Effects of Bisphenol a and Bisphenol S Exposure on Memory and Hippocampal Plasticity in Gonadally Intact Adult Male and Female Mice

Effects of Bisphenol A and Bisphenol S Exposure on Memory and Hippocampal Plasticity in Gonadally Intact Adult Male and Female Mice

Jessica Marie Woodman

A Thesis presented to The University of Guelph

In partial fulfilment of requirements for the degree of Master of Science in Biomedical Sciences (Neuroscience)

Guelph, Ontario, Canada

EFFECTS OF BISPHENOL A AND BISPHENOL S EXPOSURE ON MEMORY AND HIPPOCAMPAL PLASTICITY IN GONADALLY INTACT ADULT MALE AND FEMALE MICE

Jessica Marie Woodman Advisor(s): University of Guelph, 2021 Dr. Neil James MacLusky Dr. Elena Choleris

Bisphenol A (BPA), a well-established endocrine disrupting chemical, has been of particular focus due to its widespread prevalence and adverse effects on human and animal health. In the central nervous system, BPA exposure attenuates learning and memory processes, while also reducing the neuroplastic effects of sex steroids in the brain, especially the hippocampus. Widespread concern led to BPA’s replacement with structurally-related chemicals, such as bisphenol S (BPS). However, whether BPS is actually safer is an open question because its biological effects have yet to be fully explored. Thus, this study sought to determine the impact of BPS on spatial memory in relation to BPA, hippocampal plasticity, and endogenous hormones. We demonstrated that BPS exposure does not impact spatial memory or endogenous hormones.

However, task performance was reduced indicating a behavioural effect independent of memory processes. Thus, BPS may not be a safer alternative, at least in the context of cognitive function.

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ACKNOWLEDGEMENTS

To my advisor, Dr. Neil MacLusky: thank you for your continuous guidance and attentive support. You have provided me with countless opportunities, all of which have allowed me to flourish as a young researcher and develop confidence both in and outside the lab. Your dedication and ability to be an outstanding mentor contributed to me discovering my own passion for teaching. This opportunity has been more rewarding than ever imagined, and I am endlessly grateful for all that you have taught me. Thank you so very much Neil!

To my co-advisor, Dr. Elena Choleris: thank you for your guidance, time, and thoughtful input. You encouraged me to consider aspects of my project that may have been overlooked and for this I am very grateful. Thank you very much for all your help and allowing me to become a member of the Choleris lab.

To my advisory committee member, Dr. Laura Favetta: thank you for your valuable advice and feedback throughout my MSc. The different perspectives and ideas you provided were always helpful.

To my examination committee, Dr.Neil MacLusky, Dr. Elena Choleris, Dr. Giannina Descalzi, and Dr. Roger Moorehead: thank you kindly for taking the time to participate in this process.

To Kate Nicholson, Lauren Isaacs, and Peter Paletta: thank you for your patience, willingness to train me, as well as answering every question I had. Your wisdom, guidance and involvement in this project was paramount for its success and completion, I am extremely grateful to have had your help and mentorship!

To the MacLusky and Choleris lab members: To all the lab members I had the pleasure of spending time with, thank you for making me feel a part of a research community. Thank you for your support and light-hearted fun, you made the challenges easier to overcome.

To faculty, students and staff of the Department of Biomedical Sciences: thank you for providing a friendly and collaborative environment.

To my mentor, Dr. Melissa Perreault: My initial interest in research flourished because of you. Thank you Melissa for your continual guidance and compassion; for this I am extremely thankful.

To my family and friends: There are no words that offer justice to the amount of gratitude I have for you all. Your ceaseless support made all my successes achievable, and all of my failures bearable. This process would have been much more difficult without each and every one of you. Thank you for always cheering me on along the sidelines, moreover, thank you for everything that you have done and continue to do for me.

DECLARATION OF WORK PERFORMED

I declare that I have performed all of the work presented in this thesis except:

Kate Nicholson performed cervical dislocations followed by brain and reproductive organ collection. Kate also performed and analyzed the corticosterone ELISA in chapter 3. Lastly Kate assisted with behavioural testing and lavage cytology in both chapters 2 and 3 as well as treatment administration in chapter 3.

Melanie Williamson collected images used for dendritic spine analysis in chapter 2.

Dr. Neil MacLusky performed analysis on the absorbency data collected from the testosterone ELISA in chapter 3.

TABLE OF CONTENTS

Abstract ...... ii

Acknowledgements ...... iii

Declaration of work performed ...... iv

Table of Contents ...... v

List of Tables ...... ix

List of Figures ...... x

List of Symbols, Abbreviations or Nomenclature ...... xii

Chapter 1: Introduction and Review of the Literature ...... 1

1.1 General Introduction ...... 2

1.2 Hippocampus ...... 3

1.2.1 Introduction ...... 3

1.2.2 Anatomical organization and Circuitry ...... 3

1.2.3 Structural and Synaptic Plasticity ...... 6

1.3 Estrogens ...... 9

1.3.1 Introduction ...... 9

1.3.2 Mechanisms of Estrogen Action ...... 11

1.3.3 Estrogens and Hippocampal Synaptic Plasticity ...... 13

1.3.4 Estrogens and Behaviour ...... 17

1.4 Androgens ...... 19

1.4.1 Introduction ...... 19

1.4.2 Mechanisms of Androgen Action ...... 21

1.4.3 Androgens and Hippocampal Synaptic Plasticity ...... 21

1.4.4 Androgens and Behavior ...... 23

1.5 Bisphenol-A ...... 26

1.5.1 History and Properties ...... 26

1.5.2 Environmental Prevalence and Human Exposure ...... 27

1.5.3 Behavioural Impacts ...... 29

1.5.4 Proposed Mechanisms ...... 34

1.6 Bisphenol-S ...... 37

1.6.1 History and Properties ...... 37

1.6.2 Environmental Prevalence and Human Exposure ...... 39

1.6.3 Behavioural Impacts and Proposed Mechanisms ...... 41

1.7 Rationale ...... 45

1.7.1 Hypotheses...... 46

1.7.2 Objectives ...... 46

2 Chapter 2: A method of discreet Bisphenol A exposure in mice to prevent stress responses ...... 47

2.1 Introduction ...... 48

2.2 Materials and Methods ...... 51

2.2.1 Animals and husbandry ...... 51

2.2.2 Treatment Administration ...... 52

2.2.3 Object Placement Paradigm ...... 53

2.2.4 Behavioural Data Analysis ...... 55

2.2.5 Brain, Tissue, and Blood Collection ...... 56

2.2.6 Golgi Staining and Tissue Processing ...... 57

2.2.7 Dendritic Spines Microscopy ...... 57

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2.2.8 Statistical Analysis ...... 59

2.3 Results ...... 60

2.3.1 Object Placement Paradigm ...... 60

2.3.2 Hippocampal Dendritic Spine Data ...... 68

2.3.3 Vaginal Cytology ...... 71

2.3.4 Body Weight Measurements ...... 71

2.4 Discussion ...... 73

2.4.1 Effects of BPA Exposure on Spatial Memory ...... 73

2.4.2 Effects of BPA Exposure on Dendritic Spines ...... 77

2.4.3 Significance of Work ...... 81

3 Chapter 3: The effects of Bisphenol S exposure on spatial memory and endogenous hormones in gonadally intact male and female mice ...... 82

3.1 Introduction ...... 83

3.2 Materials and Methods ...... 84

3.2.1 Animals and husbandry ...... 84

3.2.2 Treatment Administration ...... 85

3.2.3 Object Placement Paradigm ...... 86

3.2.4 Brain, Tissue, and Blood Collection ...... 86

3.2.5 Quantification of Hormone Concentrations ...... 87

3.2.6 Statistical Analysis ...... 87

3.3 Results ...... 89

3.3.1 Object Placement Paradigm ...... 89

3.3.2 Hormone Concentrations ...... 100

3.3.3 Hippocampal Dendritic Spine Data ...... 100

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3.3.4 Vaginal Cytology ...... 101

3.3.5 Body Weight and Reproductive Organ Weights ...... 102

3.4 Discussion ...... 105

3.4.1 Significance of Work ...... 112

4 Chapter 4: General Discussion ...... 113

4.1 Conclusions and Implications ...... 114

4.2 Limitations and Future Directions ...... 115

References ...... 121

LIST OF TABLES

Table 1.1: Chemical Properties of BPS and BPA...... 38

Table 2.2: Vaginal cytology for experiment 1...... 71

Table 3.1: Vaginal cytology for experiment 2………………………………………...….. 101

LIST OF FIGURES

Figure 1.1: Hippocampal regions and strata...... 5

Figure 1.2: Hippocampal trisynaptic circuitry displaying the three main pathways for information flow...... 6

Figure 1.3: Dendritic spine morphology...... 8

Figure 1.4: Steroid biosynthesis and metabolism...... 10

Figure 1.5: Hormone levels and unstained vaginal smears characterizing the female rodent estrus cycle...... 14

Figure 1.6: Bar graph and photomicrographs depicting the influence of OVX and estradiol administration on dendritic spines...... 15

Figure 1.7: Spine synapse density in the CA1 hippocampus of male rats...... 22

Figure 1.8: Influence of BPA on CA1 dendritic spine synapse density...... 35

Figure 1.9: Concentration ratio depicting the presence of bisphenols in biological and environmental samples...... 39

Figure 1.10: Urinary concentrations of BPA, BPF, and BPS...... 41

Figure 1.11: Bisphenol S significantly influenced novelty preference in adult female zebrafish...... 43

Figure 2.1: Visual representation of experimental design and sequence of events……53

Figure 2.2: Object placement paradigm schematic...... 54

Figure 2.3: Golgi Cox stained dorsal hippocampus section...... 58

Figure 2.4: Effects of BPA on OP performance in gonadally intact male mice ...... 64

Figure 2.5: Other non-directional and directional behaviours observed during the OP paradigm for gonadally intact male mice treated with vehicle or BPA...... 65

Figure 2.6: Effects of BPA on OP performance in gonadally intact female mice...... 66

Figure 2.7: Other non-directional and directional behaviours observed during the OP paradigm for gonadally intact female mice treated with vehicle or BPA...... 67

Figure 2.8: Images of Golgi Cox stained secondary dendrites from hippocampal CA1 neurons ...... 69

Figure 2.9: Hippocampal CA1 dendritic spine density and spine length of gonadally intact male and female mice...... 70

Figure 2.10: Body weight measurements across time for male and female mice in vehicle and BPA groups...... 72

Figure 3.1: Design and sequence of events for experiment 2…………………………….85

Figure 3.2: Effects of BPS and BPA on object placement performance in gonadally intact male mice...... 93

Figure 3 3: Other non-directional and directional behaviours observed during the OP paradigm for gonadally intact male mice...... 94

Figure 3.4: Effects of BPS and BPA on object placement performance in gonadally intact female mice...... 98

Figure 3.5: Other non-directional and directional behaviours observed during the OP paradigm for gonadally intact female mice...... 99

Figure 3.6: Serum hormone concentrations at time of tissue collection...... 100

Figure 3.7: Body weight measurements across time for male and female mice in vehicle, BPA or BPS group...... 103

Figure 3.8: Weight measurements for reproductive tissue between treatment groups 104

LIST OF SYMBOLS, ABBREVIATIONS OR NOMENCLATURE

ACTH - Adrenocorticotropin hormone AKT - Protein kinase B AMPA - α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate AR - Androgen receptor ARKO - Aromatase knockout BDNF - Brain-derived neurotrophic factor BPA - Bisphenol A BPF - Bisphenol F BPS - Bisphenol S BSID - Bayley Scales of Infant Development BW - Body weight CA - Cornu ammonis CaMKII - alpha-Ca+2/calmodulin-dependent protein kinase II CNS - Central nervous system CREB - cAMP response element-binding protein DA - Dopamine, 4-(2-aminoethyl)benzene-1,2-diol DG - Dentate gyrus DHEA - Dehydroepiandrosterone DHT - Dihydrotestosterone DMSO - Dimethyl sulfoxide DOPAC - 3,4-dihydroxyphenylacetic acid E1 - Estrone E2 - 17�-estradiol E3 - Estriol EB - Estradiol benzoate EDC - Endocrine disrupting chemical ER - Estrogen receptor FDA - U.S. Food and Drug Administration FSH - Follicle stimulating hormone GABA - γ-amino-butyric acid GPER - G-protein coupled estrogen receptor GR - Glucocorticoid receptor H295R - Human adrenocortical carcinoma cell line HPA - Hypothalamic-pituitary-adrenal axis HT-22 - Mouse neuronal hippocampal cell line HVA - Homovanillic acid LH - Luteinizing hormone LTD - Long term depression LTP - Long term potentiation MAPK - Mitogen-activated protein kinase MSCA - McCarthy Scales of Children’s Abilities NMDA - N-methyl-D-aspartate OP - Object placement OR - Object recognition ORX - Orchidectomy/orchidectomized xiii

OVX - Ovariectomy/ovariectomized P450scc - Cholesterol side-chain cleavage PB - Phosphate buffer PFC - Prefrontal cortex PI - Percent investigation PI3K - Phosphoinositide 3-kinase PKA - Protein kinase A PSD-95 - Postsynaptic density protein-95 PXR - Pregnane X receptor ROS - Reactive oxygen species SGZ - Subgranular zone TDI - Tolerable daily intake TP - Testosterone Propionate UGT - uridine 5’’-diphosphoglucuronyl transferase 17�-HSD - 17�-hydroxysteroid dehydrogenase 5-HT - Serotonin, 5-hydroxytryptamine 5-HIAA - 5-hydroxyindoleacetic acid

Chapter 1: Introduction and Review of the Literature

1.1 General Introduction

Brain structure and function are delicately driven by a dynamic milieu of steroid hormones. The codependence and intricate sensitivities between the central nervous system (CNS) and endocrine system sets the stage for adverse effects of endocrine disrupting chemicals (EDCs). Bisphenol A (BPA) is one of the most widely used EDCs, which has been of particular focus for decades due to its widespread distribution and potential threat to human and animal health (Konieczna et al., 2015; Vaughn, 2010).

With heightened regulations as well as scientific and public concern, BPA is being replaced with an array of structurally similar, but inadequately investigated analogues

(Eladak et al., 2015). Bisphenol S (BPS) is the leading substitute for BPA, and is increasingly being used for industrial and consumer purposes (Chen et al., 2016). While

BPS may have once been considered a safe alternative for BPA, emerging evidence indicates detrimental effects of BPS exposure on the neuroendocrine system and subsequently behaviour (Naderi and Kwong, 2020). Hence, this review will address the current state of knowledge surrounding neurobehavioral effects of BPS and possible modes of action. However, in efforts to understand the risks of BPS exposure, I first summarize the role of the hippocampus and endogenous hormones in the context of regulating brain structure and function. As a basis for comparison, I then consider BPS’s cousin compound, BPA, highlighting its effects and interactions with the hippocampus, endogenous hormones, and ultimately behaviour. Finally, I shed light on possible misconceptions and limitations in the literature, some of which are addressed in this thesis.

1.2 Hippocampus

1.2.1 Introduction

First insights into the function of the hippocampus were made in the 1950s when patient H.M. received bilateral limbic surgery removing the hippocampus, amygdala, collateral sulcus, perirhinal cortex, entorhinal cortex, and medial mammillary nucleus in efforts to treat recurrent seizures (Scoville & Milner, 1957). However, post-surgery H.M experienced severe memory deficits; he could neither form nor retain long-term memory of events (episodic memory) and facts (semantic memory), thus suggesting a possible link between the hippocampus and memory (Scoville & Milner, 1957). Numerous studies have now refined the role of the hippocampus with regards to cognitive functions, including regulation of emotional behaviour (Adolphs et al., 1995), aspects of motor control (Graybiel et al., 1994), modulation of hypothalamic function (Diamond et al., 1996), as well as learning and memory (Squire and Zola-Morgan, 1988; Squire and

Zola-Morgan, 1991). Presently, the most established contribution of the hippocampus concerns learning and memory as it plays a profound role in consolidation of episodic and spatial memory possibly through long-term potentiation (LTP) (Day et al., 2003;

Riedel et al., 1999; Winters et al., 2004; Winters et al., 2008).

1.2.2 Anatomical organization and Circuitry

The hippocampus, located medially in the temporal lobe, is a bilaminar gray matter structure consisting of the dentate gyrus (DG), Ammon’s horn, and subiculum

(Dekeyzer et al., 2017; Duvernoy et al., 2013; Witter 2010). The general anatomy of the hippocampus is highly conserved across many species including humans, non-human primates, and rodents (Witter, 2010). The DG is composed of three visibly different

layers: (1) the stratum moleculare which receives the majority of inputs from the entorhinal cortex; (2) the stratum granulosum which consists of granular cells; and (3) the subgranular zone (SGZ), described as a major site of neurogenesis (Duvernoy et al., 2013; Witter 2010). Joining with the DG is Ammon’s horn also known as the cornu ammonis (CA) (Duvernoy et al., 2013; Witter 2010). From the ventricular cavity to the vestigial hippocampal sulcus, the CA has six divisions: alveus, stratum oriens, stratum pyramidale, stratum radiatum, stratum lacunosum, and stratum moleculare (Duvernoy et al., 2013). However, based upon heterogeneity of cellular composition the CA is described as having four regions; CA1, CA2, CA3, and CA4 (figure 1.1; Dekeyzer et al.,

2017). Pyramidal neurons are the primary cell type found in all CA regions which can be identified by prominent apical dendrites projecting towards the DG and shorter basal dendrites (Sweatt, 2004, Witter 2010). While the cell type is consistent, neuron morphology between CA regions can differ. For example, CA1 neurons are less arborized displaying a simpler branching pattern than CA3 neurons which bifurcate closer to the cell body (Witter, 2010). The third structure comprising the hippocampus is the most inferior portion, namely the subiculum. The subiculum is described as the main output region for the hippocampus, and displays complex topography owing to its connections with the perirhinal cortex, entorhinal cortex, prefrontal cortex, amygdala, and nucleus accumbens (Aggleton & Christiansen, 2015).

Figure 1.1: Hippocampal regions and strata. (A) Simplified diagram of the dorsal hippocampus and its CA major divisions. Red box depicts region in B. Abbreviations: Cornu Ammonis (CA); Dentate Gyrus (DG). (B) Image of Golgi-cox stained hippocampal slice from OVX female mouse. Stratum oriens ~ 40-60% the length of the basal dendrite, stratum radiatum ~30-50% the length of the apical dendrite, lacunosum moleculare ~80-100% the length of the apical dendrite. Modified from Sheppard et al., 2019.

The flow of information within the hippocampus is typically unidirectional and

commonly termed the trisynaptic circuit, which consists of the three main pathways: the

perforant pathway, the mossy fibre system, and the Schaffer Collateral pathway (figure

1.2; Patten et al., 2016; Hjorth-Simonsen & Jeun, 1972; O’Mara et al., 2001). The

perforant pathway provides excitatory input from Layer II of the entorhinal cortex to the

granule cells of the lateral and medial layers of the DG (Hjorth-Simonsen & Jeun, 1972;

Naber et al., 2001; Witter, Wouterlood, Naber, & Van Haeffen, 2000). The DG granule

cells process and filter the information before sending it to the apical dendrites of CA3

pyramidal neurons, which form the mossy fibre pathway (Gaarskjaer, 1986; Witter

2010). From here, the CA3 pyramidal neurons form the Schaffer Collateral pathway by

connecting with the CA1 pyramidal neurons which project to the subiculum and

entorhinal cortex (Collingridge, Kehl, & Mclennan, 1983) as well as other regions such

as the prefrontal cortex (PFC) (Witter, 2010).

Figure 1.2: Hippocampal trisynaptic circuitry displaying the three main pathways of information flow. Simplified schematic illustrating the trisynaptic circuit, which begins with layer II of the entorhinal cortex (EC) projecting to the dentate gyrus (DG) via the lateral (light blue) and medial (purple) perforant pathway, LPP and MPP respectively. Notably, the LPP and MPP can synapse directly on the CA3 pyramidal neurons, bypassing the DG. The DG granule cells synapse on the CA3 pyramidal cells in the stratum pyramidale and lucidum layers via the mossy fibre (MF) pathway (green). Axons from the CA3 project to the CA1 stratum radiatum and stratum lacunosum-moleculare layers via the Schaffer Collateral (SC) pathway (pink). CA1 pyramidal neurons project to the subiculum (S) and layers V and VI of the EC (dark blue). The subiculum can also project to the EC, layer V (yellow). Diagram from Patten et al., 2016.

