Quantitative Analysis on the Origins of Morphologically Abnormal Cells in Temporal Lobe

Quantitative analysis on the origins of morphologically abnormal cells in temporal lobe

epilepsy

A dissertation submitted to the

Division of Research and Advanced Studies of the University of Cincinnati

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

In the Molecular and Developmental Biology Graduate Program

Shatrunjai P. Singh

M.Sc. Stem Cell Biology, Manipal University

September 15th 2015

Dissertation Committee:

Steve Danzer, Ph. D. (chair)

Mark Baccei, Ph. D.

Kenneth Campbell, Ph. D

Brian Gebelein, Ph. D.

Ronald Waclaw, Ph. D ii

Abstract

Epilepsy is a common and devastating neurological disease with no real preventive or cure. In most cases of acquired epilepsy, an initial precipitating injury to the brain is followed by a silent period which eventually culminates into the development of spontaneous, recurrent seizures. This interval between the primary insult and the first seizure is referred to as the latent period of epileptogenesis and is characterized by abnormal morphological and physiological changes in the hippocampus. In the studies described herein, I aim to elucidate changes in the different phases of epileptogenesis with the end goal of deciphering critical epileptogenic mechanisms.

To study the initial stages of epileptogenesis, I employed the early kindling model. In this protocol, it is possible to administer a limited number of stimulations sufficient to produce a lifelong enhanced sensitivity to stimulus evoked seizures without associated spontaneous seizures. In these experiments, I characterized the morphology of GFP-expressing granule cells from Thy-1 GFP mice either one day or one month after the last evoked seizure. I observed several morphological changes at the one day time point, which all normalized to control levels at the one month time point. Interestingly, I did not observe the presence of basal dendrites, frequently observed in other models of epilepsy. These findings demonstrate that the early stages of kindling epileptogenesis produces transient morphological changes but not the dramatic pathological rearrangements of dentate granule cell structure seen in typical models associated with spontaneous seizures.

To study epileptogenesis after the incidence of spontaneous, recurrent seizures, I used the pilocarpine model of epilepsy. Our lab has previously used this model to show that adult hippocampal neurogenesis is profoundly altered under epileptic conditions, leading to the iii production of morphologically abnormal dentate granule cells. Under epileptic conditions, these adult generated cells migrate to ectopic locations and develop misoriented basal dendrites.

Although it has been established that these abnormal cells are newly-generated, it is not known whether they arise ubiquitously throughout the progenitor cell pool or are derived from a smaller number of bad actor progenitors. To explore this question, I describe clonal analysis experiments conducted in epileptic mice expressing the brainbow fluorescent protein reporter construct in dentate granule cell progenitors. Brain sections were rendered translucent so that entire hippocampi could be reconstructed and all fluorescently-labeled cells identified. The findings revealed that a small number of progenitors produced the majority of ectopic cells in epileptic mice, indicating that either the affected progenitors or their local micro-environments had become pathological. By contrast, granule cells with basal dendrites were equally distributed among clonal groups. These findings strongly predict that distinct mechanisms regulate different aspects of granule cell pathology in epilepsy.

The experiments described here utilize different models of epilepsy and employ cutting edge technology to provide valuable insight into the process of epileptogenesis. The results and ideas presented here are intended to advance our knowledge of epilepsy and eventually lead to better antiepileptic therapies.

Dedication

I dedicate this thesis to my family and to science enthusiasts around the world.

Acknowledgement

“In the vastness of space and immensity of time, it is my joy to spend a planet and an epoch with you”

Completing a doctoral thesis, although long and sometimes demanding, was the best decision I have ever made. My journey through graduate school has taught me a lot and has made me a better, happier person. This would not have been possible without my mentors, family, friends and colleagues, who have helped me at every step of my career. They have selflessly looked out for my best interests and have tirelessly supported me in my pursuit of happiness.

I would like to thank Dr. Steve Danzer for showing me what an ideal boss should be like.

During the course of my business classes, I had enrolled in a leadership class and every lecture made me realize that he had already showed me every technique they could possibly teach.

Steve’s scientific brilliance mixed with a high emotional quotient, empathy, perfectionism and the ability to motivate people makes him the best mentor I could have asked for. I am indebted to him for granting me the freedom to take business courses, do an internship and ultimately for molding my career. Thank you.

I would also like to thank my dissertation committee for guiding me through the ups and downs of graduate school. I am indebted to Dr. Brian Gebelein, for believing in me and writing endless recommendation letters which helped me get grants and fellowships. I am grateful to Dr. Mark

Baccei for always being forthcoming with new ideas and making himself ever available for advice and inspiration. I am obliged to Dr. Ronald Waclaw, for helping me develop the skill of asking better scientific questions and for always reassuring me that I am always on track to vii graduate. I would like to thank Dr. Kenneth Campbell, for his constant guidance on the future of my research and his unfaltering support in every committee meeting and at every poster presentation. All of them have been an integral part of my graduate school experience and I will forever be grateful for all your support.

I am thankful to my lab mates and colleagues, Mike Hester (by far the smartest guy I know),

Isaiah Rolle (for making me feel like family), Dr. Candi LaSarge (my favorite coffee conversationalist and postdoc par excellence), Bethany Hosford (for showing me how to be an excellent graduate student), Mary Dusing (for making all our lives livelier and more organized),

Dr. Shaadi Khademi (for patiently listening to all my ‘negative-result-again’ stories), Salwa

Ragab (for making the lab so much fun), Rylon Hofacer (my fellow NPR enthusiast), Dr.

Shane Rowley (for the much needed guy talk), Dongyi Tong (for the great conversations we’ve had), Victor Santos (the Brazilian who taught me how to like Indian food more), Bernadin

Joseph (the greatest immunostaining genius that has ever lived), Amen An (undergraduate rockstar whose also a Krav Maga expert) and Jules Rosen (for patiently putting up with all my jokes). I would like to specially thank Dr. Ray Pun for teaching me electrophysiology, the virtue of patience and the endless pharmacological support (coffee). I would also like to thank all my other friends (you know who you are!) for making my life so lively and complete.

My family has always been my strongest quality. I am indebted to my mother, Aprajita Singh, for her unconditional love, support and guidance at every step of my life. I thank my father,

Devendra Singh, for teaching me everything ranging from cricket to why charity makes you happy. Dad, thanks for all the things you have done for me and Tutu; we are eternally grateful to you. I would like to thank my younger brother, Dr. Vijendra Singh (Tutu), who has been my strongest supporter for as long as I can remember. Tutu, we are all so proud of you (although viii mom says she’s more proud of me). I would also like to acknowledge my grandma, because her voice makes everything better. I would like to thank my cousin, Dr. Sanjay Singh, who besides being my role model, has always been a source of inspiration and a stronghold of support.

Finally, I would like to thank my extended family that has played such a critical role in my life.

Thank you all.

I would also like to acknowledge support from the University of Cincinnati, the Cincinnati

Children’s Medical Center, the Albert J. Ryan foundation, the American Heart Association and the American Epilepsy Foundation, without which I could not have completed my studies.

Finally, I would also like to thank the MDB program for providing me the opportunity to conduct my research at this excellent institute.

Thank you,

Shatrunjai Singh

Table of contents

Cover ...... i Abstract ...... ii Dedication ...... v Acknowledgement ...... vi Table of contents ...... ix List of Figures...... xi List of Abbreviations ...... xii

Chapter 1: Introduction ...... 1 Epilepsy is a common, debilitating neurological disorder ...... 2 The hippocampus is an important regulator of memory formation plays a role in TLE ...... 3 The dentate gyrus acts as a gate which breaks down under epileptic conditions ...... 5 Adult neurogenesis in the hippocampus ...... 6 Epilepsy affects adult neurogenesis and leads to the production of abnormal DGCs ...... 8 Animal models of temporal lobe epilepsy ...... 11 Modelling epileptogenesis before and after SRS ...... 13 References ...... 15 Figures and Tables ...... 25

Chapter 2: Morphological changes among hippocampal dentate granule cells exposed to early kindling-epileptogenesis ...... 30 Abstract ...... 31 Introduction...... 32 Methods ...... 34 Results ...... 40 Discussion ...... 46 References ...... 51 Figures and Tables ...... 59

Chapter 3: Clonal analysis of newborn hippocampal dentate granule cell proliferation and development in temporal lobe epilepsy ...... 69 Abstract ...... 70 Introduction...... 71 Methods ...... 73 Discussion ...... 83 References ...... 89 Figures and Tables ...... 95

Chapter 4: Discussion ...... 107 Summary ...... 108 The transient changes produced by early kindling are features of hyper-excitability ...... 108 The battle ensues after the initial epileptic insult: A tussle between pro and antiepileptogenic morphological changes ...... 109 Are ectopic DGCs, MFS or DGCs with basal dendrites required for SRS? ...... 111 The two-hit hypothesis for the development of SRS ...... 113 Origins of abnormal cells in epilepsy: The good, the bad, and the ectopic ...... 114 Cell death and notch de-repression drives terminal differentiation in the stem cell pool .... 115 Inverted glial cells: An Ockham’s razor solution to the origin of ectopic cells ...... 116 How to keep a basal dendrite ...... 117 Conclusions...... 118 Avenues for future research: Curing epilepsy one dentate at a time ...... 119 References ...... 120 Figures ...... 126

List of Figures

Chapter 1: Figure 1.1 ...... 25 Figure 1.2 ...... 26 Figure 1.3 ...... 27 Figure 1.4 ...... 28 Figure 1.5 ...... 29 Chapter 2: Figure 2.1……………………………………………………………………………………………………………………………. 59 Figure 2.2……………………………………………………………………………………………………………………………. 60 Figure 2.3……………………………………………………………………………………………………………………………. 61 Figure 2.4……………………………………………………………………………………………………………………………. 62 Figure 2.5……………………………………………………………………………………………………………………………. 63 Figure 2.6……………………………………………………………………………………………………………………………. 64 Figure 2.7……………………………………………………………………………………………………………………………. 65 Figure 2.8……………………………………………………………………………………………………………………………. 66 Table 2.1.……………………………………………………………………………………………………………………………. 67 Table 2.2.……………………………………………………………………………………………………………………………. 68 Chapter 3: Figure 3.1……………………………………………………………………………………………………………………………. 95 Figure 3.2……………………………………………………………………………………………………………………………. 96 Figure 3.3……………………………………………………………………………………………………………………………. 97 Figure 3.4……………………………………………………………………………………………………………………………. 98 Figure 3.5……………………………………………………………………………………………………………………………. 99 Figure 3.S1.……………………………………………………………………………………………………………………… 101 Figure 3.S2.……………………………………………………………………………………………………………………… 102 Table 3.1.………………………………………………………………………………………………………………………… 104 Table 3.2.………………………………………………………………………………………………………………………… 105 Table 3.3.………………………………………………………………………………………………………………………… 106 Chapter 4: Figure 4.1………………………………………………………………………………………………………………………… 126 xii

List of Abbreviations

Acronym Full Form

AED Anti-epileptic Drug

BD Basal Dendrite

CA Cornu Ammonis

CFP Cyan Fluorescent Protein

DG Dentate Gyrus

DGC Dentate Granule Cell

EEG Electroencephalogram

GCL Granule Cell Layer

GFP Green Fluorescent Protein

IML Inner Molecular Layer

MFS Mossy Fiber Sprouting

ML Molecular Layer

OML Outer Molecular Layer

RFP Red Fluorescent Protein

SE Status Epilepticus

SGZ Sub-Granular Zone

SRS Spontaneous Recurrent Seizures

SVZ Sub-Ventricular Zone

TLE Temporal Lobe Epilepsy

YFP Yellow Fluorescent Protein

Chapter 1: Introduction

Epilepsy is a common, debilitating neurological disorder

Epidemiology Epilepsy is the fourth most common neurological disorder, affecting more than 65 million people around the world (England et al., 2012). It is estimated that 1 in 26 Americans will develop epilepsy in their lifetime (Kobau et al., 2012, Begley and Durgin, 2015).

Epidemiological studies have further indicated that children and young adults are the fastest- growing population segments diagnosed with epilepsy, and thus it is poised to be a major cost to healthcare systems around the world (England et al., 2012, Begley and Durgin, 2015).

Additionally, the social stigma associated with epilepsy often leads to depression and is associated with a significant decrease in quality of life (Jacoby and Austin, 2007).

Treatment Current antiepileptic treatments do not cure or prevent the disease but are aimed at suppressing symptomatic seizures (Beghi, 2003). Further, 30 percent of new cases of epilepsy are refractory to even these suppressive treatments, thereby leaving neurosurgery as the only alternative (Kwan et al., 2011). Newer therapies targeted at reversing the changes that occur during epileptogenesis (i.e. changes that give rise to an epileptic brain) are critically needed, and deciphering the causal mechanisms of epileptogenesis will be the key to developing these new treatment modalities. Major research efforts have thus been directed towards medial TLE, specifically focusing on the hippocampus.

Definition and Classification Epilepsy is defined as a condition characterized by two or more unprovoked seizures within 24 hours (Fisher et al., 2014). A seizure is described as increased electrical activity in the

2 brain. Although multiple types of epilepsies have been identified, the most common form of refractory epilepsy is temporal lobe epilepsy (TLE). This can be further classified as medial

TLE, arising in the hippocampus and related regions, or the less frequently observed lateral TLE, arising in the neocortex (Blumcke et al., 2013).

The hippocampus is an important regulator of memory formation and plays a role in TLE

The hippocampus plays a central role in TLE The observation of frequent sclerosis in the hippocampus of patients with TLE and the alleviation of seizure activity upon removal of the hippocampus led to the hypothesis that the hippocampus plays a central role in seizure generation (Avoli, 2007). Specifically, the hippocampal dentate gyrus was hypothesized to be an epileptic focus (region with high epileptiform activity) and an epileptogenic zone (the place of seizure origin) (Dudek and Sutula,

2007). Subsequently, major research efforts were directed to studying the role of the hippocampus under normal and disease conditions.

Structure of the hippocampus The hippocampus, located in the medial temporal lobe in primates, is made of two cortical areas extending across the anterior-posterior axis of the brain (Cajal, 1894, Amaral and

Lavenex, 2006). The first area is called the cornu ammonis (CA) and is composed of excitatory pyramidal neurons in three separate zones: CA1, CA2, and CA3 (Fig. 1.1). The second area is the dentate gyrus composed of glutamatergic dentate granule cells (DGCs) (Cajal, 1894, Amaral and Lavenex, 2006). The dentate gyrus is further divided into a hilus, a granule cell layer

(GCL), and a molecular layer. The dentate hilus is composed of interneurons, basket cells, and mossy cells, whereas the granule cell layer is comprised of the cell somas of the DGCs. The organized lamellar structure of the hippocampus serves as an excellent system to study the process of the brain and has been the focus of research in memory formation and pattern separation (Fortin et al., 2002, Bird and Burgess, 2008).

The role of hippocampus in memory formation and pattern recognition The observation that patients with bilateral hippocampal damage had compromised ability to form new explicit memories but had intact procedural memories provided the first clues a role of the hippocampus in short-term memory processing (Carlesimo et al., 2001, Holdstock et al., 2005). Subsequently, research revealed that the hippocampus is more important for episodic memory (e.g., remembering recent personal events, interactions, etc.) and less important for semantic memory (e.g., remembering facts, words, etc.) (Tulving, 2002, Steinvorth et al., 2005).

