Effects of mild traumatic brain injury on hippocampal synaptic plasticity and behaviour in juvenile rats

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Behaviour in Juvenile Rats by

Cristina Pinar Cabeza de Vaca BSc, Universitat de Barcelona, 2013 MSc, Universitat Pompeu Fabra, 2014 A Dissertation Submitted in Partial Fulfillment

of the Requirements for the Degree of DOCTOR OF PHILOSOPHY

in the Division of Medical Sciences (Neuroscience)

 Cristina Pinar, 2019 University of Victoria

We acknowledge with respect the Lekwungen peoples on whose traditional

territory the university stands and the Songhees, Esquimalt and WSÁNEĆ peoples whose historical relationships with the land continue to this day.

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Supervisory Committee

Effects of Mild Traumatic Brain Injury on Hippocampal Synaptic Plasticity and Behaviour in Juvenile Rats

by

Cristina Pinar Cabeza de Vaca BSc, Universitat de Barcelona, 2013 MSc, Universitat Pompeu Fabra, 2014

Supervisory Committee

Dr. Brian R. Christie, Division of Medical Sciences Supervisor

Dr. Leigh Anne Swayne, Division of Medical Sciences Departmental Member

Dr. Raad Nashmi, Department of Biology Outside Member

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Abstract

Supervisory Committee

Dr. Brian R Christie, Division of Medical Sciences Supervisor

Dr. Leigh Anne Swayne, Division of Medical Sciences Departmental Member

Dr. Raad Nashmi, Department of Biology Outside Member

Traumatic Brain Injury (TBI) is a global health problem and concussion, or mild TBI (mTBI), accounts for up to 75% of all brain injuries occurring annually in the US. There is also growing awareness that repeated mild traumatic brain injury (r-mTBI) can result in cumulative neuropathology and learning and memory deficits, however there is a paucity of preclinical data as to the extent these deficits manifest. R-mTBI in juvenile populations is of special interest as not only is this a high risk group, but this is also a time period when the human brain continues to mature. The hippocampus is a brain region important for learning and memory processes, and r-mTBI during the juvenile period may particularly disrupt the development of cognitive processes.

To examine this issue we used a model of awake closed head injury (ACHI), and administered 8 impacts over a 4 day period to juvenile male and female rats (P25-28). At 1 or 7 days after the last injury, a cohort of rats was used for behavioural testing to study anxiety and risk-taking behaviours and cognitive abilities. From a different cohort, hippocampal slices were generated and used for in vitro electrophysiological recordings, and the capacity for long-term depression (LTD) and long-term potentiation (LTP) was examined in the medial perforant path (MPP)-dentate gyrus (DG) synapse.

Our results showed that r-mTBI impaired hippocampal-dependent spatial learning and memory and that r-mTBI significantly impaired the capacity for LTD but not LTP in both sexes. These data are the first to describe the negative impact of r-mTBI on LTD in the juvenile DG in both males and females, and provide evidence for the delayed development of neurological deficits with r-mTBI.

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List of Tables

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List of Figures

Figure 1. The Edwin Smith Papyrus, Recto Columns 6 & 7.. ... 2

Figure 2. Location of the Hippocampus in the Human and Rat Brains. ... 15

Figure 3. The hippocampal circuitry. ... 16

Figure 4. Schematic of an overview of some input to dentate gyrus granule cells ... 18

Figure 5. Schematic representation of simplified NMDAR-LTP mechanism. ... 21

Figure 6. Schematic representation of the simplified mechanism of NMDA-R and mGLU-R dependent LTD. ... 25

Figure 7. Awake closed head injury (ACHI) model description. ... 43

Figure 8. Neurological Assessment Protocol. ... 45

Figure 9. Experimental design. ... 46

Figure 10. Repeat awake closed head injury produces acute loss of consciousness and acute neurologic impairment. ... 57

Figure 11. Repeat awake closed head injury impairs hippocampal-dependent spatial memory. ... 59

Figure 12. Global spatial learning and memory is not impaired after repeat awake closed head injury at post-injury day 1. ... 61

Figure 13. Repeat awake closed head injury does not impair global spatial learning and memory at post-injury day 7. ... 62

Figure 14. Repeat awake closed head injury did not increase anxiety or risk-taking behaviour. ... 64

Figure 15. Field in-vitro electrophysiology methodology. ... 74

Figure 16. Repeat awake closed head injury produces acute loss of consciousness and acute neurologic impairment. ... 78

Figure 17. Paired pulse plasticity is unaffected by rmTBI. ... 80

Figure 18. r-mTBI does not alter postsynaptic responsiveness to increasing stimulation. 82 Figure 19. R-mTBI reduces capacity for long-term depression at post-injury day 7 in females. ... 84

Figure 20. R-mTBI reduces capacity for long-term depression at post-injury day 7 in males. ... 85

Figure 21. R-mTBI does not affect the capacity for long-term potentiation in females. .. 87

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List of Abbreviations

ACHI awake closed head injury LOC Loss of consiousness aCSF artificial cerebrospinal fluid LPP lateral perforant path AMPA

D-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid

LTD Long term depression ANOVA Analysis of Variance LTP Long Term Potentiation ATP adenosine-triphosphate MAPK mitogen-activated protein

kinase

BM Barnes Maze Mg2+ Magnesium

CA1 Cornu Ammonis 1 mGluR metabotropic glutamate

receptor

Ca2+ Calcium ML molecular layer

CaMKII calcium/calmodulin-dependent protein kinase II

MML middle molecular layer cAMP cyclic adenosine

monophosphate

MPP medial perforant path CCI controlled cortical impact MRI Magnetic Resonance

Imaging

CRE cAMP response element mTBI Mild Traumatic Brain Injury

CREB cAMP response element binding protein

Na+ Sodium

CS conditioning stimulus NAP Neurological Assessment

Protocol

CSF cerebrospinal fluid NMDA N-methyl-D-aspartate

CT Computed tomography NMDAR N-methyl-D-aspartate

receptors

DAG diacylglycerol NLR Novel Location

Recognition

DAI Diffuse Axonal Injury NT neurotransmitters

DG Dentate Gyrus OML outer molecular layer

DTI Diffusion Tensor Imaging PCS Post-Concussive Syndrome DVI Diffuse Vascular Injury PID post-injury day

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E2 estradiol PIP2 phosphatidylinositol 4,5-biphosphate

EC Entorhinal cortex PKA protein kinase A

EPM elevated-plus maze PKC protein kinase C

ERK1/2 extracellular regulated kinase 1/2

PLC phospholipase C fEPSP Field excitatory post-synaptic

potential

PML polymorphic layer FPI fluid percussion injury PND Post-Natal Day GABA Gamma-Aminobutyric Acid PP paired pulse

GCL granule cell layer PP1 protein phosphatase 1

GCS Glasgow Coma Scale r-mTBI repeat mild traumatic brain injury

Glu Glutamate SCAT Sport Concussion

Assessment Tool HFS High Frequency Stimulation SGZ subgranular zone IML inner molecular layer STD short-term depression IP3 inositol triphosphate STP short-term potentiation

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Acknowledgments

First I would like to thank Dr. Brian Christie for taking a chance on me and fostering my growth, both personal and professional. Your support has made the past four years in the Christie laboratory an incredible opportunity to work independently and expand my skill-set and this thesis would not have been possible without it. Thank you! And thank you to my committee members, Dr. Leigh-Anne Swayne and Dr. Raad Nashmi for your feedback and suggestions on my research and your support and enthusiasm in shaping this thesis into what it is today.

