UNIVERSITY OF CINCINNATI

Date: 5-Aug-2010

I, Brian L Murphy , hereby submit this original work as part of the requirements for the degree of: Doctor of Philosophy in Neuroscience/Medical Science Scholars Interdisciplinary It is entitled: Aberrant hippocampal neurogenesis and integration in

Student Signature: Brian L Murphy

This work and its defense approved by: Committee Chair: Steve Danzer, PhD Steve Danzer, PhD

8/18/2010 999 Aberrant hippocampal granule cell neurogenesis and integration in 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 Graduate Program in Neuroscience of the College of Medicine

August 5, 2010

By

Brian Liam Murphy

Bachelor of Science, Hope College, 2004

Committee Chair: Kim Seroogy, Ph. D. Kenneth Campbell, Ph. D. Steve Danzer, Ph. D. Katherine Holland-Bouley , M.D., Ph. D. Neil Richtand, Ph. D.

ii ABSTRACT

The data from the present studies suggest that granule cells in different stages of neuronal differentiation may be differentially susceptible to changes in their cellular structure and survival following an epileptogenic stimulus. Recent work in the field has focused on the development of dendritic abnormalities with respect to immature or newly generated neurons following exposure to an epileptogenic stimulus. In Chapter 2, we show for the first time that fully differentiated granule cells are capable of dendritic rearrangement. Specifically, we have shown that previously established apical shift to the basal portion of the cell as the somata of these cells radially migrate up an adjacent primary towards the dentate molecular layer. In doing so, dendritic branches on this dendrite become a new primary dendrite. We also propose that this migration underlies the dispersion of the granule cell layer, which was previously suggested by other laboratories; however, the utility of organotypic explant cultures made from Thy1-YFP mice allowed us for the first time to observe fully differentiated granule cell migration and their contribution to the dispersion of the granule cell layer. Granule cell dispersion and distortions to granule cell dendritic structure are common pathologies of the epileptic brain. Both phenomena also occur in adult animal models of epilepsy.

Using bi-transgenic Gli-CreERT2+/-;Green fluorescent protein (GFP) reporter+/- mice, data from Chapter 3 suggests that the pool of hippocampal subventricular zone and/or subgranular cell layer neural progenitor cells active several days prior to a prolonged seizure become disrupted following early-life seizure activity. Specifically, fewer cells within the were labeled with GFP, and associated with decreased numbers of progenitor cells, immature and mature granule cells. Additionally, we are the first laboratory to show that early-life seizures disrupt the integration of granule cells, which has been a ‘hot’ topic in studies using adult

iii models of epilepsy. In Chapter 4, we used the same bi-transgenic mice, but in an adult model of epilepsy. From this study, we found that the pool of subgranular zone progenitor cells producing progeny following seizures become disrupted, and at later time points, give birth to fewer new granule cells. Additionally, this study implicates that granule cells born during the first week following a seizure may exhibit an accelerated rate of maturation. The results of these studies will hopefully spur further research into the underlying cellular signaling pathways involved in the formation of basal dendrites on dentate granule cells, etcopic localization of granule cells to the hilus and dentate molecular layers and granule cell layer dispersion.

Additionally, future studies of cell-fate mapping in the early-life seizure and adult model of epilepsy should include the use of alternate tamoxifen injection time points to determine if progenitors active before or after an epileptogenic stimulus respond differently than the conditions tested here.

iv

ACKNOWLEDGMENTS

I would like to thank Steve for the many lessons about thinking and doing science that he has provided me over the years – specifically when it comes to data mining. Additionally, I learned about the process of setting up a research lab, in particular management skills. This was a great surprise, and I think is important for any young scientist to see what their future career path entails. I know you did not plan on those lessons, but I am very grateful for being able to experience them with you. Additionally, I would like to thank you for giving me the opportunity to think and plan experiments independently during this last year. This has helped me feel ready for the next step in my scientific career. I would also like to thank the current

