CHANGES IN HIPPOCAMPAL SYNAPTIC PLASTICITY FOLLOWING EXPOSURE TO HIGH FREQUENCY HEAD IMPACTS (HF-HI)

A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience

By

Stephanie S. Sloley, B.S.

Washington, DC April 23, 2020 Copyright 2020 by Stephanie S. Sloley All Rights Reserved

ii CHANGES IN HIPPOCAMPAL SYNAPTIC PLASTICITY FOLLOWING EXPOSURE TO HIGH FREQUENCY HEAD IMPACTS (HF-HI)

Stephanie S. Sloley, B.S.

Thesis Advisors: Mark P. Burns, Ph.D & Stefano Vicini, Ph.D.

ABSTRACT

Concussions account for over 80% of all cases of TBI reported each year. Sustaining multiple concussions is associated with the development of lasting cognitive and memory impairments.

Chronic traumatic encephalopathy (CTE), a slowly developing tauopathy, has been linked to the development of these impairments; however, it is unknown if the cognitive deficits associated with

CTE can occur prior to the deposition of tau and death of neuronal cells. The overarching goal of these studies was therefore to 1) identify any tau-independent mechanisms associated with deficit development following exposure to multiple concussive impacts and 2) to assess how injury frequency contributes to deficit development.

To perform these studies, our group has developed an experimental model of high frequency head impacts (HF-HI), where mice receive 5 hits a day for 6 days (30 hits total). It recapitulates the frequency of injuries experienced by athletes engaged in high contact sports like football and boxing. Immunohistochemical analyses revealed that HF-HI did not cause cell death, inflammation, tau accumulation or excitatory synapse loss compared to sham mice. Despite this, behavioral testing at one-month post injury showed that HF-HI results in deficits in hippocampal- dependent learning and memory. I hypothesized that synaptic adaptations resulting from HF-HI were underlying the development of the deficits. To assess synaptic plasticity, I measured long- term potentiation via stimulation of the Schaffer collateral pathway, as this has been described as the cellular mechanism underlying learning and memory. I discovered a significant impairment in

iii LTP despite there being no difference in their stimulus input/output curve. Whole cell patch clamp electrophysiology was then done on individual CA1 pyramidal neurons at 24 hours and 1 month after HF-HI. Current clamp experiments revealed differences in intrinsic cellular properties and excitability 24 hours post impacts, which resolved after 1 month. Voltage clamp experiments revealed that NMDA receptor contributions to excitatory post synaptic currents were increased at

24 hours and 1 month after impacts, reinforcing the hypothesis that synaptic adaptations related to plasticity occur following HF-HI. Taken together, these changes illustrate that postsynaptic modifications to glutamatergic receptors arise following HF-HI, and are sufficient to alter plasticity, learning and memory.

iv ACKNOWLEDGEMENTS

To my mentors, Drs. Mark Burns and Stefano Vicini, thank you for all of your guidance throughout the course of this project. Mark, thank you for entrusting me with the freedom to truly make this project my own. Stefano, thank you for taking me on as one of your students, and for inspiring me with your enthusiasm for neurophysiology. I must also thank Dr. John Partridge for teaching me how to patch, and for the support, wisdom, and encouraging words that carried me through even the most frustrating experiment days.

To the Burns lab, thank you for brightening so many of my dark days with electronic dance music, excellent GIFs, movie quotes and witty banter. You’ve taught me that love is a coconut on the palm tree of life. To the Vicini lab, thank you rescuing me from so many disasters, and for feeding me when my experiments ran longer than expected. I am grateful to have worked hard with, laughed with and learned from the members of both labs.

I would like to extend my sincerest gratitude to the members of my thesis committee: Drs.

John Partridge, Katherine Conant, Jian-Young Wu and Zygmunt Galdzicki. Your guidance, both professionally and scientifically, has meant the world to me. Additionally, I am grateful to Dr.

Patrick Forcelli for his advice about statistics, science and life. Now more than ever, I am thankful for the resilience of the IPN community and the compassionate leadership of Dr. Kathy Maguire-

Zeiss.

Thank you to my family: Mom, Dad, Rich, Tiff, Matt, Naydia, Bubbeh, Dad-dad, and my love Evan. I wouldn’t have made it to this point without you. And thank you to my amazing roommates/soulmates, and my friends both within and outside of Georgetown. I’m so fortunate to have you all in my corner.

v TABLE OF CONTENTS

Chapter 1: Introduction ...... 1

Overview of Traumatic Brain Injury ...... 1

Primary and Secondary Injury in Moderate and Severe Traumatic Brain Injury ...... 3

Mild Traumatic Brain Injury ...... 11

Primary Injury and Mild Traumatic Brain Injury ...... 13

Secondary Injury and Mild Traumatic Brain Injury ...... 14

Chronic Traumatic Encephalopathy ...... 18

The Present Study ...... 24

Chapter 2: Methods ...... 29

High Frequency Head Impact (HF-HI) Model ...... 29

Pharmacological Treatments- in vivo ...... 29

Behavioral Testing ...... 30

Aβ40 ELISA ...... 31

Western Blot ...... 31

Immunohistochemistry ...... 32

Golgi Staining and Dendritic Spine Counts ...... 34

Local Field Potential Electrophysiology ...... 34

Whole Cell Recordings ...... 35

Recording Electrode Production ...... 45

Statistical Analysis ...... 45

vi Chapter 3: HF-HI elicits learning and memory deficits in the absence of pathological changes in classical injury-associated markers ...... 46

Introduction ...... 46

Results ...... 49

Discussion ...... 66

Chapter 4: Intrinsic and synaptic properties of hippocampal CA1 pyramidal neurons are acutely altered by HF-HI ...... 70

Introduction ...... 70

Results ...... 75

Discussion ...... 95

Chapter 5: Glutamatergic receptor signaling in hippocampal CA1 pyramidal neurons is altered by HF-HI ...... 102

Introduction ...... 102

Results ...... 107

Discussion ...... 124

Chapter 6: Pretreatment with memantine reverses the behavioral and electrophysiological changes associated with HF-HI ...... 130

Introduction ...... 130

Results ...... 134

Discussion ...... 143

vii Chapter 7: General discussion ...... 147

Discussion ...... 147

Limitations ...... 155

Future Directions ...... 160

Appendix: Identifying changes in calcium activity relative to synaptic plasticity induction after

HF-HI ...... 170

Introduction ...... 170

Results ...... 176

Discussion ...... 185

References ...... 190

viii LIST OF FIGURES

Figure 2.1. Calculations performed using hyperpolarizing current clamp recordings...... 39

Figure 2.2. Illustration of calculation parameters for the NMDA/AMPA ratio...... 42

Figure 3.1. HF-HI does not cause a detectable increase in apoptosis or axonal damage...... 51

Figure 3.2. Inflammation is not detectably increased after HF-HI...... 52

Figure 3.3. Accumulation of phosphorylated tau and APP was not observed in the cortex after HF-HI...... 53

Figure 3.4. Accumulation of phosphorylated tau and APP was not observed in the hippocampus after HF-HI...... 55

Figure 3.5. HF-HI does not cause detectable accumulation of Aβ-40 in the cortex and hippocampus...... 57

Figure 3.6. Chronic cognitive deficits occur after exposure to HF-HI...... 60

Figure 3.7. Long-term potentiation is impaired in the Schaffer collateral pathway 24 hours after HF-HI...... 63

Figure 3.8. Excitatory synapses are not lost after HF-HI...... 65

Figure 4.1. Presynaptic vesicular release is unaltered by HF-HI...... 76

Figure 4.2. Action potential firing rate is decreased acutely after HF-HI...... 78

Figure 4.3. Action potential firing rate normalizes 1 month after HF-HI...... 79

Figure 4.4. Inward rectification of potassium currents is altered acutely by HF-HI...... 81

Figure 4.5. Changes in intrinsic properties of CA1 cells are resolved 1 month after HF-HI...... 83

Figure 4.6. Spontaneous activity is not altered acutely by HF-HI...... 86

Figure 4.7. Spontaneous activity is not altered chronically by HF-HI...... 88

ix Figure 4.8. Action potential independent activity is altered acutely by HF-HI...... 91

Figure 4.9. Action potential independent activity normalizes 1 month after HF-HI...... 93

Figure 5.1. The NMDA/AMPA ratio is acutely increased by HF-HI...... 109

Figure 5.2. The NMDA/AMPA ratio is chronically increased by HF-HI...... 111

Figure 5.3. HF-HI does not produce detectable silent synapse formation acutely...... 114

Figure 5.4. HF-HI does not produce detectable silent synapse formation chronically...... 116

Figure 5.5. Inward rectification mediated by calcium permeable AMPA receptors is unchanged by HF-HI...... 119

Figure 5.6. Extrasynaptic and perisynaptic NMDA EPSCs are unchanged by HF-HI...... 122

Figure 6.1. Spatial learning and memory are recovered by pretreatment with memantine...... 136

Figure 6.2. Memantine pretreatment prevents deficits in hippocampal-dependent memory. .... 139

Figure 6.3. Memantine pretreatment prevents changes in the NMDA/AMPA ratio...... 141

Figure 7.1. Schematic of changes in glutamatergic receptor expression elicited by HF-HI...... 153

Figure A.1. Examples of somatic and dendritic ROIs...... 178

Figure A.2. Baseline calcium transients elicited by single stimulation in somatic ROIs...... 179

Figure A.3. Baseline calcium transients elicited by single stimulation in dendritic ROIs...... 180

Figure A.4. Acute changes in somatic calcium in response to HFS after HF-HI...... 181

Figure A.5. Acute changes in dendritic calcium in response to HFS after HF-HI...... 183

Figure A.6. Preliminary ensemble analysis of sham and HF-HI slices after HFS...... 189

x LIST OF TABLES

Table 1. List of antibodies used for western blot and immunohistochemical analyses...... 33

xi LIST OF ABBREVIATIONS

AD Alzheimer's disease ADP Adenosine diphosphate AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AO Apical obliques APOE Apolipoprotein E APP Amyloid precursor protein ATP Adenosine triphosphate Aβ Amyloid beta BBB Blood brain barrier BDNF Brain derived neurotrophic factor BMR Bicuculline methobromide CA Cornu ammonis CaMKII Calcium-dependent protein kinase II CC Concussive convulsions CCI Controlled cortical impact CHI Closed head injury Closed head impact model of engineered rotational CHIMERA acceleration CNQX Cyanquixaline CPP 3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid CREB cAMP response element binding protein CSF Cerebrospinal fluid CsMeSo4 Cesium methanesulfonate CT Computed tomography CTE Chronic traumatic encephalopathy CV Coefficient of variation DAMPS Damage associated molecular patterns DG Dentate gyrus DS Down syndrome ELISA Enzyme linked immunosorbent assay EPSC Excitatory postsynaptic current EPSP Excitatory postsynaptic potential ER Endoplasmic reticulum GABA γ-aminobutyric acid GCS Glasgow coma scale GECI Genetically encoded calcium indicator GFAP Glial fibrillary acid protein HF-HI High frequency head impacts HFS High frequency stimulation

xii ICP Intracranial pressure Ih Afterhyperpolarizing current IKR Inwardly rectifying potassium current IL Interleukin InsP3Rs Inositol 1, 4, 5-triphosphate receptors IPSC Inhibitory postsynaptic current IV Current versus voltage KA Kainate Kgluc Potassium gluconate LF-HI Low frequency head impacts LTD Long term depression LTP Long term potentiation MCP Monocyte chemoattractant protein-1 mGluR Metabotrophic glutamate receptor MMP Matrix metalloproteinase MPTP Methyl-4- phenyl-1,2,3,6-tetrahydro pyridine MRI Magnetic resonance imaging MWM Morris water maze NAC N-acetyl-cysteine NF Neurofilament NFL National football league NFT Neurofibrillary tangle NMDA N-methyl-D-aspartic acid NMDG N-methyl D-glucamine NSE Neuron specific enolase NT Neuropil threads PCS Post concussive syndrome PD Parkinson's disease PHF Paired helical filaments PPCS Prolonged post concussive syndrome p-tau Phosphorylated tau PTE Post traumatic epilepsy PVBC Parvalbumin basket cell ROI Region of interest RyR Ryanodine receptor TBI Traumatic brain injury TDP-43 TAR DNA-binding protein 43 TNF Tumor necrosis factor TTX Tetrodotoxin VEH Vehicle VGCC Voltage gated calcium channel

xiii Chapter 1

Introduction

Traumatic brain injury (TBI) is the world’s leading cause of disability, morbidity and mortality in individuals under the age of 45.1,2 Over 1.7 million TBIs occur each year in the United States, and approximately 52,000 of those incidents are serious enough to result in death.3 The economic burden of TBI in the United States alone was estimated to be $76 billion dollars in 2010, which includes both the costs that arise due to initial hospitalization and treatment, as well as costs incurred for continuing treatment needed after TBIs are sustained.4 A 2003 study found that over

43% of hospitalized survivors developed chronic disabilities or impairments resulting from their

TBIs and required either long-term care or rehabilitation.5 This makes TBI an incredibly prevalent and costly health crisis, which has life-altering effects on its survivors.

Overview of Traumatic Brain Injury

TBI is defined as an alteration in brain function or other evidence of brain pathology caused by an external force.6 The age groups that tend to be most affected are children ages 0-4, adolescents/young adults ages 15-19, and adults age 75 and over, and the incidence of TBI is higher in males than in females.7,8 The leading cause of TBIs is involvement in motor vehicle accidents, with motor vehicle occupants being the predominant sufferers in high-income countries, and pedestrians, motorcyclists and cyclists being the majority of sufferers in middle to low income countries.9

TBI is classified as either mild, moderate or severe depending on several diagnostic criteria.

One of the most common metrics to assess TBI severity is the level of consciousness of the affected individual, which is measured using the Glascow Coma Scale (GCS). The GCS was first introduced by Teasdale and Jennett in 1974, and while it remains one of the most widely used

1 scales for diagnosing TBI severity to date, 10 it is known to be a poor discriminator of mild TBI, and is difficult to assess in infants, young children and people with pre-existing neurological impairments.11 The GCS Scores are based on the individual’s ability to respond in three categories: eye opening, best verbal response and best motor response. Numerical scores are given for each category, and the overall score based on the sum of those scores is ascribed to each patient.12 A

GCS score of 3-8 corresponds to a severe TBI, while injuries that result in scores of 9-12 are classified as moderate and injuries resulting in a score of 13-15 are classified as mild.13

Severe and Moderate Traumatic Brain Injury

a. Severe Traumatic Brain Injury

Severe traumatic brain injury is usually diagnosed if patients display prolonged periods of loss of consciousness, as indicated by their GCS score. Those affected have a high likelihood of developing hypotension, hypoxaemia and brain swelling following injury, as well as increased intracranial pressure.14 The primary course of action for patients with severe TBI is to implement treatments acutely following injury to prevent the aforementioned cascades, however patient outcomes usually involve some long term disability or prolonged vegetative states.13,15

b. Moderate Traumatic Brain Injury

Moderate TBI is diagnosed in the emergency department if there is a GCS score of 9-12. Any combination of the following symptoms may also be present: confusion lasting days to weeks, and impairments (physical, cognitive and/or behavioral) lasting for months or permanently.16 Unlike severe TBI, moderate TBI presents with a variable injury progression and heterogeneous clinical outcomes which are difficult to predict. Patients may either deteriorate rapidly, or remain stable and have an overall favorable outcome once surgical interventions and therapies are applied.17

Approximately 60% of the patients diagnosed with moderate TBI are positive for intracranial

2 trauma when computed tomography (CT) scans are administered, and require either admission into the intensive care unit or surgery for a mass lesion or depressed skull fracture.18

Primary and Secondary Injury in Moderate and Severe Traumatic Brain Injury

a. Primary Injury

Moderate and severe traumatic brain injuries cause damage to the brain through both primary or secondary injury mechanisms. Primary injuries result from the actual mechanical force exerted on the brain, and occur immediately after the injury-inducing event. These injuries may be identified using structural or functional imaging.19 There are two subcategories of primary injury: focal and diffuse. Focal injuries occur most often in contusive or penetrating TBIs where there is an injury site (coup) or the formation of a lesion, and necrotic tissue as well as compromised blood flow or various kinds of hemorrhages are observed in close proximity to the site. Diffuse injuries normally arise from the forces that the head is exposed to, like rotational acceleration, which cause shearing and stretching of brain tissue and vasculature.20,21 Vascular shearing and torsion can inflict hypoxic/ischemic damage on the brain, causing tissue to become necrotic or dysfunctional.

Neuronal axons are also sensitive to diffuse injury, which has serious repercussions on axonal transport and subsequently brain function.22

b. Secondary Injury

Secondary injuries tend to have a delayed onset, as they develop from the initial mechanical insult. These injuries involve the development of conditions such as global or focal ischemia, cerebral metabolic dysfunction, oxidative stress, and inflammation.1 While primary injuries cannot be prevented at the hospitalization stage, there is a therapeutic window in which it is possible to stave off some of the deleterious effects of the secondary injury cascades.23 In patients with severe injuries, there are predictable changes in the acute phase that denote the occurrence of

3 certain kinds of secondary injury, which allows for intervention and treatment to stave off adverse outcomes. These changes are less well understood in moderate TBI, due to the heterogeneity of its presentation and the tendency of some to treat moderate and severe TBI similarly, so interventions are harder to implement.24

c. Secondary Injury Mechanisms

While there are multiple secondary injury cascades that contribute to loss of function and overall damage of the brain after injury, there are three key mechanisms that are heavily studied in the field. Excitatory amino acid/ionic imbalance, inflammation and protein dysregulation are all thought to be hallmarks of TBI. Many of the experimental therapies that move to clinical trials are focused on reducing the effects of these cascades to promote survival and maintain function, as they can cause massive damage and impair cognitive function long after the initial insult is sustained.

i. Excitatory Amino Acid and Ionic Imbalance

The excitatory amino acid glutamate can play a pivotal role in the secondary injury cascade.25 Several human and animal studies have revealed that after moderate to severe TBI, injured or dying neurons at the lesion epicenter indiscriminately release glutamate. 26 This causes increased activation of glutamatergic receptors, particularly the N-methyl-D-aspartate (NMDA) ionotropic glutamatergic receptors.27 Elevated extracellular glutamate induces a cycle in which adenosine triphosphate (ATP) is depleted, increases in intracellular calcium occur and contribute to prolonged depolarization, and an efflux of potassium ion occurs. These events perpetuate depolarization of cells and release of glutamate, and commonly result in cell death.28 This process, also called excitotoxicity, allows degeneration to continue long after the initial trauma is

4 sustained.29 In addition to excitotoxicity, glutamate imbalance can disrupt other physiological processes (described below) to contribute to loss of function and poor outcomes after TBI.

Elevated extracellular glutamate can alter synaptic plasticity, potentially hindering cognitive and motor function.26 Under normal physiological conditions, NMDA receptors mediate synaptic function by bi-directionally regulating the activation and expression of glutamatergic, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors at the post- synaptic density in a calcium dependent manner. Changes in AMPA receptor properties profoundly alter synaptic transmission by either strengthening or decreasing the synaptic coupling of neurons. These processes have been described experimentally as long-term potentiation (LTP) and long-term depression (LTD), the cellular models of experience-dependent plasticity thought to underlie learning and memory.30-32 When extracellular glutamate concentrations increase to potentially excitotoxic levels, as is observed in AD, the subsequent increase in NMDA receptor activation in response to ambient glutamate levels causes an increase in non-specific calcium ion influx, generating too much synaptic “noise” for LTP to occur.33 In moderate to severe traumatic brain injury, where elevated extracellular glutamate and excitotoxicity have been reported, studies have also demonstrated impaired LTP or LTD after injury.34

Elevated extracellular glutamate can also alter the balance between excitation and inhibition (and shift the excitation to inhibition or E:I ratio), leading to increased excitability that alters normal brain function.35 In fact, these alterations can lead to the occurrence of post traumatic epilepsy (PTE), a chronic condition that develops months to years after injuries are sustained.

Severe TBI carries with it the highest risk for developing PTE.36 One way that a shift in the E:I ratio can allow the development of PTE is through compensatory changes in receptor expression.

Decreases in GABAA receptor alpha 1 subunits occur in the hippocampus after TBI, promoting a

5 hyperexcitable state and contributing to the development of temporal lobe epilepsy after TBI.37

Following controlled cortical impact (CCI), an experimental model of TBI, reductions in GLUR1 subunits (found on glutamatergic AMPA receptors) and increases in NMDA GLUN2B subunits occurred in response to elevated extracellular glutamate.38 Decreased expression of GLUR2 subunits in AMPA receptors has also been observed following TBI, conveying permeability of calcium to these AMPA receptors and thereby promoting excitotoxicity.39 These changes in receptor expression promote increased excitability, which has lasting effects on synaptic plasticity and network function after TBI. The composition and function of AMPA and NMDA receptors is discussed more in depth in Chapter 5.

ii. Inflammation

The inflammatory response is an innate measure taken by the body to promote wound healing and neuroprotection. In TBI, particularly when there is formation of a chronic lesion, this response can become damaging.40 After sustaining a moderate or severe TBI, there tends to be an increase in intracranial pressure (ICP), which can reduce cerebral perfusion pressure (CPP) and cause secondary ischemia and edema.41 Brain edema in TBI occurs through either cellular or vasogenic mechanisms. Cellular or cytotoxic edema occurs when extracellular cations enter neurons and astrocytes and accumulate, causing a subsequent influx of anions in an attempt to restore electrical neutrality. These ionic changes prompt an osmotic response of the affected cell which ultimately leads to oncotic or necrotic cell death.42 Vasogenic edema ultimately allows for components of blood plasma that the brain is otherwise safeguarded against to enter. It involves disruption of the blood brain barrier (BBB), the brain’s safeguard, through endothelial damage, widening of tight junctions, or induction of abnormal vesicular transport.43 This disruption can last

6 anywhere between several days to a few weeks or years, with biphasic permeability occurring first within 4-6 hours after the injury and then again several days after the injury.43,44

Several cell types present in the brain after injury contribute to the inflammatory process.

Peripheral neutrophils, macrophages, natural killer cells, T helper cells, and T cytotoxic suppressor cells can all invade the brain through the compromised BBB after injury. Infiltrating leukocytes release pro-inflammatory molecules such as cytokines, cytotoxic proteases, and reactive oxygen species, which perpetuate a state of inflammation by activating the resident immune cells of the brain, microglia. Similar pro-inflammatory molecules released by microglia can prolong BBB permeability and also independently maintain inflammation in the absence of BBB disruption.45

Astrocytes are also activated following TBI, and can produce cytokines, chemokines, DAMPs and alarmins to mediate inflammation, as well as impair repair mechanisms.46 This is important for recovery after TBI as the majority of the cell types mentioned produce cytokines, and the presence of certain kinds of cytokines is associated with the outcome.

Cytokines are polypeptides that are upregulated either acutely or chronically in response to pathological situations, and help to mediate inflammatory responses as well as intercellular communication and tissue homeostasis.47 The interleukin (IL) family of cytokines has been studied extensively due to its role in impeding functionality after injury. Pro-inflammatory cytokines IL-

1B, IL-6, IL-8, and anti-inflammatory cytokine IL-10 are linked to poor outcomes and increased mortality.48 Tumor necrosis factor alpha (TNFα) has also been linked to promoting microvascular damage, edema formation, interference with tissue repair mechanisms, and poor outcomes after

TBI. Pharmacological blockades of TNFα can also be protective after injury. These effects are time dependent, as treating with TNFα prior to inducing ischemia in experimental conditions can also reduce injury severity, and the presence of TNFα can stimulate protective mechanisms when

7 administered at later time points (usually a matter of weeks after injury).49 Another cytokine with an important role in TBI is Fas ligand (FasL), which has been shown to be upregulated following injury through release from microglia and neurons, and is implicated in eliciting neuronal apoptotic processes.47 Monocyte chemoattractant protein-1 (MCP-1), another cytokine found to be upregulated after injury, and perpetuated inflammation by signaling to monocytes and resident glia to injury foci.50 In experiments using mice MCP-1 deficient mice in a stroke model, it was shown that infarct size was significantly smaller than in mice with intact MCP-1, suggesting a pathogenic role of MCP-1 after brain injury.51 Although the findings indicate that cytokines play different roles depending on many factors associated with injury, inflammatory cytokines have been shown to have an important influence on recovery after TBI.

iii. Protein Dysregulation

After sustaining a TBI, there can be changes in proteins triggered by the acute phase of the injury that contribute to chronic impairment. The majority of these proteins have also been implicated in the development of multiple neurodegenerative disorders. While dysfunction in proteins such as alpha synuclein (which plays an important role in the development of Parkinson’s

Disease) and transactive response-DNA binding protein 43 (TDP-43; implicated in frontotemporal dementia and amyotrophic lateral sclerosis) have been observed following TBI, three proteins are most commonly cited as being important for influencing outcomes after TBI.52,53

Axonal injury is common in TBI, and is a major source of the common problematic proteins. When the axonal cytoskeleton is damaged, for instance, it can cause axonal swellings in which proteins like amyloid beta precursor protein (APP) and neurofilament (NF) can accumulate.22 Cleavage of APP into amyloid beta (Aβ) by either beta or gamma secretases also occurs, and Aβ has been shown to accumulate in swollen axons in multiple models of TBI.54

8 Plaques composed of Aβ have also been found in clinical and preclinical cases of TBI, and can contribute to degeneration.55 The presence of Aβ plaques has been shown exacerbate excitotoxicity, and trigger chronic inflammatory reactions.56,57 Further, knocking out the gene responsible for coding the beta secretase enzyme or pharmacological inhibition of the gamma secretase enzyme have been shown to reduce Aβ accumulation and prevent cognitive decline in a

CCI model of TBI.58 Aβ plaques are the hallmark pathology associated with Alzheimer’s Disease

(AD), so it is not surprising that myriad research has identified TBI as a risk factor for AD.59,60

Taken together, these findings suggest that Aβ accumulation and deposition occur following moderate and severe TBI, and the presence of Aβ can exacerbate any physical or functional damage that arises.

Another protein typically found in AD and other neurodegenerative disorders that also becomes dysfunctional after TBI is the microtubule stabilizing protein tau.61 Tau has been shown to dissociate from microtubules, become hyperphosphorylated, form intraneuronal aggregates, and is aberrantly processed by cells. These aggregates can be either paired helical filaments (PHF), twisted ribbons or straight tangles.62 The tau-based end-products of this cycle can then go on to promote cell death, and affect multiple cellular functions, including apoptosis and mitochondrial homeostasis, which contributes to impaired activity after injury.63 Clinically and experimentally, it has been shown that tau can be detected in the serum following TBI and the levels increase proportionately with the injury severity.64 Neurofibrillary tangles (NFTs) formed from PHFs or other tangles containing tau were found in post-mortem patient tissue after exposure to even a single moderate or severe TBI, regardless of age. Tau oligomers were also shown to arise after

TBI was induced experimentally via CCI and fluid percussion injury, and the injection of tau oligomers into the brain accelerated the onset of cognitive deficits.65 Since the presence of NFTs

9 and aberrant tau is associated with neurodegenerative diseases that have late- life onsets, the presence of these NFTs in TBI patients suggests that sustaining a TBI can accelerate the onset of these diseases.66 These findings suggest that aberrant tau can promote decline and contribute to poor functional outcomes after moderate and severe TBI.

d. APOE4 and Moderate and Severe Traumatic Brain Injury

Apolipoprotein E (apoE) is a lipoprotein constituent which serves to transport triglyceride from the liver to peripheral tissues, and redistributes cholesterol among cells.67 It is present in multiple tissues in the body as well as throughout the CNS, and is primarily synthesized and secreted by astrocytes and macrophages.68,69 The gene locus that encodes the protein is polymorphic, and the alleles code for three major isoforms: E2, E3 and E4.70 The most commonly expressed allele is

E3, with 65-70% of the population expressing one or two copies of it. The E4 allele is a lot less commonly expressed (15-20%), but has been implicated as contributing to adverse outcomes in multiple neurodegenerative diseases and injuries.71 For example, there is a significant association between the E4 allele and the incidence of late-onset familial AD as well as sporadic AD.72 Studies have shown that the apoE4 protein interacts very efficiently with Aβ, producing more complex monofilaments than those that are observed in apoE3 carriers. 73 Additionally, carboxyl terminal truncated fragments of apoE4 are found in the brains of patients who suffered from AD, and the presence of only these fragments has been shown to elicit neurodegeneration and deficient spatial learning and memory experimentally.74 Given these connections between the E4 allele and multiple mechanisms of degeneration in AD, as well as the connection between AD and TBI, it is not surprising that APOE4 is also a risk factor for poor outcomes following TBI.

After TBI, there can be deposition of Aβ which is similar to what is observed in the brains of patients with AD. Postmortem studies have shown that if patients who have had a head injury have

10 an E4 allele, there tends to be a higher instance of Aβ deposition than in patients with any other form of the APOE allele.75 Clinical outcomes are also affected adversely by E4. A study on 6 months post-TBI outcomes found that regardless of age at the time of injury, the proportion of patients with lower GCS scores and poorer prognoses was higher in APOE4 patients than those with any other APOE alleles present.76 Patients under the age of 15 who suffered a head injury and are APOE4 carriers also have a higher propensity towards experiencing a negative outcome.77 The presence of the E4 allele is also associated with a higher incidence of moderate/severe contusional injury and severe ischemic brain damage in cases of TBI that were fatal.78 Patients that suffered from a moderate/severe TBI exhibited worse learning and memory if they possessed an E4 allele than those that did not.79 After severe TBI, levels of S-100B and neuron-specific enolase (NSE), injury markers shown to increase in blood and cerebrospinal fluid (CSF) after injury, were elevated to a much higher extent in APOE4 carriers versus patients with no APOE4 present.80 Taken together, these findings indicate that presence of one or more E4 alleles is associated with worse outcomes after moderate and severe TBI, although the precise mechanisms by which APOE4 exacerbates injury are still unclear.