The declarative theory of hippocampal function, a popular hypothesis for hippocampal memory formation, states that recent episodic memory is initially processed and stored in the hippocampal complex but is later consolidated into neocortical areas outside the medial temporal lobe (Squire, 1986, Bird and Burgess, 2008) . Recent discovery of hippocampal place cells has added interest in the functioning of the hippocampus (Ono et al., 1991, Ekstrom et al., 2003).

These place cells are known to encode a sense of location by providing a representation of position relative to environmental place cues, explaining our ability to map a terrain.

The hippocampus is also important for pattern separation (i.e. the ability to distinguish between similar objects in space and time (Wills et al., 2005). During pattern separation, similar inputs

4 are converted to less similar outputs, facilitating the brain’s ability to distinguish between similar stimuli, cues, or environments (Sahay et al., 2011). It is proposed that the selective transformation of signals required for pattern separation is realized by the gate like function of the dentate gyrus.

The dentate gyrus acts as a gate which breaks down under epileptic conditions

Experiments in the 1970’s demonstrated that the dentate gyrus suppresses the flow of excitation from the perforant pathway to the rest of the hippocampus (Andersen et al., 1971,

Alger and Teyler, 1976, Winson and Abzug, 1977). This suppression could be reversed with a stimulus of longer duration applied at an appropriate frequency. These observations led to the hypothesis that dentate granule cells act as a gate for excitatory input incident upon the hippocampus (Hsu, 2007). Properties of high resting membrane potential (Fricke and Prince,

1984) and strong GABA receptor-mediated inhibition (Coulter, 1999) made DGCs an excellent candidate for this role.

Using the kindling model, Behr et al. (1998) showed that this gate could be broken down under conditions that induce hyper-excitability, the first step towards epilepsy. Field recordings of entorhinal-hippocampal brain slices in low-magnesium conditions generated spontaneous seizure like activity, both in the entorhinal cortex and in the CA3. Kindling significantly increased the amplitude and duration of this spontaneous activity in the CA3, implying a breakdown of the dentate gate. These findings also led to development of the maximal dentate activation concept by Stringer and Lothman (1989, 1992). In a set of experiments, they stimulated the perforant

5 pathway until a saturating peak level of activity was recorded from the granule cell layer. This state, referred to as maximal dentate activation, was accompanied by a stark negative shift in the

DC potential (depolarization) of the dentate granule cells, a large increase in extracellular K+ ion concentration, and a burst of large amplitude field spikes. Stimulation above the maximal dentate activation state led to after-discharges which could persist for hours after stimulation.

This state was hypothesized to be the breaking point of the dentate gate and is now routinely used to estimate the condition of the dentate gate in epileptic animals (Hsu, 2007).

Apart from its role in memory formation, pattern separation, and signal gating, the hippocampus is now recognized as a site for post-natal neurogenesis and this has significant implications in epileptogenesis.

Adult neurogenesis in the hippocampus

Neurogenesis is the process of generating new neurons from precursor cells. This process was traditionally thought to be restricted to embryonic and perinatal stages of mammalian development. Classical experiments by Altman (1990) changed this view when he discovered the presence of new neurons in the rat hippocampus. The introduction of bromodeoxyuridine (BrdU) labelling for lineage-tracing studies established that continuous adult neurogenesis is restricted to two neurogenic niches in the mammalian brain (Shibui et al., 1989, Kuhn et al., 1996): the subventricular zone (SVZ) in the lateral ventricles, and the subgranular zone (SGZ) in the dentate gyrus of the hippocampus (Zhao et al., 2008). The new neurons generated in the SVZ migrate through the rostral migratory stream into the olfactory bulb and mature as interneurons.

Dentate granule cells are born in the SGZ and migrate to the granule cell layer where they

6 undergo functional maturation and integration. Adult neurogenesis is now recognized as a highly orchestrated process which is extremely sensitive to physiological and pathological stimuli (Zhao et al., 2008, Ming and Song, 2011, Aimone et al., 2014).

In the SGZ, newborn DGCs arise from type-1 progenitor cells which can self-renew and exhibit multipotency (give rise to both neurons and glia) (Gage, 2000, Bonaguidi et al., 2011). They are quiescent and display label retaining properties traditionally associated with stem cells (Gage,

2000, Weissman et al., 2001). Under physiological stimuli, type-1 cells give rise to type-2 progenitor cells, also called transit amplifying cells (Ming and Song, 2005). These type-2 cells can then give rise to either immature neurons (DGCs) or immature glial cells. Post mitotic, immature DGCs then migrate into the granule cell layer and undergo maturation.

During this process of maturation, granule cells undergo a number of morphological, functional, and physiological changes (Liu et al., 2000, Ming and Song, 2005, Overstreet-Wadiche and

Westbrook, 2006, Pedroni et al., 2014). DGCs travel almost 40µm from their place of origin in the SGZ to the granule cell body layer and send axons (mossy fibers) into the hilus, eventually synapsing with CA3 neurons (Jones et al., 2003). They extend apical dendrites into the molecular layer, where they make connections with inputs from the entorhinal cortex, eventually becoming a functional unit of the hippocampal circuit (Liu et al., 2000). During the process of development, immature granule cell extends a basal dendrite which is eventually retracted under normal conditions (Jones et al., 2003).

DGCs also undergo electrophysiological maturation over the first two to seven weeks after cell division (Ye et al., 2000). The input resistance (which varies inversely with the dendritic length) decreases as a cell matures and can be used to differentiate between immature and mature DGCs.

GABAergic inputs to the immature granule cells are initially depolarizing but switch to hyperpolarizing around three weeks after the last cell division (Ye et al., 2000). The characteristics of different stages of adult neurogenesis have been summarized in Fig. 1.2. These features of DGCs are dramatically altered under epileptic conditions.

Epilepsy affects adult neurogenesis and leads to the production of abnormal

DGCs

Seizures cause an acute increase in neurogenesis (Parent et al., 1997, Parent et al., 1998) and extensive pathological remodeling of the dentate gyrus (Parent et al., 1997, Pierce et al., 2005).

Adult-born DGCs are susceptible to epileptogenic insults and exhibit multiple integrational and morphological abnormalities (Tauck and Nadler, 1985, Spigelman et al., 1998, Ribak et al.,

2000, Scharfman et al., 2000, Hester and Danzer, 2013). These include mossy fiber sprouting

(MFS), ectopic DGCs, and DGCs with basal dendrites (Fig. 1.3), all of which increase excitability in the dentate.

One of the first observed morphological abnormalities was the presence of dentate granule cell axons in the inner molecular layer instead of the dentate hilus (Tauck and Nadler, 1985, Sutula et al., 1989, Nadler, 2003). This pathology was labelled mossy fiber sprouting (Fig. 1.3 C,D) and was hypothesized to create recurrent excitatory connections between the granule cells, eventually leading to hyperexcitability in the hippocampus (Sutula and Dudek, 2007). However, recent

8 research has shown that MFS may not be as critical for the development of epilepsy as initially hypothesized. Alternatively, it has also been suggested that MFS might actually be antiepileptogenic, leading to new inputs on inhibitory interneurons, which then function to decrease hyperexcitability (Sloviter et al., 2006). The exact role of MFS in epilepsy has yet to be established (Buckmaster, 2012).

Adult-generated granule cells born just before or after a seizure also display hilar basal dendrites

(Fig. 1.3.E) which can again be synaptically targeted by mossy fibers in the hilus. Our lab has previously shown that these basal dendrites arise in up to 50% of DGCs born up to 5 weeks before or after SE (Walter et al., 2007). Together with MFS, basal dendrites thus complete the epileptic recurrent excitatory circuit (Shapiro and Ribak, 2006, Shapiro et al., 2007). Indeed, mathematical modelling predicts that only a small number of DGCs with hilar basal dendrites would be enough to induce spontaneous bouts of hyperexcitability in the dentate gyrus (Morgan and Soltesz, 2008).

Another common pathology observed in the epileptic dentate is the presence of dentate granule cells in ectopic locations such as the hilus (hilar ectopic granule cells; Fig 1.3A.1, B.1) or the molecular layer (semi-lunar granule cells) (Marti-Subirana et al., 1986, Scharfman et al., 2000,

Parent et al., 2006). Hilar ectopic granule cells are connected to mossy fibers from normotopic granule cells (Pierce et al., 2005) and may contribute to recurrent excitatory loops. Research from our lab and others has shown that these ectopic cells are specifically born after status epilepticus (SE) (Parent et al., 1997, Walter et al., 2007). The hypothesis that their presence and accumulation promotes epilepsy is now corroborated by several lines of experimental evidence.

Newborn ectopic DGCs have enhanced synaptic excitation and reduced synaptic inhibition, favoring an overall excitable phenotype (Zhan et al., 2010). These newborn ectopic DGCs display spontaneous epileptiform bursts (Scharfman et al., 2000) and are active during experimentally-induced temporal lobe seizures (Scharfman et al., 2002), suggesting their direct involvement in seizure propagation. Subsequently, in a study by Scharfman and Pierce (2012), hilar ectopic DGCs were found to have extensive dendritic projections throughout the hippocampus and send axonal projections to both the CA3 (normal target) and the molecular layer (pathological target), thereby forming a hub that could potentially coordinate a seizure.

Finally, seizures have also been linked to mossy cell death (Sloviter, 1987, Hester and Danzer,

2013), dispersion of the granule cell layer (Houser, 1990), granule cell spine density changes

(Isokawa, 1998, 2000), changes in the volume of granule cell axon hillock (Wimmer et al.,

2010a), and somatic hypertrophy (Murphy et al., 2012). All these changes, along with the ones mentioned above, are hypothesized to be epileptogenic, predisposing the brain to recurrent seizures. Studies from our lab have shown strong correlations between the frequency of seizures and the degree of MFS, the number of hilar ectopic DGCs and the number of DGCs with basal dendrites (Hester and Danzer, 2013), providing further proof for this hypothesis.

Major research efforts have thus been directed at blocking neurogenesis (Jung et al., 2004, Jung et al., 2006, Pekcec et al., 2008, Cho et al., 2015) as a potential antiepileptogenic strategy.

Although promising, some of these approaches are global in their effects. Only a subset of adult- born DGCs appear to be morphologically and physiologically abnormal (Murphy et al., 2011), and blocking neurogenesis would compromise the function of normal DGCs too. Recent

10 approaches using temporary optogenetic inhibition (Berglind et al., 2014) or highly selective genetic ablation (Cho et al., 2015) of specifically newborn DGCs bypass some of these issues by specifically targeting newborn cells. Indeed, the genetic ablation of new born DGCs born after

SE leads to a reduction in seizure frequency and thus provides strong evidence for their role in epileptogenesis (Cho et al., 2015).

Another exciting avenue for antiepileptic therapy includes neural stem cell grafting. Stem cell therapy in epilepsy could potentially remodel dysfunctional neuronal circuits and be used to locally deliver neuroprotective factors (Miltiadous et al., 2013, Parent and Anderson, 2015).

However, limited knowledge of the effects of seizures on progenitor cell proliferation, differentiation, and integration has limited the applicability of this approach. Understanding how dentate gyrus progenitor cells respond to seizures is central to designing antiepileptic therapies, and I aim to explore some of these questions in this thesis.

Animal models of temporal lobe epilepsy

Understanding the various mechanisms underlying epileptogenesis and seizure initiation cannot be completely understood using human clinical studies alone, and hence the use of proper animal models is essential (Kandratavicius et al., 2014). Over the years, multiple types of animal models of epilepsy have been employed including chemoconvulsant, electrical, traumatic brain injury, hypoxia, and genetic models. For my studies, I employed the kindling (electrical) and the pilocarpine (chemoconvulsant) models to study the different phases before and after the occurrence of SRS (summarized in Fig. 1.4).

Pilocarpine model of epilepsy

Pilocarpine, a muscarinic acetylcholine receptor agonist and can induce limbic seizures on systemic or intracerebral administration (Turski et al., 1983). This chemoconvulsant model of epilepsy closely mirrors human TLE as the initial insult (like traumatic brain injury, hypoxia or status epilepticus) is followed by a latent period before the occurrence of SRS (Turski et al.,

1983, Cavalheiro et al., 1991). The latent period is hypothesized to be the phase of epileptogenesis where various morphological, electrophysiological, and cellular changes lower the threshold for seizure generation, conditioning the brain towards spontaneous seizures

(Dichter, 2009).

The pilocarpine model provides a great testing ground for potential anti-epileptic drugs (AED) for human epilepsy as it recapitulates several morphological and cellular alterations typically seen in human TLE (Leite and Cavalheiro, 1995). For example, the presence of interictal activity in the subiculum (Wellmer et al., 2002), upregulation of neurotrophins in the hippocampus

(Poulsen et al., 2004), acute increase in neurogenesis (Parent et al., 1997), cognitive memory deficiencies (Hort et al., 1999), and mossy cell death (Hester and Danzer, 2013) are common to both the pilocarpine rodent model of epilepsy and human patients with TLE. The limitations of the pilocarpine model include a high mortality rate (varying from 30% to 70%), variable frequency and severity of seizures, and hippocampal/neocortical lesions (Curia et al., 2008).

The kindling model of epileptogenesis Kindling refers to the enhancement of seizure susceptibility that occurs after repeated electrical stimulations which induce after-discharges in targeted areas of the brain. The kindling model of epilepsy is fairly robust and has been used to model different variations of seizures by

12 modulating the number of after-discharges (McNamara, 1986). A smaller number of after- discharges induce acute morphological alterations resembling partial seizures (classes 1–

3) (Racine, 1972a) which on increasing the number of after discharges (classes 4–5), evolve into secondary generalizations (Goddard et al., 1969, Racine, 1972b).

A completely kindled state can be seen after 90–100 class 5 seizures, but it is also possible to induce hyperexcitability by gentler protocols (Sloviter et al., 2007). Indeed, milder kindling protocols have been employed specifically to induce epileptogenesis without the SRS (Bertram and Lothman, 1993, Watanabe et al., 1996, Henshall and Meldrum, 2012, Singh et al., 2013) or the associated cell death (Pinel and Rovner, 1978, Pretel et al., 1997). These early kindling models provide an opportunity to study the primary phases of epileptogenesis without the confounding variables introduced by recurrent seizures.

The reproducibility of molecular and network changes seen after kindling has made it a popular choice for studying mechanisms of epileptogenesis (Sutula, 2004). Although popular, kindling models are usually expensive to produce, labor intensive, and require constant supervision

(Kandratavicius et al., 2014).

Modelling epileptogenesis before and after SRS

The overall goal of my thesis is to elucidate mechanisms of epileptogenesis, thereby providing novel opportunities for therapeutic intervention. To this end, I employed different protocols to model the changes that occur before and after the occurrence of SRS. The overall goals of this study are summarized in the Fig. 1.4.

In Chapter 2, I used the early kindling model to study the acute and chronic morphological changes that occur in the brain after a seizure. I used a Thy1-GFP constitutive reporter mouse which labels a high percentage of mature, pre-natal DGCs and only a small fraction of adult- generated DGCs. This study was designed to provide an insight into the variety, degree and timing of morphological changes after the first epileptogenic insult.

In Chapter 3, I discuss the experiments describing the first clonal analysis of postnatally- generated DGCs in the epileptic rodent brain. This study provides important clues on the clonal origins of abnormal DGCs implicated in the progression of epilepsy. I utilize the pilocarpine model of epilepsy and study these abnormal cells after the occurrence of SRS. I aim to determine whether these abnormal new born DGCs arise from a small pool of bad actor stem cells or do they arise equally from different progenitor cells in the dentate. The overall hypothesis for this study is summarized in Fig. 1.5. Together, these studies will increase our understanding of the key mechanisms of epileptogenesis.

