stria terminalis CaM: calmodulin cAMP: cyclic adenosine monophosphate CaMPKII: Ca2+/calmodulin-dependent protein kinase II CB1: cannabinoid receptor type 1 ClC-3: chloride channel 3 COC: cocaine CPP: conditioned place preference CREB: cAMP response element-binding protein D[#]R: dopamine receptor # DA: dopamine DAT: dopamine reuptake transporter DCN: dorsal cochlear nucleus xix

dH20: distilled water DLS: dorsolateral striatum DMS: dorsomedial striatum DNA: deoxyribonucleic acid DRL: differential reinforcement of low-rate responding EPSP: excitatory postsynaptic potential ERK: extracellular signal-regulated kinase FSCV: fast-scan cyclic voltammetry FR: fixed ratio G1 mGluR: group 1 metabotropic glutamate receptor GABA: γ-aminobutyric acid GluNR2A/B: subunit A/B of the NMDA glutamate receptor GPR[#] or GPCR: G-protein coupled receptor # HPC: hippocampus I.P.: intraperitoneal IPSP: inhibitory postsynaptic potential IRT: inter-response time IT: intratelencephalic tract KO: knockout LTD: long-term depression LTP: long-term potentiation MAPK: mitogen-activated protein kinase MEK: MAPK/ERK kinase MMP: matrix metalloproteinase MSN: medium spiny neuron MT: metallothionein NAc: nucleus accumbens NMDA: N-methyl-D-aspartate xx

OT: olfactory tubercle PBS: phosphate buffered saline PDE: phosphodiesterase pERK: phosphorylated ERK PFC: prefrontal cortex PKA: protein kinase A PR: progressive ratio PSD: postsynaptic density PT: pyramidal tract RNA: ribonucleic acid SAL: saline SD: standard deviation SEM: standard error of the mean SN: substantia nigra TrkB: tropomyosin receptor kinase B VGlut1: vesicular glutamate transporter 1 VTA: ventral tegmental area WT: wildtype ZIP: Zrt, Irt-like protein ZnT#: zinc transporter # 2-AG: 2-arachidonylglycerol 5-HT: serotonin

1 CHAPTER 1:

GENERAL INTRODUCTION

1.1 INTRODUCTION

Neuroplasticity is a phenomenon that has received much attention in recent years in academia as well as in the general public. The most basic definition of plasticity is the ability to change in response to experience. Many experiences, including learning and drug use, have been shown to exert effects on the brain and cause changes in both anatomy and function. Neuroplasticity can occur at different levels (i.e. whole brain, brain region, or synapse) and through different mechanisms (most notably long-term potentiation and long-term depression).

Different experiences have been found to cause changes in different brain regions and various neurotransmitters are thought to underlie these plastic changes, glutamate being one of the most thoroughly studied. For example, the striatum, which includes dorsal and ventral components, is involved in goal- directed learning, habit formation, motivation and reward; plastic changes can be found in this region in response to specific learning tasks or to drugs of abuse.

One less well-studied molecule thought to play a role in plasticity at synapses is the element zinc. Most zinc is bound up tightly in proteins; however, a subset of the zinc found in the brain is loaded into vesicles exclusively by zinc transporter 3

(ZnT3). It is this pool of zinc that has been implicated in experience-dependent plasticity. Removal of the gene that codes for this protein results in animals that lack vesicular zinc; ZnT3 knockout (KO) mice can be used to probe the function of vesicular zinc in behaviour. Little is known about the role of zinc in the striatum 2 where it is found in high concentrations. The overarching purpose of this thesis is to determine a potential role for zinc in the striatum by examining mice that lack zinc on various tasks meant to test striatal function. This chapter will provide background on zinc, the striatum, and drug-evoked plasticity.

1.2 ZINC

1.2.1 Zinc in the Body

Zinc is an essential micronutrient that we take in through our diets (Prasad,

2013). It is found in every cell of the body and has various structural and enzymatic roles (Chasapis et al., 2012).

Zinc homeostasis in the body is very important as zinc deficiency is involved in several disorders while high amounts of zinc lead to toxicity. In the body, zinc homeostasis is tightly regulated by Zrt-Irt-like proteins (ZIPs), zinc transporter proteins (ZnTs), and metallothioneins (MT). These 3 families of proteins control movement of zinc into and out of the cell and cellular compartments (ZIPs and

ZnTs) or act as a buffer to maintain homeostatic zinc levels within the cell (MTs)

(McAllister and Dyck, 2017).

ZIPs (solute-linked carrier 39) are the family of proteins (slc39 gene family) in charge of moving zinc into the cytosol, whether from outside of the cell or from cellular compartments (Kambe et al., 2015). There are 14 members of this family, some are ubiquitous while others are localized to certain regions of the body and each is typically found on (a) specific compartment(s). 3

In contrast, ZnTs (solute-linked carrier 30) are the family of proteins (slc30 gene family) involved in extracting zinc from the cytosol and moving it either out of the cell or into cellular compartments (Kambe et al., 2015). There are 10 members of this family. Most important for this thesis is ZnT3, which is found on synaptic vesicles and is solely responsible for loading zinc into synaptic vesicles.

It will be discussed in more detail below (Section 1.2.2).

Finally, there are 4 members of the MT family. MT3, in particular, is found in the cytosol of neurons and is involved in maintaining zinc homeostasis within the cell by binding to free zinc (Kambe et al., 2015). It has 7 binding sites, of which 4 are high affinity while the rest are lower affinity. It has been hypothesized that zinc may be released from MTs in an activity-dependent manner and the released zinc may contribute to signaling within the cell (Grabrucker, 2014).

1.2.2 ZnT3 and Vesicular Zinc

As mentioned above (Section 1.1), in the brain a pool of zinc known as vesicular zinc is found “free” (i.e. not tightly bound to other proteins) and loaded into synaptic vesicles by zinc transporter 3 (ZnT3) (Palmiter et al., 1996; Cole et al., 1999). In most brain areas, ZnT3 is found on vesicles that also contain glutamate; as such, zinc is usually released alongside glutamate into the synaptic cleft (Beaulieu et al., 1992; Sindreu et al., 2003). Vesicular zinc is not present in every glutamatergic neuron – only a subset of the glutamatergic neurons in the brain regions that stain for vesicular zinc actually contain zinc (Sindreu et al.,

2003). In certain brain regions, zinc is co-localized with glycine or GABA (Birinyi 4 et al., 2001; Danscher et al., 2001; Wang et al., 2001). An overview of zincergic anatomy is found in the next section.

There are certain factors in the brain that are known to interact with ZnT3 and/or vesicular zinc. Adapter protein 3 (AP-3) is involved in targeting ZnT3 to synaptic vesicles (Salazar et al., 2004). Another factor is vesicular glutamate transporter 1 (VGlut1) which is located on synaptic vesicles and is responsible for loading glutamate into the vesicle (Du et al., 2020). As zinc is usually co-localized with glutamate, VGlut1 and ZnT3 are found on the same vesicles; both, as well as chloride channel 3 (ClC-3), interact with AP-3. VGlut1 and ZnT3 have been shown to enhance the function of the other – glutamate transport enhances zinc transport and vice versa (Salazar et al., 2005).

Another factor that interacts with ZnT3 is estrogen (Lee et al., 2004a).

Estrogen has been found to reduce the expression of ZnT3 which leads to decreased vesicular zinc in the hippocampus. This is thought to be mediated through interactions with AP-3 (Lee et al., 2004a). This suggests the possibility that altering zinc levels in the brain may affect functioning differentially in males and females. Another finding that suggests sex differences in vesicular zinc may exist is the Genome Wide Association Study that linked a polymorphism in the gene for ZnT3 to schizophrenia, but more so in females than males (Maycox et al., 2009).

Lastly, changes in dietary levels of zinc have been shown, in some studies

(Takeda et al., 2003; Grabrucker et al., 2014), to affect levels of vesicular zinc in 5 the brain. Several studies have used such manipulations in rodents to examine the role of vesicular zinc in brain function and behaviour.

1.2.3 Zincergic Anatomy

Different methods of measuring zinc in the brain exist. Some methods measure all of the zinc present – bound zinc and “free” vesicular zinc – without distinguishing between the two. Other methods examine only the “free” zinc and are not sensitive to zinc that is bound to proteins (Danscher and Stoltenberg,

2005).

Vesicular zinc can be visualized using a silver-selenium method

(Frederickson et al., 1992). This involves an intraperitoneal injection of a sodium selenium compound (ex. sodium selenite or sodium selenide) which binds to free zinc in the synapse, cytoplasm, and vesicles and forms a crystal. After a short survival time (60-90 min), brains are removed and sectioned, and these zinc- selenite crystals can be visualized using a staining solution containing silver lactate (amongst other compounds). The location of these zinc-selenium crystals can then be determined using light microscopy.

Other methods to label vesicular zinc exist (Frederickson, 2003), the majority of which use fluorescent probes (Carter et al., 2014). Until recently, most of these probes had limitations, including low sensitivity for zinc, that made them ill-suited for accurate measurements of vesicular zinc. However, a new approach involving 6 acetylation of photoinduced electron transfer-based fluorescent zinc probes shows promise (Zastrow et al., 2016).

Vesicular zinc is present in most areas of the forebrain (Figure 1.1). There are particularly high concentrations in the neocortex, hippocampus, amygdala, and striatum (Frederickson et al., 1992). In the neocortex, there is a laminar distribution with high concentrations in layers II/III, V, and VI and low concentrations in layer IV. An area of interest in terms of response to drugs of abuse that shows high concentrations of vesicular zinc is the bed nucleus of the stria terminalis (BNST) (Vranjkovic et al., 2017). Vesicular zinc is also present in some areas of the diencephalon, brainstem, and spinal cord (Frederickson et al.,

1992).

In the striatum specifically, vesicular zinc is found in a patch-matrix pattern.

However, when comparing the zinc patch-matrix to acetylcholinesterase (AChE) staining (typically used to show the striosome-matrix distribution in the striatum), there is an incomplete overlap – the stains do not match up perfectly (Mengual et al., 1995). This indicates that zinc is found in both the striosomes and the matrix.

Zinc is also found in higher concentrations in the caudomedial margin of the striatum (Shu et al., 1990). The reason for high and low areas of zinc is currently unknown.

The silver-selenium method can also be used to determine the location of zincergic cell bodies. By injecting a lower dose of selenite, either intraperitoneally

(whole brain zincergic cell bodies) or intracranially (zincergic cell bodies projecting to a specific region), and increasing the survival time (~24 hours), the 7 zinc-selenite crystals will be retrogradely transported (Brown and Dyck, 2004a).

Cell bodies containing these crystals can be visualized using a silver staining method. However, if no further steps are taken, vesicular zinc will also be stained and may obscure the location of the zincergic cell bodies. This can be remedied by using a hydrogen peroxide treatment to remove the vesicular zinc and better visualize the cell bodies (Brown and Dyck, 2003a).

Most zincergic neurons are in the forebrain, almost exclusively in the cerebral cortex, hippocampus, and amygdala (Brown and Dyck, 2004b). Outside of the forebrain, zincergic neurons can be found in the dorsal cochlear nucleus

(Frederickson et al., 1988), in a few cell types of the cerebellum (Wang et al.,

2002), and in the spinal cord (Sørensen et al., 1990; Jo et al., 2000).

Of particular importance, while there are high concentrations of vesicular zinc in the synapses of the striatum, there are no zincergic cell bodies in the striatum.

This implies that the vesicular zinc present in the striatum is coming from outside the striatum. Since zinc is packaged in glutamatergic vesicles, it is likely coming from one or more of the afferent connections that use glutamate as their primary neurotransmitter – the neocortex, hippocampus, amygdala, or thalamus. Any of these areas may be using zinc to modulate striatal function. Interestingly, while the neocortex, hippocampus and amygdala show high levels of vesicular zinc, the thalamus has low to no vesicular zinc, seeming to imply that it is not likely to be using zinc (Frederickson et al., 1992). However, a study examining the location of ZnT3 mRNA found it to be present in the paraventricular nucleus of the thalamus (Palmiter et al., 1996). The paraventricular nucleus projects to the 8 shell region of the NAc (Pinto et al., 2003). Therefore, this nucleus may use zinc as a cotransporter to modulate signaling in the NAc shell.

One study used retrograde tracing with selenium to map zincergic projections to the striatum in rats (Sorensen et al., 1995). For this study, sodium selenite was injected into one of three striatal regions (dorsolateral, dorsomedial, or ventrolateral). Overall, the study found that the main zincergic projection to the striatum came from prefrontal motor cortices. Other areas that also project to striatum include the somatosensory cortex, parietal cortex, cingulate cortex and the basolateral and basomedial nuclei of the amygdala. Cell bodies were found both ipsi- and contralaterally. Most cortical regions had cell bodies located in deep layer V and superficial layer VI (Sorensen et al., 1995).

Another interesting note is that the BNST has a similar zincergic setup in that there are high amounts of vesicular zinc but no zincergic cell bodies. The relevance of this is yet unknown as the BNST is even less studied in terms of zincergic function than the striatum.

1.2.4 Zinc Signaling in the Brain

Vesicular zinc has been shown to act as a neurotransmitter/neuromodulator on several neurotransmitter receptors (Nakashima and Dyck, 2009b; Marger et al., 2014; McAllister and Dyck, 2017). It can also act on other types of channels

(e.g. voltage-gated ion channels) and can enter post-synaptic cells and affect second messenger systems (McAllister and Dyck, 2017). Many of these are present in the striatum. This chapter will focus on the potential interactions of zinc with the glutamatergic and dopaminergic systems as these two systems have the highest likelihood of being modulated by zinc given its location within glutamatergic neurons and comment briefly on other factors of interest.

Also, it is important to consider whether zinc can actually reach these targets.

As zinc is released alongside glutamate, its ability to act on those receptors in vivo is highly likely. There was a hypothesis that released zinc formed a veneer that stayed on the presynaptic membrane and did not diffuse across the synapse

(Kay et al., 2006; Kay and Tóth, 2006; Nydegger et al., 2010; Nydegger et al.,

2012); however, most research supports that zinc is able to diffuse across the synaptic cleft and have actions on the postsynaptic membrane. Whether zinc can reach the other receptors that it has been shown to modulate is a different story.

One study found that zinc diffuses out of the synapse and had actions on extrasynaptic NMDARs (Anderson et al., 2015), suggesting the possibility that it could act on receptors or transporters within the striatum that are peri- or extrasynaptic to the glutamatergic synapses it is released from. 11

1.2.4.1 Vesicular zinc as a neuromodulator

Most research on vesicular zinc has focused on its effects on glutamatergic neurotransmission due to its localization and release with glutamate (Paoletti et al., 2009). Zinc has binding sites on all three ionotropic glutamate receptors and can modulate their function in a subunit- and concentration-dependent manner with mainly inhibitory effects (McAllister and Dyck, 2017). There are zinc binding sites on the GluNR2A and GluNR2B subunits of the NMDA receptor (Vogt et al.,

2000). For GluNR2A, zinc is a high affinity voltage-independent inhibitor at low nM concentrations. Higher concentrations of zinc (1-10 μM) are required to inhibit the GluNR2B subunit. Tonic levels of zinc (<25 nM) are thought to be sufficient for inhibition of the GluNR2A subunit while activity-dependent release of zinc is necessary for inhibition of GluNR2B (Vogt et al., 2000). As mentioned above, zinc has also been found to act on extrasynaptic NMDA receptors (Anderson et al., 2015).

Less research has been done on AMPA and kainate glutamate receptors.

Findings on AMPA receptors have had mixed results, however the most recent study suggests that zinc has an inhibitory effect on AMPA and that this inhibitory effect requires activity-dependent release of zinc – the receptor is not affected by tonic zinc levels (Kalappa et al., 2015). Kainate receptors also seem to be inhibited by zinc in a subunit- and concentration-dependent manner (Mott et al.,

2008). 12

Zinc has been found to bind to sites on several members of the dopaminergic receptor families (Schetz and Sibley, 1997; Schetz et al., 1999; Schetz and

Sibley, 2001; Liu et al., 2006b), as well as to the dopamine uptake transporter

(DAT) (Richfield, 1993; Norregaard et al., 1998; Bjorklund et al., 2007; Pifl et al.,

2009). It is a negative allosteric modulator of both D1R and D2-like receptors

(Schetz and Sibley, 1997; Schetz et al., 1999). One study found that high doses of zinc administered to rats disrupted binding of dopamine to the D1R and impaired spatial memory (Turner and Soliman, 2000). Zinc has been shown to inhibit the DAT (Richfield, 1993; Norregaard et al., 1998; Bjorklund et al., 2007;

Pifl et al., 2009).

Zinc can enter postsynaptic cells through NMDA and AMPA receptors, as well as through certain voltage-gated cation channels (Sensi et al., 1997). There are several ways that zinc has been found to act within the cell. Zinc can inhibit adenylyl cyclase (AC) and can augment or reduce phosphodiesterase (PDE) in a concentration-dependent manner (Klein et al., 2004). AC forms the second messenger 3’,5’-cyclic adenosine monophosphate (cAMP) while PDE degrades cAMP. Dopamine receptors also act to either stimulate (D1R, D5R) or inhibit

(D2R, D3R, D4R) AC (Do et al., 2012). Thus, zinc has the potential to alter dopamine signaling as well as other metabotropic signaling pathways that converge on AC. cAMP has been shown to play important roles in learning and memory; alterations in levels can have drastic effects on the functioning of cells

(Lee, 2015). 13

Zinc has also been shown to affect the function of another second messenger, calcium. Most of zinc’s intracellular effects on calcium take place through its interaction with Calmodulin (CaM) (Mills and Johnson, 1985) which it potentiates. In contrast, zinc inhibits calcium/CaM-dependent protein kinase II

(CaMPKII) (Weinberger and Rostas, 1991; Lengyel et al., 2000).

In addition to its effects on AC, PDE, and CaM, zinc can also affect the

ERK/MAPK second messenger system (Azriel-Tamir et al., 2004; Nuttall and

Oteiza, 2012). Zinc deficiency leads to decreases in the MEK/ERK signalling pathway in the hippocampus (Jiang et al., 2011). In mice lacking vesicular zinc, activation of ERK1/2 MAPK is reduced in the mossy fibers of the hippocampus

(Sindreu et al., 2011).

Intracellular zinc has also been found to transactivate (i.e. activate independently) the brain derived neurotrophic factor (BDNF) receptor tropomyosin receptor kinase B (TrkB) (Huang et al., 2008). It is thought to do so by inhibiting the C-terminal of Src kinase which reduces the autoinhibition of the

Src kinases and thus increases activation. Src kinases are involved in phosphorylating TrkB. However, it is not clear whether this is happening in vivo as one study on ZnT3 KO mice found increases in TrkB phosphorylation and

BDNF content in the hippocampus which is the opposite of what one would expect (Helgager et al., 2014). However, a recent study in our lab found no differences between ZnT3 KO and WT mice in BDNF or TrkB protein levels regardless of the sex or age of the animals (McAllister et al., 2020). 14

Zinc may also affect TrkB through a BDNF-dependent mechanism. This involves matrix metalloproteinases (MMPs) which are involved in producing mature BDNF by cleaving proBDNF extracellularly (Hwang et al., 2005). Zinc increases MMP activation and the resulting increase in BDNF triggers the signaling cascades associated with TrkB including Src kinases, ERK1/2, and Akt.

MMPs are also thought to play a role in synaptic plasticity and mediate effects of drugs of abuse (Smith et al., 2015).

Two studies have examined the interaction between zinc and cocaine

(Richfield, 1993; Bjorklund et al., 2007). Zinc was found to augment cocaine’s action on the DAT, making cocaine more efficient at blocking uptake of dopamine

(Richfield, 1993; Bjorklund et al., 2007), which results in prolonged activation of dopamine receptors.

Although dopamine and glutamate are the major neurotransmitters thought to affect striatal signaling, there are numerous other systems present that also contribute to striatal function. Zinc has been shown to have binding sites and modulatory action on some of these; however, much less is known. Briefly, zinc potentiates nicotinic acetylcholine receptors (Hsiao et al., 2001) and 5-HT3 serotonin receptors (Uki and Narahashi, 1996; Hubbard and Lummis, 2000), and inhibits 5-HT1A and 5-HT7 (Barrondo and Salles, 2009; Satała et al., 2018) and

GABA A receptors (Draguhn et al., 1990; Smart et al., 1991; Hosie et al., 2003;

Kodirov et al., 2006). It has also been found to activate the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) (Bennasroune et al., 2010) which is 15 thought to be involved in cocaine sensitization and conditioned place preference

(Lasek et al., 2011).