(Ray, Hulian, Niki, Mike, Olagide and Victor) and past members (Cynthia and Stefanie) of the lab for making the laboratory a low stress and fun environement as well as all technical help when needed. I would also like to thank my committee members (Drs. Kim Seroogy, Kenneth

Cambell, Katherine Holland-Bouley and Neil Richtand) for their guidance in data interpretation of the studies in this dissertation. Also, I am greatful for University of Cincinnati’s Biomedical

Flex Program (Laura Hildreth, Dr. Robert Highsmith and Mary Joe Petersman) and

Neuroscience graduate program (Deb Cummins, James Herman and Michael Lehman) faculty and staff for initially accepting me into their graduate programs.

Also, thank you Susan Melhorn and Georgette Suidan for their company during the early years of graduate school…the late night study and drinking sessions have been missed over the last couple of years! I will also miss the friends that I made here, in and out of the program; for those of you that were or are in program, I will miss hanging out and sharing food, games, drinks and sciene talk (Jane/Peter A., Martine L./Dave Z., Amanda S., Matt L., Stefanie B.,

Katrina/Sean P., Anne/Kevin E.)! Lastly, none of this would have happened with out the encouragement of my undergraduate research advisor, Leah Chase, as well as the love and support of my parents, Maureen and Bill Murphy; sister, Eileen Murphy; aunt, Kathleen

Killoughrey and my partner Jason Oscar.

vi TABLE OF CONTENTS

Table of Contents……………………………………………………………………..……………….. 1

List of Tables and Figures……………………………..…………………………..………………… 2

Abbreviations…………………………………………….…………………………...………………... 4

Chapter 1: Introduction……………………………………………………..………….…………….. 5

Chapter 2: Somatic translocation of mature hippocampal dentate

granule cells as a novel mechanism of dendritic dysmorphogenesis and granule

cell dispersion……………………………………………………………….…..…………… 42

Chapter 3: Reduced neurogenesis of hippocampal subventricular zone progenitor cells

exposed to neonatal status epilepticus………………………………………….……… 84

Chapter 4: Genetic fate-mapping of adult-generated hippocampal dentate granule cells

In the epileptic mouse brain………...……………..……………………………..……… 124

Chapter 5: Discussion……………………………...…………………………………………..….. 163

1 List of tables and figures

Chapter 1

Figure 1. The “trisynaptic circuit.” pg. 18

Figure 2. Examples of aberrant dentate granule cells within the epileptic brain. pg. 19

Figure 3. Representative Thy1-YFP organotypic hippocampal explant culture. pg. 20

Chapter 2

Figure 1. Postmitotic age of YFP-expressing granule cells in vitro. pg. 65

Figure 2. Recurrent basal dendrite formation and granule cell layer dispersion. pg. 66

Figure 3. Branch to dendrite conversion. pg. 67

Figure 4. Extreme example of branch to dendrite conversion. pg. 68

Figure 5. Granule cells with normal morphology. pg. 69

Figure 6. Granule cell dispersion induced by IHpKA. pg. 70

Figure 7. Ectopic granule cells and recurrent basal dendrites occur in vivo. pg. 71

Figure 8. Model depicting granule cell dysmorphogenesis pg. 72

Supplemental Table 1. Recurrent basal dendrite formation and branch-to- dendrite conversion in control and kainic acid treated explants. pg. 73

Supplemental Figure 1. Dying YFP granule cell. pg. 74

Chapter 3

Figure 1. Cortical EEG recording from a seven day old Gli1-CreERT2 -/-;GFP+/- mouse. pg. 103

Figure 2. Cre-mediated recombination is efficacious and specific. pg. 104

Figure 3. GFP-expression in the adult mouse brain. pg. 105

Figure 4. GFP-expression in five cell types within the adult dentate gyrus. pg. 106