1.2.3 Structural and Synaptic Plasticity

The hippocampus is a dynamic structure exhibiting both structural and functional

plasticity. Structural plasticity is commonly defined as neurogenesis, which is the

production of new neurons and their associated structures (i.e. dendrites, spines) that

incorporate into CNS circuitry (Altman & Das, 1967; Ehninger et al., 2008); whereas,

functional (aka. synaptic) plasticity is the process by which existing neurons modify their

communicative abilities (Bliss & Collingridge, 1993). Synaptic plasticity often leads to

enhancement of synaptic efficacy, coined as long-term potentiation (Bliss &

Collingridge, 1993; Bliss & Lomo, 1973). LTP occurs when the post-synaptic neuron

receives strong excitatory glutamatergic stimulation resulting in depolarization and

subsequent binding of glutamate, allowing the activation of α-amino-3-hydroxy-5-

methyl-4-isoxazolepropionate (AMPA) receptors which produces a calcium influx (Bliss

& Collingridge, 1993; Malinow, 2003). The calcium influx depolarizes the cell and consequently removes the voltage-dependent magnesium bock from N-methyl-D- aspartate (NMDA) receptors (Bliss & Collingridge, 1993). With persistent response to a stimulus, LTP can alter dendritic structure and density through mechanisms involving: alpha-Ca+2/calmodulin-dependent protein kinase II (αCaMKII), mitogen-activated protein kinases (MAPK), protein kinase A (PKA), cAMP response element-binding protein

(CREB), and brain-derived neurotrophic factor (BDNF) (Frey et al., 1993; Bradshaw &

Emptage, 2003; Pang & Lu, 2004; Yang, Wang, Frerking & Zhou, 2008). In the hippocampus, the CA1 and CA3 are primarily investigated for their roles in synaptic plasticity as their basal and apical dendrites both present transient and stable spines

(Sweatt, 2004). Dendritic spines are small, actin-rich protrusions of the post-synaptic neuron which receive input from presynaptic neurons and are the principal area of post- synaptic excitatory synapses (Penzes et al., 2011; Risherr et al., 2014). The relative abundance of dendritic spines as well as their morphology can act as an indicator of synaptic plasticity (Bourne & Harris, 2008; Risherr et al., 2014). Regarding morphology

(figure 1.3), mushroom spines are mature and stable spines with terminal enlargements, thought to represent a less plastic synapse. In contrast, filopodial, stubby, and thin spines are characterized as immature and transient, capable of rapid turnover (Sorra &

Harris, 2000). Although perhaps an oversimplified model, it has been suggested that mushroom spines are “memory” spines and filipodia spines are “learning” spines. While both spine structures form synaptic contacts, filipodia spines are capable of experience- induced alterations contributing to immature and transient synaptic contacts which drive

memory formation and ultimately produce mature mushroom spines that form more

permanent functional synapses (Bailey et al., 2015; Bourne & Harris, 2007).

Figure 1.3: Dendritic spine morphology. A simplified graphic of the 4 main morphological dendritic spine subtypes. The morphology of spines can reflect their level of synaptic plasticity. Modified from von Bohlen and Halbach, 2009.

Importantly, dendritic spines and consequently, the hippocampus, are sensitive to a

variety of physiological and environmental factors. Physiological factors such as

circulating and locally synthesized steroids – including estrogens, androgens, and

glucocorticoids – can modulate dendritic spine density (Galea et al., 1997; Leranth,

Petnehazy, MacLusky, 2003; McEwen & Milner, 2007; Phan et al., 2012). As for

environmental factors, various stimuli, such as EDCs, can impact plasticity. Altogether,

learning, memory, and subsequent behavior are functions of the hippocampus that are

influenced, albeit partially, by its plasticity and steroid-sensitivity.

1.3 Estrogens

1.3.1 Introduction

Although estrogens are commonly known for their role in reproduction as the primary female sex hormones, they have remarkably diverse and widespread action in the body within males and females; including but limited to, the metabolic system, skeletal system, immune system, cardiovascular system, and nervous system (Cui et al., 2013; Knowlton & Lee, 2012; Nilsson et al., 2001; Turgeon et al., 2006; Wilson et al., 2006). Estrogens encompass a group of lipid soluble, steroid hormones including estrone (E1), estradiol (E2 or 17β-estradiol), and estriol (E3); all derived from a 27- carbon cholesterol precursor (Compagnone & Mellon, 2000; Hanukoglu, 1992;

MacLusky et al., 1986). Via the steroidogenic pathway (figure 1.4), aromatization of androstenedione and testosterone will produce E1 and E2, respectively. Moreover, 17β- hydroxysteroid dehydrogenase (17β-HSD) will enzymatically convert E1 to E2

(MacLusky et al., 1986; Steimer, 1993). Typically, E2 is the major product of the steroidogenic pathway in non-pregnant mammals and is the most potent estrogen; with potency defined as the ability to induce an uterotrophic effect (Cui et al., 2013; Folmar et al., 2002; MacLusky et al., 1997). E3 is the predominant circulating estrogen in human pregnancy and is enzymatically synthesized in vivo by aromatization of

16� hydroxy dehydroepiandrosterone (DHEA) (Cui et al., 2013). In addition to estrogens, the steroidogenic pathway can also produce progestogens and androgens.

Using the contemporary definition for steroid classification (Miller and Auchus, 2011), figure 1.4 classifies the type of steroid based upon the receptor(s) to which it binds rather than chemical structure.

Figure 1.4: Steroid biosynthesis and metabolism. All estrogens, androgens and progestogens are synthesized from cholesterol which is first metabolized to pregnenolone by P450scc. Pregnenolone can then be enzymatically converted to progesterone by 3�-HSD or 17�-hydroxypregnenalone by 17�-OHase. Progesterone and 17�-hydroxypregnenalone may undergo a series of reactions to produce a principal androgen namely testosterone. Testosterone can then be metabolized to DHT by 5�-reductase, or undergo an aromatization reaction and be converted to E2, which may further metabolize to form E1 or E3 via 17�- HSD. A key for enzymatic and hormone classification is included. Hormone classification is determined by which receptor(s) the steroid can bind rather than its chemical structure. OHase = hydroxylase, HSD = hydroxysteroid dehydrogenase.

Estrogens are predominantly formed in the gonads; however, they are also

synthesized de novo by adipose tissue, adrenal glands, cardiac tissue, and brain tissue

(Cui et al., 2013; MacLusky et al., 1986; MacLusky et al., 1994; Roselli et al., 1985).

Unlike gonadally-synthesized estrogens which enter circulation, extragonadally

synthesized estrogens mainly act at the site of synthesis and contribute to tissue

specific activity. This allows for varying estrogen concentrations throughout the body,

and such is the case with brain tissue (Hojo et al., 2009). The brain, like the gonads, is a

steroidogenic organ that is equipped with all of the enzymes necessary to synthesize estrogens de novo from the cholesterol precursor, including P450side-chain cleavage

(scc), 17α-hydroxylase, 17β-HSD, 3β-HSD, and P450aromatase (Compagnone &

Mellon, 2000, Hanukoglu 1992; Hojo et al., 2009 Lambeth & Stevens, 1984; Melcangi et al., 1996). Importantly, the quantity of enzymatic expression is non-uniform in the brain.

For example, the hippocampus demonstrates varying quantities of E2 between regions because of differences in aromatase expression; as a result E2 concentrations may be highest in the CA3 (Prange-Keil & Rune, 2003). Collectively, while steroidogenic enzymes are expressed in the brain, their expression may be region-specific which subsequently influences steroid-sensitive behaviours.

1.3.2 Mechanisms of Estrogen Action

Estrogen-induced-effects require the binding and subsequent activation of functional ERs such as ERα, ERβ or G-protein coupled estrogen receptor (GPER), which can act via classical or non-classic pathways through genomic and non-genomic mechanisms respectively (Cui et al., 2013; Prange-Keil & Rune, 2003). In estrogen sensitive cells, ERα and ERβ are located in the nucleus, cytoplasm, and plasma membrane while GPER can be found in the endoplasmic reticulum, Golgi apparatus, and plasma membrane (Duarte-Guterman et al., 2015; McEwen et al., 2012; McEwen &

Milner, 2007; Vasudevan & Pfaff, 2008). The locations of said receptors are driven by their structural elements which also direct receptor-ligand interactions. ERα and ERβ may act via genomic or non-genomic mechanisms while GPER has been demonstrated to act via non-genomic actions only (Cui et al., 2013; Prange-Keil & Rune, 2003). The genomic pathway is initiated when estrogens intracellularly bind to ERα or ERβ (Nilsson

et al., 2001). The ER-estrogen complex dimerizes, relocates to the nucleus and influences gene transcription by either directly binding a hormone response element or indirectly with co-activator or co-repressor interactions (Nilsson et al., 2001). Through behavioural studies, the genomic effects of ERs have been described as long-term, resulting in quantifiable changes 24-48 hours after estrogen administration (Choleris et al., 2008). In contrast, the non-genomic pathway is initiated with estrogen binding membrane bound ERα, ERβ or GPER leading to the activation of intracellular signaling cascades such as, but not limited to, the MAPK or phosphoinositide 3-kinase (PI3K)- protein kinase B (AKT) pathways. These pathways can then induce physiological and/or behavioural effects with outcomes occurring rapidly, on a minute to hour’s timescale

(Cui et al., 2013; Vasudevan & Pfaff, 2008; Woolley, 2007).

ER distribution in the brain is widespread and variable in density. For example, the cerebellum and isocortex are regions that express one or two out of the three of the

ERs (Cui et al., 2013). Meanwhile, regions such as the amygdala, hypothalamus, and hippocampus express all three ERs. Moreover, density may differ within a region. For example: ERα, ERβ, and GPER expression in the hippocampus is weak, moderate, and high respectively (Cui et al., 2013). In addition, within the hippocampus, ERβ expression appears relatively uniform, whereas ERα expression is highest in the CA3 and CA1

(Mitra et al., 2003). Furthermore, ERα and ERβ are found in the nucleus, dendrites, dendritic spines, axons, and nerve terminals of hippocampal neurons as well as in glial cells (Milner et al., 2001, Milner et al., 2005; Mitra et al., 2003; Mitterling et al., 2010;

Shughrue et al., 1997; Shughrue & Merchenthaler, 2001). However, the expression of said receptors may fluctuate in females due to their estrus cycle; typically receptor

expression is highest during proestrus when E2 levels reach their peak concentration

(Frick et al., 2015). Yet, some studies demonstrate high ER expression correlating with diestrus and metestrus which is when E2 levels are low; a potential compensatory response to hormonal fluctuations (Llorente et al., 2020; Mendoza-garcés et al., 2011).

Regarding GPER, its expression localizes to cell bodies, dendritic spines, axons and nerve terminals within pyramidal neurons, interneurons and also glia (Akama et al.,

2013; Waters et al., 2015). All in all, the distribution and properties of ERs allow their activation to induce diverse physiological and/or behavioural effects in accordance with endogenous estrogens and environmental stimuli that may interrupt estrogen signaling.

1.3.3 Estrogens and Hippocampal Synaptic Plasticity

A large and well-established body of evidence suggests that circulating estrogens alter dendritic spine density in various brain regions of female rodents, including the hippocampus (Sheppard et al., 2019). The 4-5 day estrus cycle in rodents plays an important role when discussing dendritic spine densities. As seen in figure 1.5, the cycle consists of 4 distinct phases: proestrus, estrus, metestrus, and diestrus. Each phase is characterized by vaginal cytology and circulating concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol, and progesterone

(Goldman et al., 2007). Proestrus, defined by a rise in estradiol followed by an ovulatory surge consisting of peaking LH and FSH concentrations as well as a gradual increase in progesterone, is characterized by the majority of vaginal cells being nucleated with few to no leukocytes present. Following proestrus is a rapid decline in estradiol, LH, and

FSH indicative of estrus, a phase further defined at the vaginal epithelial level as having vast amounts of keratinized cells (Goldman et al., 2007). Throughout the following

metestrus phase of the cycle, circulating concentrations of estradiol remain low and

vaginal cells consist of many keratinized cells with increasing quantities of leukocytes

(Goldman et al., 2007; Walmer et al., 1992). Subsequently, diestrus is the phase of the

cycle where estradiol concentrations gently begin to increase in preparation of the

ovulatory surge and is considered to have few keratinized cells and many leukocytes

(Goldman et al., 2007; Walmer et al., 1992).

Figure 1.5: Hormone levels and unstained vaginal smears characterizing the female rodent estrus cycle. Schematic diagram of the 4-5 day estrus cycle illustrating serum estradiol, progesterone, and luteinizing hormone (LH). Dark blocks along the x-axis of the graph indicate the dark portion of the 14:10hr light:dark photoperiod.As (M estrogen) Metestrus, con combinationcentrations of some fluctuate “needle throughout-like” cells, roundthe estrus “pavement cycle, cells” dendritic and few spinesmaller leukocytes. (D) Dietrus, leukocytes in the presence or absence of larger round epithelial cells. (P) Proestrus, granular-looking cells may appear in clumps and/or strands. (E) Estrus, classic keratinized cells that may appear “needledensities-like” or in round female with rodents jagged irregular have beenedges. demonstrated Adapted from Goldman to quantifiably et al., 2007. differ depending on

the stage of the cycle. For example, apical CA1 dendritic spine densities increase by

approximately 30% in proestrus (Frankfurt & Luine, 2015; Mendell et al., 2017; Woolley

et al., 1990). Additionally, ovariectomy (OVX), the removal of ovarian tissue, will result

in a major decrease of circulating estrogens followed by a reduction in CA1 apical

dendritic spine densities (Gould et al., 1990); which can be reversed by estradiol

administration (figure 1.6; Woolley & McEwen, 1993).

Figure 1.6: Bar graph and photomicrographs depicting the influence of OVX and estradiol administration on dendritic spines. Bar graph shows mean ± S.E.M in apical dendrite spine density in ovariectomized with estradiol benzoate (OVX + E, stippled bar, n=5) or oil vehicle (OVX + O, solid bar, n=6). ** indicates significant differences from OVX + O (p<0.01). Photomicrographs represents dendritic segments of both treatment groups. Arrows indicate some dendritic spines. Scale bar = 10�m. Adapted from Woolley & McEwen, 1993.

Moreover, further support is offered to the relationship between estrogens and dendritic

spines when studying physiological times, such as pregnancy, where circulating

estrogen levels are very high. In late pregnancy and during lactation, female rats exhibit

a dendritic spine density in CA1 pyramidal neurons greater than in any phase of the

estrus cycle (Kinsley et al., 2006). Importantly, changes in neuronal microstructure, brain connectivity, and subsequent activity – both within and between limbic structures – have been correlated with fluctuations in the estrus cycle in rodents (Blume et al.,

2017), menstrual cycle in women (Dietrich et al., 2001; Lisofsky et al., 2015), and with exogenous E2 administration in women (Bayer et al., 2018). Altogether, these findings imply that ovarian hormones may modulate dendritic spine densities and brain activity.

Similarly to the influence of estrogens in females, dendritic spine densities in the

CA1 can be altered in response to hormones in males. The changes in dendritic spine densities in males are, however, mainly driven by testosterone (Leranth et al., 2003) and not through its aromatization to E2 (MacLusky et al., 2004). However, there is evidence that E2 administration in male ex vivo hippocampal slices can increase dendritic spine density in the CA1 (Mukai et al., 2007; Murakami et al., 2006). Similarly, both E2 and testosterone in vivo administration in ORX male rodents rapidly (within 30 minutes) increased dendritic spine density in the CA1 (Jacome et al., 2016). Although few in number, these studies suggest that estrogens can increase dendritic spine densities in males as well.

Thus far we have discussed how hippocampal dendritic spines and their synaptic plasticity have a strong relationship with learning and memory. More recently, we have outlined the role that estrogens play in maintenance of hippocampal synaptic plasticity and thus their influence on behaviour reasonably follows in sequence.

1.3.4 Estrogens and Behaviour

Estrogens have been demonstrated to influence a multitude of behaviours including anxiety, aggression, learning, and memory. In brief, estrogens have been reported to have both anxiolytic and anxiogenic effects, the controversial literature is likely a result of varying estrogenic mechanisms (e.g. genomic vs. non-genomic)

(Choleris et al., 2018). Similarly, E2 administration in males can both agonize and antagonize aggressive behaviour with demonstrated photoperiod interactions (Trainor et al., 2008; Trainor et al., 2007). However, this section will focus on estrogenic effects on learning and memory as this is most relevant for the experiments presented herein.

The process of memory formation, which entails encoding, storing and eventually retrieving information, relies on a series of events. Learning is the beginning of memory formation and is typically considered the acquisition or encoding of information, whereas memory is the storage and retrieval of information (Bailey et al., 2015; Stuchlik, 2014).

Regardless, learning and memory is an adaptive processes where neuronal connections respond to stimuli through synaptic plasticity encompassing structural changes in dendrites and dendritic spines, leading to physiological plasticity described as long-term potentiation and long term depression (LTD) (Sheppard et al., 2019).

The strong relationship between dendritic spine density in the hippocampus and memory is well established in the literature through use of various behavioural assessments. Moreover, increased dendritic spine size/number is associated with LTP

(Matsuzaki et al., 2004) and decreased dendritic spine size/number is associated with

LTD (Moster et al., 1994; Conrad et al., 2012; Leuner et al., 2003). Therefore, a robust interaction between LTP, dendritic spine density and memory exists; however, since

memory can increase both LTP and spines, it is difficult to determine whether increased hippocampal spine density is the cause or consequence of improved hippocampal dependent memory in animal studies alone (Frankfurt & Luine, 2015). But, with consideration of clinical studies in Alzheimer’s patients as well as drug-related studies, data support the notion that spine plasticity induces learning and memory changes and not vice versa (Frankfurt & Luine, 2015). Therefore, in general, the brain drives behaviour while behaviour may affect the brain.

Not surprisingly, estrogens can impact learning and memory processes. Initial studies with chronic estradiol treatments in ovariectomized (OVX) rats demonstrated improved performance on the radial arm maze, Morris water maze (Daniel et al., 1999),

T-maze (Gibbs, 1999), and OR test (Walf et al., 2006). Moreover, when estrogen concentrations are high in gonadally intact cycling rats (Walf et al., 2006) and during pregnancy (Macbeth & Luine, 2010), enhanced object recognition (OR) and object placement (OP) memory is demonstrated. Importantly, OVX rats without estrogen treatment display decreased spine density and poor memory performance (Wallace et al., 2006). With regards to acute administration, estrogenic effects were consistent with chronic treatment. For example, when OVX rats were injected with 15µg/kg of E2 30 minutes prior to OP, results indicate that E2 enhanced memory (Inagaki et al., 2010).

Further support is provided with a dose-dependent increase in post-synaptic spine density, a marker for synapses, being observed within 30 minutes in the CA1 of OVX rats given E2 injections (MacLusky et al., 2005b). Additionally, both E2 administrations into the dorsal hippocampus via bilateral cannulae (Phan et al., 2015) or into general circulation via subcutaneous administration (Phan et al., 2012) in OVX CD-1 adult mice

rapidly improved learning and memory which correlated with rapid increases in immature synapses and quantity of hippocampal dendritic spines. Although few studies have been conducted in male rodents, similar relationships are displayed. Generally, orchidectomy (ORX), the removal of testicular tissue, reduces circulating levels of testosterone, and then subsequent E2 treatment typically restores learning and memory performance (Frick et al., 2015; Gibbs, 2005; Jacome et al., 2016). For example, ORX rats have been shown to respond to acute E2 administration with rapid enhancements in memory consolidation in the OP task and increased apical CA1 dendritic spine density (Jacome et al., 2016). Additionally, in a delayed matching-to-position test, ORX males demonstrated a significant impairment in acquisition, whereas E2 administration enhanced acquisition (Gibbs, 2005).

Yet, estrogens do not always augment learning and memory. Both animal and clinical literature indicates that estrogens impose an inverted U-shape influence on hippocampal-dependent memory performance, with low and high levels impairing and intermediate concentrations improving performance on a range of tasks (Bayer et al.,

2018; Sheppard et al., 2019). Taken together, the influence of estrogens on hippocampal dendrites and subsequent learning and memory is multifaceted and requires careful consideration of dose and time.

1.4 Androgens

1.4.1 Introduction

Present in both males and females, the chief androgens are testosterone and androstenedione, as well as the metabolites DHEA and dihydrotestosterone (DHT); all of which exhibit a diverse range of biological actions (Bardin & Catterall, 1981). In

males, androgens play a critical role in the development of the male reproductive system and remain important for continuous function of the penis, seminal vesicles, and prostate as well as maintenance of spermatogenesis. In females, androgens are important for breast, endometrium, and ovarian function. Additionally, in both sexes, androgens demonstrate a significant role in pubertal changes as well as in continuous function and maintenance of the cardiovascular system and musculoskeletal system

(Smith et al., 2013).