One of the two people that I am most indebted to is Dr. Christine Fontaine. For being the most supportive, patient and encouraging grandma, co-author and co-conspirator. You have always been there to provide feedback and help in every single step of this dissertation. I can’t imagine being here without you. I am also looking forward to the opportunity to keep teaming up to accomplish any goal we set together (although maybe not the 15 hours of electrophysiology ones). Thank you for answering every single question (science related or not), for being my number one supporter and for always being there for me.

To the Christie lab family, past and present, thanks for everything! Dr. Joanna Gil-Mohapel, for always being there as a mentor and role model, you truly are amazing. Katie Neale, for your advice on pens and notebooks, or on anything else. Erin Grafe, for being the future that I could only dream about. You are an amazing person and scientist, I couldn’t think about anyone better to pass my colour on (you may wear it now). – You are all fantastic!

To the Neuroscience Graduate Program student and trainee body – thank you for all the memories! The Caruncho’s OG, especially Raquel, for bringing all us together. Without your insistence we would not be the family that we are. Ben, Carla, Jenessa, Josh, Juan SA, Laura H, Mo and Luis for all the “What does the fox says”, “Chum Drum Bedrum” and “Lose Yourself”. And, of course, Katie and Essie, I could not have asked for better people

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to work with as executives of the Neuroscience Graduate Student Association. You all are amazing humans (11/10).

To the Neuroscience Graduate Program faculty members, for their dedication. You really made my role as Director of University Affairs easier than I expected. I enjoyed every guideline, strong encouragement and comma we edited. Thank you for sharing your passion for the program with me, I have learnt from every one of you. To Dr. Bruce Wright for always having your door open and saying things you will deny saying. And to Dr. Pedro Grandes for your positive attitude and interest in my own work.

To the Neuroscience Graduate Program administrative staff members, for their endless support. I can say without hesitation that I would be years behind in my studies without your incredible support. Erin Gogal, Heather Alexander, Chii Kong, Nicole Coutts and Sara Ohora, I am forever grateful for all the help you have provided me throughout my graduate studies.

To the Animal Care Staff for always being there taking care of the animals, and by always I mean every single day of the year. Thank you! And thank you teaching me everything I know about rats and ethics, you have been an amazing support and I consider myself incredibly lucky to have had you throughout my graduate studies.

To the outside the lab Canadian family, for making Victoria feel like home, you are incredible! Melissa and Shaq for their lessons on everything from how to make sausages to how not to start a boat motor and for always feeding us. And Boof, for all your loving hugs and bites. Christine, Ryan and Penny for the countless rides home, knowledge on weird diets and loud howls. Laura and Chris, for opening your families to us and allowing us to be part of your special moments, I hope we can keep sharing experiences regardless of where we all end-up. Josh and Niki, for always being there for us, for all the coffees, beers and dances and for being family. Their first born Diego, for all the loving howls and take away white fur. And, of course, Thalia, for all her hugs and kisses and for all our

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“discussions” on how things are called (eg: “manzana” vs “apple”). – To all of you, thank you! I could not have asked for a better family!

And to my Catalan/Spanish family. My parents and sister Anna, and Puça, thank you so much for everything you do. Your never-ending support have got me to where I am today and I couldn’t have done it without you. Gràcies! The Trivino-Paredes family for welcoming me into your family and always encouraging me, and for all the suitcases. Gracias! My Voll Damm family of friends for always keeping me in the loop even when being really far from it, for visiting and for always making time to see me during my visits. Gràcies! Amanda Fernandez for visiting almost every year and for those long GinTonic conversations, I can’t wait for our next one. Alexia and Katxe for being our Spanish family in Canada. For all those weekend camps in Gordon Head or View Street, you really were the pillar of our first 2 years here. Gracias!

And finally to the person I am most indebted to: Juan Trivino Paredes, the Juan and only. Thank you for all your support in the laboratory and outside. Thank you for all those scientific questions that I hated so much (well, maybe just half of them). For celebrating the victories or giving me a shoulder to cry on. Thank you for always trying to push my type A personality into Juan’s Standard Time (JST) zone. And for all the spiders. You truly are the best partner in crime I could have asked for.

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Dedication

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Chapter 1. Introduction

Traumatic Brain Injury

The first written document about head injury, known as The Edwin Smith Papyrus, was written around 1650-1550 BC (U.S. National Library of Medicine) (Figure 1). In this document, the ancient Egyptian physician exposes possible treatments of patients with head injury. Specifically, it describes how from a series of observations in head-injured patients (medical examination and signs), three verdicts (prognosis) can be reached: 1) "a medical condition I can treat;" 2) "a medical condition I can contend with;" or 3) "a medical condition you will not be able to treat." During the ancient Greek period, medical writings from Hippocrates (460- 370 BC) demonstrated how they already understood the brain to be the center of thought by describing a number of neurologic conditions, many of them resulting from battlefield head injuries.Since the ancient Greek period, many others have contributed to our current knowledge of head injury (for a review on landmarks in the history of traumatic brain injury see (Al Awar and Sustickas, 2017).

Traumatic Brain Injury (TBI) is defined as damage to the brain resulting from impulsive force transmitted to the head by an external mechanical force (Frieden et al., 2015; NCIPC, 2003). This form of trauma is a major worldwide health and socioeconomic concern, as it represents the foremost cause of mortality and disability for individuals 45 years of age and under (Cole, 2004; Ghajar, 2000). In Canada alone, 160,000 individuals experience some form of TBI each year and it is estimated that ~1.5 million Canadian lives are affected by this type of brain injury (Fu et al., 2015). These prevalent injuries (most commonly caused by falls and motor vehicle crashes (Frieden et al., 2015) can lead to persistent structural and functional damage in the brain that alter behaviour such as learning

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2 and memory, emotion, anxiety and decision-making. The severity of the damage and behavioural outcomes vary with the type of TBI as well as other factors such as patient characteristics, socioenvironmental factors and medical care. As such, the outcomes of TBI can lead to hospitalization, extensive specialist and treatment regimen that are costly to the healthcare system. The estimated annual cost of TBI in Canada is~$3 billion (Fu et al., 2015). Globally, 10 million hospitalizations and/or deaths are the direct result of TBI with an estimate of 57 million people currently living having a history of TBI (Langlois et al., 2006).

Figure 1. The Edwin Smith Papyrus, Recto Columns 6 & 7. The Edwin Smith papyrus was discovered by the Egyptologist Edwin Smith in 1862 outside of Luxor, Egypt. It describes 48 case studies of head and torso wounds and instructs to examine the patient and look for revealing physical signs that may indicate the outcome of the injury. This papyrus revealed a practical guide to management of wounds based on a rational and inductive process instead of the prevailing magic and mysticism. Thus, it is also often considered as the birth of analytical thinking in medical practices and teaching. Other panels of this papyrus are accessible at

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3 Classification and Diagnosis

TBI severity is most commonly determined on the basis of clinical severity in the emergency room (Stein and Spettell, 1995). A TBI can either be a penetrating injury or a closed-head injury. An injury classified as a penetrating TBI presents with damage to the skull, dura and brain parenchyma, while in a closed-head TBI, the skull and usually these other brain structures remain intact or do not show evident alterations (Cassidy et al., 2004). The severity of the injury is determined based on: level of consciousness (duration and severity, if lost), memory and neurological deficits and brain imaging (Table 1).