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Figures and Tables

Figure 1.1. Schematic depicting the structure of the dentate gyrus. The dentate is composed of three main layers: the hilus, the granule cell layer (GCL), and the molecular layer (ML). The dentate granule cells (DGCs) are located in the GCL and project mossy fiber axons into the hilus.

These mossy fiber axons then synapse with pyramidal cells in the Cornu Ammonis (CA) layers

(CA1, CA2, and CA3). The dentate granule cells also project apical dendrites onto the molecular layer where they receive inputs from the entorhinal cortex via the perforant pathway.

Figure 1.2: The stages of adult neurogenesis in the SGZ. The different stages of adult hippocampal neurogenesis are shown along with the average stage time, the morphology of the cells at different stages, the surface markers traditionally used to identify cells at each stage, and the known synaptic inputs associated with different cells. Type-1 progenitors are slow-cycling stem cells that give rise to transit-amplifying type-2 cells. These type-2 cells can acquire a neuronal fate and eventually mature into functional DGCs over time.

Figure 1.3: Seizures alter adult neurogenesis and lead to the production of morphologically abnormal DGCs. Shown are maximum projection images from control and epileptic (SE) animals expressing Thy1-GFP. (A, B) The overall morphology of the dentate changes after a seizure due to extensive cell death in the Cornu Ammonis (CA) regions and dentate hilus (H).

(A.1, B.1) Seizures also lead to the production of ectopic dentate granule cells (DGCs) located in the dentate hilus. (C, D) Another pathology seen after seizures in the presence of mossy fiber sprouting (MFS) in the inner molecular layer of the dentate. (E) Aberrant basal dendrites also arise from a larger number of DGCs in the epileptic dentate. Scale bars A, B: 300µm; A.1, B.1=

20µm; C, D= 250µm; E: 5µm

Figure 1.4: Summary of the model systems employed in the current thesis. In this dissertation, I used the early kindling model (Chapter 2) as well as the pilocarpine model (Chapter 3) to study different phases of epileptogenesis.

Figure 1.5: Schematic depicting the hypothesis being tested in the clonal analysis study described in Chapter 3. Different stem cells are randomly labeled by different colors of the brainbow reporter. After inducing status epilepticus (SE), I tracked the progeny arising from these different stem cells to determine if abnormal cells arise homogenously from all the progenitors or are generated from a few bad actor stem cells.

Chapter 2:

Morphological changes among hippocampal dentate granule cells exposed to early kindling-epileptogenesis

Singh SP, He X, McNamara JO, Danzer SC (2013) Morphological changes among hippocampal dentate granule cells exposed to early kindling-epileptogenesis. Hippocampus 23:1309-1320.

Abstract

Temporal lobe epilepsy is associated with changes in the morphology of hippocampal dentate granule cells. These changes are evident in numerous models that are associated with substantial neuron loss and spontaneous recurrent seizures. By contrast, previous studies have shown that in the kindling model, it is possible to administer a limited number of stimulations sufficient to produce a lifelong enhanced sensitivity to stimulus evoked seizures without associated spontaneous seizures and minimal neuronal loss. Here we examined whether stimulation of the amygdala sufficient to evoke five convulsive seizures (class IV or greater on Racine’s scale) produce morphological changes similar to those observed in models of epilepsy associated with substantial cell loss. The morphology of GFP-expressing granule cells from Thy-1 GFP mice was examined either one day or one month after the last evoked seizure. Interestingly, significant reductions in dendritic spine density were evident one day after the last seizure, the magnitude of which had diminished by one month. Further, there was an increase in the thickness of the granule cell layer one day after the last evoked seizure, which was absent a month later. We also observed an increase in the area of the proximal axon, which again returned to control levels a month later. No differences in the number of basal dendrites were detected at either time point. These findings demonstrate that the early stages of kindling epileptogenesis produce transient changes in the granule cell body layer thickness, molecular layer spine density and axon proximal area, but do not produce striking rearrangements of granule cell structure.

Introduction

Changes in the structure and function of hippocampal dentate granule cells are hallmarks of temporal lobe epilepsy (Danzer, 2012). These include changes in spine density (Isokawa, 1998,

Swann et al., 2000, Leite et al., 2005, Santos et al., 2011), granule cell layer dispersion (Houser,

1990, Bouilleret et al., 1999, Riban et al., 2002, Heinrich et al., 2006), the appearance of ectopic cells (Scharfman et al., 2000, Dashtipour et al., 2001, Scharfman et al., 2003, Cameron et al.,

2011, Zhao et al., 2012), the appearance of hilar projecting basal dendrites (Spigelman et al.,

1998, Ribak et al., 2000, Diaz-Cintra et al., 2009, Murphy et al., 2012, Sanchez et al., 2012), sprouting of granule cell mossy fiber axons (Tauck and Nadler, 1985, Sutula et al., 1989, Shibley and Smith, 2002, Nadler, 2003) and somatic hypertrophy (Murphy et al., 2012). Abnormalities have been described in numerous models of temporal lobe epilepsy. For example, changes in spine density have been observed in systemic pilocarpine (Isokawa, 1998), systemic kainic acid

(Wenzel et al., 2000), intra-hippocampal kainic acid (Suzuki et al., 1997) and lesion models

(Bundman and Gall, 1994) of epilepsy. Notably, these models are associated with both spontaneous seizures and substantial neuron loss (Gall, 1988, Golarai et al., 2001, Riban et al.,

2002, Zhang et al., 2002, Wang et al., 2008).

In the kindling model of epilepsy, electrical stimulations can be applied which initially produce only brief electrographic seizures (≈5s). Repeated stimulation, however, produces longer electrographic seizures, and eventually results in tonic-clonic behavioral seizures (Goddard et al.,

1969, Wada et al., 1974, McNamara et al., 1980, Dennison et al., 1995). This increased responsiveness to stimulation lasts lifelong (Moshe and Albala, 1982, Dennison et al., 1995).

When only a few tonic-clonic seizures are evoked (≈5), the procedure produces minimal

32 neuronal loss (Bengzon et al., 1997, Pretel et al., 1997, Zhang et al., 1998) and does not result in spontaneous recurrent seizures (Bertram and Lothman, 1993, Watanabe et al., 1996, Tuunanen and Pitkanen, 2000, Henshall and Meldrum, 2012). This “early kindling” protocol, in which only a small number of seizures are evoked is believed to model the early phase of epileptogenesis, while evoking repeated seizures may reflect later phases of the epileptogenic process (Bertram, 2007). Extended kindling can also give rise to more pronounced neuronal cell loss and spontaneous seizures (Pinel and Rovner, 1978, Cavazos and Sutula, 1990, Bengzon et al., 1997).

Early kindling provides an opportunity to reveal the persistent changes in brain function and structure which might be operative in the earliest stage of epileptogenesis in a model minimally confounded by cell loss (McNamara, 1986, Tuunanen and Pitkanen, 2000). Here, we queried whether some of the more dramatic morphological changes evident in epilepsy models associated with robust cell death would be evident at the earliest stage of kindling epileptogenesis. To explore this possibility, Thy1-green fluorescent protein (GFP) expressing transgenic mice (Feng et al., 2000) received repeated stimulations of the amygdala until 5 successive seizures with clonic and/or tonic motor convulsions of class IV or above (Racine,

1972) were observed. Animals were sacrificed one day after the last evoked seizure, to look for acute effects, and one month after the last seizure, to look for chronic effects.

Methods

Thy1-GFP Expressing Mice

Male and female mice hemizygous for the Thy1-green fluorescent protein transgene (Thy1-GFP,

M line) (Feng et al., 2000) were maintained on a C57BL/6 background. Mice from this line express GFP in a subset of hippocampal dentate granule cells (~11%). Labeled granule cells have been found to be morphologically and physiologically indistinguishable from unlabeled cells (Vuksic et al., 2008). Mice were sorted for experiments such that littermates were present in kindled as well as control groups. Analysis of granule cell giant mossy fiber bouton structure in these animals has been previously published (Danzer et al., 2010).

Kindling Model

Two to three-month-old mice were anesthetized with pentobarbital (60 mg/kg, intraperitoneal

[i.p.] injection) and stereotaxically implanted with a bipolar stimulation-recording electrode in the right amygdala. The following coordinates were used with bregma as reference: 1.0 mm posterior, 2.9 mm lateral and 4.6 mm below dura. A ground electrode was secured to the skull over left frontal cortex. One week post-surgery, a 60Hz, 60 μA, 1 s train of 1 ms biphasic rectangular pulses was used to determine the electrographic seizure threshold. Stimulation intensity was increased by 20 μA increments at 1 min intervals until an electrographic seizure lasting at least 5 s was detected (the electrographic seizure threshold). The experimental animals were subsequently stimulated twice a day at stimulus intensities 100 μA above the electrographic seizure threshold with a minimum inter-stimulus interval of at least 4 hours. Daily stimulations were administered until five seizures were induced. To be counted as one of the five seizures, the event had to last at least 12 seconds and receive a behavioral severity score of class IV or

34 above according to the Racine scale (Racine, 1972b). Surgically implanted control animals

(sham) were connected twice daily to the stimulation apparatus, but did not receive any stimulations or undergo seizure threshold determination. Mice were perfused with paraformaldehyde one day (control, N = 7 [4 male, 3 female]; kindled, N =7 [4 male, 3 female]) or one month (control, N = 8 [7 male, 1 female]; kindled, N = 7 [6 male, 1 female]) after the last evoked seizure or sham protocol.

Tissue preparation

Animals were anesthetized by i.p. injection of 100 mg/kg pentobarbital. They were perfused through the ascending aorta with ice-cold phosphate buffered saline [0.1M PBS] with 1 mM sodium orthovanadate (NaOV) and 1 U/ml heparin for 30 seconds at 10ml/min, immediately followed by 2.5% paraformaldehyde, 4% sucrose and 1mM NaOV in 0.1M PBS at 25oC for 10 minutes. Brains were removed and post-fixed in the same solution for 1 h at 4°C. Brains were cryoprotected in an increasing concentration of sucrose solutions (10%, 20%, 30%) in 1 mM

NaOV in PBS, frozen in isopentane pre-cooled to −25°C with dry ice and stored at −80°C until cryosectioning. Coronal sections of 40 µm thickness corresponding to bregma levels -1.64 to -

2.75 (figures 46 – 48, Paxinos and Franklin’s mouse atlas, 2001) were cut and mounted on charged slides (Superfrost Plus slides, Fisher Scientific, Waltham, MA). Since only sections from a portion of dorsal hippocampus were examined, thus the results may not reflect changes occurring in other hippocampal regions.

GFP immunostaining

Sections were thawed in PBS, incubated for 1 h with 0.5% Igepal + 5% normal goat serum in

PBS (blocker), and then overnight in rabbit anti-GFP (1:500, Chemicon, Billerica, MA) antibody in blocker at 4°C. Sections were rinsed in blocker and incubated for 2 h in 1:750 Alexa Fluor

488 goat anti-rabbit antibodies (Invitrogen, Eugene, OR). Sections were then rinsed in PBS, dehydrated in alcohols, cleared in xylenes and mounted with Krystalon (EMD chemicals,

Gibbstown, NJ).

Microscopy and neuronal reconstruction

The investigator was blind to treatment group during image collection and data analysis. Images of the GFP-labeled dentate granule cells were acquired using a Leica SP5 confocal system, set up on a Leica DMI6000 inverted microscope equipped with a 63X oil immersion objective

(numerical aperture 1.4). This system was used to capture three dimensional “image stacks” through the z-depth of the tissue at 0.5 μm steps with a 1X optical zoom (field size 240 x 240

m). All granule cells selected for analysis were brightly labeled with GFP and had their somata fully contained within the tissue section. To reduce variability, cells selected for this study were restricted to the upper blade of the dentate gyrus within dorsal hippocampus. On average 20 cells (range 18 – 23) were selected from each animal with an average of 2 sections per animal.

All cells in an image stack that met selection criteria were used for analyses.

Confocal z-series image stacks were imported into Neurolucida software for analysis (Version

5.65, Microbrightfield Inc.,Williston, VT). For each cell, maximum soma profile area and the number of primary apical and basal dendrites was determined. Basal dendrites were counted as those dendrites that originated from the hilar side of the soma (i.e. arising from a region below

36 the equator of the soma). Basal dendrites that projected into the hilus were defined as “hilar basal dendrites” and basal dendrites that turned back into the dentate molecular layer were termed “recurrent basal dendrites”. Dendrites arising from above the equator of the soma were scored as apical dendrites. Portions of the proximal axon contained within the granule cell body layer (GCL) were also examined to determine their mean cross sectional area. This was done by first encoding the volume of the first 8.0 - 13.0 m of the axon (lengths varied slightly due to artificial axon truncation at the tissue surface, although groups were not significantly different [at

1 day after the last kindling evoked seizure: control, 11.1 ± 0.22 m, kindled, 10.9 ± 0.15 m; p=0.26, t-test]) and then dividing by the length measured. The density of axonal varicosities along the portion of axon contained within the granule cell body layer was also determined.

Axonal varicosities were defined as expansions of the axon with a diameter greater than 0.5 µm.

Proximal axon varicosity number was normalized by dividing by the length of axon measured.

Image stacks were also used to determine the width of the granule cell body layer. Two methods were used to determine layer width: 1) distance from the hilar border to the molecular layer border at the midpoint of the upper blade; 2) area of the entire granule cell body layer in the brain section divided by the length of the entire cell body layer. Further, to correct for potential bias introduced by the angle of sectioning, the thickness of the granule cell layer was normalized relative to the thickness of the CA3 and CA1 pyramidal cell layers in same sections, using the following equations: GCL thickness/pyramidal cell layer thickness x 100. Image stacks were also used to determine the relative depth of each neuron examined within the GCL (cell distance from GCL-hilar border/GCL width).

For analyses of dendritic spine density, images of dendritic segments located within the inner and the outer molecular layers were captured using a 0.2 m step at 6X optical zoom (field size

40 x 40 m). Inner molecular layer dendritic segments were imaged by placing the lower edge of the imaging field on the granule cell body layer - molecular layer border and by scanning the initial projection into the molecular layer. Outer molecular segments were scanned by placing the upper edge of the field at the hippocampal fissure and imaging the terminal segments of the dendritic tree. Only dendritic segments fully contained within the tissue section were imaged.

Images were imported into Neurolucida software for analysis. Dendritic spines were defined as protrusions from the dendritic shaft with a length greater than 0.25 m. Spine density was determined by dividing the number of spines counted by the length of dendrite examined.

Although confocal microscopy does facilitate visualizing spines located above and below the dendritic shaft, it is likely that smaller spines are still obscured in these regions, so spine measurements likely underestimate actual values. In addition, images/analyses were not adjusted for shrinkage of the tissue, so comparisons between the present findings and work using different methodologies should be made with these caveats in mind.