Mild Traumatic Brain Injury

Mild TBI, commonly referred to as concussion, accounts for over 75% of the TBI cases reported each year.81 Clinically, concussion may present with symptoms including confusion, disorientation, loss of consciousness, short-lived post-traumatic amnesia, and neurological abnormalities like seizures.82 These symptoms are collectively referred to as post-concussive syndrome (PCS). If symptoms persist, it is classified as prolonged post concussive syndrome

(PPCS).83 Importantly, after a single mild TBI, the symptoms are commonly transient, and tend to resolve spontaneously.84

11 Studying the effects of mild TBI has posed a unique challenge for TBI research. While moderate and severe injuries have hallmark structural abnormalities that can be observed via standard imaging techniques and quantified, following mild TBI brains appear “normal”. This makes mild TBI difficult to assess clinically, as well as creates a challenge for adapting reliable animal models to study the cellular and molecular changes that it can elicit. Further, factors such as impact frequency and force can strongly influence how the brain changes after mild TBI.

a. Repetitive Mild Traumatic Brain Injury

Mild TBI is unique when compared to moderate to severe injuries, mainly because the outcomes are less predictable. They occur on a continuum, where they can range anywhere from experiencing no consequences to impaired neurocognitive performance and the presence of histopathological injury markers.85 Chronic impairments after exposure to a single mild TBI rarely occur, usually with a late onset and resulting from mechanisms resembling secondary injury. The chances of developing lasting chronic impairments is significantly higher after repetitive mild

TBIs are sustained,86 and sustaining one concussion is a risk factor for sustaining subsequent concussions.87 Exposure to multiple concussions plays a large role in the injury sequelae that ensue, with the current theory in the field being that impairment severity increases longitudinally, and in an injury frequency-dependent manner.88 Because of this, single mild TBI and repetitive mild TBI are often examined independently.

b. Subconcussive Head Impacts

In some instances, head impacts can occur that produce no visible acute neurological dysfunction or discernable symptoms, and are therefore not diagnosed as concussions. These impacts are referred to as subconcussive, and generate a risk for development of deficits when multiple are sustained.89 In fact, the likelihood of neurophysiological impairments occurring

12 increases as the number of impacts sustained increases.90 Changes such as impaired visual working memory and neurophysiological deficits in brain regions adjacent to where these impacts were sustained have been shown to occur.91 Subconcussive head impacts can also alter white matter integrity, especially at the junctions where white and grey matter intersect.92 Even in the absence of clinical symptoms, subconcussions can cause deficit development, and should therefore be taken into consideration when studying how head impacts can alter brain function.

Primary Injury and Mild Traumatic Brain Injury

The roles of primary and secondary injury in determining the degree of damage are not as well understood in mild TBI as they are in moderate to severe injuries. Concussions present seemingly without a primary injury, unlike moderate and severe TBI. There are rarely any focal injuries such as lesions, hemorrhages, obvious axonal injuries and other gross structural abnormalities detectable on either CT scans or magnetic resonance imaging (MRI) scans.93 In the absence of focal pathology, diffuse injury could underlie some of the damage sustained after a mild TBI.

Presently, it is thought that diffuse axonal injuries comparable to those observed in more severe TBIs occur after concussions and could potentially underlie any resulting deficits, although they are not easily detectable using conventional MRI and CT scans. Using T2 weighted spin-echo imaging and T2* imaging, abnormalities associated with diffuse axonal injuries may be detected.

One study found such abnormalities in 6 out of 20 patients classified as having a mild TBI with no other abnormalities in CT scans.94 Advanced imaging methods such as diffusion tensor imaging are more astute at identifying changes arising from diffuse axonal injury, however there has been very little evidence linking the changes seen on these scans to actual axonal pathology. More convincing evidence of diffuse axonal injury causing axonal pathology has been provided in

13 experimental models of injury. For instance, using a porcine rotational acceleration model of concussion, multi-focal axonal pathology was found throughout the white matter.95 It has also been shown that repetitive mild TBI increases the degree of diffuse axonal injury that can occur.

After sustaining a single mild TBI, an increase in the expression of sodium channels along axons occur that predispose the axons to undergo a higher degree of degeneration if subsequent mild

TBIs are sustained.96 While diffuse axonal injury seems to play a role in generating impairments following mild TBI, imaging studies that find no evidence of axonal injury suggest that there are likely other mechanisms contributing to deficit development.

Secondary Injury and Mild Traumatic Brain Injury

Cascades comparable to secondary injuries observed in more severe TBIs have been observed more definitively following mild TBI. A range of alterations, including changes in intra- and extracellular ionic concentrations, free radical production, inflammation and metabolic changes can occur.97 Because of their role in more severe injuries, many studies on concussions have been aimed at understanding how excitatory amino acid/ionic imbalance, inflammation and protein dysregulation contribute to chronic impairments. The distinct contribution of each of these processes to the outcomes after concussive injury, are described below.

a. Excitatory amino acid and ionic imbalance

An influx of calcium and subsequent efflux of potassium and glutamate similar to what is seen after more severe injuries has been shown to occur after concussions are sustained, though not to the extent where excitotoxicity is elicited.98-100 Perturbations of these ions and glutamate can trigger more voltage and ligand gated ion channels to open, perpetuating this dysregulated state.101

In an effort to correct this ionic imbalance, the ATP-dependent ionic pumps become hyperactive, causing metabolic dysfunction.102 Sustaining multiple mild TBIs within 24 hours of one another

14 can delay the rectification of this metabolic dysfunction, increasing the likelihood of functional impairments in neurons and the development of neurocognitive impairments.103

It has been shown that elevations in extracellular glutamate and impaired synaptic plasticity occur in moderate and severe forms of injury. Several studies focused on assessing plasticity after a single mild TBI have also shown that LTP is deficient after injury.104-108 The findings less straightforward when plasticity was examined in experimental models of repetitive mild TBI.

Some studies have reported reduced LTP, in line with findings for single mind TBI and more severe injuries. For instance, in a lateral fluid percussion injury model where rats received two injuries 48 hours apart, deficient LTP, learning and memory were observed.109 Exposure to two blast injuries, which is thought to elicit damage similar to what is observed after sports concussions, caused a reduction in LTP when the injuries were delivered 24 hours and 72 hours apart, but not 144 hours apart.110 Long term potentiation was also shown to be attenuated after mice received 1 closed head injury (CHI) daily for 4 days.111 Unlike the previous studies, a separate study that employed CHI once daily for 3 days found enhanced LTP in the ipsilateral hemisphere as compared to sham injured and single CHI mice.112 These findings suggest that while plasticity is altered after repetitive mild TBI, the directionality of the change in LTP can be variable after repetitive mild TBI. These variations could be attributed to differences in the time interval between hits or the number of hits given, but provide factors to consider when exploring how repetitive mild TBI can alter function and plasticity.

Mild TBI can also alter the E: I balance in the brain, likely through changes in excitatory and inhibitory receptor expression. Inhibition mediated by GABAB has been shown to increase

35 chronically in athletes who have had at least two concussions. Decreased GABAA inhibitory transmission has also been shown to decrease in experimental models of mild TBI.107 Excitatory

15 transmission is also altered. The GLUR2 subunit expression decreases after injury,113 as well as expression of the GLUN2B subunit.114 The net changes in the E:I ratio that occur after mild TBI are unlikely to result in the development of PTE following concussive head injury.115 Acutely, it can contribute to concussive convulsions (CC), which occur in 1 out of 70 patients and are not considered epileptic events. They present with a tonic phase occurring within 2 seconds of impact, followed by a clonic or myoclonic phase lasting up to several minutes.116 The occurrence of CC does not increase the risk of developing PTE, or long-term neurological sequelae.117

b. Inflammation

Inflammatory responses similar to that observed in more severe forms of injury have been examined as a potential origin for deficit development after mild TBI. Since infiltration of peripheral immune cells can be a source of inflammatory factors, disruption of the BBB has been studied after mild TBI. Rodents studies have yielded mixed results regarding whether the BBB was disrupted after mild TBI.118 Clinically, the results are also ambiguous. In football players exposed to multiple concussive injuries, some BBB permeability has been reported using dynamic contrast enhancing MRI.119 Studies done on boxers exposed to repetitive head impacts have shown increases in S-100-B (a biomarker of BBB integrity)120, as well as no change in S-100-B.121 The degree of immune cell response is influenced by the number of injuries sustained. An experimental model comparing single and repetitive mild TBI showed that both produced the activation of microglia and astrocytes characteristic of an inflammatory response, however the response increased as time progressed for the repetitive mild TBI but peaked at a particular time-point after injury.122 Post-mortem samples from patients that sustained repetitive mild TBIs also exhibited inflammation.123 Further, inflammatory cytokine production similar to that observed after more severe injuries can also be influenced by concussive impacts. The concentration of inflammatory

16 cytokines like TNF-α, IL-1α, IL-1β, IL-6 and IL-10 was shown to increase acutely after mild injury.124 Each of these markers of inflammation were cited as being more prevalent after repetitive mild TBI than after a single mild TBI.125

Evidence supporting the ability of subconcussive head impacts to elicit an inflammatory response has also been inconclusive. Studies done on football players and soccer players have shown BBB disruption (as a function of S-100-B levels) following subconcussive head impact exposure.126-128 Experimental models and clinical research have shown that some inflammation can occur. In a mild lateral fluid percussion injury model of subconcussive injury, a significant elevation in immunoreactivity for glial fibrillary acid protein (GFAP), a marker for activated astrocytes was found in multiple brain regions. Increased CD68 positive cells (a marker for microglia/macrophages) was also found.129 A factor to consider regarding these findings, however, is that fluid percussion injury involves creating a craniotomy in order to induce the injury, which may contribute to the inflammatory response observed. Neuroimaging studies have shown that repetitive subconcussive head impacts can elicit blood brain barrier disruption and inflammation in humans, 130,131 however these findings are not consistently observed. Therefore, it is difficult to deduce the extend to the neuroinflammatory response elicited by subconcussive injury.

c. Protein Dysregulation

Changes in proteins known to be dysregulated after other brain trauma as well as in several neurodegenerative disorders have also been shown to exhibit dysfunction after repetitive mild TBI.

Pathological TDP-43 phosphorylation and translocation occurs after repetitive mild TBI, but not after single and more severe forms of TBI.132 Increased Aβ aggregation and deposition,133,134 and hyperphosphorylation and aggregation of tau have also been observed after repetitive mild TBI.135

When changes in these proteins occur after exposure to repetitive mild TBI and in a stereotypical

17 pattern, their presence is linked to the development of the neurodegenerative disorder chronic traumatic encephalopathy. The pattern of protein accumulation and their connection to behavioral dysfunction will be discussed in greater detail in the context of chronic traumatic encephalopathy, as this disorder is thought to underlie the impairment and dysfunction associated with repetitive mild TBI.

Chronic Traumatic Encephalopathy

Chronic Traumatic Encephalopathy (CTE) is a progressive neurodegenerative disorder characterized by the presence of phosphorylated tau (p-tau) tangles in distinct regions of the brain.

There are several “hallmark patterns” of pathology noted in CTE. Astrocytic/neurofibrillary tangles are commonly present, and distributed perivascularly and within the depths of cerebral sulci. Subpial and periventricular deposits of astrocytic tangles in areas of the cerebral cortex, diencephalon, basal ganglia may also be present. Additionally, neurofibrillary tangles distributed preferentially in superficial layers of the cerebral cortex also occur.136

a. CTE Stages

Preliminary identification of four disease stages, based on stereotyped structural changes and tau pathology, has been documented in the literature and provides a means of evaluating pathological severity.136,137 In Stage I, gross changes in the brain are generally not present except occasionally in the frontal horns of the lateral ventricles. There may also be p-tau NFTs, neuropil threads and astrocytic tangles limited to discrete perivascular regions in the depths of cerebral sulci of the superior, dorsolateral, lateral and inferior frontal cortices. It is rare for p-tau pathology in this stage to occur in the locus coeruleus, hippocampus, amygdala, entorhinal cortex, medulla or cingulate gyrus. Vascular Aβ deposits and perivascular Aβ plaques are not present, but TDP-43

18 inclusions in the subcortical frontal white matter and fornix may be. There is also gliosis of the white matter commonly at the apex of gyri and junction between the deep layers of the cortex.

Stage II involves mild enlargement of the frontal horns of the lateral ventricles and third ventricle, a cavum septum, and pallor of the locus coeruleus and substantia nigra. Pathology

(including p-tau NFTs, neurites surrounding small vessels and focal subpial p-tau in astrocytes) also spreads from the regions defined in Stage I to focal epicenters to the superficial layers of the adjacent cortex. The nucleus basalis of Meynert contains moderate NFTs and neurites, and structures in the medial temporal lobe shows mild neurofibrillary pathology. While TDP-43 pathology occurs in most subjects at this stage within subcortical white matter, the medial temporal lobe and brainstem, there is no Aβ pathology.

In Stage III, there is a reduction in brain weight and mild atrophy of the frontal and temporal lobes as well as enlargement of the lateral and third ventricles. It is also common that p-tau pathology becomes widespread at this stage, and can extend throughout the frontal, temporal, parietal and insular cortices. Cortical epicenters and depths of sulci display masses of NFTs and astrocytic tangles. At this stage, there is dense NFT deposition in the nucleus basalis of Meynert, and NFTs are also present in medial temporal lobe structures (i.e. the hippocampus and amygdala).

Most of the cases also show TDP-43 pathology throughout many brain regions, and there is still no Aβ deposition although neuritic Aβ plaques are found in some patients.

Finally, in Stage IV brains weight is significantly decreased, as atrophy tends to be widespread throughout the brain. There can be thinning of the hypothalamic floor, discoloration/atrophy of the mammillary bodies, and enlargement of the lateral and third ventricles. There is also atrophy of the white matter and thinning of sections of the corpus callosum. The locus coeruleus and substantia nigra also become very pale. The medial temporal

19 lobe will also have neuronal loss, gliosis, and NFT/ astrocytic tangles, and neurofibrillary degeneration. Notably, NFTs are now present throughout all of the regions of the hippocampus, and there is severe sclerosis of CA1 as well as predominantly p-tau reactive astrocytes.

b. CTE Symptoms

Current characterizations of CTE have identified an average age of onset for each of the stages.

Stage I is thought to occur at approximately 22.2 years of age, and the progress towards/ onset of

Stage II is approximately 39.0 years of age. The average age of onset for Stage III is 44.3, and

Stage IV is 57.2 years. Additionally, death resulting from CTE can occur anywhere between 14-

98 years of age.136,138 Age can also influence the symptoms that present. Stern et al. performed an assessment of 36 subjects diagnosed with CTE after autopsy, and sought to identify common symptoms of CTE, as well as any factors that could affect how these symptoms present. The study found that while some form of cognitive dysfunction eventually occurred in all patients, there were two distinct categories of CTE symptoms: behavioral/mood symptoms, and cognitive symptoms.

Patients whose initial presentation tended to occur at a younger age showed more behavioral and mood disruptions, including explosivity, impulsivity, violence, depression and hopelessness.

Patients whose initial presentation occurred at older ages tend to exhibit more cognitive symptoms, including executive dysfunction, episodic memory impairment, and dementia.139 In addition to these symptoms, others have observed motor manifestations, including Parkinsonism and ataxia.140

c. APOE4 and CTE

The presence of the APOE4 allele can exacerbate the development of adverse outcomes and pathology in patients after mild, moderate and severe TBI, and the same can be said for CTE.

Boxers with the E4 allele and high exposure to boxing were shown to have the highest scores on

20 the Chronic Brain Injury Scale, and the frequency of the E4 allele was higher in boxers who were deemed to have either probable, moderate or severe CTE versus those that were not.141 In a study done on neuropathologically confirmed CTE patients, 50% of the patients whose genotypes were assessed had at least one copy of the E4 allele. Given that the prevalence of the allele is 15% in the general population, this suggests that presence of the allele may be a risk factor for development of CTE.142 This study also found higher levels of soluble Aβ42 in E4 carriers, and in a different study of CTE patients, the presence of the APOE4 allele was also significantly associated with Aβ deposition.143 These findings suggest a higher likelihood of developing CTE, as well as a more exaggerated pathological phenotype is associated with the APOE4 genotype.

d. CTE and Boxing

The current incidence and prevalence of CTE are unknown, as the condition and stages are diagnosed post-mortem through observation of the aforementioned pathology. Presence of clinical symptoms is not sufficient to truly diagnose the disease, however early observations of these deficits in boxers began the characterization of the disease within the boxer population. The first reports of these cognitive and behavioral abnormalities were made in 1928 by Dr. Harrison

Martland. He named the condition “Punch Drunk” syndrome due to its high-frequency of occurrence among professional boxers when compared to the general population.144 As a deeper understanding of the condition developed from access to brain samples, it was renamed “Dementia

Pugilistica,” and a distinct neurodegenerative signature associated with the condition was identified.145 It was characterized as a progressive encephalopathy, and post-mortem examinations of brains revealed the presence of plaques similar to those seen in other dementias.146 Eventually, the condition became known as CTE.146-148 It was shown to involve the steady development of symptoms including ataxia, Parkinsonism/tremors, and dementia. The presence of cerebral atrophy

21 in frontal regions, and neurofibrillary changes similar to those observed in Alzheimer’s disease in the cortex and midbrain were also observed.147 In 1973, Corsellis et al., performed a comprehensive study of the brains of 15 deceased boxers and provided, at that time, the most definitive description of the neuropathology and clinical deficits associated with CTE.149 In even the characterizations of this disorder, it is important to note that the symptoms were observed to be distinctly different from post-concussive syndrome.150

e. CTE and Professional Football

While these early characterizations of CTE were invaluable, the scope of those who were likely to be affected was somewhat limited to professional boxers. Corsellis was one of the first researchers to report the occurrence of CTE in athletes other than boxers. The key feature uniting affected athletes in their likelihood of developing CTE was their elevated risk of sustaining multiple head injuries.151 Since then, a wealth of research has been conducted on repetitive concussive sports injuries and their role as a risk factor for developing CTE. Given their predisposition to sustaining repeat mild TBIs, it is no surprise that a connection between the development of CTE and professional football players was also revealed. Dr. Bennet Omalu reported the first documented case of CTE-like changes in a retired professional NFL player. Post- mortem examination of the brain revealed no structural abnormalities, and the presence of tau- positive NFTs in the neocortex and around blood vessels, neocortical tau-positive neurites in the neuropil, and diffuse neocortical Aβ plaques. Unlike the pattern of expression common in AD, this patient did not have NFTs present in the entorhinal cortex or hippocampus.152 Soon after, a second case of CTE in a former NFL player was reported. Similar to the first case, there were no gross abnormalities or atrophy and no hemorrhages detected. Contrary to the first patient’s findings, the presence of diffuse Aβ plaques was not detected, and NFTs and neuropil threads (NTs) were

22 observed in several regions including the hippocampus. The report noted that a potential contributing factor to the differences in pathology may be that the first patient had an APOE genotype of E3/E3, and the second patient’s genotype was E3/E4.153 These cases, although variable, displayed the stereotypical pathology attributed to CTE.

f. CTE Pathology in Sports Concussion Versus Blast TBI

For military personnel, exposure to the detonation of improvised explosive devices (IEDs) elicit a specific kind of mild traumatic brain injury known as blast mild TBI.154 Blast mild TBIs can arise through primary injury mechanisms (exposure from the blast pressure wave itself that can cause shearing), secondary injury mechanisms (head impacts resulting from shrapnel or fragments propelled by the overpressure wave), tertiary injury mechanisms (head impacts resulting from propulsion of the body into objects as a result of the blast wave) and quaternary injury mechanisms (heat or gases that are injurious and originate from the explosion).155 A human postmortem study comparing pathological findings after blast mild TBI to those related to repetitive sports concussions found neurofibrillary and glial tau tangles consistent with those observed in CTE in military veterans exposed to blast TBI (a finding that was recapitulated in a mouse model of blast TBI).156 Other studies have failed to confirm a relationship between the presence of tau pathology indicative of CTE and purely blast-related mild TBI, citing that characteristic tau pathology was only present in patients that had also sustained other kinds of head impacts.157 Although blast mild TBI elicits acute symptoms similar to those observed in postconcussion syndrome, as well as chronic deficits in some cases, evidence is so far inconclusive on whether or not this kind of mild TBI conveys the same predisposition towards the development of CTE.158

23 The Present Study

The connection between repetitive head impacts and cognitive dysfunction has been studied in the neural injury field since the 1920’s.159 Dr. Omalu’s studies on professional football players, as well as news stories about several high-profile National Football League (NFL) players and professional boxers believed to be affected by CTE has recently garnered immense mainstream media attention. As public awareness and fear of CTE rose, so too did the importance of gaining a better understanding of the clinical, pathological, and functional characteristics of the disease in order to disseminate the appropriate information. Identifying the gaps in the knowledge of how exposure to repetitive head impacts leads to cognitive decline and neurodegeneration, and understanding where CTE fits into this progression will be crucial for trying to identify interventions to prevent these eventualities.

Once such gap in the knowledge exists in the understanding of how CTE progresses in vivo. A diagnosis of CTE can only be confirmed after post-mortem examination of the brain. While current research has focused on identifying biomarkers for the disease and reconciling pathology with posthumous psychological reports to correlate symptoms with pathology, the extent to which tau pathology can produce neurological dysfunction is not yet known.160 This is evident in the early stages of CTE, where there can be neuropathological changes in the absence of symptoms,161 and there can be an occurrence of behavioral abnormalities that contribute to early deaths with little to no neurofibrillary tangles found in the cerebral cortex, subcortical nuclei and brainstem

(known as “incipient CTE”).162 For these reasons, many prominent researchers in the TBI field have acknowledge that there is yet to be a consensus on the staging of CTE.163 Further preclinical and clinical research is therefore necessary in order to determine the nature of the relationship

24 between tau pathology and deficit development in vivo to further elucidate stages and disease progression.

While the field has a handle on the pathology of CTE, little is known about the effect of head impacts on the physiology of the brain. As previously described, impaired cognitive function can occur after exposure to concussive and subconcussive head impacts, which have been shown to not produce overt structural damage. Classical preclinical studies on head impacts have identified the role of common injury modulators such as inflammation, axonal injury, and protein dysregulation in generating cognitive dysfunction.164-170 However, more recently developed repetitive head impact models generate minimal changes in these modulators, but demonstrate cognitive dysfunction.171-174 These findings all suggest that in lieu of structural damage and the occurrence of common injury modulators, there could be an alternative source of cognitive impairments that is activated after repetitive head impacts are sustained.

It is known that, during a single season, American football players will absorb over 1,000 head impacts at the high school level and up to 2,492 head impacts at the college level, with multiple hits being incurred during a single game.82,134 Even with this likelihood of exposure to high-frequency impacts, there are currently few models that focus on assessing the effects of high frequency of repetitive head impacts on deficit development. The majority of the mild TBI models published do not exceed five head impacts total, and many of them separate the impacts with an interval of twenty-four to forty-eight hours, making the identification of any influence of a high frequency of impacts on altered function difficult to surmise.175 Since athletes exposed to high frequencies of head impacts (like football players and boxers) seem to be the most vulnerable to developing impairments and neurodegeneration, it is important to understand how impact frequency can influence impairment of brain function.

25 Taken together, these gaps in our understanding represent important questions that the field has yet to address. Repetitive head impacts generate functional impairments, and eventually neurodegeneration. Aberrant tau, which drives CTE progression, plays a pivotal role in this degeneration, but in the early stages of CTE its presence may not be necessary to generate cognitive and behavioral deficits. This suggests that there are alternate mechanisms, which have yet to be identified, that can generate deficits. Further, it seems that the number of head impacts, and the time between exposure can influence the degree to which function is altered by these impacts. However, the means by which frequency of impacts contributes to deficit development are still unknown. The present study sought to address these questions by identifying a tau- independent mechanism of deficit development that may underlie early symptom presentation following repetitive head impact exposure. By utilizing a model that mimics the high frequency of head impacts sustained by athletes engaged in high risk sports like football or boxing, the unique contribution of frequency to deficit development was also explored.

Previous studies have shown that in response to mild TBI, elevations in extracellular glutamate occur,98-100 and glutamatergic receptor expression is altered.39,113,114 Similar adaptations in glutamatergic receptor expression have also been observed in response to epileptic, ischemic and hypoxic insults, where extracellular glutamate is also elevated.176,177 Not only are these receptors sensitive to injury, they play an important role in mediating excitatory synaptic transmission and synaptic plasticity. Synaptic plasticity is the cellular analog of learning and memory, and when it is impaired via glutamatergic receptor blockade, cognitive function is hindered.178 After performing RNA-sequencing, subsequent gene ontology analyses revealed that the expression of genes related to synaptic transmission and plasticity were the most changed after exposure to our model of high frequency head impacts. These findings led us to believe that instead

26 of hyperphosphorylation and accumulation of tau, changes in glutamatergic receptors likely underlie any cognitive impairments that we observe in our model.

The experiments performed in this dissertation sought to test the hypothesis that tau- independent mechanisms activated by HF-HI were sufficient to drive cognitive and behavioral impairment. The first aim of this study was to characterize our model of high frequency head impact, and test our hypothesis that our model could elicit behavioral abnormalities without causing changes in classical markers associated with injury. Henceforth, “classical” markers of injury are defined as the inflammation, cell death and protein dysregulation described previously that occur as part of the canonical injury cascades. I did this by identifying any changes in cognition and related cellular plasticity mechanisms elicited by the model, and determining whether inflammation, protein dysregulation or cell death played a role in the development in these changes. Secondly, I sought to determine whether our model elicited any changes in intrinsic electrophysiological or cellular network properties that could be responsible for modulating learning and memory. While ionic dysregulation is common after concussive injury and can cause changes in excitability that effect intrinsic and network properties,97 I hypothesized that these effects would be acute and therefore not sufficient to drive any of the impairments that I observed.

The third aim of this study was to identify any changes in glutamatergic transmission in this cellular network, as I hypothesized that changes to these receptors would be instrumental in generating any dysfunction that is observed. Finally, the fourth aim was to identify a mechanism by which deficits are elicited. After identifying the glutamatergic receptor population most altered by our head impact paradigm in the third aim, I hypothesized that pretreatment with a selective antagonist prior to delivering head impacts would block the receptor mediated changes and reverse deficit development. By testing these hypotheses, I sought to provide evidence that tau-

27 independent mechanisms can generate impairments in the absence of structural damage and changes in classical injury markers, and provide a new target for preventative or palliative interventions designed to block deficit development after high frequency head impacts.

28 Chapter 2

Methods

High Frequency Head Impact (HF-HI) Model

All procedures were performed in accordance with protocols approved by the Georgetown

University Animal Care and Use Committee, as described previously with modifications.173,179 5-

12 week old male C57Bl/6J mice (Jackson laboratories, Bar Harbor ME) were anesthetized with

3% isoflurane evaporated in 1.5L/min oxygen in an induction chamber for 2-3 minutes. They were then removed from the chamber and placed on the injury platform, where anesthesia was maintained via nose cone. Impact occurred with a 7.5mm impact depth and speed of 2.5m/s using a custom made pneumatic impactor. Five successive impacts were delivered over a 5 second interval during their light cycle. The time from removal from the anesthesia chamber to initiation of impacts was 60 sec. This procedure was conducted daily, whereby five successive hits were administered each day for 6 days, so that at the culmination of the injury period mice received 30 hits. Sham mice received identical handling and anesthesia, but no impacts.

Pharmacological Treatments- in vivo

For memantine experiments, wildtype mice were randomly assigned in a blinded manner to receive either Memantine (M9292, Sigma-Aldrich, St Louis, MO) or saline, 1 hour prior to receiving head impacts. Memantine was stored as 100µL aliquots in -20°C, and administered in saline (900µL) via intraperitoneal injection at 10 mg/kg in 10 mL/kg daily, for the 6-day duration of the HF-HI protocol. The route of administration, safety and efficacy of this dosing regimen in rodents has previously been established.180,181

29 Behavioral Testing

To assess spatial learning and memory deficits, the Barnes maze, Morris water maze, and

T-maze were used as previously described by our group and others,182,183 with the following modifications. All testing was done during the light cycle by an experimenter who was blinded as it pertains to pharmacological treatment and condition.

For the Barnes Maze, we used a white plastic apparatus (San Diego Instruments) 0.914m in diameter with 20 50mm diameter holes evenly spaced around the perimeter of the maze. The target hole was randomly selected and contained an escape chamber beneath the maze floor. Four distal visual clues were used to enhance visuospatial learning and memory. For the spatial acquisition phase we used 2 trials per day for 4 consecutive days. Each trial lasted 180 seconds, and if the mouse did not find the target hole, it was placed in the target hole and allowed to remain for 10 seconds. 72h following the acquisition phase the mice were placed back on the same maze for a single 180s probe trial, but with the target hole blocked. The mice were tracked using

AnyMaze tracking system (Stoelting Co, Wood Dale IL).

For the Morris water maze a 1.2m diameter pool (San Diego Instruments, San Diego, CA) filled with 20°C water was used. Four distal visual clues were used to enhance visuospatial learning and memory, and a hidden platform (0.1m in diameter) was submerged 10 mm below the opaque water. For the spatial acquisition phase we used 2 trials per day for 4 consecutive days.

Each trial lasted 90 seconds, and if the mouse did not find the hidden platform, it was placed on the platform and allowed to remain for 10 seconds. 72h following the acquisition phase the mice were placed back in the same maze for a single 90s probe trial, but with the escape platform removed. The mice were tracked using AnyMaze tracking system. A visual probe trial (where the platform was made visible and the latency of the mouse to find the platform was recorded) were

30 conducted at the end of the probe trial to confirm the visual and motor ability of the mice to complete the task.

Hippocampal function is also an important component of the T-maze spontaneous alternation test.184 We tested spontaneous alternation in HF-HI mice using a beige mouse T-maze

(San Diego Instruments). Mice were placed in the start area and allowed to acclimatize for 30 seconds. Once the start door was opened, mice were allowed to ambulate up the starting arm and make a choice of the left or right arm. Once inside, the door to the arm was closed and the mouse confined in the chosen arm for 30 seconds. Mice were then removed from that arm of the maze, the door was reopened, and they were replaced in the start area of the maze and the test repeated.

This process was repeated 3 times on each mouse, with at least 1h between each session. The number of times that the mice chose the novel arm during the second phase of the trial

(spontaneous alternation) was recorded.

Aβ40 ELISA

Aβ40 ELISAs were conducted on cortical and hippocampal brain regions as previously described by our group.185,186 Brain regions were homogenized in 0.4% diethylamine (Sigma

#471216) in a 250mM sucrose, 20mM Tris, 1mM EDTA and 1mMEGTA buffer. Concentration of endogenous mouse Aβ40 were detected using an ELISA with an assay range of 7.81-500pg/mL

(Invitrogen #KMB3481).

Western Blot

Western blot analysis was performed as previously described.187 Frozen hippocampi and sections of cortex were homogenized in radioimmunoprecipitation assay buffer (50 mmol/L NaCl,

1% Nonidet P-40, 0.25% C24H39NaO4, 1 mmol/L NaVO3, 1 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 μg/mL protease inhibitor cocktail (20-188, Millipore,

31 Billerica, MA). 20µg of protein were added to Laemmli sample buffer (1610737, BioRad,

Hercules, CA) with 5% v/v β-mercaptoethanol (1610710, BioRad) and heated to 95°C for 5 minutes. Protein separation via SDS-PAGE gel electrophoresis was conducted, and those proteins were transferred to nitrocellulose membranes. Primary antibodies (table 1) were added to 2% w/v skim milk/TBS-T and applied to membranes for overnight incubation at 4°C. HRP-conjugated goat anti-rabbit, goat anti-mouse, or rabbit anti-goat secondary antibodies (Jackson

Immunoresearch, Westgrove, PA, 1:1000 in 2% w/v skim milk powder in PBS-T) were used before detection with SuperSignal West Pico Chemmiluminescent Substrate (34080,

Thermoscientific, Waltham, MA). Visualization of chemiluminescent signal was done with

Amersham 600 imaging machine (GE healthcare, Chicago, IL). Raw pixel intensity of bands from the blots were quantified using Image J software densitometry plugins (Version 1.47, NIH,

Bethesda, MD).