The Glasgow Coma Scale (GCS) is a universally accepted tool for clinical assessment of injury based on the level of consciousness of the patient. The score is determined by a sum score of eye, motor, and verbal responsiveness, ranging from 3-15. Injuries are classified as severe (GCS <9), moderate (GCS 9-12), or mild (GCS 13-15). Other diagnostic tools such as the Sport Concussion Assessment Tool (SCAT) are widely accepted tools to assess concussion. Indeed, the SCAT includes the GCS assessment and also memory and cognitive assessments to expand on cases where the GCS may not detect deficits. Although the GCS and the SCAT have been proven to correlate with the patient’s outcome and disability (Teasdale et al., 2014; Teasdale and Jennett, 1974a), some have had reservations about it due to confounding factors that could cause some of these test components to be untestable. These factors could include paralysis, intoxication, sedation or intubation (Balestreri et al., 2004; Middleton, 2012; Zuercher et al., 2009). Thus, these tests are not the only assessment tool used in TBI diagnosis and neuroimaging techniques such as computed tomography (CT) or magnetic resonance imaging (MRI) are used to complement the GSC (Firsching et al., 2001; Uchino et al., 2001; Zhu et al., 2009).

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4 It is important to note that, in contrast to more severe forms of TBI, a concussed brain may or may not lead to loss of consciousness or evident memory loss; moreover, it usually present with normal neuroimaging results (Table 1) which makes these injuries challenging to diagnose using current diagnostic techniques (Bigler, 2018; Eierud et al., 2014). Therefore, despite the latest advances in neuroimaging and in diagnostic testing for TBI (Ciuffreda et al., 2017), there is a need for more reliable biomarkers or other indicators to aid in the identification and classification of these types of brain injuries.

Table 1. Classification of TBIs

Mild TBI Moderate TBI Severe TBI Structural brain imaging Normal Normal or abnormal Normal or abnormal Loss of consciousness 0-30 min 30 min to 24 hrs > 24 hrs

Altered mental state ≤ 24 hrs > 24 hrs > 24 hrs Post-trauma amnesia ≤ 1 day 1 – 7 days > 7 days Glasgow Coma Scale score 13-15* 9-12* <9*

Adapted from Traumatic Brain Injuries Review (Blennow et al., 2016). *Best score obtained in the first 24 hours following the injury.

The Problem of Repeated Mild Traumatic Brain Injury

Mild TBI (mTBI), also referred to with the term “concussion”, represents the most common type of TBI (Frieden et al., 2015). According to the “2016 Berlin Consensus Conference”, concussion is defined as a traumatic brain injury induced by biomechanical forces that: (1) typically results in the rapid onset of short-lived impairment of neurological function that resolves spontaneously, (2) may or may not involve loss of consciousness (LOC) and (3) may result in neuropathological changes (McCrory et al., 2017). Clinical

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5 symptoms of mTBI may include, but are not limited to: headaches, confusion, nausea, balance problems, attention deficits, sleep disturbances, learning and memory problems, and emotional alterations (Caine et al., 2014; Kelly and Rosenberg, 1997). Symptoms are typically short-lived and resolve spontaneously in a matter of days or weeks; however, in a subset of individuals, mTBI symptoms can persist for over a year for undetermined reasons (Hall et al., 2005a; McCrea et al., 2003b). Despite these common and widespread functional impairments caused by mTBI, these injuries are nearly impossible to detect with modern in vivo imaging techniques in the clinic.

In the last decade, repeat mTBIs (r-mTBI) have been reported to produce synergistic injuries. This suggests that while a single mTBI may not cause evident or long-lasting structural or functional deficits, it may render the brain vulnerable to subsequent injuries, creating a window of susceptibility where the accumulation of multiple mild concussive events may lead to more severe cumulative damage and long-term cognitive dysfunction (Fehily and Fitzgerald, 2017; Guskiewicz et al., 2003; Prins et al., 2012). Additionally, having a history of mTBI increases the risk of sustaining additional head injuries (Barkhoudarian et al., 2011; Dams-O’Connor et al., 2013; MacGregor et al., 2011; Tremblay et al., 2013; Zemper, 2003).

Epidemiology

Mild TBI accounts for up to 80% off all head injuries (Faul et al. 2010; Frieden et al. 2015). Based on those medically reported injuries, the incidence of mTBI is currently estimated to be 100 - 300 people per 100,000 (Cassidy et al., 2004; Nguyen et al., 2016). However, mTBI is an underreported injury as many people who sustain a mTBI do not seek medical care (Setnik and Bazarian, 2007). A more accurate estimate for the incidence

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6 of mTBI is likely to be approximately 600 people per 100,000, or roughly 42 million globally (Cassidy et al., 2004). Certain sub-populations, such as athletes and military personnel, are particularly susceptible to sustain this type of injury (Cassidy et al., 2004). Moreover, r-mTBI is also common amongst victims of domestic violence (Goldin et al., 2016) or child abuse (Al-Holou et al., 2009; Duhaime et al., 1998).

Athletes who have sustained sports-related injuries make up a large proportion of the clinical r-mTBI research population. Not surprisingly, boxing and other contact sports such as hockey, soccer, and football present a higher incidence of concussion (Cantu, 1996; Koh et al., 2003; Matser et al., 1998a; Tysvaer et al., 1989). Other studies have also looked at r-mTBI incidence in martial arts athletes (such as kickboxing, taekwondo) and have found that these athletes suffer on average 70 concussions over 1000 activity sessions (including training and combats) (Koh et al., 2003; Zazryn et al., 2003). While the public visibility of professional athletes has pushed the issue of concussion into the spotlight, it is important to note that repeat head injury is not limited to professional leagues, but also impacts juvenile and amateur athletes at all levels of play (Broglio et al., 2012; Guskiewicz et al., 2003; Powell and Barber-Foss, 1999). In high school athletes, a cumulative effect of concussion was identified where repeat trauma significantly increased the severity of symptoms (Collins et al., 1999; Marar et al., 2012; McCrea et al., 2004; Powell and Barber-Foss, 1999). Similarly, in college football players, the magnitude and duration of symptoms (headache, nausea, confusion, fatigue, memory problems, attention deficits, and sleep disturbances) showed a cumulative effect after r-mTBI (Guskiewicz et al., 2000, 2003; Hootman et al., 2007; McCrea et al., 2003b). Several groups have shown that r-mTBI produces longer lasting cognitive and motor deficits (De Beaumont et al., 2007;

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7 Guskiewicz et al., 2005; Omalu et al., 2010). Interestingly, athletes that have suffered previous concussions are significantly more likely to sustain a second concussion (Cantu, 1996), especially within 7 to 10 days of the first injury (Guskiewicz et al., 2003).