Table 1. Mean percentage of GFP-labeled cell types per dentate gyrus. pg. 107

Table 2. Mean number of GFP-labeled cell types per dentate gyrus. pg. 108

2 Figure 5. Early-life SE disrupts putative neural stem/progenitor cell fate. pg. 109

Figure 6. Scatter plot of Pearson correlation results. pg. 110

Figure 7. Examples of mature dentate granule cells with abnormal integraton following early-life SE. pg. 111

Supplemental figure 1. Cre-recombinase polymerase chain reaction. pg. 112

Supplemental table 1. Mean percentage of GFP-expressing dentate granule cells possessing basal dendrites (DGC+BD) or ectopically localized in the hilus (HEC). pg. 113

Chapter 4

Figure 1. Endogenous GFP expression in the adult brain. pg. 141

Figure 2. Specificity of induction and cell labeling in Gli1-CreERT2;GFP reporter mice. pg. 142

Figure 3. GFP-expression in cells of the SGZ, dentate gyrus and aberrantly integrated dentate granule cells. pg. 143

Table 1. Mean percentage of GFP-expressing cells per dentate gyrus five weeks following SE. pg. 144

Table 2. Mean percentage of GFP-expressing cells per dentate gyrus twelve weeks after SE. pg. 145

Figure 4. Labeling of mossy fiber in stratum lucidum. pg. 146

Figure 5. Mossy fiber sprouting in epileptic mice. pg. 147

Table 3. Pearson correlation results: Are the changes in the cell-fate of GFP-expressing cells related mossy fiber sprouting 12 weeks following SE? pg. 148

Supplemental Figure 1. Cre-recombinase polymerase chain reaction. pg. 149

Supplemental Table 1. Mean number of GFP-expressing cells per dentate gyrus five and twelve weeks after SE. pg. 150

3 List of Abbreviations

Cdk5 Cyclin dependent kinase 5

Disabled 1 Dab1

Doublecortin DCX

EEG Electroencephalography

GFP Green fluorescent protein

ILAE International League Against Epilepsy

MWRST Mann-Whitney Rank Sum Test

MTLE Mesial temporal lobe epilepsy

NDE1 Nuclear distribution factor E homologue 1

Shh Sonic hedehog

SGZ Subgranular zone

SE Status epilepticus s. c. Subcutaneous

YFP Yellow fluorescent protein

4 Chapter 1

General introduction and background

5 Epilepsy is an incapacitating disease that currently lacks a cure and preventative strategies. It is one of the most common neurological disorders in the world, affecting people of varying ages (Chang and Lowenstein, 2003; Duncan et al., 2006; Henshall and Simon, 2005).

About 50 million people are affected worldwide (Henshall and Simon, 2005), and two million in the United States (Chang and Lowenstein, 2003). Additionally, it is estimated that 3% of the world’s population will develop epilepsy at some point in their life (Chang and Lowenstein, 2003;

Henshall and Simon, 2005). Epilepsy is characterized by recurrent unprovoked seizures (Haut et al., 2006) caused by the synchronous discharge of neurons (Engel, 1996a; Henshall and

Simon, 2005). These seizures can manifest as a change in mental state, tonic or clonic movements, convulsions, and various other psychic symptoms (e.g. aura, déjà vu or jamais vu)

(Blume et al., 2001).

There are many forms of characterized by differences in the type of seizure observed, which is determined by changes in electrical activity of the brain measured by electroencephalography (EEG) as well as the types of involuntary changes in behavior and/or sensations (Bancaud et al., 1981). Due to variations in these parameters, classification of seizure types and related epilepsies has been a complicated task throughout history (Bancaud et al., 1981; Commission on Classification and Terminology of the International League Against

Epilepsy, 1989; Engel and International League Against Epilepsy (ILAE), 2001; Engel, 2006).