Synthesis of androgens is similar to that of estrogens since both classes of steroids follow the steroidogenic pathway (figure 1.4) and utilize cholesterol as the parent molecule (Bardin & Catterall; 1981). Androstenedione, produced from cholesterol, can enzymatically be converted to testosterone via 17β-HSD and testosterone can subsequently be converted to DHT, a steroid metabolite 2.5 times more potent than testosterone (Mahoudeau et al., 1971). Importantly, testosterone can also be converted to estradiol via P450aromatase (Hojo & Kawato, 2018; Nilsson et al.,

2001). Alike estrogens, the brain is equipped with all the enzymes required to synthesize androgens, including: P450scc, 17α-hydroxylase, 17β-HSD, 3β-HSD, and

5α-reductase (Compagnone & Mellon, 2000, Hanukoglu 1992; Hojo et al., 2009; Hojo &

Kawate, 2018; Lambeth & Stevens, 1984; Melcangi et al., 1996). 17α-hydroxylase and

5α-reductase expression is not sexually differentiated and exhibits no fluctuation with the rodent estrus cycle (Hojo et al., 2014; Kato et al., 2013; Kimoto et al., 2010), however, circulating concentrations of testosterone are sexually differentiated (Charles

& Alexander, 2011). Comparatively, females generally exhibit levels of testosterone that are 10%-40% of the testosterone levels in males (Charles & Alexander, 2011; Labrie et

al., 2009; Maliqueo et al., 2013; Sfikakis et al., 2014; Zhao et al., 2005). Additionally, serum titers illustrate testosterone following the rise and fall of ovarian hormones

(mainly estradiol) in cycling females (Burger, 2002; Enea et al., 2008).

1.4.2 Mechanisms of Androgen Action

Unlike estrogens, androgenic effects are mediated through a single intracellular/nuclear androgen receptor (AR) (Palvimo, 2012). In addition to being located elsewhere in the CNS, ARs are present in the hippocampus (Kerr et al., 1995;

Abdelgadir et al., 1999), more specifically, they are primarily located in the pyramidal neurons of the CA1 (Clancy et al., 1992; Kerr et al., 1995). In rat CA1 neurons, ARs are present in nuclei, dendritic spines, and axon terminals (Tabori et al., 2005). The current model of androgen action is a multi-step process beginning with the entry of testosterone in the target cell, followed by testosterone or its metabolite, DHT, binding to cytoplasmic AR (Saatcioglu, 2011). Ligand bound AR then undergoes a conformational change resulting in translocation to the nucleus where AR dimerizes and binds to DNA inducing activation or repression of gene transcription (Saatcioglu, 2011).

It has also been suggested that AR may act non-genomically via Erk-2, Raf-1, and Src pathways (Kousteni et al., 2001; Michels & Hoppe, 2008).

1.4.3 Androgens and Hippocampal Synaptic Plasticity

The significant role of androgen in hippocampal synaptic plasticity is well established. In males, testosterone secretion from the testes plays a major role in maintenance of hippocampal synaptic density and, as demonstrated with ORX in both rodents and non-human primates, the number of CA1 dendritic spine synapses are significantly reduced in testosterones absence (Leranth et al., 2003; Leranth et al.,

2004b MacLusky et al., 2004). Leranth and colleagues (2003), reported that ORX rats treated with E2 cannot restore dendritic synapse density, suggesting that the male hippocampus does not respond to E2. Moreover, because of the apparent response in

ORX rats treated with testosterone or DHT combined with the lack of response to E2, testosterone is thought to induce spine synapse formation in the hippocampus via AR mechanisms (figure 1.7; Leranth et al., 2003). In further support, hormonal manipulation

– although acute in nature (2 injections 24 hours a part) – was evaluated for effects 48 hours after the last injection, implying that the underlying mechanisms for said results are likely genomic (Leranth et al., 2003).

Figure 1.7: Spine synapse density in the CA1 hippocampus of male rats. Bar graph depicts the spine synapse density in the CA1 stratum radiatum of male rats in control, gonadectomized (GDX), gonadectomized + testosterone propionate administration (GDX + TP), gonadectomized + dihydrotestosterone administration (GDX + DHT), and gonadectomized + estradiol administration (GDX + E2). The spine synapse densities in the GDX and GDX + E2 rats are significantly (p<0.001) lower than control animals. Adapted from Leranth et al., 2003.

Androgens have also been shown to impact CA1 spine synapse density in females. In a study conducted by Leranth and colleagues (2004a), OVX rats were

treated with testosterone or DHT in combination with a non-steroid aromatase inhibitor namely letrozole. Compared to the OVX controls, testosterone reversed the loss of CA1 spine synapses and DHT treatment produced similar, but not as potent results (Leranth et al., 2004a). Moreover, letrozole treatment almost completely blocked the response to testosterone, but not to DHT, signifying that synaptic activity of testosterone reflects a large contribution of intrahippocampal estrogen biosynthesis (Leranth et al., 2004a).

Yet, aromatization is not essential as DHT can induce spine synapse formation in both sexes (Hajszan et al., 2008; Leranth et al., 2004a). Therefore, analogous to the estrogenic effects on synaptic plasticity and behaviour, androgens may greatly impact behaviour as well.

1.4.4 Androgens and Behavior

A body of evidence suggests that testosterone may influence performance on cognitive tasks. In fact, positive associations have been demonstrated between cognitive performance and levels of circulating androgens in men (Barrett-Connor et al.,

1999; Neave et al., 1999; Yaffe et al., 2002) and women (Barrett-Connor & Goodman-

Gruen, 1999). Yet the clinical literature is inconsistent (Celec et al., 2015) and, thus, animal studies allow researchers to investigate androgenic effects to achieve clarity. In adult male rats, ORX impairs performance in various maze tasks including the T-maze

(Kritzer et al., 2001) and radial arm maze (Daniel et al., 2003; Harrell et al., 1990), suggesting that testosterone plays a role in spatial memory. Moreover, in object recognition tasks, gonadally intact and ORX + testosterone rodents significantly outperform their ORX counterparts, which cannot discriminate between novel and familiar objects (Ceccarelli et al., 2001; Aubele et al., 2008). Testosterone also improves

delay-dependent memory as ORX rats display lower retention scores compared to intact controls (Sandstrom et al., 2006). With one exception (i.e. Gibbs & Johnson,

2008), exogenous testosterone treatment reversed the cognitive deficiencies that resulted from ORX (Aubele et al., 2008; Ceccarelli et al., 2001; Gibbs, 2005; Havens &

Rose, 1992; Kritzer et al., 2001; Sandstrom et al., 2006). Furthermore, exogenous testosterone treatment in gonadally intact male rodents enhanced memory-related task performance (Alexander et al., 1994; Flood et al., 1992; Vazquez-Pereyra et al., 1995).

Importantly, testosterone may mediate learning and memory through aromatization to

E2 therefore, methods such as aromatase knockout (ArKO) mice, DHT administration rather than testosterone, aromatase inhibitors (AI), or AR blockers (i.e. flutamide) are often utilised to target androgenic pathways. In ArKO male mice, Martin and colleagues

(2003) found that Y-maze performance was significantly worse than wildtype controls.

Then, ORX diminished between group differences as the ORX wildtype controls exhibited poor performance whereas the ORX ArKO mice resembled that of the ArKO group (Martin et al., 2003). This highlights the importance of testosterone being aromatized to E2 for memory processes in males. Notably, ArKO mice lack aromatase throughout their lifespan and thus, the effects of E2 deficiency at critical time periods, including development vs. adulthood, cannot be discerned. In contrast, findings from studies using intact male rats suggest that AI (i.e. letrozole or anastrozole) treatment may improve working memory possibly by supressing E2 synthesis (Alejandre-Gomez et al., 2007; Moradpour et al., 2006). For example, anastrozole (0.25, 0.5, 1 µg/0.5 µL or DMSO) administration via bilateral cannulae into the CA1 of adult male rats demonstrated dose dependent decreases in escape latency and traveled distance in the

Morris water maze task when compared to controls, thus suggesting improved acquisition of spatial learning and memory (Moradpour et al., 2006). Similarly, microinjection of DHT (0.25, 0.5, and 1 µg/0.5 µL or DMSO) into the CA1 of adult male rats significantly reduced escape latency and traveled distance in the Morris water maze task (Babanejad et al., 2012). Meanwhile, in aged male rats, silastic capsules of testosterone, but not DHT, improved working memory when assessed in the water- escape radial arm maze task in comparison to aged, vehicle sham counterparts

(Bimonte-Nelson et al., 2002). The contradicting results with DHT treatment may result from differences in experimental design (adult vs. aged, microinjection vs. silastic capsule, and Morris water maze vs. water-escape radial arm maze). Finally, Cherrier and colleagues (2003) demonstrated that hypogonadal men provided testosterone or

DHT exhibit significant improvements in verbal memory and spatial memory respectively when compared to baseline testing. Collectively, these studies implicate testosterone in learning and memory processes both through androgenic and estrogenic mechanisms.

When considering females, the influence of androgens on the CNS and behaviour is not well characterized. Yet, significant levels of circulating testosterone have been reported in women, female monkeys, and female rats; rising to as high as 40% of male values (Charles & Alexander, 2011; Labrie et al., 2009; Maliqueo et al., 2013; Sfikakis et al., 2014; Zhao et al., 2005). Alike males, testosterone may impact female cognition by directly binding AR, before or after metabolization to DHT, or by binding estrogen receptors following conversion to E2 (Edinger & Frye, 2007; Handa et al., 2011). To tease apart the effects of E2 and testosterone, Taylor and colleagues (2017) assessed

spatial memory in OVX rats administered testosterone propionate (TP) with or without an aromatase inhibitor (i.e. letrozole) compared to gonadally intact cycling females.

Results indicate that spatial performance is superior with coadministration of E2 and TP, and though E2 and TP can both independently improve memory, TP partially compensated for spatial performance when E2 bioavailability was low (Taylor et al.,

2017). Moreover, as evidence supports the role of DHT metabolites in ER activation

(Handa et al., 2008; Handa et al., 2009; Mahmoud et al., 2016), it is feasible that superior spatial performance seen with high levels of testosterone and E2 (Taylor et al.,

2017) is driven by indirect effects of DHT.

Collectively, the role of testosterone in memory and learning is likely a delicate balance of androgenic and estrogenic actions in females, while heavily skewed to androgenic actions in males. Nevertheless, the vital role hormones play in hippocampal synaptic plasticity and behaviour sets the stage for potentially diverse and sex-specific impacts of environmental stimuli that interact with the endocrine system, such as endocrine disrupting chemicals.

1.5 Bisphenol-A

1.5.1 History and Properties

Bisphenol A, also referred to as 2,2-bis(4-hydroxyphenyl)propane or 4,4'- dihydroxy-2,2-diphenylpropane, is a small organic compound with the following molecular formula, (CH3)2C(C6H4OH)2 (National Center for Biotechnology Information

[NCBI], n.d.) . BPA is comprised of two phenolic rings linked by carbon atoms which possess methyl groups (NCBI, n.d.). The first reported synthesis of BPA, from phenol and acetone, was in 1905 by Thomas Zincke of the University of Marburg, Germany

(Huang et al., 2011; Vaughn, 2010). Zincke described physical properties of BPA such as molecular weight (228.3 Da), melting point (150-155ºC), and water solubility (120-

300mg/L), but failed to suggest any application (NCBI, n.d.; Richter et al., 2007;

Vaughn, 2010). In the late 1950s, commercial applications for BPA were established in the development and manufacturing of polycarbonate plastics and epoxy resins

(Vaughn, 2010). Nowadays, BPA serves a variety of industrial and consumer uses. For example, polycarbonate plastics are used in the production of automobiles, household appliances, sports safety equipment, consumer electronics, water bottles, medical devices, children toys, and eye glasses (Konieczna et al., 2015; Vaughn, 2010). As for epoxy resins, they are used in adhesives, thermal paper, can linings, industrial floorings, sealants, furniture finishes, automotive primers and so on (Konieczna et al., 2015;

Vaughn 2010). All in all, BPA represents one of the most widely used synthetic compounds. In 2015, global consumption of BPA was 7.7 million metric tonnes and is projected to increase to 10.6 million metric tonnes by 2022 (Lehmler et al., 2018).

1.5.2 Environmental Prevalence and Human Exposure

With BPA’s large production rates, anthropogenic pollution was an anticipated environmental issue. In fact, BPA has been detected in water, sewage, air, soil, and sediment samples worldwide (Huang et al., 2011; Lehmler et al., 2018). BPA is released into the environment during production of polycarbonate plastics, epoxy resins and their respective products as well as in the disposal of such products after use (Huang et al.,

2011; Yamamoto et al., 2001). While contamination is widespread, the levels of BPA are generally low due to fairly rapid and complete degradation of BPA in the environment. BPA in surface water is degraded by bacteria, resulting in a half-life of a

couple days (Vaughn, 2010; Huang et al., 2011). Atmospheric degradation occurs by hydroxyl radicals with a rapid half-life of a few hours (Vaughn, 2010; Huang et al.,

2011). Soil and sediment concentrations of BPA are often the highest due to a half-life of several days (Vaughn, 2010; Huang et al., 2011). Regardless, with significant production and utilization, BPA contamination is widespread; making it an important pollutant with incessant exposure rates.

The primary source of BPA exposure in humans is food and water contamination

(Konieczna et al., 2015). BPA contaminates drinking water by leaching from products in landfills, direct release from manufacturing facilities, and small unreacted quantities present in PVC pipes (Huang et al., 2011). Regarding food, BPA contaminates dietary sources by leaching from food packaging and reusable plastic food containers as well as livestock and animal by products if housed in polluted areas (Huang et al., 2011;

Konieczna et al., 2015; Vaughn, 2010). Although oral intake is the primary source of

BPA exposure, secondary sources include permeation and absorption of BPA through inhalation and dermal contact (Vom Saal and Hughes, 2005). As is the case in the environment, the terminal half-life of BPA in the human body is short, approximately 6 hours (Völkel et al., 2002). BPA is often subjected to hepatic first pass metabolism resulting in the production of glucuronide via uridine 5”-diphosphoglucuronyl transferase

(UGT) and subsequently eliminated in urine (Konieczna et al., 2015; Völkel et al., 2002).

While glucuronide is the major metabolite of BPA, trace amounts are converted to bisphenol-3,4-quinone and BPA-sulfate (Konieczna et al., 2015). Even though BPA is metabolized and eliminated from the human body quickly, BPA exposure is continuous.

Ninety percent of Canadians are exposed to BPA on a daily basis and ninety-five

percent of North Americans have detectable concentrations of BPA in their urine

(Lehmler et al., 2018). Thus, considering the numerous sources of BPA, it can be considered a very important global pollutant with chronic rates of human exposure.

1.5.3 Behavioural Impacts

A growing body of evidence supports the notion that BPA exposure at any given time in one’s lifespan may impact behaviour. Numerous rodent and some non-human primate studies have shown behavioural and functional effects of BPA exposure on memory, anxiety, locomotion, exploratory activities, social behaviour, and reproductive behaviour. Many of these effects are demonstrated across early development (gestation and /or lactation), adolescence, and adulthood. However, a large portion of BPA literature focuses on developmental stages and so, the experiments presented herein investigate bisphenol exposure in adult rodents rather than developmental periods.

Importantly, the effects of EDCs may differ substantially in critical periods vs. adulthood and thus having both models in the literature is crucial. Additionally, alike prior sections which emphasize hippocampal microstructure and function this review will follow in suit by focusing on the learning and memory effects of BPA exposure during adulthood.

Studies that have focused on adult male mice have demonstrated the following: chronic (90 days) oral BPA exposure at doses of 4mg/kg bw/d and 40mg/kg dw/d enhanced acquisition and retention of fear memory (Zhang et al., 2014); chronic (84 days) oral gavage of 0.4, 4, or 40mg/kg bw/d worsened spatial (platform location) memory determined by increased escape path length in the Morris water maze (linear dose-response) and enhanced retention of fear memory determined via decreased step-down latency 24h after footshock in the step-down passive avoidance test (non-

linear dose-response) (Xu et al., 2013); and chronic (14 days) oral gavage of 20mg/kg bw/d worsened spatial memory determined by higher latencies to platform and increased escape path length in the Morris water maze test (Kim et al., 2011). Notably, some studies (i.e. Kim et al., 2011 and Xu et al., 2013) measured behavioural effects via latency which can be confounded by non-specific effects such as locomotor abilities.

Additionally, the above studies used doses of BPA that are high enough to exert estrogen agonist effects which may partially explain some of the behavioural improvements (i.e. Xu et al., 2013 and Zhang et al., 2014) Moreover, while these studies indicate that BPA exposure may influence learning and memory processes, all the doses used lack translational relevance. In contrast, studies mentioned below utilize doses equal to or below 50µg/kg bw/d which are much more translationally relevant as this dose corresponds with the U.S. Environmental Protection Agency safe daily limit.

Fan and colleagues (2013) chronically exposed male Wistar rats to BPA (50µg/kg bw/d) and assessed spatial memory with the Morris water maze task. They found that the

BPA-treated rats swam faster, longer distances, and spent more time trying to find the hidden platform (Fan et al., 2013). Then, on the final testing day when the platform was removed, the BPA-treated rats spent less time in the platform quadrant when compared to controls; suggesting that BPA significantly impaired both acquisition and retention of spatial memory (Fan et al., 2013). In another study, in male Sprague Dawley rats, acute

BPA exposure at 40µg/kg impaired OR and OP performance when both paradigms had a 2 hour inter-trial delay (Eilam-Stock et al., 2012). This indicates that BPA exposure can impact both spatial and visual memory (Eilam-Stock et al., 2012). Finally, in male

St. Kitts African vervet monkeys, 30 days of BPA exposure at 50µg/kg bw/d via an

injected subcutaneous osmotic minipump demonstrated deficits in working memory accuracy in the two-choice spatial delayed response task after 1 week exposure, but interestingly not after 4 weeks, suggesting a compensatory mechanism at play

(Elsworth et al., 2015). Collectively, these studies suggest that BPA exposure in males will impair cognition via visual and spatial memory.

Fewer studies have addressed the behavioural effects of BPA exposure in adult females. Inagaki and colleagues (2012) first investigated acute BPA exposure in both

OVX and gonadally intact Sprague Dawley rats. OVX rats were administered 0, 0.4, 1,

4, 40, or 400µg/kg BPA alone or in combination with 17β- or 17α-E2, whereas intact rats were administered 0 or 40µg/kg BPA alone or in combination with 17β-E2 (Inagaki et al., 2012). The results indicate that BPA rapidly blocks E2-induced enhancements of spatial and non-spatial memory in a dose-dependent manner in OVX females, whereas only supressing OR memory in proestrus of intact females (Inagaki et al., 2012).

Moreover, in the absence of E2, BPA did not affect memory performance suggesting the importance of estradiol’s presence for BPA effects (Inagaki et al., 2012). In contrast,

Xu and colleagues (2015b) investigated the impact of chronic BPA exposure (40 and

400µg/kg/d) on performance in the Morris water maze and step-down passive avoidance test in both OVX with estradiol benzoate (EB) (10µg/kg/d) and sham operated (intact) females. The study demonstrated the following: (1) the rescue effect of

EB on OVX-induced memory impairment and (2) in OVX mice with estrogen deficiency,

BPA exposure improved learning and memory which was observed by a shortened escape path length and increased time spent in the target quadrant of the Morris water maze task as well as prolonged step-down latency in the passive avoidance task (Xu et

al., 2015b). Moreover, no significant effects of BPA were found in the sham-operated, cycling females (Xu et al., 2015b). Collectively, these two studies suggest divergent effects of BPA exposure on memory in adult females, indicating that outcomes may be influenced by circulating concentrations of estrogens. Notably, BPA exposure before the onset of ovarian hormone cyclicity in adolescent rodent models impaired spatial memory in OP and increased anxiety (Bowman et al., 2015; Diaz Weinstein et al.,

2013). Therefore, this suggests that while BPA effects may not require estrogenic interactions, it may be vulnerable to potential organizational and activational effects of

E2 which may, albeit partially, facilitate differences between the prepubertal and adult female brain (Bowman et al., 2019).

Although most potential health hazards of BPA are deduced from animal studies, epidemiological studies offer support when considering the link between BPA exposure and cognitive performance in children (Casas et al., 2015; Hong et al., 2013). Casas and colleagues (2015) assessed prenatal BPA exposure by urinary measurements in the first and third trimester of pregnancy in 438 pregnant Spanish women. Cognitive and psychomotor abilities of the children were evaluated at 1 and 4 years of age with age- appropriate standardized neuropsychological tests including the Bayley Scales of Infant

Development (BSID – 163 items) and McCarthy Scales of Children’s Abilities (MSCA – general cognitive index and 5 subscales) respectively (Casas et al., 2015). A significant decrease in psychomotor ability was observed at 1 year of age when comparing prenatal urinary concentrations of BPA in the highest-tertile and lowest-tertile; no association was observed with motor score at age 4 (Casas et al., 2015). Regarding cognition (BSID and MSCA scores), no association was observed between prenatal

urinary BPA concentrations and cognitive scores at either age 1 or 4 (Casas et al.,

2015). Similarly, Hong and colleagues (2013) assessed BPA exposure via urinary concentrations in 1008 children aged 8-11 years in five different regions of Korea and evaluated learning performance with a parent-reported questionnaire (Learning

Disability Evaluation Scale) and an intelligence scale (Korean Educational Development

Institute Wechsler Intelligence Scales for Children). A significant adverse association was observed between urinary BPA concentrations and learning performance, whereas, no association was observed between BPA concentrations and intelligence scores

(Hong et al., 2013).