R-mTBI in juvenile populations

The juvenile developmental period is one of dynamic change as the brain undergoes the final stages of maturation and myelination prior to young adulthood. This is often regarded as somewhat of a critical period with significant capacity for change, but may also be a period of particular vulnerability to damage. Indeed, studies show that children are more likely to sustain a head injury than adults (Frieden et al., 2015; Rutland-Brown et al.), and young males between the ages of 15-24 represent the most at risk population given they sustain mTBI 2-3 times more frequently than females (Bernstein, 1999; Weight, 1998). Additionally, sports-related injuries are the most prevalent cause of mTBI in children aged 10 – 19 (Frieden et al., 2015), and are of special consideration as they may result in r-mTBI with cumulative effects (Collins et al., 1999; Matser et al., 1998b). It is possible that r-mTBI early in life, during these critical final periods of development, can lead to long-term dysfunction across the lifespan.

To note, while the dynamic nature of this developmental stage may increase vulnerability it may also present a unique window during which therapeutic intervention can have the greatest capacity to improve function long-term. Thus, expanding our knowledge of the effects of r-mTBI in this population is important.

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8 Biophysical Mechanisms of mTBI

Understanding how the mechanical energy from the external force is transferred to the brain and the effects of this physical stimulus on the living tissue and neural/glial networks is critical for understanding concussive injuries.

Despite the protection that the skull and cerebrospinal fluid (CSF) provide, head injury—even without skull fracture—can damage fragile brain tissue via acceleration and deceleration forces. Due to its physical properties, the brain tissue shows nonlinear behavior in response to the applied loading rate (Arbogast et al., 1997; Donnelly and Medige, 1997; Miller and Chinzei, 1997; Prange-Kiel et al., 2003; Takhounts et al., 2003). Thus, as brain tissue is mostly composed of water, it is resistant to changing its shape when subjected to pressures. However, it deforms easily in response to shear forces compared with other biologic tissues. Several studies have investigated the impact of shear deformation in comparison to other forces and have led to the idea that shear deformation is the main cause of injury in concussion (Adams et al., 1982; Gennarelli et al., 1982; Unterharnscheidt and Higgins, 1969). The anatomical location and structure of different brain regions can make certain areas more susceptible to the shearing forces. For instance, clinical studies using MRI have reported the hippocampus as one of the most vulnerable regions to shearing forces after moderate and severe TBI (Bigler, 2018; Bigler et al., 2002; Kotapka et al., 1992; Serra-Grabulosa et al., 2005; Tate and Bigler, 2000; Tomaiuolo et al., 2004). The mechanical forces of the injury can ultimately lead to physiological changes that are explained in the next section.

Neurobiology of mTBI

Neurological damage occurs not only at the moment of impact (immediate), but evolves afterwards (minutes to months). Therefore, the damaging effects of mTBI can be

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9 divided into two main phases: primary and secondary injury (see (Blennow et al., 2012; Pekna and Pekny, 2012) for a review). The initial impulsive forces are the cause of the primary injury during which the brain tissue sustains a sudden mechanical stress that may induce necrosis, diffuse axonal injury (DAI) and diffuse vascular injury (Pekna and Pekny, 2012; Ray et al., 2002; Werner and Engelhard, 2007). These initial events subsequently trigger the secondary injury characterized by multiple alterations in several cellular processes including alterations in energetic metabolism, redox status, neuronal structure, inflammation and homeostasis of glutamate (Glu) and Ca2+ (Ca2+ ) (Fineman et al., 1993; McIntosh et al., 1997; Pekna and Pekny, 2012; Sun et al., 2008; Walker and Tesco, 2013; Weber, 2012).

Pathophysiological Changes Post mTBI: Primary Injury

The biomechanical forces of mTBI generate intracranial pressure gradients that lead to shearing and tearing of neurons, glial cells, and blood vessels in the brain (Blennow et al., 2012; Pekna and Pekny, 2012). These forces seem to be higher in areas of changing tissue densities and rigidity such as the grey matter-white mater interface. Axons extend great distances within the brain and thus are more susceptible to this stretching, which leads to DAI. Disruption of the axolemmas (axons’ cell membrane) increases their permeability (Pettus et al., 1994; Povlishosk and Pettus, 1996), Ca2+ influx, and mitochondrial swelling (Mata et al.; Maxwell et al., 1997). Microtubule disorganization post-injury has been identified as a consequence of axon stretching where ultrastructural analysis has shown breakage and folding of microtubules after TBI, triggering microtubule disassembly (Maxwell et al., 1997; Povlishosk and Pettus, 1996; Tang-Schomer et al., 2010). This causes accumulation of organelles in the axon and axonal swelling, with eventual

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10 disconnection and axotomy (Barkhoudarian et al., 2011; Christman et al., 1994; Giza and Hovda, 2014; Johnson et al., 2013). DAI has been identified in patients with TBI (Oppenheimer, 1968), although the severity of DAI has been found to be proportional to the deceleration force of impact (Elson and Ward, 1994). Until recently, DAI pathology following mTBI was not detectable using conventional neuroimaging techniques. However, advances with diffusion tensor imaging (DTI) have now identified DAI in mTBI patients (Bazarian et al., 2007; Mayer et al., 2010; Miles et al., 2008) and even in response to sub-concussive blows (Bazarian et). Moreover, some studies have also shown, using DTI, that the extent of DAI after mTBI is related to post-concussion cognitive problems (Lipton et al., 2008; Niogi et al., 2008; Wilde et al., 2008). Therefore, the disruption of axonal fibers caused by mTBI mechanical forces can lead to synaptic transmission alterations and neuronal circuit dysfunction. In addition, blood vessels are also structures vulnerable to shear forces. Indeed DAI is usually accompanied by microbleeds in the same locations which can be referred to as diffuse vascular injury (DVI) (Gentry et al., 1988; Onaya, 2002; Pittella and Gusmão, 2003). Rupture of several capillaries, a phenomenon known as multiple petechial hemorrhages, is commonly observed in TBI patients with different severities (Mckee and Daneshvar, 2015). The hypoxic event caused by this halt in blood flow can contribute to the immediate functional dysfunctions following mTBI. Moreover, it has been reported that following TBI, the cerebrovascular reactivity (brain ability to elevate blood flow above baseline) is compromised (Adams et al., 2018; Amyot et al., 2018). This means that the brain capacity to modulate metabolic demands caused by neuronal activity is deficient and can alter the normal functioning of the brain. In addition,

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11 the extravasation of blood contents can trigger or exacerbate several events described in the secondary injury phase (Kurland et al., 2012).

Pathophysiological Changes Post mTBI: Secondary Injury

One of the events that characterizes the secondary injury is the opening of voltage-dependent potassium (K+) channels (Farkas et al., 2006). This disruption causes an unregulated amount of ion flux, specifically K+ efflux and sodium (Na+) influx at the cellular level, and a subsequent dysregulated release of neurotransmitters (NT), particularly the excitatory amino acid glutamate.