Clinicians and investigators have generally categorized seizure disorders/epilepsies into two main groups, generalized and partial, each with numerous subtypes of epilepsies (Bancaud et al., 1981; Berg et al., 2010). The criteria used to distinguish these two categories stems from where in the brain the electrical discharges are most synchronous. Commonly, generalized epilepsies is used to distinguish seizures that are thought to originate simultaneously in both cerebral hemispheres; whereas partial epilepsies encompass seizures that are thought to originate in localized foci in one hemisphere, which tend to spread to the rest of the brain

6 (Bancaud et al., 1981). It is important to note, however, that this classification scheme is currently under revision (Engel and International League Against Epilepsy (ILAE), 2001; Engel,

2006). The etiologies of most seizures and epilepsy syndromes are largely unknown. However, a leading hypothesis for the development of epilepsy is that it is initiated by an insult to the brain, either genetically, developmentally or environmentally produced. Thus, some forms of epilepsy are thought to be primarily due to an underlying genetic component, such as gene mutations of ion channels and receptors (Lu and Wang, 2009; Mullen and Scheffer, 2009) while other forms of epilepsy are thought to result from a central insult, such as traumatic brain injury, infection and tumors (Avila and Graber, 2010; Chang and Lowenstein,

2003; Lee et al., 2010; Solbrig and Koob, 2004).

Currently, treatment of seizures and epilepsies utilize drugs that act to prevent or at least decrease the frequency of seizures in epileptic patients, thereby making unmanageable seizure occurrences more manageable. The most common of these drugs, termed antiepileptic drugs, target voltage-gated or ligand-gated ion channels, such as voltage-activated Na+ and Ca2+ channels or GABAA receptors, which act to alter the physiology of these channels so that neuronal hyperexcitability throughout the brain is largely suppressed (Armijo et al., 2005;

Meldrum and Rogawski, 2007; Rogawski and Loscher, 2004). However, there are many patients that are non-responsive to such drugs, leading to so-called drug-resistant or pharmacologically intractable epilepsy (Hauser, 1992). The most common pharmacologically intractable epilepsy is mesial temporal lobe epilepsy (MTLE) (Cavazos and Cross, 2006;

Henshall and Simon, 2005). In order such cases, surgical resection of the epileptogenic zone is frequently used as an alternative treatment (Engel, 1996b).

MTLE is characterized by seizures originating in mesial temporal limbic structures, and very commonly within the itself (Engel, 1996a; Mathern et al., 1995a). Although

7 the etiology of MTLE remains largely unknown, epidemiological data from patients diagnosed with this epilepsy disorder have led to several theories about what may underlie the source of seizures in the affected brain regions. These include a history of initial precipitating events such as prolonged febrile seizures, generalized status epilepticus (SE), head trauma, intracerebral infections, brain tumors and stroke (Engel, 1996a; Mathern et al., 1995b; Mathern et al., 1996).

Additionally, an increased incidence of family history has been noted, suggesting an underlying genetic predisposition to MTLE (Engel, 1996a). Clinically, patients diagnosed with MTLE generally present with an initial aura that may occur in isolation or shortly followed by seizure- induced behavioral manifestations, including freezing behavior or motor arrest, staring, oroalimentary automasims (e. g. lip-smacking, chewing) and unilateral posturing of the upper limb that is contralateral to the seizure activity (Engel and International League Against Epilepsy

(ILAE), 2001). This type of seizure is termed complex partial since the seizure activity commonly originates within the mesial temporal area, (which is possibly responsible for the initial aura experienced). Seizure-induced behavioral manifestations are thought to occur when the seizure activity spreads more globally (Engel and ILAE, 2001; Lieb et al., 1986). Following the seizure episode, the patient usually seems disoriented or confused along with no recollection of the event (Engel, 1996a).