Recently, zinc has been shown to be the sole activator of GPR39 – a G protein coupled receptor utilizing Gq signaling (Holst et al., 2007; Besser et al.,

2009). One study has shown that GPR39 protein in medium spiny neurons

(MSNs) of the striatum decreases when dopamine decreases and increases with increasing doses of L-DOPA, indicating that GPR39 is present within the striatum

(Heiman et al., 2014). Activation of GPR39 by zinc results in the synthesis of the endocannabinoid 2-arachidonylglycerol (2-AG). 2-AG then acts as a retrograde signal to activate presynaptic CB1 receptors and reduce the probability of vesicle release (Perez-Rosello et al., 2013). Activation of GPR39 also results in increased phosphorylation of CaMKII and ERK1/2 (Besser et al., 2009). Although the Heiman et al. (2014) study suggests that GPR39 is located in the striatum, data from the Allen Mouse Brain Atlas suggests that there is little to no GPR39

RNA within the striatum. Therefore, whether GPR39 is playing a role in the functioning of the striatum is questionable.

Another orphan receptor that zinc has been found to activate is GPR83. In contrast to GPR39, data from the Allen Brain Institute, as well as other studies, shows relatively high amounts of RNA in the striatum (Pesini et al., 1998; Muller et al., 2013b; Gomes et al., 2016). Less is known about the functioning of GPR83 compared to GPR39; however, it was determined to act through Gq/11 signaling

(Muller et al., 2013a). Initially zinc was thought to be the sole activator of GPR83, but recent research has found it is also activated by the neuropeptide PEN 16

(Fakira et al., 2019). There is evidence that GPR83 in the nucleus accumbens

(NAc) and ventral tegmental area (VTA) impacts dopamine and reward-related behaviour (Fakira et al., 2019). In addition, knockdown of GPR83 in the NAc of mice showed differences in dopamine response to morphine, as measured by fast-scan cyclic voltammetry, and in conditioned place preference to morphine; these differences presented in a sex-dependent manner with females requiring higher doses of morphine to see effects (Fakira et al., 2019).

To summarize, vesicular zinc can modulate neuronal function by acting on both ionotropic and metabotropic postsynaptic receptors, in addition to being able to enter postsynaptic cells and directly affect second messenger systems within the neuron. Due to its actions on NMDA receptors and on the endocannabinoid system, zinc may affect synaptic plasticity mechanisms. The next section discusses a role for vesicular zinc in these processes.

1.2.4.2 Vesicular zinc in synaptic plasticity

A thorough review of synaptic plasticity, in general, is beyond the scope of this thesis. However, I will provide a brief overview of synaptic plasticity before discussing potential roles for vesicular zinc in this process. Section 1.3.2 will discuss synaptic plasticity in the striatum and Section 1.4 will examine how drugs of abuse can alter synaptic plasticity. 17

Plasticity at synapses can be either short-term or long-term with each timeframe having different underlying mechanisms. Short-term plasticity typically involves changes in calcium levels and neurotransmitter release, as well as modulation of proteins already present at the synapse while long-term plasticity involves structural changes involving protein synthesis (Citri and Malenka, 2008).

There is evidence that zinc may be involved in both types of synaptic plasticity.

There are thought to be many different forms of long-term synaptic plasticity; however, the best characterized and most studied are long-term potentiation

(LTP) and long-term depression (LTD) (Citri and Malenka, 2008). LTP and LTD are opposing mechanisms, with LTP leading to a strengthening of synapses and

LTD leading to a weakening of synapses (Zucker and Regehr, 2002). Both require calcium inflow and typically involve changes in AMPA receptor phosphorylation and trafficking. LTP typically increases AMPA phosphorylation leading to improved receptor function and results in more AMPA receptors being inserted into the postsynaptic membrane. LTD does the opposite – reduces

AMPA phosphorylation and causes removal of AMPA receptors from the postsynaptic membrane.

There are several types of LTP and LTD – some forms involve the NMDA receptor while others occur independently of NMDA receptors (Kauer and

Malenka, 2007). The best characterized forms of LTP are NMDA-dependent and presynaptic LTP. There is also NMDA-dependent LTD. NMDA-independent LTD involves either activation of the metabotropic glutamate receptor (mGluR) or retrograde signaling by endocannabinoids (eCB). 18

This section will examine the behavioural findings suggesting an involvement of zinc in experience-dependent synaptic plasticity, the electrophysiological studies of zinc in LTP and LTD, and conclude with a discussion of a role for zinc in stabilizing the postsynaptic density.

1.2.4.2.1 In behaviour

It has been proposed that vesicular zinc is involved in experience-dependent plasticity (Nakashima and Dyck, 2009b). It has also been proposed that it is involved in the fine tuning of the circuits where it is found (Grabrucker et al.,

2014; Anderson et al., 2017).

One way to examine a role for vesicular zinc in experience-dependent plasticity is to alter an animal’s environment and look for changes in vesicular zinc staining. Most studies use the vibrissae/somatosensory (barrel) system to examine this type of plasticity. This system consists of vibrissae (whiskers) that are organized in a very specific and consistent way on the face. Each vibrissa has a corresponding “barrel” in the somatosensory cortex forming a somatotopic map. Thus, single vibrissa can be manipulated, and effects can be seen in specific locations in the brain. In particular, vesicular zinc shows characteristic staining in Layer IV of the barrel cortex with higher amounts of zinc in the septa between barrels with little zinc normally found in the middle of the barrel (Brown and Dyck, 2002). Plucking or trimming of vibrissae leads to rapid increases in vesicular zinc in the corresponding barrel in somatosensory cortex (Brown and

Dyck, 2002; Brown et al., 2003; Nakashima and Dyck, 2010). This change is 19 more pronounced in females (Nakashima et al., 2010) and after exposure to a complex environment (Nakashima and Dyck, 2008). The opposite also seems to occur – vibrissae stimulation results in decreases in vesicular zinc in the corresponding barrel (Brown and Dyck, 2005).

Another way to examine whether vesicular zinc is involved in experience- dependent plasticity is to alter zinc levels and determine whether the animal responds differently on various behavioural tasks. There have been several behavioural studies using dietary zinc manipulations. Some of the findings in these studies suggest that the striatum may be affected by the loss of zinc. For example, female mice who experienced prenatal zinc deficiency show deficits in nest building and marble burying (Grabrucker et al., 2016) – tasks that supposedly examine repetitive behaviours (Angoa-Perez et al., 2013) and are thought to reflect basal ganglia dysfunction (Lewis and Kim, 2009). Young (4-5 weeks old) male ZnT3 knockout mice show increased repetitive behaviours (Yoo et al., 2016). Also, adult female ZnT3 knockout mice do not show the expected improvement in accuracy on a skilled reach task, which may be indicative of basal ganglia disruptions (Thackray et al., 2017).

In addition, studies with human subjects suggest that zinc deficiency may be implicated in disorders that involve striatal dysfunction (Grønli et al., 2013). One of these studies was a genome-wide association study that found a single nucleotide polymorphism in the gene that codes for ZnT3 was associated with a higher incidence of schizophrenia in females (Maycox et al., 2009). We cannot know for sure that the zinc deficiency is specifically affecting striatal function and 20 not another brain region in these studies; however, it suggests the possibility that zinc neurotransmission may play a role in the etiology of these disorders. Further research is required to determine how zinc signaling is altered and whether it is altered in all brain regions or only specific regions.

1.2.4.2.2 In LTP and LTD

Because of its presence in glutamatergic synapses and its actions on NMDA and AMPA receptors, zinc is in an ideal location to modulate mechanisms of synaptic plasticity, such as LTP and LTD.

Most research on the role of zinc in LTP/LTD has been done using the hippocampus. More is known about the role of zinc in LTP; only one study to date has examined zinc in LTD (Izumi et al., 2006). Findings have been recently reviewed by McAllister and Dyck (2017).

Exogenous application of zinc disrupts hippocampal LTP (Xie and Smart,

1994; Izumi et al., 2006). Disruptions in NMDA-independent LTP in the hippocampus have been found after chronic dietary zinc deficiency (Lu et al.,

2000) and zinc chelation (Lu et al., 2000; Li et al., 2001). Zinc chelation also disrupts LTP at corticoamygdala synapses (Kodirov et al., 2006). Additionally,

ZnT3 knockout mice are impaired in the induction of a presynaptic form of LTP

(Pan et al., 2011). The one study that examined LTD suggests that LTD may be more susceptible to disruption as it is inhibited by lower concentrations of zinc

(Izumi et al., 2006). 21

The most recent studies focus on the role of vesicular zinc in the dorsal cochlear nucleus (DCN) in the brainstem. They found zinc-dependent LTD in response to high frequency stimulation and zinc-dependent LTP in response to low frequency stimulation; these changes occurred independently of NMDARs and were instead dependent on Group 1 metabotropic glutamate receptors (G1 mGluR) (Vogler et al., 2020). This was the first study to show a role for G1 mGluRs in zincergic plasticity. This same study proposes that these zinc- dependent forms of synaptic plasticity may contribute to other forms of LTP and

LTD in brain regions that contain high amounts of vesicular zinc and, through this, contribute to metaplasticity, the idea that early modifications can affect how later experiences affect the brain (Abraham and Bear, 1996).

Together, these studies suggest that alterations in zinc levels – increases or decreases – can affect synaptic plasticity and that there are different mechanisms through which zinc can act.

1.2.4.2.3 Zinc in the postsynaptic density

The postsynaptic density (PSD) is the network of proteins that are involved with detecting, transducing, and integrating the signals being sent through a synapse (Sheng and Kim, 2011). Proteins present in the PSD include receptors and scaffolding molecules involved in anchoring the receptors in place as well as in sending signals both intracellularly and presynaptically (Sheng and Kim, 2011).

As discussed above (Section 1.2.4.1), zinc can act on NMDARs and AMPARs present in the PSD of excitatory synapses. In addition, zinc can also interact with 22 other proteins involved in the PSD and has been suggested to help with stabilization of glutamatergic synapses. One of the proteins zinc interacts with is the scaffolding protein Shank3 (Arons et al., 2016). Altering zinc levels affects expression of ProSAP/Shank complexes and can affect stability of synapses

(Grabrucker et al., 2014). Since the PSD has been shown to respond to experiences by increasing or decreasing in size (Sheng and Kim, 2011), lack of stability may drastically affect the synapse and, in turn, behaviour.

1.2.5 Genetic Manipulations and ZnT3 Knockout Mice

There are two genetic manipulations to date that have been shown to affect brain zinc levels. One way to assess the role of vesicular zinc is using the mocha mouse which has a mutation that inactivates AP-3 (Lane and Deol, 1974). As mentioned above (Section 1.2.2), AP-3 is the protein responsible for positioning

ZnT3 in the vesicles (Salazar et al., 2004). Without it, there is significantly less vesicular zinc. These mice have altered locomotor and coordination abilities, hyperactivity, and enhanced auditory gating (Miller et al., 1999).

The other genetic manipulation that affects vesicular zinc is to examine zinc transporter 3 (ZnT3) knockout (KO) mice. ZnT3 KO mice lack the slc30A3 gene that codes for the ZnT3 protein which is solely responsible for loading zinc into synaptic vesicles (Palmiter et al., 1996); therefore, ZnT3 KO mice have a complete absence of vesicular zinc (Cole et al., 1999). 23

Since their creation in 1999 (Cole et al., 1999), the ZnT3 KO mice have been examined on several tasks in an attempt to characterize a behavioural phenotype. They have been examined on many aspects including sensory and motor function (e.g. olfaction, auditory threshold, motor coordination), emotional regulation (e.g. fear conditioning), learning and memory (e.g. Morris water task, alternation in a T-maze), and social behaviour (Cole et al., 2001; Martel et al.,

2010; Martel et al., 2011; Sindreu et al., 2011; Thackray et al., 2017). Subtle behavioural differences have been found between ZnT3 wildtype (WT) and KO mice. These differences include deficiencies in conditioned (but not innate) fear memory (Martel et al., 2010; Sindreu et al., 2011); in the Morris water task that involves alternating platform location (Martel et al., 2011); in the t-alternation maze (Sindreu et al., 2011); in accuracy on a skilled reach task (Thackray et al.,

2017); in discrimination of fine textures using their whiskers (Wu and Dyck,

2018); and in performance in the Morris water task of older ZnT3 KO mice compared to younger ZnT3 KO mice (Adlard et al., 2010).

Due to the lack of apparent findings in abnormalities of the ZnT3 KO mice under standard conditions, recent studies in our lab have examined the ZnT3 KO mice using environmental enrichment housing and chronic social defeat stress models to determine whether there were behavioural differences under these non-standard circumstances. Results of the enrichment study found that the

ZnT3 KO mice do not show the expected benefits of being in that environment, including the increased neurogenesis and cell survival and reduced cell death seen in WT mice in the same environment (Chrusch, 2015). ZnT3 KO mice in the 24 enriched environment also did not show the improvements in spatial object recognition and the Morris water task seen in WT mice (Chrusch, 2015). Effects of chronic social defeat stress on ZnT3 KO mice were more difficult to determine as the mice seemed to show resilience to stress in social interaction, but enhanced effects of stress (compared to stressed WT mice) on cued fear conditioning (McAllister et al., 2018).

As the most recent research on ZnT3 KO mice suggests they exhibit a lack of plasticity in certain tasks dependent on the hippocampus and amygdala, it is possible that they will show similar deficits in plasticity in tasks dependent on the striatum.

1.3 STRIATUM

This section will delve into the anatomy (both macro and micro) and function of the striatum.

1.3.1 Striatal Anatomy

All brain regions can be examined in terms of their connections with other brain regions (afferent and efferent) and in terms of the connections within said brain region. I will begin this section by discussing the macrocircuitry that the striatum is involved in, then discuss the microcircuitry within the striatum. A closer examination of synapses and the neurotransmitters present in the striatum is also included. 25

1.3.1.1 Macrocircuitry

The striatum is the input nucleus for the basal ganglia. It consists of the caudate nucleus, the putamen, the nucleus accumbens (NAc), and olfactory tubercle (OT). Anatomically, the striatum is divided into dorsal (caudate nucleus and putamen) and ventral (NAc and OT) regions. Each region serves a different functional purpose (expanded on below).

Afferent connections to the striatum form topographical maps within the striatum that are maintained throughout the basal ganglia circuits (Alexander and

Crutcher, 1990) (Figure 1.2). Most of the afferent connections use glutamate or dopamine as their main neurotransmitter. Glutamatergic input comes from the neocortex, hippocampus (HPC), amygdala (AMY), and thalamus. Dopaminergic input comes from the substantia nigra (SN), ventral tegmental area (VTA), and retrorubral area. The striatum also receives cholinergic input from two brainstem nuclei, the pedunculopontine nucleus and the lateral dorsal tegmentum, as well as serotonergic input from the dorsal raphe nuclei (Steinbusch, 1981).

Most regions of the neocortex project to the striatum. Connections from the motor cortex can either be part of the pyramidal tract (PT) or intertelencephalic tract (IT). The IT tract projects only within the telencephalon and contains both ipsi- and contralateral projections. While the PT projects principally to the brainstem and spinal cord with collaterals to other cortical and subcortical regions, PT tract projections are all ipsilateral. These pathways have been shown 26 to have differing electrophysiological properties, use different neuromodulators, and have different molecular profiles (Shepherd, 2013).

Efferent projections are to other basal ganglia areas (Alexander and

Crutcher, 1990). Most of the neurons in the striatum are part of either the direct pathway or the indirect pathway. The direct pathway neurons project directly to the basal ganglia output neurons – the globus pallidus internal segment and the substantia nigra pars reticulata. As indicated by its name, the indirect pathway takes a longer route; these neurons first project to the globus pallidus external segment and the subthalamic nuclei which then project to the basal ganglia output nuclei. A small group of neurons project to the ventral tegmental area and the substantia nigra pars compacta, acting as a negative feedback loop. The output nuclei project to nuclei in the thalamus which then project back to the cortex, maintaining the topographical map at each step of the pathway.

Figure 1.2 Striatal Circuitry. An overview of the macrocircuitry of the striatum including afferent projections and direct vs indirect efferent projections. 28

1.3.1.2 Microcircuitry

GABAergic medium spiny neurons (MSNs) are the main projection neuron of the striatum and make up ~95% of the neurons located there (Matamales et al.,

2009). The other ~5% are interneurons. Traditionally, the interneurons were divided into 4 categories; however, recent evidence suggests a more complex setup (Silberberg and Bolam, 2015). Interneurons either use acetylcholine or

GABA as their primary neurotransmitter. Many of the GABAergic interneurons use co-transmitters and can be divided into classes based on their firing properties (Silberberg and Bolam, 2015). The interneurons function as modulators of MSN firing.

In addition to connections with interneurons, MSNs have also been found to show lateral inhibition – they synapse onto neighbouring MSNs and can modulate their function. Findings have been recently reviewed by Burke et al.

(2017).

Within the striatum, there are anatomical distinctions beyond the direct/indirect pathways. One of these is the division of the striatum into patch

(also called striosome) and matrix compartments (Brimblecombe and Cragg,

2017). Each compartment contains neurons from both direct and indirect pathways, but are distinguished from one another by the staining for various proteins – the most commonly used being AChE, which is high in the matrix

(Graybiel and Ragsdale, 1978), and the mu opioid receptor, which is high in striosomes (Brimblecombe and Cragg, 2017). 29

1.3.1.2.1 Striatal synapses

As mentioned above (Section 1.3.1.1), the major inputs to the striatum are glutamatergic from the cortex, hippocampus and amygdala and dopaminergic from VTA/SN. Glutamatergic axons synapse onto spines on the dendrites of

MSNs and interneurons. There are two families of glutamate receptors: ionotropic (NMDA, AMPA, and kainite) and metabotropic (mGluRs). All appear to be present within the striatum and are depolarizing in nature (Wüllner et al.,

1994).

Dopaminergic axons typically synapse onto the spine necks or dendritic shafts of glutamatergic spines to modulate glutamatergic neurotransmission

(Calabresi et al., 1997). There are also 2 families of dopamine receptors, D1-like and D2-like, all of which are G protein coupled receptors (GPCRs) (Jaber et al.,

1996). The D1-like family consists of D1 and D5. They are coupled to either Gs or Golf proteins and thus cause increases in adenylyl cyclase (AC) activity.

Increased AC activity results in increases in the second messenger cyclic adenosine monophosphate (cAMP) which activates the cAMP-dependent kinase

(PKA). This causes a cascade of cellular effects. The D2-like family consists of

D2, D3, and D4. They are coupled to GI/O proteins which inhibit AC activity.

Therefore, the second messenger system is not activated.

Other receptors of interest in the striatum, particularly with respect to zinc, include GABA receptors, ACh receptors, serotonin (5-HT) receptors, and the

BDNF receptor TrkB. As mentioned above, most interneurons in the striatum use 30

GABA or ACh as their primary neurotransmitter; therefore, GABA and ACh receptors can be found on MSNs. Also, lateral inhibition from other MSNs uses

GABA as a neurotransmitter. Nicotinic cholinergic receptors (nAChRs) are found on dopamine terminals (Grady et al., 2007). An overview of receptors of interest, their probable location within the striatal synapses, and how they respond to zinc can be found in Figure 1.3.

Figure 1.3 Striatal Synapse. A model synapse in the striatum showing the various cell types and receptors that are present. 32

1.3.2 Striatal Plasticity

Plastic changes are thought to underlie many aspects of learning and memory. Although the focus of studies on learning and memory has traditionally been on the hippocampus, plastic changes also occur within the striatum

(Kreitzer and Malenka, 2008). This section will focus on plasticity in the striatum in general. Section 1.4 will cover drug-evoked plasticity mechanisms and the role of the striatum in response to drugs of abuse.

1.3.2.1 LTP and LTD

Although research on LTP/LTD has traditionally focused on the hippocampus, more studies are starting to examine LTP/LTD in the striatum

(Calabresi et al., 1997; Calabresi et al., 2007; Lerner and Kreitzer, 2011; Cerovic et al., 2013). LTD is better characterized as LTP has been difficult to elicit reliably. Striatal LTD seems to use endocannabinoid (eCB) mediated mechanisms and requires D2R, L-type calcium channels, Gq coupled group 1 mGluRs, and CB1 receptors, but not NMDARs (Kreitzer and Malenka, 2008). In contrast to other brain regions, dopamine is critically important for LTP/LTD induction in the striatum (Calabresi et al., 2007). It seems to have differential effects on the direct versus indirect pathway, at least with respect to LTD in which high levels of DA block LTD at the direct pathway but enhance inhibition at the indirect pathway (Kreitzer and Malenka, 2008). 33

While many types of synapses in the striatum have been examined in terms of synaptic plasticity, the most relevant to this thesis are the corticostriatal synapses. As mentioned above (Section 1.2.3), most areas of the cortex contain high concentrations of vesicular zinc and zincergic cell bodies; the striatum contains vesicular zinc but no cell bodies, while the thalamus, apart from the paraventricular nucleus, contains neither vesicular zinc nor zincergic cell bodies.

Therefore, zinc is most likely acting at corticostriatal synapses, but not others within the striatum. Although, since the best characterized type of LTD in the striatum does not seem to be reliant on NMDAR, of which zinc is a potent inhibitor, it is difficult to say how much of an effect zinc may have on this type of synaptic plasticity within the striatum – it may play a larger role in stabilization of synapses following LTP/LTD or act through the newly discovered G1 mGluR- mediated mechanisms similar to the DCN (Vogler et al., 2020).