While epidemiological studies in young populations offer some support for the potential influence of BPA exposure on learning and memory, the literature remains insufficient when considering the impact of BPA on adults. Although not exhaustive, animal studies offer evidence to support the notion that BPA exposure impairs spatial and non-spatial memory performance (Mhaouty-Kodja et al., 2018). Yet, some studies indicate no effect or even enhanced performance; the causes of these discrepancies have been subjected to detailed scrutiny by other Authors (Beronius et al., 2013) and remain difficult to address given the range of experimental conditions (i.e. dose, route of administration, period of exposure, sex and reproductive status of subjects, etc.) among studies. Regardless, the accumulating evidence progressively points towards an effect of BPA on memory and learning, though the mechanisms producing said effects are not fully understood.

1.5.4 Proposed Mechanisms

The behavioural effects elicited by BPA can be explained, albeit partially, with parallel changes observed at the cellular and molecular levels. With developmental exposure to BPA, the following mechanistic outcomes have been reported in the brain: changes in expression of ERα and ERβ (Mahoney & Padmanabhan, 2010; Xu et al.,

2010), alterations in glutamatergic NMDA receptors and aminergic or serotoninergic systems (Adewale et al., 2010; Matsuda et al., 2010; Tian et al., 2010; Xu et al., 2010), modifications in NO system (Martini et al., 2010), changes in expression of neuropeptides such as oxytocin and kisspeptin (Adewale et al., 2010; Naule et al.,

2014), and modifications in neural differentiation (Rubin et al., 2006; Patisaul & Fortino,

2007). Data addressing the mechanisms of BPA action are not as extensive in the adult model, however, numerous studies suggest that BPA exposure impairs synaptic plasticity. In male non-human primates and rodents, BPA exposure has been demonstrated to decrease synaptic density in the hippocampus and PFC (Eilam-Stock et al., 2012; Elsworth et al., 2015; Xu et al., 2013). Furthermore, studies in male rodents also show BPA exposure down regulating receptor subunits GluN1 and GluR1 in NMDA and AMPA respectively (Xu et al., 2013; Zhang et al., 2014) as well as decreasing postsynaptic density protein -95 (PSD-95), a synaptic marker in the hippocampus

(Eilam-Stock et al., 2012). Importantly, in ORX or sham operated male rats treated with

BPA (300µg/kg), testosterone propionate (1.5mg/kg) or vehicle, the number of spine synapses in the PFC and hippocampus were reduced with a compensatory increase in astroglia density; therefore this study suggests that BPA may act as an anti-androgen as it prevented the synaptogenic response to testosterone (Leranth et al., 2008).

Furthermore, studies have revealed that BPA antagonizes androgen receptor-mediated

transcriptional activities (Satoh et al., 2004; Sohoni & Sumpter et al., 1998; Xu et al.,

2005). Therefore, BPA, commonly referred to as an “environmental estrogen”, may

interfere with testosterone responses through androgenic mechanisms suggesting that

its primary endocrine disrupting properties extend beyond estrogenicity (Wetherill et al.,

2007).

Meanwhile, data in females are more convoluted. Rodent studies indicate that

BPA in OVX females impairs the E2-induced increase in spine density in the

hippocampus (Inagaki et al., 2012; figure 1.8a, MacLusky et al., 2005a; Xu et al.,

2015b), but not in the PFC (Inagaki et al., 2012); whereas, spine density is unaffected

by BPA exposure in gonadally intact females (Xu et al., 2015b). Additionally, as seen in

figure 1.8b, the response to BPA in OVX rodents is dose-dependent.

Figure 1.8: Influence of BPA on CA1 dendritic spine synapse density. (A) BPA inhibits the enhancing effects of 17�-E2 on dendritic spine synapse density. *Significantly higher synapse density compared to vehicle rats without BPA (p<0.05). **Significantly reduced synapse density compared to vehicle rats without BPA (p<0.05). (B) The dendritic spine synapse density response to 17�-E2 is inhibited in a dose-response manner to BPA. Data represented by mean ± SD of 3 independent observations. **Significant difference in synapse density from vehicle-treated rats without BPA (p<0.05). From MacLusky et al., 2005a. 35

Moreover, expression of protein synapse I, PSD-95, and receptor NMDA receptor

GluN2B is not affected by BPA treatment in gonadally intact, but inhibited in OVX and

OVX + EB female rodents (Xu et al., 2015b). Collectively, BPA seemingly antagonizes synaptic remodeling in females, however, this effect is dependent on the levels of endogenous hormones (OVX vs. gonadally intact).

In sum, in vitro studies classified BPA as an EDC due to its binding affinity with estrogen receptors and, thus, this has become a screening method to determine the disruptive nature of similar compounds (Bonefield-Jorgensen et al., 2007; Lee et al.,

2003; Sohoni & Sumpter, 1998; Xu et al., 2015b). However, mechanisms eliciting BPA’s effects and subsequent safety screening methods may have been prematurely concluded. This is rooted in the observed in vivo effects being diametrically opposite to what would be predicted for a substance with estrogen-agonist activity, alluding to the possibility that BPA in addition to being a weak estrogen-agonist, may act as an estrogen-antagonist and/or independent of estrogen receptors. There is evidence suggesting that BPA may exert its effects by: disrupting androgen-related systems; interfering with thyroid-related systems and acting via thyroid receptors; acting via secondary metabolic and pharmacokinetic actions; influencing the immune system; and imparting generational effects via epigenetic mechanisms (Wetherill et al., 2007). Due to various mechanisms proposed for BPA action, narrowly defining BPA as an environmental estrogen is inaccurate. BPA is an EDC in the broad sense of the definition; animal studies demonstrate profound and diverse impacts of BPA exposure, indicating an overall negative influence on human and animal health. As a result, multiple governments intensified regulations surrounding the use of BPA in consumer

products as well as declared BPA as a potentially dangerous substance; this led to chemical companies seeking BPA substitutes, such as Bisphenol S (Eladak et al, 2015;

EPA, 2010).

1.6 Bisphenol-S

1.6.1 History and Properties

In the early 2000’s, BPA regulations began to tighten, particularly to protect against exposure during development and critical time periods. For example, the use of

BPA in baby bottles was banned in 2008 in Canada, in 2010 in France, and in 2011 in the European Union (Eladak et al., 2015). The current tolerable dose intake (TDI) in

Canada is 25µg/kg/d and 50µg/kg/d in the US (Eladak et al, 2015; EPA, 2010).

Remarkably, the European Food Safety Authority reduced their TDI to 4µg/kg/d in 2015

(Eladak et al, 2015). Therefore, in response to the increasing pressures from regulatory agencies, concerned researchers, and environmental groups, the chemical industry introduced a total of 16 bisphenol analogues to replace BPA (Chen et al., 2016).

However, these analogues underwent minimal testing and are presently in use while unregulated (Chen et al., 2016; Eladak et al., 2015). Among these BPA alternatives is one of the most commonly used substitutes, BPS, also known as 4,4’-sulfonyldiphenol, bis-(4-hydroxyphenyl)-sulfone. BPS is manufactured through the reaction of phenol and sulfuric acid (Fink, 2014) and due to the presence of strong O=S=O double bonds, displays several advantages over BPA. For example, BPS has greater thermal and light stability, making BPS less leachable from plastics and other consumer products (Chen et al., 2013; Wu et al., 2018).

Table 1.1: Chemical Properties of BPS and BPA. Adapted from Naderi & Kwong, 2020. Compound Chemical Structure Molecular Molecular Boiling Melting Water Formula weight point point solubility (g/mol) (ºC) (ºC)

Bisphenol S C12H10O4S 250.27 240-241 245-250 1100mg/L (at 20 ºC)

Bisphenol A C15H16O2 228.29 220 158-159 120 mg/L (at 25 ºC)

As a result, BPS occupies a significant role in the global market. According to the

European Chemical Agency 1000 to 10,000 million metric tonnes of BPS are manufactured or imported annually into Europe alone (Lehmler et al., 2018). BPS is commonly used as an additive in thermal paper and currency bills (Bjornsdotter et al.,

2017; Liao et al., 2012b), an anticorrosive agent in epoxy glues (Vinas et al., 2010), and a preservative in canned foodstuffs (Yang et al., 2014). Unfortunately, BPS-containing products are marketed as “BPA-free” and are assumed to be safe. However, many BPA alternatives have yet to undergo the same scrutiny that BPA was subjected to and thus, in some cases, BPA substitutes may be just as harmful as BPA – or more so – and have been coined potential “regrettable substitutions”. Ideally, chemical substitutes would be inert, or at least far less toxic than the original chemical. Yet, because of structural similarities between BPA and BPS (Table 1.1), it is hypothesized the BPS will share similar adverse health effects through endocrine disruption (Rochester and

Bolden, 2015).

1.6.2 Environmental Prevalence and Human Exposure

As BPS increasingly replaces BPA, its prevalence in the environment is an emerging concern. Studies suggest that BPS is less amendable to microbial and photochemical degradation in water as well as equally persistent in soil and sediment when compared to BPA (Danzl et al., 2009; Ike et al., 2006; Sakai et al., 2007). There is also considerable evidence for its increasing concentration in worldwide samples of water, indoor dust, soil, sediment, and sewage (figure 1.9, Chen et al., 2016; Lehmler et al., 2018; Qui et al., 2019).

Figure 1.9: Concentration ratio depicting the presence of bisphenols in biological and environmental samples. Ratio determined by the concentration of the bisphenol analogue present in the sample divided by the sum of the eight analogue concentrations. Samples are from human urine, food, indoor dust, sediment, sludge, water. Data for human urine A and B is from Yang et al., (2014) and Asimakopoulos et al., (2015) respectively. From Chen et al., 2016.

In addition to environmental BPS exposure, humans are further exposed to BPS by use of BPS-containing products as well as consuming contaminated food (Chen et al, 2016).

Notably, dietary intake appears to be a major source of BPS exposure, with BPS identified in various food samples from China, USA, and Europe, including but not limited to: canned food, cereals, meat, seafood, eggs, milk, vegetables, fruits, and beverages (Qui et al., 2019).

Although limited in number, studies have begun to assess human exposure to bisphenol analogues through analysis of urine samples. While single spot urine samples primarily evaluate bisphenol exposure occurring shortly before collection, a sufficient sample size may offer adequate interpretation of population exposure. A multinational study incorporating the U.S. and 7 Asian countries reported BPS in 81% of human urine samples at concentrations up to 21ng/mL (Liao et al., 2012a). BPS concentrations were highest in Japanese urine samples followed by U.S. samples (Liao et al., 2012a).

Another study exclusive to the U.S. population reported the detection of BPA, BPS, and bisphenol F (BPF) in 95.7, 89.4, and 66.4% respectively from urine samples of 1808 adults and 868 children taken in 2013 and 2014 (figure 1.10, Lehmler et al., 2018).

Median levels of BPA, BPS, and BPF were 1.24, 0.37, and 0.35µg/L respectively in adults and 1.25, 0.29, and 0.32µg/L respectively in children (Lehmler et al., 2018).

Furthermore, the study reported higher urinary concentrations of BPA (p=0.002), BPS

(p= 0.08), and BPF (p=0.03) in males than in females (Lehlmer et al., 2018). Even though some differences are not statistically significant, these biomonitoring data are in line with other worldwide studies demonstrating greater urinary concentrations of bisphenols in men than in women (Lehlmer et al., 2018). Regardless, this trend is

noteworthy, as it may reveal slightly higher daily intakes of BPS in men, as well as

highlight sexual differences in bisphenol toxicokinetics (Kim et al., 2003; Waxman &

Holloway, 2009).

Figure 1.10: Urinary concentrations of BPA, BPF, and BPS. Subjects participating in the National Health and Nutrition Examination Survey are grouped by age. From Lehmler et al., 2018.

1.6.3 Behavioural Impacts and Proposed Mechanisms

Available in vitro and in vivo studies have demonstrated various effects of BPS

exposure potentially exhibiting acute toxicity, endocrine disruption, neurotoxicity,

immunotoxicity, and reproductive and developmental toxicity (Qui et al., 2019).

Focusing on endocrine disruption, in vitro studies provide evidence that BPS can alter

hormone levels and expression of hormonal genes in several cell lines. For example,

murine MA-10 Leydig cells exposed to 10µM of BPS for 6 hours demonstrated altered

hormone secretion and expression of genes involved in the steroidogenic pathway

(Roelofs et al., 2015). Comparably, BPS affected progesterone, cortisol, and

testosterone synthesis as well as related gene expression in a human adrenocortical

carcinoma cell line (H295R) (Feng et al., 2016; Rosenmai et al., 2014). Moreover, BPS has exhibited hormone regulation and weak estrogenic activity in yeast-based transcriptional assays (Chen et al., 2002), embryo-larvae zebrafish (Zhang et al., 2017), human primary adipocytes (Verbanck et al., 2017), and pig oocytes (Zalmanova et al.,

2017). In a recent study, the agonistic and/or antagonistic effects of BPA along with eight analogues (BPAF, BPAP, BPB, BPE, BPF, BPP, BPS, and BPZ) were characterized in vitro against human nuclear receptors, including estrogen receptors, androgen receptors, glucocorticoid receptors (GR), pregnane X receptor (PXR), and constitutive androstane receptor (Kojima et al., 2019). All the analogues, except for

BPP, showed AR- and GR- antagonistic activity as well as PXR-, ERα- and ERβ- agonistic activity (Kojima et al., 2019). While BPS demonstrated weaker potency in some instances, it was able to elicit multiple effects on the human nuclear receptors alike BPA (Kojima et al., 2019). In addition to receptor action, BPS potentially elicits effects through oxidative stress as it has been shown to increase the activity of antioxidant enzymes (Qui et al., 2018a, 2018b, Ullah et al., 2017) as well as induce lipid peroxidation, suggesting a compensatory response to an increase in reactive oxygen species (ROS) production (Michalowicz et al., 2015).

When considering the impact of BPS exposure on the CNS and behaviour, data are insufficient to draw concrete conclusions, however, available studies suggest that

BPS may be neurotoxic and alter animal behaviour. In vitro, BPS demonstrates the ability to impair neuronal differentiation of mouse embryonic stem cells (Yin et al.,

2019), as well as induce oxidative stress and inhibit cell proliferation in a mouse hippocampal cell line (HT-22) at doses comparable to human plasma and urine

concentrations (Pang et al., 2019). In vivo studies suggest that BPS exposure is disruptive as well. For example, as seen in figure 1.11 chronic BPS exposure in adult female Zebrafish at 10µg/L (OR only) and 30µg/L – a dose relevant to aquatic environments – reversed the usual preference for novelty yielding a preference for familiarity (Naderi et al., 2020). Moreover, due to the investigation ratios significantly differing from chance performance, motivation underlying exploratory behaviour is likely being modified rather than recognition per se (Naderi et al., 2020). Although reasons for said changes in behaviour are not yet understood, it is possible that BPS exposure may elicit affective behaviours (i.e. anxiety, fear) where a preference for familiarity as opposed to novelty is common (Naderi et al., 2020; Wong et al., 2010).

Figure 1.11: Bisphenol S significantly influenced novelty preference in adult female zebrafish. Schematic of object recognition (OR) and object placement (OP) paradigms to evaluate non-spatial and spatial memory performance respectively in adult, female zebrafish. T1 and T2 are training and test phase respectively. Graphs illustrate the investigation ratio for each treatment group (n=28). The investigation ratio is the time spent exploring the novel or displaced object divided by the total time spent exploring both objects. The dashed line at 0.5 indicates chance performance. # Significant difference from chance (p<0.05), * Significant difference in investigation ratio compared to the control group (p<0.05).

Presently, 7 rodent studies have investigated the effects of BPS exposure on neurobehavioral outcomes, all of which utilized a developmental model of prenatal or postnatal exposure. Although behavioural outcomes varied, these studies indicate that

BPS exposure in a developmental model produces the following effects in offspring: impaired social behaviour (Kim et al., 2015), elevated anxiety (da Silva et al., 2019; Kim et al., 2015; Mornagui et al., 2019), increased locomotor activity in males and decreased exploratory behaviour in females (da Silva et al., 2019), altered feeding behaviour such that there is decreased nursing behaviour (LaPlante et al., 2017) and increased food intake coinciding with an augmented preference for a fat-enriched diet in adulthood (da Silva et al., 2019; Regz et al., 2018). Additionally, dams exposed to BPS demonstrate impaired maternal care toward pups (Catanese & Vandengerg, 2017;

LaPlante et al., 2017; Shi et al., 2019). Evidently, BPS exposure during critical development periods may impact behaviour but, due to the current lack of experiments in a non-developmental model, it is not possible to ascertain if BPS will elicit the same effects in adults.

1.7 Rationale

BPA is known to be an endocrine disrupting chemical capable of producing deficits in learning and memory performance. One of the mechanisms correlated to said effect is modulation of hippocampal dendritic spine and synapse densities (Eilam-Stock et al.,

2012; Elsworth et al., 2015; Inagaki et al., 2012; MacLusky et al., 2005a; Xu et al.,

2015b; Xu et al., 2013). However, dendritic responses, albeit partially, are driven by gonadal hormones which are sexually differentiated. Therefore, mechanisms underlying

BPA action and subsequent effects may be sexually differentiated as well; highlighting the importance of investigating bisphenol exposure in both sexes. Importantly, the research field surrounding BPA is transitioning to bisphenol analogues as regulatory agencies and societal pressures have advocated for the cessation in BPA use.

However, with no clearly defined mechanisms for the adverse effects of BPA, testing analogues for their safety parameters is challenging. BPS is one of the primary analogues replacing BPA. Yet, whether BPS is a safer alternative is an open question as it has not been subjected to the same degree of scrutiny as BPA. Furthermore, because of the structural similarity between BPA and BPS as well as biomonitoring data illustrating a positive trend for world-wide BPS exposure, environmental and human health concerns are rapidly increasing. Therefore, in this thesis, the effects of BPS exposure on OP performance – which is a hippocampal-dependent task – as well as hippocampal microstructure were explored in comparison to BPA with emphasis on sex differences and endogenous hormones.

1.7.1 Hypotheses

Bisphenol S will impair hippocampal-dependent memory performance as well as reduce dendritic spine density in magnitude similar to that of Bisphenol A.

1.7.2 Objectives

1. To determine an appropriate method that maximizes translational relevance

when investigating BPA and BPS exposure.

• These results will establish a method by which bisphenol exposure can

occur discreetly to minimize stress responses.

2. To investigate the effects of BPA and BPS exposure on hippocampal-dependent

memory in both gonadally intact adult male and female mice.

• These results will determine whether bisphenols impair learning and

memory processes and if effects are sexually differentiated.

3. To assess the effects of BPA and BPS exposure on dendritic spine density in the

hippocampus of both sexes.

• These results may correlate dendritic spine densities with learning and

memory processes in BPA- and BPS-exposed mice.

4. To characterize the concentrations of endogenous hormones in the context of

BPA and BPS exposure.

• These results will determine if BPA and/or BPS exposure influences

endogenous hormones which may correlate to potential behavioural

effects.

2 Chapter 2: A method of discreet Bisphenol A exposure in mice to prevent stress responses

2.1 Introduction

There is great consensus in the literature that stress is a potent modulator of cognitive function and, more specifically, learning and memory processes (Sandi &

Pinelo-Nava, 2007). One of the many ways to biochemically characterize stress is measuring circulating concentrations of glucocorticoids such as cortisol (corticosterone in rodents), which is synthesized from a series of P450 and oxidoreductase enzymatic conversions much alike androgens and estrogens. Under normal conditions, glucocorticoids provide negative feedback to the hypothalamus and to the anterior pituitary to inhibit the release of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH), attenuating the adrenal release of glucocorticoids and terminating the physiological stress response modulated by the hypothalamic pituitary adrenal (HPA) axis (Herman et al., 2016). However, when an individual experiences persistent psychological or emotional disturbance, a chronic physiological state of stress may manifest, where the negative feedback mechanism is dysregulated causing sustained HPA axis activation producing prolonged elevation of glucocorticoids and, thus, ample opportunity for glucocorticoids to bind and enable stress responses through glucocorticoid (GR) and mineralocorticoid receptors (MR) (Hanukoglu, 1992;

Herman et al., 2016). Aside from chronic stress often supressing the immune and reproductive systems, prolonged activation of GRs in the brain causes deficits in learning and memory (de Kloet et al., 1999; Lupien & McEwen, 1997; Wang et al,

2017).