Statistical Analysis

All statistical analyses were performed using Sigma Plot (version 12.0). For each parameter, data from each animal was averaged to generate individual animal means. Animal means were used for determination of significance instead of cell numbers, as cells from the same animal are not completely independent and may result in artificially low p-values. Significance was determined using two-tailed Students t-tests for data that met assumptions of normality and equal variance. The Mann-Whitney rank sum test was used for non-normal data. Values presented are

38 means ± standard error of the mean (SEM) or medians (range), as appropriate. Pearson product moment correlation was used for correlations. Since both male and female animals were included in the study, data were examined for effects of sex. No significant effects of sex were found for any parameter (data not shown). Removing the female animals from the one month groups and analyzing only males did not change statistical outcomes either (data not shown). For these reasons, male and female animals were binned and analyzed together.

Figure Preparation

Maximum projections from confocal z-series stacks were prepared using Leica’s LAS AF

Confocal software (1.3.1 build 525). Background artifact reduction was accomplished using an erosion filter run for one iteration with a three pixel radius (Leica software). Contrast, brightness, montage adjustments and figure preparation were done using Adobe Photoshop CS5

(version 12.0). Identical filtering and adjustments to brightness and contrast were done for images used for comparison.

Results

Part I: Morphological analyses one day after the last kindling-evoked seizure

Gross hippocampal morphology is unchanged in kindled mice

Granule cells in the Thy1-GFP line, brightly labeled with GFP, clearly revealed the axonal and dendritic structure of each labeled neuron (Figure 2.1). Each kindled animal experienced 5 evoked seizures, with cumulative electrographic seizure duration of 527 ± 101 seconds/mouse.

The mean number of stimulations required to evoke 5 class IV or above behavioral seizures was

22.0 ± 3.4 stimulations/mouse. The number and distribution of GFP-expressing granule cells in kindled and controls animals was statistically similar between groups, as demonstrated in previous work with the mice used for the present study (Danzer et al., 2010). The morphological architecture of the hilus, the granule cell layer and the molecular layer was also comparable between control and kindled mice. No evidence of gross neuronal loss or overt neuronal injury

(e.g. dendritic beading or retraction) was observed.

Granule cell body layer thickness is increased one day after the last kindling-evoked seizure

Status-epilepticus models of epilepsy can produce dramatic granule cell dispersion and somatic hypertrophy (Houser, 1990, Murphy et al., 2012). Here, we observed a modest increase in the width of the granule cell body layer in kindled mice compared to controls. At the midpoint of the upper blade, control animals had an average granule cell body layer width of 46.9 ± 2.7 m

(N=7 mice, 2 sections/mouse) compared to 63.0 ± 6.5 m (N=7 mice, 2 sections/mouse) in kindled animals (p= 0.02, t-test, Figure 2.2). Similar results were obtained by dividing the entire area of the granule cell body layer by the entire length of the granule cell body layer (control,

48.9 ± 2.4 μm, N=7 mice, 2 sections/mouse; kindled, 61.1 ± 4.5 m, N=7 mice, 2

40 sections/mouse; p=0.03, t-test, Figure 2.2). This latter finding suggests that the increase in width affects most of the layer. This difference in the width of the granule cell body layer also remains significant when normalized to the width of CA1 and CA3 (Table 2.1), indicating that the angle of sectioning did not interfere with our results. Although non-significant, a tendency for granule cell somata to be larger in kindled mice likely contributed to the increase in layer width. The average soma area in cells from kindled animals was 95.3 ± 8.8 m2 (N=7 mice, 140 cells from 2 sections/mouse) compared to 78.9 ± 6.3 m2 (N=7 mice, 133 cells from 2 sections/mouse) in control animals (p= 0.15, t-test, Figure 2.2). There was no significant change, however, in the relative location of GFP-expressing granule cell somata within the granule cell layer (cell distance from GCL-hilar border/GCL width) (Table 2.1) suggesting that the observed expansion of the granule cell body layer is relatively uniform. We did not observe a difference in the mean width of either CA3 or CA1 at this time point (Table 2.1).

Apical and basal dendrites

Changes in the number of apical dendrites (Murphy et al., 2011) and the de novo appearance of basal dendrites projecting into the dentate hilus (Spigelman et al., 1998) have been described in multiple models of epilepsy. Whether and to what extent these changes are a feature of the early phases of kindling epileptogenesis is, however, unclear. To explore these questions, apical and basal dendrite number was assessed among granule cells from control and kindled mice one day after the last kindling-evoked seizure. No differences were found in the number of apical or basal dendrites in the kindled mice relative to controls (Table 2.1). All granule cells in the study had at least one apical dendrite, with a few cells having as many as four. Hilar basal dendrites,

41 on the other hand, were only rarely found on granule cells from control animals, and this did not change significantly in kindled mice (Table 2.1). No recurrent basal dendrites were observed.

Reduced dendritic spine density in kindled mice

Spine density along dendritic segments located within the inner and outer molecular layers was significantly decreased in kindled animals relative to controls one day after the last evoked seizure. Within the inner molecular layer, spine density was reduced 30% from 1.81 ± 0.13 spines/m (N=7 mice, 133 cells from 2 sections/mouse) in controls to 1.26 ± 0.09 spines/m

(N=7 mice, 140 cells from 2 sections/mouse) in kindled mice (p=0.005, t-test, Figure 2.3A).

Similar reductions were observed in the outer molecular layer, where spine density dropped by

21% from 3.57 ± 0.18 spines/m (N=7 mice, 133 cells from 2 sections/mouse) in controls to

2.81 ± 0.16 spines/m (N=7 mice, 140 cells from 2 sections/mouse) in kindled mice (p=0.01, t- test, Figure 2.3A). Finally, we conducted a correlation analysis between mean inner and outer molecular later spine density for each animal, and found that the values were positively correlated with each other across the entire group of mice (controls and kindled combined, R=

0.609, p=0.0207; Figure 2.3B). This finding indicates that animals with a decrease in inner molecular layer spine density are likely to exhibit a concomitant reduction in the outer molecular layer spine density.

Increased volume of the proximal axon one day after the last kindling-evoked seizure

To determine whether axonal swelling occurs in the kindling model, we measured the mean cross sectional area of the proximal axon of GFP-expressing granule cells. One day after the last seizure, cross sectional area of the proximal axon was increased in kindled animals relative to

42 controls. Proximal axon segments from kindled animals had an average cross sectional area of

0.75 ± 0.08 µm2 (N=7 mice, 113 cells from 2 sections/mouse) compared to 0.54 ± 0.03 µm2

(N=7 mice, 90 cells from 2 sections/mouse) in control animals (p=0.04, t-test, Figure 2.4). By contrast, no difference was found between groups in the density of axonal varicosities along the portion of the axon located within the granule cell body layer (Table 2.1).

Part II: Morphological analyses one month after last kindling-evoked seizure

Animals examined one month after the last of 5 kindling-evoked seizures experienced a cumulative electrographic seizure duration of 397 ± 69 seconds. The mean number of stimulations required to evoke 5 class IV or above behavioral seizures was 17.0 ± 2.2 stimulations/mouse. Similar to animals examined one day after the last seizure, the pattern of

GFP expressing cells and the gross structure of the dentate gyrus were qualitatively similar between control and kindled animals (Figure 2.5). Kindled animals did not show any gross neuronal loss and the morphological architecture of the hilus, hippocampal pyramidal cell layers and molecular layers were qualitatively similar between groups.

Granule cell body layer width normalizes one month after the last kindling-evoked seizure

The increase in granule cell layer width observed one day after the last kindling-evoked seizure was no longer detectable one month later. Kindled animals had an average granule cell body layer width of 49.9 ± 4.2 m (N=7 mice, 2 sections/mouse) compared to 49.5 ± 3.9 m (N=8 mice, 2 sections/mouse) in control animals (p= 0.94, t-test, Figure 2.6). Similarly, no difference was observed in the area divided by length of the granule cell body layer, either for the raw

(kindled, 56.1 ± 4.3 m, N=7 mice, 2 sections/mouse; control, 49.8 ± 4.9 m, N=8 mice, 2

43 sections/mouse; p=0.35, t-test) or normalized values (Table 2.2). The trend towards increased soma area observed at one day after the last kindling-evoked seizure was also absent at this time point. The average soma area from kindled animals was 63.8 µm2 (range: 57.8 - 116.8 µm2, N=7 mice, 184 cells from 2 sections/ mouse) compared to 65.9 µm2 (range: 57.9 - 102.4 m2, N=8 mice, 122 cells from 2 sections/mouse) in control animals (p=0.83, t-test, Figure 2.6). Similar to the results obtained one day after the last kindling-evoked seizure, relative somata position within the granule cell body layer remained unchanged between control and kindled animals

(Table 2.2). Likewise, we did not observe any difference in the thickness of CA1 or CA3 (Table

2).

Apical and basal dendrites

Control and kindled animals had comparable numbers of apical and basal dendrites one month after the last kindling-evoked seizure (Table 2.2). Basal dendrites were rare, with only 2.4 ±

1.2% of granule cells (3 of 122 cells from 2 sections/mouse, N=8 mice) from control animals and

2.7 ± 3.4% of granule cells (5 of 184 cells from 2 sections/mouse, N=7 mice) from kindled animals possessing a basal dendrite. Only hilar basal dendrites were observed.

Kindled animals exhibit normal spine density one month after the last kindling-evoked seizure

In comparison to the results at one day, at one month after the last kindling-evoked seizure smaller reductions of dendritic spine density were evident in the inner (23% reduction in kindled animals) and outer molecular layers (13% reduction in kindled animals). In the inner molecular layer, kindled animals had 1.39 ± 0.16 spines/m (N=7 mice, 184 cells from 2 sections/mouse), compared to 1.79 ± 0.30 spines/m (N=8 mice, 122 cells from 2 sections/mouse) in control

44 animals. These differences were not statistically significant (p=0.279, t-test, Figure 2.7).

Similarly, in the outer molecular layer, the kindled animals had 3.52 ± 0.19 spines/m (N=7 mice, 184 cells from 2 sections/ mouse), compared to 4.06 ± 0.36 spines/m (N=8 mice, 122 cells from 2 sections/mouse) in control animals; also non-significant (p=0.21, t-test, Figure 2.7).

Further, no correlation was observed between spine densities in the inner and outer molecular layers of kindled and control animals at this time point (p=0.39, r=0.235).

Proximal axon cross sectional area is indistinguishable from control one month after the last kindling-evoked seizure

The cross sectional area of the proximal axon did not show a significant difference between kindled and control animals one month after the last kindling-evoked seizure. Proximal axon segments from kindled animals had an average cross sectional area of 0.55 ± 0.03 µm2 (N=7 mice, 148 cells from 2 sections/mouse) compared to 0.61 ± 0.05 m2 (N=8 mice, 122 cells from

2 sections/mouse) in control animals (p=0.36, t-test, Figure 2.8). Similarly, the density of axonal varicosities along the portion of the axon located within the granule cell body layer did not change significantly between control and kindled groups (Table 2.2).

Discussion

In the present study, hippocampal granule cells were examined either one day or one month after early kindling, in which five generalized, tonic-clonic seizures were evoked. We observed an increase in the thickness of the granule cell body layer, a decrease in the number of apical dendrite spines in the inner and outer molecular layers and an increase in the area of the proximal axon one day after the last kindling-evoked seizure. Apart from a persistent non-significant reduction in spine density, these changes had remitted in animals examined one month later.

Morphological features seen in other models of temporal lobe epilepsy, such as ectopic granule cells or granule cells with hilar-projecting basal dendrites were not observed in kindled animals examined at either time point.

Significance of increased granule cell body layer thickness

The increase in granule cell body layer thickness observed one day after the last kindling-evoked seizure could reflect a variety of processes, such as increased neurogenesis, somatic hypertrophy or reactive astrogliosis. All of these phenomena have previously been demonstrated in different models of epilepsy (Houser, 1990, Ribak and Dashtipour, 2002, Jessberger et al., 2005, Shapiro et al., 2007, Kraev et al., 2009, Murphy et al., 2012). Increased neurogenesis following seizure activity could increase granule cell layer thickness (Ribak and Dashtipour, 2002). The transient nature of the increase, however, favors other explanations, although it is possible that new (or old) cells might undergo apoptosis, thus restoring granule cell width to control values at one month. Reactive gliosis has been reported in the kindling model of epilepsy (Kraev et al., 2009) and increased number and thickness of astroglial fibers might also contribute to the changes observed in the present study. Finally, the trend towards increased somata area described here

46 likely contributes to the observed increase in granule cell layer thickness. Acute increases in cell volume can occur following osmotic changes caused by neuronal activity (Andrew et al., 1989,

Ostby et al., 2009) . In addition, work by Kato and colleagues (Kato et al., 2001) described dendritic hypertrophy a few hours after the last kindled seizure. Electron microscopy studies of these animals revealed an increase in stable polymerized microtubules in these dendrites, raising the possibility that cytoskeletal changes might contribute to increased granule cell layer width.

Development of hilar-projecting aberrant basal dendrites has been described in numerous models of temporal lobe epilepsy (Spigelman et al., 1998, Ribak et al., 2000, Murphy et al., 2012) and is hypothesized to promote epileptogenesis by creating recurrent excitatory circuits. In the present study, no increase in the incidence of basal dendrites was observed either one day or one month after the last kindling-evoked seizure. This finding is consistent with findings by Wood and colleagues (Wood et al., 2011), who used a retroviral approach to label adult-generated granule cells after rapid-kindling, but contrasts with work by Pekcec and colleagues (Pekcec and

Potschka, 2007, Pekcec et al., 2011) where an increase in basal processes following 15 kindling- evoked seizures was observed among granule cells labeled with doublecortin. Differences in the kindling protocols used could account for the divergent results, with the number of evoked Class

IV or greater seizures (15 vs. 5 in the present study) and the kindling protocol used (rapid vs. standard kindling) being likely candidates. Alternatively, the labeling strategy and time points examined could also contribute to the observed differences. Doublecortin is transiently expressed by developing granule cells (≈1-2 weeks old) (Brown et al., 2003), while Thy1-driven

GFP is only expressed in mature granule cells (>5 weeks) (Walter et al., 2007, Vuksic et al.,

2008, Murphy and Danzer, 2011, Santos et al., 2011). The retroviral strategy used by Wood and

47 colleagues targeted developing granule cells generated after rapid kindling; however, labeled cells were examined ≈2 months later, similar to the present study. The three studies, therefore, examined cells of different ages at different time points after kindling. Significantly, therefore, work by Walter and coworkers (Walter et al., 2007), using the same Thy1-GFP expressing mouse line examined here, found that 20% of Thy1-GFP expressing granule cells exhibited basal dendrites one month after pilocarpine induced status epilepticus; indicating that the neuronal population examined in the present study can develop basal dendrites under the right conditions.

Whether more robust seizures, associated cell death or other differences between the pilocarpine status epilepticus and kindling models of epilepsy account for the contrasting morphological responses remains to be determined.

Significance of spine density changes

Dendritic spines are post-synaptic specializations which house the synaptic machinery that allows a neuron to respond to excitatory pre-synaptic input. Since most of the excitatory inputs received by principal neurons are targeted to dendritic spines, spine counts provide an indirect means to assess the density of excitatory synapses on a neuron. Spines are highly plastic and can exhibit rapid changes in structure and number in response to a variety of physiological and pathological changes (Gray, 1959, Drakew et al., 1996, Fiala et al., 2002,

Kasai et al., 2010, Gonzalez Burgos et al., 2012, Kulkarni and Firestein, 2012).

One plausible mechanism contributing to the lifelong enhanced propagation of seizure activity from the stimulated amygdala to hippocampus and extra-limbic sites in kindled animals is increased numbers of excitatory synapses formed between principal neurons. This proposal

48 predicts an increase in the density of spines on the apical dendrites of the dentate granule cells.