1.3.3 Striatal Function

Due to the topographical organization of the striatum, different regions of the striatum are thought to be involved in distinct functions (Liljeholm and O'Doherty,

2012). The dorsal striatum is involved in motor function generally with the lateral portion (dorsolateral striatum, DLS) thought to be involved more in habit formation and the medial portion involved in goal-directed learning (dorsomedial striatum, DMS). In particular, the DMS has been implicated as being critical for instrumental conditioning in which animals must learn action-outcome associations (Balleine et al., 2009). A proposed model of function in instrumental 34 conditioning involves two streams, similar to those in the visual system, in which the dorsal stream (DMS) learns what to do and a ventral stream (ventral striatum) determines when and where to do it (Hart et al., 2013). As such, the ventral striatum/NAc acts as a “motor-limbic interface” – the region where limbic functions/emotions are able to govern behavioural responses (Mogenson et al.,

1980). It has also been proposed that the basolateral amygdala (another region high in vesicular zinc) mediates the interaction between these 2 streams (Hart et al., 2013). The NAc is also considered part of the so-called reward circuit that is involved in motivation and response to rewarding stimuli. This particular role will be expanded in Section 1.4 below.

The striatum is thought to be one of the main brain regions involved in stereotyped behaviours (Langen et al., 2011b; Langen et al., 2011a). In rodents, this includes behaviours such as excessive digging, grooming, or rearing and head weaving/bobbing. These behaviours can be elicited with high doses of certain drugs, including psychomotor stimulants (Rebec and Segal, 1980).

Several neurotransmitter systems, including glutamate, interact within the striatum and imbalances between the systems may play a role in the production of stereotyped behaviours (Langen et al., 2011b; Langen et al., 2011a).

In addition, the various divisions of the striatum – direct/indirect pathways and striosome/matrix compartments – are thought to have different functions.

The direct pathway is thought to promote wanted behaviours while the indirect pathway suppresses unwanted behaviours (Freeze et al., 2013). Less research has examined the roles of the striosome/matrix compartments in behaviour. 35

However, it has been suggested that imbalances between these compartments can lead to certain diseases (Crittenden and Graybiel, 2011).

Dysfunction of the striatum, particularly dopamine within the striatum, is associated with motor disturbances in diseases like Parkinson’s disease, involving difficulty initiating movements, and Huntington’s disease, involving excessive unwanted movements (Gittis and Kreitzer, 2012). Striatal dysfunction is also implicated in several neuropsychiatric disorders including drug addiction, schizophrenia, autism spectrum disorder, and obsessive-compulsive disorder

(Shepherd, 2013). The remainder of this chapter will focus on drug addiction.

1.4 DRUG-EVOKED BEHAVIOUR AND PLASTICITY

Many drugs have been found to produce profound changes in behaviour, physiology, and anatomy. These changes are thought to be due to a drug’s ability to hijack normal plasticity in the brain. This thesis will focus on changes produced by cocaine and will start with an overview of circuits and neurotransmitter systems thought to be involved in the response to drugs of abuse, then discuss the behavioural changes commonly produced by drugs and the synaptic changes thought to underlie them.

1.4.1 Behavioural Plasticity

There are 3 main paradigms used to study rodent models of addiction: psychomotor sensitization, conditioned place preference, and self-administration. 36

In normal mice, acute cocaine exposure leads to increased locomotor activity

(Pijnenburg et al., 1976), while repeated administration additionally results in behavioral sensitization (Landa et al., 2014) and alterations in morphology of

MSNs in the striatum as well as in neurons in the cerebral cortex (Robinson and

Kolb, 1999b).

The increase in locomotor activity after cocaine administration is linked to the cocaine-induced increase in dopamine in the synapse – as dopamine increases, locomotor activity increases (Pijnenburg et al., 1976). The exception is at extremely high doses of cocaine where increased dopamine can lead to stereotyped behaviours, which cause a decrease in locomotor activity (discussed further below). In rodents, a video camera and computer software can be used to track movement in an open field to determine changes in their activity after cocaine administration.

Behavioural sensitization has been found to occur in response to several drugs of abuse, including cocaine (Steketee and Kalivas, 2011). Sensitization in the context of drugs can be defined as an increased response to the same dose of drug. Behavioural, specifically locomotor behaviour, sensitization occurs in rodents who have had a drug withheld for a period of time after receiving repeated administration of it. When the drug is reintroduced, the rodents show an enhanced response, travelling farther distances than when they initially received the drug. Sensitization can occur with locomotor response (Kalivas and Stewart,

1991) and with the incentive-motivation properties of drugs (the "wanting" aspect of drug addiction) (Robinson and Berridge, 1993, 2000). Sensitization has been 37 found to occur following a single injection of a drug. However, it is more robust following repeated administration of and withdrawal from a drug (Steketee and

Kalivas, 2011).

There are two phases involved in sensitization: induction and expression.

The induction of sensitization involves changes in dopamine neurons in the VTA, while expression involves changes within the NAc (Steketee and Kalivas, 2011).

LTP/LTD mechanisms are thought to underlie sensitization; however, different forms are involved in the different phases. Induction of sensitization is thought to be NMDAR-dependent, while expression of sensitization is thought to involve

NMDAR-independent mechanisms (Kauer and Malenka, 2007). Blocking LTD in the NAc prevents the expression of behavioural sensitization (Brebner et al.,

2005). Normal behavioural sensitization involves both the dopaminergic and glutamatergic neurotransmitter systems (Landa et al., 2014).

High doses of cocaine can also result in stereotyped behaviours (Flagel and

Robinson, 2007; Tilley and Gu, 2008). These behaviours are typically repetitive and can be an exaggeration of a normally occurring behaviour (such as grooming or digging) or be a new behaviour that emerges as a result of the drug (such as head weaving or circling).

Another type of behaviour that can be examined in regard to drugs of abuse is conditioned place preference (CPP). CPP is used to examine motivational aspects of drugs, rewarding or aversive (Cunningham et al., 2006). It does so using Pavlovian conditioning, by pairing a specific environment with a drug. The drug acts as an unconditioned stimulus (US) that produces an unconditioned 38 response (UR), and the environment acts originally as a neutral stimulus that becomes the conditioned stimulus (CS) after repeated pairings. More information is provided about CPP in Chapter 4.

A final behavioural task, typically the gold standard of animal models of addiction research, is self-administration (Chistyakov and Tsibulsky, 2006). This involves surgical implantation of a cannula to deliver drugs to the rodent and uses instrumental conditioning chambers, allowing the rodent to make a response to receive infusion of the drug. Arguably, this is more similar to what is seen in human drug-takers compared to the other tests in which the drug is controlled and administered by the experimenter. As ZnT3 KO mice have not been examined with respect to response to cocaine, and self-administration is methodologically more complicated in mice (Thomsen and Caine, 2007) than behavioural sensitization and CPP, this thesis does not examine self- administration.

1.4.2 Circuits and Neurotransmitters

As mentioned in Section 1.3.3, cortico-striatal circuits are important in habit formation, learning, and control of actions, including responses to drugs of abuse

(Haber, 2016). In models of addiction, the prefrontal cortex is involved in the initial decision to take a drug, the dorsomedial striatum is involved in goal- directed actions to take the drug, and, after multiple experiences with the drug, the dorsolateral striatum is involved with the habitual or compulsive taking of the drug (Lipton et al., 2019). 39

The mesolimbic DA system, projecting from the VTA to the NAc, plays a large role in response to drugs of abuse (Pierce and Kumaresan, 2006). Most drugs of abuse cause increases in dopamine in the striatum, either directly or indirectly (Pierce and Kumaresan, 2006). As proposed by the incentive- sensitization theory, dopamine is the neural correlate of wanting or craving

(Robinson and Berridge, 1993, 2000). Increases in striatal dopamine are linked to the learning of drug-associated cues, which can later produce craving in the absence of the drug. The opioid system is involved with the “liking” aspect of the drug (Laurent et al., 2015). Orexin is also necessary for drug-evoked changes, particularly within the VTA (Baimel et al., 2015).

1.4.3 Cellular Mechanisms of Plasticity

Several studies have found that drugs of abuse can alter the normal synaptic plasticity processes in areas involved in motivation and reward, including the striatum, and it has been suggested that these alterations contribute to the etiology of drug addiction (Wolf et al., 2004; Hyman et al., 2006; Kauer and

Malenka, 2007; Lüscher and Malenka, 2011). Studies have traditionally focused on the NAc and largely ignored the dorsal striatum. However, there are more recent studies starting to consider the role that the dorsal striatum might play in response to drugs of abuse (Gremel and Lovinger, 2017).

Several classes of drugs can cause alterations in normal LTP and/or LTD in the NAc (Lüscher and Malenka, 2011). In addition to LTP/LTD, drugs of abuse 40 have also been shown to cause structural alterations in the striatum. Changes in the branching of dendrites and spine density in the striatum have been shown to occur. Psychomotor stimulants, like amphetamine and cocaine, have been found to increase both (Robinson and Kolb, 1997; Robinson and Kolb, 1999a;

Robinson and Kolb, 1999b; Robinson et al., 2001).

1.4.3.1 Cocaine-specific mechanisms

Cocaine is one highly abused drug whose effects are well characterized, both in humans and in rodents. Cocaine exerts its effects mainly by acting on

DAT, blocking the removal of dopamine from the synapse (Kahlig and Galli,

2003), leading to continued activation of dopamine receptors.

Cocaine produces many changes in the brain (Figure 1.4). Administration of cocaine has been found to alter signaling pathways, particularly those discussed earlier in this chapter (Sections 1.2.4.2 and 1.3.2) that are involved with plasticity.

Specifically, it affects glutamatergic signaling, ERK signaling, and BDNF signaling (Thomas et al., 2008; Li and Wolf, 2015). It has also been suggested that cocaine induces metaplasticity within the brain (Thomas et al., 2008; Lee and Dong, 2011).

Glutamatergic signaling is thought to be affected by cocaine in both the VTA and the NAc with changes in the VTA needing to occur prior to any changes in the NAc (Thomas et al., 2008). Glutamatergic receptors are redistributed in the

VTA following cocaine exposure, and a single dose of cocaine can produce 41 potentiation of synaptic strength for at least 5, but not 10 days. Within the NAc, dopamine and glutamate converge and facilitate the trafficking of AMPARs into the PSD at specific glutamatergic synapses (Gardoni and Bellone, 2015).

Cocaine responses rely on the interaction between D1Rs and NMDARS and involve ERK signaling pathways (Lu et al., 2006; Thomas et al., 2008). Cocaine has also been found to induce changes in NMDARs (Ortinski, 2014).

ERK signaling pathways can be activated in many ways (see previous and following paragraphs) and are involved with regulating gene expression (Jain et al., 2018). Changes in ERK phosphorylation and in levels and/or activity of pathway components accompany responses to cocaine (Lu et al., 2006; Thomas et al., 2008). Blocking ERK phosphorylation prevents behavioural sensitization,

CPP, and drug-evoked synaptic plasticity (Thomas et al., 2008).

BDNF is critical in its involvement in neuronal plasticity (Lu et al., 2014). It acts by promoting phosphorylation of cAMP Response Element Binding protein

(CREB), which regulates gene expression as well as activates the Ras/ERK/Rsk pathway (Lu et al., 2014). Levels of BDNF are typically low in the striatum but higher in the PFC, VTA, BLA, and BNST (Meredith et al., 2002; Graham et al.,

2007). When cocaine is on board, BDNF expression is altered (Li and Wolf,

2015); in particular, forced administration (i.e. not self-administered) causes increases in BDNF in the striatum (Zhang et al., 2002; Liu et al., 2006a).

Acute and chronic cocaine administration have also been found to initiate epigenetic changes in DNA methylation, histone modifications, and microRNA

(Heyer and Kenny, 2015; Sadri-Vakili, 2015). 42

As mentioned above (Section 1.2.4.1), zinc can modulate all three of the pathways that are affected by cocaine, placing it in a unique location where alterations in vesicular zinc levels may affect response to cocaine by affecting glutamatergic, BDNF, or ERK signaling pathways. A final consideration is whether drugs act similarly in males and females.

NMDA receptor.

1.4.4 Sex Differences

There is a lot of evidence supporting differences in how males and females respond to various drugs of abuse (Castner et al., 1993; Becker, 1999; Becker et al., 2001; Becker and Hu, 2008). Some evidence stemmed from findings that male and female rodents had different baseline levels of DA in the striatum

(Castner et al., 1993); however, a recent meta-analysis using rat studies found no baseline sex differences in DA in either the dorsal striatum or NAc, as well as no sex differences in drug-induced dopamine changes, although all drugs examined increased DA in both regions (Egenrieder et al., 2020). Therefore, changes in DA may not be the main underlying mechanism behind sex differences in response to cocaine or other abused drugs.

Another proposed mechanism behind sex differences involves interactions between estrogen and BDNF (Barker et al., 2015). However, there is little evidence to support this.

1.5 RATIONALE AND OVERVIEW OF EXPERIMENTS

As mentioned above (Section 1.2.3), there are high concentrations of vesicular zinc in the striatum (dorsal and ventral) and in the neocortex as well as in other brain regions that project to the striatum (Frederickson et al., 1992)

(Figure 1.4). Although little is known about the role of vesicular zinc, specifically in the striatum, research on other brain regions suggests it plays a role in synaptic plasticity. Several in vitro studies have found that altering zinc levels 45 disrupts LTP. Although most of these studies took place in the hippocampus, it seems likely that the role of zinc in LTP/LTD will be similar in other brain areas, including the striatum. Therefore, altering zinc levels should affect LTP/LTD in the striatum and disrupt behaviours that rely on LTP/LTD mechanisms for encoding. Additionally, since zinc has been found to play a role in stabilizing excitatory synapses through its action on Shank3, altering zinc will likely affect the stability of synapses. Increasing zinc may result in increased stability, while decreased zinc may result in a loss of stability. Both could potentially cause issues in behaviour. While there is no guarantee that the role of vesicular zinc is identical in every brain region, it is probable that its action on specific channels is similar regardless of brain region (i.e. if it inhibits the GluN2A subunit in the hippocampus, it is likely to also inhibit that subunit in the striatum).

The presence of vesicular zinc in the dorsal striatum, a structure involved in goal-directed and habitual learning, suggests that it may modulate the learning of goal-directed tasks such as instrumental conditioning. Its presence in brain regions involved in drug-evoked plasticity, especially in the NAc and prefrontal cortex, suggests that vesicular zinc may contribute to responses to drugs of abuse mediated by these areas.

Figure 1.5 Circuits Under Investigation. A sagittal section of a mouse brain stained for vesicular zinc showing the circuits under investigation in this thesis. Solid white arrows indicate the pathway affected by drugs of abuse. Dashed white arrows are the pathway involved in instrumental conditioning (and somewhat with addiction).

The yellow dashed lines are other brain regions involved in modulating those circuits. AMY: amygdala, DSTR: dorsal striatum, HPC: hippocampus, NAc: nucleus accumbens, PFC: prefrontal cortex, SN: substantia nigra, THAL: thalamus, VTA: ventral tegmental area. 47

As stated above (Section 1.1), the purpose of this thesis was to examine the potential role(s) of vesicular zinc in the striatum using ZnT3 KO mice. My hypothesis is that vesicular zinc is critical for striatal plasticity; therefore, the

ZnT3 KO mice will exhibit differences in instrumental learning as well as in cocaine-induced behavioural, anatomical, and physiological changes.

Chapter 2 examines the behaviour of the mice in instrumental conditioning tasks, including differential-low-rate-of-responding (DRL) and progressive ratio

(PR) protocols used to examine impulsivity and motivation, respectively. Chapter

3 examines the effects of cocaine on behaviour (dose response and sensitization), physiology (fast-scan cyclic voltammetry), and neuronal morphology (Golgi-Cox stained tissue). Chapter 4 examines conditioned place preference to cocaine. Chapter 5 examines duration of behavioural effects of cocaine. Lastly, Chapter 6 is a general discussion of all of the findings included in this thesis.

ACKNOWLEDGEMENTS

I would like to thank Sarah Bryden for creating the original version of Figure 1.3 which has been modified for this thesis.

2 CHAPTER 2:

IMPULSIVITY AND MOTIVATION IN MICE THAT LACK VESICULAR ZINC

2.1 INTRODUCTION

The element zinc is an important divalent cation in all cells, playing various structural and functional roles (Chasapis et al., 2012). A subset of zinc in the brain acts as a neurotransmitter – it is loaded into synaptic vesicles by zinc transporter 3 (ZnT3) (Cole et al., 1999) and released into the synapse in an activity- and calcium-dependent manner where it acts on many neurotransmitter receptors (reviewed by McAllister and Dyck, 2017).

This so-called vesicular zinc is found in high concentrations in most regions of the forebrain (Frederickson et al., 1992) and has been studied in depth in the hippocampus and somatosensory (barrel) cortex where it has been shown to be involved in synaptic plasticity mainly due to its ability to bind to and inhibit the glutamatergic NMDA receptor (as reviewed by Nakashima and Dyck, 2009a).

ZnT3 knockout (KO) mice, which lack the slc30A3 gene that codes for the

ZnT3 protein, show a complete absence of vesicular zinc (Cole et al., 1999).

ZnT3 KO mice have been used to study the role of vesicular zinc in the brain.

Most behavioural studies have examined basic sensorimotor tasks, spatial learning and memory, and fear conditioning (Cole et al., 2001; Martel et al., 2010;

Martel et al., 2011; Sindreu et al., 2011; Wu and Dyck, 2018).

One area of the forebrain that is considerably understudied with regards to vesicular zinc is the striatal complex (including both dorsal and ventral striatum). 50

Staining for vesicular zinc indicates a high concentration of vesicular zinc in both dorsal and ventral portions. These structures are important in instrumental learning tasks (Balleine et al., 2009) as well as in response to rewarding stimuli

(Haber, 2016). The function of the striatum can be tested using various versions of instrumental conditioning, which involve learned associations and, in rodents, typically involve learning that a specific response (i.e. pressing a lever or sticking their nose into a specific port) will result in delivery of a food (e.g. food or sugar pellet) or liquid (e.g. sweetened milk) reward. To date, ZnT3 KO mice have not been examined in instrumental conditioning tasks.

In addition, a previous study that examined skilled reach behaviour in female

ZnT3 KO mice suggested that these mice may be more impulsive than wildtype mice (Thackray et al., 2017). Certain instrumental tasks can be used to measure impulsivity in mice.

The purpose of this study was to examine the behaviour of ZnT3 KO mice in two instrumental conditioning tasks – namely, differential reinforcement of low rate of responding (DRL) and progressive ratio – to probe a role for vesicular zinc in these processes. DRL can be used to study impulsive behaviour, and PR is used to study motivated behaviour.

2.2 METHOD

2.2.1 Animals

Male and female ZnT3 wildtype (WT) and KO mice were generated from breeding pairs heterozygous for the slc30A3 allele. Mice were housed in same- sex littermate groups of two or three in a temperature- and humidity-controlled room on a 12 hr light/dark cycle with lights on at 7am. They had ad libitum access to water. For a portion of the experiment, they were food-restricted (see below), but for the remainder, they had ad libitum access to food. All procedures were approved by Life and Environmental Sciences Animal Care Committee at the University of Calgary and followed the guidelines for the ethical use of animals provided by the Canadian Council on Animal Care.

2.2.2 Training

2.2.2.1 Food restriction

Mice were 2-3 months old at the start of training. During the training session, mice were food-restricted until they reached 85 ± 5% of their initial body weight.

Mice were weighed every day to ensure that they remained within this weight range. Mice were fed 2 g of standard lab chow food per mouse per day. Two days prior to training, sugar pellets (Dustless Precision Pellets, 45 mg, Sucrose,

Bio Serv, Flemington, NJ, USA) that were later used in the conditioning boxes were placed in the bottom of the mouse cages to familiarize the mice to the pellets.

2.2.2.2 Conditioning boxes

The conditioning boxes (24x30x30cm; Med Associates Inc., St. Albans, VT,

USA) were located in sound-attenuating cupboards that had outside noise masked by ventilation fans. Each box had a pellet dispenser and food cup in the center of one side of the box with a nose poke hole on either side; one nose poke was active (i.e. would result in a reward) and the other inactive (i.e. would not result in a reward; which side was active was counterbalanced across mice).

Med Associates software (Med-PC, Med Associates Inc., St. Albans, VT, USA) controlled the boxes and recorded the data. All sessions started with illumination of the house light. The software scored nose pokes and food cup entries when the pre-set infrared photo-beam was broken.

2.2.2.3 Training procedure

Fifteen mice per group (male and female, WT and KO) were put through the training sessions. Each session lasted 30 min and followed a fixed ratio 1 (FR1) schedule (i.e. every nose poke resulted in a sugar pellet). To meet the inclusion criteria for the DRL protocol, mice had to meet the following criteria 3 days in a row: a minimum of 5 pellets (45 mg) and a 3:1 active/inactive nose poke ratio (i.e. the mouse had to make at least 3 times as many responses on the active side than on the inactive side).

Mice were kept on the training protocol for a minimum of seven and a maximum of 17 days. Once a mouse met the inclusion criteria, but not earlier than day seven, it was switched to the DRL schedule (see below and Figure 2.1) 53 on the following day. Mice that did not pass training were removed from the experiment.