In order to restore the ionic balance, the Na+_/K+_{adenosine-triphosphate (ATP)}

dependent pumps activity is increased, which results in a depletion of the energy stores creating a metabolic crisis. In order to restore the energy reservoir, the system mobilizes intracellular glucose to generate more ATP causing hyperglycolysis. Impaired oxidative metabolism may result in decreased ATP production, thereby exacerbating the energy crisis, ionic imbalance and further contributing to hyperglycolysis. Following this trauma-induced hyperglycolysis, there is an accumulation of lactate, resulting in acidosis, increased membrane permeability and cerebral edema (Kalimo et al., 1981). Studies have shown an increase in glucose metabolism as early as 5 minutes post-TBI and lasting up to 4 hours in rats (Gardiner et al., 1982), and this is followed by a period of hypometabolism of variable duration dependent upon injury severity (Peskind et al., 2011).

In addition to these energy perturbations, excessive extracellular glutamate binds to post-synaptic N-methyl-D-aspartate (NMDA), D-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and kainate receptors causing further regional depolarization (Faden, 1992). Consequently, activated NMDA receptors (NMDARs) flux Ca2+ into the

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12 cell. This Ca2+ acts as a second messenger triggering numerous pathways. For instance, increased Ca2+ _{following trauma may cause cell death via over activation of calpains}

(Kampfl et al., 1997; Roberts-Lewis and Siman, 1993), phospholipases (Farooqui and Horrocks, 1991), or protein kinases (Verity, 1992). Moreover, the large influx of Ca2+ via NMDARs accumulates in the mitochondria resulting in impaired oxidative metabolism (Xiong et al., 1997). This mitochondrial dysfunction leads to decreased production of ATP, thereby worsening the energy predicament (Vagnozzi et al., 2007; Xiong et al., 1997). All these events following the initial insult are believed to be the cause of acute post-injury deficits (Barkhoudarian et al., 2016; Giza and Hovda, 2014b).

In summary, the biomechanical forces induced by the impact cause pathophysiological changes through the primary and secondary injury mechanisms. These changes, not surprisingly, can lead to a spectrum of functional limitation and reduced quality of life. Some of these effects are compiled in the following section.

Neurobehavioural consequences of r-mTBI

As mentioned, psychological and neurologic disorders can develop following mTBI. These usually resolve spontaneously within 2-3 weeks (Lovell et al., 2003; McCrea et al., 2003a), whereas repeat injuries may cause symptoms to persist for extended periods of time (Arciniegas et al., 2005; Halstead and Walter, 2010; Pellman et al., 2003). The term “Post-Concussive Syndrome” (PCS) is commonly used in literature to refer to the neurobehavioral sequelae associated with r-mTBI when symptoms persist for more than three months (Hall et al., 2005b; Hsu et al., 2015; Voormolen et al., 2018). Researchers and clinicians have identified some factors contributing to the development of long term PCS, including: age, female sex, prior head injury, lower education, personality disorder

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13 and cognitive dysfunction pre-injury (Bazarian et al., 2005; Polinder et al., 2018; Scopaz and Hatzenbuehler, 2013)

Clinical evidence of patients with a history of multiple concussions have identified increased learning and memory disabilities (Bijur et al., 1996; Matser et al., 1998a; Wall et al., 2006), anxiety, personality changes, depression and post-traumatic epilepsy (Agrawal et al., 2006; Hart et al., 2011; Lowenstein et al., 1992; Rosenthal et al., 1998). Moreover, r-mTBI may actually increase the risk of developing dementia (Guskiewicz et al., 2005) and neurodegenerative diseases (Masel and DeWitt, 2010b; McKee et al., 2009).

Effects of r-mTBI can manifest, in pediatric and juvenile populations, as academic failure, chronic behavior problems, social isolation, and difficulty with employment, relationships, and, in some cases, difficulty with the law (Ewing-Cobbs et al., 2004; Gerrard-Morris et al., 2010; Hendryx and Verduyn, 1995; Huw Williams et al., 2010). Because of the age of these individuals, however, these changes might be mistakenly attributed to other causes ranging from lack of motivation and laziness, adolescent-like behaviour to bad parenting (Wade et al., 2006), making r-mTBI even harden to diagnose in the juvenile population.

Animal models of r-mTBI

As it is challenging to study the effects, pathophysiological mechanisms, and treatments of r-mTBI only in human patients, a translational research approach involving animal models has been a common strategy in an attempt to provide insight into TBI. There are several mTBI injury models including the weight drop (WD) model (Feeney et al., 1981; Foda and Marmarou, 1994), the fluid percussion injury (FPI) model (Dixon et al., 1987; Gennarelli, 1994), the controlled cortical impact (CCI) model (Dixon et al., 1991;

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14 Lighthall, 1988; Lindner et al., 1998), the blast injury model (Cernak et al., 1996; Leung et al., 2008) and the awake closed head injury (ACHI) model (Petraglia and Huang, 2013) used for this dissertation (see (Petraglia et al., 2014a; Shultz et al., 2016; Xiong et al., 2014) for extensive reviews on different models of TBI). Compared to other models, to use the ACHI model no craniotomy surgery or skull exposure is performed and impacts are delivered to fully conscious, restrained animals without the need of anaesthesia, which might be neuroprotective (Jiang et al., 2017). These factors could be considered benefits in comparison to other previously developed experimental models as they likely make the ACHI model more representative of clinical r-mTBI.

R-mTBI and the Hippocampus

Certain brain regions tend to be more vulnerable to damage due to their anatomical location and structure. In the clinical population, concussions are commonly a result of an impact to the frontal or temporal lobes, making the hippocampus a region of susceptibility to mTBI (Geddes et al., 2003; McCarthy, 2003). Studies of severe TBI have shown decreased hippocampal volume in both adults (Kim et al., 2008) and juveniles (Tasker et al., 2005). Moreover, this structure is closely related to emotional processes and learning and memory, all reported to be affected after mTBI in humans. Therefore, with the cognitive and emotional impairments that go along with r-mTBI, and considering the functions in which the hippocampus is involved, it is reasonable to infer that damage to this brain region can underlie some of these deficits.

The Hippocampal Formation

The hippocampal formation, or hippocampus, is a bilateral structure found in the medial temporal lobe in the mammalian brain (Figure 2). The term hippocampus originates

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15 from the Greek words “hippos” meaning “horse” and “kampos” meaning “sea monster” and was originally used to name this brain structure by Arantius in 1587. However, it was not until many years later that a possible function was described. In 1957, Scoville and Milner described memory loss following bilateral medial temporal lobe resection in the patient HM (Scoville and Milner, 1957). Further exploration of this structure, memory and the brain pinpointed the hippocampus as the critical structure for memory related to facts and events. In the past two decades, however, it has become apparent that the cortical areas surrounding the hippocampal formation also play critical roles in memory. For an excellent review of hippocampal literature see (Anderson et al., 2007).

The hippocampal formation consists of the dentate gyrus (DG), the Cornu Ammonis (CA) 1 and CA3 regions (also known as the hippocampus proper) and the subiculum (reviewed in (Blumenfeld, 2010).

Information Flow in the Hippocampus

Figure 2. Location of the Hippocampus in the Human and Rat Brains.

The hippocampal formation (red) sits below the surface of the neocortex with a linear shape and horizontal orientation in the human brain (top), but is more c-shaped and vertically oriented in the rat brain (right).