Dentate gate hypothesis of mesial temporal lobe epilepsy

Dentate granule cells participate in the classical hippocampal “trisynaptic circuit” along with CA3 pyramidal cells and CA1 pyramidal cells (Anderson et al., 1971). An oversimplified description of this circuit is that under normal conditions, the first in this circuit comes from entorhinal cortical projections along the perforant path, which transmits excitatory input to dentate granule cells (Fig. 1; long dashed arrow, 1). The second synapse transmits excitatory information to CA3 pyramidal cells via dentate granule cell axons or mossy fibers (Fig. 1; dashed-dot arrow, 2), and the third synapse carries excitatory input to CA1 pyramidal cells from

8 CA3 pyramidal cells via schaffer collaterals (Fig. 1; dotted arrow, 3). Next, excitatory information is transmitted to the subiculum (Amaral et al., 1991) and out of the hippocampal formation to the cortex. Since the initial entry point of information flow into the hippocampus is through the dentate granule cells, they are thought to act as a filtration system for the amount of excitatory transmission coming into the hippocampus from the (Collins et al.,

1983; Hsu, 2007). This filtering capability is primarily attributed to the fact that dentate granule cells also synapse on within the hilus and stratum lucidum en route to CA3 pyramidal cells, providing feedforward inhibition to CA3 pyramidal cells. When dentate gating function is compromised, it is suggested that excitatory input into the hippocampus overcome this basal inhibitory activity, allowing more synaptic transmission to CA3 pyramidal cells, and thus through the hippocampus into surrounding cortex. This increased amount of neurotransmission is hypothesized in leading to hyperscynchronous discharges within the brain, and ultimately behavioral convulsions (Behr et al., 1998; Collins et al., 1983; Gloveli et al., 1998;

Heinemann et al., 1992).

Following an insult to the brain, such as a prolonged seizure, is a latent period during which maladaptive changes in the brain occurs, culminating with the later appearance of spontaneous seizures (Pitkanen et al., 2002). During this latent period, changes to affected brain regions occur, including brain inflammation (Auvin et al., 2010), hyperexcitability of neurons (Martin and Sloviter, 2001) and cell death (Henshall and Simon, 2005). Since MTLE is also the most common epilepsy treated by surgical resection (Engel, 2001; Ojemann, 1997) it has been the target of numerous investigations. Studies of this tissue, specifically the hippocampus, have shown several pathological abnormalities present relative to non-epileptic control tissue. These abnormalities include: hippocampal neuronal loss known as hippocampal sclerosis (Babb et al., 1984), reorganization of mossy fibers in the dentate gyrus (Babb et al.,

1991; Franck et al., 1995; Houser et al., 1990; Isokawa et al., 1993; Represa et al., 1989),

9 dispersion of granule cells to ectopic locations within the hippocampus (Houser et al., 1990;

Houser, 1990) and distortions to granule cell dendritic structure (Scheibel ME and Scheibel AB,

1973; von Campe et al., 1997).

It is hypothesized that aberrant granule cell plasticity following seizure activity compromises the filtering capabilities of the dentate by creating feed-forward loops within granule cell circuitry (Lynch et al., 2000; Molnar and Nadler, 1999; Okazaki et al., 1999; Sutula et al., 1989; Wuarin and Dudek, 1996; Wuarin and Dudek, 2001). Normally, dentate granule cells have a highly polarized structure with dendrites extending from the apical portion of the cell body into the dentate molecular layer and an (mossy fiber) extending from the basal portion of the cell body into the hilus (Fig. 2, a; green cell; white arrowhead denotes axon). In the epileptic brain, however, several common morphological changes have been observed.

Firstly, mossy fibers sprout collaterals (Fig. 2, a; light purple) that extend back into the inner molecular layer where they form on granule cell apical dendrites (Molnar and Nadler,

1999; Wuarin and Dudek, 1996). In this way, mossy fiber sprouting contributes to increasing network excitability (Babb et al., 1991; Okazaki et al., 1995; Okazaki et al., 1999; Represa et al.,