2.2.3 Differential Reinforcement of Low Rate Schedule

The differential reinforcement of low rate (DRL) of responding schedule was used to assess impulsivity. Mice had to learn to withhold responding for a certain period of time before responding again; if they responded too early, the clock would reset to zero.

They were started on a DRL-6 schedule, meaning they had to wait 6 sec between responses, and were kept on this schedule for 4 days. The interval was increased in increments of 6 sec following the same pattern of 4 days for each schedule up until DRL-36. Success rate, number of sugar pellets earned, number of active nose pokes, and active/inactive nose poke ratio were averaged for each

DRL schedule. Frequency of inter-response time (IRT) less than 3 sec and less than DRL time was also determined.

Figure 2.1 Experiment Timeline. Mice underwent 7-10 days of training on a FR1 schedule of reinforcement until they met the inclusion criteria. After passing the training, mice were started on a DRL schedule of reinforcement that began with a 6 sec delay and advanced by 6 sec every 4 days. Following 24 days on the DRL schedule, mice were kept on a PR schedule for 5 days. 55

2.2.4 Progressive Ratio (PR)

Following completion of the DRL procedure, mice completed 5 days under a progressive ratio (PR) schedule. The progressive ratio schedule utilized the

j/5 nj = 5e -5 equation (i.e.1, 2, 4, 6, 9, 12, etc.) (Richardson and Roberts, 1996).

Breakpoint was determined for each mouse.

2.2.5 Statistical Analyses

Repeated measures analysis of variance (ANOVA) were used to examine differences between DRL schedules or session (PR task). Paired-samples t-tests were used to follow up significant differences between days. One-way ANOVAs were used to compare individual tests. Statistics were computed using IBM

SPSS v.24 (Armonk, NY: IBM Corp.). Figures were created using GraphPad

Prism version 8.4.2 for Windows (GraphPad Software, San Diego, California

USA, www.graphpad.com).

2.3 RESULTS

2.3.1 Training

A similar training paradigm (FR1) is used by many other researchers.

Generally, 75% of mice pass within 7-10 days, 95% within 15-17 days, and 5% never meet the criteria (Sharma et al., 2012). Chi square tests were used to compare this expected outcome with how many mice in each group passed 56 training (note: sample sizes are too low for this to be completely accurate).

Based on chi square results, male WT followed the expected outcome [χ2(2) =

2.87, p = .239]. However, male KO [χ2 (2) = 39.02, p <.001], female WT [χ2 (2) =

25.87, p <.001], and female KO [χ2 (2) = 14.87, p <.001] did not follow the expected outcome, with more mice failing training than expected (Figure 2.2).

As mice were food-restricted, weight was carefully monitored during training.

Weight from the day restriction started was compared to weight on the first day of testing (Figure 2.3). As hunger provides motivation to perform in this task, mice who failed to pass training may not have lost sufficient weight to be motivated to learn the task.

Weight change for mice that did not meet criteria and were excluded from the study. 59

2.3.2 DRL Schedule

One female WT and one female KO were removed from analysis; the WT was determined to be maloccluded, which resulted in it not eating food pellets but scarfing down sugar pellets (and therefore having data that is more than 2 SD away from the mean); the KO had data more than 2 SD from the mean for unknown (to us) reasons.

Success rate was examined using a 2x2x6 (sex x genotype x DRL)

ANOVA. No significant differences were found [3-way interaction: F(5,185) =

1.12, p = .353] (Figure 2.4A & B).

A 2x2x6 (sex x genotype x DRL) ANOVA examining number of pellets earned found a significant effect of DRL [F(5,185) = 22.59, p< .001] (Figure 2.4C

& D). Follow-up tests found significant difference between DRL-6 and all other

DRL times [DRL-12: F(1,37) = 36.75, p<.001; DR-18: F(1,37) = 55.13, p <.001;

DRL-24 :F(1,37) = 47.19, p <.001; DRL-30: F(1,37) = 39.12, p < .001; DRL-36:

F(1,37) = 49.53, p <.001]. The DRL x genotype interaction approached significance [F(5,185)= 2.61, p = .060, Greenhouse-Geiser  = .558]; however, no statistically significant difference was observed in any of the follow-up tests. Both sexes showed statistically significant differences in DRL mimicking that found in the overall test [males: F(5,100) = 13.76, p <.001; females: F(5,85) = 9.85, p

<.001]. In males, the DRL by genotype interaction approached significance

[F(8,100) = 2.47, p = .083, Greenhouse-Geisser  = .500] 60

Number of active nose pokes was examined using a 2x2x6 (sex x genotype x

DRL) ANOVA where a statistically significant effect of DRL was found [F(5,185) =

9.85, p <.001] (Figure 2.4E & F). Follow-up tests found significant differences between DRL-6 and all other DRL times [12: F(1,37) = 15.13, p<.001; 18: F(1,37)

= 24.50, p<.001; 24: F(1,37) = 27.92, p<.001; 30: F(1,37) = 19.01, p<.001; 36:

F(1,37) = 21.84, p<.001]. DRL x genotype approached significance [F(5,185) =

2.70, p = .052, Greenhouse-Geiser  = .573]; follow-up tests did not have any significant differences. There were no other statistically significant main effects or interactions.

A 2x2x6 (sex x genotype x DRL) ANOVA was used to examine active/inactive ratio and found a statistically significant effect of DRL [F(5,185) =

3.51, p = .027, Greenhouse-Geiser  = .473] (Figure 2.4G & H). Follow-up tests found a significant difference between DRL-6 and DRL-12 [F(1,37) = 21.07, p

<.001]; 6 and 36 [F(1,37) = 10.10, p = .003]. When separated by sex, only males have a significant effect of DRL [F(5,100) = 3.49, p = .006] and only DRL-6 vs

DRL-12 was significant [F(1,20) = 13.69, p = .001]. There were no other statistically significant main effects or interactions.

IRTs less than 3 sec indicate impulsive responding. A 2x2x6 (sex x genotype x DRL) ANOVA was not significant [F(5,185) = 1.49, p = .209] (Figure 2.5A & B).

No differences were found for individual sexes either. There were no other statistically significant main effects or interactions. 61

IRTs less than DRL time also indicate impulsive responding and a lack of learning. A 2x2x6 (sex x genotype x DRL) ANOVA had a significant effect of DRL

[F(5,185) = 2.28, p = .049] (Figure 2.5C & D). Follow-up tests found statistically significant differences between DRL-6 and DRL-18 [F(1,37) = 10.52, p = .003] as well as DRL-6 and DRL-30 [F(1,37) = 10.20, p = .003]. There were no other significant main effects or interactions. Figure 2.5 also shows a histogram of frequencies of IRTs divided into 3 sec bins for the last day of the DRL-36 schedule of reinforcement for each group (Panels E-H). 62

Figure 2.4 . DRL Results. Average success rate (A & B), number of pellets earned

(C & D), nose pokes in the active hole (E & F), and active/inactive nose poke ratio

(G & H) for male (A, C, E, G) and female (B, D, F, H) mice on each stage of DRL reinforcement. Number of pellets earned and number of nose pokes decreased across sessions for both male and female mice. The active:inactive ratio decreased significantly between DRL6 and DRL12 in males but was otherwise unaffected. Error bars represent ± SEM. *: p < .05; ***: p <.001. 63

Figure 2.5 Inter-Response Time. Average frequency of IRTs less than 3 sec (A & B) and IRTs less than DRL time (C & D) for male (A, C) and female (B, D) mice. IRTs less than DRL time significantly increased in both male and female mice on DRL18 and DRL30 compared to DRL6. Panels E-H show histograms of IRTs on the last day of DRL-36 for each group. Error bars represent ± SEM. *: p < .05; ***: p <.001. 64

2.3.3 PR Schedule

Breakpoint is the point in the PR schedule at which mice cease responding.

A 2x2x5 (sex x genotype x day) ANOVA had a significant effect of day [F(4,144)

= 36.41, p <.001] (Figure 2.6). Follow-up tests found differences between days 1 and 2 [F(1,37) = 62.28, p <.001]; 1 and 3 [F(1,37) = 37.93, p <.001]; 1 and 4

[F(1,37) = 89.93, p <.001]; 1 and 5 [F(1,36) = 99.22, p <.001; 2 and 5 [F(1,36) =

10.19, p = .003]; and 3 and 5 [F(1,36) = 12.51, p = .001]. There were no statistically significant effects of sex or genotype or any interactions.

Figure 2.6 Progressive Ratio Results. Breakpoint for each day on the PR schedule for male (A) and female (B) mice. Both male and female mice showed significant decreases in breakpoint over time; however, there were no differences between genotypes. Error bars represent ± SEM. *: p < .05; ***: p < .001. 66

2.4 DISCUSSION

This study examined the performance of zinc transporter 3 (ZnT3) knockout

(KO) mice on two instrumental conditioning tasks, namely differential reinforcement of low rate of responding (DRL) and progressive ratio (PR), to determine whether they exhibit changes in impulsivity or motivation, respectively, compared to wildtype (WT) mice. Perhaps the most intriguing finding of this study is that ZnT3 KO mice of both sexes and female WT mice did not pass the training schedule at the same rate as male WT mice when compared to other studies that use similar testing protocols. One study found that approximately

75% of mice passed training within 7-10 days and 95% of mice passed within 17 days; only 5% of mice failed to meet the inclusivity criteria (Sharma et al., 2012).

In the current study, 40% of male KO, 33% of female WT, and 27% of female KO failed to pass the training. This may relate to a lack of motivation due to not having lost enough weight during the food restriction. However, when examining

Figure 2.3 Panels B (passed) and C (failed), this explanation works for the two male WT that failed training and 2 of the 6 male KO that failed but does not explain why the others did not pass training. It is noteworthy that three male KO mice did not seem to lose any weight but were still able to meet the inclusion criteria, suggesting an involvement of other factors.

Once mice successfully passed the training phase, no statistically significant effects of sex or genotype on any other measures were observed (e.g. number of pellets earned, number of active nose pokes, breakpoint, success rate, active/inactive ratio, IRTs). Lack of differences between genotypes indicates that 67

ZnT3 KO mice show similar amounts of impulsivity and motivation as ZnT3 WT mice.

One interesting observation from this study involves the active/inactive ratio.

To “pass” training and inclusivity criteria, mice had to have an active/inactive ratio of 3:1 for 3 consecutive days. However, Panels G and H in Figure 2.4 suggest that this ratio was not maintained throughout the duration of the experiment.

Most mice were maintaining closer to a 2:1 ratio. This may indicate that when the initial strategy they learned (nose poke in the active hole) was not getting them what they wanted (i.e. the sugar pellet), they switched strategies.

Another observation involves the IRT histograms from the last day of DRL-

36. Other studies have found that when an animal has learned the DRL task sufficiently, they will show an increase in responses that peak around the DRL time (in this case 36). A lack of this increased responding suggests that the ZnT3 mice, WT and KO, did not learn the DRL task and were performing randomly.

Therefore, the lack of findings may have more to do with the lack of learning the task properly, and we cannot conclusively say that the ZnT3 KO mice are less impulsive based exclusively on their performance on this DRL task.

There were a few methodological issues that may have impacted the findings of this study. One factor is the size of the conditioning boxes used. Rather than the more commonly used mouse-sized conditioning boxes, in this experiment, larger rat-sized boxes were used. While mice were still able to acquire the conditioning, it is possible that the size of the box impacted the mouse’s response. In particular, the reason that the passing rate was low for male KO and 68 female WT and KO mice may have more to do with the size of the box than with their actual ability to perform the task.

Type of reinforcer and type of action to receive the reinforcer may also affect responding. There are different types of reinforcers that can be used for instrumental conditioning tasks. In our study, we used 45 mg sugar pellets, as these were the correct size for our dispensers; however, in other studies with mice, smaller pellets (20 mg) or liquid reinforcers such as sweetened milk were used. While we adjusted the number of reinforcers the mice needed to achieve in a given session based on the weight of the pellets, it is possible that the mice would respond better to smaller portioned reinforcers or to liquid reinforcers.

Also, some studies use lever pressing while others use nose pokes. It has been suggested that when both are available, levers should be used as they produce more reliable responses (Sharma et al., 2012). However, it has also been determined that this does not affect performance in certain strains of mice (Haluk and Wickman, 2010). In our case, we only had rat levers available, and the mice were not heavy enough to activate them.

Another potential factor impacting the outcome was that food restriction was not maintained throughout the course of testing. Food restriction, or more accurately the hunger produced by the food restriction, is the standard protocol used to motivate the animal to respond. It is possible that the decrease in responding seen throughout the course of testing was due to a lack of hunger or motivation to work for the sugar pellet. This may have had more of an effect on the PR schedule than the DRL schedule. 69

In conclusion, while fewer male KO and female mice met the inclusivity criteria for the DRL and PR tasks, once the criteria are met, WT and KO mice perform similarly across the sessions. This suggests that ZnT3 KO mice do not have deficits in motivation or impulsivity, however, methodological limitations make it difficult to determine for certain.

2.5 ACKNOWLEDGEMENTS

I would like to thank Katy Celina Sandoval, Selena Fu, Angela Pochakom, and

Nicole Niewinski for assisting with data collection.

3 CHAPTER 3:

VESICULAR ZINC MODULATES THE EFFECTS OF ACUTE AND CHRONIC

COCAINE EXPOSURE

3.1 INTRODUCTION

Zinc is a cation found in all cells of the body where it performs various structural and functional roles (Chasapis et al., 2012). In the brain, a subset of zinc, called vesicular zinc, is loaded into synaptic vesicles by zinc transporter 3

(ZnT3) where it can then be released in a calcium- and activity-dependent manner (McAllister and Dyck, 2017).

Staining for vesicular zinc reveals high amounts in almost all areas of the forebrain (Frederickson et al., 1992). Of note, there are high amounts in most areas of the so-called reward pathway, including the striatum, the nucleus accumbens, and prefrontal cortex, but to date, no research has examined its role there. In other brain areas, like hippocampus and somatosensory (barrel) cortex, vesicular zinc is thought to play a role in modulating synaptic plasticity due to its ability to inhibit NMDA and AMPA receptors (Nakashima and Dyck, 2009b;

McAllister and Dyck, 2017).

ZnT3 knockout (KO) mice lack the slc30A3 gene that codes for the ZnT3 protein and can be used to determine the role of vesicular zinc in the brain (Cole et al., 1999). Drugs of abuse can be used to test the function of the reward system. Cocaine, in particular, has well-characterized effects on anatomy, physiology and behaviour in rodents (Robinson and Kolb, 1999b; Heien et al., 72

2005; Flagel and Robinson, 2007). As a stimulant drug, cocaine exerts most of its effects by blocking the dopamine (DA) transporter (DAT) leading to increases in DA in the synapse (Amara and Sonders, 1998). However, it is also capable of blocking the norepinephrine and serotonin transporters (Amara and Sonders,

1998).

Many studies have found that zinc is capable of modulating DA receptors as well as DAT (Norregaard et al., 1998; Meinild et al., 2004; Bjorklund et al., 2007;

Pifl et al., 2009). One in vitro study examined the combined effects of zinc and cocaine and found that zinc also bound to the DAT and augmented cocaine’s actions leading to further and prolonged increases of DA (Richfield, 1993).

Due to its location in the reward pathway and its purported role at the DAT as well as its effects on NMDAR and AMPARs, zinc is in an ideal position to modulate plastic responses to cocaine.

This study aims to determine a role for vesicular zinc in the modulation of the response to cocaine by examining behavioural, physiological, and anatomical outcomes in both ZnT3 KO and wildtype (WT) mice following exposure to cocaine. Behavioural sensitization has been found to occur in rodents following a drug-free period after previous repeated exposure to a drug and can be used to examine the effects of cocaine (Steketee and Kalivas, 2011). Fast-scan cyclic voltammetry (FSCV) is used to measure dopamine release in the striatum before and after cocaine exposure in mice that have been acutely or chronically exposed to cocaine. The Golgi-Cox stain is used to examine dendritic branching and spine density in the striatum following acute or chronic exposure to cocaine 73 as changes in these measures have been previously reported following repeated cocaine exposure (Robinson and Kolb, 1999b; Kolb et al., 2003).

3.2 METHOD

3.2.1 Animals

The male and female ZnT3 wildtype (WT) and knockout (KO) mice used in this study were bred from mice heterozygous for the slc30A3 gene on a mixed

C57BL/6 x 129/SvEv genetic background. All mice were group housed (2-5 same-sex littermates per cage) in standard laboratory cages under a 12 h light/dark cycle in a temperature- and humidity-controlled room (22C; 25-30% humidity) and provided tap water and food (LabDiet Mouse Diet 9F, #5020) ad libitum. Mice were 2-3 months old at the start of testing. All procedures were approved by the Life and Environmental Sciences Animal Care Committee of the

University of Calgary and conformed to the guidelines set out by the Canadian

Council on Animal Care.

3.2.2 Drugs

Cocaine hydrochloride (Medisca Pharmaceutique Inc., St-Laurent, Quebec,

Canada) was dissolved in 0.9% saline and administered intraperitoneally (I.P.) at doses of 5, 10, 15, 20, 25, and 30 mg/kg. Control animals were given equivalent doses of 0.9% saline. 74

3.2.3 Behaviour

3.2.3.1 Habituation

Mice were moved to the testing room 30 min before testing began. All mice were weighed at the beginning of the test day in the testing room. Weight was recorded and used for dosing purposes. There were 3 days of habituation to the testing arenas, as well as to intraperitoneal (I.P.) injections (Figure 3.1). A single trial consisted of placing the animal in the test arena (40x40x40 cm box made from white corrugated plastic with a thin layer of bedding on the bottom) and tracking its movement with an overhead camera (Basler acA1300-60gm GigE,

Basler AG, Germany) and computer software (Ethovision, Noldus Information

Technology, Wageningen, the Netherlands) for 15 min. The mouse was then removed briefly, given an I.P. injection of saline (0.9%; 0.01 ml/g body weight) and returned to the test arena to be tracked for an additional 45 min. At the end of the 45 min period, the mouse was removed and returned to its home cage.

Four mice were tested (in separate arenas) concurrently. Boxes and bedding were cleaned with 70% EtOH between mice and bedding was changed between sexes.

3.2.3.2 Dose response

Dose response testing took place over 10 days with a two day break half-way through (Figure 3.1). Mice were assigned to either the cocaine (COC) experimental group (male: n = 13 WT, 12 KO; female: n = 13 WT, 14 KO) or to a saline (SAL) control group (male: n = 12 WT, 12 KO; female: n = 10 WT, 10 KO).

A single trial was as described above; however, the I.P. injection was either saline (0.9% 0.01 ml/g body weight) or cocaine (mg/kg body weight), depending on which group the mouse was assigned to. Cocaine was administered starting with a sub-threshold dose (5 mg/kg) to determine whether ZnT3 mice are more sensitive to cocaine. The dose was increased in increments of 5 mg/kg over the

10 days of testing (with some doses being administered more than once; see

Figure 3.1) to a final dose of 30 mg/kg.

Within session data were also examined graphically using 15 min bins (4 total: 1 pre-drug, 3 post-drug) with total distance travelled in each bin determined.

Average response for each dose is presented in the Appendix.

Figure 3.1 Dose Response and Sensitization Schedule. Male and female ZnT3 WT and KO mice were habituated to the test arena and received I.P. saline injections for

3 days to determine baseline locomotor activity (BL1-BL3). Mice were then assigned to either a saline control group or to the cocaine group. Mice in the cocaine group received escalating doses of cocaine (5-30 mg/kg) over 10 days while mice in the control group received equivalent injections of saline. Each session involved a 15 min habituation period followed by the injection and tracking of movement for 45 min post-injection. Following a two-week incubation period, all mice were given 10 mg/kg cocaine and tracked for 45 min.

3.2.3.3 Locomotor sensitization

Behavioural sensitization has been shown to occur in rodents that have received repeated administration of cocaine followed by an incubation period.

After the two weeks of dose response activity, mice were left undisturbed in their home cages for a period of two weeks. Following this was a cocaine challenge day, on which all mice, irrespective of initial drug group, received an I.P. injection of 10 mg/kg cocaine and activity was recorded as stated above.

3.2.3.4 Stereotyped behaviours

High doses of cocaine have been shown to induce stereotypy. Therefore, video recordings made during the dose response portion of the experiment were examined for evidence of stereotyped behaviours. In cases where the mice received the same dose on more than 1 day, only the first day of administration was scored. Based on the methodology used by Rebec and Segal (1980), mice were observed for 1 min periods beginning 5 min after drug administration and continuing every 10 min until the end of the testing session (i.e. 4 intervals).

During each 1 min period, we examined individual components of stereotypy

(sniffing, head and limb repetitive movements, and oral behaviors such as licking and biting) for their intensity (1 = mild; 2 = moderate; 3 = intense) and duration (1

= discontinuous; 2 = continuous). For each behaviour, a single score in each interval was determined by multiplying the intensity score by the duration score.