Immunohistochemistry

Mice were perfused with 1x phosphate buffered saline (PBS), and brains were extracted and fixed in four percent paraformaldehyde solution. Brains were then sliced into 20 µm sections using a microtome. Slices were incubated in 0.3% H2O2, and blocked with 3% normal goat serum in PBS with 0.25% Triton X-100 before undergoing overnight incubation with primary antibody in PBS, 0.25% Triton X-100 and 1% normal goat serum (table 1). For cleaved caspase 3, sections were pretreated for antigen retrieval with citrate buffer. Biotinylated goat anti-rabbit or anti-mouse secondary antibodies (1:2000, Vector laboratories, Burlingame, CA; catalog number BA-100) in a 0.25% Triton X-100 and PBS solution. They were then incubated in a Vectastain avidin/biotinylated enzyme complex kit (1:400, Vector Laboratories; catalog number PK-6100) and a 3, 3’-diaminobenzidine (Sigma-Aldrich, St. Louis, MO) to visualize the primary antibodies.

32 Table 1. List of antibodies used for western blot and immunohistochemical analyses.

Antibody Supplier Secondary Dilution Phospho-Tau (Ser199, 1:1000 Invitrogen (44-768G) Anti-Rabbit HRP Ser202) Phospho-Tau (Ser396) 1:1000 Cell Signaling (9632) Anti-Mouse HRP (PHF13) Phospho-Tau (Ser416) 1:1000 Cell Signaling (15013) Anti-Rabbit HRP (D7U2P) 1:5000 β-Actin Sigma-Aldrich (A5441) Anti-Mouse HRP

Amyloid Precursor 1:1000 Invitrogen (36-6900) Anti-Rabbit HRP Protein (APP) 1:1000 Iba1 Anti-Rabbit HRP Wako (019-19741) 1:1000 CD68 Serotec (MCA1957T) Anti-Mouse HRP 1:1000 GFAP Dako (Z-0334) Anti-Rabbit HRP 1:5000 Cleaved Caspase 3 Cell Signaling (9661) Anti-Rabbit HRP

33 Golgi Staining and Dendritic Spine Counts

In order to examine and quantify dendritic spines, Golgi staining was performed on hippocampal sections using the FD Rapid Golgi Stain Kit (FD NeuroTechnologies, Ellicott City,

MD) as previously described.173,188 Bright-field microscopy images of pyramidal neurons in the

CA1 were captured by a person blinded to treatment group, and the number of dendritic spines on apical oblique (AO) dendrites were quantified. We counted spines in a 20μm section of the primary

AO dendrites between 30-100μm away from the apical dendrite. Slides and resultant images were coded, and dendritic spines were counted in a blinded fashion using Image J Software (NIH).

Local Field Potential Electrophysiology

a. Slice Preparation

In a separate cohort of animals, LTP experiments were conducted. Animals were euthanized via decapitation twenty-four hours after the last day of impacts. Brains were extracted, and 350

µm horizontal sections of the hippocampus were made via a vibratome using a 4°C cutting solution containing (in mM):3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 MgSO4, 2 CaCl2, 10 dextrose and 252 sucrose. Following cutting, slices were incubated for 4 hours at room temperature in artificial cerebrospinal fluid (in mM: 132 NaCl, 26 NaHCO3, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2 and

10 dextrose) with constant saturation in 95% O2 and 5% CO2. Recordings were performed while slices were maintained in a constant perfusion of artificial cerebrospinal fluid with 95% O2 and

5% CO2.

b. Long Term Potentiation Recordings

A unipolar stimulating electrode was placed in the stratum pyramidale layer of hippocampal

CA3, and a borosilicate glass electrode filled with 0.9% NaCl internal solution (1MΩ tip resistance) was placed in the stratum radiatum layer of CA1 to record field excitatory postsynaptic

34 potentials (fEPSPs). The threshold for response was determined by identifying the lowest stimulation required to elicit a response. Then, a stimulus input/ output curve was generated by exposing the slice to a randomized series of stimulations (10 pulses total for 1 second each, with a

10 second interstimulus interval) that ranged from below the identified threshold to the stimulation that evokes the maximum response. Using the stimulus that elicited 1/3 of the maximum response, a baseline was recorded for 5 minutes (10 stimulations total, at 1 second each with a 30-second interstimulus interval). The same stimulus was used in a high frequency stimulation (HFS) paradigm to elicit LTP, whereby 4 tetanic trains were delivered at 100Hz, 1 second each, with an interstimulus interval of 10 seconds. Thirty seconds after the tetanic pulses were delivered, LTP was recorded for one hour, during which stimulation occurred every 30 seconds (for 1 second) using the same stimulus.189 Two slices per animals were recorded each day, and their data were normalized between days for comparison.

Whole Cell Recordings

a. Slice Preparation

In a separate cohort of animals, whole cell patch clamp recordings were conducted. Twenty- four hours after the last day of impacts, mice were maintained under isoflurane anesthesia and transcardially perfused using ice-cold N-methyl D-glucamine (NMDG) solution.190 The solution contained (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 dextrose, 10 sucrose, 5 ascorbic acid, 2 thiourea, 3 sodium pyruvate, 5 N-acetyl-L-cysteine (NAC), 10 MgSO4

7H2O, and 0.5 CaCl2 2H2O. The solution was brought to a pH of 7.3- 7.4, and an osmolarity of

300-310 mOsM, and was bubbled with 95% O2 and 5% CO2 30 minutes prior to perfusion. Brains were extracted and 300 µm horizontal sections of the hippocampus were made via a Vibratome

3000 Plus sectioning system (Vibratome, St. Louis, MO) using ice-cold NMDG solution bubbled

35 constantly with 95% O2 and 5% CO2. Hemisected slices were incubated for 12 minutes each in

NMDG solution maintained at a temperature of 32°C and bubbled constantly with 95% O2 and 5%

CO2. Following cutting, individual slices were then transferred to separate mesh-bottom wells in chamber and submerged in an incubation solution consisting of (in mM): 92 NaCl, 2.5 KCl, 1.2

NaH2PO4, 30 NaHCO3, 20 HEPES, 25 dextrose, 5 ascorbic acid, 2 thiourea, 3 sodium pyruvate 5

NAC, 2 MgSO4 7H2O, and 2 CaCl2 2H2O. The solution had a pH of 7.3-7.4 and an osmolarity of

300-310 mOsm/kg, and was constantly bubbled with 95% O2 and 5% CO2. The slices were allowed to incubate at room temperature (22-24°C) for approximately 4 hours prior to being used for recordings. Recordings were done using a constant perfusion (2-3 ml/min) of aCSF solution containing (in mM): 124 NaCl, 4.5 KCl, 1 MgCl2 2H2O, 1.2 NaH2PO4, 10 dextrose, 26 NaHCO3, and 2 CaCl2 (pH 7.4, bubbled constantly with 95% O2 and 5% CO2).

b. Potassium Gluconate Recordings

To record the paired pulse ratio, spontaneous excitatory post synaptic currents and miniature excitatory post synaptic currents (sEPSCs and mEPSCs), and hyperpolarizing and depolarizing current clamp recordings from CA1 pyramidal cells, a potassium gluconate (kgluc) internal solution was used. The potassium gluconate solution contained (in mM): 145 potassium gluconate,

10 HEPES, 5 EGTA, 5 ATP∙Mg 0.2 GTP∙Na, osmolarity 305 mOsm, adjusted to pH 7.20. For all recordings, pharmacological compounds were dissolved in aCSF and delivered via Y-tube.191

i. Paired Pulse Ratio

Paired pulse ratio recordings were performed with kgluc internal solution. To study the paired pulse ratio, a stimulus that elicited a moderate response was identified, and two pulses were delivered in rapid succession (50ms apart) while 25µM bicuculline methobromide (BMR) was being delivered via y-tube.192-194 Ten recordings per cell with the stimulus administration were

36 made, and the ratio of the second pulse to the first pulse was calculated from the average of the ten recordings per cell for both the sham and the HF-HI groups.

ii. sEPSCs and mEPSCs

Recordings of sEPSCs were conducted in the presence of 25µM BMR, and spontaneous mEPSCs, were recorded in 1µM tetrodotoxin (TTX) and 25µM BMR administered (via Y- tube).195 Cells were held in the voltage clamp configuration at -70mV holding potentials, and spontaneous currents were recorded for 3 minutes per cell. Stock solutions of BMR and TTX were purchased from Abcam Biochemical (Cambridge, MA), prepared in deionized water, and diluted in 1:1000 aCSF. For both recordings, templates were made from the data set of stereotypical sEPSC and mEPSC events using Clampfit 10.7 software (Molecular Devices LLC, Sunnyvale,

CA). These templates were then used to automatically identify and measure the properties of these events (including the amplitude and frequency) for all recordings, and validated with manual event detection.

iii. Current Clamp Recordings

Current clamp configuration was used to look at voltage responses to current injections.

First cells were injected with slow current to bring them to a membrane potential of -70mV. Once the current required to keep the cell at that potential was determined, a constant injection of current with that value was used. Then, hyperpolarizing and depolarizing current was injected into the cell at 20pA steps. The voltage responses from each set of steps were recorded. From the depolarizing current clamp injections, the firing rate of action potentials and the approximate rheobase were calculated. From the hyperpolarizing current injections, the capacitance and input resistance were calculated. Approximations of the afterhyperpolarizing (Ih) current and the inward rectification of

37 potassium currents (IKr) were also be calculated from the hyperpolarizing current clamp results

(Fig. 1).

38

Figure 2.1. Calculations performed using hyperpolarizing current clamp recordings.

The input resistance, IKr rectification and Ih sag were calculated from the voltage traces highlighted in Figure 1. Colored arrows indicate where the measurements of each property were taken from. IKr was measured at the peak and Ih was measured at the end of the voltage response.

The green lines indicate where cursors were placed along the trace for analysis in Clampfit. A trace corresponding to the first depolarizing current injection has been added to the top of the hyperpolarizing current clamp recording. Input resistance was calculated from the depolarizing current clamp injections as well as the hyperpolarizing injections, and those values were compared to ensure that they were similar.

39 c. Cesium Methanesulfonate Recordings

Recordings aimed at measuring excitatory input to the CA1 pyramidal cells were done as

196 previously described. Briefly, a cesium methanesulfonate (CsMeSo4) internal solution containing (in mM): 120 cesium methanesulfonate, 5 NaCl, 10 tetraethylammonium chloride, 10

HEPES, 4 QX314, 1.1 EGTA, 5 MgATP, and 0.2 NaGTP, osmolarity 305 mOsm, with a pH adjusted to 7.25 using CsOH was used. Cesium was used to block K+ channels, and methanesulfonate was used to maintain chloride at physiological levels. Stimulation was delivered approximately 300µm from the CA1 pyramidal cells in the stratum radiatum layer (to stimulate

Schaffer Collateral afferents) using a Tungsten bipolar stimulating electrode (Microprobes for Life

Sciences Inc., Gaithersburg, MD).

i. NMDA/AMPA Ratio

To assess the NMDA/AMPA ratio, stimulation of the Schaffer Collateral pathway occurred at an intensity that would evoke EPSCs between 100-400pA in amplitude in the presence of 25µM

BMR delivered via y-tube. First, cells were clamped at -70mV to estimate the AMPA mediated

EPSC, and the peak amplitudes were recorded. Then, using the same stimulus intensity, the cells were clamped at +40mV, and the peak amplitude of the EPSC 60ms after the stimulus artifact was recorded in order to estimate only the NMDA mediated contribution. One single stimulation and preceding 5mV test pulse were recorded at each holding potential (10 sweeps). To confirm that the NMDA response was measured at the appropriate time following the stimulus artifact, a subset of cells from both groups was exposed to 10uM CNQX administered via y-tube in order to eliminate any AMPA mediated EPSC and isolate the NMDA EPSC. The ratio of the average peak amplitude of the NMDA response to the average peak amplitude of the AMPA response for both the sham and HF-HI groups was then calculated. A detailed diagram of the time-points used for

40 the calculation of current size for AMPA and NMDA EPSCs is provided (Fig. 2). The decay of the NMDA EPSC was also calculated using a double exponential fitting (selected to minimize

/ error). The equation for the double exponential was 푓(푡) = ∑ 퐴푒 + 퐶, where C was fixed at 0. The values obtained from the double exponential equation (A1, A2, tau1 and tau2) were then used to calculated the weighted tau (τw), where τw = tau1*(A1/(A1 + A2)) + tau2*(A2/(A1+A2)).

41

Figure 2.2. Illustration of calculation parameters for the NMDA/AMPA ratio.

The recordings illustrate the currents analyzed to obtain the NMDA/AMPA ratio. The current recorded at -70mV (black) has been inverted in order to illustrate where the AMPA EPSC decays relative to the NMDA EPSC. The larger black current is the combined AMPA and NMDA EPSC.

Green lines indicate where the peak amplitude was calculated (60ms after the stimulus artifact, where the AMPA EPSC should have mostly decayed). The red current indicated the NMDA only response with AMPA EPSC subtracted. The average peak amplitude of the response at 40mV was divided by the average peak amplitude of the response at -70mV (AMPA EPSC) to assess the

NMDA/AMPA ratio.

42 ii. Minimal Stimulation Recordings

Minimal stimulation recordings were done (as previously described, but with some modifications) order to identify potential silent synapses in sham and HF-HI groups.197-199 Using

CsMe internal solution, CA1 pyramidal cells were patched and held at -70mV. Stimulation of the

Schaffer Collateral pathway was delivered, and a stimulus intensity was identified that elicited a

10-30pA response was identified. Using this intensity, 50 stimulations were given. This recording was considered to be the AMPA mediated EPSC response to the stimulation. Then, cells were held at 40mV, and the same stimulus was given 50 times. At each membrane potential, the number of failures for AMPA and NMDA were calculated by visually identifying any responses that did not appear to deviate from the baseline of the recordings, and quantifying them.

iii. Inward Rectification Mediated by Non GLUR2 Subunit Containing AMPARs

To determine whether HF-HI elicited changes in AMPA receptor subunit-specific contributions to the EPSCs related to calcium permeability, we examined inward rectification.

Recordings were conducted as previously described, but with some modifications.200,201 In the presence of 25µM of the GABAA antagonist BMR and 40 µM of the NMDA antagonist (R)-CPP

(Abcam Biochemical (Cambridge, MA) delivered via y-tube, 10 EPSCs between 100-400pA were evoked using stimulation of the Schaffer Collateral pathway at -70mV and -40mV holding potentials. Then, the cells were held at 40mV and stimulated using the same intensity. An I-V curve was plotted using the peak amplitude of the EPSC response to the stimulations at each of the holding potentials for sham and HF-HI mice. A rectification index for each group was also generated by calculating the ratio of the peak EPSC responses at the holding potential of 40mV to

-40mV.

43 iv. Extrasynaptic and Perisynaptic Receptor EPSCs

Recordings were performed as previously described202 in order to identify any changes in

NMDA receptor-mediated EPSC decay indicative of changes in extrasynaptic and perisynaptic receptor expression or composition. Schaffer Collateral afferents were given single stimulations

(one every 20 seconds) to elicit a response between 100-400pA at a holding potential of 40mV (to record NMDA mediated EPSCs). Then, a high frequency stimulation was performed (7 stimulations at 200Hz) to generate glutamatergic “spillover” out of the synapse and engage the extrasynaptic NMDA receptor mediated response. The decay (τw) of this response was calculated using a double exponential fitting as previously described. The percent charge transfer (%Q) from extrasynaptic NMDA receptors as compared to synaptic NMDA responses to single stimulations were measured for both sham and HF-HI groups. The %Q was calculated by dividing the area under the curve of the extrasynaptic/perisynaptic response by the area under the curve of the response to the single stimulation (both normalized to their own mean amplitude of the peak of the response).

v. Tonic NMDA Current

Tonic currents are primarily mediated by extrasynaptic receptors responding to ambient glutamate. To assess the tonic current, I performed stimulation of the Schaffer Collateral pathway while the cells were held in voltage clamp at 40mV membrane potential. First, 20 sweeps were recorded with a constant perfusion of 25µM BMR. Then, 40µM (R)-CPP was added and another

20 sweeps were recorded at the same membrane potential. The tonic NMDA current was calculated by subtracting the leak current of the recordings after (R)-CPP was added from the leak current of the recording prior to the addition of (R)-CPP (to isolate the NMDA-receptor mediated contribution to the leak current).

44 Recording Electrode Production

Recording electrodes made from borosilicate glass were pulled using a vertical heating puller (PP-83, Narishige, Tokyo, Japan). Only those with 3-5MΩ tip resistance were used for whole cell recordings. For local field potential recordings, a 1MΩ tip resistance was used. Patch clamp recordings were performed using a MultiClamp 700B amplifier, with signals being filtered using a low-pass (2 kHz) filter and sampled at 5kHz. The signals were acquired using a Digidata

1440A (Molecular Devices, Sunnyvale, CA) on a Dell Optiplex 990 PC using Clampex

(Molecular Devices LLC, Sunnyvale, CA).

Statistical Analysis

Electrophysiology data analysis was performed using Clampfit v10.7 software (Molecular

Devices LLC, Sunnyvale, CA). For local field potential recordings, the stimulus threshold was examined to make sure that the threshold for response of slices varied no more than 0.1mA. For all whole cell patch clamp experiments, access resistance was monitored for each cell such that they were excluded if a change more than 20% was observed. All data was analyzed using Graph

Pad Prism Software™ (version 7.0). The data are presented in mean + SEM, and analyzed using either One-way ANOVA with Dunnett’s post hoc test, a Two-way ANOVA with Bonferroni multiple comparison’s post-hoc test, a Two-way ANOVA with repeated measures with Bonferroni multiple comparison’s post-hoc test, or an unpaired t-test where applicable. The criteria for statistical significance was p<0.05 for all experiments, and a G*Power analysis (G*Power 3.1,

Germany) was used to determine statistical power required for each experiment.

45 Chapter 3

HF-HI elicits learning and memory deficits in the absence of pathological changes in

classical injury-associated markers

Introduction

Exposure to repetitive concussive and subconcussive head impacts is associated with the initiation of multiple injury cascades. Elevated and persistent inflammation occur in experimental models of TBI and in human post-mortem samples.203 Aberrant phosphorylation and/or aggregation of proteins such as Aβ, and tau have also been linked to dysfunction after repeat concussion, and contribute to impairments in cognition and function.204 Protein dysregulation is thought to arise from diffuse axonal injury that occurs as a direct result of the injury.205 All of these cascades can promote apoptotic cell death after injury cessation, and in so doing contribute to any damage resulting from repetitive mild TBI. This damage can have deleterious consequences, and is thought to be related to the chronic impairments in cognition and alterations normal brain function experienced after repetitive head impacts are sustained.

Cognitive impairment is one of the chronic deficits that arise after exposure to repeat concussions. Progressive declines in memory, cognition, and executive function, in particular, are commonly reported. Even in the early stages of CTE, the disease thought to be caused by repetitive head impacts, some form of learning and memory impairment has been noted, regardless of the extent of protein pathology.206 In humans and animals, the hippocampus has been identified as being important for memory encoding and retrieval,207,208 and the prefrontal cortex has been implicated in learning,209 as well as in executive function.210 In many neurodegenerative diseases where cognitive impairments are a hallmark, pathology within these regions is correlated with learning and memory deficits.211,212 Lesions of the hippocampus have also been shown to reliably

46 impair memory.213 Assessing changes in function and the development of any pathology in these regions after exposure to repetitive mild TBI is therefore essential to understand how cognitive deficits develop, given their importance in these functions and vulnerability to injury.

Cognitive function can be examined in animal models using several behavioral paradigms.

One of the most well-known assessments (particularly for rodent models) is the Morris Water

Maze (MWM). It is used to identify deficits in spatial learning and memory, and performance in the MWM has been shown to correlate with changes in hippocampal circuitry (i.e. ability of LTP to be induced and changes in NMDA receptor signaling).214 The Barnes Maze (BM) is also used to assess spatial learning and memory experimentally. In studies where damage is imposed on the hippocampus prior to testing, performance in the maze has been shown to be correlated with hippocampal function.215 Both paradigms capitalize on the innate motivation of animals to escape an undesirable situation, and rely on them using visual spatial cues to learn and remember the location of an escape hole or hidden platform. Another common method of assessing spatial memory experimentally is to examine spontaneous alternations in a T-Maze. This test capitalizes on a preference of novelty and alternation behavior, wherein rodents will use working memory to recall which arm they previously explored so that they can explore the novel arm. It is considered spontaneous, as no reward or punishment is involved to elicit alternation.216 Spontaneous alternation is also very sensitive to hippocampal damage or circuitry changes.217,218 Together, these tests are important tools for behaviorally assessing impairment in hippocampal-dependent learning and memory.

Experimental models of repeat concussion have demonstrated impairments in these learning and memory tasks. In a closed head injury model of single and repeat concussion (5 injuries delivered at 48hr intervals), learning and memory were impaired for both in the Barnes

47 maze over a week after injury cessation.219 A weight-drop model of repetitive mild TBI caused impairments in MWM performance, depending on the interval between injuries.220 Repeated lateral fluid percussion injury set to mimic mild TBI also elicited deficits in learning and spatial memory acquisition assessed via MWM.109 Repetitive mild TBIs elicited by the Closed Head

Impact Model of Engineered Rotational Acceleration (CHIMERA) also produced deficits in

Barnes Maze performance.221 Despite these models varying in the manner by which mild TBIs are elicited, each of them produce cognitive impairments that are detectable with these behavioral paradigms.

Factors that impair synaptic plasticity have also been shown to affect performance in hippocampal-dependent tasks, providing a link between the cellular analog of learning and memory and behavioral memory assessments. In one of the earliest studies aimed at examining the connection between LTP and behavior, chronic infusion of aminophosphonovaleric acid (D,

L-AP5), an NMDA antagonist shown to block the induction of LTP, was shown to impair spatial learning in the MWM.222 Additionally, reductions in NR2A subunit expression in the hippocampus were also shown to impair LTP and spatial memory in the MWM.223 Aging produced impaired spatial memory in the MWM, which was observed concurrently with deficits in the late phase of hippocampal LTP.224 Mice with mutations in gene that encodes calcium-dependent protein kinase

II (CaMKII), a protein essential for LTP induction, were unable to form spatial memory in the

Barnes maze when presented with normal LTP inducing stimulation. The mutation in the CaMKII gene shifted the frequency at which LTP could be induced out of the normal range, but did not impair LTP, showing that LTP induction at a specific frequency of stimulation was essential for normal spatial memory-based performance.225 Given the association between normal LTP and performance on hippocampal-dependent learning and memory tasks, repetitive head impacts that

48 can produce quantifiable deficits in spatial memory should also show deficient synaptic plasticity.

Therefore, synaptic plasticity assessments are essential in understanding how cognitive impairments arise after exposure to repeat concussion.

The high frequency head impact (HF-HI) model was produced by our lab in order to study how injury frequency may influence deficit development, and to identify any tau-independent mechanisms underlying these impairments. In order to study these mechanisms, my first aim was to characterize the model. Given the propensity of some repetitive head impacts models to elicit inflammation, aberrant protein phosphorylation/aggregation, cell death and axonal injury, I began by trying to determine whether these changes were also features of HF-HI. Cognitive impairment is also known to arise after exposure to repetitive head impacts, therefore my second objective was to determine whether HF-HI caused impairments in hippocampal-dependent behavioral tests.

Finally, to identify a potential cellular origin of cognitive deficits, I tested LTP after HF-HI to see if synaptic plasticity was impaired. In all of these experiments, I chose to focus primarily on the cortex and hippocampus, as these are two of the most important regions of the brain responsible for normal cognition and memory.

Results

a. Classic Acute TBI pathology Is Absent in HF-HI Cortex or Hippocampus

To determine if HF-HI induced classical primary or secondary injury cascades we analyzed key cell death and inflammation markers using immunohistochemistry 24 hours post injury. There was no visible damage to the skull or gross brain of HF-HI mice (data not shown). No difference in cleaved-caspase 3 or APP staining was observed between groups, in either the corpus callosum or hippocampus (Fig 3A, B). In addition, Iba1 and GFAP staining reveal no changes in microglial or astrocyte responses in HF-HI mice compared to sham counterparts (Fig 4). We next determined

49 if HF-HI altered Aβ40 or p-tau levels 24 hours after the last day of impacts. Western blot analyses for multiple phosphorylated tau epitopes revealed no differences between HF-HI and sham animals in the cortex (Fig 5) or hippocampus (Fig 6). Using an ELISA, we also identified no changes in

Aβ40 levels in HF-HI cortex or hippocampus (Fig. 7A, B). These findings suggest that HF-HI does not result in classical injury cascades and the development of commonly observed TBI pathology.

HF-HI did result in chronic inflammation of the optic tract, as previously reported.179 This inflammation is also seen in our low frequency head impact (LF-HI) model,173 and other mouse models of repetitive mild TBI,174,226,227 suggesting that the optic nerve and tract is particularly vulnerable to head impact in rodent models.

50

A

B

Figure 3.1. HF-HI does not cause a detectable increase in apoptosis or axonal damage.

To assess cell death and inflammation in the hippocampus and corpus callosum (cc) we compared sham and HF-HI brains at 24h post-impact. Pathology from controlled cortical impact (CCI) mouse brain (1.5mm impact depth, 5.25 m/s impact speed, 24h post-injury) is shown as a positive control. (A) Apoptotic cell death and axonal injury markers are absent in HF-HI brains. Cleaved

Caspase-3, a marker of apoptosis, reveled sparse cell death throughout the brain of sham or HF-

HI mice, but frequent neuronal cells undergoing apoptosis in the CCI brain. (B) APP, a marker of axonal injury, is absent in white matter tracts of sham or HF-HI brain, but highly prevalent outside of cell bodies in the CCI brain. Area shown for cleaved caspase-3 focuses on the hippocampus, for

APP the corpus callosum (cc). Scale bar is 50µm.

51 Sham HF-HI CCI

A

Iba1

B

CD68

C

AP

F G

Figure 3.2. Inflammation is not detectably increased after HF-HI.

Neuroinflammation markers are unchanged in HF-HI mouse brains 24 hours after the last day of impacts. (A) Iba1 staining for microglia in CA1 from sham, HF-HI and CCI brain slices. (B)

CD68 staining for activated microglia in CA1 from sham, HF-HI and CCI brain slices. (C) GFAP staining for activated astrocytes in CA1 from sham, HF-HI and CCI brain slices.

Immunohistochemical staining reveals that while morphological profiles are unchanged between

Sham and HF-HI mice, CCI injury results in increased staining of these markers compared to

Sham. Images representative of n=6 per group. Scale bar is 50µm

52

A

B

C

Figure 3.3. Accumulation of phosphorylated tau and APP was not observed in the cortex after HF-HI.

Western blot analyses of tau phospho-epitopes 24 hours after the last day of impacts and concurrent densitometry. β-Actin is included as a control, and densitometry is calculated as intensity of protein of interest divided by the intensity of . β-Actin. (A) Levels of p-tau epitope serine 199,202 are not

53 increased after HF-HI. Representative image and densitometry show no difference between HF-

HI and Shams. (B) Levels of p-tau epitope serine 416 is not increased after HF-HI. Representative image and densitometry show no difference between HF-HI and Shams. (C) Levels of p-tau epitope serine 396 and APP are not increased after HF-HI. Representative image and densitometry show no difference between HF-HI and Shams. n = 7 Sham, n = 8 HF-HI, unpaired t-test.

54

A

B

C

Figure 3.4. Accumulation of phosphorylated tau and APP was not observed in the hippocampus after HF-HI.

Western blot analyses of tau phospho-epitopes 24 hours after the last day of impacts and concurrent densitometry. β-Actin is included as a control, and densitometry is calculated as intensity of protein of interest divided by the intensity of . β-Actin. (A) Levels of p-tau epitope serine 199,202 are not

55 increased after HF-HI. Representative image and densitometry show no difference between HF-

HI and Shams. (B) Levels of p-tau epitope serine 416 is not increased after HF-HI. Representative image and densitometry show no difference between HF-HI and Shams. (C) Levels of p-tau epitope serine 396 and APP are not increased after HF-HI. Representative image and densitometry show no difference between HF-HI and Shams. n = 7 Sham, n = 8 HF-HI, unpaired t-test.

56

A B

Figure 3.5. HF-HI does not cause detectable accumulation of Aβ-40 in the cortex and hippocampus.

ELISA assays were performed 24 hours after the last day of impacts for Aβ-40. (A) In the cortex, there was no significant difference in Aβ-40 levels between sham and HF-HI mice. n = 7 Sham, n

= 8 HF-HI, unpaired t-test. (B) In the hippocampus, there was no significant difference in Aβ-40 levels between sham and HF-HI mice. n = 7 Sham, n = 8 HF-HI, unpaired t-test. There were also no differences in p-tau or Aβ40 at 1m post-impact (not shown).

57 b. HF-HI Causes Chronic Cognitive Deficits in Mice

We have previously reported that thirty head impacts delivered with low frequency (1 impact per day) does not produce cognitive dysfunction.173 To assess the chronic effect of a higher frequency of impacts on cognitive function, we exposed three different cohorts of mice to HF-HI and tested them 1 month after the last day of impacts in three separate cognitive tests – Barnes

Maze, Morris Water Maze, and the T-Maze. In the Barnes maze, mice exposed to HF-HI showed significant deficits during the acquisition phase compared to sham animals (Fig. 8A; *P<0.05,

Two-way RM ANOVA with Bonferroni post-hoc test). To test spatial memory, we conducted a probe test 72h after the final training session. HF-HI mice displayed a reduction in time spent in escape quadrant as well reduced entries into escape hole and different search strategy as compared to sham counterparts (Fig. 8B-D; *P<0.05, Two-way ANOVA with Bonferroni post-hoc test & unpaired t-test). No differences between the groups were detected in total distance travelled or mean speed during the probe day (Fig. 8E, unpaired t-test). HF-HI mice had a lower percent distance traveled (over total distance) and a lower percent of total time spent in the escape quadrant than sham mice (Fig. 8F, G; *P<0.05, **P<0.01. Two-way ANOVA with Bonferroni post-hoc test). In the MWM, HF-HI mice display increased latency to the hidden platform during the acquisition phase (Fig. 8H; *P<0.05, Two-way RM ANOVA with Bonferroni post-hoc test).

During the 72h probe trial, HF-HI mice had a less focused strategy when searching for the platform as compared to shams (Fig. 8I). HF-HI mice also had fewer platform crossings, as well as fewer entries, less time spent and reduced distance travelled in the target quadrant as compared to sham counterparts (Fig. 8J-M; *P<0.05, **P<0.01, ****P<0.0001, Two-way ANOVA with Bonferroni post-hoc test or unpaired t-test). As a control, a visual probe using a visible platform, HF-HI mice maintained a swimming latency and distance similar to that of sham animals (data not shown),

58 indicating their visual perception and swimming capabilities were normal across groups. In the T-

Maze, HF-HI mice display fewer spontaneous alternations compared to sham mice (Fig.8N;

*P<0.05, unpaired t-test).