1989; Sutula et al., 1989; Wuarin and Dudek, 2001). Secondly, hilar basal dendrites (Fig. 2 a; red) are present on granule cells in the epileptic brain. These dendrites originate from the basal pole of the cell and project inappropriately into the hilus (Buckmaster and Dudek, 1999; Okazaki et al., 1999; Spigelman et al., 1998). The formation of hilar basal dendrites is also thought to increase network excitability through their development of dendritic spines, innervations by neighboring granule cell mossy fibers and subsequent bursting activity (Austin and Buckmaster,

2004; Ribak et al., 2000; von Campe et al., 1997). Thirdly, ectopically localized dentate granule cells in the hilus (Fig. 2, b; light blue cell) (Parent et al., 1997; Parent et al., 2006) are also implicated in creating recurrent excitatory loops due to abnormal innervations of their apical dendrites from neighboring granule cells axons (Pierce et al., 2005), and exhibit bursting

10 properties uncharacteristic of granule cells within the granule cell layer (Scharfman et al., 2000;

Scharfman et al., 2002; Scharfman et al., 2003). Related to this is the dispersion of the normally compact granule cell layer (Fig. 2, b; note the location of Prox1-immunoreactive granule cells relative to figure 2, a) (Houser et al., 1990; Houser, 1990), which might also lead to changes to neuronal excitability due to the migration of granule cells to ectopic locations. More recently, another type of basal dendrite, termed recurrent basal dendrites, has been described

(Ribak et al., 2000). Recurrent basal dendrites (Fig. 2, b; yellow) also originate from the base of the granule cell body, at or near the origin of the axon, but rather than projecting into the hilus, they curve back towards the granule cell layer to enter the molecular layer, like their apical dendrite counterparts (Ribak et al., 2000; Yan et al., 2001). Neighboring granule cell mossy fibers have been found to synapse on recurrent basal dendrites, thus forming a recurrent excitatory circuit (Dashtipour et al., 2002). These abnormalities are found in both human epileptogenic tissue as well as animal models of epilepsy, and recent work in the field has focused on discerning whether these changes occur on mature, newly-generated granule cells or both in order to develop future therapeutics that target the development of these abnormailities.

Development of the dentate gyrus and neurogenesis

Dentate granule cells are some of the last neurons generated during the development of the brain. Granule cell neurogenesis begins several weeks prior to birth and continues throughout the postnatal period in animals and humans (Altman and Bayer, 1990a; Altman and

Bayer, 1990b; Bayer, 1980; Eriksson et al., 1998; Knoth et al., 2010; Schlessinger et al., 1975;

Seress et al., 2001). In rodents, granule cell neurogenesis peaks around postnatal day seven

(Altman and Bayer, 1990a; Bayer, 1980; Schlessinger et al., 1975). Dentate granule cells, like other neurons, originate from neural stem and progenitor cells, and populate the dentate in six steps (for review see Li and Pleasure, 2005). (1) Late embryonically, around E16, dividing

11 neural stem/progenitor cells first populate an area of the brain adjacent to the dentate notch called the secondary dentate matrix (Altman and Bayer, 1990a; Altman and Bayer, 1990b; Li and Pleasure, 2005; Pleasure et al., 2000). More recently, it is has also been referred to as the hippocampal subventricular zone (Navarro-Quiroga et al., 2006). It is here where the first granule cells are generated, and the stem/prognitor pools expand (Altman and Bayer, 1990a;

Altman and Bayer, 1990b). (2) At E18, these granule cells begin to migrate along previously formed radial glial fibers to the dentate anlage and (3) begin populating the primordial dentate gyrus along this radial glial scaffolding (Eckenhoff and Rakic, 1984; Liu et al., 2000; Pleasure et al., 2000). (4) During this time, the stem/progenitor cells also begin to migrate towards this primordial dentate gyrus to take up residence in the hilus and form the tertiary dentate matrix

(Altman and Bayer, 1990a; Altman and Bayer, 1990b; Pleasure et al., 2000) where they continue to divide and produce the majority of the dentate granule cell population over the first postnatal week (Muramatsu et al., 2007). This initial wave of stem/progenitor cell migration continues until approximately postnatal day five (Navarro-Quiroga et al., 2006). (5) By the end of the first postnatal week, the hilar matrix becomes reorganized so that proliferating progenitor cells reside in a region between the granule cell layer and hilus called the subgranular zone (Pleasure et al., 2000). (6) By postnatal day ten, the radial glial cells have also migrated from the tertiatry matrix and taken up residence in this subgranular zone (SGZ)

(Forster et al., 2002), and are thought to exhibit neural stem cell characteristics (Gage et. al.,

2000). Thus, it is in the SGZ that neurons are continually generated throughout adulthood.