The scores were summed to have a single value for behavior at each dose. The observer was blind to the condition of the mice. 78

3.2.4 Fast Scan Cyclic Voltammetry

Fast-scan cyclic voltammetry (FSCV) is one method that can be used to measure dopamine release. Mice were left undisturbed in their home cages following the cocaine challenge day for at least one week prior to commencement of FSCV surgeries.

3.2.4.1 Electrode fabrication

Carbon fibers were inserted into glass pipettes using a vacuum. Pipettes were pulled to create two electrodes of equal length. Carbon fibers were cut to a length of 120-140 µm using a scalpel blade under 100x magnification.

Connecting wires were created by soldering a gold pin to the end of a 30-gauge wire wrapped with blue insulation. Shrink wrap was used to secure the wire to the pin. Silver print was applied to the exposed wire which was then carefully inserted into the carbon fiber-containing glass pipette. Shrink wrap was used to secure the connection between the glass and the wire.

Reference electrodes were created by inserting a 1 cm piece of silver wire into a gold pin. Epoxy was used to secure the wire to the pin. Immediately prior to surgery, a reference electrode was chlorinated by electroplating in 1 N HCl.

3.2.4.2 Electrode calibration

Each recording electrode was assigned a number and calibrated to determine its sensitivity to DA. Electrodes were hooked up to a flow cell that contained a chlorinated reference electrode and that allowed continuous flow of artificial cerebrospinal fluid (aCSF) and addition of either 250 nM or 1000 nM DA.

Wires were used to connect the electrode to the computer. TarHeel CV software

(UNC Electronics Facility, Chapel Hill, NC) was used for data collection. The software was set to create a triangular waveform scanning from +1.3 V to -0.4 V at a rate of 400 V/s. The waveform was applied at 60 Hz and the electrode was lowered into the aCSF in the flow cell. To ensure the electrode was functioning properly and that it was lowered to the correct depth (i.e. into the aCSF flow), an oscilloscope was used. Any electrodes not having the characteristic shape on the oscilloscope were rejected and replaced. Electrodes displaying the characteristic shape were cycled (had the triangular waveform applied) at 60 Hz for approximately 10 min. The frequency was then lowered to 10 Hz for calibration.

For each electrode, 3 recordings were made for each of aCSF only, 250 nM DA, and 1000 nM DA. These were later used in analysis of successful surgeries.

3.2.4.3 Surgeries

Mice were anaesthetized with 5% urethane dissolved in sterile dH2O approximately 1 hour prior to surgery. Fur was removed from the top of the head and the mouse was placed securely into the stereotaxic instrument using the ear bars and nose piece. Lidocaine was injected under the scalp. Scalp was then 80 removed using scissors to reveal the skull. Burr holes were drilled over dorsal striatum (AP: +0.9; ML: +1.8; DV: -2.0) and ventral tegmental area (AP: -3.2; ML:

-0.6; DV: -4.0) of the right hemisphere for placement of the recording and stimulating electrodes, respectively, and over cortex (AP: -2.2; ML: -3.2) of the left hemisphere for placement of the reference electrode. A chlorinated reference electrode was carefully inserted into its burr hole and cemented into place with

Metabond (Parkell, Inc., Edgewood, NY). Once dry, the recording electrode was attached to the stereotactic arm, wires were connected to both reference and recording electrodes, and TarHeel software was setup as stated above

(triangular waveform, cycling at 60 Hz). The oscilloscope was opened, and the recording electrode was carefully lowered into the brain to DV -2.0. It was cycled at 60 Hz for 20-30 min then at 10 Hz for 10-15 min. If at any point there appeared to be a problem with the electrode (e.g. broken, too sensitive, not sensitive enough) that may affect recording, it was replaced with a new electrode.

While the recording electrode was cycling, the stimulating electrode (4mm

Bel Ped Elect, 8mm proj, HRS Scientific, Anjou, QC, Canada) was setup. After ensuring the electrode was straight from all angles, it was lowered to DV -3.8 and left there until recording commenced.

Once the recording electrode finished cycling, the TarHeel software was set to deliver stimulating pulses (150 µA, 4 ms monophasic current; 60 pulses delivered at 60 Hz). The stimulating electrode was lowered in 0.2 mm increments until robust dopamine release was detected at the recording electrode. 81

When both electrodes were in position for optimal dopamine detection, training set files were recorded for use in analysis. Eight files were collected, altering the frequency and number of pulses of the stimulating electrode (60 Hz +

60 p; 60 Hz + 24p; 60 Hz + 12 p; 60 Hz + 6 p; 30 Hz + 6 p; 30 Hz + 12 p; 30 Hz +

24 p; 30 Hz + 60 p).

To mimic what was done during the behavioural testing, mice were injected intraperitoneally with saline and recorded for 15 min to determine baseline dopamine release, then injected with 10 mg/kg cocaine and recorded for 45 min.

TarHeel software was set to collect 300s files, stimulating at 60 Hz, 24 pulses, 5 s after initiation of the file. Thus, the mouse received a stimulation every 5 min. At the end of recording, the mouse was removed from the stereotaxic apparatus and perfused transcardially with 0.9% saline (see section 3.2.5.1).

A subset of mice were used for FSCV; however, only mice that had a successful surgery were included in the analysis (male WT SAL = 6; KO SAL = 6;

WT COC = 3; KO COC = 7; female WT SAL = 3; KO SAL = 6; WT COC = 6; KO

COC = 5).

3.2.4.4 Analysis

FSCV data was analyzed using High Definition Cyclic Voltammetry software

(HDCV; University of North Carolina, Chapel Hill, NC). Briefly, slope was determined for each individual recording electrode using the information collected during calibration to determine sensitivity to DA at 250 nM and 1000 nM DA 82

(GraphPad software Inc., San Diego, CA, USA). Slope data were used as a measure of electrode sensitivity to DA and entered into the HDCV software for each mouse. Training set data were also loaded into HDCV for each mouse to create a DA response profile. HDCV data converter software was used to convert and concatenate the recorded files into longer HDCV files for analysis. DA concentration was determined by chemometric analysis in HDCV software, which used principle components analysis. Data were exported into excel files for group analyses.

3.2.5 Morphology

3.2.5.1 Procedure

Immediately following completion of FSCV surgery, mice were perfused transcardially with 0.9% saline. Brains were removed and, if the surgery was successful, cut in half along the midsagittal line. The right hemisphere (where the electrodes were located during surgery) was placed in formalin for 24 hours before being moved to a glycerol solution. The left hemisphere, or the whole brain for unsuccessful surgeries, was placed into Golgi-Cox solution for a period of 10 days. After the 10 days, brains were switched to a 30% sucrose solution for

3-7 days. Brains were then sliced either sagittally (for half brains) or coronally (for whole brains) on a vibratome into 250 µm sections. Sections were mounted onto

1.5% gelatin-coated slides and pressed down gently using bibulous paper dampened with distilled H2O (dH2O). Slides were left in a humidity chamber for 2- 83

5 days before staining. The slides were stained as follows: 1 min in dH2O; 30 min in ammonium hydroxide under a dark box; 1 min in dH2O; 30 min in Kodak Fix for

Film diluted 1:1 with dH2O under a dark box; 1 min in dH2O; 1 min in each of

50%, 70%, 95% ethanol; 3 x 5 min in 100% ethanol; 10 min in equal parts chloroform/xylene/100% ethanol; 2 x 15 min in xylene. Slides were then cover- slipped with Permount.

3.2.5.2 Quantification

Medium spiny neurons (MSNs) make up ~95% of the neurons and are the main projection cells in the striatum. Dorsolateral striatum (DLS) is the region of the striatum associated with motor function and was therefore chosen for an examination of MSNs for morphological changes caused by cocaine. Cells were selected for analysis if the full cell was visible within the section and did not overlap greatly with nearby cells. Selected neurons (n = 4/brain) were traced at

400x magnification (40X/0.75 objective) using a microscope (Zeiss Axioskop 2) with a camera lucida attachment. Arborization was examined using Sholl analysis, which consists of placing a transparency with a series of concentric rings centered over the cell soma and counting the number of dendrites crossing each ring. This allows for an estimation of total dendritic length.

Spine density of MSNs in the DLS was also examined. Neurons were selected in a similar way as above and sections of terminal dendrites (n =

8/brain) were traced at 1000x magnification (100x/1.30 objective). Length of branch was determined using ImageJ software (ImageJ 1.51j8; National 84

Institutes of Health, Bethesda, MD, USA) and number of spines was counted.

These measurements were used to calculate spine density (# of spines/length of dendrite).

3.2.6 Vaginal Lavage and Cytology

As stage of estrus cycle has been shown to affect behaviour, female mice were subjected to vaginal lavage to determine estrus cycle stage.

A subset of female mice (n = 7/group) were subjected to vaginal lavage every day (beginning on habituation day 1 until the last day of dose response testing) following testing or around the time testing would normally finish on days when no testing was scheduled. They also had vaginal lavage performed on the cocaine challenge day. To perform vaginal lavage, ~30-50 µl of sterilized dH2O was flushed into and out of the vaginal canal using a micropipetter. The collected fluid was placed onto a glass microscope slide and allowed to dry. Once dry, slides were stained using 1% crystal violet (Sigma-Aldrich, Oakville, ON,

Canada). Slides were examined under a microscope (Zeiss Axioskop 2) at 100x magnification and estrus stage was determined based on which cell types were present. There are four stages of the estrus cycle: proestrus (characterized by high amounts of nucleated epithelial cells), estrus (characterized by cornified squamous epithelial cells), metestrus (characterized by a mix of cells including a high amount of leucocytes and a few nucleated epithelial and cornified squamous epithelial cells), and diestrus (characterized by high amounts of leucocytes and several nucleated epithelial cells) (McLean et al., 2012). 85

3.2.7 Statistical Analyses

Repeated measures analysis of variances (ANOVAs) were used to examine dose effects with genotype and group (SAL or COC) as factors. 2x2 (genotype x group) ANOVAs were used to examine effects at a single dose. Protected paired t-tests were used to follow-up main effects of dose. Tukey’s HSD or protected independent t-tests were used to follow-up significant main effects other than dose. All statistics were performed using IBM SPSS Statistics Version 24.0

(Armonk, NY: IBM Corp.). Figures were created using GraphPad Prism software version 8.4.2 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com).

3.3 RESULTS

3.3.1 Behaviour

3.3.1.1 Dose response

In males, there was a significant three-way interaction between dose, genotype, and drug interaction [F(5,225) = 3.26, p = .034, Greenhouse-Geisser 

= .479], as well as a significant interaction between dose and drug [F(5,225) =

22.53, p <.001] and finally a significant main effect of dose [F(5,225) = 31.21, p

<.001] (Figure 3.2A). The interaction between dose and genotype, and main effects of genotype and drug were not significant. 86

Both saline [F(5,110) = 6.79, p = .001, Greenhouse Geisser  = .473] and cocaine [F(5,115) = 29.13, p <.001] groups had significant effects of dose. Within the saline group, although the “dose” of saline stayed constant, there were significant differences between days when 5 mg/kg and 30 mg/kg were administered (p = .001) and when 15 mg/kg and 30 mg/kg were administered (p

= .001). There was a significant difference between genotypes on the 20 mg/kg day (p = .040). Within the cocaine group, 10, 15, and 20 mg/kg doses were not different from 30 mg/kg; 15 vs 20 and 20 vs 25 mg/kg were also not significant.

All other dose comparisons were significant. Also in the cocaine group, the interaction between dose and genotype interaction approached significance

[F(5,115) = 2.65, p = .071, Greenhouse-Geisser  = .474]. There was a significant difference between genotypes at 5 mg/kg (p = .047, Cohen’s D = .84) and a non-significant difference at 10 mg/kg but a medium effect size (Cohen’s D

= .62).

In females, there was a significant interaction between dose and drug

[F(5,215) = 23.50, p <.001] and a significant main effect of dose [F(5,215) =

42.56, p <.001] (Figure 3.2B). Both the saline [F(5,90) = 5.31, p = .020,

Greenhouse Geisser  = .282] and cocaine [F(5,125) = 49.66, p <.001,

Greenhouse-Geisser  = .607] groups had a significant effect of dose. Within the saline group, there was only a significant difference between the days the cocaine group received 5 and 10 mg/kg (p = .002). Within the cocaine group, the interaction between dose and genotype approached significance [F(5,125) =

2.56, p = .061, Greenhouse-Geisser  = .607]. Also, in the cocaine group, there 87 was a trend towards a significant effect of genotype [F(1,25) = 3.81, p =.062].

There was a significant difference between genotypes at 25 mg/kg (p = .024,

Cohen’s D = .92) with WT mice traveling greater distances than KO mice. Doses of 5, 20, and 30 mg/kg had non-significant differences, but medium effect sizes

(Cohen’s D = .63, .74, .78, respectively).

Distance travelled was determined. Male (C) and female (G) ZnT3 WT and KO mice in the cocaine group were compared on distance travelled the first time they received

10 mg/kg cocaine and on the challenge day – the second time they received 10 mg/kg cocaine. Panels D & H show oral stereotypic behaviours; Panels E & I show repetitive stereotypic behaviours; and Panels F & J show sniffing stereotypic behavior. Error bars represent ± standard error of the mean (SEM). *: p < .05; ***: p <.001; a: medium effect size; aa: large effect size. 89

3.3.1.2 Sensitization

Whether the mice showed sensitization to cocaine was determined by comparing locomotor activity on day 3 with day 26 – on both days mice received

10 mg/kg cocaine. Independent samples t-tests were used to compare between groups, and paired samples t-tests were used to compare activity within each group. As mice in the saline group would not show sensitization, only the cocaine groups were examined.

In males, the WT cocaine group showed a significant increase in activity on day 26 compared to day 3 [t(12) = 7.31, p < .001]; the KO cocaine group did not

[t(11) = 1.45, p = .175] (Figure 3.2C). Comparing the fold increase (day 26 divided by day 3 distance travelled), there was a significant difference between the genotypes with WT mice showing a greater increase [t(23) = 2.29, p = .032,

Cohen’s D = .91].

In females, both WT [t(12) = 4.36, p = .001] and KO [t(13) = 6.65, p <.001] cocaine groups showed significant increases in activity (Figure 3.2G). There was no significant difference between genotypes.

3.3.1.3 Focused stereotypy

For oral behaviours in males, there was a significant interaction between dose and drug [F(5,225) = 3.54, p = .019, Greenhouse-Geisser  = .561] with the cocaine group showing greater reductions in oral behaviours as dose increased, as well as significant main effects of dose [F(5,225) = 18.39, p <.001] with 90 behaviours decreasing as dose increased and drug [F(1,45) = 19.17, p <.001] with cocaine groups showing fewer oral behaviours at the higher doses (Figure

3.2D). Following up the dose effect, 5 mg/kg was significantly different than all other doses and between 10 and 20, 10 and 30 mg/kg. There were no other differences between doses. In the saline groups, there were significant differences between 5 and 20 and 10 and 20 mg/kg; in the cocaine groups, there were significant differences between 5 mg/kg and all other doses, 10 and 30, 15 and 30 mg/kg.

For repetitive behaviours in males, there was a significant interaction between dose and drug [F(5,225) = 3.64, p = .003] with mice in the cocaine groups showing reductions in repetitive behaviours as dose increased while saline mice stayed consistent, as well as significant main effects of dose

[F(5,225) = 2.65, p =.024] and drug [F(1,45) = 49.69, p <.001] (Figure 3.2E).

Following up the effect of dose, 5 mg/kg was significantly different than 20 mg/kg, but there were no other differences between doses. This effect was caused by the cocaine group, as the saline groups did not have any significant differences between dose while it was significant in the cocaine groups.

For sniffing behaviours in males, there was a significant interaction between dose and drug [F(5,225) = 4.29, p =.003, Greenhouse-Geisser  = .791] with mice in the saline groups showing increases over time while mice in the cocaine groups remained relatively consistent, as well as a significant main effect of dose

[F(5,225) = 6.69, p <.001] (Figure 3.2F). This was consistent when examining saline and cocaine groups separately. In the saline group, there were significant 91 differences between 10 and 25, 10 and 30, 15 and 25, 15 and 30 mg/kg. In the cocaine group, there was a difference between 20 and 25 mg/kg.

For oral behaviours in females, there was a significant effect of dose

[F(5,220) = 8.26, p <.001] with all groups showing reductions as dose increased and a significant main effect of drug [F(1,44) = 14.65, p <.001] with mice in the cocaine groups showing fewer incidences of sniffing behaviours (Figure 3.2H).

Following up the dose effect, there were significant differences between 5 and

20, 25, 30 mg/kg and between 10 and 25 and 30 mg/kg. Notably, the saline groups did not have any significant differences between dose while these differences were significant in the cocaine groups.

For repetitive behaviours in females, there was a significant effect of dose

[F(5,220) = 17.40, p <.001] with reductions in behaviour occurring as dose increased and a main effect of drug [F(1,44) = 84.51, p <.001] with mice in the cocaine group showing few repetitive behaviours at higher doses (Figure 3.2I).

Following up the effect of dose, 5 mg/kg was significantly different than all other doses, but there were no other differences between doses. Notably, the saline groups did not have any significant differences between dose while these differences were significant in the cocaine groups. In the cocaine groups, at doses of 5, 15, and 25 mg/kg, genotype differences approached significance [p =

.078, p = .073, p = .054, respectively] with medium to large effect sizes [Cohen’s

D = .71, .72, .80, respectively].

In females’ sniffing behaviours, there was a significant dose by drug interaction [F(5,220) = 2.88, p =.026, Greenhouse-Geisser  = .778] with mice in 92 the cocaine groups remaining relatively consistent while mice in the saline groups increased sniffing behaviours over time and a significant effect of dose

[F(5,220) = 8.57, p <.001] with increased incidences of sniffing as dose increased

(Figure 3.2J). This is consistent when examining saline and cocaine groups separately. In the saline group, there were significant differences between 5 and

25, 15 and 25 mg/kg. In the cocaine group, there was a difference between 15 and 30 mg/kg.

3.3.2 Fast Scan Cyclic Voltammetry

In males, when examining the response over 5 min intervals, there was a significant effect of time [F(8,112) = 49.44, p <.001]. The interaction between time and drug approached significance [F(8,112) = 2.78, p = .075, Greenhouse-

Geisser  = .268] (Figure 3.3A). There were significant differences between early time points (up until 20 min) with the DA response increasing over time, followed by non-significant differences as the response leveled out (from 20-40 min).

Similar differences were seen when examining the drug groups separately.

Maximum DA response was determined for both the saline trials and the cocaine trials. As expected, there was a significant increase in response post- drug compared to pre-drug [F(1,18) = 106.70, p <.001] (Figure 3.3B).

Additionally, there was a marginally significant interaction between time (pre-, post-drug) and drug [F(1,18) = 3.63, p = .073]. Examining the drug groups separately found similar changes (i.e. significant increase in DA response post- drug). Paired sample t-tests for the groups separately found that WT SAL [t(5) = 93

6.21, p = .002], KO SAL [t(5) = 6.72, p = .001], and KO COC [t(6) = 6.01, p =

.001] all had significant increases post-drug; however, the WT cocaine group approached significance [t(2) = 3.71, p = .066]. There were no differences found between the individual groups.

Total DA release was also determined for pre- and post-drug trials by calculating the Area Under the Curve (AUC). There was a significant increase in response post-drug [F(1,18) = 65.02, p <.001] (Figure 3.3C). Similar to the maximum response, this was significant for both saline groups and the KO cocaine group, but not for the WT cocaine group. We also examined the fold increase in both maximum response and AUC; however, there were no significant differences in males.

In females, when examining the response over 5 min intervals, there was a significant effect of time [F(8,104) = 26.49, p <.001] (Figure 3.3D). There were significant differences between early time points (up until 20 min) with the DA response increasing over time, followed by non-significant differences as the response leveled out (from 20-40 min). Similar differences were seen when examining the drug groups separately.

Maximum dopamine response was determined for both the saline trials and the cocaine trials. As expected, there was a significant increase in response post-drug compared to pre-drug [F(1,16) = 72.69, p <.001] (Figure 3.3E).

Examining the drug groups separately found similar changes (i.e. significant increase in DA response post-drug). However, in the cocaine groups, the main effect of genotype approached significance (p = .080). Paired sample t-tests for 94 the groups separately found that KO SAL, WT COC and KO COC had significant increases post-drug [t(5) = 6.39, p = .001; t(5) = 4.99, p = .004.; t(4) = 3.76, p =

.020, respectively]; however, the WT saline group approached significance [t(2) =

3.84, p = .062]. When examining differences between genotypes, pre- and post- drug maximum response approached significance [t(9) = 2.09, p =.067; t(9) =

1.91, p = .088].

Total DA release was also determined for pre- and post-drug trials by calculating the AUC. There was a significant increase in response post-drug

[F(1,16) = 56.59, p <.001] (Figure 3.3F). Similar to the maximum response, this was significant for both cocaine groups and the KO saline group, but not for the

WT saline group. When examining differences between genotypes, there was a significant difference between WT and KO mice in the cocaine group on the AUC prior to drug injection [t(9) = 2.63, p = .027]. We also examined the fold increase in both maximum response and AUC; however, there were no significant differences.