Within stress sensitive brain regions such as the hippocampus, chronic stress may precipitate neuroinflammation, dendrite atrophy, and reduced dendritic spine and

synapse density often associated with behavioural implications (Chen et al., 2008;

Frank et al., 2012; Watanabe et al., 1992). For example, male rats subjected to chronic restraint stress demonstrate significant impairments in spatial memory when tested in a radial arm maze task which corresponded with significant CA3 dendritic atrophy when compared to unstressed controls (Luine et al., 1994). Similar effects are seen in hippocampal-dependent tasks such as the Morris water maze, Y-maze object recognition task, and object placement task (Conrad et al., 2017); however, these behavioural impairments as well as dendritic effects can often be reversed with time

(Luine et al., 1994). Stressful stimuli have also been shown to reduce hippocampal dendritic arborization, spine density, and cell proliferation in the DG (Gould et al., 1992;

Gould et al., 1998; Watanabe et al., 1992). Moreover, the decline in DG neurogenesis directly correlated with stress and depressive-like behaviours in mice (Mitra et al.,

2006). With stress capable of modulating hippocampal neurogenesis, LTP, and neuronal structure and function, it is evident that glucocorticoids can greatly impact learning and memory processes. Therefore, when investigating learning and memory behaviour in an animal model, it is crucial that potential stressors are not overlooked.

When investigating the neurobehavioural effects of bisphenols, the majority of the literature utilizes administration methods, surgical procedures, and behavioural paradigms that incorporate innate stressors. The most common methods for bisphenol exposure in animal models are subcutaneous injections or oral gavages (Richter et al.,

2007); and while these methods allow for administration of precise quantities, they introduce a restraint stress and/or injection stress (Benedetti et al., 2011; Gärtner et al.,

1980; Gong et al., 2015; Jones et al., 2016; Stuart & Robinson, 2015). Moreover, if

treatment is chronically administered in a stressful manner, it is possible – especially for hormone-sensitive endpoints – that stress may confound the study (Benedetti et al.,

2011; Gärtner et al., 1980; Jones et al., 2016; Stuart & Robinson, 2015). Although not well adopted, some researchers have chosen to introduce bisphenol exposure through diet or drinking water ad libitum; which removes the potential of administration stress however, assuring daily treatment doses are achieved may present its own unique set of logistical challenges. In addition to administration methods, most bisphenol studies that involve adult female animals tend to control for the estrus cycle through surgical procedures, specifically ovariectomy followed with hormone replacement via silastic capsules or injections (Strom et al., 2012). Undoubtedly, surgical procedures induce physical stress that can affect the structure and function of the hippocampus (Isaacs,

2019; Jacobsen et al., 2012). In fact, patients have reported memory, attention, and information processing deficits following major surgery (Steinmetz et al., 2017).

Moreover, in rodents, previous work conducted in our laboratory has demonstrated significant alterations in CA1 and CA3 dendritic structure 1- and 2-months post gonadectomy surgery (Isaacs, 2019). Therefore, if surgical procedures are introduced into the experimental design, proper controls are essential to ensure the effects of bisphenol exposure are not in combination with surgical stress. Similarly, extra care should be taken when considering behavioural paradigms since some tasks are innately more stressful than others. For example, the Morris water maze task will likely be more stressful than an object placement task yet, both tests evaluate hippocampus- dependent function (Harrison et al., 2009; Morris et al., 1986; Mumby et al., 2002). To completely abolish all potential stressors from experimental design is not feasible,

however greater care can be taken to ensure treatment effects are not confounded by stress especially when studying hippocampus-dependent behaviour given the stress- sensitivity that this brain region exhibits (Conrad et al., 2017; Sandi & Pinelo-Nava,

2007). Accordingly, the present experiment aimed to establish a non-invasive method, a dietary bolus, for bisphenol exposure in mice.

2.2 Materials and Methods

2.2.1 Animals and husbandry

Two cohorts – composed of 26 (total n=52) CD-1 mice aged 2-3 months old consisting of 13 males and 13 females – were acquired at different dates (cohort 1: 26 male and female mice acquired in August, cohort 2: 26 male and female mice acquired in January) from Charles River Laboratories (Kingston, NY, USA). Upon arrival, mice were housed in same-sex groups of 3 on a reversed light/dark cycle (12:12h, lights on at 2000h) at 23±2ºC. To minimize background exposure to bisphenols beyond treatment regimen, mice were housed in clear polyethylene cages (26cm × 16cm ×12cm) with corncob bedding, environmental enrichment (paper nesting material and polyethylene hut) with ad libitum access to bisphenol-free rodent chow pellets (14% Protein Rodent

Maintenance Diet, Harlan Teklad, WI) and tap water (held in high-temp polysulfone containers and BPA-free AISI 316 stainless steel drinking spouts). Animal care and experimental procedures were conducted in accordance with the Canadian Council on

Animal Care and approved by the University of Guelph’s Animal Care and Use

Committee.

2.2.2 Treatment Administration

Four days post-arrival mice were individually housed for the commencement of daily treatment; at this time bodyweight was measured. To reduce the quantity of potential stressors, the route of administration was voluntary oral consumption of either

1.5mg/kg bodyweight of vehicle [Nuts to You Organic Smooth Peanut Butter, packaged in bisphenol free material (Goodness Me, Guelph, ON, Canada)] or BPA mixed in vehicle [50ug/kg bodyweight of ≥99% BPA (Sigma-Aldrich, Oakville, ON, Canada)].

Treatments were supplied and stored in bisphenol-free packaging (i.e. glass containers). The vehicle dose was selected to provide a small quantity (40-60mg, dependent upon body weight) of peanut butter and the dose of BPA was selected to correspond with the U.S. Environmental Protection Agency safe daily limit for human exposure (EPA, 2010). Treatment was provided between the hours of 0830h to1200h in a clear, glass dish exclusive to each subject so that odor cues would not be transmitted.

To determine if peanut butter was palatable to mice and if voluntary oral consumption would be successful, both cohorts began with 8 days of vehicle treatment. Some mice did not consume their day 1 (4 males) and/or day 2 (2 males) treatment. From day 3 onwards, all mice were readily consuming their treatment. On day 9, all the mice in cohort 1 switched to BPA treatment; there were no neophobic reactions. The mice in cohort 2 continued with vehicle treatment. On day 9, both cohorts were weighed and necessary treatment adjustments based on 1.5mg/kg body weight/day of vehicle with or without 50ug/kg bodyweight of BPA determined by group were made to compensate for weight changes. See figure 2.1 for schematic of study design.

Figure 2.1: Visual representation of experimental design and sequence of events.

2.2.3 Object Placement Paradigm

The evening prior to behavioural testing, mice were moved into the testing room to acclimate. To establish home territory, cages were not cleaned for at least 3 days prior to testing. OP testing occurred on day 8 and 16 in the home cage with nesting material, environmental enrichment, food, and water removed. Since prior work suggests that BPA exposure in mice via oral bolus administration or dietary administration will reach peak serum concentrations at 1 hr and 6 hrs respectively (Sieli et al., 2011), we considered 4 hours post treatment to be appropriate for testing close to maximum circulating concentrations of BPA. The paradigm from Phan et al., 2012 was adapted by changing the quantity of habituation phases, phase duration, and rest phase intervals. This was to facilitate an “easy paradigm” where the control mice would demonstrate learning/memory, to test for impairing effects of BPA treatment. The paradigm consisted of 3 habituation sessions and a test, each 4 minutes in duration and separated by 3 minute rest intervals where objects where removed from the cage (figure

2.2). Habituation and test phases were recorded under red light, during the dark phase of the light cycle.

Figure 2.2: Object placement paradigm schematic. Paradigm began 4 hours (240mins) after administration and was completed within 4hr25mins (265mins) of administration. Two identical objects are introduced during habituations (H) 1, 2, and 3. In the test phase, one of these objects was moved to a novel location. Each phase was separated by a 3 minute rest interval where objects were removed. Selected objects and their locations were counterbalanced among the mice.

Two identical objects were attached via velcro 12-14cm apart on one of the two long-side walls of the home cage. During habituations, object locations were consistent whereas in the test phase, one of the objects was moved 12-14cm to a novel location, directly across from its placement during habituation. Objects used were plastic hairclips

(4 × 3 × 3 cm3) and glass cubes (4 × 4 × 4 cm3), which were tested to ensure no object type preference among mice (Phan et al., 2011). Objects were washed and dried for each mouse using both odorless detergent (Sparkleen 1; Fisher Scientific, Ottawa, ON,

Canada) and baking soda to ensure consistent novelty of stimuli and no lasting odor cues. Object selection, location for habituations, and novel location were counterbalanced between mice. Similarly, object selection and locations differed for each mouse during behavioural testing 1 (day 8) and 2 (day 16). Upon completion of the paradigm, nesting, environmental enrichment, food, and water were returned to the home cage. All the mice were weighed and vaginal smears were taken from the female mice. The mice were then returned to colony room.

2.2.4 Behavioural Data Analysis

Ten behaviours (Table 2.1) were recorded during the paradigm using The

Observer XT15 software (Noldus Information Technology, Wageningen, Netherlands) by a trained observer and later analyzed.

Table 2.1: A descriptive list of the behaviours collected and analyzed for the object placement paradigm.

Behaviour Description

Sniff Stimulus Active sniffing/nose twitching within 1-2mm of object

Bite Stimulus Biting object

Sit/Climb on Sitting/climbing on object with all four paws off the cage floor Stimulus Forepaws propelling bedding in posterior direction. May be object Dig oriented.

Bury Forepaws pushing bedding away from body in anterior direction. May be object oriented. Horizontal Walking, non-stimulus sniffing and exploration of the cage Exploration

Vertical Activity Rearing, both forepaws off the cage floor

Inactivity Includes behaviours such as sit, lay down, freeze, and sleep

Self-groom Grooming/scratch face and body

Strange repetitive behaviours (>3) such as spin turns, jumps, back Stereotypies flips, lid chews, and head shakes.

Due to the natural tendency for mice to explore novel stimuli over familiar stimuli, we calculated a percent investigation (PI) representative of investigative preference (PI

= × 100); where N is the time spent investigating the novel or displaced stimulus

(or during habituations, the stimulus that will be displaced) and F is the time spent investigating the familiar stimulus. The mean PI was calculated for habituations 1, 2, and 3 to reduce ambiguity that may result from small, random fluctuations in investigative behaviour (Phan et al., 2012, 2011). It was expected that during habituations, investigative behaviour would be evenly distributed between stimuli. It was predicted that if discrimination was present, the test PI will be statistically greater than the mean habituation PI; and thus a non-statistically significant result indicates non- discrimination (Phan et al., 2012). Animals excluded were those with total investigation durations less than 10 seconds (0 mice) during the test phase and outliers [>2SD ± mean; 4 female mice and 4 male mice (2 mice/treatment group)].

2.2.5 Brain, Tissue, and Blood Collection

On day 17 (24 hours after behavioural testing was completed which corresponded with 4 hours after the final treatment administration), bodyweight measurements were recorded and then mice were sacrificed via cervical dislocation followed by decapitation. Immediately following sacrifice, the following tissues/organs/samples were collected: brain, whole blood, ovaries, and vaginal cells.

The brains were removed and either (1) dissected in half along the rostro-caudal midline or (2) flash frozen in liquid nitrogen and stored at -80ºC as whole brain. The half brains, alternating left and right hemispheres to avoid lateralization effects, were either placed in Golgi Cox solution for morphological analysis (see section 2.2.8 for details) or flash frozen and stored at -80ºC for use in another study. Ovaries were extracted, immersion fixed, and stored at -80ºC for future analysis of corpus luteum if necessary. Trunk blood

was collected and centrifuged at 3000rpm for 10 minutes. Serum was extracted and stored at -80ºC for subsequent hormone analysis.

2.2.6 Golgi Staining and Tissue Processing

Golgi Cox solution [1% potassium dichromate (Fisher Scientific), 0.8% potassium chromate (Sigma-Aldrich), 1% mercuric chloride (Sigma-Aldrich) was prepared, filtered

(Whatman grade 1 filter paper; Fisher Scientific) and stored at room temperature in the dark until time of use (within 2 days of preparation). Following sacrifice, half brains were immediately placed in 20mL scintillation vials containing 18mL of Golgi Cox solution and stored for 28 days at room temperature protected from light exposure. They were then transferred to 30% sucrose 0.1M phosphate buffer (PB) for 48-72 hours at 4ºC.

Samples were then sliced at 300µm in 30% sucrose PB using a Leica VT1000S vibrating blade microtome (Leica Biosystems, Concord, ON, Canada). Free floating sections were then placed in 6% sucrose PB at 4ºC for 24 hours. Sections were sequentially processed by rocking at room temperature in 2% paraformaldehyde

(Sigma-Aldrich) for 15 minutes, ammonium hydroxide (Sigma-Aldrich) for 15 minutes, and Kodak Rapid Fixative A (Sigma-Aldrich) for 25 minutes with 5-minute Milli-Q washes between each processing step. Sections were float mounted onto gelatin- coated microscope slides and serially dehydrated in 50%, 75%, 95% ethanol for 2 minutes each followed by 2 separate steps of 100% ethanol and xylene for 5 minutes each. Then, slides were cover-slipped using Permount mounting medium (Fisher), left to dry for 24-48 hours and sealed with clear nail polish to prevent oxidation.

2.2.7 Dendritic Spines Microscopy

Golgi-stained sections were used to investigate dendritic spine density in

pyramidal neurons within the CA1 of the dorsal hippocampus (see figure 2.3). Neurons

were selected based on the following criteria: (1) neuron must be fully contained within

the specified hippocampal region; (2) basal and apical dendrites are neither broken nor

damaged; (3) neuron must be fully impregnated by the Golgi Cox staining; (4) relatively

isolated from surrounding neurons; and (5) dendrite is continuously traceable from the

cell body with at least 10µm of well-delineated dendrite. Selected neurons were then

imaged by an observer blind to treatments using Zeiss Axio Imager.D1 (Carl Zeiss

Canada Ltd., Toronto, ON, Canada). Three segments of the dendritic tree were

captured, proximal (10-30% out from the cell body), medial (40-60%), and distal (70-

90%) with 5-6 images of each segment per animal (n=7-11 animals/group). A second

observer blind to treatments analyzed the images using ImageJ software (version Fiji,

National Institutes of Health) and calculated dendritic spine density as the average

number of spines per 10µm length of dendrite. Then, the mean density per dendritic

segment (i.e. proximal, medial, distal) was calculated per mouse and statistically

evaluated for treatment and sex group differences.

Figure 2.3: Golgi Cox stained dorsal hippocampus section. (A) Golgi Cox stained mouse brain section near bregma -3.12mm, sections such as this were selected for morphological analysis of the hippocampus. (B) Dorsal hippocampus section indicating the 3 major sub-regions; CA1, CA3, and DG. Neurons from CA1 are imaged for morphological analysis. 58

2.2.8 Statistical Analysis

Statistical analysis was performed using SigmaPlot 10.0 (Systat Software Inc.,

San Jose, CA, United States) or SPSS statistics 27 (IBM, Armonk, NY, United States).

The limits of statistical significance were set at p < 0.05.

Prior to statistical analysis of OP data, outliers were removed and determined as >

2SD ± mean (8 mice). To reduce type I errors, α priori paired t-tests were planned in the statistical model to assess differences in PI between habituation and test phases within each treatment group, and α priori independent samples t-tests were planned to compare PI at test between treatment groups. Percent investigation data were divided by 100 and arscine transformed (figures represent raw PI data).

Total investigation duration and additional behaviours (Table 2.1) were separately analyzed for each sex using two-way repeated measures ANOVAs, phase as the repeated measure and treatment as the between groups factor. Body weight measurements were also analyzed using two-way repeated measures ANOVA, body weight as the repeated measure and treatment as the between groups factor. If

Mauchly’s test revealed a violation of the sphericity assumption (p <0.05), a

Greenhouse-Geisser correction was utilised. Post-hoc Bonferroni pairwise comparisons were employed to assess significant findings associated with phase effects. Post-hoc independent samples t-tests were employed to assess significant findings associated with treatment effects.

Dendritic spine data, which represented mean density per dendrite region per mouse, were analyzed using α priori independent samples t-tests to assess main effects

of treatment on spine density and spine length. If the Shapiro-Wilk normality test and/or

Levene’s equality of variances test were violated, a Mann-Whitney Rank Sum test was utilised.

2.3 Results

2.3.1 Object Placement Paradigm

BPA administration demonstrated a significant treatment effect in the object placement task of both male (figure 2.4A) and female mice (figure 2.6A). For males, an independent samples t-test revealed that BPA-treated mice had significantly lower percent investigations during test than vehicle-treated mice (t(21)=3.988, p<0.001).

Similarly for females, BPA-treated mice had significantly lower percent investigations during test than vehicle-treated mice (t(21)=2.131, p=0.045). Percent investigation was significantly higher at test than at habituation for male vehicle (t(10)=-17.651, p<0.001), female vehicle (t(10)=-6.167, p=<0.001), and female BPA-treated (t(11)=-5.349, p<0.001) mice, indicating they successfully performed the task whereas the male BPA- treated (t(11)=-1.124, p=0.285) mice could not. A significant effect of phase was demonstrated for total investigation duration in both males (F(2.151, 49.482)=5.148, p=0.008) and females (F(2.370, 54.519)=6.878, p=0.001). For each group, except the male BPA-treated mice, total investigation duration through habituation phases 1 to 3 decreased, and total investigation duration for the test phase increased (figure 2.4B and figure 2.6B). Post hoc pairwise comparisons using Bonferroni analyses in the male mice revealed that total investigation duration in habituation 1 significantly differed from habituation 3 (p=0.043) and test (p=0.003). Moreover, a significant interaction between phase and treatment was demonstrated (F(2.151, 49.482)=6.327, p=0.003) where the

male BPA group spent significantly more time investigating either object in habituation 2 compared to the vehicle group (t(14.22)=-2.650, p=0.019). In the females, post hoc pairwise comparisons using Bonferroni analyses revealed that total investigation duration in habituation 1 significantly differed from habituation 3 (p=0.043) and test

(p=0.003). Overall, there was no treatment effect on total investigation duration in either sex.

For the male mice, significant treatment effects were presented for a few of the object-oriented behaviours: BPA treatment decreased biting, burying, and digging responses towards the objects. Significant phase effects were also demonstrated for both directional and non-directional behaviour. A two-way repeated measure ANOVA demonstrated a significant effect of phase for the following behaviours: sitting (F(1.892,

41.622)=6.428, p=0.004) and burying (F(1.845,36.895)=5.291, p=0.011) the novel object, horizontal activity (F(3,66)=57.793, p<0.001), vertical activity (F(3,72)=5.977, p=0.001), inactivity (F(2.082, 43.722)=9.634, p<0.001), and self-grooming (F(1.381,

26.246)=7.519, p=0.006). For sitting on the objects, post hoc pairwise comparisons using Bonferroni analyses revealed that habituation 1 significantly differed from sitting on the novel object in test (p=0.04). For burying the objects, post hoc pairwise comparisons using Bonferroni analyses revealed that burying in habituation 2 was significantly higher than burying the novel object in test (p=0.048). Moreover, a significant interaction between phase and treatment was demonstrated (F(1.845,

36.895)=6.326, p=0.005) where the BPA group spent significantly less time burying the objects in habituation 2 compared to controls (t(20)=2.425, p=0.033). For horizontal activity, post hoc pairwise comparisons using Bonferroni analyses revealed that

habituation 1 was significantly higher than habituation 2 (p<0.001), habituation 3

(p<0.001), and test (p<0.001). For vertical activity, post hoc pairwise comparisons using

Bonferroni analyses revealed that habituation 1 was significantly higher than habituation

3 (p=0.048) and test (p=0.018). For inactivity, post hoc pairwise comparisons using

Bonferroni analyses revealed that habituation 1 was significantly less than habituation 3

(p=0.004) and test (p=0.024). For self-grooming, post hoc pairwise comparisons using

Bonferroni analyses revealed that habituation 1 and 2 were significantly less than habituation 3 (p=0.035) and test (p<0.001). Two-way repeated measures ANOVAs and post-hoc independent samples t-tests also demonstrated a significant effect of treatment for biting of the objects in habituation 1 where the BPA group exhibited the behaviour significantly less than controls (t(20)=2.863, p=0.014). Similarly, a significant treatment effect was observed for digging of the objects in habituation 1 (t(23)=3.499, p=0.003) and non-novel object in test (t(23)=2.397, p=0.035) where the BPA group exhibited the behaviour significantly less than controls.

For the female mice, no significant treatment effects were presented for directional or non-directional behaviours; however, significant phase effects were demonstrated. A two-way repeated measure ANOVA demonstrated a significant effect of phase for the following behaviours: sitting on the novel object (F(1.672,

38.457)=7.177, p=0.004), horizontal activity (F(1.867, 44.808)=36.585, p<0.001), vertical activity (F(2.177, 52.237)=3.846, p=0.025), inactivity (F(3, 63)=8.670, p<0.001), and self-grooming (F(1.762, 38.768)=3.858, p=0.034). For sitting on the objects, post hoc pairwise comparisons using Bonferroni analyses revealed that habituation 1 significantly differed from sitting on the novel object in test (p=0.028). For horizontal

activity, post hoc pairwise comparisons using Bonferroni analyses revealed that each consecutive phase exhibited significantly (p<0.001) less horizontal activity than the prior phase, except for habituation 3 and test (p=0.106). For vertical activity, post hoc pairwise comparisons using Bonferroni analyses revealed that habituation 2, which was similar in value to habituation 1 and 3, was significantly higher than test (p=0.021). For inactivity, post hoc pairwise comparisons using Bonferroni analyses revealed that habituation 1 exhibited significantly less inactivity than habituation 3 (p=0.001) and test

(p=0.013). For self-grooming, post hoc pairwise comparisons using Bonferroni analyses revealed that habituation 1 was significantly less than habituation 2 (p=0.041), and close to significantly less than test (p=0.07). The two-way repeated measure ANOVAs did not demonstrate significant treatment effects or phase × treatment interactions.