59

Figure 3.6. Chronic cognitive deficits occur after exposure to HF-HI.

Cognitive assessment one-month post HF-HI. (A) Latency to platform during the acquisition phase of the Barnes maze. Data from two trials per day on four consecutive days. (n = 20, Two-Way

ANOVA and Bonferroni post-hoc). (B) Time spent in Barnes maze quadrants during the probe

60 trial at 72h post-acquisition (n = 20, Two-Way ANOVA and Bonferroni post-hoc). (C) Heat map detailing time spent in Barnes maze during the probe trial. (D) Number of entries into escape target during the probe trial (n = 20, unpaired t-test). (E) Distance traveled and speed of travel (n = 20, unpaired t-test). (F) Percent of distance traveled in each of the maze quadrants over total distance traveled during probe trial Barnes maze (n = 20, Two-Way ANOVA and Bonferroni post-hoc).

(G) Percent of time spent in each of the quadrants during the Barnes maze probe trial (n = 20,

Two-Way ANOVA and Bonferroni post-hoc). (H) Latency to platform during the acquisition phase of the Morris water maze (MWM). Data from two trials per day on four consecutive days (n

= 20, Two-Way ANOVA and Bonferroni post-hoc). (I) Heat map detailing time spent in MWM during the probe trial. (J) Number of platform crossings during the probe trial (n = 20, unpaired t- test). (K) Number of entries in each of the quadrants during the probe trial of the MWM (n = 20,

Two-Way ANOVA and Bonferroni post-hoc). (L) Time spent in MWM quadrants during the probe trial at 72h post-acquisition (n = 20, Two-Way ANOVA and Bonferroni post-hoc). (M)

Distance travelled in each of the quadrants of the MWM (n = 20, Two-Way ANOVA and

Bonferroni post-hoc). Data in A,B,F,G,H,K,L, Mare mean ± s.e.m.,n = 20/group,****= P <

0.0001; **= P < 0.01;* = P < 0.05,Two-Way ANOVA withBonferroni post-hoctest.Data in D, E,

Jare mean ± s.e.m.,n = 20/group,**= P < 0.01;* = P < 0.05, unpaired t-test. (N) T-Maze spontaneous alternations (n = 13 sham and 18 HF-HI, unpaired t-test).

61 c. Long-term Potentiation is Deficient After HF-HI

To assess determine whether impaired cognitive function was related to altered synaptic plasticity, I investigated LTP 24 hours after HF-HI. I chose to focus on the Schaffer collateral/CA1 pyramidal cell synapses in the hippocampus, as the characteristics of their plasticity and associated synaptic changes are a well understood.228 Also, the changes in AMPA receptor expression and activation that characterize altered plasticity in CA1 are dependent on NMDA receptor activation.229 Further, if NMDA receptor activation and subsequent LTP induction is blocked in this pathway, spatial learning and memory in the MWM is impaired.230

In HF-HI mice, the peak amplitude of the field excitatory postsynaptic potential (fEPSP) is shifted downward, indicating that LTP is significantly reduced compared to sham mice (Fig.

9A, *** P<0.001, Two-Way RM ANOVA with Bonferroni post-hoc test). This change was not driven by dendritic damage or a lack of synaptic recruitment, as the fEPSP peak amplitude response on stimulus input/output curves were identical for sham and HF-HI mice (Fig. 9B; Two-

Way RM ANOVA with Bonferroni post-hoc test). Furthermore, no changes in dendritic spine number is observed between groups (Fig. 10A-C; unpaired t-test), suggesting that synaptic plasticity is altered after HF-HI based on changes in synaptic function, and not physical loss of dendrites or synapses.

62

A

B

Figure 3.7. Long-term potentiation is impaired in the Schaffer collateral pathway 24 hours after HF-HI.

63 Long-term potentiation (LTP) was assessed in acute hippocampal slice preparations 24 hours after the last day of impacts via local field potential recordings of the CA1, with extracellular electrical stimulation of the Schaffer collateral pathway in CA3. (A) Normalized peak amplitudes of field

EPSPs indicate that while the baseline recordings are similar between groups, high frequency stimulation meant to elicit LTP produces deficient plasticity in HF-HI mice compared to shams (n

= 8 animals per group, average of 2 slices per animal. Two-Way RM ANOVA with Bonferroni post-hoc test). (B) The stimulus input/output curve indicates that the normalized peak amplitude of the field EPSP is the same for both groups with increasing stimulus intensities (n = 8 animals per group, average of 2 slices per animal. Two-Way ANOVA with Bonferroni post-hoc test).

64

A B

C

Figure 3.8. Excitatory synapses are not lost after HF-HI.

Twenty-four hours after the last day of impacts, Golgi staining and subsequent quantification of dendritic spines was performed in the apical oblique region of the CA1 to determine whether spines were lost as a result of HF-HI. (A) The average number of spines per individual neuron was not altered by HF-HI (n = 48 sham and 49 HF-HI). (B) The average number of spines per animal is not altered by HF-HI (n=5, average of 9-10 neurons per mouse). (C) Representative image of the dendrites and spines in the CA1 apical oblique region for sham and HF-HI animals. Scale bar =

20µm.

65 Discussion

Here, we show that the HF-HI model produces cognitive deficits in the absence of changes associated with classical injury markers. In contrast to what is observed in moderate, severe and some iterations of mild TBI, we found no differences in microglial/monocyte activation between sham and HF-HI mice. We also observed no indication of increased astrocytic activation following

HF-HI. While inflammation is a mediator of lasting disruption after injury cessation in more severe forms of TBI, it does not seem to influence functional deficits after HF-HI. Inflammation responses have been shown to be either present or absent, depending on the length of the interval allowed between head impacts in several models.168,231-238 We also find no differences between sham and

HF-HI in cleaved caspase-3 staining, indicating that apoptotic cell death (a feature of injury observed in other forms of TBI239) is not being elicited by our model. This is similar to what is observed in repeat concussive and subconcussive injury models that closely replicate human concussive injury.175

When exploring the contribution of protein dysregulation to functional impairment after impacts, we found no differences between the sham and HF-HI groups in the amount of Aβ in the cortex or hippocampus. Aβ accumulation is a common pathological hallmark in multiple neurodegenerative disorders and TBIs of increased severities, and an increased amount is positively correlated with more negative outcomes. The Aβ protein is 1-43 amino acids long, and cleavage can generate multiple isoforms.240 The two isoforms of Aβ thought to be most neurotoxic are Aβ40 and Aβ42, which are found in the fibril-rich neuritic plaques thought to be the most damaging in AD. Diffuse plaques, which are representative of “immature” lesions, are primarily composed of Aβ42.241 For this reason, we decided to look specifically for Aβ40 after HF-HI. The fact that Aβ40 levels were not altered by HF-HI indicated that any functional changes observed

66 were not caused by accumulation and aggregation of this protein. In TBI specifically, the presence of Aβ is linked to axonal damage sustained by the insult.55 Our findings are, therefore, unsurprising given that when we stained for APP (a marker for axonal damage and the precursor to Aβ) in major white matter tracts such as the corpus callosum, we saw no differences between the sham and HF-HI groups. The chance of impairments arising from axonal damage after HF-HI is unlikely, and supports the hypothesis that functional disruption, and not physical damage, is the origin of deficit development in this model.

We wanted to explore whether the model elicited any changes in the accumulation of phosphorylated tau, given its inclusion in pathological aggregates known to contribute to degeneration in injury and disease. Aberrant phosphorylation of tau at specific sites creates epitopes that are stereotypically found in filaments and tangles at discrete stages of tau pathology formation, and in different regions relative to neurons. Tau phosphorylated at serine 199, 202 is common in early-NFTs, and in intracellular and extracellular NFTs, serine 396 in intracellular and

(most often) extra-extracellular NFTs,242 and serine 416 is found in the neuronal soma.243 We chose to look at the amounts of several tau epitopes found in all stages of aggregate formation, and despite the wide array of epitopes that we assayed, we found no differences between sham and

HF-HI mice in any of them. This indicates that tau is not playing a role in deficit development in

HF-HI. These findings are similar to those seen in other models of repetitive mild TBI, where cognitive deficits arose in the absence of tau pathology.244-246 Even in mice expressing human transgenic tau (h-tau), tau pathology was not observed up to a year after repetitive CHIMERA injury.247 Transgenic h-tau mice express the same isoforms of tau that humans do, which are not present endogenously mice. The effects of injury in these models are more applicable to the human condition, because unlike endogenous mouse tau, h-tau mice tend to exhibit accumulation and

67 form aggregates with age.248 Taken together, the findings from our group and others support the idea that tau-independent mechanisms of deficit development arise after repetitive head impacts are sustained, and may be sufficient to generate impairment.

Mice exposed to HF-HI demonstrated impaired spatial learning and memory, as assessed using several different behavioral paradigms. As previously mentioned, behavioral dysfunction in rodent models of repetitive mild TBI are well documented. The MWM, Barnes maze, and T-Maze are also accurate measures of hippocampal dysfunction in rodent models, and deficits in these tasks can be correlated with deficient plasticity in the Schaffer Collateral pathway in the hippocampus.225 We also observed deficient LTP in this pathway after HF-HI, suggesting that deficient plasticity plays a role in the deficits that we observe. This is despite the fact that there are no differences between sham and HF-HI groups at any of the stimulus intensities tested in the stimulus input/output curve, indicating that signal transduction and synaptic recruitment are likely not affected. The impairment in LTP is also not a result of the loss of excitatory synapses, because dendritic spine counts are not significantly different between sham and HF-HI groups. After LF-

HI, fluctuations in dendritic spine numbers were reported, with an initial increase observed 1 hour after impact cessation, followed by a decrease in spines lasting up to 24 hours.173 Given that HF-

HI does not elicit changes in spine numbers, the frequency of the impacts could be prompting a response to sustain spines in the face of repetitive impact exposure. That, coupled with the finding of deficient synaptic plasticity in the absence of other common injury cascades led us to believe that the mechanism of deficit development is likely modification of the glutamatergic NMDA and

AMPA receptors present at these synapses.

Overall, our findings suggest that the HF-HI model elicits cognitive impairments in the absence of cell death/ physical damage, inflammation, or protein dysfunction. These findings are

68 important, as they highlight the presence of a mechanism outside of the common pathology associated with TBI that is sufficient to generate cognitive impairment. This mechanism appears to be sensitive to the frequency of head impact exposure, as our group found a similar absence of pathology (except in the optic tract) but with no cognitive impairment in the LF-HI equivalent of the model.173 Identifying any mechanisms that are impact frequency specific is important to understanding how impairments develop, because the populations that have the highest risk of having lasting impairment and neurodegeneration are those exposed to high frequencies of impacts

(such as boxers and football players). The remainder of this study was, therefore, aimed at characterizing changes in neuronal function to identify novel mechanisms of deficit development.

69 Chapter 4

Intrinsic and synaptic properties of hippocampal CA1 pyramidal neurons are acutely

altered by HF-HI

Introduction

The hippocampus is a medial temporal lobe structure shown to be important for learning and memory. It has been extensively characterized in the literature, therefore a brief overview of its structure and function will be provided. It is composed of multiple distinct sub-regions, including the dentate gyrus (DG), and Cornu Ammonis (CA) 1, 2, 3, and 4.249 The sub-regions of the hippocampus can be identified based on unique cellular morphology, composition and functional properties. For example, the DG is primarily composed of granule neurons, while the

CA regions are mainly composed of glutamatergic pyramidal neurons. Within the CA field, cell morphology can also help distinguish between regions. The pyramidal cells in CA1 tend to be small with single, long apical dendrites; the pyramidal cells in CA2 and CA3 tend to be much larger with one to two shorter apical dendrites.250 Firing properties can also distinguish cells belonging to distinct regions. In particular, while CA3 neurons are known to fire single spikes,

CA1 pyramidal cells are known to fire action potentials either individually, or in high-frequency bursts.251 These bursts are important for evoking postsynaptic spikes to promote reliable synaptic communication252 and inducing synaptic plasticity.253 Using these properties, specific regions of the hippocampus can be differentiated and examined.

Further division of the hippocampus into layers can be done based on features of the pyramidal cells. The alveus is the layer containing the axons on pyramidal cells that project to the fimbria or the subiculum. The next layer, known as the stratum oriens, is the layer between the alveus and the somas of the pyramidal cells, and contains basal dendrites of the pyramidal cells as

70 well as basket cells. The layer containing the somas of the pyramidal cells is called the stratum pyramidale. Directly underneath the stratum pyramidale is the stratum radiatum, where proximal dendrites of the pyramidal cells are located. Finally, the distal dendrites are located in the stratum lacunosum moleculare.254

Throughout all of these layers, inhibitory interneurons make contact with specific regions of pyramidal cells. Axo-axonic chandelier cells (which send axons throughout the oriens and pyramidale layers) inhibit at the axon initial segment of pyramidal cells, while basket cells (located in the pyramidale and radiatum layers) synapse on cell bodies and proximal dendrites, and bistratified cells (heavily ramified throughout the radiatum and oriens layer) synapse inhibit proximal and distal dendrites.255,256 These inhibitory interneurons are important for regulating the activity of pyramidal cells via inhibition, and do so based on their strategically positioned synaptic connections.

The interplay between interneurons and pyramidal cells is important for generating oscillations to integrate information. Two kinds of oscillations are prominent in the hippocampus: theta and gamma. Theta rhythms arise as a result of activation of ensembles of pyramidal cells and interneurons, and are essential for spatial and episodic memory processing as well as coding and decoding active neuronal ensembles. These oscillations are also thought to be involved in promoting synaptic plasticity.257 Processes important for working memory are also reliant on theta oscillations.258 Within the hippocampus, theta rhythms occur more regularly in CA1 of the hippocampus, though they are also found in DG and CA3.257 Gamma oscillations can co-occur with theta oscillations, although they are generated independently in either CA3 or DG. They are important for coordinating neuronal activity at fast time-scales to create functional ensembles, which has implications for memory encoding, memory retrieval, working memory and

71 representations of spatial sequences.259 Generation of both rhythms, and their relationship with one another are dependent on inhibitory drive via interneuronal manipulation of pyramidal cell activity.260,261

The ability of oscillations to promote functional relationships between sub-regions of the hippocampus is reliant on the complex system of connectivity between the regions. A review authored by Knierim (2015) succinctly describes this circuitry, and the evolution of our understanding of the connectivity between them. In initial characterizations of the hippocampus, a unidirectional, feed-forward trisynaptic loop was proposed. In this loop, the entorhinal cortex funnels cortical input into the hippocampus through the perforant pathway to the DG. The DG then primarily projects to the CA3 region through the mossy fiber pathway, and CA3 projects to CA1 through the Schaffer Collateral pathway. The CA1 then projects back to the entorhinal cortex.

Although once thought to be unidirectional, studies have revealed more complex connections between the sub-regions. It is now known that CA3 neurons send recurrent collateral projections onto other CA3 neurons (which may convey auto-associative properties262), as well as projections back to the DG. The entorhinal cortex has also been shown to connect directly to CA1 and CA3.263

Each of these connections plays an important role in information processing and formation of different kinds of hippocampal-dependent memory.

The hippocampus is also thought to be divided longitudinally, based on its connectivity to other regions of the brain, and the function of these areas of the hippocampus with respect to these connections. The roles of these hippocampal regions are also highly conserved, and are comparable in rodents and humans.264 The dorsal hippocampus connects to the retrosplenial and anterior cingulate cortices, which are involved in visuospatial and memory information processing265, navigation and exploration.266,267 Several lesion studies aimed at identifying the contribution of the

72 dorsal hippocampus to spatial learning and memory have found that disruption of dorsal but not ventral hippocampal function resulted in deficient learning in multiple maze paradigms.268 The ventral hippocampus has strong connections to olfactory areas, the amygdala, and nucleus accumbens.266,269 This makes it more heavily implicated in emotional and anxiety related processes, such as fear conditioning. 270,271 The distinct roles of these hippocampal regions are important to consider when assessing normal and interrupted function after injury.

Spatial learning and memory is modulated by the dorsal hippocampus, which encodes spatial information through firing of certain pyramidal cells in response to specific aspects of the environment in order to produce a spatial map. These cells became known as “place” cells, as their firing patterns were heavily correlated with specific places in space, known as “place fields.”272,273

A subset of these place cells are also responsible for encoding episodic, context-dependent and non-spatial memory through the formation of engrams, and those cells can be distinguished from spatially encoding place cells via their firing rates.274 While place cells occur in CA3 and CA1, and encoding of place cell information occurs along the CA3 to CA1 pathway, it seems that

NMDA receptor activation in CA1 is necessary for proper spatial learning and memory to occur.

A study done using an NR1 knockout that inhibited disrupted NMDA receptor function in CA1 specifically found that MWM performance was impaired, and although CA1 place cells could still form stable place fields, the fields were larger (indicating reduced spatial specificity) and ensemble coding was disrupted.275 CA3 place cells are important for rapid formation of highly-tuned CA1 place cells, but their ablation does not preclude the formation of place field formation.276 The CA3 to CA1 pathway, and specifically CA1 pyramidal cells, are therefore important mediators of spatial learning and memory.

73 In the previous chapter, I detailed the cognitive deficits elicited by the HF-HI model.

Briefly, mice exposed to HF-HI showed significant impairment in hippocampal-dependent and spatial learning and memory tasks, such as the Barnes Maze, MWM, and T- Maze. When synaptic plasticity was assessed in the Schaffer Collateral pathway, HF-HI mice showed deficient LTP as compared to shams. Because of these findings, I developed a second aim that sought to determine whether neuronal function and network properties within the hippocampus were being disrupted by HF-HI, thus contributing to cognitive and plasticity impairments. To explore this, I employed whole cell patch clamp electrophysiology to interrogate single cells and identify functional changes. I chose to focus on CA1 pyramidal cells, because of their role in regulating spatial learning and memory, and because they are the postsynaptic partners of the Schaffer Collateral pathway that I examined previously. Since spatial learning and memory were impaired, I also chose to focus on more dorsal aspects of the hippocampus. The cognitive impairments observed in our behavioral assessments occurred 1 month after the last day of impacts, and the lack of changes in common injury markers were observed 24 hours after the last day of impacts (Fig. 1-

5) as well as 1 month after (data not shown). These findings at each of the time-points used made time a crucial variable to explore, as they illustrate both acute and chronic changes elicited by HF-

HI. Therefore, I performed the electrophysiology experiments 24 hours and 1 month after the last day of impacts. I hypothesized that since subconcussive and concussive head impacts can elicit changes in ionic homeostasis and metabolism,101 both of which can alter cellular function, intrinsic properties of the cells and excitability of the cells (and network) would be altered by HF-HI.

74 Results

a. Presynaptic Vesicular Release Probability is Not Altered by HF-HI

The release of vesicles of neurotransmitter from the presynaptic terminal plays a crucial role in information processing as well as regulating bidirectional changes in synaptic strength.277 To assess one aspect of presynaptic vesicular release after HF-HI, I performed paired pulse ratio recordings. This paradigm measures a presynaptic form of short-term plasticity, and has been shown to be inversely correlated with vesicular release probability.278 Two pulses of the same stimulus intensity were delivered 50ms apart, and the peak amplitude of each response were recorded. The ratio of the peak amplitude of the response to second pulse divided by the peak amplitude of the response to the first pulse (the paired pulse ratio) was compared for both sham and HF-HI groups. At 24 hours after the last day of impacts, there was no difference between the two groups in paired pulse ratio (Fig. 11A, B; unpaired t-test). When the paired-pulse ratios were compared at 1 month after the last day of impacts, there was also no difference between sham and

HF-HI groups (Fig. 11C, D; unpaired t-test).

75

A C

B D

Figure 4.1. Presynaptic vesicular release is unaltered by HF-HI.

Paired pulse ratio (PPR) recordings were performed in CA1 pyramidal cells to determine whether

HF-HI elicited changes in presynaptic vesicular release probability. The PPR is the ratio of the peak amplitude of the second EPSC divided by the peak amplitude of the first EPSC. First, PPR was recorded at 24 hours after the last day of impacts. (A) Representative trace of EPSC responses to stimuli delivered 50ms apart. Sham is on the left, and HF-HI is on the right. (B) There is no significant difference between sham and HF-HI groups in PPR (n = 5 mice, 1 CA1 cell per mouse, unpaired t-test). PPR was also measured 1 month after the last day of impacts. (C) Representative trace of EPSC responses to stimuli delivered 50ms apart. Sham is on the left, and HF-HI is on the right. (D) There is no significant difference between sham and HF-HI groups in PPR (n = 9 mice,

1 CA1 cell per mouse, unpaired t-test).

76 b. HF-HI Causes an Acute Decrease in Neuronal Excitability

The efficacy of computational functions of neurons is maintained through regulation of neuronal excitability.279 Homeostatic changes in excitability are reflective of leakage conductance and the activity of voltage gated ion channels in response to network activity, and changes in these properties can alter neuronal signaling.280 To determine whether HF-HI altered neuronal excitability, I performed current clamp experiments that utilized injections of depolarizing and hyperpolarizing currents in 20pA steps. First, I looked at action potential firing in response to depolarizing current steps. The firing rate was significantly reduced in HF-HI animals as compared to shams 24 hours after the last day of impacts, despite the fact that the approximate rheobase, or minimum current required to generate an action potential, was unchanged (Fig. 12A-C; **P<0.01,

Two-way RM ANOVA with Bonferroni post-hoc test; unpaired t-test). This effect was not a chronic response to HF-HI, however, as the firing rate was not significantly different between groups at 1 month after the last day of impacts (Fig. 13A-C Two-way RM ANOVA with

Bonferroni post-hoc test). This suggests that the excitability of CA1 pyramidal cells is reduced acutely after HF-HI.

77 A B

C

Figure 4.2. Action potential firing rate is decreased acutely after HF-HI.

Twenty-four hours after the last day of impacts, whole cell patch clamp recordings were performed on CA1 pyramidal cells. Depolarizing current injections in 20pA steps were performed, and the number of action potentials fired at each step was used to determine the firing rate. (A)

Representative traces from sham (top) and HF-HI (bottom) slices. (B) Firing rate of action potentials was decreased in HF-HI cells as compared to shams (** = P < 0.01, Two-Way ANOVA and Bonferroni post-hoc; n = 9 sham mice and 7 HF-HI mice, 1 CA1 cell/mouse). (C) Approximate rheobase, measured as the minimum current step required to elicit an action potential, was not significantly different for sham and HF-HI groups (n = 11 sham mice and 9 HF-HI mice, 1 CA1 cell/mouse, unpaired t-test).

78 A B

C

Figure 4.3. Action potential firing rate normalizes 1 month after HF-HI.

One month after the last day of impacts, whole cell patch clamp recordings were performed on

CA1 pyramidal cells. Depolarizing current injections in 20pA steps were performed, and the

number of action potentials fired at each step was used to determine the firing rate. (A)

Representative traces from sham (top) and HF-HI (bottom) slices. (B) Firing rate of action

potentials was similar for both groups, (n =12 slices Sham (1 cell/slice), n = 11 slices HF-HI, (1

cell/slice), 2 Way RM ANOVA with Bonferroni Post hoc test). (C) Approximate rheobase,

measured as the minimum current step required to elicit an action potential, was not significantly

different for sham and HF-HI groups (n = 12 sham mice and 10 HF-HI mice, 1 CA1 cell/mouse,

unpaired t-test).

79 c. Active Current Properties Are Altered Acutely After HF-HI

Since neuronal excitability is heavily regulated by intrinsic neuronal properties, I then sought to interrogate passive properties of the neurons. These properties can be derived from voltage responses of the cells to hyperpolarizing current injections. When assessed 24 hours after the last day of impacts, HF-HI and sham slices did not differ in their input resistance or capacitance (Fig.

14A-C; unpaired t-test). The responses of the cells to hyperpolarizing current injections also allowed for the approximation of the hyperpolarization-activated (Ih) current, as well as the inward rectifying potassium currents (IKr). In HF-HI slices, the IKr was significantly increased as compared to shams, while Ih was unchanged 24 hours after impact cessation (Fig. 14D, E;

*P<0.05, unpaired t-test). These effects were also acute, as the difference in IKr in the HF-HI group was resolved at 1 month after injury cessation (Fig. 15A-E; unpaired t-test). These findings suggest that potassium current properties are altered as a part of the acute response to HF-HI.

80

A

B C

D E

Figure 4.4. Inward rectification of potassium currents is altered acutely by HF-HI.

Twenty-four hours after the last day of impacts, current clamp recordings were performed.

Hyperpolarizing current injections were performed using 20pA steps, and intrinsic properties of the neurons were calculated based on the voltage responses to these current injections. (A)

Representative traces from sham (left) and HF-HI (right) animals. (B) The input resistance of HF-

81 HI cells was not significantly different from sham cells (n = 12 cells sham, n = 14 cells HF-HI,

1cell/animal, unpaired t-test). (C) The capacitance of the cell membrane in sham cells was not significantly different from the capacitance observed in HF-HI cells (n = 11 cells sham, n = 9 cells

HF-HI, 1 cell/animal, unpaired t-test). (D) Inward rectification of potassium currents was significantly increased in HF-HI cells as compared to sham cells (* = P < 0.05, unpaired t-test; n

= 12 sham mice and 14 HF-HI mice, 1 CA1 cell/mouse). (E) Sag of the voltage response indicating

Ih current was not significantly different between the two groups ( n = 12 sham mice and 14 HF-

HI mice, 1 CA1 cell/mouse).

82 A

B C

D E

Figure 4.5. Changes in intrinsic properties of CA1 cells are resolved 1 month after HF-HI.

One month after the last day of impacts, current clamp recordings were performed.

Hyperpolarizing current injections were performed using 20pA steps, and intrinsic properties of the neurons were calculated based on the voltage responses to these current injections. (A)

Representative traces from sham (left) and HF-HI (right) animals. (B) The input resistance of HF-

83 HI cells was not significantly different from sham cells (n = 12 cells sham, n = 11 cells HF-HI,

1cell/animal, unpaired t-test). (C) The capacitance of the cell membrane in sham cells was not significantly different from the capacitance observed in HF-HI cells (n = 12 cells sham, n = 11 cells HF-HI, 1 cell/animal, unpaired t-test). (D) Inward rectification of potassium currents was not significantly different in HF-HI cells as compared to sham cells ( n = 12 sham mice and n = 11

HF-HI mice, 1 CA1 cell/mouse, unpaired t-test). (E) Sag of the voltage response indicating Ih current was not significantly different between the two groups (n = 12 sham mice and n = 11 HF-

HI mice, 1 CA1 cell/mouse, unpaired t-test).

84 d. Spontaneous Activity is Unchanged by HF-HI

Network and synaptic activity are important for regulating intrinsic plasticity and excitability in neurons.281 Given the changes that I observed in excitability of CA1 pyramidal cells after HF-

HI, I next sought to identify whether HF-HI altered any synaptic activity at acute and chronic time-points. By examining sEPSCs, information about the spontaneous spiking of presynaptically coupled neurons, such changes in sEPSC frequency that could indicate altered excitability in presynaptic cells, can be determined.282 To study sEPSCs, I employed the voltage clamp technique.

I held cells at a membrane potential of -70mV, and delivered 25uM of the GABAA antagonist

BMR via y-tube to block the inhibitory currents and isolate excitatory currents. Recordings of sEPSCs revealed that the inter-event interval (which is inversely proportional to the frequency) and the amplitude of the spontaneous currents were not significantly different between groups at

24 hours after the last day of impacts (Fig. 16A-E; unpaired t-test or cumulative fraction). The inter-event interval and amplitude of sEPSCs did not differ between groups 1 month after injury as well (Fig. 17A-E; unpaired t-test or cumulative fraction). These findings indicate that presynaptic activity culminating in CA1 is not altered by HF-HI.

85

A

B D

C E

Figure 4.6. Spontaneous activity is not altered acutely by HF-HI.

Twenty-four hours after the last day of impacts, voltage clamp recordings were performed on CA1 pyramidal cells. (A) Example traces of spontaneous excitatory postsynaptic current (sEPSC) for sham (left) and HF-HI (right). Recordings were performed at -70mV in the presence of 25µM

BMR. (B) Mean inter-event interval of sEPSCs per cell and (C) cumulative fraction of inter-event

86 interval per group (n = 11 mice per group, 1 CA1 cell/mouse, unpaired t-test). (D) Mean amplitude of sEPSCs per cell and (E) cumulative fraction of amplitudes per group (n = 11 mice per group, 1

CA1 cell/mouse, unpaired t-test).

87

A

B D

C E

Figure 4.7. Spontaneous activity is not altered chronically by HF-HI.

One month after the last day of impacts, voltage clamp recordings were performed on CA1 pyramidal cells. (A) Example traces of spontaneous excitatory postsynaptic current (sEPSC) for sham (left) and HF-HI (right). Recordings were performed at -70mV in the presence of 25µM

BMR. (B) Mean inter-event interval of sEPSCs per cell and (C) cumulative fraction of inter-event interval per group (n = 15 mice per group, 1 CA1 cell/mouse, unpaired t-test). (D) Mean amplitude

88 of sEPSCs per cell and (E) cumulative fraction of amplitudes per group (n = 16 sham, n = 15 HF-

HI per group, 1 CA1 cell/mouse, unpaired t-test).

89 e. Action Potential Independent Activity is Altered Acutely Following HF-HI

While synaptic activity seemed to be unaltered by HF-HI, changes in information processing related to action potential-independent signaling may be affected. Specifically, changes in the frequency and amplitude of responses to the release of single quanta of action potentials can reveal changes in synapse number and postsynaptic receptors. To study the action potential-independent responses to spontaneous, single quantal release from presynaptic cells, I recorded mEPSCs 24 hours and 1 month after the last day of impacts. The inter-event interval of mEPSCs was increased in HF-HI animals 24 hours after the last day of impacts (Fig. 18A-C; *P<0.05, unpaired t-test, cumulative fraction). The amplitude of mEPSCs was not significantly different between groups

(Fig. 18D, E; unpaired t-test, cumulative fraction). The effect on mEPSC inter-event was acute, and at 1 month after impacts there were no differences between the groups (Fig. 19A-C; unpaired t-test or cumulative fraction). There was also no difference between the two groups in amplitude

1 month after impact cessation (Fig. 19D, E; unpaired t-test or cumulative fraction). These data, when compared to other findings, suggest an acute loss of synaptic connectivity was brought about by HF-HI.

90 A

B D

C E

Figure 4.8. Action potential independent activity is altered acutely by HF-HI.