Due to proposed sex differences in basal and drug-induced dopamine levels, we also compared males and females on maximum dopamine response and

AUC before and after cocaine administration. The only significant difference found was between males and females in the WT group that originally received saline (during the dose response test) on AUC after cocaine administration during the FSCV surgery [t(7) = 2.52, p = .040] with males having close to double the amount of DA release.

Figure 3.3 Fast-scan Cyclic Voltammetry Results. Male (Panels A-C) and female

(Panels D-F) ZnT3 WT and KO mice in saline and cocaine groups were injected

(I.P.) first with saline (0.9%) then with 10 mg/kg cocaine. The VTA was stimulated every 5 min and dopamine concentration was determined. Color plots (Panel G) and Concentration versus time plots (Panel H) show dopamine response following saline or cocaine injection.

3.3.3 Morphology

Examining dendritic length of medium spiny neurons in the dorsal striatum

(Figure 3.4A-C) in males using Sholl analysis revealed a trend toward an interaction between genotype and drug, [F(1,34) = 2.99, p = .093] (Figure 3.4D).

There were no differences between genotypes in the cocaine group; in the saline group, genotype difference approached significance with a large effect size [t(16)

= 1.94, p = .070, Cohen’s D = .94]. There were no significant main effects or interactions for spine density in the dorsal striatum (Figure 3.4E).

Examining dendritic length in females using Sholl analysis, there were no differences in length between groups for the dorsal striatum (Figure 3.4F). There were also no significant main effects or interactions for spine density in the dorsal striatum (Figure 3.4G).

3.3.4 Estrus Cycle Effects

The stage of the estrus cycle did not appear to affect locomotor response

(data not shown). However, several mice did not seem to be cycling properly (did not follow the proestrus – estrus – metestrus – diestrus pattern), irrespective of genotype. A possible reason for this is that the mice were not exposed to a male mouse in adulthood, something proposed to be necessary to kick-start the estrus cycle in female mice.

Dendritic length as measured by Sholl analysis is shown for males (D) and females

(F). Spine density (number of spines/length of dendrite) is shown in Panels E and G for males and females respectively. Error bars represent ± standard error of the mean

(SEM). aa: large effect size. 98

3.4 DISCUSSION

The purpose of this study was to examine the effects of cocaine on mice lacking vesicular zinc to determine whether zinc plays a role in cocaine-induced plasticity in the brain. Female KO mice showed reduced locomotor response to higher doses of cocaine (significant at 25 mg/kg) and normal sensitization, while male KO mice were no different than WT mice on locomotor response to most doses but did not sensitize to cocaine.

The reduced locomotion seen in female KO mice at higher doses of cocaine does not appear to be caused by changes in the amount of stereotyped behaviours they exhibit as there were no significant differences between the genotypes. This suggests that vesicular zinc does not play a role in stereotyped behaviours but is reducing locomotor activity through another mechanism.

Locomotor activity correlates with DA release (Pijnenburg et al., 1976).

Higher levels of DA release typically lead to higher amounts of motor activity

(Pijnenburg et al., 1976). Previous studies have found that vesicular zinc is not present in the ventral tegmental area (VTA) or the substantia nigra pars compacta (SNc) where the dopaminergic cell bodies are located (Frederickson et al., 1992; Brown and Dyck, 2004b). Therefore, if zinc is able to affect DA release, and therefore locomotor activity, it must be doing so either by acting on the presynaptic terminals of VTA/SNc neurons in the striatum or by affecting the neurons that project to these nuclei to modulate DA release. 99

One possible mechanism by which zinc may affect striatal function is through its actions on the glutamatergic system. Zinc is co-released with glutamate and inhibits NMDA and AMPA receptors (Paoletti et al., 2009). Since glutamate activates NMDA and AMPA receptors, zinc is effectively the “brake” on glutamate. Without zinc, glutamate should be better able to activate its receptors.

However, if those receptors are found on the inhibitory GABAergic medium spiny neurons (MSNs) – the main projection neuron in the striatum - increased glutamate activation may result in increased inhibition of the cortico-basal ganglia-thalamic circuit. This, in part, depends on which type of MSNs are affected by the lack of zinc. There are two types of MSNs: those forming the direct pathway and those forming the indirect pathway (Smith et al., 1998). If zinc is acting equally on both pathways, a lack of zinc should not affect the overall functioning of the system. However, if zinc acts more strongly on one pathway than the other, an imbalance in the circuit occurs, resulting in either higher activation (if zinc more strongly activates the direct pathway) or higher inhibition

(if zinc more strongly affects the indirect pathway).

Another possible mechanism of action is through zinc binding to the DA transporter (DAT). Zinc augments cocaine’s action on the DAT (Richfield, 1993); without it, cocaine is less effective. However, this begs the question, if this is the case, then why do we not see similar decreases in locomotor activity in male KO mice?

A potential reason for the sex differences in our results is the interactions occurring between zinc and estrogen. Estrogen has been shown to decrease 100 vesicular zinc and ZnT3 protein (Lee et al., 2004b), and zinc-deficient ovariectomized rats had lower estrogen levels than zinc-sufficient and zinc- supplemented ovariectomized rats indicating that zinc and estrogen do interact

(Sunar et al., 2009). Because estrogen decreases ZnT3 and vesicular zinc, it is possible that lower levels of zinc caused by higher levels of estrogen play a part in the increased activity in WT females. Although the sexes were not compared statistically in our study, many previous studies have found that females show enhanced locomotor activity in response to cocaine compared to males (Walker et al., 2001). However, if that were the case, we would expect the male KO mice to be more sensitive to cocaine and indeed they are trending towards this at the lowest level of cocaine. Additionally, the male KO mice have the highest response to the 10 mg/kg cocaine dose on the challenge day. Although it is not significantly higher than the COC groups, it is an unexpected finding given that the COC groups should show higher responses because of behavioural sensitization. It is possible that the sensitivity of the male KO mice to cocaine is being masked by the fact that all mice in the COC groups receive repeated doses of cocaine. Giving naïve mice single doses of 5-30 mg/kg cocaine may be necessary to reveal differences in cocaine sensitivity in males.

Most mice, regardless of genotype or group showed all 3 categories of stereotypic behaviour at the lowest doses of cocaine (Figure 3.2). However, as cocaine dose increased, fewer mice were exhibiting repetitive and oral behaviours. Other stereotyped behaviours typically elicited by high doses of cocaine or amphetamine include head bobbing and weaving (Flagel and 101

Robinson, 2007) and rearing (Tilley and Gu, 2008). These were not examined in this study as the camera angle and quality of the video did not allow for accurate assessment.

The main purpose of this study was to examine locomotion (distance travelled) after cocaine administration. It was not done with the intention of examining stereotyped behaviours induced by cocaine. One reason for this is due to the tracking software and camera available for our use; there was a trade- off between comprehensive tracking capability and overall video quality. Since the purpose was to examine locomotion, priority was given to high quality tracking. As such, the video recordings were not high quality and, in some cases, this made quantifying stereotyped behaviours difficult. In addition, the videos were a top-down view of the mice, meaning we may miss certain stereotyped behaviours, such as head bobbing, simply because of the view used for scoring.

A future study might strictly examine cocaine-induced stereotypy using several camera angles to ensure all stereotyped behaviours are being recorded.

Results from fast-scan cyclic voltammetry (FSCV) indicate that cocaine is increasing DA release in the dorsal striatum as expected. DA levels rise continuously until about 15 min after the cocaine injection before plateauing.

Levels do not drop significantly within the timeframe of our recording (45 min).

There were no significant differences found between genotypes or group (SAL or

COC). However, group sizes were relatively small and there was high variability between mice. In addition, the parameters used may not have been wide enough. It may be possible that there is a genotypic difference in how long it 102 takes DA levels to return to pre-drug levels. However, we did not record long enough to observe DA levels in response to cocaine returning to saline levels. A future study should examine length of time before DA levels return to normal and whether this is affected by genotype. It may also be interesting to examine DA levels in the same mice at different time points by implanting cannulas for the

FSCV electrodes. This would allow us to determine how single versus repeated doses of cocaine affect DA release and whether basal DA levels change over time with repeated administration of cocaine.

Unlike what is typically found in the dorsal striatum in response to cocaine, there were no significant differences in dendritic length or spine density. A potential explanation for the lack of differences may be that the mice were not immediately euthanized following cocaine exposure as this study included a washout period between final exposure and the FSCV surgery. Alternatively, these differences may lie in other areas such as the nucleus accumbens or prefrontal cortex, which may show differences that the dorsal striatum does not.

Finally, this study lacked a true control group for Golgi analysis as all of the mice had at least 2 exposures to cocaine (on day 26 and in the FSCV surgery). There is previous research suggesting that a single dose of cocaine can affect functioning at glutamatergic synapses (Ungless et al., 2001), which may explain the lack of difference between the saline and cocaine groups.

In conclusion, absence of vesicular zinc in ZnT3 KO mice affects response to cocaine in a sex-dependent way. Differences found in locomotor activity do not seem to be caused by changes in stereotyped behaviours. Due to current group 103 sizes, it is difficult to determine conclusively whether changes in DA release or in morphology contribute to the changes seen in activity.

104

3.5 ACKNOWLEDGEMENTS

I would like to thank Sarah Bryden for her enormous help in planning and preparing for the experiment as well as in data collection for this experiment. In addition, I’d like to thank Nicoline Bihelek and Selena Fu for their help in running the behavioural component of the experiment. I would also like to thank Andrea

Herzog for helping to score the stereotypic behaviours. Vedran Lovic, a former co-supervisor, helped with the planning of the experiment and in FSCV training.

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4 CHAPTER 4:

CONDITIONED PLACE PREFERENCE TO COCAINE IN MICE LACKING

VESICULAR ZINC

4.1 INTRODUCTION

Elemental zinc is a metal found in all cells of the body where it has essential structural and functional roles (Chasapis et al., 2012). In the brain, a pool of zinc, termed vesicular zinc, acts as a neurotransmitter – it is loaded into synaptic vesicles by the zinc transporter 3 (ZnT3) protein, is released into the synapse in a calcium- and activity-dependent manner, and can bind to and modulate receptors on the postsynaptic membrane or enter postsynaptic cells to affect downstream signaling (McAllister and Dyck, 2017).

Localizing vesicular zinc in the brain using Timm’s silver-sulfide method reveals high concentrations of vesicular zinc within most structures of the forebrain (Frederickson et al., 1992). Most research on vesicular zinc has focused on its role in the hippocampus and somatosensory (barrel) cortex in mice, where it has been shown to be critical for synaptic plasticity (Nakashima and Dyck, 2009b). However, vesicular zinc is also found in high concentrations in regions of the so-called reward pathway in the brain including the dorsal striatum, ventral striatum including nucleus accumbens, and prefrontal cortex (Shu et al.,

1990; Frederickson et al., 1992; Mengual et al., 1995) where its role is less well understood. 106

Two decades ago, knockout (KO) mice that lack the gene slc30A3 that codes for the ZnT3 transporter and thus show an absence of vesicular zinc were created (Cole et al., 1999). Original behavioural studies of these mice found few deficits (Cole et al., 2000; Cole et al., 2001). However, further studies using ZnT3

KO mice and manipulations of zinc levels provide accumulating evidence that vesicular zinc is involved in experience-dependent synaptic plasticity (Brown and

Dyck, 2002, 2003b, 2005; Nakashima and Dyck, 2008, 2010; Nakashima et al.,

2011). It can be hypothesized that zinc plays similar roles in plasticity in response to rewarding stimuli as that found in the hippocampus and somatosensory cortex.

Conditioned place preference (CPP) paradigm is one of the simplest ways to examine reward-related behaviours in rodents (Cunningham et al., 2006). This paradigm involves pairing one environment with a drug of abuse (such as cocaine) and a different environment with drug absence (a vehicle). The animal is given a choice between the two environments and if the animal finds the drug rewarding, it will spend more time in the drug-paired environment than in a vehicle-paired environment (Cunningham et al., 2006).

Cocaine acts as a dopaminergic agonist by blocking dopamine transporters and prolonging dopaminergic action at the synapse. Cocaine has well- characterized effects in rodents, including the ability to produce a CPP in mice in response to repeated pairings. Zinc and cocaine have been shown to interact at the dopamine reuptake transporter (Norregaard et al., 1998; Meinild et al., 2004;

Bjorklund et al., 2007; Pifl et al., 2009), with zinc augmenting cocaine’s action on 107 this transporter (Richfield, 1993). Lack of vesicular zinc, as found in ZnT3 KO mice, may affect the potency of cocaine and disrupt its ability to produce a CPP.

The present study examines the role of vesicular zinc in a CPP response to cocaine using ZnT3 KO mice compared to wildtype (WT) mice with normal zinc levels. We hypothesize that the lack of vesicular zinc will disrupt the ability to associate a specific environment with cocaine.

4.2 METHOD

4.2.1 Animals

Mice on a mixed C57BL/6x129/Sv genetic background were generated from breeding pairs heterozygous for the slc30A3 allele. Male and female wildtype

(WT) and ZnT3 KO mice 2-3 months old were used. Mice were housed in standard cages in a temperature- and humidity- controlled room on a 12:12 light/dark cycle in same-sex sibling groups from weaning (postnatal day 21) until testing and provided ad libitum access to food and water. All procedures were approved by the Life and Environmental Sciences Animal Care Committee at the

University of Calgary and followed the guidelines for the ethical use of animals provided by the Canadian Council on Animal Care.

4.2.2 Drugs

108

Cocaine hydrochloride (Medisca Pharmaceutique Inc., St-Laurent, Quebec,

Canada) was dissolved in 0.9% saline and administered intraperitoneally (I.P.) at a dose of 20 mg/kg. Control animals were given an equivalent amount of 0.9% saline.

4.2.3 Conditioned Place Preference

The place preference boxes were rectangular (42x15x15 cm) with a wall placed in the middle to create 2 chambers (21x15x15 cm); one chamber had diagonal black stripes on 3 walls while the other had different coloured 1inch dots in a 3x4 grid pattern on 3 walls (Figure 4.1A). The central divider had a small opening on the pretest and testing days to allow mice to move between the chambers. During the pairing sessions, the wall was solid to contain the mice to one chamber. Mice were tracked using an overhead camera (Basler acA1300-

60gm GigE, Basler AG, Germany) and Ethovision XT video tracking system

(Noldus Information Technology Inc., Leesburg, VA, USA). Five to six mice were tested in separate arenas concurrently.

A pretest was used to determine whether mice had a pre-existing preference for one chamber. If a preference was found (i.e. mice spent more than 800 s of the total 1200 s in one chamber), that chamber was used for saline pairings, while the non-preferred chamber was used for cocaine pairing. Because the

ZnT3 KO mice have been shown to have deficits in other tests with minimal training but perform normally with more experience (i.e. fear conditioning, Morris water task) (Martel et al., 2010; Martel et al., 2011; Sindreu et al., 2011), tests of 109 place preference were performed after 2 drug-chamber pairings and 4 drug- chamber pairings. Mice received saline in one chamber and cocaine in the other chamber in an alternating fashion (i.e. day 1 was saline, day 2 was cocaine, etc.) for a total of 4 days (i.e. 2 days of saline pairings and 2 days of cocaine pairings) for 30 min each day (Figure 4.1B). With the exception of mice showing a preference for one chamber during the pretest, mice were randomly assigned to receive cocaine in either the dots or the stripes chamber. First pairing (saline vs cocaine) was also randomized such that half of the mice had the saline pairing on the first day while the other half had the cocaine pairing the first day.

On day 5, the wall dividing the chambers had an opening allowing the mice to move between the compartments. Mice were placed near the center of the box and allowed to explore for 20 min; time spent in each chamber was determined.

On days 6-9, the mice received 2 more pairings each of saline and cocaine continuing the alternating pattern. This was followed by another test on day 10.

Mice were then left undisturbed in their home cages for 1 week. They were placed back in the box for a final test session to determine whether they retained the memory of the drug-chamber pairings. Figure 4.1B shows the timeline of the experiment.

4.2.4 Statistical Analyses

Repeated measures ANOVAs were used to compare between tests for all groups and the sexes individually. Paired-samples t-tests were used to follow-up significant differences between days. One-way ANOVAs were used to compare 110 individual tests. Effects sizes are presented as Cohen’s D values (small effect =

.2, medium effect = .5, large effect = .8). Statistics were computed using IBM

SPSS v.24 (Armonk, NY: IBM Corp.). Figures were created using GraphPad

Prism version 8.4.2 for Windows (GraphPad Software, San Diego, California

USA, www.graphpad.com).

111

Figure 4.1 . Experimental Setup. Panel A shows a mock-up of the conditioned place preference boxes used; the door connecting the two chambers was absent during conditioning days and present during test days. Panel B shows the timeline for the experiment. Mice were counterbalanced in terms of whether they received the saline or cocaine pairing first and which chamber was paired with cocaine. 112

4.3 RESULTS

4.3.1 Distance Travelled under Influence of Cocaine

Days where the tracking program lost the mouse for more than 10% of samples taken were removed from analysis.

A 3-way repeated measure ANOVA examining the 8 days of drug pairing found a significant effect of day [F(7,182) = 68.57, p < .001, Greenhouse-Geiser

 = .349]. There was a significant difference between average saline response and average cocaine response [t(35) = 13.45, p < .001] (Figure 4.2). Because cocaine is known to increase locomotion, saline pairings and cocaine pairings were also examined separately. There was a significant effect of day in saline pairings, [F(3,87) = 7.68, p =.001, Greenhouse-Geiser  = .676]. Follow-up tests found a significant difference between days 1 and 3 [t(32) = 3.60, p = .001] and 2 and 4 [t(35) = 3.10, p = .004]. For the cocaine pairings, there was also a significant effect of day [F(3,87) = 3.69, p =.039, Greenhouse-Geiser  = .565].

Follow-up tests were not significant.

As sex differences have been suggested to occur in the ZnT3 KO mice previously (Thackray et al., 2017), we also examined the sexes separately. Male and female mice had significant effects of day [F(7,91) = 27.63, p < .001,

Greenhouse-Geiser  = .305; F(7,91) = 48.00, p < .001, Greenhouse-Geiser  =

.386, respectively]. For the saline pairings, male, but not female, mice had a significant effect of day, although females did approach significance [F(3,45) =

5.50, p = .009, Greenhouse-Geiser  = .659; F(3,45) = 2.62, p = .064, 113 respectively] (Figure 4.2A). For the cocaine pairings, female, but not male, mice had a significant effect of day [F(3,45) = 2.91, p = .044; F(3,42) = 2.02, p = .170,

Greenhouse-Geiser  = .450, respectively] (Figure 4.2B). Follow-up tests were not significant.

114

Figure 4.2 Distance Travelled During Conditioning Sessions. Panel A (male) and

Panel B (female) show distance traveled over the 30 min pairing sessions under the influence of saline or cocaine. Shaded areas represent cocaine pairings while unshaded areas represent saline pairings. Mice travelled significantly farther following the cocaine injection compared to the saline injection. Error bars represent ± SEM. * represents p < .05; *** represents p < .001. 115

4.3.2 Conditioned Place Preference

For all test days, only the first 10 minutes were used for analysis as mice tended to habituate to the chambers over the duration of the test. A 3-way repeated measures ANOVA comparing all mice on all testing days (Pretest, Test

1/Day 5, Test 2/Day 10, Test 3/Day 17) found a significant effect of test [F(3,96)

= 29.05, p <.001, Mauchly’s W (5)= .79, p = .206] (Figure 4.3). Follow-up tests showed significant differences between the Pretest and Test 1/Day 5 [F(1,32) =

55.40, p < . 001], Test 2/Day 10 [F(1,32) = 45.84, p < . 001], and Test 3/Day 17

[F(1,32) = 25.98, p < . 001]. There was also a significant difference between Test

1/Day 5 and Test 3/Day 17 [F(1,32) = 10.08, p = .003] and Test 2/Day 10 and

Test 3/Day 17 [F(1,32) = 11.02, p = .002]. There was no significant difference between Tests 1/Day 5 and 2/Day 10.

As mentioned above, sex differences have been found in ZnT3 KO mice in other tests, therefore an a priori hypothesis was that male and female mice would show differences in CPP. Males and females had a significant effect of test,

F(3,48) = 14.63, p<.001, and F(3,48) = 16.34, p<.001, respectively. There was a significant difference between the Pretest and Tests 1/Day 5, 2/Day 10, and

3/Day 17 for both males [F(1,16) = 28.14, p <.001; F(1,16) = 23.28, p < .001,

F(1,16) = 11.01, p =.004, respectively] (Figure 4.3A) and females [F(1,16) =

30.97, p < .001; F(1,16) = 25.08, p < .001, F(1,16) = 20.57, p <.001, respectively]

(Figure 4.3B), but no interaction or difference between genotypes. When comparing Tests 1/Day 5 & 2/Day 10 and 2/Day 10 & 3/Day 17 and 1/Day 5 &

3/Day 17, there were no significant differences between tests. 116

Additionally, each test has the potential to have differing effects based on genotype. There were no differences between genotypes during the Pretest or

Test 1/Day 5 in either sex. In Test 2/Day 10, male KO mice spent significantly less time in the cocaine-paired chamber than male WT mice [F(1,16) = 6.40, p =

.022, Cohen’s D = 1.20], while in Test 3/Day 17 male KO showed a similar trend

[F(1,16) = 2.94, p = .105, Cohen’s D = .81].