Figure 2.4: Effects of BPA on OP performance in gonadally intact male mice. (A) Bar graph illustrating the percent investigation for vehicle (n=11) and BPA-treated (n=12) male mice (light grey bars represent the average percent investigation of the three habituation phases ± SE ; dark grey bars represent the average percent investigation of the test phase± SE). BPA-treated mice did not discriminate between novel and familiar object locations which demonstrates memory impairment. **p < 0.001 (B) Line graph illustrating the mean total investigation duration of both stimuli in each habituation and test phase ± SE.

* **

Figure 2.5: Other non-directional and directional behaviours observed during the OP paradigm for gonadally intact male mice treated with vehicle or BPA. Data represented as mean ± SE. * p <0.05, ** p <0.005 BPA- vs. vehicle-treated mice for that phase of the paradigm.

Figure 2.6: Effects of BPA on OP performance in gonadally intact female mice. (A) Bar graph illustrating the percent investigation for vehicle (n=11) and BPA-treated (n=12) female mice (light grey bars represent the average percent investigation of the three habituation phases ± SE; dark grey bars represent the average percent investigation of the test phase ± SE). Both vehicle- and BPA-treated mice discriminated between novel and familiar object locations, signifying no memory impairments. * p <0.05, ** p < 0.001 (B) Line graph illustrating the mean total investigation duration of both stimuli in each habituation and test phase ± SE.

B 66

Figure 2.7: Other non-directional and directional behaviours observed during the OP paradigm for gonadally intact female mice treated with vehicle or BPA. Data represented as mean ± SE. No significant treatment effects.

2.3.2 Hippocampal Dendritic Spine Data

Independent samples t-tests, and Mann-Whitney Rank Sum tests used when the normality assumption was not met, demonstrate no significant treatment effects on dendritic spine density in the CA1 of male (distal: U=56.0, p=0.124; medial: t(16)=1.236, p=0.234; proximal: t(16)=0.540, p=0.597) and female (distal: t(14)=1.203, p=0.249; medial: t(13)=0.0145, p=0.989; proximal: U=27.0, p=0.786) gonadally intact mice, as shown in figure 2.9. Similarly, spine length data demonstrates no significant treatment effects in the CA1 of male (distal: t(15)= -0.132, p=0.897; medial: U=46.0, p=0.526; proximal: t(16)=1.673, p=0.114) and female (distal: t(14)= -0.801, p=0.436; medial: t(13)= 1.316, p=0.211; proximal: t(14)= -0.105, p=0.918) mice, as shown in figure 2.9.

Figure 2.8: Images of Golgi Cox stained secondary dendrites from hippocampal CA1 neurons from female and male mice treated with vehicle or BPA. Columns indicate sex and treatment: A – Vehicle treated female mice, B – BPA treated female mice, C – Vehicle treated male mice, and D – BPA treated male mice. Rows indicate location of secondary dendrite: distal (70-90% out from cell body), medial (40- 60%), or proximal (10-30%). Scale bar: 2µm

Vehicle BPA

Figure 2.9: Hippocampal CA1 dendritic spine density and spine length of gonadally intact male and female mice. No significant differences are demonstrated in spine density or spine length between vehicle- and BPA-treated mice of either sex. Light and dark grey bars represent vehicle and BPA groups respectively. Bars represent mean ± SE.

2.3.3 Vaginal Cytology

Vaginal cells were collected on day 8, 16, and 17. The majority of female mice in each treatment group presented in the estrus phase of the cycle for each collection date. The proportion of mice in each phase of the estrus cycle is shown in table 2.2.

Table 2.2: Vaginal cytology prior to BPA administration (day 8), as well as at time of behavioural testing (day 16) and tissue collection (day 17).

Stage of Day 16: Behavioural Day 17: Tissue Day 8 Cycle Testing Collection

Vehicle crossing Vehicle Vehicle BPA Vehicle BPA over to BPA

Diestrus 3 0 3 1 1 4

Proestrus 1 5 1 1 3 4

Estrus 6 8 6 11 9 5

Metestrus 3 1 3 1 0 1

2.3.4 Body Weight Measurements

A two-way repeated measures ANOVA revealed no statistically significant effects or interactions between body weight and treatment for the male mice (Weight: F(1.395,

29.293)=1.888, p=0.082; Interaction: F(1.395, 29.293)=2.604, p=0.107). In contrast, a two-way repeated measures ANOVA revealed a significant increase in weight across time regardless of treatment effect for the female mice (Weight: F(1.451,

36.283)=17.182, p<0.001; Interaction: F(1.451, 36.283)=1.607, p=0.217). Post hoc

Bonferroni pairwise comparisons for the female mice reveal that body weight

measurements on day 17 are significantly higher than day 1 (p<0.001) and day 8

(p<0.001), see figure 2.10.

Figure 2.10: Body weight measurements across time for male and female mice in vehicle and BPA groups. BPA treatment begun on day 9, from day 1 to 8 all mice received vehicle to acclimate to the route of administration. Male mice do not exhibit any significant changes in body weight. Both vehicle and BPA exposed female mice demonstrate a significant increase in body weight. All data points are represented as mean ± SEM. n= 13-14 mice/group. ** p < 0.001 versus day 1 and 8 body weight measurements within group.

2.4 Discussion

The purpose of this experiment was to evaluate the impact bisphenol A exposure may elicit on learning and memory as well as hippocampal dendritic spine densities, while minimizing potential stress responses. The findings from this study demonstrated that BPA exposure impacts spatial memory in a sex-specific manner and does so irrespective of hippocampal CA1 dendritic spine densities.

2.4.1 Effects of BPA Exposure on Spatial Memory

BPA exposure impaired spatial memory in a sex-specific manner. In male mice, 8 days of BPA exposure resulted in impaired memory performance in the home cage object placement paradigm. This finding is consistent with prior work in adult male rats demonstrating BPA exposure at a dose of 50µg/kg bw/day impairing memory performance in OP and OR (Eilam-Stock et al., 2012) as well as in the Morris Water maze task (Fan et al., 2013). In contrast, in female mice, 8 days of BPA exposure did not result in impaired memory performance in the home cage object placement task; signified by a statistically significant increase in PI from habituation to test. However, there was a significant treatment effect where the vehicle-treated mice outperformed the

BPA-treated mice, indicating reduced task performance. Consistent with our findings,

Xu and colleagues (2015b) reported that sham-operated, cycling female mice exposed to 40 or 400µg/kg bw/day of BPA do not exhibit spatial memory impairments in the

Morris Water maze task compared to their vehicle counterparts. Additionally, OVX induced significant impairments in task performance which were rescued with OVX +

EB. Interestingly, OVX + BPA eliminated the difference between cycling females and

OVX females whereas co-treatment of BPA (400µg/kg bw/day) + EB in OVX females

partially eliminated the rescue effect of EB (Xu et al., 2015b). This suggests that BPA may improve learning and memory deficits caused by estrogen deficiency. However, since EB and BPA co-administration in OVX females partially eliminated the rescue effect of EB, BPA may act as an antagonist or a competitive agonist, thus reducing the potency of EB’s estrogenic effects. Importantly, due to BPA exposure demonstrating no effect in cycling females – which present estrus cycle fluctuations in E2, progesterone, and testosterone (Nilsson et al., 2015) – it is suggested that BPA’s effects may be influenced by circulating hormone levels. This offers support to the current study where

BPA treatment did not impact learning and memory suggesting that the endogenous hormonal background in gonadally intact cycling females may protect against potential antagonistic actions of BPA. Altogether, BPA treatment impaired memory performance in a sex-specific manner where males are impaired and females are not; however, female mice demonstrated reduced task performance when compared to their vehicle counterparts suggesting that behaviour independent from learning and memory processes may be influenced.

In addition to object investigation, we analyzed several other behaviours during the habituation and testing phases, both directional (stimuli oriented) and non-directional.

Importantly, we found no significant effects of BPA treatment on total investigation duration, horizontal activity, vertical activity, or inactivity in male or female mice which suggests that the results were not a consequence of non-specific effects of BPA exposure on locomotion. Moreover, BPA-treated female mice did not exhibit any statistically significant differences in non-directional or directional behaviours when compared to their vehicle-treated counterparts. Yet, BPA treatment in females produced

a significant reduction in task performance without impairing memory; therefore, there may be a behavioural effect that did not reach statistical significance especially since the total duration of behaviours such as biting, burying, and digging demonstrate differing trends when comparing BPA- and vehicle-treated female mice across paradigm phases. In contrast, male mice demonstrated significant phase × treatment interactions for object-oriented biting and digging in habituation 1 as well as object-oriented burying in habituation 2, where BPA treatment decreased said behaviours. As biting and digging/burying responses may indicate aggression and anxiety-like behaviour respectively (Broekkamp et al.,1986; Deacon, 2006; Kuchiiwa & Kuchiiwa, 2014; Miczek et al., 2001), our results suggest that BPA exposure may elicit an anxiolytic-like response in gonadally intact adult male mice in comparison to their vehicle counterparts when foreign objects are encountered in home territory. Yet, this speculation contrasts prior work which demonstrates BPA exposure exhibiting a J-shape curve with aggression and a U-shape curve with anxiety-like behaviour; more specifically, low doses of BPA treatment in a developmental model increased aggression and anxiety- like behaviour in rodents (Richter et al., 2007). While a large portion of the literature focuses on BPA exposure during developmental periods, Xu and colleagues (2015a) addressed exposure in adulthood and reported that BPA exposure at 40 and 400 µg/kg bw/day via oral gavage causes anxiety- and depressive-like behaviour in adult male mice by use of an open-field test, elevated plus maze task, and forced swim test.

Additionally, the BPA exposed female mice were either unaffected or exhibited behaviours inversely related to the males, thus eliminating or reversing the sex differences of these emotional behaviours (Xu et al., 2015a). Notably, the chosen route

of administration, oral gavage, is inherently more stressful as a physical restraint is required (Gärtner et al., 1980; Gong et al., 2015; Stuart & Robinson, 2015). Therefore it is possible – especially for hormone-sensitive endpoints – that stress may confound the

Xu et al (2015a) study. Overall, with contrasting results to prior work and less research conducted in adult mice without hormone manipulation, the underlying reasons behind said object-oriented behaviours potentially expressing an anxiolytic-like phenotype with

BPA exposure are presently unknown; highlighting an area of future investigation.

Our results also demonstrate phase effects on total investigation and other directional and non-directional behaviours. Overall, the subjects demonstrated higher total investigation and active behaviours (i.e. horizontal and vertical activity) during the earlier habituation phases which paralleled the opposite increase in inactive behaviours

(i.e. inactivity and self-grooming) during later paradigm phases. This inverse relationship between active and inactive behaviours is common in this paradigm and considered to result from novelty surrounding experimental conditions (Kunn, 2021; Phan et al., 2012).

Importantly, these phase effects were consistent across treatment groups and thus, do not impact the interpretation of BPA’s effects on memory performance and other behaviours.

Interestingly, spatial memory tasks are often sexually differentiated where the males outperform their female counterparts (Chen et al., 2020), yet our vehicle males and females are very comparable. This finding may reflect the easiness of the rapid learning paradigm employed. In the present study, there is a brief (3 minute) delay between habituation and test phase. With brief delays (< 1 hour), studies employing spatial tasks typically do not demonstrate sex differences as the protocol emphasizes acquisition

(Cost et al., 2012) whereas, longer delays (> 1 hour) allow sex differences to precipitate as the protocol emphasizes differences in memory consolidation and storage (Bowman,

2005; Labuda et al., 2002). Another sex difference is that stress differentially impacts performance in spatial tasks where stress exposed females can outperform their male counterparts and thus eliminating, or even reversing, prior sex differences in spatial task performance (Bowman, 2005). This highlights an interesting approach to understanding

BPA. Under the hypothesis that BPA inflicts a physiological state indicative of stress, we would expect BPA treatment to mimic the stress-induced differences in cognitive function; where stress exposed female mice outperform their male counterparts

(Bowman, 2005). The present study demonstrated memory deficits in BPA-treated males but not BPA-treated females indicating that effects of BPA treatment may parallel spatial task performance sex differences seen in stress-exposed mice. Importantly, this study did not include a “stressed” control group for comparison nor did we determine the circulating levels of corticosterone which limits the associations and subsequent conclusions that we can draw about stress responses.

2.4.2 Effects of BPA Exposure on Dendritic Spines

Our results demonstrate that BPA exposure does not impact CA1 dendritic spine densities in gonadally intact adult male or female mice.

Prior work in male mice is inconsistent with our findings, however; these differences may result from experimental design. For example, Eilam-Stock and colleagues (2012) associated memory deficits with a 10-25% reduction in dendritic spine densities in the

CA1 when memory consolidation was involved. Although our control animals exhibit spine densities that are consistent in absolute value to Eilam-Stock (2012), our study

does not demonstrate a reduction in spine density with BPA treatment nor does the experimental design involve memory consolidation. Eilam-Stock and colleagues (2012) administered BPA post-trial during memory consolidation and collected brain tissue within 40 minutes of BPA exposure suggesting that BPA can rapidly effect spine densities correlated to memory consolidation. In contrast, our study collected brain tissue 24 hours post-behavioural testing and 4 hours after the final daily dose of BPA treatment. Importantly, the findings from a follow up study by Eilam and colleagues investigating the effects of BPA administration on CA1 dendritic spine density without memory consolidation agrees with our findings. They reported that brain tissue collected

4 hours after acute BPA exposure does not exhibit a treatment effect (Eilam-Stock et al., 2012). Altogether chronic BPA exposure in adult male mice elicits memory deficits that may not be associated with lasting effects on CA1 dendritic spine densities since

BPA administration and collection of brain tissue in a rapid or delayed time course offers contrasting results.

Similarly to the males, our results demonstrate that BPA exposure does not impact

CA1 dendritic spine densities in gonadally intact adult female mice. This finding agrees with the study by Xu and colleagues (2013), who found no significant differences in synapse density of cycling mice 3 days post BPA exposure compared to controls.

However, unlike the present study, Xu and colleagues (2013) administered BPA at a high dose (400µg/kg bw/day for 12 weeks) via oral gavage which is a route of administration that tends to provoke physiological stress responses in mice (Benedetti et el., 2011; Jones et al., 2016; Walker et al., 2012). Importantly, corticosterone and/or it’s metabolites were not analyzed by Xu an colleagues (2013), and thus, conclusions

surrounding potential stress responses cannot be made. Simultaneously, tissue extraction occurred 3 days after the final oral gavage of BPA, potentially allowing stress responses associated with the procedure to dissipate (Xu et al., 2013). Overall, the present study addressed these limitations by administering a low dose of BPA in a non- stressful manner which yielded further evidence that BPA exposure does not influence

CA1 dendritic spine density in cycling mice. Moreover, given the dose differences between these two studies, the similar findings may indicate an inverted U-shape response to BPA which is a common dose-response relationship for hormones (Bayer et al., 2018; Sandi & Pinelo-Nava, 2007). Importantly, findings in OVX rodents are inconsistent with our results; BPA exposure in OVX rodents with and without E2 co- treatment demonstrates reduced hippocampal dendritic spine densities (Inagaki et al.,

2012; MacLusky et al., 2005a; Xu et al., 2013). Potential explanations for this disparity are (1) the ability for BPA to antagonize synaptic remodelling may depend on the circulating concentrations of endogenous hormone, and (2) surgical stress may be a confounding variable. In adult rodents, OVX has been shown to produce significant reductions in dendritic spine density and quantity of spine synapses in the hippocampus and PFC (Gould et al., 1990; Wallace et al., 2006). Importantly, these findings can be mitigated with E2 or E2+Progesterone treatment (Gould et al., 1990). However, Nilsson and colleagues (2015) demonstrated that gonadally intact adult rats and their OVX+E2 counterparts significantly differ in their quantity of estradiol, testosterone, DHT, and progesterone; indicating that OVX+E2 may not represent an appropriate model for cycling female rodents. Therefore, with differing hormonal backgrounds, the ability for

BPA to reduce hippocampal dendritic spine densities in OVX+E2 may result from

interactions with differing E2 levels associated with hormone replacement. Additionally, even though progesterone and testosterone levels fluctuate throughout the estrus cycle

(Nilsson et al., 2015), such hormone replacement is commonly absent in OVX models; again suggesting a lack of validity when comparing gonadally intact adult rats to their

OVX counterparts. Furthermore, there is an alternative explanation for said reductions in synapse and spine density following OVX, that being the stress of surgery. Previous work conducted in our laboratory has demonstrated significant remodeling in CA1 and

CA3 dendritic structure 1- and 2-months post sham-surgery in male adult rats (Isaacs,

2019). Moreover, prolonged glucocorticoid treatment – in the absence of surgical stress

– partially reflected the hippocampal remodelling demonstrated in sham-surgery rats

(Isaacs, 2019). Therefore, it is likely that glucocorticoids are elevated post-surgery and contribute to changes presented in hippocampal dendrites. Altogether, comparisons between OVX and gonadally intact cycling rodents lack validity. Therefore, while our results indicate that BPA may not impact CA1 dendritic spine density in cycling females, it is possible that experimental manipulation (i.e. OVX) of endogenous hormones yields an environment conducive for BPA’s effects to prevail.

While our dendritic spine findings are supported by prior work, the data possesses limitations which hinders the comprehensiveness of available conclusions. To start, only hippocampal CA1 pyramidal cell spine densities were investigated and thus it is possible that dendritic spine densities are influenced in other brain regions such as the

PFC. Additionally, it is difficult to concretely discern the effect of BPA on spine densities, in both males and females, without analysis of dendritic arborization. BPA may influence dendrite length and/or branching patterns which may alter spine distribution. Therefore,

without full morphological analysis we cannot conclusively determine whether hippocampal spine densities are impacted by bisphenols and even so, dendritic spines densities may be influenced elsewhere in the brain.

2.4.3 Significance of Work

The body of work presented in this section contributes to the understanding of how chronic BPA exposure impacts spatial memory and dendritic spine densities in gonadally intact adult male and female mice when experimental conditions attentively minimize potential stress responses. Prior work has investigated the effects of BPA on hippocampal-dependent learning and memory tasks, however; a large emphasis has been placed on critical time periods such as gestational development and puberty.

Additionally, prior work often incorporated experimental conditions that are innately stressful. Therefore, our results add to a growing body of literature attempting to understand the effects and mechanisms of BPA action in adulthood. Moreover, this work highlights that BPA exposure may influence behaviour in a sex-specific manner irrespective of hippocampal CA1 dendritic spine densities when endogenous hormones are not manipulated by experimental conditions (i.e. ORX/OVX, hormone replacements, stress-inducing paradigms and treatment administration).

3 Chapter 3: The effects of Bisphenol S exposure on spatial memory and endogenous hormones in gonadally intact male and female mice

3.1 Introduction

BPA is an industrial chemical extensively used in the production of polycarbonate plastics and epoxy resins which are incorporated into numerous consumer products and thus results in sustained low levels of human exposure (Lehmler et al., 2018; Vaughn,

2010). BPA has been a large concern for decades due to its endocrine disrupting properties, including effects on metabolic processes, immune system, reproductive system, and most importantly for the experiment presented herein, the central nervous system (Richter et al., 2007). Low dose BPA exposure attenuates spatial – which is hippocampal-dependent (Morris et al., 1986; Mumby et al., 2002) – and non-spatial memory performance (Eilam-Stock et al., 2012; Inagaki et al., 2012), while also reducing the neuroplastic effects of sex steroids in the brain, particularly in the hippocampus (Inagaki et al., 2012; Leranth et al., 2008; MacLusky et al., 2005a). These effects have prompted replacement of BPA with structurally-related chemicals, such as

BPS (Chen et al., 2016). However, whether BPS is actually safer is an open question because its biological effects have not been fully explored. Environmental and biological evidence indicates that BPS exposure is increasing among human and animal populations (Chen et al., 2016; Lehmler et al., 2018). Yet, research surrounding the neurobehavioural effects of BPS exposure is still in its infancy. Available in vitro studies indicate that BPS has the potential to be an EDC as it exhibits acute toxicity, immunotoxicity, reproductive toxicity, and neurotoxicity (Qui et al., 2019). Moreover,

BPS demonstrated similar human nuclear receptor binding assays to BPA (Kojima et al., 2019). With respect to in vivo studies, there are presently 7 rodent studies that have investigated the neurobehavioral effects elicited by BPS exposure; all of which utilised a

developmental model (prenatal and/or postnatal exposure) (Naderi & Kwong, 2020).