Twenty-four hours after the last day of impacts, voltage clamp recordings were performed on CA1 pyramidal cells. (A) Example traces of miniature excitatory postsynaptic currents (mEPSC) for sham (left) and HF-HI (right). Recordings were performed at -70mV in the presence of 25µM

91 BMR and 1 µM TTX. (B) Mean inter-event interval of mEPSCs per cell is significantly increased by HF-HI (* = P < 0.05, unpaired t-test; n = 8 mice, 1 CA1 cell/mouse). (C) Cumulative fraction of inter-event interval per group. (D) Mean amplitude of mEPSCs per cell and (E) cumulative fraction of amplitudes per group (n = 8 mice per group, 1 CA1 cell/mouse, unpaired t-test).

92

A

B D

C E

Figure 4.9. Action potential independent activity normalizes 1 month after HF-HI.

One month after the last day of impacts, voltage clamp recordings were performed on CA1 pyramidal cells. (A) Example traces of miniature excitatory postsynaptic currents (mEPSC) for sham (left) and HF-HI (right). Recordings were performed at -70mV in the presence of 25µM

93 BMR and 1 µM TTX. (B) Mean inter-event interval of mEPSCs per cell is not significantly different between sham and HF-HI groups (n = 10 mice, 1 CA1 cell/mouse, unpaired t-test). (C)

Cumulative fraction of inter-event interval per group. (D) Mean amplitude of mEPSCs per cell and

(E) cumulative fraction of amplitudes per group (n = 10 sham, n = 11 HF-HI mice, 1 CA1 cell/mouse, unpaired t-test).

94 Discussion

Many of the intrinsic properties of the cells remained unchanged by HF-HI. The input resistance, capacitance, and Ih currents remain the same for both groups at all time-points tested, signifying that changes to the neuronal membrane or dendritic complexity are unlikely after HF-

HI. Spontaneous EPSC amplitude and inter-event interval, as well as mEPSC amplitude and paired pulse ratio were also unchanged after HF-HI. These findings indicate that the activity of presynaptic partners to the CA1 pyramidal cells is also not altered by HF-HI, and supports the possibility that HF-HI does not affect network transmission under baseline conditions. Changes to synaptic plasticity in the absence of baseline network alterations could indicate that the deficits produced by HF-HI are mediated by postsynaptic receptors and not network excitability.

Exposure to HF-HI elicited decreased excitability and increased IKr 24 hours after exposure. It has been previously shown that sustaining concussive injuries is associated with excitatory amino acid neurotransmitter release and increased ion efflux. In particular, there are increases in extracellular potassium (K+) ion concentration, which can depolarize neurons and perpetuate ionic and neurotransmitter imbalances.97 Elevated extracellular K+ changes the conductance of K+ channels in neurons, including increasing the conductance of inwardly rectifying K+ channels.283 The activity of these channels is directly related to the intrinsic excitability of neurons,284,285 as they set and maintain the resting membrane potential.286

Knockouts of specific K+ channels that display inward rectification have been shown to increase excitability.285In HF-HI mice we found that the conductance of inwardly rectifying K+ channels was increased in CA1 pyramidal cells, which could have the opposite effect and ultimately decrease excitability. I expected this change in inward rectification of K+ channels and excitability to be acute. Sustaining concussive head impacts within 24 hours of one another can delay the

95 recovery of cerebral metabolism,103 however several studies on concussive injuries have shown that increased K+ and subsequent metabolic dysfunction resulting from the activation of ion pumps to clear elevated extracellular K+ is very short-lived, and tends to resolve spontaneously after impact cessation.97,287 Therefore it is unsurprising that this effect was present at 24 hours but not

1 month after HF-HI.

The changes in excitability after HF-HI in the absence of changes in rheobase and input resistance were unexpected. Often, changes in rheobase and input resistance are inversely proportional to one another, such that if one increases, the other decreases. Changes in either intrinsic property (or both) also greatly affect excitability, and are usually modulated by changes in K+ channel properties.288 The increase in IKr provided evidence that K+ dysregulation occurs after HF-HI. In general, several K+ channels (not just those with inward rectification properties) have been implicated in maintaining homeostatic excitability. In situations when increased NMDA activation and subsequent calcium influx occur from elevations in extracellular glutamate, such as in all severities of TBI, several key K+ channels can provide neuroprotection. Inward rectifying channels that are strongly inhibited by ATP (containing the Kir 6.2 subunit), can be rapidly activated following an event where ATP levels decrease (such as an ischemic insult289) to transiently hyperpolarize the membrane, reduce action potential firing, and promote neuroprotection.290,291 Calcium influx can also activate calcium dependent BK channels, which allow efflux of K+ to occur when intracellular calcium increases. Activation of BK channels has been shown to decrease excitability in CA1 pyramidal cells.292 In a similar way, Kv2.1 channels open in response to increased intracellular calcium, and can provide neuroprotection by reducing neuronal excitability after insults. Their activation has a longer duration than BK channels, and their position near postsynaptic membranes make them particularly adept to responding to aberrant

96 NMDA activation. In fact, extrasynaptic NMDA activation and subsequent calcium signaling has been shown to potentiate Kv2.1 activity, and extrasynaptic NMDA receptors are thought to play a pivotal role in modulating neuronal injury severity.293 Finally, G-protein coupled inwardly rectifying potassium (GIRK) channels (Kir3) are inward rectifying channels that are usually coupled with GABAB receptors and help to facilitate slow inhibitory postsynaptic potentials

(IPSCs). These slow IPSCs, as well as GIRK expression, have been shown to increase upon increased calcium signaling through NMDA receptor activation, and provide inhibitory drive to reduce neuronal excitability.294,295 The activity of all of these K+ channels, and their ability to provide neuroprotection by reducing excitability have not been studied in HF-HI. Further, the effect that impact frequency has on their expression and regulation is not known. Exposure to intermittent, high-frequency insults, such as those delivered by our unique model of head impacts, could produce a phenotype in CA1 pyramidal cells where excitability is increased without the changes in rheobase or input resistance. Additionally, the ability of K+ activity to influence

GABAB mediated IPSCs should also be explored, as chronic inhibitory changes could play a role in modulating excitability, and only GABAA was inhibited during these studies. A time-course experiment to characterize K+ channel conductance/ expression changes and inhibitory influence at each stage of impact progression (along the 6 days of impacts) would be needed to elucidate how the net effect that we observe was achieved.

Spontaneous mEPSCs were also altered, as HF-HI mice had a higher inter-event interval

(indicating a lower frequency) than the sham mice 24 hours after impact cessation. This effect was acute, as it was not present at 1 month after HF-HI. Under the experimental conditions used (see methods chapter for details), the mEPSCs recorded are thought to be primarily mediated by AMPA receptors. The use of BMR throughout the recording also precludes the influence of GABAA

97 mediated inhibition. A decrease in the frequency of mEPSCs is thought to potentially arise from a reduction in presynaptic vesicular release. The changes in mEPSC frequency that I observed in the absence of changes in sEPSC frequency or amplitude could be attributed to the existence of synaptic niches that are dominated by either spontaneous or evoked release.296 I did not observe changes in the presynaptic vesicular release probability when I performed PPR experiments, which indicated that evoked vesicular release probability is likely unaltered by HF-HI. Further, sEPSCs include action potential-mediated vesicular release, which are an evoked response and could thus arise from activation of combinations of these different vesicular pools. Taken together, my findings could indicate that while evoked release is unaltered by HF-HI, spontaneous vesicular release could be. Future studies must be done in order to determine whether changes in presynaptic components responsible for vesicular release and characteristic of either spontaneous or evoked synaptic niches are effected by HF-HI. Another potential contributor to changes in mEPSC frequency is changes in synapse number. Golgi staining to quantify dendritic spines in CA1 revealed no loss of excitatory synapses after HF-HI, making it unlikely that synaptic loss is mediating this effect. Factors affecting mEPSC frequency are also commonly observed concurrently with changes in mEPSC amplitude, which was unchanged after HF-HI. Several studies have found similar results, and attribute these findings to multiple potential origins. For instance, some evidence suggests that an increase in the ratio of αCaMKII to βCaMKII can decrease mEPSC frequency in Schaffer Collateral/CA1 synapses.297 Synaptic plasticity is dependent on CaMKII, and these enzymes differ in their selectivity for calcium levels.298 An important future direction would therefore be to examine the activity and expression of these enzymes to see if they are altered after HF-HI. The frequency of mEPSCs can increase in the absence of any significant effects on amplitude when silent synapses are “unsilenced”.299-301

98 Therefore an increase in silent synapses could cause the opposite effect, and decrease the frequency without changing the amplitude. To explore this as a possible mechanism for my findings, the effect of HF-HI on silent synapse formation was explored further in the next chapter. Another study found decreased mEPSC frequency with no change in paired pulse ratio, amplitude of mEPSCs, or synapse number, which was reported to occur with a simultaneous increase in the amount of docked synaptic vesicles.302 These findings were attributed to synaptic dysfunction that preceded any discernable morphological degeneration. I did not explore changes in the number of docked vesicles in this study, nor was I able to assess dendritic spine morphology, but both of those things may be altered by HF-HI. Future directions of the lab are to use electron microscopy to interrogate the pre- and postsynaptic terminals to look at vesicle number and postsynaptic composition. Additionally, biocytin filling and fluorescent imaging of CA1 pyramidal cells to examine dendritic spine morphology is also a priority for future studies of HF-HI, as spine size and shape can impact AMPA receptor expression and detectable, available synapses responding to neurotransmission at resting membrane potentials, subsequently altering mEPSC properties.303,304 I believe that similar synaptic dysfunction, mediated by glutamatergic receptor changes, is occurring after HF-HI. These changes may alter the signaling capabilities of these cells and contribute to the development of lasting functional impairments.

Studies done using other models of repetitive concussion have found varying results when examining intrinsic properties. A common factor observed in single and repetitive models of mild

TBI is altered excitability. After a single closed head injury, a transient lasting increases in excitability were reported.305,306 After a single, mild fluid percussion injury, a reduction in excitability was observed.307 Decreased excitability was also produced in mild lateral fluid percussion injury. 308 The firing of place cells during task performances was also decreased after

99 single mild TBI.309 In a pediatric weight drop model, changes in intrinsic properties and excitability were not elicited after repetitive mild TBI.310 Many of these studies employed field potential recordings to assess excitability, and looked at impact frequencies much lower than those used in HF-HI, so differences in excitability may be influenced by these factors.

Synaptic properties after mild and repetitive mild TBI are also variable different models of repeat concussion. Similar to what was observed after HF-HI, paired pulse ratio was also not altered in an in vitro, axonal stretch model of repetitive mild TBI.311 In a comprehensive assessment of the effects of repetitive closed head injury, no changes in paired pulse facilitation was observed, but increased sEPSC frequency and decreased sEPSC amplitude, as well as an increase in mEPSC frequency and amplitude were observed.112 Probability of vesicular release is similar between the HF-HI model and this repetitive closed head injury paradigm, however the synaptic findings vary greatly. This could be due to the difference in impact frequency. Another factor to consider is the lack of inflammation incurred by HF-HI that is present in this model, as inflammation can influence synaptic function in the hippocampus.312 These findings highlight important injury related mechanisms that can influence synaptic function in concussive injury and should be considered when comparing the effects of HF-HI to other models.

Taken together, these results suggest that HF-HI elicits acute changes in intrinsic properties and synaptic transmission of CA1 pyramidal cells. A number of repetitive head impact models have found changes in both cellular and network properties, but the results are very variable. This could highlight the importance of impact frequency when assessing these factors, or the unique lack of contribution of classical injury sequelae in determining functional alterations after HF-HI.

The intrinsic changes are not present at 1 month after impacts, so it is unlikely that they are contributing to the plasticity changes that underlie the cognitive deficits that we observe. In the

100 next chapter, the possibility that the mEPSC inter-event interval increase that we observed is indicative of glutamatergic synapse alterations caused by HF-HI is explored. Glutamatergic function is essential in determining synaptic plasticity, cell survival after insult, and learning and memory efficacy, so lasting alterations in these receptors could underlie impairments after HF-

HI.313

101 Chapter 5

Glutamatergic receptor signaling in hippocampal CA1 pyramidal neurons is altered by

HF-HI

Introduction

Glutamate is the primary excitatory neurotransmitter of the central nervous system. It exerts its actions through distinct classes of receptors. Ionotropic receptors, in particular, are important for fast excitatory transmission as the binding of the ligand to the receptor directly opens a pore that specific ions can flow through to depolarize the membrane or mediate downstream signaling processes. In the Schaffer collateral/CA1 pathway of the hippocampus, there are three main ionotropic glutamatergic receptor subtypes: AMPA, Kainate (KA), and NMDA.314,315 The

KA receptors expressed postsynaptically on CA1 pyramidal cells are functional, however they contribute very little to the EPSCs in that region.316,317 Therefore studies on glutamatergic signaling in CA1, including the present study, focus primarily on AMPA and NMDA receptors.

AMPA receptors are heteromeric structures, usually built with a combination of four distinct subunits: GLUR1-4.318 In CA1, the majority of the AMPA receptor complexes consist combinations of either GLUR1/2 or GLUR2/3 subunits.319 The movement of AMPA receptors into and out of the membrane or changes in their phosphorylation state is important for synaptic efficacy and plasticity. Endocytosis of AMPA receptors occurs as a result of an influx of specific amounts of calcium that interacts with the calcium/ calmodulin dependent phosphatase, calcineurin. This process results in weakening of a synapse, or LTD.320 Conversely, exocytosis of

AMPA receptors in response to calcium influx and the interaction of calcium with CaMKII can strengthen the synapse, resulting in LTP.321 Additionally, phosphorylation of GLUR1 at serine

831 by CaMKII is essential for LTP, whereas dephosphorylation of GLUR1 on serine 845 by

102 protein phosphatases 1/2a is important for LTD. Cycling of AMPA receptors into and out of the membrane in response to synaptic activity also helps to maintain the homeostatic balance of synapses as well.322 Studies have shown that surface expression of GLUR1/2 containing AMPA receptors is specifically involved in plasticity, whereas GLUR2/3 containing AMPA receptors are continuously turned-over to maintain homeostasis at synapses that already contain AMPA receptors.323 The highly mobile nature of these receptors, and the roles that specific AMPA receptors play in experience and activity dependent plasticity make them an important receptor population to study in order to identify mechanisms that could be mediating the functional changes elicited by HF-HI.

A small percentage of AMPA receptors lack a GLUR2 subunit, or contain a switch at the

Q/R site that codes for the amino acid arginine instead of glutamine in the GLUR2 subunit, making them permeable to calcium and zinc.324,325 They are identified electrophysiologically because they exhibit inward rectification, meaning that at positive membrane potentials they have reduced conductance in the presence of polyamines.326 Expression of these AMPA receptors has been shown to be influenced by insults to the brain, and perpetuate calcium dysregulation that can cause degeneration. After a global ischemic insult, mRNA coding for GLUR2 subunits is significantly decreased in CA1 pyramidal cells, and AMPA-elicited increases in calcium occur.327,328 When calcium influx is increased via increased calcium permeable AMPA receptor expression after ischemia, an increase in neuronal degeneration occurs in the hippocampus.329-331 In multiple models known to elicit seizures, calcium permeable AMPA receptor expression also increases and allows for a toxic increase in calcium that promotes degeneration.332-335 The same is true in some models of injury, where increases in calcium permeable AMPA receptors occur and promote degeneration and cell death.113,336,337 These findings suggest that calcium permeable AMPA

103 receptors may mediate key degenerative processes, and are therefore important to study after HF-

HI.

Another important glutamatergic receptor implicated in both excitotoxicity and plasticity processes is the NMDA receptor. Their function in these processes is usually attributed to the fact that, unlike the majority of the AMPA receptors present in the hippocampus, NMDA receptors are highly permeable to calcium.338 They are either di- or tri-heteromeric receptors, composed of one obligatory GLUN1 subunit, and combinations of either GLUN2A-D or GLUN3A&B subunits.339

The majority of NMDA receptors found in the adult hippocampus are comprised of two GLUN1 subunits and two GLUN2 subunits, either GLUN2A and/or GLUN2B.340,341 When synapses mature, there is a switch from primarily GLUN2B mediated EPSCs to GLUN2A mediated

EPSCs.342,343 GLUN2B-containing NMDA receptors have slower decay kinetics than GLUN2A- containing receptors, thus allowing more calcium into the cell once opened.344,345 GLUN2A subunits are primarily found in the synapse, whereas the GLUN2B subunit is found predominantly extrasynaptically. GLUN2B can also be found in the synapse, but in much smaller amounts when compared to GLUN2A. This could partially be attributed to their mobility, as in vitro studies have shown that GLUN2B containing NMDA receptors can move easily between synaptic and extrasynaptic sites.313 The GLUN2D subunit is present on CA1 pyramidal cells as well as interneurons in the hippocampus of humans and rats in adulthood, and in juvenile mice. It has the longest decay of all of the GLUN2 subunits, and is usually found extrasynaptically.202,345 The subunit composition of NMDA receptors alters their functionality, thus altering their relative contributions to synaptic plasticity as well as degeneration in injury or disease.346,347

Specific NMDA receptors have been implicated in modulating the response to injury. In hypoxia, ischemia, seizures, and brain injury, an increase in extracellular glutamate can cause

104 depolarization, and an increase in intracellular calcium in adjacent cells.348-350 This increase in intracellular calcium is linked to cell death.351,352 Activation of NMDA receptors by increased extracellular glutamate is thought to be the primary route through which aberrant calcium enters cells, making them a target for many studies focused on mitigating this process.353 For over a decade, there has been some debate as to whether the subunit composition or the location of the

NMDA receptors play a role in determining if protective or degenerative processes are elicited when faced with an insult. As previously mentioned, GLUN2A subunits tend to be located at synaptic sites, while GLUN2B subunits are found most often at extrasynaptic sites, so teasing apart the contribution of subunits and locations has been particularly difficult. In a study performed using experimental ischemia, Liu et al., showed that, when both synaptic and extrasynaptic

GLUN2A subunit containing receptors were activated, it promoted cell survival, while activation of both populations of GLUN2B containing receptors caused neuronal apoptosis.354 Several other studies using multiple models of insult have found similar relationships between specific subunits and excitotoxicity or cell survival.355,356 These findings were called into question by a study performed by Hardingham et al., which implicated receptor location in mediating cell death or survival. Extrasynaptic NMDA receptor stimulation was shown to promote the shut off of cAMP

Response Element Binding Protein (CREB), a protein shown to promote cell survival, as well as reduce the amount of brain derived neurotrophic factor (BDNF) expressed. Loss of mitochondrial membrane potential, and subsequent cell death occurred as a result. Conversely, synaptic NMDA receptor activation induced CREB activity, increased BDNF production, and served to promote anti-apoptotic processes.357 In fact, blockade of extrasynaptic receptors preferentially by

Memantine has been shown to stop excitotoxic cell death.358 Location of receptors in relation to their roles in excitotoxic situations has also been extensively studied.359 Taken together, these

105 findings emphasize the ability of discrete populations of NMDA receptors to contribute to neural injury. Based on this idea, I sought to explore NMDA receptor signaling as a mechanism for HF-

HI mediated functional deficits.

The interplay between NMDA and AMPA receptors is important for normal function, synapse structure and plasticity. Under physiological conditions, calcium influx via NMDA receptors mediates LTP and LTD through alterations in AMPA receptor expression and phosphorylation.31,32 Dendritic spines in stratum radiatum of CA1 have an almost uniform expression of NMDA receptors. The quantity of NMDA receptors is directly related to the size of the spine, such that larger spines contain more NMDA receptors. AMPA receptor density is variable, and is not tightly correlated with spine size, but tends to be lower than the density of

NMDA receptors.360 This is, perhaps, reflective of the varying propensities for plasticity of individual spines. Spines that have NMDA receptors but lack functional AMPA receptors are referred to as “silent synapses” because they do not respond at the resting membrane potential.361

They are thought to be substrates for LTP, as strong stimulation that elicits sufficient depolarization to open the NMDA receptors will elicit AMPA receptor insertion and “unsilencing” of these synapses.199,362 Under pathological conditions in the adult brain, it is thought that silent synapses can contribute to impairment and neurodegeneration. In addiction and some forms of brain injury, De novo generation of silent synapses, which are then unsilenced, have been shown to contribute to aberrant synaptic reorganization. Additionally, silencing of functional synapses can eventually lead to synapse loss and neurodegeneration.363 Silent synapses could therefore be an important contributor to deficit development following HF-HI.

Glutamatergic receptors play an integral role in normal function, as well as in providing neuroprotection or perpetuating degeneration after insults to the brain. Previously, I showed that

106 processes directly linked to glutamatergic receptor function were altered by HF-HI. Specifically,

HF-HI caused deficient LTP, and impairments in learning and memory. Given the lack of changes that I observed in classical injury cascades (p-tau, Aβ, inflammation, and cell death) after HF-HI, as well as the unaltered network and intrinsic cellular properties in CA1 pyramidal cells, I theorized that the deficits resulted from HF-HI induced glutamatergic receptor changes that arose in response to injury. I decided to employ whole cell patch clamp electrophysiology again to examine these receptor properties in CA1 pyramidal cells. I also chose to evaluate these properties at 24 hours and 1 month after the last day of impacts, in order to characterize any acute and chronic changes

(respectively). I hypothesized that HF-HI would elicit changes in receptor expression, particularly

NMDA receptors as they are more susceptible than AMPA receptors to aberrant activation when extracellular glutamate is elevated. I also hypothesized that calcium permeable AMPA receptor expression may increase, given that different kinds of injury can suppress GLUR2 expression.

Further, since extrasynaptic receptors have been implicated in excitotoxicity, I theorized that extrasynaptic receptor function or expression may be altered by HF-HI. Finally, given the mEPSC frequency decrease that I previously observed and it’s connection to silent synapse formation, I also theorized that silent synapses might be altered by HF-HI. If these changes in receptor function or expression occur, they could underlie the cognitive deficits observed in this head impact model.

Results

a. The NMDA/AMPA Ratio is Chronically Altered by HF-HI

The ratio of NMDA to AMPA at the synapse plays a major role in the summation of synaptic currents, as well as the amount of calcium admitted into the postsynaptic cell in response to presynaptic activity.364 In order to identify any changes in glutamatergic receptor expression or function elicited by HF-HI, I first sought to determine the NMDA to AMPA ratio. As previously

107 mentioned, the KA receptor contribution to EPSCs in CA1 in minimal,316,317 therefore components of the EPSC with decay kinetics characteristic of AMPA receptors was considered to be primarily mediated by AMPA. These recordings revealed that at 24 hours after the last day of impacts, the

NMDA/AMPA ratio was increased in HF-HI mice (Fig. 20A,B; *P<0.05, unpaired t-test). The same is true after 1 month after the last day of impacts (Fig. 21A,B; *P<0.05, unpaired t-test).

These results suggest that HF-HI causes a chronic increase in the ratio, either via an increase in

NMDA receptor expression, or a decrease in AMPA receptor expression. To determine whether

NMDA EPSCs were driving this shift, I assessed their properties. Since the subunit composition of NMDA receptors can alter the decay kinetics and possibly the contribution of NMDA receptors to the EPSC, I analyzed the decay (weighted tau) of the averaged evoked NMDA EPSC elicited during the NMDA/AMPA ratio recordings. There was no significant difference between the two groups, indicating that subunit composition of the receptors was likely unchanged by HF-HI (Fig.

20C; 21C; unpaired t-test). To determine whether decreases in AMPA receptor expression could likely be mediating the observed shift in the NMDA/AMPA ratio, I next assessed the peak amplitudes for AMPA and NMDA in the cells assessed. While different stimulus intensities were used in each slice to elicit the responses measured, there is no significant difference between the two groups in the intensity used (Fig. 20D; 21D; unpaired t-test). At 24 hours, the peak amplitude for AMPA EPSC was higher in sham cells than HF-HI cells (Fig. 20E; *,#P<0.05, Two-way RM

ANOVA with Bonferroni post-hoc test). At 1 month, the peak amplitude of the NMDA EPSC was higher in HF-HI cells than sham cells (Fig. 21E; *P<0.05, Two-way RM ANOVA with Bonferroni post-hoc test).

108

A

B C

D E

Figure 5.1. The NMDA/AMPA ratio is acutely increased by HF-HI.

Twenty-four hours after the last day of impacts, the NMDA/AMPA ratio was recorded in CA1 pyramidal cells. The peak amplitude of the NMDA EPSC recorded at 40mV was divided by the

109 peak amplitude of the AMPA EPSC at -70mV to determine the ratio. (A) Representative traces from sham (left) and HF-HI (right) CA1 pyramidal cells. (B) Quantification of the NMDA/AMPA ratio shows a significant increase in HF-HI cells as compared to shams (*P <0.05, unpaired t-test; n = 10 cells sham, n = 10 cells HF-HI, 1 CA1 cell/animal). (C) Weighted tau, representative of decay of the NMDA EPSC is not significantly different between sham and HF-HI groups (n = 9 cells sham, n = 8 cells HF-HI, 1 CA1 cell/animal, unpaired t-test). (D) Stimulus intensities used to elicit currents at -70mV and 40mV were not significantly different (n = 11 cells sham, n = 11 cells

HF-HI, 1 CA1 cell/animal). (E) Peak amplitudes of EPSCs recorded at -70mV and 40mV (n = 10 cells per group, 1 CA1 cell/animal; *,#P <0.05 Two-way RM ANOVA with Bonferroni post-hoc test).

110

A

B C

D E

Figure 5.2. The NMDA/AMPA ratio is chronically increased by HF-HI.

One month after the last day of impacts, the NMDA/AMPA ratio was recorded in CA1 pyramidal cells. The peak amplitude of the NMDA EPSC recorded at 40mV was divided by the peak

111 amplitude of the AMPA EPSC at -70mV to determine the ratio. (A) Representative traces from sham (left) and HF-HI (right) CA1 pyramidal cells. (B) Quantification of the NMDA/AMPA ratio shows a significant increase in HF-HI cells as compared to shams (*P <0.05, unpaired t-test; n =

11 cells per group, 1 CA1 cell/animal). (C) Weighted tau, representative of decay of the NMDA

EPSC is not significantly different between sham and HF-HI groups (n = 11 cells per group, 1

CA1 cell/animal, unpaired t-test). (D) Stimulus intensities used to elicit currents at -70mV and

40mV were not significantly different (n = 12 cells sham, n = 12 cells HF-HI, 1 CA1 cell/animal).

(E) Peak amplitudes of EPSCs recorded at -70mV and 40mV (n = 11 cells per group, 1 CA1 cell/animal; *P <0.05, Two-way RM ANOVA with Bonferroni post hoc test).

112 b. Silent Synapse Formation is Not Detected After HF-HI

Next, I sought to determine whether changes in the NMDA/AMPA ratio resulted from a decrease in AMPA receptors at functional synapses. These AMPA silent synapses can be detected using a protocol called “minimal stimulation.” These recordings involve using a stimulus intensity that elicits an AMPA mediated response at -70mV, but has an approximate failure rate (where failures are described as a lack of current response to the given stimulus) of 60%. The same stimulus is then applied at 40mV to examine the NMDA mediated response, and the number of failures are counted. If there is an increase in AMPA silent synapses, the failures will be higher at

-70mV than at 40mV, because synapses containing only NMDA receptors will be silent when the cell is held close to the resting membrane potential, but will respond at more depolarized potentials where the magnesium blockade is removed.365 When minimal stimulation was performed 24 hours after the last day of impacts, there was no significant difference between the sham and HF-HI groups in the number of failures at each membrane potential, or the ratio of failures at -70mV and

40mV (Fig. 22A,B; Two-way RM ANOVA with Bonferroni post-hoc test). There was also no significant difference between the groups in any of the aforementioned measurements at 1 month after the last day of impacts (Fig. 23A,B; Two-way RM ANOVA with Bonferroni post-hoc test).

The coefficient of variation (CV) was calculated for AMPA and NMDA by dividing the standard deviation by the mean of the peak amplitudes recorded from 50 sweeps. There was no significant difference between the sham and HF-HI groups in CV 24 hours after the last day of impacts (Fig.

22C; Two-way RM ANOVA with Bonferroni post-hoc test). The same was observed at 1 month after the last day of impacts (Fig. 23C; Two-way RM ANOVA with Bonferroni post-hoc test).

Both minimal stimulation and the CV calculations for both sham and HF-HI groups at acute and chronic time points reveal no discernable increase in silent synapses after HF-HI.

113

A

B C

Figure 5.3. HF-HI does not produce detectable silent synapse formation acutely.

A minimal stimulation protocol was used to determine whether HF-HI elicited silent synapse formation 24 hours after the last day of impacts. A stimulation at an intensity that elicits 10-30pA responses 60% of the time was used to determine the number of failures that occur at -70mV and

40mV holding potentials. (A) Representative traces from sham (left) and HF-HI (right) cells at

40mV (top trace) and -70mV (bottom trace). (B) The number of failures at each membrane potential, and the ratio of failures at -70mV to 40mV for each group was not significantly different

(n = 13 cells per group, 1 cell/animal; Two-way RM ANOVA). (C) CV for each receptor subtype

114 (calculated as the standard deviation divided by the mean of the peak amplitude of responses for each membrane potential) is not significantly different between either group for either receptor subtype (n = 13 cells sham, n = 12 cells HF-HI, 1 cell/animal; Two-way RM ANOVA).

115

A

B C

Figure 5.4. HF-HI does not produce detectable silent synapse formation chronically.

A minimal stimulation protocol was used to determine whether HF-HI elicited silent synapse formation 1 month after the last day of impacts. A stimulation at an intensity that elicits 10-30pA responses 60% of the time was used to determine the number of failures that occur at -70mV and

40mV holding potentials. (A) Representative traces from sham (left) and HF-HI (right) cells at

40mV (top trace) and -70mV (bottom trace). (B) The number of failures at each membrane potential, and the ratio of failures at -70mV to 40mV for each group was not significantly different

116 (n = 10 cells per group, 1 cell/animal; Two-way RM ANOVA). (C) CV for each receptor subtype

(calculated as the standard deviation divided by the mean of the peak amplitude of responses for each membrane potential) is not significantly different between either group for either receptor subtype (n = 11 cells sham, n = 10 cells HF-HI, 1 cell/animal; Two-way RM ANOVA).

117 c. Calcium Permeable AMPA Receptor Expression is Likely Unchanged by HF-HI

GLUR2-lacking AMPA receptor expression can be increased by different forms of injury.

Given their permeability to calcium, they can have a major effect on synaptic plasticity, as well as on cellular responses to elevated extracellular glutamate. To determine whether the expression of these calcium permeable receptors is altered by HF-HI, I recorded current responses to stimulation of the Schaffer Collateral pathway at various positive and negative membrane potentials while

25µM BMR and 40µM (R)-CPP were constantly perfused via y-tube. These recordings were performed 24 hours after the last day of impacts. I found no difference between the sham and HF-

HI groups in the rectification index, or the ratio of the average peak amplitude of current at -40mV to the average peak amplitude of the current recorded at 40mV (Fig. 24 A,B; unpaired t-test).