In order to show a place preference, the mice must spend more time in the cocaine-paired chamber. Paired sample t-tests were used to compare time spent in the cocaine-paired chamber to time spent in the saline-paired chamber (Table

4.1 and Figure 4.3C-J). Notably, male mice spent significantly more time in the cocaine-paired chamber during Test 1/Day 5 regardless of genotype. Female WT mice did not spend significantly more time (medium effect size) in the cocaine- paired chamber during Test 1/Day 5; however, female KO mice did (p = .045). In

Test 2/Day 10, male WT and KO (large effect sizes) and female WT (large effect size) mice spent significantly more time in the cocaine -paired chamber; female

KO mice did not (medium effect size). In Test 3/Day 17, none of the groups spent significantly more time in the cocaine-paired chamber; however, the WT mice

(male and female) had medium effect sizes (Cohen’s D = .79 and .61, respectively). Both male and female KO mice had a small effect size (Cohen’s D

= .27 and .37, respectively) on Test 3/Day 17.

117

F) and females (Panels G-J). The solid (Panels A & B) and dotted (Panels C-J) lines represent equal time spent in the two chambers. Male mice of both genotypes spent significantly more time in the cocaine-paired chamber during tests 1 (Panel D) and 2 (Panel E). Female WT mice spent significantly more time in the cocaine-paired chamber on Test 2 (Panel I) while female KO mice spent significantly more time in Test 1 (Panel H). Error bars represent ±SEM. * represents p < .05; ** represents p <.01; *** represents p < .001. 118

Table 4.1 P values and effect sizes for cocaine value compared to the saline value (i.e. do they spend significantly more time in the cocaine compartment than the saline compartment?) for all tests. * represents p < .05, ** represents p

< .01; a represents medium effect size, aa represents large effect size. 119

4.4 DISCUSSION

The goal of this study was to examine conditioned place preference to cocaine in male and female ZnT3 WT and KO mice. Cocaine is known to be a potent psychomotor stimulant (Pijnenburg et al., 1976). As expected, the ZnT3 mice, both WT and KO, respond to it with a significant increase in distance travelled compared to saline (Figure 4.2).

Overall, mice spent significantly more time in the cocaine-paired chamber during Tests 1/Day 5 & 2/Day 10 compared to the pretest, suggesting that a CPP has formed. For Test 3/Day 17, there was a significant difference between the

Test and Pretest in females, but not males, suggesting that females retain the

CPP one week following pairings while males do not.

Paired sample t-tests examining place preference for each group showed that there were sex differences in terms of amount of time spent in the cocaine- paired chamber. Male mice spent significantly more time in the cocaine-paired chamber on Tests 1/Day 5 & 2/Day 10; however, there was a trend suggesting a difference between genotypes in Test 2/Day 10 and 3/Day 17 with KO mice spending less time in the cocaine-paired chamber (effect sizes also support this).

In Test 3/Day 17, neither genotype spent significantly more time in the cocaine- paired chamber, but effect sizes suggest that WT mice spent more time in the cocaine-paired chamber while KO mice spent equal time in both chambers.

Female KO mice spent significantly more time in the cocaine-paired chamber in

Test 1/Day 5, WT mice spent significantly more time in the cocaine-paired chamber in Test 2/Day 10, and while neither genotype spent more time in the 120 cocaine-paired chamber on Test 3/Day 17; effect sizes suggest that both genotypes spent more time in the cocaine-paired chamber during Test 1/Day 5 while only WT mice spent more time in Test 3/Day 17.

As previous research with ZnT3 mice has found subtle differences with weak/minimal training (Martel et al., 2010; Martel et al., 2011; Sindreu et al.,

2011), one of our hypotheses was that ZnT3 KO mice may require more drug- chamber pairings to show a preference for the drug-paired chamber. This was not the case for male mice, as both WT and KO showed a significant place preference after both 2 and 4 drug-chamber pairings (Tests 1/Day 5 & 2/Day 10, respectively), suggesting, at least for this test, that KO mice are able to learn associations between a drug (i.e. cocaine) and a specific environment that it has been paired with. This may have more to do with the dose of the drug, as 20 mg/kg is a moderate to high dose (Brabant et al., 2005). It is possible that using a lower dose may produce different results.

In female mice, 2 drug-chamber pairings (Test 1/Day 5) was not enough to produce significant differences in time spent in the 2 chambers for the WT mice.

After 4 drug-chamber pairings (Test 2/Day 10), only female WT mice spent significantly more time in the cocaine-paired chamber. However, KO mice had high variability for this test and effect sizes suggest that increasing power may change the results of this test.

Test 3/Day 17 was included to determine how long a potential CPP would last. Results show that while none of the groups spent significantly more time in the cocaine-paired chamber on Test 3/Day 17, effect sizes suggest that both 121 male and female WT mice may benefit from increasing power, but KO mice, particularly male KO mice would not. This suggests that the KO mice, especially the males, do not retain the drug-chamber pairing memory.

A possible mechanism underlying the effects of lack of vesicular zinc on cocaine-induced conditioned place preference may involve the ERK/CREB pathway (Thomas et al., 2008). Studies have shown that this pathway underlies response to cocaine and learned associations involving cocaine and other cues, including environmental cues used in CPP paradigms. Disruption of this pathway disrupts formation or expression of the CPP. Several studies have found that zinc is also capable of modulating this pathway; although most of these studies focus on the hippocampus (Jiang et al., 2011; Sindreu et al., 2011; Nuttall and

Oteiza, 2012). Altering levels of zinc, both nutritionally and genetically, to produce deficiency has been associated with deficits in ERK1/2 signaling with concurrent deficits in spatial memory (Sindreu et al., 2011).

An important finding in our results is the differences found between the sexes. Many drugs have differences in terms of how males and females respond

(Becker, 1999; Becker et al., 2001; Becker and Hu, 2008). In addition, there have been sex differences found in how cocaine affects ERK/CREB signalling pathways with females having lower activation of the pathway in response to cocaine (Nygard et al., 2013).

A limitation of this study was that only a moderate dose of cocaine was used; lower (10-15 mg/kg) or higher (25-30 mg/kg) doses of cocaine may produce different effects. The female mice that did not form a CPP after 2 exposures to 122 the drug-chamber pairing may show a CPP if we had used a higher dose. There may also be a threshold difference in the males in terms of the effects of cocaine which may be discovered by using lower doses of cocaine. Another limitation could be the environments used in the study. There were only subtle differences between the environments (i.e. different patterns on the walls) which may have resulted in difficulties distinguishing the 2 environments for the female mice which required more drug-chamber pairings to form the CPP. Using environments with clearer distinctions (i.e. different flooring or scents) may result in different findings for CPP in the female mice.

In conclusion, vesicular zinc appears to play a critical role in forming CPPs to cocaine; however, it does so in a sex-dependent manner. In females, it is critical for the formation of a CPP in general, while in males, it is needed to maintain the

CPP beyond the scope of the drug-chamber pairing sessions.

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4.5 ACKNOWLEDGEMENTS

I would like to thank Ali Abdullah, Kira Palanca, and Katy Celina Sandoval for helping with data collection.

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5 CHAPTER 5:

ACUTE AND CHRONIC BEHAVIOURAL EFFECTS OF COCAINE IN MICE

LACKING VESICULAR ZINC

5.1 INTRODUCTION

Zinc is an important structural and functional component of all cells, including cells in the nervous system (Chasapis et al., 2012). A subset of neurons in the brain contain a transporter (zinc transporter 3, ZnT3) necessary to load zinc into synaptic vesicles (Cole et al., 1999; McAllister and Dyck, 2017). This vesicular zinc can be released into the synapse in a calcium- and activity-dependent manner where it exerts effects on many postsynaptic receptors, but notably glutamatergic NMDA and AMPA receptors (McAllister and Dyck, 2017).

Staining for vesicular zinc reveals high levels in brain regions that make up the reward pathway, including the nucleus accumbens and prefrontal cortex

(Frederickson et al., 1992). While vesicular zinc in other regions of the forebrain has been studied, in particular the hippocampus and somatosensory (barrel) cortex, much less is known about the role of zinc in the so-called reward pathway.

A simple way to test the function of the reward pathway is to administer drugs of abuse that are known to act on this pathway; cocaine is one such drug whose effects are fairly well characterized in rodent models and include strong locomotor activation (Flagel and Robinson, 2007). Cocaine is also of interest as 125 studies have shown that it interacts with zinc at the dopamine reuptake transporter (DAT), with zinc augmenting cocaine’s actions on the DAT (Richfield,

1993).

ZnT3 knockout (KO) mice lack the slc30A3 gene which codes for the ZnT3 transporter and thus have an absence of vesicular zinc in their brains (Cole et al.,

1999). These mice can be compared to wildtype (WT) mice with normal zinc levels to determine the effects of cocaine.

The finding that zinc augments cocaine’s action suggests that mice lacking vesicular zinc may show differing effects of cocaine. Cocaine may be less efficient in the KO mice, producing either a shorter duration of action (i.e. the effects within a session wear off faster than in WT mice) or an overall reduction in locomotion in response to cocaine.

The purpose of this study was to characterize the behavioral effects of cocaine in ZnT3 KO mice; a secondary purpose was to examine molecular changes in response to cocaine, but this has been delayed due to the COVID-19 situation.

5.2 METHOD

5.2.1 Animals

Mice were generated from breeding pairs on a mixed C57BL/6x129/Sv genetic background heterozygous for the slc30A3 allele. They were housed in 126 standard cages with same-sex littermate groups from weaning (postnatal day 21) until testing in a temperature- and humidity- controlled room on a 12:12 light/dark cycle. Mice were provided ad libitum access to food and water. All procedures were approved by the Life and Environmental Sciences Animal Care Committee at the University of Calgary and followed the guidelines for the ethical use of animals provided by the Canadian Council on Animal Care.

Two to three-month old male and female wildtype (WT) and ZnT3 knockout

(KO) mice were used for all tests. In the experiment presented in Chapter 3, we found that most mice had not returned to baseline locomotor response within the

45 min session (see Appendix). Due to possible interactions of zinc and cocaine,

90 min sessions were used to determine whether there was a difference between

WT and KO in duration of acute action of cocaine.

Following testing, brains were removed for zinc staining (Group 1, see below) or for use in qPCR and Western blots (Group 2). Figure 5.1 shows the experimental overview.

5.2.2 Drugs

Cocaine hydrochloride (Medisca Pharmaceutique Inc., St-Laurent, Quebec,

Canada) was dissolved in 0.9% saline and administered intraperitoneally (I.P.) at a dose of 20 mg/kg for all experiments. Control animals were given an equivalent dose of 0.9% saline

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5.2.3 Behaviour

Mice were assigned to either the saline control group or the chronic cocaine group. Open field boxes (40x40x40 cm) with an even layer of bedding (same type as in home cage and only enough to cover the floor of the box) were used for all behavioral tests; bedding was included to reduce anxiety to the test environment.

Four mice were tested in separate arenas concurrently. To reduce olfactory cues between rounds, boxes were sprayed with 70% ethanol and bedding was thoroughly mixed with the ethanol and returned to an even layer; bedding was changed between sexes. An overhead camera (Basler acA1300-60gm GigE,

Basler AG, Germany) and Ethovision XT video tracking software (Noldus

Information Technology Inc., Leesburg, VA, USA) was used to track the movement of the mice during testing. Sessions took place at roughly the same time every day (between 8 am and 4:30 pm); mice were given 30 min to acclimatize to the testing room.

There were a total of 7 sessions, one per day, taking place over 7 consecutive days. All mice were given a 15 min habituation time to the testing arena during which time their movements were tracked; following habituation, they were briefly removed, given an I.P. injection consistent with which group they were assigned to, then placed back into the open field for 90 min and tracked for the duration of this time. At the end of the testing, mice were returned to their home cages. Total distance traveled was determined for each session. 128

To examine duration of cocaine action within each session, data were compiled into 30 sec bins representing distance travelled in that time; average baseline locomotion was determined for each mouse based on the 15 min habituation period at the beginning of each session. To determine the time at which each mouse returned to baseline, 30 sec bins were compared to the baseline average; mice were considered as having returned to baseline when they had been at or below the average baseline for 10 consecutive bins (5 min).

Maximum distance traveled was also determined as the 30 sec bin with the highest cm moved. Within session data are presented in 15 min bins as a ratio of distance travelled in the respective 15 min period relative to distance travelled in the 15 min habituation period prior to drug administration to determine the strength of the locomotor effect.

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(40x40x40 cm) for 15 min habituation, given an I.P. injection then replaced for 90 min. On day 8, brains were removed for further analyses. 130

5.2.4 Brain Processing

On the eighth day, mice in Group 1 were administered 15 mg/kg sodium selenite (Sigma-Aldrich Corp., St. Louis, MO, USA) and left singly in a standard mouse cage for 60-90 min. Following this, they were deeply anaesthetized with

2.5% isofluorane in oxygen until nonresponsive. The head was removed with scissors, the brain dissected out and immediately frozen on dry ice; brains were stored in -20°C freezer.

Mice in Group 2 were deeply anaesthetized with 2.5% isofluorane in oxygen until nonresponsive; the head was removed with scissors and the brain dissected out; left and right nucleus accumbens and dorsal striatum were dissected out, weighed, placed in tubes and stored in -80°C for further analysis.

Due to the pandemic, brains have not yet been processed for zinc staining or molecular analyses (qPCR and Western blots).

5.2.5 Statistical Analyses

Repeated measures analysis of variance (ANOVA)s were used to compare across days. 2x2 factorial and one-way ANOVAs, and independent t-tests were used to follow-up significant effects. Effects sizes are presented as Cohen’s D values (small effect = .2, medium effect = .5, large effect = .8). Statistics were computed using IBM SPSS v.24 (Armonk, NY: IBM Corp.). Figures were created using GraphPad Prism version 8.4.2 for Windows (GraphPad Software, San

Diego, California USA, www.graphpad.com). 131

5.3 RESULTS

The purpose of this study was to characterize the response of male and female ZnT3 KO mice to cocaine. When analyzing all of the data together, there was a significant effect of drug [F(1,72) = 171.92, p <.001], as expected, with mice in the cocaine groups showing more locomotor activity (Figure 5.2). There was also a significant drug by genotype interaction [F(1,72) = 4.96, p = .029].

Follow-up tests found a significant difference between sexes in the saline groups,

[F(1,36) = 6.23, p = .017] with females showing increased locomotor activity compared to males. There was also a significant difference between genotypes in the cocaine groups [F(1,36) = 4.25, p = .046], with KO mice showing reduced locomotor response.

Males and females were also analyzed separately as sex differences have often been found in this strain of mice. When examining the results across the 7 days of drug administration, both sexes had the expected increase in locomotion in response to cocaine [males: F(1,36) = 105.44, p <.001; females: F(1,36) =

68.67, p <.001] (Figure 5.2A & I).

In males (Figure 5.2A), examining the saline and cocaine groups separately, there were significant differences between genotypes in the saline group on days

1 [t(18) = 2.17, p = .043] and 2 [t(18) = 2.47, p = .024] with days 3, 4, 5, and 6 having medium effect sizes [Cohen’s D = .69, .52, .53, .54, respectively], although not being significantly different. For the cocaine groups, there was a significant difference between genotypes on day 2 [t(18) = 2.37, p = .029] with 132 day 1 showing a large effect size (.82) and day 3 showing a medium effect size

(.61) while not being statistically significant.

In females (Figure 5.2I), no differences between genotype or day were found in the saline group. In the cocaine groups, the main effect of day approached significance [F(6,108) = 2.46, p = .095, Greenhouse-Geiser  = .360]. When examining each day individually for the cocaine groups, days 3 [t(18) = 1.90, p =

.073, Cohen’s D = .85] and 4 [t(18) = 2.00, p = .060, Cohen’s D = .90] approached significant differences between genotypes, both having large effect sizes. Days 5, 6, and 7 had medium effect sizes (Cohen’s D = .58, .62, .52, respectively) while not being statistically significant. There is also a trend towards decreasing locomotor activity across the days in both WT and KO mice in the cocaine groups. Although not significant, this may suggest that tolerance to cocaine is developing.

We were also interested in examining how ZnT3 mice respond to cocaine within a session (i.e. how long the effects of the drug last, how long it takes before the mice reach their maximum response to the drug, etc.). For this within trial data, distance travelled was binned in 15 min segments and a ratio for each segment was determined by dividing the distance travelled in that segment by the distance travelled in the 15 min habituation phase prior to receiving the I.P. injection (Figure 5.2B-H & J-P).

As previously stated, there was a day by drug interaction with mice in the cocaine groups showing significantly more locomotor activity than those in the 133 saline groups. This was found on every day of testing as well. Therefore, for the within day analysis, we will examine the saline and cocaine groups separately.

In the male saline groups, there was a significant effect of time on the first 6 days (but not on day 7) with mice showing reductions in locomotor activity as time in the testing box increased (Figure 5.2B-H). This decrease in locomotion over time was also found in the male cocaine groups, suggesting that the effects of the drug are wearing off over the course of the 90 min sessions. On day 1 in the male cocaine groups, the time by genotype interaction approached significance [F(5,90) = 2.79, p = .088, Greenhouse-Geisser  = .319]. Two-tailed t-tests following up each time point found significant differences between WT and

KO at 30 min (p = .020) and 45 min (p = .048) with KO mice showing less distance travelled. On day 2, there was a significant difference between genotypes at 15 min (p = .030) but not any other timepoint. And on day 3, There was a significant difference between the genotypes at 60 min (p = .037) and it approached significance at 75 min (p = .057) but not at any other timepoint.

In the female saline groups, there was a significant effect of time on days 1,

2, 3, 5, and 6 with mice showing reduced locomotor activity over time; this was not significant on days 4 or 7 (Figure 5.2J-P). For the cocaine groups, there was a significant effect of time on all days with mice showing reductions in locomotion across the session.

For time to maximum response and time to return to baseline, only groups receiving cocaine were examined (Table 5.1). Data were binned into 30 sec bins across the 90 min session. The bin with the highest distance travelled was 134 determined as the maximum response time. Return to baseline time was determined as the first bin of 10 consecutive bins that were at or below the average baseline time determined during the daily habituation period (i.e. the mouse showed baseline locomotor activity for a minimum of 5 min).

With respect to the maximum response, when examining all of the groups, there was a significant effect of day [F(6, 210) = 2.19, p = .045] with mice reaching their maximum response to the drug sooner as the days progressed; for females only, this approached significance [F(6,108) = 2.28, p = .074,

Greenhouse-Geisser  = .622]. For males alone, the main effect of genotype approached significance [F(1,17) = 4.10, p = .059] (Table 5.1).

For time to return to baseline, when examining all of the groups on time to return to baseline, the day by sex by genotype interaction approached significance [F(6, 216) = 2.38, p =.050, Greenhouse-Geisser  = .711]. For females alone, the main effect of genotype approached significance [F(1,18) =

4.29, p = .053] and there was a significant difference on day 7 [F(1,18) = 5.59, p

= .030]. In males alone, there was a significant difference between genotypes on day 2 [F(1,18) = 8.69, p = .009] (Table 5.1).

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Figure 5.2 Behavioural Results. Panels A & B show the response across the 7 days of testing in male and female mice, respectively. Panels C-I (male) and J-P (female) show the response over time within each session across the 7 days as a ratio compared to the baseline response prior to injection. The dotted line represents baseline. Error bars represent ± SEM. *: p < .05; a: medium effect size; aa: large effect size. 136

Table 5.1 Times to reach the maximum response to cocaine and time taken to return to baseline in mice in the cocaine groups over the 7 days of testing. Data are presented as M ± SD (Median).

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5.4 DISCUSSION

The purpose of this study was to characterize the acute and chronic response of ZnT3 KO mice to cocaine, using a dose of 20 mg/kg for all sessions.

One prominent finding is that male and female mice respond differently to cocaine, something that has been shown previously (Becker, 1999; Becker et al.,

2001; Becker and Hu, 2008). In particular, the male and female ZnT3 KO mice respond differently relative to the ZnT3 WT mice with male KOs showing reduced responses initially but no differences in later sessions and female KOs mirroring the response of the WT but showing slightly less locomotor activity (Figure 5.2A

& I).

There are two features often found to occur with repeated exposure to drugs of abuse: tolerance and sensitization (Robinson and Berridge, 2000). Tolerance typically develops with frequent administration of a drug while sensitization develops to intermittent administration. Both require repeated administration of the drug and can be seen in different components of the drug effects (i.e. physiological effects, locomotor effects, etc.). In rodent models using dopamine agonists, including cocaine, psychomotor sensitization is often used (Kõks,

2015).