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16 The main flow of information in the hippocampus is the well-known trisynaptic circuit. Briefly, the entorhinal cortex (EC) projects to the granule cells of the DG via the perforant pathways, the mossy fibers from the DG project to activate the CA3 pyramidal neurons, and the CA3 Schaffer collaterals synapse on the CA1 pyramidal neurons. Finally, the CA1 pyramidal neurons project to the EC and the subiculum. Theses major pathways in the hippocampus are excitatory and unidirectional. Generally, the subiculum is the primary source of subcortical projections whereas the EC is the primary source of neocortical projections. It is important to note, however, that there are direct projections from the EC, the CA3, CA1 and subiculum in addition to other more complex connections. For the purposes of this dissertation, the rest of this section will focus on the DG region of the hippocampus.

Figure 3. The hippocampal circuitry. The dentate gyrus (DG) receives most of its inputs from layer II of the entorhinal cortex through the medial and lateral perforant pathways (MPP and LPP, respectively). The axons of the granule cells extend towards the pyramidal cells of the cornu ammonis (CA) 3 region, forming the mossy fibers. The projections of the CA3 pyramidal cells form the Schaffer Collaterals, which establish synapses with dendrites of the CA1 pyramidal cells. Finally, CA1 neurons complete the hippocampal circuitry by projecting their fibres to the deep layers of the entorhinal cortex. CA1 also receives direct input from layer III of the entorhinal cortex through the temporoammonic pathway (TA). Used with permission from (Pinar et al., 2017). Abbreviations: DG: dentate gyrus; CA1: cornu ammonis 1; CA2: cornu ammonis 2; CA3: cornu ammonis 3; CA4: cornu ammonis 4; LPP: lateral perforant path; MPP: medial perforant path.

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17 Dentate Gyrus

The basic structure of the DG has been well-characterized and is described in detail by a variety of book chapters and review papers (Amaral and Lavenex, 2006; Anderson et al., 2007; Frotscher and Seress, 2007). The DG can be sub-divided into three lamina: the molecular layer (ML), the granule cell layer (GCL), and the hilus or polymorphic layer (PML) (Figure 4).

The ML contains dendrites of granule cells and associated excitatory and inhibitory cells as well as the axons of the perforant path. This layer is itself divided into thirds: the outer molecular layer (OML), the middle molecular layer (MML) and the inner molecular layer (IML). The EC provides the primary cortical input to the DG through the perforant pathways. Perforant path fibers originating in the lateral entorhinal area create the LPP and occupy the OML, whereas the fibers originating from the medial EC create the medial perforant path (MPP) and occupy the MML (Hjorth-Simonsen, 1972; Hjorth-Simonsen and Jeune, 1972). The inner third of the molecular layer contains intrinsic connections as well as inhibitory connections with interneurons (Buckmaster et al., 1992, 1996; Frotscher et al., 1991; Laurberg and Sørensen, 1981).

The dentate granule layer is where the somas of mature and newly born granule cells are located. The dendritic arbours of these cells extend into the ML and receive glutamatergic inputs from the MPP and the LPP and modulation from variety of NT from the other two layers (ML and PML). The granule cells give rise to the mossy fibers (named by Ramon y Cajal), which synapse with the mossy cells of the PML and with the CA3 pyramidal cells of the hippocampus. Importantly, inhibitory interneurons, named pyramidal basket cells for their basket-like plexus, are also found in the GCL. (see (Amaral and Lavenex, 2007; Ribak and Shapiro, 2007) for a review of DG anatomy).

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18 The PML contains both inhibitory and excitatory neurons, including the mossy cells. These glutamatergic cells get their name from receiving input from the mossy fibers as well as for their large and dense number of spines. It is also important to mention that the PML and the GCL are separated by the subgranular zone (SGZ). This is one of the few regions in the adult brain that retains the capacity for neurogenesis (Altman, 1962; Altman and Das, 1967; Bruel-Jungerman et al., 2007a, 2007b; Cameron and Mckay, 2001; Kempermann et al., 1997; Kuhn et al., 1996; van Praag et al., 2002).

Hippocampal Synaptic Plasticity

The capacity for alterations in the nature, strength, or number of interneuronal synaptic connections between neurons was described and referred to as synaptic plasticity Figure 4. Schematic of an overview of some input to dentate gyrus granule cells. Granule cells (light green) of the dentate gyrus receive excitatory input primary from the entorhinal cortex via the lateral and medial perforant paths (arrows, upper right), which also provide input to the inhibitory basket cells (right, dark green). Mossy cells (lower left) also provide glutamatergic input to local granule cells. The pyramidal basket cells (and other inhibitory interneurons) play a critical role in plasticity in this region given that they can modulate granule cell activity. Synapses are indicated with red circles.

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19 by Konorski (Konorski and Jerzy, 1948). Years later, the unifying hypothesis of memories being stored as alterations in the strength of synaptic connections between neurons in the CNS was first introduced by the Canadian researcher Donald Hebb in the mid-1900s (Hebb, 1949; Konorski and Jerzy, 1948). Hebb published his seminal formulation as what is now generally known as Hebb’s postulate:

“When an axon of cell A … excites cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells so

that A’s efficiency as one of the cells firing B is increased. (Hebb, 1949)”

Since Hebb’s seminal paper, synaptic plasticity and its functional and molecular correlation to learning and memory have been extensively studied and both short and long term forms of plasticity (both described below) have been identified. Indeed, hippocampal synaptic plasticity is considered one of the top candidates for the mechanisms underlying behavioural learning and memory.

Long-Term Potentiation

The seminal report by Bliss and Lomo in 1973 (Bliss and Lomo, 1973), was the first in describing that tetanic stimulation, at the MPP-DG synapse of the rabbit hippocampus, yielded an increase in the amplitude of their recorded field excitatory post-synaptic potentials (fEPSPs). Later, this phenomenon was named “Long Term Potentiation” (LTP) (Douglas and Goddard, 1975).

LTP is a long-lasting increase in synaptic efficacy as a result of prior synaptic activation at a high frequency. Over four decades later, and after many excellent reviews of the literature (Abraham, W. C. & Williams, 2003; Bear and Malenka, 1994; Bliss et al., 2007; Bortolotto et al., 2011; Larkman and Jack, 1995; Malenka and Bear, 2004; Stevens

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20 and Sullivan, 1998), the basic mechanisms of long-term potentiation in the healthy and diseased brain remain vague. The form of LTP observed can vary depending on the conditioning stimulus (CS) used, which itself can vary in terms of their intensities, frequencies, durations and patterns.

NMDA-mediated LTP is the most commonly described and the best characterized form of LTP in the hippocampus (Figure 5). After the repetitive presynaptic activation and release of glutamate, the postsynaptic cell is depolarized through the activation of AMPAR and the subsequent flux of Na+ and K+ in and out of the cell. Consequently, the NMDAR Mg2+ block is released and allows Na+ and Ca2+ to flow into the cell through the NMDAR. Once the NMDAR pore is open it fluxes important Ca2+ ions in addition to Na+ into the postsynaptic cell. It should also be noted that AMPARs that do not contain the GluA2 subunit are also capable of fluxing Ca2+ _{although to a lesser extent (see ((Isaac et al., 2007)}

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21 Typically, LTP is broken down into stages based on the temporal order of expressed mechanisms. However, these phases are not exclusive from each other and may overlap (Sweatt, 2010). For a literature review on phases of LTP see (Baltaci et al., 2019). The first of these phases is known as short-term potentiation (STP) and refers to a potentiation of fEPSPs lasting on the order of 5-10 minutes post CS (Malenka, 1994). Different mechanisms have been proposed as being responsible for STP, one of them being increases in Ca2+ and its effects on the phosphorylation of synapsin I by calcium/calmodulin-dependent protein kinase II (CaMKII) (Greengard et al., 1993; Rosahl et al., 1995). However, others have shown that while the induction of STP can be NMDA dependent, it is independent of protein kinase activity (Sweatt, 2010).