Given the inwardly rectifying properties of calcium permeable AMPA receptors, an increase in their expression would be reflected by a reduced peak amplitude of current at positive membrane potentials (40mV). Because no difference between the two groups in rectification was observed, it is likely that HF-HI does not increase the expression of these receptors. Additionally, the recordings were not repeated 1 month after the last day of impacts because their absence at 24 hours made it unlikely that there would be any changes in the expression of these receptors at a more chronic time-point.

118

A B

Figure 5.5. Inward rectification mediated by calcium permeable AMPA receptors is unchanged by HF-HI.

Recordings were performed 24 hours after the last day of impacts at either -40mV or 40mV holding potential in the presence of 25µM BMR and 40µM (R)-CPP. (A) Current versus voltage (IV) curve for both groups show no differences in current at positive holding potentials (n = 9 cells per group,

1 cell/animal, unpaired t-test). (B) There is no significant difference in the rectification index (the ratio of the peak amplitude of the current at 40mV to -40mV) between sham and HF-HI groups (n

= 9 cells per group, 1 cell/animal, unpaired t-test).

119 d. Extrasynaptic and Perisynaptic Receptor Mediated EPSCs are Unaltered by HF-HI

When glutamate is allowed to spill over into extrasynaptic sites, slower EPSCs than those generated with synaptic NMDA receptor activation are observed, and they are thought to be mediated by activation of extrasynaptic/perisynaptic receptors.366 Extrasynaptic and perisynaptic receptors in CA1 pyramidal cells are primarily composed of GLUN2B and (in humans and some animals) GLUN2D subunits.367 These subunits are known to convey longer decay times than the other GLUN2 subunits, giving the characteristically slower decay to EPSCs observed with glutamate spillover.345 Therefore, decay characteristics can be used to examine changes in extrasynaptic/perisynaptic NMDA receptor function, subunit composition and expression, which is important given that extrasynaptic receptor activation promotes cellular vulnerability in excitotoxic environments.368 Using a protocol adapted from Lozovaya et al., 2004, I sought to determine whether extrasynaptic/perisynaptic receptors were altered by HF-HI. I examined extrasynaptic/perisynaptic receptor signaling 24 hours after the last day of impacts, and used a stimulation protocol of 7 stimuli delivered at 200Hz. This stimulation frequency is similar to physiological stimulus patterns, and shown to convey considerable facilitation of the EPSC, as well as generate the characteristic extrasynaptic/perisynaptic EPSC slow decay.202,369 Each cell’s response to this stimulation protocol at 40mV was compared to its own response to single stimuli at 40mV delivered every 20 seconds. The decay of the extrasynaptic/perisynaptic response was not altered by HF-HI (Fig. 25A,B; unpaired t-test). The percent charge transfer (%Q) was calculated by dividing the area under the curve of the extrasynaptic/perisynaptic response by the area under the curve of the response to the single stimulation (both normalized to their own mean amplitude of the peak of the response). There was no significant difference between sham and HF-

HI mice in the %Q 24 hours after the last day of impacts (Fig. 25C; unpaired t-test). I also recorded

120 the tonic NMDA current 24 hours after the last day of impacts as they have been shown to be mediated primarily by extrasynaptic NMDA receptors.370 It was assumed that any changes in the tonic current at 24 hours would likely be the same at 1 month after impacts. There was no difference between the two groups (Fig. 25D; unpaired t-test).These results indicate that HF-HI does not alter extrasynaptic receptor expression or function. Since no changes were observed at the 24 hour time-point, I did not repeat the experiment at 1 month after the last day of impacts.

The chronic changes in these extrasynaptic/perisynaptic receptor mediated EPSCs will have to be determined.

121

A

B C

D

Figure 5.6. Extrasynaptic and perisynaptic NMDA EPSCs are unchanged by HF-HI.

Twenty-four hours after the last day of impacts, single and high frequency stimulation (7 stimuli given at 200Hz) were given at 40mV holding potential in the presence of 25µM BMR, and the

122 decay for each was measured. (A) Representative traces of sham (left) and HF-HI (right) currents elicited in response to single (red) and high frequency stimulation (black). (B) The ratio of the weighted tau (decay) of the extrasynaptic component (slow NMDA EPSC) to the synaptic (NMDA

EPSC) component is not significantly different between both groups (n = 12 sham, n = 12 HF-HI,

1 cell/animal, unpaired t-test). (C) Normalized percent charge transfer (%Q) is not significantly different between sham and HF-HI slices (n = 14 sham, n = 13 HF-HI, 1 cell/animal, unpaired t- test). (D) Tonic NMDA current (measured as the difference in leak current value before the addition of 40µM (R)-CPP minus the current value after the addition of (R)-CPP). For this experiment, n = 9 cells, 1 cell/animal/group, unpaired t-test.

123 Discussion

The findings from these experiments indicate that HF-HI alters the function and expression of glutamatergic receptors. The NMDA/AMPA ratio is elevated both acutely and chronically after

HF-HI, despite the fact that the decay of the NMDA currents remain unchanged. At 24 hours after the last day of impacts, the peak amplitudes measured at -70mV (reflecting the AMPA EPSC) was decreased in HF-HI as compared to shams, while the NMDA EPSC (measured at 40mV) was unchanged. At 1 month, the opposite effect was observed, in that the NMDA EPSC was increased in HF-HI as compared to shams, while the AMPA EPSC was not changed. These data were recorded using different stimulus intensities between slices to elicit a response within a distinct range, however there is no significant difference between the groups in the strength of stimulus used, suggesting that strength of stimulus is not mediating the trends observed in peak amplitude values for each group. These findings might indicate that changes in different receptor populations contribute to the skewed NMDA/AMPA ratio at both time-points. Further studies must be done to identify synaptic expression changes in order to determine exactly how each receptor population changes acutely and chronically in response to HF-HI. The decay of perisynaptic/extrasynaptic

NMDA receptors, perisynaptic/extrasynaptic NMDA receptor charge transfer, and NMDA tonic current were also unchanged by HF-HI. This suggested that changes in AMPA receptor properties are primarily underlying the NMDA/AMPA ratio shift.

Given the strong evidence that receptor expression is altered by HF-HI, I sought to identify a mechanism for these changes. One of the mechanisms that can underlie a shift in the

NMDA/AMPA ratio is the formation of synapses that contain only NMDA receptors, commonly referred to as silent synapses. To determine whether HF-HI caused an increase of silent synapses,

I employed a minimal stimulation protocol. I found that there were no significant changes in the

124 ratio of failures at -70 and 40mV, suggesting that silent synapses were not increased at either chronic or acute time-points. I also calculated the CV of the EPSCs from this experiment, in an attempt to try to confirm decreases in AMPA receptor expression using another method. The CV of AMPA EPSCs and NMDA EPSCs can has been shown to reveal information about their expression.361 This measurement is larger during basal transmission for AMPA than NMDA

ESPCs, indicating that there are less AMPA receptors.371 Further, the CV is shown to be altered by LTP and LTD, reflecting plasticity mediated changes in AMPA receptor expression.372 These calculations also did not reveal a difference in AMPA receptor expression at 24 hours or 1 month after impacts. Of course, it is possible that HF-HI does not produce silent synapses, and could instead alter AMPA receptor expression through changing dendritic spine turnover or altering spine morphology. Both of these mechanisms could decrease AMPA receptor expression without producing synapses that completely lack AMPA receptors.304,373 As previously mentioned, experiments aimed at using fluorescent labelling to asses spine morphology are currently underway, and will give a more definitive idea of whether altered spine morphology is contributing to the NMDA/AMPA ratio phenotype observed after HF-HI. However, given that a fair amount of data collected suggest that silent synapse formation is occurring after HF-HI, another explanation is that the protocol that I used to assess failure rates and CVs for AMPA EPSCs and NMDA EPSCs may not be sensitive enough to detect silent synapses.

To try to examine silent synapse formation, I performed multiple iterations of the minimal stimulation experiment. Each iteration involved troubleshooting with different stimulation parameters used to achieve the desired failure rate, and different methods of analysis to standardize and automate response detection (ruling out any subjective observations). The findings from each of these experiments were inconsistent in both sham and HF-HI slices. The data from some

125 experiments suggested a trend of increased silent synapses in the HF-HI group, but with no statistical significance. Kirchner and Nicoll (2008) highlighted an important factor that can confound results from minimal stimulation recordings. They proposed that somatic recordings from pyramidal cells in the hippocampus in response to extracellular electrical stimulation have been shown to provide inconsistent results between sweeps due to an inability to control for certain factors. It is difficult to have complete control over synaptic connections to the cell of interest and which inputs to the cell are being stimulated because of the nature of the hippocampal circuitry, therefore it is hard to hold pre- and postsynaptic factors constant in a manner that allows one to derive straightforward conclusions.198 One way to provide focused stimulation and limit glutamatergic spillover onto neighboring synapses that could influence current responses is to perform paired recordings. In a paired recording, a CA3 pyramidal cell and its synaptically coupled partner (a CA1 pyramidal cell) are both patched. Direct current injection of the CA3 cell to produce infrequent postsynaptic responses in its binding partner can allow for a more direct assessment of a discrete population of synapses, a proportion of which may be better identified as silent.374 I would therefore want to repeat this experiment using paired recordings of CA3 and CA1 pyramidal cells (which are difficult, but can be done in slice preparations 375) to apply a more focused method of detecting changes in silent synapses, and hopefully better elucidate the effects of HF-HI on silent synapse formation.

Exposure to HF-HI did not seem to elicit an increase in GLUR2-lacking AMPA receptors, as the current versus voltage (IV) curve and rectification index was not changed by HF-HI. The inward rectification characteristic of these calcium permeable AMPA receptors was not more readily apparent in the IV relationship at positive membrane potentials in HF-HI mice as compared to sham mice. A caveat to this experiment is that these recordings were performed with a

126 pharmacological blockade of GABAA and NMDA receptors extracellularly to isolate the AMPA

EPSC, but spermine was not included in the intracellular solution. Instead, I relied on the endogenous polyamines to convey the voltage-dependent inwardly rectifying properties,376 with the idea that if HF-HI produced discernable changes in the IV curve, I would then perform subsequent experiments to further characterize these change in calcium permeable AMPA receptor expression. It has previously been reported that there is a time-dependent decrease in this rectification when whole cell configuration is used, and the use of intracellular spermine can prevent the decrease from occurring.377 Therefore, it is possible that if HF-HI elicited any effects resulting from a change in calcium permeable AMPA receptor expression, they were not detected due to this time-dependent effect.

Extrasynaptic and perisynaptic NMDA receptors are thought to promote the activation of cell death pathways when faced with insult-induced increases in extracellular glutamate.378 While expression of these receptors is thought to be relatively more stable than AMPA receptors, they can translocate between synaptic and extrasynaptic sites.379 Extrasynaptic NMDA receptor expression changes have been reported in the visual cortex.380 Increased extrasynaptic NMDA receptor expression has been reported in the striatum in response to the expression of the mutant

Huntington gene, and as a result, neurons were more sensitive to glutamate induced neurotoxicity.381 Changes in their expression or composition could, therefore, greatly influence cellular function when sub-toxic elevations in glutamate occur following HF-HI. To determine whether extrasynaptic/perisynaptic receptors played a role in altering function after HF-HI, I assessed extrasynaptic receptor mediated decay 24 hours after the last day of impacts and found no difference between sham and HF-HI groups. This indicates that the subunit composition, or the number of extrasynaptic receptors is unchanged by HF-HI. These findings were not surprising,

127 given that we do not observe increases in cell death after HF-HI. While expression may not be altered, there may be some nuances in extrasynaptic receptor activity that could underlie the glutamatergic receptor changes that I observe. It has previously been shown that activation of

GLUN2B containing NMDA receptors, such as those found at extrasynaptic sites, have the ability to reduce synaptic AMPA receptor expression.382 Activation of these extrasynaptic receptors via increases in ambient glutamate has also been shown to inhibit LTP.383 Therefore, modulating extrasynaptic receptors during HF-HI to block activation, or identifying and modulating extrasynaptic receptor activation-dependent proteins involved in downstream cascades related to

AMPA receptor expression may prevent the changes observed in HF-HI.

Changes in glutamatergic receptor expression and function have been reported in other models of concussive and subconcussive head impacts. For example, after 3 CHI, whole-cell recordings suggested an increase in AMPA receptors seven days after the last day of injuries.112 In another 3 hit CHI model, decreases in GLUR1&2, and GLUN2A&2B were observed, while

GLUN1 was increased. There were also deficient LTP, and spatial learning and memory deficits.384 Repetitive mild lateral fluid percussion injury also elicited spatial learning and memory deficits concurrently with deficits in LTP and reduced NMDA receptor mediated currents.164

Repetitive closed head injury (4 hits, 1 hit per day) also induced a reduction in GLUN2B and

GLUN1, as well as deficient LTP and spatial learning and memory.111 Mice exposed to a repetitive weight drop model, in which they received a total of 7 hits over the course of 9 days, had increases in the phosphorylated form of the AMPA subunit GLUR1 (with no change in total GLUR1) in the cortex at both 3 days and 5 weeks after injury. They also showed a decrease in the GLUN2B subunit at 3 days after injury in the cortex, and both receptor changes were correlated with deficits in learning and memory.385 The same change in phosphorylated GLUR1 was observed after a

128 single lateral fluid percussion injury.386 After a single CHI, an increase in GLUR1 and an increase in phosphorylated GLUN2B were also observed.387 As previously mentioned, increases in calcium permeable AMPA receptor expression were observed, as well as increases in phosphorylated

GLUR1 in an in vitro model of mild mechanical injury.336,388 Many of these models also displayed some inflammation and/or axonal damage, and none of the aforementioned models had as high frequency of an impact as ours. While changes in glutamatergic receptor expression (in either direction) seems to be a common finding in mild TBI, the frequency of impacts in our model may allow for these receptor changes to occur in the absence of other pathology. The HF-HI model may, therefore, serve as evidence that synaptic changes are sufficient to drive spatial learning and memory deficits.

129 Chapter 6

Pretreatment with memantine reverses the behavioral and electrophysiological changes

associated with HF-HI

Introduction

Many studies have shown that aberrant activation of NMDA receptors, and subsequent calcium influx, is a key mediator in perpetuating neuronal death. In fact, glutamatergic receptor over activation has been implicated in multiple neurodegenerative disorders, including

Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, HIV-associated dementia, multiple sclerosis and amyotrophic lateral sclerosis.389 Because of this, therapeutic interventions aimed at blocking NMDA receptors at different stages of disease/ degeneration progression have been widely explored.

The NMDA uncompetitive antagonist memantine (1-amino-3,5-dimethyladamantane) is a popular compound that has been widely studied as a potential treatment for various neurodegenerative diseases and injuries.390 Memantine is an open channel blocker, and as such it hinders NMDA receptor-mediated ion flow into cells by binding to a site within the ion channel only after receptors are activated and the channel has opened.391 Unlike other NMDA antagonists, memantine does not block normal NMDA receptor activity, preserving physiological function.

This reduces the likelihood that it will produce psychomimetic side effects, making it a desirable therapeutic candidate.392 The mechanism underlying the ability of memantine to not produce these side effects is associated with the properties of the NMDA receptors that it interacts with.

Memantine has been shown to become increasingly effective as extracellular glutamate levels increase.393 In cases of injury or illness where extracellular glutamate levels are shown to become elevated, it is thought that deleterious cascades arise through selective activation of extrasynaptic

130 NMDA receptors.357 The efficacy of memantine in blocking neurotoxic cascades in response to elevated glutamate was therefore thought to occur because memantine selectively blocked extrasynaptic NMDA receptors.394 More recently, it has been shown that location of the receptors is not the primary factor mediating memantine’s efficacy, rather it is the likelihood of the receptor to reach a desensitized state that determines memantine’s affinity. Extrasynaptic receptors tend to be most effectively blocked by memantine because their location makes them more vulnerable to increased activation and calcium influx from extracellular glutamate, and thus more likely to reach a calcium dependent desensitized state that memantine was shown to stabilize.395,396 Many studies have utilized these characteristics to explore memantine’s neuroprotective effects.

The ability of memantine to prevent neurodegeneration and cell death in the hippocampus has been explored in various preclinical degenerative models. Using quinolinic acid, an NMDA receptor-specific model of excitotoxic brain damage, it was shown that pretreatment with memantine could preserve neuronal viability in the hippocampus.397 It was also shown that administration of memantine one hour prior to ischemic or hypoxic insult protected the CA1 subfield from incurring cellular damage.398 After focal ischemic injury was elicited, treatment with memantine was shown to reduce the neuronal damage incurred after circulation is restored.399

Memantine has also been shown to protect CA1 in the hippocampus from degeneration caused by

AB-40, a protein known to be dysregulated in several kinds of dementias.400

Spatial learning and memory has also been shown to be rescued by memantine treatment.

Pretreatment with memantine reduced the escape latency and distance traveled in the MWM in rats after the induction of global ischemia.401 Sub-chronic delivery of memantine in an APP/PS1 transgenic mouse model of Alzheimer’s disease decreased the escape latency during the acquisition phase of MWM as well as the time spent in the outer zone during the probe trials.402 In

131 a pentylenetetrazol (PTZ) kindled rat model of chronic epilepsy, memantine delivered chronically during kindling increased the number of platform crossings during the probe trial as compared to vehicle treated, kindled rats.403 The neuronal effects of Down syndrome (DS), which can include neurodegeneration, aberrant NMDA receptor function, inflammation and oxidative stress, can also be ameliorated by memantine.404 In a transgenic mouse model of DS, performance on the win- stay radial arm maze (to assess hippocampal-dependent spatial memory) was rescued by chronic memantine treatment.404 In each of these models, which have the common feature of extracellular glutamate elevations and subsequent NMDA receptor activation increases preceding neurodegeneration, memantine was able to improve spatial learning and memory when administered chronically and/or prophylactically.

A possible mechanism for the improvement in spatial learning and memory mediated by memantine is its effects on hippocampal LTP in conditions where NMDA receptors activation is increased. In trying to model these conditions, one study used low magnesium solution to increase

NMDA receptor activation, and found that LTP in CA1 was restored in slices pretreated with memantine for 4 hours (as compared to untreated slices).405 In another study, administration of

NMDA 15 minutes prior to LTP induction suppressed LTP in the CA1, while performing the same incubation with NMDA and memantine preserved LTP.406 These results have been shown after in vivo administration of memantine as well. Acute hippocampal slice preparations were made from rats that had undergone chronic pretreatment with memantine, and were then exposed to synthetic

Aβ42. Memantine treated slices displayed LTP that was not significantly different from sham slices (that were not exposed to Aβ42), but was significantly higher than LTP induced in Aβ42- exposed slices pretreated with saline only (which were shown to have deficient LTP).407 In a 1- methyl-4- phenyl-1,2,3,6-tetrahydro pyridine (MPTP) mouse model of Parkinson’s disease,

132 pretreatment with memantine 1 hour prior to MPTP injections rescued the reduction in LTP observed after theta burst stimulation in non-memantine treated mice up to a month after MPTP administration.408 The ability of memantine to restore LTP in these conditions makes it an important tool for assessing NMDA receptor-mediated contributions to spatial learning and memory impairment in degenerative and post-insult conditions.

Memantine has been explored as a potential therapeutic agent to preserve neuronal viability after TBI. After moderate CCI, administration of memantine reduced cell death in the hippocampus resulting from the injury.409 Similarly, in moderate fluid percussion injury treatment with memantine acutely following injury reduced cerebral infarct volume, improved neuronal survival, and reduced inflammation.410 In a cold-induced traumatic brain injury, reductions in

DNA fragmentation and inflammatory cytokines were observed in memantine treated animals as compared to injured animals that received vehicle (saline) treatment.411 Similar findings were observed in models of concussive and subconcussive injuries. In a closed head, weight drop injury model where mice received 1 hit a day for 4 days, memantine was administered 1 hour prior to the last hit. A partial rescue of injury induced LTP deficits was observed as well as a reduction in the inflammatory response.111 In a repetitive stretch injury paradigm that mimicked mild TBI in organotypical hippocampal slice cultures, memantine treatment one hour following stretch rescued deficient LTP and prevented cell death (as compared to vehicle treated, injured cultures).412 Taken together, these findings suggest that memantine could be effective in mitigating the degenerative and functional effects of different severities of TBI.

Given memantine’s ability to improve outcomes and provide neuroprotection after exposure to several kinds of injury, I sought to determine whether pretreatment with memantine could reverse the behavioral and synaptic phenotype elicited by HF-HI. The changes in the NMDA/AMPA ratio

133 that occur following HF-HI are thought to be an adaptive response to exposure to a high frequency of impacts specifically. This shift in the ratio likely underlies the deficient synaptic plasticity (LTP) and spatial learning and memory impairments that we observe. As previously mentioned, these factors have shown to be improved following chronic or prophylactic memantine treatment. We decided to employ a pretreatment strategy with memantine in order to see if we could prevent the adaptations in glutamatergic receptors from occurring. One hour prior to administering hits (on all

6 days of the paradigm), we delivered 10 mg/kg of memantine via intraperitoneal injection.

Twenty-four hours after the last day of impacts, I performed whole cell patch clamp electrophysiology to determine whether memantine pretreatment could reverse the effects of HF-

HI on the NMDA/AMPA ratio. To assess behavioral effects of memantine pretreatment, we performed hippocampal-dependent learning and memory tasks, including the Barnes maze and T-

Maze one month after the last day of impacts. Since studies have shown that elevations in extracellular glutamate occur after repetitive exposure to concussive and subconcussive head impacts, extrasynaptic NMDA receptors may become aberrantly activated. Extrasynaptic receptors have been shown to influence AMPA receptor expression at the synapse,382 I hypothesized that by preventing this activation via HF-HI with memantine, frequency-mediated changes in synaptic receptor expression may be prevented, and cognitive impairments will not occur. Memantine could help to identify a mechanism through which HF-HI alters cognition and function in the absence of changes in classical injury markers.

Results

a. Memantine Pretreatment Reverses Learning and Memory Deficits Elicited by HF-HI

The ability of memantine treatment to reverse the cognitive deficits observed in multiple injury models has been well documented. I chose to employ memantine’s modulatory effects on NMDA

134 receptors as a pretreatment to prevent their activation in response to any elevations in extracellular glutamate elicited by HF-HI. Since I believe that changes in synaptic receptor expression result from the influence of elevated glutamate on these receptors, I wanted to determine if blocking this effect would be sufficient to rescue the learning and memory deficits that we have observed. One month after the last day of impacts, acquisition in the Barnes maze was assessed. The HF-HI group treated with only saline had a significantly increased latency to learn the location of the escape hole, while memantine treated HF-HI and both sham groups did not differ in their latencies (Fig.

26A; *P<0.05, Two-way RM ANOVA with Bonferroni post-hoc test). Treatment with memantine also influence the search strategy of HF-HI animals, as heat maps depicting frequency of visits to areas of the maze more closely resembled shams in memantine treated versus vehicle treated mice

(Fig 26B). The number of entries into the escape hole, and the time spent in the escape hole were also increased in HF-HI memantine mice versus HF-HI vehicle treated mice (Fig 26C, D; *,+ P <

0.05; ++P < 0.01, Two-Way ANOVA and Bonferroni post-hoc). The time spent in the escape quadrant was also increased by memantine treatment in HF-HI animals (Fig. 26E; ***P <

0.001;*,+ P < 0.05, Two-Way ANOVA and Bonferroni post-hoc ). While memantine induced improvements in Barnes maze performance in the HF-HI mice, it did alter sham performance, nor did it effect the distance traveled or mean speed of either group (data not shown). These results suggest that memantine pretreatment can reverse chronic deficits in spatial learning and memory observed after HF-HI.

135

A B

C D

E

Figure 6.1. Spatial learning and memory are recovered by pretreatment with memantine.

One month after the last day of impacts, Barnes maze testing was performed on mice treated with either saline (vehicle) or memantine. (A) Latency to platform during the acquisition phase of the

Barnes maze. Data from two trials per day on four consecutive days. (Sham n=14, HF-HI=15,

136 * = P < 0.05, Two-Way ANOVA and Bonferroni post-hoc). (B) Representative heat maps illustrating the search strategies of each of the groups in the Barnes Maze probe trial. (C) Number of entries into the escape target during the Barnes Maze probe trial (Sham n=14, HF-HI=15, += P

< 0.05, Two-Way ANOVA and Bonferroni post-hoc). (D) Time spent in the escape target during the Barnes Maze probe trial (n = 15, * = P < 0.05; ++ = P < 0.01;Two-Way ANOVA and

Bonferroni post-hoc). (E) Time spent in each of the quadrants of the Barnes Maze(Sham n=14,

HF-HI=15, *** = P < 0.001;* = P < 0.05;+= P < 0.05, Two-Way ANOVA and Bonferroni post- hoc).

137 b. Hippocampal-dependent T-maze Performance is Rescued Acutely and Chronically by

Memantine

Spontaneous alternations are a hippocampal-dependent behavior that is shown to be disrupted after injury and in many neurodegenerative diseases.184 We have previously shown that HF-HI disrupts spontaneous alternation using the T-maze apparatus. In order to determine whether pretreatment with memantine can rescue spontaneous alternation, we performed T-maze testing both 24 hours and 1 month after the last day of impacts. Sham and HF-HI groups were treated with either saline (vehicle) or memantine during all 6 days of hits, and were subjected to 4 runs through the maze with 1 hour in between each run. Memantine treatment significantly increased spontaneous alternation in HF-HI mice as compared to saline treated HF-HI mice at both time- points (Fig. 27A, B; *P<0.05, Two-way ANOVA with Bonferroni post-hoc test). There was no significant difference between sham groups (saline and memantine treated) and the HF-HI memantine group. These data suggest that memantine pretreatment can prevent deficits in hippocampal-dependent memory elicited by HF-HI.

138

A B

Figure 6.2. Memantine pretreatment prevents deficits in hippocampal-dependent memory.

Mice pretreated with either saline (vehicle) or memantine were tested in the T-maze apparatus at

24 hours and 1 month after the last day of impacts. Each mouse was tested 4 times, with an hour in between each run. (A) Spontaneous alternations for each group during T-Maze testing performed 24 hours after the last day of impacts (n = 15, * = P < 0.05, Two-Way ANOVA and

Bonferroni post-hoc). (B) Spontaneous alternations for each group during T-Maze testing performed one month after the last day of impacts (n = 15, * = P < 0.05, Two-Way ANOVA and

Bonferroni post-hoc).

139 c. Changes in Glutamatergic Receptor Contributions is Restored By Memantine Pretreatment

Next, I sought to determine whether pretreatment with memantine could prevent the alteration in the NMDA/AMPA ratio I observed previously after HF-HI. To explore this, I performed whole cell patch clamp electrophysiology on CA1 pyramidal cells 24 hours after the last day of impacts.

I found that the elevated NMDA/AMPA ratio was preserved in saline (vehicle or VEH) treated

HF-HI mice as compared to sham mice treated with saline. The HF-HI mice treated with memantine had an NMDA/AMPA ratio that was not significantly different from shams (Fig.

28A,B; *P<0.05, One-way ANOVA with Bonferroni post-hoc test). These findings suggest that memantine pretreatment can prevent changes in glutamatergic receptor expression that can alter the NMDA/AMPA ratio. The peak amplitudes of the AMPA and NMDA EPSCs suggest that

NMDA EPSCs are increased in HF-HI, and after memantine treatment, they return to approximately sham levels (Fig. 28C; Two-way RM ANOVA with Bonferroni post-hoc test).

These changes in the peak amplitudes occur in the absence of a significant difference in the stimulus intensities used to elicit the EPSCs for all groups (Fig. 28D; One-way ANOVA with

Bonferroni post-hoc test).

140

A

C B

D

Figure 6.3. Memantine pretreatment prevents changes in the NMDA/AMPA ratio.

Twenty-four hours after the last day of impacts, whole cell patch clamp recordings were performed on CA1 pyramidal cells in acute hippocampal slices taken from either saline (vehicle) treated sham

141 and HF-HI mice, or memantine treated HF-HI mice. The ratio was determined by dividing the peak amplitude of the NMDA EPSC recorded at 40mV by the peak amplitude of the AMPA EPSC at -70mV. (A) Representative traces from vehicle treated sham (left), vehicle treated HF-HI

(middle) and memantine treated HF-HI (right) CA1 pyramidal cells. (B) Quantification of the

NMDA/AMPA ratio shows a significant increase in vehicle treated HF-HI cells as compared to vehicle treated shams (*P < 0.05, One Way ANOVA with Tukey’s Multiple Comparison Test).

There is a significant decrease in the NMDA/AMPA ratio in memantine treated HF-HI cells as compared to vehicle treated HF-HI cells (*P < 0.05, One Way ANOVA with Tukey’s Multiple

Comparison Test). There is no significant difference between vehicle treated sham and memantine treated HF-HI cells (n = 8 cells VEH Sham, n = 9 cells VEH HF-HI, n = 10 cells MEM HF-HI, 1 cell/animal). (C) Peak amplitudes of the EPSCs at -70mV and 40mV (n = 8 cells VEH Sham, n =

9 cells VEH HF-HI, n = 11 cells MEM HF-HI, 1 cell/animal). (D) Stimulus intensities used to elicit the currents measured for the NMDA/AMPA ratio and peak amplitudes were not significantly different between the groups (n = 8 cells VEH Sham, n = 9 cells VEH HF-HI, n =

11 cells MEM HF-HI, 1 cell/animal).

142 Discussion

Memantine, the uncompetitive NMDA receptor antagonist, has previously been used to rescue the cognitive deficits and alterations in synaptic plasticity observed in multiple injury models. In this set of experiments, I showed that pretreatment with memantine reversed the hippocampal dependent learning and memory impairments, as well as the functional changes that may indicate altered glutamatergic receptor expression induced by HF-HI. It is important to note that in many of the studies that used memantine to reverse deficits, it was administered chronically as a therapeutic agent. I chose to administer memantine as a pretreatment to help elucidate a mechanism for deficit development, and not necessarily to evaluate its therapeutic potential.

Because we observed no pathology, inflammation, or cell death in the HF-HI model, it was important to examine synaptic changes, as they may underlie the changes that occur. Memantine helped to further understand how HF-HI affects normal function, as it allowed us to specifically target a receptor extrasynaptic population that was responding to impact exposure at the high frequency of impacts used. Blocking extrasynaptic NMDA receptors during the impacts reversed the NMDA/AMPA ratio change that HF-HI normally elicits, and the behavioral impairments that

I observed were normalized. Further experiments will need to be done using the LTP paradigm in order to determine whether memantine can also reverse the plasticity deficits, but these results solidify NMDA receptor activation as a mechanism for deficit development in HF-HI.