In the male mice, tolerance seems to be developing in the WT mice as a decrease in locomotor response can be seen across the days. The opposite seems to occur in the male KO mice who seem to show a slight increase in activity (i.e. sensitization). In female mice, both WT and KO seem to show tolerance to the locomotor effects of the drug across days. 138

When examining the within trial data (Figure 5.2B-H & J-P), both male and female mice have a 2.5-3 fold increase in locomotor activity in response to cocaine compared to the pre-drug habituation period. As mentioned above, this is slightly lower in male KO mice which show only a 2-2.5 fold increase.

Although there is much variability between mice even within the groups – consistent with studies in rats (Gulley et al., 2003) and different strains of mice

(Downing et al., 2003) - most mice reached a maximum response to cocaine within the first 20 min on the first day. This maximum was reached much faster on subsequent days, with median values suggesting that the maximum response for most mice in the male and female WT occurred within the first 5 min on days

2-7. Female KO mice also show this from day 3 onwards while male KO mice do not show this rapid onset of activity until day 6. This suggests that either the effects of cocaine become faster with repeated exposure to the drug in the WT mice or that the WT mice have formed associations between cues and the drug effects and are responding to those cues instead of the actual drug. Regardless of which explanation is correct, this process seems to occur slower in the KO mice. Brain levels of cocaine have been shown to peak in mice approximately 5 min after an I.P. injection at various doses and has a half life of 16 min (Benuck et al., 1987). This is consistent with what we found in WT mice on days 2-7.

However, future studies examining brain levels of cocaine in ZnT3 KO mice are necessary to determine whether the slower onset of action is due to a later peak of cocaine levels or to another factor. 139

In terms of the length of drug action, there are differences in male and female mice as well. In general, females seem to return to their baseline activity levels faster than male mice (8-18 min faster) suggesting that the drug has longer- lasting effects in the males (Table 5.1). This is similar to what was found in a study by Arenas et al. (2019). Although this study used a lower dose of cocaine

(10 mg/kg) and only tracked locomotion for 30 min, they found that OF1 strain male mice continued to show high locomotor responses at the end of the session, while female mice had almost returned to baseline responses (Arenas et al., 2019).

There were also some interesting genotype differences in each of the sexes.

In males, the WT mice seem to return to baseline quicker as the number of sessions increased suggesting that the drug effects were not lasting as long with repeated exposure. The opposite was seen in KO mice with the drug seemingly having longer lasting effects with repeated exposure; this may explain the sensitization-like effect seen across days. The female WT mice had relatively consistent times to return to baseline across the first 4 days with shorter durations on days 5 & 6 and a longer duration on day 7. Female KO mice have the shortest effects overall and return to baseline an average of 10 min faster than WT.

A potential mechanism to explain why male and female ZnT3 KO mice respond differently has to do with the interactions of ZnT3 and zinc in general with estrogen receptors (Lee et al., 2004a). Little is known about this interaction; 140 however, it has been shown that altering estrogen levels can alter ZnT3 and zinc levels (Lee et al., 2004a).

One limitation of this study is that we only used one relatively high dose of cocaine – results may be different with lower or higher doses. Also, this study used non-contingent administration of cocaine (i.e. the mice were not in control of their drug intake). Use of contingent drug administration, such as self- administration of cocaine, may provide more information about how the ZnT3 KO mice respond to cocaine. In particular, because the KO mice seem to have reduced locomotor responses to cocaine in general, that may affect how much drug they take in a self-administration task or their potential for relapse.

In conclusion, lack of vesicular zinc does seem to impact the effects of cocaine on locomotor behaviour, specifically by decreasing the effectiveness of cocaine in producing locomotor behaviour. The specific effect is sex dependent with male and female ZnT3 KO showing slightly different changes relative to WT mice.

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5.5 ACKNOWLEDGEMENTS

I would like to thank Selena Fu for her help with planning the experiment and data collection.

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6 CHAPTER 6:

GENERAL DISCUSSION

The original purpose of this thesis was to determine a role for vesicular zinc in the striatum. Specifically, as zinc is thought to be involved in experience- dependent synaptic plasticity in other brain regions (i.e. hippocampus and somatosensory cortex), the goal was to examine the role of zinc in striatal plasticity. As such, the experiments included involved learning (i.e. Chapters 2 and 4) and administration of a drug (i.e. Chapters 3, 4, 5); both involve synaptic plasticity and are thought to involve the striatum. However, prior to discussing the findings of this thesis, there are a couple of caveats that need to be discussed.

While the original goal of this thesis was to examine the role of zinc in the striatum, many of the experiments included in this thesis involve more brain regions than just the striatum. Instrumental learning, while typically thought to rely on the striatum, particularly the dorsomedial striatum, also involves the hippocampus and prefrontal cortex (Balleine et al., 2009). As these areas also typically have high concentrations of vesicular zinc, the differences in meeting inclusion criteria may have more to do with loss of zinc in regions other than the striatum.

Also, since cocaine was administered intraperitoneally, it will be affecting all brain regions that have DAT present. Therefore, other brain regions involved in the so-called reward pathway need to be considered as well. While not all of these contain vesicular zinc, several have relatively high concentrations, including the prefrontal cortex and bed nucleus of the stria terminalis, and it may 143 be that the lack of vesicular zinc in these areas is having a greater impact on behaviour than loss of zinc in the striatum.

In addition to the many brain regions that may be involved in these tasks, zinc has been shown to modulate many different neurotransmitter systems, many of which interact within the striatum and likely in other brain regions as well. An overview of these systems within the striatum and whether zinc has been shown to potentiate or inhibit each receptor/pathway is provided in Figure 6.1.

Another caveat involves the use of germline knockout mice. Since there are high concentrations of vesicular zinc in many regions of the forebrain and ZnT3

KO mice are missing all of this pool of zinc, we cannot confirm that the changes in behaviour compared to WT are caused by the loss of zinc in the striatum – it may be caused by loss of zinc in other areas. There also may be compensatory mechanisms taking place in the KO mice as they have not had vesicular zinc since conception.

These caveats need to be kept in mind while discussing the findings of this thesis. As the findings of the instrumental conditioning tasks were somewhat inconclusive, they will only be discussed briefly. The rest of the discussion will focus on the results of the drug response studies. There are 3 main groups of findings that I will discuss in detail below. There were differences found between

WT and KO mice in the acute response to cocaine (i.e. locomotor response and duration of drug action), in the long-term effects of cocaine (i.e. sensitization and

CPP), as well as differences between sexes. 144

145

6.1 INSTRUMENTAL CONDITIONING

Based on the high concentrations of vesicular zinc in the striatum and the fact that the striatum is thought to be critical for instrumental conditioning, it seemed likely that lack of vesicular zinc in the ZnT3 KO mice would affect their abilities on these tasks. While fewer KO mice met the inclusion criteria, the fact that many were able to meet the criteria suggests that vesicular zinc is not critical for the learning of the action-outcome association. There were, however, many limitations of this experiment (discussed in Chapter 2) that make it difficult to conclude with absolute certainty that the ZnT3 KO mice are normal in terms of motivation and impulsivity. In the future, instrumental conditioning studies should be conducted in smaller, mouse-sized conditioning boxes and mice should be maintained on a food restricted diet for the duration of the testing in an attempt to maintain sufficient motivation.

6.2 DIFFERENCES IN SHORT-TERM RESPONSE

The short-term responses to cocaine included the locomotor activity following cocaine administration within a trial (i.e. duration of action and maximum response) and how that did or did not change over trials on consecutive days. I will also discuss the CPP tests that occurred within 24 hours of drug-chamber pairings in this section. Longer-term effects, discussed in the next section, include experiments with “extinction” periods of 1 or more weeks like sensitization and Test 3/Day 17 of CPP. 146

In general, there was a reduction in locomotor response to cocaine in the

ZnT3 KO mice. This was found more often in females (in Chapter 3 dose response, Chapter 4 conditioned place preference, and Chapter 5 duration of action) than in males (in Chapter 4 conditioned place preference and Chapter 5 duration of action for the first 3 days) and was not always statistically significant, but present nonetheless. While this thesis did not examine underlying mechanisms, potential mechanisms underlying these differences can be proposed based on findings from previous studies.

To start, based on previous findings on the interactions between zinc and the dopaminergic system, decreasing vesicular zinc should lead to decreased inhibition on DAT and DARs. A loss of inhibition on DAT should mean that DA is cleared from the synapse faster since zinc normally inhibits its function. However, while in the synapse, DA should bind easier to its receptors. Also, given that zinc was found to augment the action of cocaine on the DAT (Richfield, 1993), a lack of zinc may result in cocaine – and possibly other drugs which also act on DAT - having a reduced effect. The overall result should be faster clearance of DA in the synapse. As dopamine levels are correlated with locomotor activity

(Pijnenburg et al., 1976), this should result in reductions in the distance travelled over a certain period of time or a quicker return to baseline activity (i.e. the effects of the cocaine wear off faster). This does seem to be the case as ZnT3

KO mice show reductions in their response to 20 mg/kg cocaine (in Chapter 3 dose response, Chapter 4 conditioned place preference, and Chapter 5 duration of action) as well as 25 and 30 mg/kg cocaine (in Chapter 3 dose response). 147

ZnT3 KO mice also seem to return to baseline activity ~10 min sooner than WT mice on average.

In the studies that did not find a reduction in locomotor response, namely the dose response (females at 5-15 mg/kg and males at all doses) and the later days of Chapter 5 (males only), it is possibly due to metaplasticity. Cocaine is thought to induce metaplasticity (Thomas et al., 2008) and a single dose of cocaine is enough to cause changes in how subsequent administration is received (Ungless et al., 2001). Thus, it is possible, particularly in the dose response study, that the lower, earlier doses are causing changes in how the higher, later doses are affecting behaviour. This may be occurring in all of the studies involving repeated exposure to cocaine and future studies may want to examine different groups of

ZnT3 KO mice on different doses of cocaine to get a more accurate idea of the effects.

In the FSCV experiment in Chapter 3, female KO mice in the cocaine group had lower basal levels of DA compared to female WT mice. As DA levels correlate with activity levels (Pijnenburg et al., 1976), this reduction in DA matches the reduction in locomotor activity seen in the female KO mice at higher doses of cocaine. This also suggests that zinc interacts with DA in acute response to cocaine, in females.

Acute responses to cocaine may also be affected by loss of zinc action on the glutamatergic receptors. Decreasing vesicular zinc should lead to decreased inhibition on NMDA and AMPA receptors. A loss of inhibition of NMDAR and

AMPAR means that glutamate should be more efficient at activating these 148 receptors. Both receptors cause depolarization making the cell more likely to fire.

If glutamate is more efficient at activating AMPAR, it is more likely that the

NMDAR will be activated. Since NMDARs are involved in synaptic plasticity, there should be changes in NMDA-dependent forms of plasticity.

NMDA-dependent plasticity is not the only, or even necessarily the main form of plasticity within the striatum. As mentioned in Chapter 1, a common type of plasticity in the striatum is NMDA-independent and involves G1 mGluRs, amongst others (Kreitzer and Malenka, 2008). In particular, G1 mGluRs are thought to be involved in plastic changes that occur in response to drugs of abuse (Bird and Lawrence, 2009). A specific type of zinc-dependent LTD in the

DCN has recently been shown to be reliant on G1 mGluRs (Vogler et al., 2020).

It is possible that this type of zincergic signaling is also important within the striatum.

Whether the lack of vesicular zinc affected the functioning of NMDAR and

AMPAR was not directly determined in this thesis. However, it would be interesting to conduct electrophysiology studies in the future examining baseline physiological differences in the striatum as none have been conducted to date.

As well as examining whether the lack of zinc in the presence of cocaine affects channel functioning.

A recent study found an association between the NMDA receptor and ZnT1, a plasma membrane transporter involved in transporting zinc from the cytoplasm to the extracellular space (Mellone et al., 2015). Blocking ZnT1 resulted in a lack of zincergic inhibition on the NMDA receptor in the absence of vesicular zinc 149 suggesting that it is zinc being extruded from the postsynaptic cell through ZnT1 that is most critical for NMDA modulation (Krall et al., 2020). Therefore, it is possible that NMDAR signaling is not adversely affected in the ZnT3 KO mice which may explain the lack of deficits on short-term effects including normal CPP

24 hours following drug-chamber pairings.

6.3 DIFFERENCES IN LONG-TERM EFFECTS

While the short-term effects of cocaine on ZnT3 KO mice likely involve the interactions between zinc (or lack thereof), dopamine, and cocaine, the long-term effects, such as sensitization and formation of a CPP, are more likely caused by different mechanisms.

Sensitization involves induction and expression components which are mediated by different brain regions. Because induction of sensitization involves changes in the VTA where there is no vesicular zinc, it is likely that induction of sensitization is not affected in ZnT3 KO mice. However, expression of sensitization may be affected. Female ZnT3 KO mice showed the expected sensitization to cocaine following a 2-week washout period, while male ZnT3 KO mice did not. This indicates that different mechanisms may be involved in male and female ZnT3 KO mice (discussed more in Section 6.4) and that male mice may be more affected by the lack of vesicular zinc.

The results of the CPP experiment suggest that the ZnT3 KO mice can form associations between a drug and environmental cues. However, this association 150 seems to be short-lived. ZnT3 KO mice, particularly the males, spend more time in the cocaine-paired chamber on Tests 1/Day 5 & 2/Day 10 as is expected for a psychomotor stimulant drug, but spend equal time in both chambers after a one- week washout/extinction period. In contrast, the WT spent more time in the cocaine-paired chamber on Test 3/Day 17, although not significantly so.

Together, these results suggest, at least for male mice, that vesicular zinc is required for drug-evoked “memories” to form. This implies that zinc may be more critical in its intracellular signaling roles and in stabilizing the PSD rather than in its modulation of receptors. Cocaine induces changes in BDNF and ERK signaling pathways that correlate with psychomotor sensitization and CPP

(Thomas et al., 2008). If signaling in these pathways is blocked, sensitization and

CPP do not occur. Zinc has also been found to affect these same pathways. My results suggest that vesicular zinc modulates cocaine’s effects on these pathways and that the lack of zinc produces similar results as blocking the pathways altogether, at least in male mice.

6.4 SEX DIFFERENCES

Sex differences have been found frequently in responses to drugs of abuse, including cocaine. It has also been suggested that sex differences exist in the

ZnT3 KO mice in their response on certain tasks (Thackray et al., 2017). This section will discuss possible mechanisms underlying these differences. 151

There are certain factors thought to underlie sex differences in general in response to cocaine, such as differences in DA levels or in ERK/MAPK pathway signaling. Many studies have examined basal and drug-evoked DA levels in specific brain regions, typically the NAc and/or dorsal striatum and found sex- specific differences in both. However, these findings have been refuted by a recent meta-analysis which found no sex differences in basal or drug-evoked dopamine levels in rats (Egenrieder et al., 2020). In my FSCV experiment in

Chapter 3, female KO mice in the cocaine group had lower basal levels of DA compared to female WT mice; this was not found in the males. When comparing male and female DA response, the only significant difference was in the WT saline group after cocaine administration. These findings indicate that basal levels of DA are similar but drug-evoked levels differ in WT mice that have been exposed to a single dose of cocaine. Also, while basal and drug-evoked DA levels may not exhibit sex differences in KO animals, there are sex differences in terms of zinc function, particularly in the interactions between zinc and DA.

In terms of ERK signaling, cocaine has been found to elicit differential effects on male and female rats with different levels of phosphorylated ERK and downstream signaling molecules in different brain regions (Nygard et al., 2013).

Other factors may underlie differences in vesicular zinc function in male vs female mice including estrogen, GPR83, and ALK. Estrogen has been shown to reduce ZnT3, and consequently, vesicular zinc levels (Lee et al., 2004b). It is therefore possible that zinc modulation is less critical in females than in males and that females may utilize other mechanisms of plasticity more than males. It 152 has also been suggested that estrogen may modulate BDNF levels which may contribute to sex differences (Barker et al., 2015); however, there is no direct evidence of this.

GPR83 and the tyrosine kinase receptor ALK are both found in the striatum, can be modulated by zinc, and have been implicated in reward-related behaviours in response to cocaine (ALK) and morphine (GPR83). The Alk gene is responsive to estrogen. Estrogen receptor α knockout mice show increases in

Alk as well as enhanced CPP and sensitization to cocaine (Lasek et al., 2011).

Unfortunately, the study was only done with male mice, therefore, no sex comparison can be made. However, the involvement of estrogen suggests a potential mechanism for sex differences. GPR83 knockdown in the NAc resulted in differences in dopamine response to morphine, as well as differences in CPP to morphine with females requiring higher doses for the same effect (Fakira et al.,

2019). While morphine acts differently than cocaine, it is possible that similar effects may be seen in response to many drugs of abuse.

Overall, there are many potential mechanisms that may underlie sex differences in drug response. However, more research is needed to parse out these mechanisms and determine whether the same mechanisms are involved in response to all drugs of abuse or if different mechanisms are used for different classes of drugs. Also, it remains to be determined what specific role vesicular zinc may be playing in these mechanisms, as it seems clear that sex differences exist in ZnT3 KO mice.

153

6.5 FUTURE DIRECTIONS

As mentioned at the beginning of this chapter, there are several caveats that limit interpretation of findings. One of these involves the use of germline knockout mice which lack vesicular zinc throughout the entire brain and for their entire life.

Therefore, deficits cannot be attributed to the lack of zinc in a specific brain region and lack of deficits may reflect compensatory mechanisms at play rather than truly normal functioning. One way to address these issues is to use a conditional knockout model where ZnT3 can be removed from specific brain regions at specific times. ZnT3-cre mice have recently been generated by

Michael Michaelides at National Drug Institute of America. These mice could be used in future studies to determine which brain region is most critical in terms of vesicular zinc function in response to drugs of abuse. ZnT3 could be knocked out of neurons projecting to either dorsal striatum or NAc to differentiate the role of zinc in each area.

Another future direction would be to examine the ZnT3 KO mice (germline or conditional) on a drug self-administration task. As I mentioned briefly in Chapter

1, self-administration tasks are considered the gold standard for rodent drug addiction studies (Chistyakov and Tsibulsky, 2006) because they provide more insight into motivation to take drugs and can be used to examine relapse potential. While self-administration is doable in mice, it is more complicated than in rats (Chistyakov and Tsibulsky, 2006) and requires specialized equipment that we do not have access to in our lab. However, given some of the findings of this 154 thesis, it would be interesting to see whether the ZnT3 KO mice have similar motivation to self-administer cocaine and whether they would relapse at similar rates as WT mice. Since the KO mice in general show reduced locomotor response to cocaine, would they be less motivated to self-administer it? Also, since the KO mice, particularly the males, do not seem to show long-term

“memories” of the drug (as evidenced by lack of CPP following 1-week of extinction and lack of sensitization), would they relapse or might the lack of vesicular zinc confer some sort of resilience to relapse?

These studies, in addition to the studies proposed earlier in this chapter, will help provide a more accurate idea of the importance of vesicular zinc in acute response to drugs of abuse and its potential importance to addiction.

6.6 CONCLUSIONS

Several research groups have suggested a role for zinc in fine tuning of synapses (Grabrucker, 2014; Anderson et al., 2015; Kumar et al., 2019). The findings presented in this thesis support a role for zinc in the fine tuning of drug- evoked synaptic plasticity.

Overall, short-term differences seen in behavioural responses to cocaine in the ZnT3 KO mice appear to be mediated by the loss of interaction between zinc and cocaine at the dopamine reuptake transporter – without zinc, cocaine becomes less efficient at producing locomotor activation. Long-term changes, including sensitization and delayed CPP, are more likely caused by the lack of 155 zinc in the postsynaptic neuron. As zinc has been found to stabilize the postsynaptic density and affect several signaling cascades involved in drug- evoked plasticity, its loss results in less stable synapses and failure to form a memory of the drug interaction. These findings are consistent with other studies that have found that the ZnT3 KO mice appear relatively normal in the short- term/acute responses but are deficient on long-term measures requiring protein synthesis and/or structural changes in synapses in response to experiences.

156

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185

APPENDIX

In Chapter 3, several aspects of cocaine response were examined in ZnT3

WT and KO mice including dose response, sensitization, dopamine response, and morphological changes. In addition to the outcomes discussed in Chapter 3, we also examined how the mice responded acutely within each session/dose during the dose response component of the experiment. Each session was binned into 15 min segments and total distance travelled during that time was determined. For doses that mice experienced twice (i.e. 5, 10, 20, and 30 mg/kg), an average response was calculated. Based on the within trial data (Figures A.1 and A.2), it is clear that neither male nor female mice have returned to baseline locomotor response at the end of the 45 min testing session, particularly at higher doses of cocaine. Females show reductions in response, but have not completely returned to baseline levels, while males do not show reductions in response and are still having a strong locomotor response during the last 15 min of the session. This information was used in the planning of the experiment presented in Chapter 5.

186

FIGURES

Figure A.1 Within Trial Data for Males. Distance travelled in 15 min intervals for each dose of cocaine and for the sensitization day (day 26). Data are presented as mean ± SEM. 187

Figure A.2 Within Trial Data for Females. Distance travelled in 15 min intervals for each dose of cocaine and for the sensitization day (day 26). Data are presented as mean ± SEM.