The early phase of LTP (E-LTP) sets in around 10-30 minutes post induction and can last for 2-3 hours. The increase in intracellular Ca2+ _{is believed to be a critical component}

Abbreviations: AMPAR: a-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid; Ca2+_:

calcium; CaMKII: calcium-calmodulin-dependent protein kinase II; LTP: long-term potentiation; Mg2+_{: magnesium; Na}+_{: sodium; NMDAR: N-methyl-D-aspartate receptor.}

Figure 5. Schematic representation of simplified NMDAR-LTP mechanism. When high-frequency stimulation is applied, glutamate (green 4-point stars) bind to both AMPARs and NMDARs. Sodium (Na+₎

influx through the AMPAR causes sufficient depolarization of the membrane to expel the Mg2+_{block in the NMDAR, allowing it to}

flux both Na+_{and Ca}2+_{. This causes an}

increase on intracellular Ca2+_{that activates}

CaMKII among other second messenger signalling pathways that ultimately lead to the insertion of AMPARs into the postsynaptic membrane and potentiation of the fEPSP.

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22 for this phase of the LTP through the activation of intracellular cascades that eventually lead to the enhancement of AMPARs mediated currents and the insertion of additional AMPA receptors into the plasma membrane. The rapid rise in intracellular Ca2+ activates calmodulin, which in turn activates the CaMKII. This kinase can maintain LTP by phosphorylating AMPA and NMDA receptor among other functions. This increases their activity, receptor activity and channel opening probability, resulting in a further depolarization of the cellular membrane. Briefly, CaMKII is composed of regulatory and catalytic subunits. These subunits separate upon binding with the calcium-bound calmodulin complex and the catalytic subunit phosphorylates a variety of substrates including the AMPAR. CaMKII can also autophosphorylate, thus remaining activated long after Ca2+ levels have decreased to baseline. CaMKII can also aid in the insertion of additional AMPA receptors into the plasma membrane. Other molecules such as protein kinase C (PKC), protein kinase A (PKA), mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate (cAMP) have also been implicated in LTP and may work alongside CaMKII to enhance NMDA and AMPA receptor activity and deactivate phosphatases.

Finally, the late phase of LTP (L-LTP) also known as long-lasting LTP, requires protein synthesis at synaptic and/or dendritic sites (Winder, 1999). Mitogen activated protein kinase (MAPK) pathways have been shown to be key players for L- LTP through the phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and ultimately the activation of the transcription factor CREB and CRE-mediated gene expression (Klann, 2008, Abraham, 2008;Davis, 2000;Lynch, 2004;Silva, 2003).

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23 In addition to the above mentioned postsynaptic mechanisms, there is also evidence for presynaptic modifications in the induction and maintenance of LTP, such as changes in transmitter release probability (Lu, 1999).

Long-Term Depression, the flipside

Once LTP was experimentally characterized, a new question arose within the researchers: what happens when all synapses are maximally potentiated? If synapses can be potentiated for very long periods of time, eventually all synapses would be driven to their maximum synaptic strength and there would be no further capacity for that synapse to participate in synaptic-plasticity-dependent processes. Moreover, this unidirectional mechanisms would only allow memory storage, but not information processing. It was then suggested that, in order for LTP to be part of a mechanism for information processing, other forms of plasticity must exist to maintain synaptic balance.

Long term depression (LTD) is a long-lasting decrease of synaptic strength was first described by Lynch in 1977 as a process that occurred at adjacent synapses to those being potentiated (Dunwiddie and Lynch, 1978; Lynch et al., 1977). This is known as heterosyanptic, or non-associative, LTD. In 1992, a type of LTD occurring in stimulated synapses was described by Serena Dudek and Mark Bear (Dudek and Bear, 1992). This LTD, known as homosynaptic, or associative, LTD was discovered in the CA1 in response to low frequency stimulation (LFS) CS and is now believed to be the counterpart to LTP in learning processes and memory formation (Dudek and Bear, 1992; Mulkey and Malenka, 1992a). Contrary to LTP, LTD remains under-investigated, however, various mechanisms have been identify to play a potential role in each form of LTD.

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24 Similarly to STP, short-term depression (STD) refer to a temporary decrease in the size and slope of the fEPSP that follow the delivery of a low frequency CS and decays quickly. STD lasts on order of 5-10 minutes post CS and the mechanisms behind it are considered to be related to a depletion of the readily releasable pool of vesicles (Schneggenburger et al., 2002) or to a reduction in presynaptic Ca2+ influx leading to reduced transmission (Kamiya and Zucker, 1994; Regehr, 2012; Xu and Wu, 2005; Zucker and Regehr, 2002).

The best-characterized form of this type of plasticity in the DG is the NMDAR-dependent LTD (Figure 6A). Similarly to the LTP mechanism, in this form of LTD, Ca2+ influx though NMDA receptors, or through voltage gated Ca2+ channels, triggers a signalling cascade. This cascade is initiated by the activation of calcineurin (also known as protein phosphatase 2B (PP2B)) by the calcium-calmoludin complex. Calcineurin then dephosphorylates inhibit 1 (I-1) and thus activates protein phosphatase 1(PP1) (Mulkey and Malenka, 1992b). PP1 acts by dephosphorylating various targets, including AMPA receptors in the postsynaptic membrane thereby reducing transmission through AMPA receptors and leading to AMPA receptor internalization. Additionally, the neuronal Ca2+ sensor protein hippocalcin is activated by modest levels of Ca2+ _{and through downstream}

mechanisms results in the removal of AMPA receptors from the plasma membrane (see (Collingridge et al., 2004) for a review on receptor trafficking) (Figure 4).

Another prominent form of LTD is the mGluR-dependent LTD (Figure 6B). However, the underlying mechanisms of these forms of LTD are not as established as the NMDAR-dependent ones, and vary depending on the mGluR activated subtype (see (Sanderson et al., 2016) for review). Group I mGluR (mGluR1 and mGlur5) activation

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25 relies on the activation of phosphoinositide-specific phospholipase C (PLC). PLC then hydrolyzes phosphatidylinositol 4,5-biphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 elicits the release of Ca2+ from intracellular stores and DAG can activate various kinases such as MAPK, ERK, PKC and phosphoinositide 3- kinase (PI3K). The later can phosphorylate the serine-880 site of the GluA2 subunit of the AMPAR which can lead to lateral diffusion and internalization of the AMPAR (Gladding et al., 2009; Lüscher and Huber, 2010). It is also important to mention that presynaptic forms of mGluR-LTD have also been investigated (Fitzjohn et al., 2001).

What differentiates electrically induced NMDAR-LTD from mGluR-LTD (Huber et al., 2000) remains unresolved.