Repetitive mild TBI models that have employed memantine previously to reverse the injury effects have some striking dissimilarities with our model that may influence the cascades that memantine modulates. In a weight drop model of repetitive mild TBI, memantine was shown to improve histopathological findings and partially restore LTP. This model also demonstrated upregulated APP and accumulation of phosphorylated tau (which is indicative of axonal injury),

143 as well as reductions in GLUN2B subunit expression and increased microglial activation. Further, their spatial learning and memory impairments were worsened by memantine treatment.181 In a modified version of the previously mentioned model, mice received a higher number of impacts

(5 hits instead of 4). These mice had reduction in the number of mature, myelinating oligodendrocytes, myelin basic protein, and neurofilament light chain, as well as altered axonal structure at subacute time-points. Treatment with memantine reversed these effects, likely by preventing excitotoxic damage to oligodendrocytes (which express NMDA and AMPA receptors).413 Once again, the key pathology in this model was axonal injury. In each of these models, hits were delivered once a day. Despite the higher number of impacts used in HF-HI, our model seems to be milder than these models, as we do not find any evidence that HF-HI causes axonal injury, loss of oligodendrocytes and myelination or inflammation. This suggests that frequency of impacts is an important factor in determining the impairments that arise. We also see improvement in spatial learning and memory after memantine pretreatment, thus memantine- mediated effects are likely influencing different cascades to improve our outcomes. When comparing preclinical studies to findings from patients, these factors are important to consider.

HF-HI more closely resembles exposure to a high number of subconcussive and possibly concussive head impacts within a short period of time, as is common in high contact sports, where overt injury may not occur. Our findings emphasize the importance of considering functional changes as a mechanism for deficit development, and memantine treatment highlights that changes in a extrasynaptic NMDA receptor activation that do not result in cell death are an important potential therapeutic target.

We chose to use memantine not only because of its proven neuroprotective ability in situations where extracellular glutamate is elevated, but because of its well established safety

144 profile. As previously mentioned, it does not produce the psychomimetic effects that some other

NMDA receptor antagonists do.392 In fact, it is already in use in the clinic for neurodegenerative disorders. It was used in Europe for the treatment of Parkinson’s disease and spasticity.398 In 2003, memantine was approved by the Food and Drug Administration of the United States for use in patients as a treatment for moderate to severe Alzheimer’s disease.414 It is also prescribed for the treatment of mild cognitive impairment in Alzheimer’s disease, however there is a lack of evidence to support its efficacy at this stage of the disease.415 Since then, several clinical trials have been dedicated to evaluating the safety and efficacy of memantine in amyotrophic lateral sclerosis,416 frontotemporal dementia,417 Lewy body dementia,418 vascular dementia,419 Down syndrome420 and other dementias accompanying neurodegenerative disorders.421 In some instances, such as in vascular dementia, memantine is currently an approved treatment. However many of the studies find little to no effect of memantine treatment on symptom severity or progression.

Memantine has also been tested clinically in TBI. After moderate TBI, patients were given

30mg memantine twice daily and outcome measures such as neuron specific enolase (NSE; a marker for neuronal damage) in cerebral spinal fluid and GCS score were monitored. After day 7,

NSE levels were significantly reduced in the memantine group (as compared to controls) and GCS scores improved on day 3.419 Clinical trials.gov has also reported the results of several trials that used memantine to alleviate the cognitive effects of mild and moderate TBI, and many of them report modest improvements (if any) with memantine treatment. Given the lack of convincing results encountered in these clinical trials, as well as a lack of understanding of how long term memantine treatment could affect brain function, it is unlikely that prophylactic memantine treatment would be recommended as a potential treatment for repetitive mild TBI. We utilized memantine to try to understand how NMDA receptor activation can contribute to functional

145 changes, and ultimately cognitive impairment after exposure to impacts. These studies are significant, as they highlight the effects of impacts delivered at a higher frequency that that used by many of the current repetitive mild TBI models, and identified a source for the effects that we observe that does not involve tau, Aβ, inflammation, or cell death. These studies have set a course for future studies on the HF-HI model aimed at teasing apart the downstream mechanisms in

NMDA receptor mediated pathways that contribute to these changes, in order to determine a more specific target for therapeutic modulation.

146 Chapter 7

General discussion

Discussion

Exposure to repetitive mild TBI has been deemed a significant risk factor for the development of cognitive impairment, behavioral abnormalities and neurodegenerative pathology.

The neurodegenerative disorder most commonly associated with repetitive mild TBI is CTE.142

The primary pathology underlying CTE is the deposition of hyperphosphorylated tau as neurofibrillary tangles, astrocytic tangles and neurites concentrated within sulcal depths and perivascular/periventricular regions.422 The exact role of hyperphosphorylated tau in the development of symptoms is not well understood, however, as there are cases of behavioral and cognitive abnormalities occurring in professional boxers and football players that do not display these pathological features.88,423 Current research efforts are dedicated to identifying the relationship between pathology and impairment, but this lack of predictability in symptom manifestation has illuminated the need for exploration of causes of behavioral deficits beyond the protein aggregation spectrum.

Our group has proposed that sustaining head impacts at a high frequency, comparable to that experienced by populations deemed most at risk for developing impairments, could uniquely contribute to deficit development by engaging a mechanism independent of protein aggregation.

To study this, we created a model of high-frequency head impacts in which mice received 5 hits a day for 6 days, culminating in a total of 30 hits. This frequency is applicable to the exposure endured by football players, as it has been shown that in a single week, college football players can sustain between of 20.6 and 41.3 head impacts.424 We chose to employ a closed head model to better approximate the head impact characteristics of those sustained in contact sports. Using

147 this paradigm, I set out to test the hypotheses that (1) HF-HI could elicit cognitive impairments in the absence of changes in classical injury markers, (2) intrinsic and network properties of neurons would be affected by possible ionic dysfunction caused by HF-HI, but these effects would not be lasting (3) sustained changes in glutamatergic receptors occur after HF-HI, and (4) preventing the changes in glutamatergic receptors will be sufficient to stop the development of cognitive deficits.

Each chapter of this dissertation aimed to test one of these hypotheses, and results from each allowed me to identify a new mechanism responsible for the cognitive impairments that arise after exposure to repetitive head impacts.

To begin to identify injury mechanisms unrelated to protein dysregulation, I first set out to determine whether classical injury markers were altered by HF-HI. I found no evidence of aberrant tau or Aβ accumulation, and no inflammation, cell death, or loss of excitatory synapses after HF-

HI. Despite the absence of changes in these injury markers, I found cognitive impairment.

Performance on the Barnes maze, MWM, and T-maze 1 month after the last day of impacts revealed that hippocampal-dependent learning and memory were chronically deficient after HF-

HI. To identify a mechanism for these behavioral impairments, I sought to determine if synaptic plasticity was altered. I found that LTP in the Schaffer collateral pathway of the hippocampus was deficient after HF-HI, which I believed could be driving the learning and memory deficits that I observed.

The lapse in synaptic plasticity that I observed illuminated glutamatergic receptors as potentially being responsible for the cognitive impairments elicited by HF-HI. Through patch clamp electrophysiological assessment of the postsynaptic partners of the Schaffer collateral pathway, the CA1 pyramidal cells, I found that although intrinsic properties such as excitability were changed by HF-HI, but these effects were acute and thus unlikely to be driving behavioral

148 impairments at 1 month post impacts. Lasting increases in the NMDA to AMPA ratio were discovered in CA1 pyramidal cells, indicating that NMDA receptors (either via decreased AMPA

EPSC or increased NMDA EPSCs) are likely mediating the impairment of plasticity. The NMDA receptor complex is important for regulating synaptic plasticity via calcium ion influx through their associated ion channel, and their aberrant activation exacerbates pathological states in multiple illnesses and forms of injury.313 Specifically, extrasynaptic NMDA receptors are vulnerable to excess activation when extracellular glutamate levels increase.425 Activation of extrasynaptic NMDA receptors has also been shown to decrease synaptic AMPA receptor surface expression.426 Therefore, I sought to selectively block this NMDA receptor population to determine whether limiting their activation will prevent the receptor changes and cognitive impairments that I observed. With pretreatment using the NMDA receptor antagonist memantine,

I was able to prevent the changes in relative glutamatergic receptor contributions to the EPSCs elicited by HF-HI, and rescue spatial learning and memory deficits. My findings support the hypothesis that, in a model of high frequency head impacts, deficit development arises through alterations in glutamatergic receptor expression to produce impaired function (Fig. 29A & B).

Many preclinical models have been employed in order to identify key components of the pathophysiological cascade that link repetitive mild TBI exposure to impairment. Different modalities for eliciting mild TBI are used to assess specific characteristics of injury, as the kind of injury used can influence how subsequent sequelae progress to contribute to impairment. There are several permutations of the closed head injury model that have been used to examine repetitive mild TBI. The weight drop model, which was popularized for use as a model for diffuse injury by

Marmarou, allows for variation in the severity based on the weight being used or the distance from the skull when dropped.427 Some iterations of the weight drop model have involved the use of a

149 protective helmet or skull exposure, however more recent versions of the model do not require additional protection of the head.166 As the model developed, there has also been some allowance of head movement to assess how that can effect injury.171 Repetitive weight drop models have employed as many as 12 hits (3 per day) with several hours inter-impact interval.428 The CCI model has also been used to study closed head, repetitive mild TBI.234,231,232,236,429 Additionally, force- based repetitive mild TBIs have also been explored. The CHIMERA model has been developed to specifically focus on rotational acceleration as a mediator of resulting injury.430 Pendulum models of repetitive mild TBI have also been used to study the effects of force exertion onto the brain.431,432

To model the injury type most commonly experienced by members of the military, blast wave exposure has also been used to elicit mild TBI.154,433 Paradigms that require a craniotomy are also widely used to study mild TBI. The severity of lateral fluid percussion injury can be modulated based on the pendulum height used to produce the pressure pulse, and a mild injury can be elicited.164,434 The velocity and impact depth of the pneumatic impactor used to deliver CCIs can also be adjusted to produce mild injuries. The caveats for these craniotomy-based injuries are that both of these models can produce a more focal injury, and the use of a craniotomy alone may be sufficient to induce an inflammatory response.435 The heterogeneous nature of the circumstances under which many human concussive head impacts are sustained calls for a wide array of experimental models that can help us understand how the characteristics of impacts can influence the deficits that result from injury. While each of these models has been instrumental in progressing towards an understanding of how deficits develop, many of them still have yet to address some specific factors that can influence the progression of impairments.

The aforementioned preclinical models implement the use of various injury frequencies for studying repetitive mild TBI. The two characteristics that determine injury frequency are the inter-

150 injury interval used and the number of injuries delivered. A recent review of widely used closed- skull repetitive mild TBI models found that the inter-injury interval typically used was 1 day, however the span of intervals ranged from 3 minutes to 30 days.436 This is an important factor, as the time between impacts can greatly alter the severity of deficits that arise after exposure to repetitive mild TBI.437 Further, the number of injuries used in most repetitive mild TBI models rarely exceeds 5, despite the finding that increased injury numbers can increase the longevity and severity of cognitive and behavioral deficits.175 This leaves the consequences of sustaining a high frequency of head impacts relatively unexplored. Frequency of head impacts is an important factor to consider, as athletes engaged in contact sports like football and boxing have a high likelihood of sustaining repetitive head impacts, and seem to have the highest risk of developing behavioral and cognitive deficits over time.

The only other group that has utilized a comparable number of head impacts is Petraglia et al., whose model involved giving mice 6 hits a day for 7 days, for a total of 42 hits.246 Mice with repetitive mild TBI elicited by this model displayed inflammation and accumulation of hyperphosphorylated tau.172,246 As reported in chapter 3 of this dissertation, we found no indication of inflammation or changes in hyperphosphorylated tau. The Petraglia et al., model used a modified CCI device, and the impacts were delivered with a 1cm depth at 5m/s, with a 2 hour inter-injury interval. Our impacts were delivered using a pneumatic impactor, and impacts occurred at 2.35m/s with a 7.5mm depth and approximately 60 seconds inter-injury interval. The

HF-HI model also does not necessarily accommodate acceleration-deceleration forces, whereas the Petraglia model does. These differences in injury parameters, or the slightly higher number of impacts could deliver a more severe injury than that which is elicited by the HF-HI model. Both models did not cause cell death, deformation of the tissue, or changes in gross brain morphology.

151 Cognitive deficits (observed in the MWM) were also present after both paradigms. This is indicative of high frequency eliciting a cognitive impairment. To my knowledge, Petraglia et al. have not reported synaptic changes resulting from their impact paradigm. Changes in glutamatergic signaling after their impacts would not be surprising, considering the changes that I have found after HF-HI.

Taken together, my findings emphasize the importance of examining factors such as impact frequency in influencing deficit development. We use one of the highest frequencies of impacts found in the current repetitive mild TBI literature, and our data reveal interesting parallels and divergences from those studies. The HF-HI model produces behavioral impairments typically observed in other repetitive models, but without changes in classical injury markers. Unlike many of the models currently being used to study repetitive concussive and subconcussive head impacts, we find that synaptic deficits alone are sufficient to elicit hippocampal-dependent learning and memory impairment. Our group has also performed unbiased transcriptomic analyses that reinforce these findings, as the highest degree of RNA-level changes (identified in Gene Ontology analyses) occur in cellular compartments associated with synaptic transmission. Based on these studies, we believe that we have identified an important mechanism related to impairment that should be further studied as a potential therapeutic target. Although we do not know yet how synaptic changes coincide with the development and progression of CTE, it may be an important facet of injury that underlies the presence of deficits despite the ambiguous relationship between tau pathology and impairment observed in Stage I.

152 A

B

Figure 7.1. Schematic of changes in glutamatergic receptor expression elicited by HF-HI.

The schematic representation of the HF-HI effect, which creates a phenotype that includes changes in glutamatergic receptor expression and spatial learning and memory deficits, is described above.

(A) We propose that under normal circumstances, extracellular glutamate levels are low, and

153 mature spines that make up the postsynaptic portion of an excitatory synapse express a certain number of AMPA and NMDA receptors. When HF-HI is elicited (1) extracellular glutamate levels increase, likely from ionic dysregulation resulting from the impacts, which may cause metabolic imbalance, and an overall increase in neuronal depolarization. This increase in extracellular glutamate leads to (2) increased activation of perisynaptic and extrasynaptic NMDA receptors.

The increased activation of these perisynaptic and extrasynaptic receptors in response to the theorized, sustained sub-toxic elevation of extracellular glutamate elicited by HF-HI, prompts adaptations in synaptic AMPA and NMDA expression (3). These adaptations occur in the form of decreases in AMPA and NMDA receptor expression at the synapse, that are detectable even after impact cessation (4). Eventually, extracellular glutamate levels may normalize, as metabolic deficits and ionic homeostasis are restored (5). Despite this, the synaptic adaptations remain, and contribute to deficient synaptic plasticity, and subsequent learning and memory deficits. (B) When extracellular glutamate concentrations increase (1), activation of extrasynaptic NMDA receptors is prevented by memantine pretreatment prior to each day of impacts (2). This perisynaptic and extrasynaptic NMDA receptor blockade prevents the adaptations in synaptic AMPA and NMDA receptors mediated by their receptor activation (3 & 4). Extracellular glutamate levels eventually normalize (5), while synaptic expression and plasticity remains unaltered by HF-HI because the synaptic adaptations elicited by the head impacts were blocked with memantine.

154 Limitations

There are several limitations of the present study that could influence the impact of the current findings. The limitations of some of the specific experiments performed were addressed in their respective chapter discussions, therefore the broader limitations of these studies will be discussed here.

One of the major limitations to the experiments performed in this dissertation is that CA1 pyramidal cells were assesses regardless of their subtle differences in connectivity and roles in facilitating episodic memory. It has been shown that subpopulations of CA1 pyramidal cells that are segregated based on their position in either the deep and superficial layers show different propensities towards being modulated by oscillations like theta, gamma or sharp wave ripples during sleep and wakefulness, as well as towards bursting, firing rates and forming place fields.438

These deep and superficial CA1 pyramidal cells also vary in their morphology, and their interactions with inhibitory neurons.439,440 It is thought that these two subpopulations of CA1 pyramidal cells form parallel, non-uniform sub-circuits, wherein deep cells have more flexible spatially related firing and are more closely linked to encoding the location of sensory stimuli, while superficial cells show more stable spatially related firing and are more closely linked to encoding global spatial context.441,442 I distinguished the CA1 pyramidal cells from interneurons based on their morphology, location, their firing properties, and their intrinsic properties, but I did not parse apart the results based on whether the pyramidal cells were superficial or deep. Since the purpose of these studies was to perform a preliminary characterization of the effects of HF-HI on cellular function, I wanted to determine whether there was a gross effect on CA1 cells first without focusing on discrete populations and their individual roles. Since these cells have different functional properties, it will be important to perform subsequent characterization of these cells

155 based on whether they are deep or superficial in order to see if HF-HI might specifically effect the function of one population more than the other.

Many of the studies performed in this dissertation were done using electrical stimulation of acute hippocampal slices. I used a bipolar microelectrode to stimulate the Schaffer Collateral pathway. It was placed in stratum radiatum, in a location through which afferent projection of these collaterals occurred. While electrical stimulation is effective, it does not allow for stimulation of discrete populations of cells.443 It is likely that stimulation of off-target cell types adjacent to the electrode occurred as well, which could have altered the response that I recorded in CA1.

Pharmacological manipulations, such as the addition of BMR during testing, reduced the potential influence of stimulation of GABAergic cells on the experiments. Previous studies have shown that in rodents, CA2 pyramidal cells project to CA1.444,445 The CA3 pyramidal cells also have recurrent collaterals, and their activation can influence normal oscillatory behavior and spatial memory processed by feedforward circuits to CA1.446 Therefore, stimulation of excitatory cells in proximity to the electrode that are not part of the Schaffer Collateral pathway could also influence the responses of CA1 pyramidal cells. Given the location of In Chapter 5, I suggested using paired recordings to perform minimal stimulation experiments as a means of refining synaptic inputs to cells of interest. While this would introduce specificity in stimulation by allowing direct current injection in presynaptic cells to stimulate firing of postsynaptic cells, identification of synaptically paired cells in this process can be very difficult. Another way to refine the stimulation of synaptic inputs to the CA1 pyramidal cells for future studies is by using an optogenetic approach. A CRE- dependent system could be implemented in conjunction with a retroviral vector injection into dorsal CA1 that results in the expression of channelrhodopsin2 specifically in synaptically coupled

CA3 pyramidal cells (regulated by a promoter like Thy-1 that ensures that only pyramidal cells in

156 CA3 are labelled). 445 That way, excitation of these cells through photostimulation with ~470nm blue light in slice can be performed to more precisely study the effects of HF-HI glutamatergic synaptic transmission.447

Some aspects of injury were not addressed in the characterization of the HF-HI model in these studies. After many forms of TBI, the blood brain barrier (BBB) is disrupted. This disruption can promote inflammation through invasion of peripheral immune cells as well as recruitment of microglia in response to the pro-inflammatory cytokines, cytotoxic proteases and reactive oxygen species produced by the invading cells.448 Upregulation of matrix metalloproteinases (MMPs) after

TBI also occurs, and can degrade the neurovascular basal lamina, increasing permeability of the vessels and contributing to edema.45 The activity of MMPs is important to consider when evaluating injury sequelae, specifically MMP-9, which has been directly linked to adverse outcomes after TBI. In patients that have experienced mild TBI, MMP-9 levels are significantly increased in plasma up to 24 hours after injury.449 After CCI, dendritic spines numbers declined, and the remaining spines were shown to become more filopodia-like in the dentate gyrus of the ipsilateral cortex, an effect that is eliminated when MMP-9 is knocked out.450 This effect has important implications for excitatory signaling, as dendritic spines are the sites of excitatory synapses. Studies have also shown that MMP-9 directly effects NMDA receptors, increasing their surface trafficking, and influence LTP.451,452 In our initial characterization of the effects of the HF-

HI model, we found no evidence of edema at any time-points studies. The immunohistochemical analyses performed in this dissertation, as well as the RNA-sequencing and ELISAs for inflammatory cytokines performed (data not shown), reveal no changes in inflammatory markers at acute or chronic time-points. Because we did not see edema or inflammation, we did not assess the upstream the mediators of these conditions, BBB permeability or changes in MMP activity.

157 However these factors could still be altered, but at a level that does not produce pathological effects. Given MMP-9’s connection to TBI, NMDA receptor signaling and LTP, a gelatin zymography study should be done to confirm that HF-HI does not alter the activity of this

MMP.453,454 The BBB should also be assessed, as experiments performed in preclinical concussion models have also shown that it can be selectively permeable to some ions, including sodium and chloride.455,456 Further, hyperexcitability caused by astrocytic activation and dysregulation of excitatory amino acids is also linked to increased BBB permeability.45 To completely narrow down the culprit associated with behavioral abnormalities resulting from HF-HI to synaptic changes, these factors must be studied to determine whether they are affected.

Finally, while animal models are incredibly important for identifying mechanism of injury that contribute to poor outcomes after mild TBI, there are several facets of experimental injury in rodents that are hard to translate to the human condition. Closed head impact models and midline fluid percussion injury produce diffuse injuries comparable to human mild TBI, and are mild enough to be adapted to explore the repetitive effects of mild TBI, however the behavioral assessments used to understand the effects of these injuries has been shown to not correlate closely with clinical presentations in patients exposed to these injuries.457 Therefore, direct relationships between head impacts and long-lasting behavioral symptoms such as post-traumatic stress disorder, irritability, stress and sleep disorders are hard to derive.457 Microglial responses

(including cytokine production, nitric oxide production, and proliferation of microglia) are also slightly different in humans versus rodents, making it difficult to surmise the exact effects of these injuries on inflammatory mediators.458 Additionally, the use of anesthetics in most of these mild rodent models (to comply with ethical parameters set forth by Animal Care and Use committees) can confound some of the results, as they have been shown to affect glutamatergic and GABAergic

158 signaling, mitochondrial function, cerebral blood flow and apoptosis.459 Some animal models are moving away from the use of anesthesia, and adapting repetitive impacts in awake rodents to circumvent these effects.172,246 Another major concern in the use of rodent models is that their brains are lisencephalic. They lack a gyrencephalic brain, the location of certain structures differs as compared to humans, and the brain size is much smaller relative to humans. These anatomical differences can increase the vulnerability to injury of specific regions of the brain, like the hippocampus in rodents as compared to humans, making it harder to accurately approximate injury effects.460 To more closely model the human brain, porcine models have been adopted. The relative size, gyrification index, and susceptibility to rotational acceleration that is not easily recapitulated in humans, allows for a closer approximation of injury mechanisms contributing to deficit development after injury.461 The aforementioned factors present caveats that should be considered with the work performed in this dissertation. The focus of my project was on the synapse, a basic unit whose function is well conserved across species despite the fact that synaptic protein expression in rodents and humans may differ.462 Because of this, I believe that the findings of this dissertation will prove to be a translatable foundation for identifying mechanisms of deficit development. Since we do not seem to elicit an inflammatory response with the HF-HI model, we hope that it likely does not play a big enough role that differences in the human inflammatory response would alter my findings. Further, the exposure of the sham animals to anesthesia will hopefully control for the impact of anesthesia until the lab is able to move towards an anesthesia- free model. The mechanism identified in this dissertation will therefore play a significant role in the design of therapeutic interventions to prevent functional deficits that arise after HF-HI.

159 Future Directions

Manipulation of the frequency of head impacts has illuminated a mechanism that is independent from protein aggregation, and that can be explored further to exploit as a potential therapeutic target. Specifically, the findings in this dissertation implicate glutamatergic receptor mediated synaptic changes as being sufficient to engender cognitive impairments after exposure to high frequency head impacts. The immediate future studies that should be performed in order to improve the current understanding of the repercussions of the acute and chronic changes resulting from HF-HI were explored in this dissertation in the respective chapter discussions. In each chapter, the experiments used to characterize the changes elicited by HF-HI were performed at the level of the single cell, but to truly understand how these changes impair hippocampal- dependent learning and memory, function must be assessed at a higher level. The hippocampus is a complex structure, with intricate functional connections throughout it. Activity on the micro- and macro-circuit level are important for maintaining proper function. The future directions discussed in this chapter will utilize the results obtained from these works as the foundation for subsequent studies aimed at further characterizing the higher-level functional changes that occur. By examining how key cell populations interact with CA1 pyramidal cells, and how the firing properties of the circuits formed between these cells that are associated with spatial learning and memory are altered, a complete picture of how episodic memory is impaired in the hippocampus in response to high frequency repetitive concussive and subconcussive head impacts can be garnered.

The studies that I performed in this dissertation characterized the functional changes that occur specifically in excitatory cells, however these results do not paint a complete picture of the changes that HF-HI can elicit. Maintaining the excitatory/ inhibitory balance is essential for normal

160 physiological signaling in the hippocampus to occur. Interneurons drive the oscillations in the region by tightly regulating excitatory cell firing.463 Specifically, a subset of interneurons known as parvalbumin positive basket cells (PVBCs) perisomatically inhibit pyramidal cells in CA1 to provide the rhythmogenesis necessary to facilitate learning and memory.440,464 These PVBCs display some preference in functional connectivity to CA1 pyramidal cells, such that they receive more excitation from superficial CA1 pyramidal cells, and more strongly inhibit deep CA1 pyramidal cells.441 During associative plasticity elicited experimentally via high frequency stimulation of the Schaffer Collaterals, it has been shown that increased intrinsic excitability of

PVBCs occurs concurrently with synaptic strengthening and potentiation of CA1 pyramidal cells, inducing a persistent increase in feedforward inhibition of pyramidal cells by PVBCs.465 Spatial memory is also dependent on PVBC input. Blocking the inhibitory function of PVBCs in CA1 is also sufficient to disrupt spatial working memory.466 It is known that PVBCs express NMDA receptors, which are important for generating gamma oscillations and the formation of hippocampal-dependent memory.467 In instances where extracellular glutamate is elevated, increased activation of NMDA receptors on inhibitory interneurons is less detrimental to the survival of the cells than in pyramidal cells.468 While cell death may not increase as a function of elevated extracellular glutamate after HF-HI, the activation of these receptors may affect PVBC function. Therefore, in order to completely comprehend how HF-HI can alter learning and memory, I would want to perform whole cell patch clamp assessments of the intrinsic and synaptic properties of the inhibitory side of the PVBC/pyramidal cell microcircuit. These experiments would be performed in acute, hippocampal slices in conditions similar to those used in this thesis.

To conduct these experiments, I would use a transgenic mouse line that expresses a fluorescent indicator specific to parvalbumin positive interneurons.469 To distinguish the PVBCs from other

161 parvalbumin expressing interneurons, a procedure similar to that employed by Caccavano et al., would be used.470 I would employ their biocytin-filling protocol in the parvalbumin positive cells that I have recorded from, and use post-hoc morphological reconstruction to make sure that only those cells that have the characteristic perisomatic axonal inputs to pyramidal cells are included in the analyses. I would first measure the spontaneous inhibitory postsynaptic currents (sIPSCs) by patching pyramidal cells using a potassium chloride internal solution, paying close attention to the differences in superficial versus deep CA1 pyramidal cells to see if HF-HI changes their connectivity. While I would not be able to isolate the specific inhibitory inputs to the pyramidal cells that are coming from PVBCs, this would give me an idea of the overall inhibitory drive to these cells. Further, assessing the miniature IPSCs (mIPSCs) in the presence of TTX could provide some information about small quanta release of inhibitory vesicles, which (depending on the changes encountered) could inform about whether GABAA receptor number, presynaptic innervation or vesicular release has been altered by HF-HI. By performing whole cell recordings on PVBCs specifically, I could also use current clamp experiments in order to enquire as to whether HF-HI alters excitability, or firing properties of these cells. Changes in their excitability at baseline could have adverse effects when plasticity is elicited via high frequency stimulation, which could have serious repercussions for the induction and maintenance of associative plasticity.465 In neurodegenerative diseases like Alzheimer’s disease, PVBC mediated gamma oscillations and inhibitory currents have been shown to be dysfunctional, which is also linked to impaired cognition.471,472 The importance of PVBC function in maintaining normal cognition makes it an important cell population to assess in order to understand the effects of HF-HI.

If further studies on the repercussions of HF-HI suggest that the function of both PVBC and pyramidal cell types is altered, then the next step would be to assess how these alterations can

162 influence place field formation and maintenance. The place cells of CA1 are the primary mediators of spatial learning and memory. Their firing is heavily regulated by interneurons, and during the formation or remapping of place fields, coupling or uncoupling of pyramidal cell firing to interneuron firing is necessary.473 In particular, PVBCs control the spike rate and theta phase locking of place cells, making them essential for maintaining the firing pattern associated with a place field.474 Since I have shown that NMDA receptor-dependent synaptic plasticity in CA1 pyramidal cells is altered by HF-HI, place field formation would be important to explore as it, too, relies on NMDA-receptor dependent plasticity.475,476 Further, assessing their function in vivo could prove useful for tracking the development of deficits after HF-HI. In Alzheimer’s disease, changes in glutamatergic receptor expression and plasticity have also been observed,477 and it has also been shown that place fields are larger (and therefore less specific), place cell firing rates decrease, and the number of active cells decreases. These changes illustrate that examining place field dynamics and place cell firing can provide a real-time functional marker of deterioration that can be used to assess the progression of memory impairments and the efficacy of therapeutic interventions.478,479

Ideally, I would measure place cell activity in vivo, as this technique would allow me the ability to observe the aforementioned place cell/field characteristics, as well as how HF-HI can alter other important cellular firing properties related to place field dynamics as animals are exposed to spatial learning and memory tasks. In particular, changes in the ability of CA1 place cells to produce

NMDA receptor-dependent complex spike bursts, or the spatially-tuned bursts of action potentials that allow them more effectively induce the synaptic plasticity necessary for learning and memory, would be best detected through the use of an in vivo whole cell patch clamp electrophysiology paradigm.480,481 Based on the data presented in this dissertation, I believe that place cell/ place field characteristics are likely changed by HF-HI. If I can track how these alterations impact function at

163 key points of task acquisition and memory formation, a more complete understanding of the specific stage of memory formation that is impaired by these synaptic changes may be gained and a direct link to cognitive impairment can be identified. Further, if this paradigm is established and differences in function are discovered between sham and HF-HI mice, then these recordings can be performed after HF-HI is delivered concurrently with memantine pretreatment to see exactly how NMDA receptor blockade changes function and rescues learning and memory.

If it is found that HF-HI alters place field dynamics, observing oscillations (one of the net result of normal function in these microcircuits) would be an important next step. Network-level oscillations are important for preserving spatial learning and memory capabilities in the hippocampus. One particularly important type of oscillation is the sharp wave ripple, which are composed of an aperiodically occurring field potential transient (the sharp wave component) and a superimposed fast ripple oscillation (usually between 150-200Hz).482 They are triggered by a population burst in CA3 pyramidal cells after disinhibition, and occur in CA1 via transmission through the Schaffer collateral pathway.483 These ripples replay compressed sequences of neuronal firing that correspond to hippocampal representations of learned information, and in so doing help to consolidate memory.484 Specifically, place cell firing patterns are replayed during sharp wave ripples that occur either during sleep or periods of wakefulness. The coincident replay of both in synaptically connected CA3 and CA1 cells triggers LTP to allow for memory consolidation.485 Awake sharp wave ripples, in particular, replay representations of past experiences that promote the learning of spatial tasks. When interrupted, that learning becomes deficient, despite the fact that place field activity remains intact.486 Deficient sharp wave ripples caused by neurodegenerative disorders can be used as a predictor of learning and memory deficits.