Collectively, the studies indicate that BPS exposure during critical development periods may elicit adverse effects on feeding behaviour (da Silva et al., 2019; LaPlante et al.,

2017; Rezg et al., 2018), social behaviour (Kim et al., 2015), locomotion (da Silva et al.,

2019; Kim et al., 2015), and anxiety-like behaviour (da Silva et al., 2019; Kim et al.,

2015; Mornagui et al., 2019), as well as impair maternal care (Catanese & Vandengerg,

2017; LaPlante et al., 2017; Shi et al., 2019). Therefore, given BPS demonstrates (1) structural similarity to BPA; (2) similar human nuclear receptor binding assays to BPA; and (3) adverse neurobehavioural effects in the developmental model, it is hypothesized that BPS will impair learning and memory processes in a magnitude similar to that of

BPA. Thus, the present experiment aimed to directly compare the effects of BPA and

BPS exposure on learning and memory performance – specifically hippocampal- dependent spatial memory – hippocampal microstructure, and endogenous hormone concentrations in gonadally intact adult mice; based on the “stress-free” method produced in chapter 2.

3.2 Materials and Methods

3.2.1 Animals and husbandry

Five cohorts – each composed of 18 CD-1 mice aged 2-3 months old consisting of 9 males and 9 females – were acquired in successive dates from Charles River

Laboratories (Kingston, NY, USA). Housing conditions and procedures for the mice were the same as described in chapter 2.

3.2.2 Treatment Administration

Administration occurred in the same manner as described in chapter 2 with the

following exceptions: (1) treatment (day 1) began 6 days post arrival; (2) daily, voluntary

oral consumption occurred from 830h-1230h, with an individual mouse receiving

treatment at the same time every day; (3) treatment groups were 1.5mg/kg bodyweight

of vehicle [Nuts to You Organic Smooth Peanut Butter (Goodness Me, Guelph, ON,

Canada)], BPA mixed in vehicle [50µg/kg bodyweight of ≥99% BPA (Sigma-Aldrich,

Oakville, ON, Canada)], or BPS mixed in vehicle [50µg/kg bodyweight of ≥99% BPS

(Sigma-Aldrich, Oakville, ON, Canada)]. While a regulatory TDI for BPS is not yet

established (Eladak et al., 2015), the selected dose of 50µg/kg bw/d was chosen to

correspond with the U.S TDI for BPA (50µg/kg bw/d) (EPA, 2010). Each cohort had 3

male and 3 female mice per treatment (n=15). See figure 3.1 for visual representation of

study design.

Figure 3.1: Design and sequence of events for experiment 2.

3.2.3 Object Placement Paradigm

Behavioural testing was conducted as previously described in chapter 2 with the following exception: testing occurred on day 9. An additional treatment day was added prior to behavioural testing so that each mouse received at least 7 days and maximum 9 days of treatment before testing. This is because chapter 2 revealed inter-subject variability in willingness to consume treatment on day 1 and 2. Similar to chapter 2, some mice did not consume their day 1 (24/90 mice – males: 10 VEH, 4 BPA, 4 BPS; females: 2 VEH, 2 BPA, 2 BPS) and/or day 2 (6/90 mice – males: 2 VEH, 2 BPS; females: 2 BPS) treatment however, by day 3 all mice were readily consuming their treatment. Analysis of behavioural data occurred in the same manner as described in chapter 2. Animals excluded were those with total investigation durations less than 10 seconds (0 mice) during the test phase and outliers determined as >2SD ± mean

(males: 3 VEH, 2 BPA, and 3 BPS; females: 3 VEH, 0 BPA, 1 BPS).

3.2.4 Brain, Tissue, and Blood Collection

Animals were sacrificed on day 10 (24 hours after behavioural testing was completed which corresponded with 4 hours after the final treatment administration) and sample collection occurred in the same manner as previously described in chapter 2. In cohorts 1-4, brains were extracted and dissected in half along the rostro-caudal midline with alternating left and right hemispheres utilized for dendritic spine analysis following

Golgi Cox staining and processing (see section 2.2.8 for details) and western blot analysis in another study. Moreover, reproductive organs (ovaries, uterus, testes, and prostate) and trunk blood were collected for analysis of wet weight and hormone

concentrations respectively. Cohort 5 mice were perfused with whole brains and ovaries collected for use in another study.

3.2.5 Quantification of Hormone Concentrations

Serum samples collected from each animal at the time of sacrifice were analyzed with a Mouse/Rat/Human/Baboon corticosterone competitive enzyme-linked immunosorbent assay (ELISA; ab 108821, Abcam, Cambridge, MA, USA) to determine circulating levels of corticosterone in each treatment group. Male serum samples collected from each animal at the time of sacrifice were also analyzed with a

Mouse/Rat/Human Testosterone competitive enzyme-linked immunosorbent assay

(ELISA; ab 108666, Abcam) to determine circulating levels of testosterone in each male treatment group. All samples and standard curves were conducted according to manufacturer’s instructions and read with absorbance set to 450nm on a SynergyTMHT

Multi-Detection Microplate Reader (BioTek Instruments Inc., Winooski, Vermont, USA) with Gen5TM software (BioTek). Mean absorbance values were calculated and hormone concentrations (ng/mL) were determined from the standard curve.

3.2.6 Statistical Analysis

The main statistical model was performed using SPSS statistics 27 (IBM), and α priori tests with SigmaPlot 10.0 (Systat Software Inc.). The limits of statistical significance set at p < 0.05, and data represented as mean ± SEM.

Prior to statistical analysis of behavioural data, outliers were removed and determined as >2SD ± mean (8 males and 4 females). The percent investigation data were divided by 100 and arcsine transformed to correct for non-homogeneity of

variance, thus, Levene’s Test of Equality of Error of Variance was able to be met for all cases. To analyze percent investigations, repeated measures ANOVAs were performed with phase as the repeated measure, and treatment as a between groups factor. To reduce type I errors, α priori paired t-tests were used to assess differences in percent investigation between mean habituation and test phase within each treatment group.

Furthermore, priori one-way ANOVAs were used to compare percent investigation at test between treatment groups followed by Tukey’s test for an all pairwise multiple comparison. Finally, PI data were analyzed using a two-way ANOVA to assess main effects of sex and treatment.

Additional behaviors (Table 2.1) were analyzed using two-way repeated measures

ANOVAs, phase as the repeated measure, and treatment as the between groups factor.

If Mauchly’s test for the two-way repeated measures ANOVA revealed that the sphericity assumption was violated (p<0.05), a Greenhouse-Geisser correction was utilised. Bonferroni post hoc tests were used to assess significant findings for the within- subjects factor, phase. Tukey post hoc tests were employed to assess significant findings for the between-subjects factor, treatment.

Reproductive tissue data were analyzed using one way ANOVA. If the Shapiro-

Wilk normality test or Levene’s equality of variances test were violated, a Kruskal-Wallis one way ANOVA on Ranks test was utilised. Body weight data were analyzed using two-way repeated measures ANOVAs, body weight as the repeated measure, and treatment as the between groups factor.

To determine effects of treatment on serum testosterone and corticosterone levels, data were reciprocally transformed – when necessary – and analyzed using two way ANOVAs, treatment and sex as the between group factors.

3.3 Results

3.3.1 Object Placement Paradigm

BPS and BPA administration demonstrated a significant treatment effect on percent investigation in the object placement task of both males (figure 3.2A, p=0.005) and females (figure 3.4A, p<0.001) with a nonsignificant effect of sex (p=0.967).

3.3.1.1 OP – Male Mice

For males, the repeated measures ANOVA revealed a main effect of Phase

(F(1,34)=82.604, p<0.001), a main effect of Treatment (F(2,34)=6.265, p=0.005), and an interaction between Phase and Treatment (F(2,34)=8.619, p=0.001) on PI. A one- way ANOVA with post hoc Tukey analysis demonstrated a significantly lower PI during test in BPA (p=0.003) and BPS (p<0.001) treated mice when compared to vehicle- treated mice. A priori binary mean comparisons, paired t-tests, demonstrated significantly higher percent investigation at test than at habituation for vehicle treated mice (t(11)=-9.702, p<0.001), BPA-treated mice (t(12) =-3.995, p=0.002), and BPS- treated mice (t(11)=-3.167, p=0.009). This indicates that each group of males successfully completed the paradigm even though treatment dependent reductions in

PIs are evident.

There were no effects of treatment on total investigation times (total time spent investigating either stimuli) as shown in figure 3.2B as well as on other directional

behaviours (digging, burying, biting, sitting) and most non-directional behaviours

(vertical activity, grooming, digging, burying) – with the exception of horizontal activity and inactivity (see figure 3.3). A significant treatment × sex interaction was also demonstrated for horizontal activity (p<0.001) and inactivity (p=0.045). Collectively, this suggests that the aforesaid results may be influenced by treatment effects on locomotion.

A two-way repeated measures ANOVA demonstrated a significant effect of

Phase and Treatment for horizontal activity (phase: F(3,114)= 97.827, p<0.001; treatment: F(2,38)=6.150, p=0.005) and inactivity (phase: F(1.2.136,74.754)=35.712, p<0.001; treatment: F(2,35)=7.362, p=0.002). Mauchly’s test for the two-way repeated measures ANOVA revealed that the sphericity assumption was violated for inactivity

(p<0.05) and thus, a Greenhouse-Geisser correction was utilised. Post hoc Tukey tests revealed that horizontal activity was significantly higher during habituation 2 (p=0.016) and at test (p=0.001) in the BPA mice when compared to control mice. Although nonsignificant, BPS mice demonstrated similar trends. Inactivity was significantly lower during habituation 1 (p=0.003), and 3 (p=0.050) in BPS-treated mice compared to controls, as well as in habituation 3 (p=0.025), and during test (p<0.05) in BPA-treated mice compared to controls.

A significant main effect of Phase was found for total investigation

(F(3,108)=31.77, p<0.001); post hoc Bonferroni analyses revealed that total time spent investigating either objects was significantly higher (p<0.001) during habituation 1 than in habituation 2, habituation 3, or test. A significant main effect of Phase was found for horizontal activity (F(3,114)=97.827, p<0.001); post hoc pairwise comparison using

Bonferroni analyses revealed that habituation 1 was significantly higher than habituation

2 (p<0.001), habituation 3 (p<0.001) and test (p<0.001). Furthermore, habituation 2 was significantly higher than habituation 3 (p<0.001) and test (p<0.001). A significant main effect of Phase was found for vertical activity (F(3,114)=9.296, p<0.001); post hoc pairwise comparison using Bonferroni analyses revealed that habituation 1 and habituation 2 were significantly higher than test (p<0.001). A significant main effect of

Phase was found for inactivity (F(2.14,74.75)=35.712, p<0.001); post hoc pairwise comparison using Bonferroni analyses revealed that habituation 1 was significantly lower than habituation 2 (p=0.009), habituation 3 (p<0.001) and test (p<0.001).

Furthermore, habituation 2 was significantly lower than habituation 3 (p<0.001) and test

(p<0.001). A significant main effect of Phase was found for self-grooming (F(1.44,

58.93)=7.286, p=0.004); post hoc pairwise comparison using Bonferroni analyses revealed that habituation 1 was significantly lower than habituation 2 (p=0.001), habituation 3 (p=0.004) and test (p=0.013). A significant main effect of Phase was found for biting (F(2.34, 81.92)=3.571, p=0.026); post hoc pairwise comparison using

Bonferroni analyses revealed that test was significantly higher than habituation 1

(p=0.041) and habituation 3 (p=0.05). A significant main effect of Phase was found for non-directional bury (F(3,108)=6.762, p<0.001); post hoc pairwise comparison using

Bonferroni analyses revealed that habituation 1 was significantly lower than habituation

2 (p=0.015), habituation 3 (p=0.005) and test (p=0.002). A significant main effect of

Phase was found for directional digging (F(2.34, 84.06)=4.316, p=0.012); post hoc pairwise comparison using Bonferroni analyses revealed that habituation 1 was significantly lower than habituation 2 (p=0.003) and habituation 3 (p=0.014). A

significant main effect of Phase was found for sitting on novel object

(F(2.37,85.27)=7.629, p<0.001) and non-novel object(F(3,108)=3.168, p=0.027); post hoc pairwise comparison using Bonferroni analyses revealed that time spent sitting on the objects was significantly higher in habituation 2 and test than habituation 1 and habituation 3 (p=0.025); all other pairwise comparisons did not significantly differ.

Figure 3.2: Effects of BPS and BPA on object placement performance in gonadally intact male mice. (A) Bar graph illustrating the percent investigation for vehicle (n=12), BPA-treated (n=13), and BPS-treated (n=12) male mice (light grey bars represent the average percent investigation of the three habituation phases ± SE; dark grey bars represent the average percent investigation of the test phase ± SE). Within group differences, between habituation and test phase, are significant; indicating recognition of novel object placement. * p=0.003 ** p<0.001 versus vehicle group. # p<0.05 ## p<0.001 habituation phase versus test phase within group. (B) Line graph illustrating the mean total investigation duration of both stimuli in each habituation and test phase ± SE. 93

* **

Figure 3 3: Other non-directional and directional behaviours observed during the OP paradigm for gonadally intact male mice. Data represented as mean ± SE. * p≤0.05, ** p≤0.001 BPA- vs. vehicle-treated mice for that phase of the paradigm. # p≤0.05, ## p≤0.001 BPS- vs. vehicle-treated mice for that phase of the paradigm.

3.3.1.2 OP – Female Mice

For females, the repeated measures ANOVA revealed a main effect of Phase

(F(1,38)=110.091, p<0.001), a main effect of Treatment (F(2,38)=12.297, p<0.001), and an interaction between Phase and Treatment (F(2,38)=13.803, p<0.001) on PI. A one- way ANOVA with post-hoc Tukey analysis demonstrated a significantly lower PI during test in BPA (p<0.001) and BPS (p<0.001) treated mice when compared to vehicle- treated mice. A priori binary mean comparisons, paired t-tests, demonstrated a significantly higher percent investigation at test than at habituation [vehicle (t(11)=-

14.005, p<0.001), BPA (t(14)=-2.165, p=0.0048), and BPS (t(13)=-6.182, p<0.001) groups]; indicating that each group of females successfully completed the paradigm even though treatment dependent reductions in PIs are evident.

There were no effects of treatment on total investigation times (total time spent investigating either stimuli) as shown in figure 3.4B as well as on other directional behaviours (digging, burying, biting, sitting) and non-directional behaviours (vertical activity, horizontal activity, inactivity, grooming, digging, burying; see figure 3.5). This suggests that the aforesaid results were not caused by treatment effects on directional and non-directional behaviours. However, stereotypy was a behaviour exclusively exhibited in the bisphenol treated female mice, 20% of the BPA-treated and 6.7% of the

BPS-treated mice. Post hoc Pearson Chi-square analysis revealed a nonsignificant association between treatment and stereotypy expression (X(2)=3.841, p=0.146) with moderate association strength signified by Cramer’s V=0.292.

A significant main effect of Phase was found for total investigation

(F(3,123)=14.183, p<0.001); post hoc Bonferroni analyses revealed that total time spent

investigating either objects was significantly higher (p<0.001) during habituation 1 than habituation 2 (p<0.001), habituation 3 (p=0.002), or test (p=0.007). A significant main effect of Phase was found for horizontal activity (F(3,105)=80.002, p<0.001); post hoc pairwise comparison using Bonferroni analyses revealed that habituation 1 was significantly higher than habituation 2 (p<0.001), habituation 3 (p<0.001) and test

(p<0.001). Furthermore, habituation 2 was significantly higher than habituation 3

(p<0.001) and test (p<0.001); and habituation 3 was significantly higher than test

(p=0.015). A significant main effect of Phase was found for vertical activity

(F(3,108)=16.240, p<0.001); post hoc pairwise comparison using Bonferroni analyses revealed that habituation 1 was significantly higher than habituation 3 (p<0.001) and test

(p<0.001). A significant main effect of Phase was found for inactivity

(F(2.49,86.96)=26.816, p<0.001); post hoc pairwise comparison using Bonferroni analyses revealed that each subsequent phase was significantly higher than the former phase (p<0.001). A significant main effect of Phase was found for self-grooming

(F(1.71,66.80)=8.254, p=0.001); post hoc pairwise comparison using Bonferroni analyses revealed that habituation 1 was significantly lower than habituation 2

(p=0.002), habituation 3 (p<0.001), and test (p=0.01). Furthermore, habituation 2 was significantly lower than habituation 3 (p=0.012). A significant main effect of Phase was found for biting (F(1.93, 63.78)=4.262, p=0.021); post hoc pairwise comparison using

Bonferroni analyses revealed that habituation 1 was significantly lower than habituation

3 (p=0.002) and test (p=0.005). A significant main effect of Phase was found for burying of the objects (F(3,108)=3.651, p=0.015); post hoc pairwise comparison using

Bonferroni analyses revealed that habituation 1 was significantly lower than habituation

2 (p=0.041). A significant main effect of Phase was found for directional digging towards the objects (F(2.38, 81.06)=4.663, p=0.008); post hoc pairwise comparison using

Bonferroni analyses revealed that digging of the non-novel object at test was significantly lower than habituation 2 (p=0.009) and habituation 3 (p=0.006). A significant main effect of Phase was found for time spent sitting on the objects (F(1.86,

66.83)=7.471, p=0.002); post hoc pairwise comparison using Bonferroni analyses revealed that sitting on the novel object at test was significantly higher when compared to habituation 1 (p=0.023) and habituation 3 (p=0.006).

Figure 3.4: Effects of BPS and BPA on object placement performance in gonadally intact female mice. (A) Bar graph illustrating the percent investigation for vehicle (n=12), BPA-treated (n=15), and BPS-treated (n=14) female mice (light grey bars represent the average percent investigation of the three habituation phases ± SE; dark grey bars represent the average percent investigation of the test phase ± SE). Within group differences between habituation and test are significant; indicating recognition of novel object placement. ** p<0.001 versus vehicle group. # p<0.05 ## p<0.001 habituation phase versus test phase within group. (B) Line graph illustrating the total investigation duration of both stimuli in each habituation and test phase ± SE.

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Figure 3.5: Other non-directional and directional behaviours observed during the OP paradigm for gonadally intact female mice. Data represented as mean ± SE. No significant main effects of treatment.

3.3.2 Hormone Concentrations

A two way ANOVA on reciprocally transformed corticosterone data revealed a

significant effect of sex on corticosterone levels (F(1,43)=4.165, p=0.048; see figure

3.6A), a nonsignificant treatment effect on corticosterone levels (F(2,43)=0.421,

p=0.660), and a nonsignificant sex × treatment interaction (F(2,43)=0.104, p=0.902).

Corticosterone data was reciprocally transformed to meet Levene’s Test of Equality of

Error of Variances (p>0.05). A one way ANOVA revealed no significant treatment effect

on testosterone levels in males (F(2,18)=1.169, p=0.336, see figure 3.6B).

A B

Figure 3.6: Serum hormone concentrations at time of tissue collection (day 10). (A) Circulating free corticosterone levels in adult male and female mice (n=7-9 mice/group). Statistical analysis revealed a significant main effect of sex, a nonsignificant main effect of treatment, and a nonsignificant interaction. (B) Circulating testosterone levels in gonadally intact male mice (n=4-8 mice/group). Data represented as mean ± SE.

3.3.3 Hippocampal Dendritic Spine Data

Research restrictions and time constraints associated with the Coronavirus

pandemic have presently prevented this portion of the experiment from being complete.

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3.3.4 Vaginal Cytology

Vaginal cells were collected after behavioural testing on day 9 and immediately post mortem on day 10. The majority of female mice in each treatment group presented in the estrus phase of the cycle for each collection date, except for the BPS group on day 10 which demonstrated an equal number of mice in estrus and diestrus phases.

Importantly, the estrus phase of the cycle sustains low levels of estradiol alike metestrus and diestrus, and thus are very comparable (Goldman et al., 2007). The proportion of mice in each phase of the estrus cycle is shown in table 3.1.

Table 3.1: Vaginal cytology at time of behavioural testing and tissue collection for VEH, BPA and BPS treated female mice. 5 vaginal smears removed from analysis due to poor sampling (i.e. urine contamination and/or few to no cells in sample).

Stage of Cycle Day 16: Behavioural Testing Day 17: Tissue Collection

Vehicle BPA BPS Vehicle BPA BPS

Diestrus 1 2 2 0 2 6

Proestrus 2 2 5 2 5 1

Estrus 10 8 7 10 7 6

Metestrus 2 0 1 2 1 1

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3.3.5 Body Weight and Reproductive Organ Weights

Two-way repeated measures ANOVAs revealed no statistically significant effects or interactions between body weight and treatment for the male (Repeated measure:

F(1,24)=0.00, p=0.987; Interaction: F(2,24)=0.013, p=0.987) and female (Repeated measure: F(1,24)=0.834, p=0.370; Interaction: F(2,24)=0.039, p=0.962) mice, shown in figure 3.7.