Paradoxically, significant decreases in spine density were observed in both the inner (30% reduction) and outer (21% reduction) molecular layers one day after the last evoked seizure. The magnitude of these reductions had diminished by one month after the last evoked seizure; statistically significant decreases were no longer detected. The reductions in spine density observed here suggest that associational/commissural excitatory input to the inner molecular layer and lateral entorhinal cortex excitatory input to the outer molecular layer are reduced. The direction of the change, namely a decrease, together with its time course support the idea that these reductions underlie a homeostatic response aimed at reducing excitability following repeated seizures. Alternatively, since spines are an indirect measure of synapse number, it is also possible that spines are lost, but excitatory synapses are preserved. Conversion of excitatory spine synapses to direct synaptic innervation of dendritic shafts could also account for the present results, and would alter the electrotonic properties of the affected synapses by eliminating the thin spine necks. Distinguishing between these possibilities will require more direct measures of synapse number, such as electron microscopy techniques.

Finally, studies of spines and excitatory synapses in the kindling model have revealed both decreases and increases (Geinisman et al., 1992, Teskey et al., 2001). We suspect these complex changes can be accounted for in part by the population of synapses studied, with different neuronal populations potentially exhibiting distinct responses. Additional factors likely to be important include the stimulation paradigm used (Zhao et al., 2012) and time points examined

(Teskey et al., 2001, Teskey et al., 2006). The time point examined also appears to be important for status epilepticus models of epilepsy, in which reductions are observed at early time points

(Olney et al., 1983, Pyapali and Turner, 1994, Isokawa, 1998) while partial recovery can occur at later time points (Isokawa, 1998).

Significance of changes in the volume of the proximal axon

The axon initial segment is located within the proximal axon. The axon initial segment is the site at which the action potential originates, and is therefore critical to neuronal function (Palay et al.,

1968). Marked swelling and dilation of the proximal axon has previously been seen in tissues from human epileptic patients (Chronister et al., 1995), and recent studies of the initial segment reveal changes in response to neural activity (Grubb and Burrone, 2010, Grubb et al., 2011) and seizures (Wimmer et al., 2010b). Whether the proximal axon swelling observed here reflects changes in the composition of the initial segment, or is merely an extension of the trend towards swelling in the somatic compartment is not known. Even transient swelling, however, could alter action potential initiation if the density of ion channels relative to internal axon volume was substantially changed.

Conclusions

Our findings demonstrate that early kindling produces acute, transient changes in the thickness of the granule cell layer, molecular layer spine density and the area of the proximal granule cell axon. Early kindling did not, however, produce the striking rearrangements of granule cell structure seen in other models of epilepsy, at least for the cell populations examined. Whether the absence of robust restructuring in these animals accounts for the absence of spontaneous seizures remains to be determined.

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Figures and Tables

Figure 2.1: Confocal maximum projections of GFP-expressing hippocampal dentate granule cells from Thy1-GFP mice. The endogenous GFP signal was enhanced by immunostaining for

GFP. Sections from either control (A) or kindled (B) animals are qualitatively similar. Scale bar,

250 µm.

Figure 2.2: One day after the last kindling-evoked seizure, there was a significant increase in granule cell layer thickness in kindled animals (B) in comparison to control (A) animals. There was also a trend towards increased somata area in kindled animals. Quantification of granule cell layer thickness and somata area are shown graphically in (C) and (D). * t-test, p<0.05. Scale bar for (A) and (B), 20m. Note: mossy cells axons, also labeled with GFP in the Thy1-GFP line, are present in the molecular layer of both the control and kindled animals (A & B, examples marked by blue arrowheads).

Figure 2.3: Images shown in (A) are confocal maximum projections showing segments of GFP- expressing granule cell apical dendrites in the inner (IML) and outer (OML) molecular layers.

One day after the last kindling-evoked seizure, spine density in both regions was significantly decreased in the kindled mice compared to control animals. Scale bar, 5 µm. The difference is depicted graphically below. * t-test, p<0.05. (B) Correlation between inner and outer molecular layer spine density for each animal in the study (kindled, red dots; control, green squares).

Figure 2.4: Proximal axon cross sectional area was quantified using Neurolucida software by calculating the volume of the first ≈10 µm of axon and dividing by the length measured. A confocal maximum projection of a sample neuron is shown in A, with the neurolucida tracing of the cell shown in B. One day after the last kindling-evoked seizure, the cross sectional area of the proximal axon was significantly increased in kindled mice (D) relative to controls (C).

Arrows in each image delineate the portion of the proximal axon that was quantified. Bar graphs

(E) show means ± SEM for each group. * t-test, p<0.05. A= Cross sectional area of the axon, V=

Volume of the axon, L=Measured length of the axon segment. All scale bars, 5 µm.

Figure 2.5: Confocal maximal projections showing GFP immunostaining in the hippocampal dentate gyrus of control and kindled mice one month after the last kindling-evoked seizure. The gross morphology of the dentate was similar between groups. Scale bar, 250 m.

Figure 2.6: Confocal maximum projections showing GFP-expressing granule cells within the granule cell body layer. The thickness of the cell body layer was similar between control (A) and kindled (B) animals one month after the last kindling-evoked seizure. Granule cell somata area was also similar between groups. Quantification of granule cell layer thickness and somata area are shown in the bar graphs in (C) and (D). Scale bar, 10m. Note: mossy cells axons, also labeled with GFP in the Thy1-GFP line, are present in the molecular layer of both the control and kindled animals (A & B, examples marked by blue arrowheads).

Figure 2.7: Images are confocal maximum projections showing segments of GFP-expressing granule cell apical dendrites in the inner (IML) and outer (OML) molecular layers. One month after the last kindling-evoked seizure, spine density in both regions was statistically similar between control and kindled mice. Bar graphs show means ± SEM for each group. Scale bar, 5

µm.

Figure 2.8: One month after the last kindling-evoked seizure, the cross sectional area of granule cell proximal axons was statistically indistinguishable from control values. Arrows delineate the portion of the proximal axon that was quantified. Bar graphs show means ± SEM for each group. Scale bar, 5 µm.

Table 2.1: Additional parameters compared between control and kindled mice one day after the last evoked seizure.

Table 2.2: Additional parameters compared between control and kindled mice one month after the last evoked seizure.

Chapter 3:

Clonal analysis of newborn hippocampal dentate granule cell proliferation and development in temporal lobe epilepsy

Shatrunjai P. Singh, Amen An, John J. McAuliffe and Steve C. Danzer

(Submitted to eNeuro, August 2015)

Abstract

Hippocampal dentate granule cells are among the few neuronal cell types generated throughout adult life in mammals. In the normal brain, new granule cells are generated from progenitors in the subgranular zone and integrate in a stereotypical fashion. During the development of epilepsy, DGC integration is profoundly altered. The new cells migrate to ectopic locations and develop misoriented “basal” dendrites. Although it has been established that these abnormal cells are newly-generated, it is not known whether they arise ubiquitously throughout the progenitor cell pool or are derived from a smaller number of “bad actor” progenitors. To explore this question, we conducted a clonal analysis in epileptic mice expressing the brainbow fluorescent protein reporter construct in dentate granule cell progenitors. Brain sections were rendered translucent so that entire hippocampi could be reconstructed and all fluorescently- labeled cells identified. Our findings reveal that a small number of progenitors produce the majority of ectopic cells in epileptic mice, indicating that either the affected progenitors or their local micro-environments have become pathological. By contrast, granule cells with “basal” dendrites were equally distributed among clonal groups. This indicates that these progenitors can produce normal cells and suggests that global factors sporadically disrupt the dendritic development of some new cells. Taken together, these findings strongly predict that distinct mechanisms regulate different aspects of granule cell pathology in epilepsy.

Introduction

Mammalian hippocampal dentate granule cells (DGCs) are generated throughout life from progenitor cells located in the subgranular zone, a proliferative region located between the granule cell body layer and the hilus. An important progenitor type in this region expresses the

Gli1 transcription factor, a Krüppel family zinc-finger protein activated by the sonic hedgehog- signal transduction cascade. Sonic hedgehog signaling is known to regulate cell proliferation

(Lai et al., 2003). Gli1-expressing type-1 progenitor cells are morphologically characterized by the presence of a radial process that projects into the dentate inner molecular layer. They exhibit the stem cell characteristics of self-renewal and multipotency and give rise to postnatally- generated dentate granule cells and astrocytes (Ahn and Joyner, 2005, Palma et al., 2005).

Adult-born DGCs are especially vulnerable to epileptogenic insults. Cells born in the weeks before and after an insult develop morphological and functional abnormalities (Ribak et al.,

2000, Scharfman et al., 2000, Walter et al., 2007, Danzer, 2012, Huusko et al., 2015). Following epileptogenic insults, adult-born DGCs populate the dentate hilus (hilar ectopic granule cells), a region they rarely occupy in normal animals (Scharfman et al., 2000). Afferent inputs to these ectopic DGC are abnormal, and the cells can exhibit atypical bursting properties (Scharfman et al., 2000, Jung et al., 2004, Zhan et al., 2010, Galanopoulou, 2013). DGC with basal dendrites are also common in the epileptic brain, a feature typically absent from non-epileptic rodent

DGCs. Basal dendrites are hypothesized to form recurrent circuits and promote hyperexcitability within the hippocampus (Ribak et al., 2000, Shapiro et al., 2008, Galanopoulou, 2013).

Although it is well established that abnormal DGC are derived from adult-progenitor cells, it is not known whether all progenitors contribute equally to the production of abnormal cells or whether distinct subsets of progenitors preferentially produce them. This is an important question as it will provide novel insights into the mechanisms underlying aberrant granule cell accumulation. Equal participation among progenitors would suggest systemic changes in the factors regulating granule cell development, while unequal participation would suggest regional disruption of the neurogenic niche or intrinsic changes within the progenitor itself. Here, we utilized a conditional brainbow reporter line driven by an inducible Gli1-CreERT2 promotor to lineage-trace clones arising from Gli1-expressing type-1 cells, in the pilocarpine model of epilepsy. Brains were rendered translucent using a novel clearing agent, and hippocampi imaged in their entirety to identify and characterize groups of daughter cells, known as a “clonal cluster”, originating from a single, labeled progenitor.

Methods

Animals

All methods involving animals were approved by the Institutional Animal Care and Use

Committee of the Cincinnati Children’s Hospital Research Foundation and conform to NIH guidelines for the care and use of animals. For the present study, hemizygous Gli1-CreERT2 mice

(Ahn and Joyner, 2004, 2005) were crossed to mice homozygous for a Gt(ROSA)26Sortm1(CAG-

Brainbow2.1)Cle/J “brainbow” reporter construct (Cai et al., 2013) to generate double transgenic

Gli1-CreERT2::Brainbow mice. All animals were on a C57BL/6 background. A total of 17 double transgenic mice were used for the present study (5 control and 12 pilocarpine-treated).

Postnatal tamoxifen treatment of these mice restricts CreERT2 expression to Type-1 cells in the hippocampal subgranular zone (Ahn and Joyner, 2005, Murphy et al., 2011, Cai et al., 2013,

Galanopoulou, 2013). Tamoxifen induced activation of Cre recombinase causes random excision and/or inversion between multiple pairs of lox sites, leading to the expression of one of four possible different fluorescent proteins in progenitor cells and all their progeny (Livet et al.,

2007). To facilitate morphological analyses, only cells expressing the cytoplasmic red or yellow fluorescent proteins (RFP or YFP) were examined. Cells expressing cyan fluorescent protein

(CFP) were excluded because morphological details were poorly revealed by this membrane bound protein. GFP-expressing cells were not observed in any of the animals, consistent with prior work (Calzolari et al., 2015).

Tamoxifen induced cell labelling and pilocarpine treatment

To achieve sparse labelling of progenitor cells, mice were given three injections of tamoxifen

(250 mg/kg, s.c.) on alternate days during postnatal week seven (Fig. 3.1A). At eight weeks of

73 age, all mice received methyl scopolamine nitrate in sterile saline (1 mg/kg, s.c.) followed fifteen minutes later by either pilocarpine (420 mg/kg, s.c.; n=12) or saline solution (controls, n=5).

Animals were monitored behaviorally for seizures and the onset of status epilepticus (defined as continuous tonic-clonic seizures). Following three hours of status epilepticus mice were given two injections of diazepam, ten minutes apart (10 mg/kg, s.c.), to alleviate seizure activity. Mice were given sterile Ringers solution as needed to restore pretreatment body weight and were then returned to their home cages, where they were provided with food and water ad libitum. Mice were housed under a 14/10 hour light/dark cycle, which is standard practice in the CCHMC vivarium to optimize breeding (Fox J.G., 2007).

Tissue preparation and optical tissue clearing

At 16 weeks of age, animals were euthanized by i.p. injection of 100 mg/kg pentobarbital. The mice were perfused through the ascending aorta with ice-cold phosphate buffered saline (0.1M

PBS) containing 1 U/ml heparin for 30 seconds at 10 ml/min, immediately followed by a 2.5% paraformaldehyde and 4% sucrose solution in 0.1M PBS at 25oC for ten minutes. Brains were removed, dissected into left and a right hemispheres and post-fixed in the same solution overnight at 4°C. Brain hemispheres were cryoprotected in 10%, 20% and 30% sucrose in PBS for 24, 24 and 48 hours, respectively. The hemispheres were then frozen in isopentane cooled to

−23°C with dry ice and stored at −80°C until sectioning. Brain hemispheres were thawed in PBS and 300 µm coronal sections were cut on a tissue slicer (Campden Instruments, Lafayette, USA).

Sections were transferred to 24 multiwell tissue culture plates (Becton Dickinson, New Jersey,

USA), maintaining their septo-temporal order. Sections were incubated for optical clearing in

ScaleA2 for two weeks at 4°C (Hama et al., 2011). Either one or both hemispheres were analyzed as needed to generate at least five clones/animal for analysis.

Confocal Microscopy

ScaleA2 cleared hippocampal sections were imaged on a Nikon AIR GasAsP confocal system attached to a motorized Nikon Eclipse Ti inverted microscope (Nikon Instruments, New York,

USA). This system was used to capture 3-dimensional “image stacks” through the z-depth of the tissue at 1 μm steps using a 10X Plan Apo λ objective (NA=0.25) at 1X optical zoom (field size

1024 x 1024 pixels, 1.23 pixels/µm). These 10X image stacks were used to identify clonal clusters, defined here as cells expressing the same fluorophore and contained within a 150 μm radius of the clone center. Identified clonal clusters were then imaged using 60X Plan Apo IR

DIC- Water Immersion objective (NA=1.3) at 1X optical zoom (field size 1024 x 1024 pixels,

0.31 pixels/um). All cells selected for analysis were brightly labeled with RFP or YFP and had their somas fully contained within the tissue section. The investigator was blind to treatment group during all image collection and data analysis.

3-dimensional hippocampal reconstructions

Confocal z-series image stacks were converted into 8 bit RGB tiff files, and Reconstruct

Software (John C. Fiala, the National Institutes of Health) (Lu et al., 2009) was used to septo- temporally align sections (10X images) for each hippocampus (Fig. 3.1B). Aligned z-stacks were imported into Neurolucida software for analysis (Version 11.01, Microbrightfield Inc.,Williston,

VT). Borders of the granule cell body layer were traced at z-intervals of 100 μm to recreate the

75 whole hippocampus. Three dimensional, whole-hippocampus reconstructions were created using the 3D visualization feature in Neurolucida at 300 frames/second (Fig. 3.1B; movie 1 and 2).