In an APOE4 knock-in model of Alzheimer’s disease, for instance, early declines in sharp wave

164 ripple abundance (measured in vivo) predicted poor performance in the MWM up to 10 months later.487 These findings provide evidence that sharp wave ripple oscillations are another important aspect of memory to study in order to truly elucidate how impairments in spatial learning and memory produced by HF-HI develop. I have already shown that LTP in the Schaffer collaterals is deficient, and since this plasticity is tied to sharp wave ripples and spatial learning and memory,485 it stands to reason that sharp wave ripples are also affected. Sharp wave ripples can also be recorded in vivo during hippocampal-dependent behavioral tasks such as the W-track, in which their properties have been shown to correspond to learning of the task.486,488 Performance on the W-track relies on spatial learning and memory, and can be used to identify changes in neuronal representations of spatial sequences in the CA1 in vivo.489 If replay of place cell ensembles concurrent with sharp wave ripples, or the properties of the ripples (i.e. frequency or amplitude of the ripples) is altered by HF-HI, this could provide more information about the stages of learning and memory formation/consolidation that are most vulnerable to the synaptic changes produced by HF-HI.

In this dissertation, whole cell patch clamp recordings were performed on CA1 pyramidal cells, irrespective of their differing roles in supporting hippocampal-dependent memory. Aside from place cells that encode spatial location through the formation of place fields, there exists a subset of CA1 pyramidal cells that are recruited into engrams. While they are also place cells, these engram cells do not maintain their firing in the same environment across time, a characteristic of a distinctly spatially-tuned cell.274 Spatially-tuned place cells produce a stable, spatial memory encoded by this location specific firing, while engram cells encode less stable spatial information but their net effect reliably reflects contextual information.490 It is thought that engram cells and place cells encoding place fields work together within a larger ensemble to support two distinct

165 aspects of episodic memory- the spatial component and the component necessary for contextual indexing.274 Therefore, in order to fully understand how HF-HI impairs episodic memory, the CA1 cells that provide contextual characteristics as they are recruited into engrams must also be studied.

To identify these engram cells, fluorescent markers tied to immediate early genes are used, as these genes are expressed immediately when the cell becomes active to encode a memory.491 Mice are exposed to a relevant behavioral task, such as contextual fear conditioning, and the expression of these genes allows for the recruitment of cells into an ensemble corresponding to the contextual memory to be visualized and tracked.492 The finding that HF-HI impairs LTP in the Schaffer

Collateral pathway is important when evaluating engram dynamics. Recent studies have shown that synaptic strengthening akin to LTP occurs in pre- and postsynaptic engram cells (CA3 to CA1) that are recruited into ensembles after contextual fear conditioning to allow for memory formation, as AMPA receptor expression increases in CA1 engram cells. These studies concluded that the strength of synaptic connections between engram cells is directly proportional to the strength of a memory.493 To truly understand how HF-HI effects this aspect of memory, I would want to employ a contextual fear conditioning protocol in mice that conditionally express fluorescent indicators via an inducible system which utilizes the promoter for the immediate early gene c-fos.494 That would ensure expression of the fluorescent indicator only the cells that are active during task- dependent memory formation, and thus, part of the relevant task-related engram. Although I propose to conduct the examination of place field dynamics in vivo, these engram experiments should first be conducted ex vivo to identify discrete characteristics of cellular function within this subpopulation of CA1 cells that are altered. Specifically, the ex vivo preparation would allow me to compare the synaptic profiles of engram versus non-engram cells in CA1 after HF-HI, to see if the changes in plasticity related to glutamatergic receptor expression that I previously observed

166 could selectively impair engram-mediated memory strength. Further, increases in the recall- induced excitability of engram cells has been linked to decreased expression of Kir 2.1 channels and decreased inward rectifying K+ currents in dentate gyrus cells.495 Given the acute decrease in excitability of CA1 pyramidal cells and increased inward rectification of K+ channels that I observe after HF-HI, I would also want to see if these alterations may occlude the excitability increase necessary for recall in CA1 engram cells as well. The phenotype that I observed in CA1 cells after HF-HI seems to oppose that of normally functioning engram cells, and could therefore indicate that this specialized population of cells are particularly vulnerable to HF-HI. If these functional differences are observed in engram-specific cells in the ex vivo hippocampal slices, I would then move towards an in vivo model that includes channelrhodopsin expressed in c-fos positive cells that are recruited into engrams in response to contextual fear conditioning.491 These in vivo experiments would be important, because they could show that artificially increasing excitability through optogenetic manipulation at key points during recall could rescue any memory deficits observed. These studies would provide another route through which synaptic and acute intrinsic changes elicited by HF-HI would be sufficient to produce impaired learning and memory, and illuminate more avenues for therapeutic targets.

After assessing the effects of HF-HI on hippocampal function, the next step would be to explore how factors that have been shown to worsen the progression of sequelae elicited by TBI influence functional changes after HF-HI. As previously mentioned, the adverse outcomes that occur after sustaining a TBI of any severity are exacerbated by the expression of APOE4. While most astrocytes express APOE at baseline, hippocampal neurons will express APOE in response to excitotoxic conditions.496 In these conditions, neuronal APOE4 production is associated with an increased susceptibility to cell death in vivo.497 Additionally, neurotoxicity caused by APOE4 was

167 shown to occur via interaction with NMDA receptors to increase intracellular calcium in hippocampal neurons, either through indirect cascades that promote phosphorylation of the receptor or increasing depolarization of the membrane to relieve the magnesium blockade.498

Under sub-toxic conditions, APOE4 has also been shown to reduce neuronal surface expression of the receptor Apoer2 (which is widely expressed on neurons and mediates both APOE and Reelin based signaling), as well as AMPA and NMDA receptor expression, facilitating the destabilization of synaptic strength.499 In addition to reducing NMDA receptor expression, APOE4 can also down- regulate NMDAR mediated signaling via CaMKII, CREB and BDNF under certain conditions.500

The strong influence of APOE4 on NMDA receptor signaling begs the question of how HF-HI may impact this relationship. I have shown with memantine pretreatment that NMDA receptors are primarily responsible for the changes in glutamatergic receptor-mediated contributions to the

EPSC that underlie the tau- and Aβ-independent development of cognitive impairments after HF-

HI. I believe that a critical next step would be to evaluate whether APOE4 exacerbates the synaptic phenotype that I observed. The exposure of transgenic mice that express human APOE3 and

APOE4 to HF-HI and subsequent ex vivo electrophysiological characterization of function in the hippocampus may help to further elucidate the downstream signaling cascades that contribute to the synaptic phenotype that I found in wild type mice. Further, our group has collected data that suggests that APOE4 expression in professional boxers is significantly higher than the prevalence observed in the general population. The propensity of boxers to sustain a high frequency of head impacts, as well as their high likelihood of developing behavioral and cognitive abnormalities, makes the effect of APOE4 on glutamatergic signaling and deficit development in response to HF-

HI an important factor to study.

168 The sum of the data obtained in this dissertation coupled with the proposed future studies should hopefully provide a complete understanding of the cascade of events that can hinder normal function in the hippocampus following exposure to a high frequency of repetitive concussive and subconcussive head impacts. In particular, the finding that HF-HI is sufficient to produce a synaptic phenotype that can impair behavior should provide evidence that reinforces the need for more clinical trials focused on assessing rest times and return-to-play intervals after sports concussions. Rest intervals are common practice in sports with increased head impact risk, however very few randomized clinical trials have actually been conducted to assess the efficacy of this practice.501 By studying how reducing the frequency of impacts may prevent the development of this phenotype, protective measures could be more precisely applied for athletes that are most at risk (like football players and boxers). In terms of treatment, clinical trials implementing numerous NMDA antagonists have not been successful in treating multiple severities of TBI.502,503 This dissertation was unable to provide the mechanisms by which NMDA receptor activation in response to HF-HI produces these changes, however studies based on these experiments may help to identify targets downstream of NMDA receptor signaling that can be more effectively pharmacologically manipulated, to stop the changes that head impacts elicit. Our group’s work to identify tau-independent mechanisms of impairment has yielded evidence that glutamatergic signaling needs to be explored beyond its ability to cause excitotoxic damage in order to understand the many facets of functional impairment induced by head impacts. With this knowledge, therapeutic interventions can be more precisely designed in order to prevent cognitive and behavioral impairments for those at risk of exposure to these kinds of head impacts.

169 Appendix

Identifying changes in calcium activity relative to synaptic plasticity induction after HF-HI

Introduction

Calcium signaling plays a pivotal role in neuronal function and synaptic transmission.

Processes ranging from the induction of synaptic plasticity to the initiation of apoptotic cell death are all reliant on dynamic rises in intracellular calcium levels. Because of this, intracellular free calcium is heavily regulated and maintained at around 100nM. This is accomplished through buffering by cytosolic calcium binding proteins, or by uptake into mitochondria and subsequent energy-dependent calcium pump extrusion.504,505 The calcium pumps clear out excess ions by relocating them to internal stores like the endoplasmic reticulum (ER), or by moving them to extracellular spaces.506 These regions can then be tapped to provide the amount of ions necessary to initiate cellular processes as needed.

Activation of inositol 1, 4, 5-trisphosphate receptors (InsP3Rs) or ryanodine receptors

(RyRs) on the ER can liberate calcium from internal stores to be used for signaling.507 The RyRs are calcium gated calcium channels that are heavily expressed in the dendrites of CA1 pyramidal cells, and also bind cyclic ADP ribose.508 For these receptors, calcium binding to the receptor on the cytosolic side will open the channel to allow calcium flow out of the ER.509 The opening of

RyR-activated channels is dependent on calcium concentration. They were shown to open when calcium levels were between 100nM and 1µM, and are inhibited at concentrations of 10µM and

506 above. Like RyRs, InsP3Rs signaling is also calcium dependent, however they require the binding of IP3 (a regulator of intracellular free calcium) in order to allow calcium efflux from the

ER. They are also expressed throughout the brain, primarily in the soma and proximal dendrites.510

170 The activity of these receptors is therefore essential for the facilitation of calcium dependent processes in neurons.

Calcium can also enter the cell from extracellular spaces through specific ion channels in the plasma membrane. Voltage gated calcium channels (VGCCs) are a major source of calcium influx, and specific kinds have been shown to be expressed in discrete locations in the neuron. The currents typical of VGCCs found in neurons are L-, N-, P-, Q-, R-, and T-type, and each can be inhibited by specific compounds (which led to their characterization).505 Nowycky et al. (1985) characterized three of these distinct calcium currents. L-type calcium currents were found to display a slow, voltage dependent inactivation, and are important mediators of gene expression, integration of synaptic input, and initiation of neurotransmitter in specific sensory cell populations.

T-type currents are activated at more negative membrane potentials, and have more transient openings than the L-type channel. The N-type currents display properties between T and L, as it has intermittent voltage dependence as well as a rate of inactivation faster than L-type but slower than T-type currents.511 L-type and T-type currents can be found in the proximal dendrites and neuronal somata.512,513 The N-, P-, Q-, and R-type calcium currents were later characterized. They were found to be most prominent in neurons, and are important for regulating dendritic calcium transients and neurotransmitter release.514 The channels that conduct these currents are composed of multiple subunits that form a pore, and the alpha 1 (α1) subunit is thought to convey the diversity in the currents ascribed to each channel. There are three main subfamilies of channels encoded by

10 genes: Cav1.1-1.4 channel family conducts L-type currents, while Cav2.1-2.3 conduct P/Q-, N-

515 and R-type currents and Cav3.1-3.3 conduct T-type currents. These calcium channels can therefore influence calcium activity and signaling in neurons.

171 Calcium levels can also be increased by the activity of various kinds of glutamatergic receptors. As previously mentioned, NMDA receptors are highly permeable to calcium. In fact, they are the primary channel that mediates subthreshold calcium signals in dendritic spines.516 If mRNA editing of the GLUR2 subunit to convert a glutamine codon to an arginine does not occur, or AMPA receptors are assembled without GLUR2 subunits, AMPA receptors can also be permeable to calcium.517 In these scenarios, ionotropic glutamate receptors allow calcium influx, using external calcium to increase intracellular levels. Metabotropic glutamate receptor (mGluR) subgroups mGluR1α and mGluR5 can also elicit an increase in intracellular free calcium, acting

518 through release from intracellular sinks via activation of the InsP3Rs. Glutamatergic receptors are, therefore, an important part of mediating calcium activity in neurons.

Examining changes in free calcium concentrations can provide information about the calcium-dependent biochemical processes that are initiated in cells, as well as relative measurements of synaptic activity.519 The subcellular localization in which calcium accumulates is indicative of the specialized functions that it can mediate within the cell. Presynaptic terminals rely on calcium influx to allow for neurotransmitter release, while calcium levels within postsynaptic dendritic spines can induce different kinds of synaptic plasticity.520 Further, calcium accumulation resulting from action potentials (APs) can regulate excitability differentially in the soma and dendrites of the neuron. Calcium activated potassium channels (SK channels) regulate excitability in the soma by contributing to afterhyperpolarization, while dendritic excitability is regulated by calcium- mediated activation of SK channels during backpropagation of APs and R- type VGCCs.521 This regulation is important, as the coordination of excitatory postsynaptic potentials and backpropagating APs can influence the likelihood of the induction of synaptic plasticity.522 Changes in the baseline-to-peak amplitude of backpropagating APs resulting from

172 EPSP activity are important for bringing the membrane potential to the optimum voltage for calcium to enter the cell through NMDA receptors and induce plasticity changes.523 The development of research tools that provide the resolution necessary to explore calcium activity in these specific regions is an invaluable tool for understanding how calcium dynamics can alter neuronal function and plasticity. Changes in calcium dynamics can provide important insights into how function can be altered by HF-HI.

Advances in optical monitoring technology have made it possible to assess neuronal activity by imaging intracellular calcium tagged with fluorescent indicators. These indicators are designed to change their fluorescent intensity when bound to calcium, allowing for relative estimation of the amount of free calcium present based on the level of fluorescence emitted.524

There are two classes of indicators: chemical and genetically encoded. Chemical indicators are synthetic compounds usually loaded into a cell of interest prior to imaging. These compounds can vary in their affinity for calcium., which can influence the reliability of the signal. High affinity indicators can produce brighter emissions, but tend to buffer calcium and become saturated when low-level increases occur. Low affinity indicators are less likely to buffer calcium, and can be used in subcellular compartments to look at rapid changes in calcium.525 Many of the well-known chemical indicators were produced by Tsien et al.526-529 Unlike chemical indicators, genetically encoded calcium indicators (GECIs) allow for chronic, non-invasive imaging of calcium in discrete neuronal populations.530 The GECIs are usually introduced into the cells of interest via gene transfer techniques such as a viral vector delivery, where genes that encode the indicator proteins are transcribed and translated within the cells into proteins that include a calcium binding domain (like calmodulin) fused with fluorescent proteins.531 Although the methods of delivery

173 were less invasive for GECIs, they were thought to be less sensitive than synthetic calcium indicators until the invention of the GCaMP family.532

The GCaMP family was developed in order to address the issues associated with classical

GECIs. One of the major concerns with GECIs was that they had an inferior signal-to-noise ratio, response linearity, and photo stability as compared to chemical indicators. GCaMP3 was therefore designed with a higher signal to noise ratio that did not elicit cell death and toxicity or behavioral changes, as well as reduced the potential of calcium buffering by requiring lower expression levels in order to perform.531 Since then, many improvements have been made in the design of GCaMPs to improve their imaging capabilities. The GCaMP6 line has shown great capabilities for reliably detecting events as small as single action potentials and transients in dendritic spines, as well as calcium activity in large populations of neurons, and has demonstrated persistent expression over long periods of time.532 Within the GCaMP6 family, there are several variants. GCaMP6s is a more sensitive version that has higher affinity for calcium, and is therefore slower than the less sensitive

GCaMP6f (with faster kinetics). This means that while GCaMP6s is ideal for imaging transients for longer periods of time, GCaMP6f can allow for the detection of faster transients and rapidly occurring action potential.533 The most recent iteration of the GCaMP family are the jGCaMP7 indicators, with specialized modifications for distinct signaling requirements. A few of the different versions of the indicator include jGCaMP7s (sensitive and slow), jGCaMP7f (fast kinetics), jGCaMP7b (brighter baseline fluorescence). These jGCaMP7 isoforms also have a better signal-to-noise ratio than GCaMP6 and GCaMP6f, making them more efficient when imaging at high rates or at lower magnification to view larger amounts of neurons.534 While jGCaMP7 is the most recent and most developed of the popular GECIs, GCaMP6f is still a widely used, powerful tool for assessing calcium activity on subcellular and network scales.

174 To determine whether HF-HI had any functional consequences for calcium activity, I performed calcium imaging experiments in acute hippocampal slice preparations from transgenic mice that expressed the GCaMP6f calcium indicator under the Thy-1 promoter (to limit expression to CA1 pyramidal cells). These studies were performed 24 hours after the last day of impacts for

HF-HI and sham mice. I recorded calcium activity via confocal imaging, with concurrent local field potential recordings and used the LTP protocol that I employed previously to determine if calcium activity related to plasticity had been altered 24 hours after the last day of impacts. This protocol allowed me to match changes in calcium activity to stages in plasticity induction, to gain a better understanding of how synaptic transmission changes may impact calcium dynamics. Since much of the focus of this project was on identifying changes on the single cell level, I was interested in going a step above that to assess the sum of cellular interactions. Therefore, I chose to perform these experiments at low magnification (20x) to assess network wide changes. That way, I could identify changes in ensemble composition before and after high frequency stimulation was delivered to elicit LTP. I also wanted to be able to examine calcium activity in both the soma and apical dendrites, because (as previously mentioned) calcium responses to action potentials in both areas of the cell vary, and changes in either field can alter plasticity.523 I, therefore, analyzed changes in calcium activity in somatic and dendritic regions of interest (ROIs) separately. I hypothesized that since I observed changes in excitability acutely, changes in glutamatergic receptor activity at chronic and acute time-points, and deficient synaptic plasticity acutely, these factors will alter calcium activity in HF-HI slices. Namely, at key points in LTP, I theorized that calcium activity will be lower in HF-HI slices than sham slices. While it is not possible to identify the exact source of calcium that was affected (i.e. narrow down the source of any changes to glutamatergic receptors specifically), especially at low magnification, these recordings can help

175 elucidate a mechanism for how plasticity may be altered on a network level. The data presented in this appendix are preliminary, as the statistical power necessary to derive any conclusions about the changes in calcium activity was not accomplished with the sample size that I was able to acquire.

Results

Here I describe the preliminary results from the calcium imaging experiments (Fig. A1-

A4) that show calcium transient responses at baseline and 10 minutes after high frequency stimulation (HFS). Slice images taken with a confocal microscope were imported into FIJI ImageJ

(NIH) using the bio-formats plugin (Open Microscopy Environment), converted into TIF stacks, and analyzed based on previously described parameters, with some modifications.470 The results are separated based on measurements taken from somatic and dendritic ROIs (respectively) which were manually defined in FIJI ImageJ based on their spontaneous and evoked changes in fluorescent intensity (Fig. A1). The change in fluorescent intensity divided by the baseline fluorescent intensity (ΔF/F) for each ROI was calculated using a custom plugin for FIJI ImageJ after built-in bleach correction was applied to each slice. These results, along with Clampfit files containing the concurrent local field potential recordings from each slice were then analyzed and compared in MATLAB using a custom script. Representative traces (indicative of the output from the MATLAB analysis) show calcium transients, matched with simultaneous local field potential recordings and raster plots indicating events that exceeded the threshold for detection in somatic and dendritic fields during baseline recordings are shown (Fig. A2&A3). The number of active

ROIs is similar for both groups in somatic and dendritic regions at baseline and 10 minutes after

HFS, indicating no loss of baseline activity associated with HF-HI (Fig. A4A & A5A). The amplitude of the calcium transients in response to stimulation at baseline and after HFS in somatic

176 and dendritic ROIs is not significantly different (Fig. A4B & A5B). Spontaneous responses, which occurred in between periods of stimulation, did not differ significantly in amplitude, frequency or duration between sham and HF-HI groups in either somatic or dendritic ROIs (Fig. A4C-E &A5C-

E). Local field potentials recorded simultaneously with calcium imaging show a trend towards a reduced percent increase after HFS in HF-HI mice as compared to shams (Fig A4F &A5F).There are trends in the somatic and dendritic fields indicating that differences may develop between the two groups, however more data needs to be collected in order to determine whether these trends will become significant.

177

A B

Figure A.1. Examples of somatic and dendritic ROIs.

Horizontal slices taken from mice that express the fluorescent calcium indicator GCaMP6f in CA1 pyramidal cells were used for this study. Cells that were continuously fluorescent (and whose activity did not change in response to stimulation) were excluded from the analyses. (A) Example of the somatic ROIs drawn over cell bodies that showed spontaneous calcium activity, as well as activity in response to single stimulations. (B) Example of the dendritic ROIs drawn over dendrites that showed spontaneous calcium activity, as well as activity in response to single stimulations.

178

A B

Figure A.2. Baseline calcium transients elicited by single stimulation in somatic ROIs.

Slices taken from mice that express the fluorescent calcium indicator GCaMP6f under the Thy-1 promoter, to restrict expression to CA1 pyramidal cells, were used for this study. Each somatic

ROI (represented by the green trace) was manually defined based on its activity during and between stimulation (10 single stimulations with a 10 second inter-stimulus interval). The change in fluorescent intensity divided by the baseline fluorescent intensity (ΔF/F) are depicted for each

ROI. Blue lines indicate local field potentials that were recorded concurrently with calcium imaging (downward deflections represent responses to stimuli). Raster plots indicate the events that exceeded the threshold defined to indicate a true calcium response. (A) Sham somatic ROI responses to baseline stimulation. (B) HF-HI responses to baseline stimulation.

179

A B

Figure A.3. Baseline calcium transients elicited by single stimulation in dendritic ROIs.

Slices taken from mice that express the fluorescent calcium indicator GCaMP6f under the Thy-1 promoter, to restrict expression to CA1 pyramidal cells, were used for this study. Each dendritic

ROI (represented by the green trace) was manually defined based on its activity during and between stimulation (10 single stimulations with a 10 second inter-stimulus interval). The change in fluorescent intensity divided by the baseline fluorescent intensity (ΔF/F) are depicted for each

ROI. Blue lines indicate local field potentials that were recorded concurrently with calcium imaging (downward deflections represent responses to stimuli). Raster plots indicate the events that exceeded the threshold defined to indicate a true calcium response. (A) Sham dendritic ROI responses to baseline stimulation. (B) HF-HI dendritic ROI responses to baseline stimulation.

180

A B

D C

E F

Figure A.4. Acute changes in somatic calcium in response to HFS after HF-HI.

Twenty-four hours after the last day of impacts, calcium imaging/ local field potential recording experiments were performed. A stimulus input/output curve was used to identify a stimulus intensity that elicited a response that was approximately 1/3 of the maximal response in local field potential and changes in fluorescent calcium activity (ΔF/F). That stimulus was then used to record

181 a baseline (with single stimulations occurring every 10 seconds), HFS (4 trains, 100Hz, 10 second intervals between trains), and then a post HFS response (single stimulations every 10 seconds).

(A) Number of active somatic ROIs in CA1 at baseline and 10 minutes after HFS. (B) Amplitude of ΔF/F of calcium transients recorded in response to stimulation at baseline and 10 minutes after

HFS. The amplitude (C) and frequency (D) of ΔF/F of spontaneous calcium transients that occur between stimulations. (E) Changes in the ΔF/F response duration in somatic ROIs. (F) Changes in local field potential recordings (represented as percent change from baseline) at baseline and 10 minutes after HFS. For these experiments, n = 7 mice, 1 slice per mouse in the sham group, and n

= 10 mice, 1 slice per mouse in the HF-HI group.

182

A B

C D

E F

Figure A.5. Acute changes in dendritic calcium in response to HFS after HF-HI.

Twenty-four hours after the last day of impacts, calcium imaging/ local field potential recording experiments were performed. A stimulus input/output curve was used to identify a stimulus intensity that elicited a response that was approximately 1/3 of the maximal response in local field potential and changes in fluorescent calcium activity (ΔF/F). That stimulus was then used to record a baseline (with single stimulations occurring every 10 seconds), HFS (4 trains, 100Hz, 10 second

183 intervals between trains), and then a post HFS response (single stimulations every 10 seconds).

(A) Number of active dendritic ROIs in CA1 at baseline and 10 minutes after HFS. (B) Amplitude of ΔF/F of calcium transients recorded in response to stimulation at baseline and 10 minutes after

HFS. The amplitude (C) and frequency (D) of ΔF/F of spontaneous calcium transients that occur between stimulations. (E) Changes in the ΔF/F response duration in dendritic ROIs. (F) Changes in local field potential recordings (represented as percent change from baseline) at baseline and 10 minutes after HFS. For these experiments, n = 9 mice, 1 slice per mouse in the sham group, and n

= 11 mice, 1 slice per mouse in the HF-HI group.

184 Discussion

In these experiments, changes in calcium activity at key time-points of a plasticity inducing paradigm were assessed. The changes in fluorescent intensity at baseline were compared to those occurring at 10 minutes after HFS in order to test the hypothesis that calcium activity would be reduced in HF-HI slices. I believed that, with an increased number of experiments, I would be able to demonstrate a reduction in calcium activity could contribute to the deficient LTP that I observed previously in HF-HI slices. While the statistical power does not allow for the observation of significant differences between sham and HF-HI slices, some trends exist in the current data that warrant further exploration.

It is promising that the local field potential recordings in this experiment show a trend similar to that observed in the previous LTP experiments. In sham slices, the HFS induced an increase in the local field potential recording 10 minutes after HFS as compared to the baseline, indicative of potentiation, while that increase seems to be reduced in the HF-HI slices. Further, the

ΔF/F recordings indicate that there are a comparable number of somatic and dendritic ROIs active at baseline and 10 minutes after HFS, which further supports previous findings that overall cell health and signaling capabilities are not impaired by HF-HI. If these findings remain stable after the n is increased, they would support our theory that synaptic function is altered although signaling is not impaired.

The findings from these experiments also highlight differences between the two ROI fields examined. The somatic calcium transients had a higher amplitude spontaneous and evoked ΔF/F at baseline and after HFS than those recorded in dendritic ROIs. These data are in line with previous studies, which have shown that dendritic calcium transient amplitude is generally lower than the amplitudes observed at the soma due to the dynamics of dendritic action potential

185 backpropagation.522 In my experiments, the somatic ROI amplitudes also seemed to trend towards being increased in HF-HI slices at baseline and more so after HFS, but were not visibly changed in dendritic ROIs. The spontaneous frequency of these transients were relatively similar in both fields. While changes in somatic ΔF/F properties will be interesting to further analyze using this paradigm in order to identify an HF-HI effect, this approach is limited in its ability to provide information about the intricacies of dendritic calcium activity. Examining the effects of HF-HI on dendritic calcium activity may require a different approach, given the smaller amplitude of the dendritic ΔF/F, as well as the high propensity of dendritic calcium signal to decrease at branch points in response to trains of stimulation similar to the HFS used to elicit LTP.535 A higher magnification should be implemented to increase the visualization of dendritic arbors belonging to active cells. This way, characteristics such as branching, and origin of calcium increase (i.e. from somatic action potentials or dendritically derived calcium spikes) can be considered separately. Backpropagating action potentials and calcium influx are important for plasticity, because their strength and timing are correlated with the ability to induce LTP. 522 In particular, branch spikes have been shown to ensure the spatial precision of place cell firing, likely through their ability to potentiate synapses and/or maintain the strength of potentiated synapses.536

Therefore, it is important to understand how dendritic calcium activity specifically may be effected by HF-HI, as it will allow for a better understanding of where in the flow of calcium activity related to plasticity mechanisms HF-HI may elicit the largest deficits.

Calcium imaging has allowed for the intricacies of network signaling in the hippocampus to be explored in depth. It is well known that while the activity of single cells can be altered, ensemble encoding of groups of hippocampal cells is essential for processing and storing spatial information.537 The formation of ensembles of CA1 pyramidal cells has been studied using calcium

186 indicators to explore how they change to encode spatial information. Using a deconvolution-based detection paradigm for action potential associated calcium events, it was shown that neuronal ensembles form between spatially clustered cells as a result of plasticity processes, and that at any given time ensembles in acute hippocampal slices, there can be almost a dozen active ensembles in CA1 with a minimum of four cells active for each.538 Recruitment of different ensembles in response to synaptic plasticity can also be dynamic. Calcium imaging studies have shown that lasting associative plasticity occurs through redistribution of the active neurons representing specific afferent inputs to CA1 rather than by loss or gain of net synaptic strength. Plasticity- inducing mechanisms caused the generation of new ensembles that are composed of cells that were previously active in pre-existing, associated ensembles.539 An important next step in understanding how calcium dynamics are effected by HF-HI would thus be to track how ensembles of cells may be altered. To do so, individual ROI analyses from baseline and post HFS recordings should be analyzed and plotted using a pairwise Jaccard similarity matrix, which allows for the visualization of correlation in ensembles before and after HFS to be for comparison. Preliminary work has already been performed in order to understand ensemble changes (Fig. A6). If correlations in cells before and after HFS are more substantially changed in HF-HI than shams, it would suggest that recruitment or attrition in ensembles in response to evoked plasticity might be altered, and could thus provide more information about how network processing is altered following HF-HI.

Taken together, these works suggest that further experiments must be done in order to understand how calcium activity is altered by HF-HI. Along with adding enough n to properly statistically power the experiment, a more focused approach to assessing changes in dendritic calcium must be performed. Changes in ensemble properties should also be examined, as that can indicate how network level processing in the hippocampus may be effected. These data serve as a

187 foundation through which calcium activity changes associated with plasticity can be further assessed, and the influence of exposure to repetitive concussive and subconcussive head impacts can be better understood. Future studies using memantine pretreatment can also be performed once calcium signaling is characterized, to see if the selective blockade of NMDA receptors can also rescue and aberrant calcium activity detected during important stages of plasticity induction. These studies provide a scope that goes beyond the single cell level, and allows for an assessment of network functionality in the hippocampus to better understand how episodic memory can be impaired.

188

A B

Figure A.6. Preliminary ensemble analysis of sham and HF-HI slices after HFS.

Somatic ROIs active before and after HFS were drawn as separate masks, and overlaid on the slice to illustrate the cells that were active before and after HFS. The green somatic ROIs represent cells that were active at baseline, and the red somatic ROIs represent cells that were active 10 minutes after HFS. Overlapping cells were active at both time-points. (A) Sham somatic ensemble changes resulting from HFS. (B) HF-HI somatic ensemble changes resulting from HFS.

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