One way ANOVA tests and Kruskal-Wallis one way ANOVA on Ranks test for the testicular tissue which failed the Shapiro-Wilk Normality test, revealed no statistically significant differences in mean weight of testicular (H=2.993, p=0.224), prostate

(F(2,26)=1.926, p=0.168) ovarian (F(2,24)=2.054, p=0.152) or uterine (F(2,25)=0.187, p=0.831) tissue across treatment groups, shown in figure 3.8.

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Figure 3.7: Body weight measurements across time for male and female mice in vehicle, BPA or BPS group. No significant effects were found.

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Figure 3.8: Weight measurements for reproductive tissue between treatment groups. No significant effects are demonstrated for reproductive tissue weight.

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3.4 Discussion

The purpose of this experiment was to evaluate the effects of BPS exposure on learning and memory, endogenous hormones, and hippocampal dendritic spine densities in comparison to BPA exposure, while minimizing potential stress responses through methods developed in chapter 2. The findings from this study demonstrated that BPA and BPS exposure at this dose does not impact spatial memory in either sex; yet BPA and BPS both significantly reduce the key index of task performance, PI.

Additionally, no significant treatment effects were found on body weight, reproductive organ weight, serum testosterone levels, or serum corticosterone levels. Hippocampal dendritic spine density work is presently incomplete.

Interestingly, BPA exposure in the current study produced conflicting results with work discussed in chapter 2. In chapter 2, BPA exposure at the same dose level produced memory impairments in male mice; however, in chapter 3, memory impairments are not evident with BPA or BPS treatment in either sex. The lack of an impairment is indicated by successful discrimination between the novel and familiar object locations represented by a statistically significant difference in PI between habituation and test phase within subjects. The reason for this discrepancy is not yet understood; it is possible that differences in mice batches, experimental design and/or treatment time course may influence animal predispositions and welfare. For example, mice in chapter 2 were housed individually for 18 days which was then shortened to 12 days in chapter 3; this modification occurred due to the switch from a cross-over design in chapter 2 to a parallel design in chapter 3. Studies report individual housing influencing animal welfare; highlighting the importance of avoiding such housing

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conditions in social species (Arndt et al., 2009). Therefore, this modification may have contributed to our differences in behavioural outcomes. Despite that, BPA and BPS exposure in chapter 3 demonstrated a significant treatment effect, like chapter 2, where performance in the test phase of the object placement paradigm was reduced in both males and females compared to their control counterparts. Furthermore, while not statistically significant, BPA treatment appears to result in a stronger reduction in performance when compared to BPS. These findings are likely a result of bisphenol treatment influencing behaviours independent from learning and memory processes.

Through analysis of the several other directional and non-directional behaviours, we revealed no significant effects of BPA or BPS treatment on total investigation duration in either sex. In the females, no significant treatment effects were found on horizontal activity, vertical activity, or inactivity which suggests that the results were not a consequence of bisphenol exposure influencing locomotion or anxiety-related responses. Changes in these behaviours were exhibited in the male mice, which will be discussed shortly. Importantly, said non-directional behaviours are not established models of anxiety so speculations require further investigation with recognized measures of anxiety-like behaviour. Interestingly, the female BPA- and BPS-treated mice exhibited stereotypies whereas their vehicle and male counterparts did not.

Stereotypies are abnormal repetitive behaviours (>3) such as spin turns, jumps, back flips, lid chews, and head shakes which are thought to reflect impaired welfare.

Importantly, previous research suggests that implications associated with stereotypies will depend on the species and animal strain as well as the form of stereotypy and level of expression (Novak et al., 2016). Regarding the form of stereotypy, our CD-1 females

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exhibited back-flipping behaviour which has been previously shown in CD-1 mice to be associated with a negative cognitive bias reflecting a negative affective state (Novak et al., 2016). In relation to level of expression, the BPA and BPS treated female mice were the only groups to present stereotyping behaviour. Moreover, the BPA group demonstrated the behaviour in greater amounts compared to the BPS group.

Interestingly, the same trend is reflected in the corticosterone levels, where BPA-treated mice have greater and more variable levels of serum corticosterone. While no significant treatment effects were demonstrated, the high amount of variability suggests that there may be individual differences in susceptibility of the HPA axis to stress (Engel et al., 2011; Meaney, 2001). Moreover, levels of variability in the corticosterone data may result from an estrus phase effect since the majority of BPA-treated mice presented in estrus or proestrus while VEH- and BPS-treated mice were predominantly in the estrus or diestrus phase of the estrus cycle (Gong et al., 2015). Importantly, serum was collected at time of sacrifice and thus, the corticosterone levels presented may not accurately represent levels during behavioural testing. Regardless, the positive relationship between corticosterone levels and expression of stereotyping behaviour suggests that BPA treatment may result in maladaptive behaviour indicative of stress and/or anxiety. Contrastingly, BPS treatment produced a downward trend in corticosterone levels compared to BPA-treated and control mice. Therefore, this may, albeit partially, explain the lesser expression of stereotypy in the BPS-treated mice.

Importantly, unlike the females, the BPA- and BPS-treated male mice exhibit significant augmentation of horizontal activity in parallel with a significant reduction in inactivity when compared to their vehicle counterparts. Moreover, while not statistically

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significant, the bisphenol-treated male mice typically demonstrated more vertical activity in each phase, when compared to controls. The relationship between these behaviours indicates that BPA and BPS may provoke an unfocused and anxiogenic state in male mice. However, it is important to bear in mind that these non-directional behaviours are not recognized measures of anxiety-like behaviour in mice and therefore, tests specific to anxiety-like behaviour are needed to support this conclusion. However, the notion of bisphenols eliciting an anxiogenic-like state is consistent with prior literature (Peluso et al., 2014). Xu and colleagues (2015a) reported that BPA exposure at 40 and 400 µg/kg bw/day via oral gavage elicits anxiety- and depressive-like behaviour in adult male mice by use of an open-field test, elevated plus maze task, and forced swim test. Additionally, the BPA exposed female mice were either unaffected or exhibited behaviours inversely related to the males, thus eliminating or reversing the sex differences of these emotional behaviours (Xu et al., 2015a). Importantly, Xu and colleagues (2015a) associated this behavioural effect with decreased levels of serum and whole brain testosterone in the

BPA mice receiving doses of 400, 4000 and 40000 µg/kg bw/day. Although our findings with a lower dose of BPA (50µg/kg bw/day) are statistically nonsignificant, the levels of testosterone in our bisphenol-treated male mice trend downwards when compared to their vehicle counterparts. This may, albeit partially, explain the anxiety-related phenotype presented in the male mice as testosterone has mood-enhancing properties and anti-depressant effects in men (Kanayama et al., 2007). Moreover, the unaffected and low levels of serum corticosterone illustrate that anxiety-related behaviour is likely not a result of experimental conditions or bisphenol treatment impacting the HPA axis.

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Another explanation for anxiety-related behavioural effects, although not biochemically investigated in the present study, is that bisphenols may dysregulate serotonin (5-HT) transmission. Serotonin has been strongly implicated in neuronal regulation of mood, aggression, and anxiety. In fact, commonly used anti-anxiety and anti-depressant medications target the 5-HT system. Recently, Ni and colleagues

(2021) reported that dietary supplementation of BPA at 5000µg/kg bw/day significantly reduced hippocampal levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-

HIAA) in male mice but not in female mice. This effect was also associated with decreased protein levels of hippocampal monoamine oxidase (MAO-A) in the male mice. MAO-A is an enzyme responsible for degradation of amine neurotransmitters such as serotonin, dopamine, and norepinephrine (Ni et al., 2021). Interestingly, the study demonstrated an overall state of sex-specific neuronal and colonic inflammation which corresponded with altered microbiota populations as well as memory deficits exhibited by impaired Y-maze and OR performance (Ni et al., 2021). However, the dose level of BPA was 100-fold higher than the present experiment, which may explain the discrepancy on learning and memory effects. Overall, this work illustrates that the serotonergic system may be affected in a sex-specific manner and, thus, possibly explaining the non-directional and stereotyping sex differences among our male and female bisphenol-treated mice.

Simultaneously, aspects of the dopaminergic system may modulate sex differences in non-directional and stereotyping behaviour among the bisphenol treated mice. Dopamine (DA) plays a fundamental role in reward and movement regulation; and has been implicated in various CNS disorders, such as addiction and Parkinson’s

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disease (Juárez et al., 2016). Aside from metabolism, the dopamine transporter (DAT) is a main mechanism for maintaining homeostatic DA levels in the brain (Fox et al.,

2013). DAT knockout (DAT-KO) mice display major elevations in extracellular DA concentrations, alterations in dopamine 1 (D1) and dopamine 2 (D2) receptors as well as several behavioural changes such as hyperactivity, inattention, and increased perseverative and stereotypical behaviours (Fox et al., 2013). Moreover, studies investigating effects of amphetamine-induced DA release demonstrate sex differences in behaviour where females show more intense and prolonged stereotypical behaviours

(Becker, 1999). Therefore, the increased stereotypies in bisphenol-treated female mice

– and not male mice – highlights how alterations of the dopaminergic system may be an underlying mechanism of bisphenol action driving behaviours exhibited in females. In fact, low dose exposure to BPA, BPS, and BPF was shown to alter transcript profiles of genes linked with DA and 5-HT systems in juvenile female rats (Castro et al., 2015).

Moreover, Yao and colleagues (2020) reported that perinatal BPA exposure (10 and

100�g/kg/d) significantly decreased brain and serum levels of DA and its metabolites –

3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) – in adult female mice offspring. Additionally, DA turnover dramatically decreased in the brain but, was enhanced in serum along with increased ratios of DOPAC:DA and HVA:DA (Yao et al.,

2020). Importantly, while the above studies investigated outcomes in adult rodents, bisphenol exposure was perinatal. Therefore, effects may differ in an adult model of bisphenol exposure implying that further studies are required for clarification.

Nevertheless, prior work suggests that bisphenols may disturb the dopaminergic system

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in female rodents and thus, the stereotypical behaviour presented in our study may result from alterations in DA expression and metabolism.

Another explanation for sex-specific non-directional and stereotyping behaviour is that γ-amino-butylic acid (GABA) neurotransmission and expression of ionotropic

GABA(A)α2 receptors may be sex-specifically regulated by BPA exposure. The α2- containing GABA(A) receptor is highly expressed in limbic regions such as the hippocampus and implicated in pathophysiology of anxiety and depression disorders

(Mohler, 2012). In fact, the anxiolytic effect of benzodiazepine drugs is mediated through GABA(A) α2 receptors (Lӧw et al., 2000). Additionally, evidence supports

GABA(A) receptor agonism (muscimol) in the hippocampus inhibiting anxiety-like responses in animals, whereas antagonism (bicuculline) does not (Engin & Treit, 2007).

To complicate matters, steroid hormones may mediate some of GABA’s anxiolytic properties as well (McHenry et al., 2014). Regardless, Xu and colleagues (2015a) demonstrated that BPA exposure at 400 and 40000µg/kg bw/day in adult female mice significantly upregulated GABA(A)α2 receptor expression and inversely downregulated expression at 4000 and 40000µg/kg bw/day in adult male mice when compared to same sex controls; and this finding correlated with anxiety-related behaviour. Decreased

GABA(A)α2 receptor expression is implicated in the etiology of anxiety and diverse mood disorders (Smith & Rudolph, 2012) and, thus, offers a possible mechanism of bisphenol action. Therefore, sex-differences in GABA(A)α2 receptor expression resulting from bisphenol exposure may be an underlying mechanism aiding in facilitation of the sex-specific non-directional and stereotyping behaviour.

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While the possible effects of BPA and BPS on serotonin, dopamine, and/or

GABA are not explored in the experiments presented herein, the literature offers support for these mechanisms underpinning the sex differences observed in non- directional and stereotyping behaviour. Therefore, these systems (i.e. 5-HT, DA, GABA) may provide insight into the mechanisms of BPA action with further investigation.

3.4.1 Significance of Work

The body of work presented in this section contributes to the understanding of how

BPA and BPS influence (1) behaviour in the object placement paradigm, and (2) endogenous hormone concentrations of gonadally intact adult male and female mice when experimental conditions attentively minimize potential stress responses. Only until recently has work begun to investigate the effects of BPS on hippocampal-dependent learning and memory tasks; and even less focuses on the role of BPS exposure mediating anxiety and stress. To our knowledge, these findings are the first to demonstrate that BPS elicits similar behavioural effects to BPA that are sexually differentiated. This suggests that the most widely used BPA replacement, BPS, may not be a safer alternative, at least as far as cognitive and other behavioural effects are concerned.

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4 Chapter 4: General Discussion

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4.1 Conclusions and Implications

These experiments were designed to test the hypothesis that BPS would impair learning and memory, reduce hippocampal dendritic spine plasticity, and influence endogenous hormone concentrations in a manner similar to that of BPA. While investigation of hippocampal dendritic spine density is incomplete due to time restraints inflicted by the ongoing Coronavirus disease pandemic, the obtained data has been inconsistent with our hypothesis. Our data suggest that low dose BPA and BPS exposure does not impair learning and memory processes in gonadally intact mice.

Although consistent with prior work in cycling females, these findings are inconsistent with prior work in gonadally intact male mice. This may be attributed to differences in treatment administration where food exposure is less stressful as well as results in decreased bioavailability and greater subject variability in plasma levels (Gayrard et al.,

2013). However, concurrent with no memory-related deficits in chapter 3, BPA and BPS exposure reduced our index of OP performance suggesting a behavioural effect that may be independent from memory processes. While further investigations are required for clarification, significantly increased horizontal activity in parallel with significantly reduced inactivity as well as augmented, albeit statistically nonsignificant, vertical activity and digging responses signify an anxiogenic-like response with bisphenol exposure in the male mice. Meanwhile, behaviour in female mice appears more convoluted as significant differences in non-directional and directional behaviours between vehicle, BPA, and BPS mice are lacking. While nonsignificant, BPA- and BPS- exposed female mice exhibit stereotyping behaviour, which their vehicle counterparts do not. Additionally, and although not consistent among all the analyzed behaviours, BPA-

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and BPS-treated female mice demonstrate opposing behavioural responses. For example, BPA-treated females exhibit directional and non-directional digging behaviours more than their vehicle counterparts whereas BPS-treated females exhibit this behaviour less than their vehicle counterparts. Finally, sex differences are not apparent when considering learning and memory processes, however, differing responses are exhibited in non-directional and stereotyping behaviours in male and female mice. Collectively, this work has been instrumental in demonstrating similarities in BPA and BPS, suggesting that neither are ideal for use in consumer products when cognitive function is of concern. Thus, considering that the vast majority of human and animal populations are exposed to bisphenols, we should be ceaselessly striving to better understand their behavioural and biological effects. With better knowledge, a standardized test can be created, allowing the industrial sector to pre-screen chemicals that may act similarly to BPA and, consequently, exclude such chemicals from consumer products, while focusing their resources on identifying safer alternatives.

4.2 Limitations and Future Directions

As we set out to contribute to a growing body of literature understanding the health implications of bisphenol exposure, we recognize that there will always be limitations to our work, many of which we are aiming to address with further investigation.

Beginning with chapter 2, we sought to develop a method of bisphenol exposure that minimized potential stress responses in mice. Providing a palatable food was a non-invasive method which removed daily handling and injection-related stress associated with treatment administration. However, to confirm each mouse successfully consumed their treatment as well as to prevent aggressive responses, the mice were

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individually housed. Individual housing opposes typical recommendations to group house mice due to their social predispositions (Arndt et al., 2009). Unfortunately, research investigating the effects of social housing conditions on behaviour and stress responses are inconsistent. Studies report both ends of the spectrum, anxiolytic-like and anxiogenic-like effects as well as no effects of individual housing in both male and female mice (Arndt et al., 2009). Although we agree individual housing should be avoided in social species, a risk-to-benefit analysis based on prior literature and the desire to provide treatment in an unchallenged territory outweighed the potential stress of short-term individual housing. Notably, the time spent in individual housing differed between the two experiments outlined in this thesis, which may explain behavioural differences. The initial design individually housed mice for 18 days which was then shortened to 12 days in the latter experiment.

In both chapter 2 and 3, the treatment stock solution of BPA and BPS integrated into peanut butter was carefully produced to provide a dose of 50�g/kg bw, however, the serum or tissue levels of BPA and BPS in our bisphenol-treated mice are not yet confirmed. As suggested, dietary bisphenol exposure results in less bioavailability and greater subject variability in biological fluids and tissues (Gayrard et al., 2013); thus inter-subject metabolic variability may mask the less noticeable effects of BPA and BPS exposure. However, current approaches for determining bisphenol concentrations in brain tissue and/or serum are limited and not yet verified in mice: this is a limitation our lab is aiming to address.

Similarly, there is a need for a standardized approach in obtaining reliable and valid measurements of E2 and P levels in small quantities of mouse serum. This is

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another limitation our lab is aiming to address, however, until then, potential mechanistic explanations for the behavioural effects in our BPA- and BPS-treated female mice are underdeveloped. During behavioural testing, the majority of our mice presented in the estrus phase of the cycle which sustains low levels of estradiol alike metestrus and diestrus in comparison to proestrus. Importantly, prior work demonstrates that even though spatial tasks, such as OP, can be influenced by experimental manipulations of estrogens, they are typically not influenced by the estrus cycle (Inagaki et al., 2012).

Therefore, under the assumption that bisphenol treatment did not alter endogenous levels of E2 and P, it is likely that our results are not confounded by the estrus cycle and its fluctuating hormone levels. However, the majority of mice also presented in the estrus phase of the cycle 24 hours after behavioural testing and because the estrus cycle in rodents is typically 4 days in length, it was expected that metestrus would be the predominant stage among the mice. This finding suggests that the vehicle- and bisphenol-treated mice may be experiencing irregularities and/or cessation in ovulatory cycles often characterized by persistent vaginal cornification and a twofold increase in

E2:P ratio (Felicio et al., 1984; Nelson et al., 1981). Interestingly, individually housed females tend to cycle more regularly than their group-housed counterparts, which is thought to result from group housed mice excreting inhibitory chemosignals in urine (Ma et al., 1998). Our work contradicts this finding; however, reasons for possible estrus persistency are not yet understood. Simultaneously, these findings represent 2 days out of the 4-5 day estrus cycle, and thus, daily lavage data would better predict cyclicity patterns. Moreover, measuring serum concentrations of E2 and P as well as counting

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the number or corpora lutea in immersion fixed ovaries could potentially elucidate our current data.

Finally, an inherent limitation many others (Antunes & Biala, 2012; Blaser &

Heyser, 2015) have acknowledged is that a battery of learning paradigms may tap into mechanisms of novelty preference as opposed to short term learning and memory.

Although chapter 2 employs the same learning paradigm twice, neither experiment showed treatment effects for stimuli investigation during habituation phase 1, wherein novelty of both stimuli is the greatest. Therefore, an effect of preference for novelty per se is unlikely.

While the results in this thesis provide insight into the effects of low dose voluntary oral consumption of BPA and BPS on spatial memory and endogenous hormones in gonadally intact adult male and female mice, it leads to many novel and unresolved questions that require further investigation. Our results indicate that spatial memory deficits do not emerge following bisphenol treatment at 50�g/kg bw/day in adult female mice; meanwhile memory impairments may (chapter 2 – BPA only) or may not (chapter

3) be presented in adult male mice. As discussed in chapter 3, this discrepancy may result from differences in mice batches, and/or experimental design which could influence animal predispositions and welfare. Even so, behavioural effects that reduce task performance are apparent. As previously mentioned, this may be the result of bisphenol treatment impacting systems pertinent to anxiety-like behaviour. Therefore, conducting a similar experiment with integration of behavioural tests such as the elevated plus maze to assess measures of anxiety-like behaviour may elucidate the

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mechanisms responsible for reduced OP task performance following BPA and BPS treatment.

Additionally, several dose-dependent relationships are demonstrated following bisphenol exposure (Peluso et al., 2014). The possibility exists that doses above or below 50�g/kg bw may impact learning and memory processes in gonadally intact male and female adult mice. Importantly, countries are lowering their tolerable daily intake doses for BPA. While the U.S dose remains 50µg/kg bw/d, Canada is now 25µg/kg bw/d, and the European Food Safety Authority has reduced to 4µg/kg/d (Eladak et al,

2015). Thus, a future experiment should establish a dose response curve surrounding all the current TDIs: 1, 5, 25, 50µg/kg bw/d doses. Plus, as BPA is increasingly being replaced, it is best to investigate other common alternatives, such as BPF. Notably, when comparing BPS and BPF structures to BPA, BPS possesses fewer structural similarities. Therefore, BPF is hypothesized to act more alike BPA, than BPS.

Regardless, investigating a dose-response relationship with other BPA alternatives will provide critical safety information surrounding the CNS and possible effects elicited by

BPA replacements, many of which are not yet regulated.

Therefore, although the results generate several questions that remain unresolved, the novel information presented in this thesis supports the notion that while

BPA and BPS may not impact learning and memory processes as well as circulating hormone concentrations, BPS can produce behavioural effects resembling those of

BPA. This suggests that the two chemicals may act similarly and, thus, BPS may not be a safer alternative to BPA.

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