Figure 6. Schematic representation of the simplified mechanism of NMDA-R and mGLU-R dependent LTD. (A) Nmethyl- D-aspartate receptor (NMDAR)-dependent LTD at glutamatergic terminals. Low frequency stimulation (LFS) leads to low Ca2+ _{influx and the formation of the}

Ca-calmodulin complex, which activates protein phosphatase 2 B (PP2B), or calcineurin. PP2B activation subsequently leads to activation of protein phosphatase 1 (PP1), which dephosphorylates the GluA1 subunit of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid receptor (AMPAR) at serine 845, leading to lateral diffusion of this receptor followed by its internalization.

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26 (B) In metabotropic glutamate receptor (mGluR)-dependent LTD, LFS leads to activation of phosphoinositide-specific phospholipase C (PLC), which hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2), generating Inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to Ca2+ _{receptors on the endoplasmic reticulum causing Ca}2+ _{release from internal stores, while}

DAG can results in the activation of several kinases, including, protein kinase C (PKC). This can phosphorylate the GluA2 subunit of the AMPAR at serine 880 and lead to its lateral diffusion and internalization. This form of LTD also activates protein tyrosine phosphatases (PTPs) that can dephosphorylate the GluA2 subunit of the AMPAR, also resulting in its internalization. Figure adapted with permission from (Pinar et al., 2017).

Abbreviations: AMPAR: a-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid; Ca2+ _:

calcium; DAG: diacylglycerol; IP3: inositol triphosphate; mGluR: metabotropic glutamate receptor; NMDAR: N-methyl-D-aspartate receptor; PIP2: phosphatidylinositol 4,5-biphosphate; PKC: protein kinase C; PLC: phospholipase C; PP1: protein phosphatase 1; protein phosphatase 2B; PTP; protein tyrosine phosphatase.

Ca2+ signals can initiate both LTP and LTD however, the affinity of CaMKII and calcineurin for the calcium-calmodulin complex is what determines the subsequent effect on synaptic plasticity. PP2B, or calcineurin, has a higher affinity for the calcium-calmodulin complex than does CaMKII, meaning that it will be preferentially activated at lower Ca2+ concentrations (see (Xia and Storm, 2005) for a review of the role of calmodulin in plasticity). Moreover, calcineurin itself can also reduce the open time of NMDARs thereby reducing further Ca2+ entry into the postsynaptic cell, which may also contribute to LTD (Shi et al., 2000). Through these mechanisms, and likely other, postsynaptic signalling pathways LTD, instead of LTP, is induced.

Paired Pulse Plasticity

The term paired-pulses (or twin-pulses) refers to two close in time action potentials in the presynaptic cell that produce two close in time fEPSPs in the postsynaptic cell. Experimentally, paired pulse (PP) plasticity has been used as a relative indicator of presynaptic release probability. Plasticity is typically measured as the ratio of the slope of

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27 the second fEPSP relative to the slope of the first: when the second fEPSP has a greater slope than the first one (ratio > 1) is called paired pulse facilitation, and when the second fEPSP has a smaller slope than the first (ratio < 1), it is paired pulse depression (see (Regehr, 2012; Zucker and Regehr, 2002) for review).

Generally, in the first pulse of stimulation vesicles in the readily releasable pool in the presynaptic terminal will exocytose their contents (NT) into the synaptic cleft to subsequently lead to a measurable fEPSP on the post-synaptic neuron. In the second pulse, however, depending on the release probability of the presynaptic terminal (the amount of NT already released during the first pulse) the second fEPSP will be, on average larger or smaller. Briefly, when a terminal exhibits a high release probability, more vesicles are released during the first pulse which can lead to fewer vesicles docked and in the readily releasable pool to be released during the second pulse, leading to a smaller second fEPSP and therefore paired pulse depression. Meanwhile in terminals with a lower release probability, fewer vesicles may be available for release upon the first stimulation and residual Ca2+ in the presynaptic terminal may facilitate vesicle release upon the second pulse leading to a larger second fEPSP and therefore paired pulse facilitation (Katz and Miledi, 1968).

It should be noted however that, although for this dissertation, paired-pulse ratio will be used as a measure of presynaptic release probability, the above is a relatively simplistic explanation of a complex process and other presynaptic factors can be involved in affecting paired pulse plasticity.

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28

Sex Differences in the Brain

Biological differences between males and females are ubiquitous. Biological sex has been shown to influence several processes in the brain such as neurogenesis, neuronal morphology, synaptic plasticity and behaviour (for review see (Choleris et al., 2018; Sheppard et al., 2019). Despite this evidence, the effect of biological sex in the brain has been historically underestimated. Specifically, the female sex has been historically understudied and there continues to be a gap in the inclusion of females in pre-clinical studies of TBI and in neuroscience in general. Fortunately, social and political development in the last few years has helped increase our understanding of how the healthy and diseased male and female brain differ.

Sexual Maturation: Puberty

Steroid hormones such as estrogens, progestogens, and androgens are involved in driving the development and subsequent regulation of sexually different structures, function, and behavior throughout life. Developmental (also termed “organizational”) actions of hormones lead to the often sexually different life-long epigenetic regulation of genes (reviewed in (McCarthy and Nugent, 2015). The so called “activational” effects of hormones are also seen at puberty (Schulz and Sisk, 2016) and in adulthood, as hormones continue to regulate and modulate multiple behaviors and cognitive functions (reviewed in (Arnold et al., 2017; Ervin et al., 2013; Gillies and McArthur, 2010).

Phoenix et al. (1959) reported the first evidence on the effects of steroid exposure during early development (Phoenix et al., 1959). Within this report, the authors also defined two phases of this effect: organizational and activational effects. The former are permanent and can only occur early in life (around birth), while the later are transient and usually occur in adulthood. Following this hypothesis, during many years, pubertal secretions were

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29 thought as “activators” of neural circuits that were “organized” during the perinatal period. However, thanks to more recent research, others have been able to proposed a two-stage model of development in which hormone-driven adolescent organization is viewed as a refinement of the sexual differentiation that occur during perinatal neural development (Schulz et al., 2009; Schulz and Sisk, 2006; Sisk et al., 2003; Sisk and Zehr, 2005). During the adolescent phase of organization, steroid-dependent refinement of neural circuits results in long-lasting structural changes that modify adult behavioral responses to hormones and socially-relevant sensory stimuli.

In normal development, puberty occurs between P30-40 in the rat (Schwarz, 2016) and can be roughly approximated by the onset of the first estrus and vaginal opening (Gaytan et al., 2017) and in males by preputial separation (Gaytan et al., 2009). Although females typically enter pubertal stages ahead of males in this approximate range. Puberty (and adolescence in general) is characterized by marked changes in social behavior and cognition (including learning and memory) (Spear, 2000; Steinberg, 2005) (for a review see (Choleris et al., 2018)). This process requires significant development of the adolescent brain and often involves many of the same developmental processes used during initial construction of the nervous system, including (but not limited to) neurogenesis (Ahmed et al., 2008; Eckenhoff and Rakic, 1988; He and Crews, 2007; Pinos et al., 2001; Rankin et al., 2003) and elaboration and pruning of dendritic arborizations and synapses (Andersen et al., 1977; Huttenlocher and Dabholkar, 1997; Lenroot and Giedd, 2006; Sowell et al., 2004; Zehr et al., 2006).