Morphological classification

Higher magnification images (60X) were used to categorize cells within each cluster as follows:

1) Type-1 cell, with a small cell body located in the subgranular zone and a single, radial process that projects through the granule cell layer and terminates in the inner molecular layer. 2)

Type-2 cell (transient amplifying cell), with a cell body located in the subgranular zone and short, aspiny processes projecting parallel to the plane of the granule cell body layer. 3)

Immature granule cells, with a cell body in the granule cell body layer and aspiny dendrites that project radially through the granule cell body layer, but terminate prior to reaching the hippocampal fissure (typically with growth cones at the tips). These cells occasionally possessed short, aspiny basal dendrites. 4) Normal mature granule cells, defined as cells with their somas located in the granule cell body layer and spine-coated dendrites projecting to the hippocampal fissure. 5) Hilar ectopic granule cells, with spiny dendrites and a cell body located in the hilus

(at least two cell body distances, ≈20 μm, away from the granule cell layer-hilar border). 6)

Mature granule cells with basal dendrites, possessing all the feature of normal mature granule cells (see 4), but with at least one dendrite originating from the hilar side of the soma (i.e. arising from a region below the midline of the soma). Cells with basal dendrites projecting into either the dentate hilus, or the dentate molecular layer (recurrent basal dendrites), were included in this measure. Further, only granule cells with clearly visible axons were scored for basal dendrites.

Basal dendrites are often thin and difficult to visualize in deeper regions of the tissue, so this criteria ensured that a basal dendrite would be detected if present. 7) Astrocytes were defined as

76 cells with a small cell body located anywhere within the dentate gyrus possessing numerous thin, aspiny process projecting outwards in a stellate fashion.

Statistical Analysis

Microsoft SQL Server (version 2012) was used to query the dataset for different clone compositions, and statistical analysis was performed using R (Version 0.98.109) or Sigma Plot

(version 12.5). Significance was determined using a two-tailed Student’s t-test for data that met assumptions of normality and equal variance. The Mann-Whitney rank sum test was used for non-normal data. Proportions were compared using z-tests. Values presented are means ± SEM or medians [range], as appropriate.

The statistical analysis for the frequency/distribution of ectopic cells and basal dendrites was performed using the binomial distribution (to compute probabilities of combinatorial events).

The experiment-wise error was conservatively set at 0.001 (Cumming, 2010). Corrections for multiple comparisons were done using a Bonferroni correction. For clusters containing ectopic cells, the resultant p-value for significance for the pilocarpine treated animals was calculated to be 4.27x10-6 (0.001/234). Similarly, for clusters containing DGCs with a basal dendrites, the probability of a single trial success was 0.0541 and the critical p-value was calculated to be

5.20x10-6 (0.001/192).

Figure Preparation

Maximum projections from z-series stacks were prepared using NIS-Elements Ar Microscope

Imaging Software (version 4.0). Contrast, brightness, montage adjustments and figure

77 preparation were done using Adobe Photoshop CS5 (version 12.0). Identical filtering and adjustments to brightness and contrast were done for images meant for comparison. Tableau

(version 8.0) and Microsoft Excel (version 2013) were used to create graphs, tables and visualizations.

In vivo lineage-tracing of individual Gli1-expressing progenitor cells in the adult mouse hippocampus

To study the proliferative activity of a cohort of Gli1-expressing granule cell progenitors in

T2 epilepsy, we treated double transgenic Gli1-CreER ::Brainbow reporter mice with tamoxifen at post-natal week seven to lineage-trace these cells. A small cohort of animals was perfused two days later, revealing an average of two type-1 cells per 300 μm hippocampal coronal section

(Fig. 3.S1), an optimal labeling sparsity for identifying individual clones (Bonaguidi et al.,

2011). Gli1 expression has been shown to exclusively label multipotent type-1 stem cells that can give rise to type-2 stem cells and other differentiated progeny (Encinas et al., 2011). Animals in the main study groups received either saline or pilocarpine one week after tamoxifen treatment

(Fig. 3.1A). Pilocarpine induces acute status epilepticus and the subsequent development of epilepsy a few weeks later (Galanopoulou, 2013, Harty et al., 2013). Animals were killed two months after pilocarpine treatment – when spontaneous seizures are typically frequent (Castro et al., 2012, Galanopoulou, 2013). Hippocampi were imaged in their entirety to identify all fluorescently-labeled cells. Brainbow fluorophore expression was strictly tamoxifen dependent and was limited to dentate granule cells, glial cells and sub-granular zone progenitor cells (Fig.

3.1C). Quantification of the number of clonal clusters per hippocampi revealed that status epilepticus did not significantly alter the number of YFP or RFP-expressing progenitors, although a slight trend towards increased numbers of clusters was evident in the epileptic mice

(control, n=6 hippocampi from 5 mice, 8.5±2.94 clusters/hippocampus; epileptic, n=14 hippocampi from 12 mice, 17.0±2.88 clusters/hippocampus; t-test, p = 0.095). Since the experimental design ensured equivalent numbers of labeled progenitors in each group at the time of pilocarpine treatment, the trend towards increased clusters in epileptic animals suggests a survival effect. Apoptosis of quiescent progenitor cells or entire clonal groups in control animals would reduce the number of clonal clusters.

Status epilepticus doubles the average number of cells per clonal cluster

Increased hippocampal neurogenesis and cell survival have been consistently demonstrated in epilepsy models (Bengzon et al., 1997, Parent et al., 1997, Gray and Sundstrom, 1998, Parent et al., 1998). The present work revealed that this increase is likely due to increased proliferation among individual progenitors and/or reduced apoptosis of their progeny. Specifically, epileptic mice exhibited increased numbers of YFP and RFP-labeled cells relative to controls (controls, n=6 hippocampi from 5 mice, 12.5 [4-118] cells/hippocampus; epileptic, n=14 hippocampi from

12 mice, 68.5 [6 to 262] cells/hippocampus; Mann-Whitney test, p = 0.035). This increase in the total number of labeled cells/hippocampus was associated with a doubling in median cluster size, from 2 cells/cluster in controls (n = 51 clusters, range 1 to 19) to 4 cells in epileptic mice (n =

238 clusters, range 1-24; Mann-Whitney test, p = 0.007; Fig. 3.1D).

Status epilepticus promotes terminal differentiation of hippocampal progenitor cells

While neurogenesis is increased after an acute epileptogenic injury, it has been shown to decrease in chronic epilepsy (Hattiangady et al., 2004, Danzer, 2012). A growing literature

79 demonstrates that progenitor cell pools can be depleted as progenitors proceed through multiple rounds of division, ultimately leading to terminal differentiation (Ledergerber et al., 2006).

Reduced neurogenesis in chronic epilepsy, therefore, could be a direct consequence of increased progenitor cell activity early in the disease.

To assess whether this might be the case, we used morphological criteria to classify cells as type-1 progenitors, type-2 progenitors, immature granule cells, mature granule cells or astrocytes

(Fig. 3.2A). This lineage analysis revealed a significant reduction in the progenitor cell pool in epileptic animals. Compared to clusters from control animals, clusters from epileptic animals exhibited an 85% reduction in type-1 cells (Fig 3.2A, Table 3.1; z-test, p<0.001) and a 49% reduction in type-2 cells (Fig. 3.2A, Table 3.1; z-test, p=0.014). Overall there was a 63% reduction in the number of clusters containing either type-1 or type-2 progenitors in the epileptic animals (Fig. 3.2C, Table 3.2; z-test, p<0.001).

We also examined this change using the more biologically relevant approach of categorizing clusters as quiescent, actively self-renewing or fully-differentiated. Quiescent clusters were defined as having only one type-1 cell, actively self-renewing clusters were defined as having one type-1 cell and any other cell type and fully differentiated clusters contained no type-1 cells.

We observed an 82% decrease in the quiescent cluster pool (Fig. 3.2D, Table 3.3; z-test, p=0.005) and a 68% decrease in self-renewing cluster pool in epileptic animals relative to controls (Fig. 3.2D, Table 3.3; z-test, p=0.047). Symmetric self-renewing clusters (composed of two type-1 cells) decreased from 6% in control animals to nil in epileptic animals (Table 3.3).

This significant shift from quiescent and self-renewing progenitors in epileptic animals was mirrored by a 50% increase in the number of completely differentiated clusters, defined as clusters composed only of neurons or glial cells (Fig. 3.2D, Table 3.3; z-test, p<0.001). This increase in differentiated clusters was paralleled by a significant increase in the percentage of mature cells in each cluster (Fig. 3.2A; Table 3.1; z-test, p<0.001).

Ectopic DGCs appear in clonal clusters in which majority of the cells are ectopic

Hilar ectopic granule cells are a common pathology seen in temporal lobe epilepsy. These neurons are newly-generated, arising after an epileptogenic brain insult (Walter et al., 2007) and are implicated in the development of epilepsy (Galanopoulou, 2013). The percentage of newborn cells found in the hilar region of epileptic animals was significantly increased relative to controls

(Fig. 3.3A; Epileptic, 64 of 1240 DGCs (5.16%); Control, 1 out of 192 cells (0.52%); p=0.007, z-test), consistent with previous studies (Parent et al., 2006). Clonal analysis revealed that ectopic cells were concentrated within a small number of clusters. Specifically, the 64 identified ectopic cells were contained within only 15 clusters (Fig. 3.3B). Within these clusters, 77.10% of all cells were ectopic (Fig. 3.3B).

The probability of finding a set number of ectopic cells, S, in a cluster containing a total of N cells was computed for all values of S from 1 to N (e.g. when S=N, 100% of the cells in the cluster are ectopic). The minimum number of ectopic cells S, at which the p-value reaches the target p-value for significance (p=4.27x10-6; see methods for calculations) was determined for

81 every cluster and compared to the observed number of ectopic cells. We found that six of the 15 clusters with ectopic cells exceeded the threshold p-value for significance (binomial p-value <

4.27x10-6; Fig 3.3B). The binomial probability of finding six of 234 clusters exceeding this value is statistically minute (less than 1.33x10-21). Restated, finding one cluster that exceeded the value would indicate that ectopic cells are not randomly distributed, and in the present study six such clusters were observed. These results provide overwhelming evidence that the accumulation of ectopic cells in certain clusters is not a random event.

DGCs with basal dendrites occur in clonal clusters in which majority of the cells do not have a basal dendrite

Another common pathology observed in the epileptic brain is the presence of dentate granule cells with basal dendrites (Spigelman et al., 1998, Buckmaster and Dudek, 1999). In the current study, 5.42% (43 of 792 cells) of granule cells from epileptic animals possessed basal dendrites

(41 of 43 of these basal dendrites were hilar projecting). By contrast, cells with basal dendrites were rare in control animals (1 of 112 cells; p=0.048, z-test). The 43 basal dendrite-possessing granule cells from epileptic mice were distributed among 31 of 192 clonal clusters (Fig. 3.4A and 3.4B). Among these 31 clusters that contained a cell with a basal dendrite, 22 had only a single basal dendrite-possessing cell, 5 had two, 2 had three and 1 had five cells with basal dendrites (Fig. 3.4B). Using the significance criterion described in statistical analysis (similar to that used for ectopic cells), there were no clusters that contained a significant number of DGCs with basal dendrite (Fig. 3.4B). Indeed, even the biggest cluster, containing 17 DGCs with five harboring a basal dendrite, failed to reach significance even if the experiment-wise alpha is relaxed to 0.05 (p= 0.054; threshold p value for α=0.001, 5.20x10-6; for α=0.05, 2.60x10-4).

Discussion

Abnormal hippocampal granule cells are common in animal models of temporal lobe epilepsy

(Rolando and Taylor, 2014) and in tissue from patients with the disease (Sutula et al., 1988,

Parent et al., 2006). Prior studies have established that many of these abnormal cells are adult- generated (Parent et al., 1997, Danzer, 2012). In the present study, we queried whether two important abnormalities – ectopic DGCs and DGCs with basal dendrites – are derived with equal likelihood from the entire progenitor pool, or whether they are preferentially produced by a subset of progenitors. We found that ectopic granule cells were highly concentrated within distinct clonal clusters – many containing only ectopic cells. By contrast, cells with basal dendrites were relatively evenly distributed among clones (Fig. 3.5). These findings strongly suggest the existence of distinct mechanisms regulating ectopic cell migration and basal dendrite formation.

A second key finding provides new insights into the bimodal changes in neurogenesis rates observed in epileptic animals. Acutely, neurogenesis increases following an epileptogenic insult, however, animals with chronic epilepsy exhibit reduced neurogenesis. Depletion of the progenitor pool, potentially as a direct consequence of the early increase in neurogenesis, has been hypothesized to account for these changes (Hattiangady et al., 2004). Our data provide direct evidence that this is occurring, with a doubling in the number of daughter cells/clone and a corresponding decrease in the percentage of actively dividing and self-renewing clones (Fig.

3.5).

Clonal analysis of adult SGZ neurogenesis in an epileptic brain

In the present study we employed an in vivo, genetic, sparse-labeling approach to mark stem cells for lineage-tracing. This approach has been used previously to study neural stem cell behavior in the sub-ventricular (Calzolari et al., 2015) and sub-granular (Suh et al., 2007,

Bonaguidi et al., 2011) proliferative zones in healthy animals. We combined this approach with recently developed tissue clearing protocols, allowing us to generate 3-dimensional reconstructions of the entire epileptic rodent hippocampus, and the Brainbow reporter line, allowing us to separate clonal groups by fluorochrome expression. Poor recombination with the brainbow reporter limited our study to two colors, rather than the potential 7 colors evident in other tissues (Livet et al., 2007, Cai et al., 2013). Even with two colors, however, the strategy provided sufficient spatial resolution for the study.

An additional advantage of the genetic approach is that it avoids disturbing the target tissue with direct brain injections, as is needed for retroviral strategies (Hope and Bhatia, 2011, Ming and

Song, 2011). Retrovirus also targets Sox2+ Type-2 progenitor cells (Suh et al., 2007), while the

Gli1 driver used here targets the parent Type-1 cells (Cai et al., 2013), so the different strategies provide complimentary data. One limitation of the present approach was the inability to use immunostaining to identify labeled cells; however the morphological classification of the different stages of dentate granule cell development is well documented and provides an accurate replication of immunophenotyping (Zhao et al., 2006, Hui Yin et al., 2013, Lenck-Santini, 2013).

Localized regulation of ectopic granule cell formation

Ectopic granule cells have been observed in a number of different epilepsy models. They are hyper-excitable (Scharfman et al., 2000), have atypical connections within the hippocampal network (Scharfman and Pierce, 2012) and their numbers correlate with the severity and duration of seizures (Galanopoulou, 2013). The mechanisms underlying ectopic cell migration, however, are unknown. A mechanistic understanding would provide new insights into the development of therapeutic strategies for epilepsy. A putative mechanism that could account for the current results is mislocation of progenitor cells from sub-granule zone to the hilus during epileptogenesis (Parent et al., 2006). Alternatively, epileptogenic stimuli could activate

“dormant” progenitors trapped in the hilus during development (Gaarskjaer and Laurberg, 1983,

Scharfman et al., 2007). If one further presumes that daughter cells produced by hilar progenitors would not have access to the necessary cues directing them to migrate into the granule cell layer, the presence of entirely ectopic clonal groups could be accounted for. Alternatively, seizures might lead to the localized disruption of migratory cues, like reelin (Teixeira et al., 2012).

Progenitor cells active in regions with disrupted cues would produce daughter cells that fail to migrate correctly, while progenitors in regions with intact cues would produce normal offspring.

In support of this possibility, Parent and colleagues observed trains of cells migrating on glial scaffolds into the hilus after seizures (Parent et al., 2006), suggesting that localized changes in non-neuronal cells might play a role. Additional studies will be needed to distinguish among these possibilities.

An additional key finding of the present study is that clonal groups with ectopic cells tended to be made up of entirely ectopic cells. This observation is consistent with a Markov chain model

(Lange, 2003), in which there are two states a progenitor cell can assume; progenitors in state 1 give rise to DGCs correctly located in the cell body layer, whereas progenitors in state 2 give rise to ectopic DGCs. For the model, we assumed that at every mitotic cycle cells could either stay in the same state, or transition between states. The transition matrix specifies the probabilities of these transitions (Fig. 3.S2). Our data show that the probability of a progenitor switching states is very low, whereas the probability that a progenitor will remain in the same state is close to 100

%, implying that cells that begin producing ectopic cells will continue to do so, and cells that initially produce normal cells also will continue to do so. Transitions between states appear to be very rare (only 2 out of 234; Fig. 3.S2).

Temporal/global regulation of basal dendrite formation

Basal dendrites were distributed close to the predicted ratio among clonal clusters, and tended to be present in clusters in which the majority of cells lacked this feature. Notably, progenitors that produce DGCs with basal dendrites can also produce morphologically normal DGCs. These observations suggest a mechanism that could impact the development of daughter cells from any progenitor, while also leaving most daughter cells unaffected. Such a mechanism might affect the entire hippocampus, but only some of the time. Seizure activity clearly meets these criteria, as seizures are episodic in nature, but affect the entire brain when they occur.

The idea that seizure activity might drive basal dendrite formation is supported by the work of

Nakahara and colleagues (Nakahara et al., 2009). They demonstrated that increasing neuronal activity in hippocampal slice cultures stabilized the normally transient basal dendrites typically present on immature granule cells. Under low activity conditions, developing granule cells

86 briefly possess basal dendrites around one-two weeks after their birth, but subsequently reabsorb these processes as they mature. Increasing neuronal activity, however, allowed these processes to persist through cell maturity, perhaps through a neurotrophin dependent mechanism (Danzer et al., 2002). Whether a similar process occurs in vivo remains to be determined, but the present findings are consistent with the idea that episodic increases in activity, including seizures, might similarly stabilize granule cell basal dendrites – but only among granule cells that happen to be at this particular developmental stage at the time of the event.

Depletion of the granule cell progenitor pool

In the current study we found a decrease in progenitor cell numbers and an increase in differentiated cells. A chronic decrease in neurogenesis has been observed previously in epileptic animals (Hattiangady et al., 2004). Reduced neurogenesis could be the result of increased progenitor cell quiescence, loss of functional progenitor cell division, decreased survival of daughter cells or an overall loss of progenitors. Our results provide evidence for the division- coupled loss of type-1 progenitor cells as a key contributor to the chronic decline in neurogenesis. We observed a decrease in quiescent and actively self-renewing progenitors, but an increase in fully differentiated clonal groups. Activation and terminal differentiation of quiescent progenitors would account for these observations. Indeed, Encinas and colleagues

(Encinas et al., 2011) observed a similar loss of stem cells during the normal aging process in the mouse hippocampus. Using a genetic label they showed that type-1 cells act as ‘disposable stem cells’; Once activated they tend to terminally differentiate rather than continuing to cycle.

Therefore, epileptic stimuli might accelerate the age-related loss of progenitor cells from the dentate. Amanda and colleagues observed a similar reduction in the progenitor cells pool

87 following intrahippocampal injection of the convulsant kainic acid. In contrast to the present results, however, they observed terminal differentiation of type-1 cells into astrocytes (Sierra et al., 2015), rather than mature granule cells as described here. This difference likely reflects the very different pathological responses, and impacts on neurogenesis, between the two epilepsy models (Murphy et al., 2012).

Concluding Remarks

Our results strongly suggest different mechanistic origins for ectopic DGCs and DGCs with basal dendrites. Ectopic DGCs are highly localized to specific clonal clusters, implicating the parent progenitor cell or the neurogenic niche in which the progenitor resides. By contrast, basal dendrites appeared to be randomly distributed among clones, suggesting transient changes acting throughout the hippocampus drive this pathology. Both abnormalities are implicated in the development of epilepsy and associated comorbidities, and separate therapeutic strategies will likely be required to mitigate these different abnormalities.

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Figures and Tables

Figure 3.1: (A) Timeline depicting the experimental paradigm used. To induce fluorophore expression, mice were injected with tamoxifen three times during postnatal week 7 and subsequently underwent either a pilocarpine or saline treatment on postnatal week 8. Mice were sacrificed on postnatal week 16. (B) Example of a 3-dimensional reconstruction of the mouse hippocampus. The Scale cleared 300 µm sections were imaged, aligned and reconstructed into a

3-dimensional reconstruction of the hippocampus with single cell resolution. (C.1) Brainbow fluorophore expression was absent from animals not treated with tamoxifen. (C.2). Clonal clusters were observed in both control (C.3) and pilocarpine (C.4) treated animals. (D) The number of cells per cluster increased in pilocarpine treated animals. Scale bar for B (3D reconstruction) = 600 µm; C.1 and C.2 = 250 µm; C.3 and C.4= 200 µm.

Figure 3.2: (A) Cells present in clonal clusters were classified based on morphology (see methods) into either (A.1) type-1 progenitor cells, (A.2) type-2 progenitor cells, (A.3) immature

granule cells, (A.4) mature granule cells with (A.5) spiny apical dendrites or (A.6) glial cells.

(B) Graphs show the composition of cell types in clonal clusters from control and epileptic

animals. There was a decrease in the number of type-1 cells and an increase in the number of

mature cells in epileptic animals. (C) Graphs show the decrease in the number of clusters with

progenitor cells and the increase in clusters without progenitor cells in an epileptic brain. (D)

Clonal clusters were classified as either quiescent (containing only type-1 cells), actively self- renewing clusters (composed of a type-1 stem cell along with other stem cells or a mature cell or

a glia) or differentiated (composed of only mature neurons or glia). We observed a decrease in

quiescent and actively self-renewing clusters and an increase in differentiated clusters in the epileptic animals. R= type-1 stem cell, N= mature DGC, G=glial cell. Scale bars for A = 50 µm.

Figure 3.3: (A) Ectopic dentate granule cells are derived from a small number of clonal clusters.

Shown is an image of clonal cluster composed entirely of hilar ectopic dentate granule cells

(higher magnification image in purple inset). (B) Quantification of all the clusters which contained ectopic cells. Additionally, for comparison, shown are some randomly selected clusters

that contain no ectopic cells. Orange bars show the total number of cells in the cluster whereas

the blue bars show the number of ectopic cells. Ectopic DGCs tended to occur in clusters in which majority of the cells are ectopic. GCL= granule cell layer, H= hilus. Scale bar for A =150

µm, Scale bar for inset of A= 40 µm.

Figure 3.4: (A) Dentate granule cells with basal dendrites arise from diverse of clonal clusters.

Shown is an example of a DGC with basal dendrites (white arrows) within a clonal cluster. The axon is denoted by the arrowhead (B) Quantification of all the clusters which contain DGCs with basal dendrites. Additionally, for comparison, shown are some randomly selected clusters that contain only normal DGCs without basal dendrites. The orange bar represents the total number of cells in different clusters whereas the blue bar represents the number of DGCs with basal dendrites within these clusters. DGCs with basal dendrites tended to be spread out across multiple clusters (in contrast to ectopic DGCs). GCL= granule cell layer, H= hilus,

ML=molecular later. Scale bar for A = 10 µm.

Figure 3.5: Summary of the key findings of the study. The first panel shows five, type-1 progenitor cells labeled with either RFP (red) or YFP (yellow), numbered from 1 to 5. (A) Under control conditions most of the type-1 cells remain quiescent (progenitors 1, 2, 4 and 5 remain quiescent) and (B) however, a small proportion of type-1 cells will enter the mitotic cycle to give rise to differentiated cells (only progenitor 3 undergoes terminal differentiation). After epileptogenesis, three key changes occur: (i) The number of clusters containing Type-1 cells decreases in epileptic animals relative to controls, and the number of clusters composed of differentiated DGCs and astrocytes increases (progenitors 1, 2, 4 and 5 terminally differentiate);

(ii) Progenitor cells either produce all correctly located offspring, or ectopic offspring

(progenitor 5 gives rise to a cluster composed entirely of ectopic DGCs) and (iii) progenitors that produce correctly located offspring occasionally produce cells with a basal dendrite, but mostly produce cells with normal dendrites (progenitors 1 and 2 give rise to clusters which contain

DGCs with basal dendrites and normal DGCs).

100

Supplementary Figure 3.S1: Quantifying the number of type-1 cells labelled for different.

concentrations of tamoxifen. Gli1-CreERT2::Brainbow mice were injected with different

concertation of tamoxifen, three times in postnatal week 7 and were sacrificed 2 days after the last injection. The number of RFP or YFP labelled type-1 cells per 300µm section of the dentate

are shown in the graph.

101

Supplementary Figure 3.S2: Markov Model: Application to the generation of clusters with

all/no/or mixed normal and ectopic cells. The first part of the figure shows a simple two state

Markov process. The system in state1 can remain there at the next step or transition to state2; the probability of remaining in the same state, or transitioning to the other state, is governed by

the transition matrix. In the example shown, the probability of remaining in the same state

(diagonal elements of matrix) is 0.99. The likelihood of transition to the other state is 0.01. At

each step the probability of moving from one state to another is governed by the transition

probabilities for the current state; the system has no “memory” of past events.

The second part demonstrates how mixed clusters can be generated by the Markov process. The

system starts with a progenitor in the normal location. It generates two cells, one terminally differentiated (Palesh et al.) and the other replicating (bright). The process continues for 4 steps.

At step 3, the low probability event of a state1 to state2 transition occurs resulting in the

102

formation of ectopic cells. The values shown in the transition matrix are representative of the

data. The probability of a 13-step process generating no ectopic cells for the matrix shown is

0.886. The p-value for number of large clusters with no ectopic cells, given a single trial success

rate of 0.886, is 0.044 (non-significant without Bonferonni correction).

103

Control Epileptic % change from control p-value

(% of 192 total (% of 1240 total ((control- (z-test)

cells) cells) Epileptic)/control)*100

Type-1 stem 11.98 1.69 85.89315526 1.00E-

cells 03

Type-2 stem 9.38 4.76 49.25373134 0.014

cells

Immature 9.9 2.42 75.55555556 1.00E-

cells 03

Mature Cells 65.63 85.56 -30.36721012 1.00E-

Glial Cells 3.13 5.56 -77.63578275 0.216

Ectopic cells 0.52083 5.08 -875.3662423 8.00E-

Table 3.1: The composition of clusters from control and status epilepticus animals. The

percentage change in composition and p-values after a z-proportions test.

104

Cluster type Control (n=51) Epileptic (n=238) % change p-value

Clusters with stem cell 47.05882353 17.22689 63.39286 1.00E-03

Clusters without stem cells 52.94117647 82.77311 -56.3492 1.00E-03

Table 3.2: The proportion of clusters with and without stem cells from control and status

epilepticus animals. The percentage change in composition and p-values after a z-proportions

test.

105

Cluster type Control % Epileptic % % Change

(n=51) (n=238) (control-Epileptic/control)*100

Total Clusters 51 238

R 27.4509804 5.04201681 81.63265

R+R 5.88235294 0 100

R+N 3.92156863 2.5210084 35.71429

R+G 0 1.2605042 NA

R+N+G 1.96078431 0 100

R+R/N/G 11.7647059 3.78151261 67.85714

N/G 60.7843137 91.1764706 -50

Table 3.3: The proportion of clusters with different self-renewal potential from control and

status epilepticus animals. Shown are clusters composed of either only a single Type-1 progenitor cells (R) or two type-1 progenitor cells (R+R), Type-1 stem cell with a mature DGC,

Type-1 progenitor cell with a glial cell (R+G), Type-1 progenitor cell with a mature DGC and a glial cell (R+N+G), Type-1 progenitor cell with another cell which can be either a type-1 cell or

a mature DGC or a glial cell (R+R/N/G), and clusters composed of only mature DGCs or glial cells (N/G). The percentage change in composition and p-values after a z-proportions test. . R=

type-1 progenitor cell, N= mature DGC, G=glial cell.

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Chapter 4: Discussion

107

Summary

In this dissertation I described the epileptogenic morphological changes and their origins, before and after the occurrence of SRS. The early kindling study described in chapter 2, defined the transient nature of changes seen in the absence of SRS. These changes appear to be homeostatic and self-correcting. It also augments the importance of the classical DGC abnormalities, like ectopic DGCs and basal dendrites, implicated in the generation of epilepsy. In the clonal analysis study, I study the origins of these classical abnormalities and describe the heterogeneity present within the progenitor pool that generates them.

Together, these results reveal the transient nature of alterations in the dentate morphology during the earliest phases of epileptogenesis and describes the genesis of abnormal DGCs observed in the later stages. In the current section, I discuss some ideas and hypothesis directly or indirectly supported by the data presented in the last two chapters.

The transient changes produced by early kindling are features of hyperexcitability

In Chapter 2, I described the transient nature of the changes produced by the early kindling protocol, which specifically models the early stages of epileptogenesis. In this study, I observed significant differences in dendritic spine densities, granule cell soma area, width of the granule cell body layer, and the volume of the axon initial segment one day following the last seizure, but all of these changes normalized to control levels a month later.

108

An interpretation of these results is to classify the observed transient morphological alterations as features of hyperexcitability rather than the harbingers of TLE. These pathologies were transient and normalized to pre-kindling levels after the gradual decrease in excitability in the brain.

Although these changes also occur in models with SRS, the results show that they alone do not produce the circuitry changes required for the generation of spontaneous seizures. In fact, these fleeting changes might actually be antiepileptogenic as has been suggested by some groups.

Another factor that should be considered when discussing these results is that in the Thy1-GFP mouse model, a majority of the cells analyzed were born prenatally, with postnatal neurogenesis contributing only to a small fraction of labelled cells. Mature DGCs are robust and resist/repair minor damages by hyperexcitability and thus may not show extensive damage post kindling. The subthreshold kindling insult used in my experiments did not affect neurogenesis enough to produce basal dendrites supporting a central role of disrupted neurogenesis in the genesis of

SRS.

The battle ensues after the initial epileptic insult: A tussle between pro and antiepileptogenic morphological changes

The kindling study also highlighted the presence of homeostatic processes in the dentate which are activated after the primary insult and may counteract the increased excitability.

Several lines of evidence now show that acute changes, such as decreased spine density, loss of mossy cells, increased neurogenesis, and decreased DGC excitability, might actually be

109 antiepileptogenic. In a recent study, Tajeda et al. (2014) used a computational model to stimulate the epileptic dentate and found that even a small decrease in DGC spine density decreased hippocampal hyperexcitability considerably.

Similarly, other changes seen just after the primary insult, might also be antiepileptogenic. The increase in neurogenesis seen 24–72 hours after a seizure may also be responsible for producing hypoexcitable DGCs. Jakubs et al. (2006) found that newborn DGCs born in an epileptic environment are inherently less excitable and have a lower excitatory input. The authors suggested that increased neurogenesis might be an attempt to reestablish the dentate gate.

Increased neurogenesis also delays the onset of kindling-induced seizures (Auvergne et al., 2002) and prevents cell apoptosis (Young et al., 1999). On similar lines, Harvey and Sloviter (2005) saw an increase in c-Fos expression specifically in interneurons and not in mature DGCs, immediately after a seizure. These results points at a compensatory decrease in the activity of pre-existing mature DGCs under epileptic conditions.

This would also predict that blocking neurogenesis post-seizure would have a negative outcome on the progression of epilepsy. Recently, Iyengar and coworkers (2015) provided evidence for the same, when they observed an increase in seizure severity and duration by an irradiation- mediated blocking of neurogenesis, in a kainic acid model of epilepsy. All of these mechanisms seem to be aimed at reducing hyper-excitability, restoring the dentate gate, and preventing further damage within the hippocampus. Similar process might be induced after our early kindling protocol, thereby normalizing the damage at the later time point.

110

It is also plausible that there is a threshold for maximal damage (similar to maximal dentate activation hypothesis described in Chapter 1) that these mechanisms can counteract, after which proepileptogenic morphological changes associated with SRS (e.g., DGCs with basal dendrites and ectopic DGCs) are activated. After this threshold is breached, the initial antiepileptogenic damage control mechanisms might actually become proepileptogenic.

Are ectopic DGCs, MFS or DGCs with basal dendrites required for SRS?

The importance of morphological changes specifically associated with newborn cells

(basal dendrites and ectopic DGCs) in generating recurrent seizures has been highly debated. In the early kindling model, I did not observe the presence of DGCs with basal dendrites or ectopic

DGCs, as is typically seen in models with SRS. Previous studies have shown the presence of

MFS in milder kindling protocols similar to the one described in Chapter 2 (Sutula et al., 1988,

Cavazos et al., 1991). My results also favor the hypothesis that ectopic DGCs and basal-dendrite- harboring DGCs play a key role in the generation of SRS.

Hilar ectopic DGCs have been hypothesized to form an excitatory hub coordinating the development of seizures (Scharfman and Pierce, 2012), and computational models show that a small number of such hubs are sufficient to significantly lower the threshold for seizure initiation

(Morgan and Soltesz, 2008). The numbers of ectopic DGCs correlate with the duration and intensity of seizures (Hester and Danzer, 2013), and a reduction in ectopic DGCs is associated with a decrease in the number of seizures (Jung et al., 2004). In a recent study by Cho et al.

(2015), genetic ablation of newborn cells led to a reduction in hilar ectopic DGCs and also led to

111 a reduction in spontaneous seizures, further providing evidence for the central role that these cells play in generating seizures. However, definitive proof implicating ectopic DGCs in seizure generation and propagation is still missing (Scharfman et al., 2007). Experiments specifically targeting ectopic cells using an in-vitro optogenetic silencing approach, combined with templates to specifically expose only he hilus to the optogenetic stimuli, could theoretically be employed to determine the exact role of these hilar ectopic DGCs.

Granule cells with basal dendrites arise from immature granule cells which have failed to retract their basal dendrites after SE (Jones et al., 2003). Computational modelling (Morgan and Soltesz,

2008) and correlative data (Hester and Danzer, 2013) have suggested that these basal-dendrite- harboring DGCs play a central role in creating a recurrent excitatory loop which eventually leads to SRS. Similar to ectopic DGCs, the extent of their contribution to epileptogenesis still remains to be determined, though it is quite clear that they play a key role in the development of epilepsy.

In contrast to ectopic DGCs and basal dendrites, a growing body of evidence suggests that MFS might not be as critical for the development of epilepsy as was previously hypothesized (Sutula and Dudek, 2007). Buckmaster and Lew (2011) showed that rapamycin can block MFS, but does not affect the development of epilepsy. Studies from our own lab have also shown that MFS was not required for the development of epilepsy in a PTEN knockout model (Pun et al., 2012).

Thymidine-kinase-ganciclovir-mediated ablation of newborn cells in a pilocarpine model of epilepsy reduced SRS but had no effect on MFS, further dissociating this phenomenon from

112 epileptogenesis (Cho et al., 2015). Thus, MFS might be an effect and not a cause of epileptoformic activity.

The two-hit hypothesis for the development of SRS

Knudson’s two-hit hypothesis for cancer, i.e. two mutations (hits) to DNA are required for cancer progression (Knudson, 1971), has proved relevant to other disorders (Williams et al.,

2013, Catuzzi and Beck, 2014, Fiebich et al., 2014, Chan et al., 2015). According to this hypothesis, the primary hit does not result in the disease itself, as compensatory homeostatic mechanisms can repair this initial damage. A second hit, however, pushes the damage beyond the capabilities of the homeostatic systems, giving rise to the disease condition (Knudson, 1971).

A similar mechanism could also be applied to the development of SRS.

In the case of epileptogenesis, the first hit in the form of kindling, pilocarpine treatment, hypoxia, or traumatic brain injury induces hyperexcitability in the brain and thereby triggers the compensatory mechanisms described above. This hit itself, although not adequate enough to drive epilepsy, disrupts the development, migration, and integration of newborn DGCs.

Functional integration of these abnormal newborn DGCs provides the second hit, leading to the irreversible development of epilepsy. This hypothesis would suggest that therapeutic interventions should be aimed at preventing the second hit (i.e. the functional integration of cells born just before and after a seizure) to prevent the development of epilepsy. To design such interventions, it is important to understand the origins of these abnormal granule cells in detail, and my studies described in Chapter 3 shed light on this aspect.

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Origins of abnormal cells in epilepsy: The good, the bad, and the ectopic

As discussed in Chapter 3, I performed a clonal analysis to query the origins of abnormal newborn DGCs observed after epilepsy. I recreated the whole epileptic hippocampus and found that ectopic DGCs tend to be generated from a handful of bad actor progenitor cells. In contrast,

DGCs with basal dendrites arise from a larger cohort of progenitor cells. I also observed a dramatic loss of the stem cell pool and an increase in the number of mature DGCs within each clonal cluster. I also found that epilepsy hastened the terminal differentiation of the stem cell pool into mature DGCs and glia. As discussed previously, this might be a homeostatic mechanism to counter cell loss and normalize the increased excitability within the dentate.

During this process of increased neurogenesis, a large portion of progenitors occasionally give rise to DGCs with basal dendrites. In contrast, only a select few progenitors also give rise to the majority of ectopic DGCs found in epileptic animals. Thus, a differential response to the epileptic stimulus is observed between the progenitor type-1 cells.

This heterogeneity in response could be attributed to inherit differences within the progenitors or differences in the niches that surround them. Studies over the last decade now point to a mixture of both of these scenarios. In a recent study, Jhaveri et al. (2015) showed the presence of functionally different sub populations within the quiescent Nestin+ type-1 progenitor cells. These subpopulations were activated by either a GABA-dependent high-KCl depolarization or by a corticosterone-norepinephrine pathway. They were sensitive to different stimuli, invoked dissimilar molecular pathways, and even gave rise to different proportions of neuron/glia, implying a complex inherit heterogeneity within the hippocampal stem cell pool. Along similar lines, Lugert et al. (2010) have identified distinct types of stem cells which can switch between

114 active and inactive states based on the type of pathophysiological stimuli. Subsequently, studies have shown that the molecular players which form the stem cell niche play a bigger role in determining the fate of stem cells than was previously hypothesized. Notch, transforming growth factor- β (TGF-β), brain derived neurotrophic factor (BDNF), and even increased Mg++ have all been implicated in the loss of quiescence in progenitor cells under pathophysiological conditions

(Breunig et al., 2007, Kandasamy et al., 2010, Thakker-Varia et al., 2014, Kikuchihara et al.,

2015). Of these hypotheses, terminal stem cell differentiation due to the loss of notch signaling in the epileptic dentate, has been garnishing support from multiple studies.

Cell death and notch de-repression drives terminal differentiation in the stem cell pool

Notch signaling is a master regulator of neuronal cell fate decisions (Breunig et al., 2007,

Kopan and Ilagan, 2009, Pierfelice et al., 2011, Perdigoto and Bardin, 2013). It plays a diverse role in stem cells from different organ systems (Perdigoto and Bardin, 2013), sometimes promoting proliferation and terminal differentiation (e.g., fly interstitial stem cells) (Riccio et al.,

2008, Pellegrinet et al., 2011) and other times maintaining quiescence (e.g., mouse hematopoietic stem cells) (Milner et al., 1996, Lauret et al., 2004). During mammalian embryonic neurogenesis, notch is responsible for regulating stem cell self-renewal (Louvi and Artavanis-Tsakonas, 2006,

Ehm et al., 2010), stem cell survival (Androutsellis-Theotokis et al., 2006), suppressing pro- neural differentiation (Lutolf et al., 2002), and regulating type-2 cell migration (Lutolf et al.,

2002, Louvi and Artavanis-Tsakonas, 2006). After birth, it maintains the stemness of type-1 progenitors (Ehm et al., 2010) and also controls dendritic arborization (Breunig et al., 2007).

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Hes-5, a notch target gene, used as a readout for notch activity, exclusively labels type-1 cells in the nonepileptic dentate and possibly maintains the quiescence of these cells (Ishibashi et al.,

1994, Ishibashi et al., 1995, Lugert et al., 2010). After SE, Hes-5 expression is significantly reduced in the hippocampus (Elliott et al., 2001), and this loss of notch activity has been shown to increase proliferation/terminal differentiation in-vitro (Breunig et al., 2007). Thus, it could be hypothesized that cells in the SGZ, forming the stem cell niche, provide notch contact signaling and keep the type-1 cells in a quiescent state. Extensive hilar cell death and vascular remodelling observed after SE could result in the loss of this notch inhibition, leading to the proliferation and terminal differentiation of these progenitors as seen in our results. This dysregulated notch signaling could also be involved in the generation of ectopic hilar DGCs, as it is a direct regulator of reelin signaling (Hashimoto-Torii et al., 2008). However, experimental evidence validating the exact role of notch post-SE is still needed.

Inverted glial cells: An Ockham’s razor solution to the origin of ectopic cells

Although I have discussed multiple hypotheses regarding the origin of ectopic DGCs (see

Chapter 3 discussion), one observation might provide the simplest explanation. During the clonal analysis experiments, I frequently observed the presence of an inverted radial glial cells, which, although morphologically similar to a type-1 progenitor cell, was oriented upside-down and was located in the dentate hilus (Fig. 4.1). Immuno-characterization revealed that a proportion of these inverted glial cells expressed type-1 progenitor cell markers like nestin and GFAP (glial fibrillary acidic protein) but were negative for sox2 (type-2 cell marker). However, a large

116 proportion of these cells only expressed GFAP and not nestin, consistent with a pure glial phenotype. In one hippocampal slice, these inverted glial cells were seen dividing from type-1 cells. These unknown cells could possibly be the product of an aberrant type-1 cell symmetric division (again, due to the loss of notch signaling) followed by faulty migration into the hilus.

Notch signaling is required for reelin expression, and the loss of notch signaling has been shown to cause significant migration defects (Hashimoto-Torii et al., 2008). The presence of stem cell markers (nestin and GFAP positive) indicate that these cells may still hold potential for pluripotency and thus could give rise to DGCs within the hilus. This hypothesis could be tested using a similar clonal analysis at earlier time points (2 weeks after SE), thereby increasing the probability of observing these inverted glial cells in the process of giving rise to ectopic DGCs in the hilus.

How to keep a basal dendrite

Basal dendrites are a common feature seen during the maturation of adult-born DGCs

(Ribak et al., 2004), which are often retracted on maturation. Under epileptic conditions, changes in local neurotrophic factors and their surface receptors might inhibit the retraction of these basal dendrites and additionally cause the generation of new ones, leading to their presence in mature

DGCs. Importantly, this phenomenon of basal dendrite projection and retraction occurs in the post-mitotic immature DGC and is therefore unlikely to be seen in other cells derived from the same progenitor cell. BDNF plays an important role in the generation of basal dendrites in DGCs

(Danzer et al., 2002). Increased excitation within the granule cell layer induces the activity of L- type Ca2+ channels on DGCs, which then increase BDNF mRNA levels. This leads to synaptic modelling and possibly the formation of new basal dendrites and inhibition of the retraction

117 process of the ones already present (Koyama et al., 2004, Dudek and Sutula, 2007). During the process of maturation, these DGCs form excitatory connections with neighboring granule cells and establish a recurrent excitatory positive-feedback circuit (Ribak et al., 2000, Thind et al.,

2008, Ribak et al., 2012). Thus, a potential therapeutic option to block the formation of basal dendrites during epileptogenesis may include targeting BDNF or L-type Ca2+ channels.

Conclusions

During the course of this doctoral thesis, I have explored the nature of epileptogenesis using different models and techniques. With the kindling study, I discovered that the changes that occur after brief seizures are dissimilar to the ones seen after SE. This provides valuable clues regarding the differences in morphological changes that occur after hyperexcitability compared to those after SE. It also provides insight into the differences in changes within pre- and postnatal DGCs in the epileptic dentate. Further, it cautions us against the comparison between different models of epilepsy as they may substantially differ in the induced morphological and physiological changes.

With the clonal analysis study, I was able for the first time to determine the exact origin and clonality of the abnormal granule cells observed after epilepsy. I also recreated the entire epileptic mouse hippocampus for the first time, to examine these abnormal DGCs in greater spatial context. The results of this clonal study contribute to understanding of these specific cellular abnormalities and will enable improved targeting of therapies to combat them. These

118 insights into the process of epileptogenesis will provide a platform for further critical inquiry and hopefully lead to the generation of more effective treatments for this debilitating disease.

Avenues for future research: Curing epilepsy one dentate at a time

‘’Science never solves a problem without creating ten more”

Even though a host of astonishing discoveries in the field of epilepsy have been made in the last decade, much still remains to be known. The advent of spectacular new techniques such as optogenetics, quantum-biology, designer receptors, and big data analysis are set to exponentially increase our understanding of the process of epileptogenesis.

Some experiments, based on the results obtained during this dissertation study would further clarify the mechanisms of epileptogenesis. One such experiment is deciphering the role of notch signaling in the changes seen in adult neurogenesis post-SE. Experiments designed to probe the levels of notch and its ligands, before and after SE, would add valuable insight into the role of notch signaling in the whole process. Additional data with notch inhibitors or introducing notch beads into the epileptic dentate would expose its affects post-SE. Experiments designed to characterize inverted glial cells and their role in the generation of ectopic DGCs could also answer mechanistic questions about the origin of these abnormal cells. Using a similar low recombination clonal analysis strategy with earlier or later time points may also provide evidence of their exact role in epileptogenesis.

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Figures

Figure 4.1: Inverted glial cells in the dentate: In hippocampal sections from Gli1-Tdtomato animals which underwent pilocarpine treatment on week 4 and were sacrificed 5 weeks later, I observed the presence of inverted radial glial cells in the hilus. These cells were positive for neural stem cell markers B) Nestin and C) GFAP but were negative for oligodendrocyte marker E) IBA-1. A composite of Nestin+GFAP+Tdtomato is shown in D). Scale bars for A) =100µm; B),C),D), and E) = 10µm; H=Hilus, GCL=Granule Cell Layer.

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