After their birth, newborn granule cells then migrate into the granule cell layer where they become fully differentiated postmitotic neurons (Kempermann et al., 2004; Seri et al., 2001; Seri et al., 2004).

The late development and continued generation of new granule cells into adulthood raise concerns regarding the effects of seizures on neurogenesis. Several laboratories have

12 shown that seizures in adult models of epilepsy lead to an increase in neurogenesis (Bengzon et al., 1997; Gray and Sundstrom, 1998; Parent et al., 1997; Parent et al., 1998; Scott et al.,

1998). However, studies using various models of early-life seizures remain inconclusive on this topic, demonstrating both increases and decreases in neurogenesis following early-life seizures.

These conflicting results may be due to differences in animal age at the time of the initial seizure episode, frequency of seizures evoked and the model used (Bender et al., 2003; Dong et al.,

2003; Holmes et al., 1998; McCabe et al., 2001; Wasterlain, 1976). Additionally, previous studies from our laboratory (Walter et al., 2007) and others have demonstrated that seizures in adult animals disrupt the integration and structure of adult-generated granule cells. Specifically, these new neurons were found to exhibit hilar basal dendrites (Jessberger et al., 2007; Shapiro et al., 2005; Walter et al., 2007) and become ectopically localized to the hilus (Jessberger et al.,

2005; Parent et al., 2006; Walter et al., 2007) and exhibit mossy fiber sprouting (Danzer, 2008;

Jessberger et al., 2007; Kron et al., 2010). Interestingly, recent studies suggest that granule cells are an integral component to principal hippocampal functions such as learning and (Jessberger et al., 2009; Whitfield and Chakravarthy, 2009). Additionally, newly generated granule cells are implicated in the formation of new (Deng et al., 2009;

Snyder et al., 2005). Therefore, aberrant synaptic connections including those discussed here may also contribute to deficits in memory and cognition that are associated with seizures

(Jessberger et al., 2007).

Models of seizures and MTLE

Animal models are frequently used to investigate underlying cellular mechanisms of neurological disorders and diseases due to the difficulty in obtaining human specimens. The studies outlined in this dissertation utilize in vitro and in vivo models of mesial temporal lobe seizures and epilepsy. In all of these studies, we used mouse models due to the vast array of tools available for the manipulation of the mouse genome.

13 The first experiment in Chapter 2 utilizes organotypic entorhinal-hippocampal explant cultures, which is an in vitro seizure model. These cultures are advantageous in studying cellular mechanisms because they preserve the neuronal connections within the hippocampus such that transmission input and output is maintained (Fig. 3; Holopainen, 2005). The preparation of these cultures reproduces characteristic pathologies of the epileptic brain, such as mossy fiber sprouting (Bender et al., 1998; Coltman et al., 1995; Gutierrez and Heinemann,

1999; Zimmer and Gahwiler, 1984) and granule cell layer dispersion (Stoppini et al., 1991).

Treatment of these cultures with excitotoxic agents also reveals hallmarks of in vivo models of

MTLE and human epileptogenic tissue, including epileptiform activity (Benedikz et al., 1993;

Routbort et al., 1999), neuronal cell loss (Benedikz et al., 1993; Holopainen et al., 2004; Rimvall et al., 1987; Routbort et al., 1999) and mossy fiber sprouting (Routbort et al., 1999).

Additionally, they can be treated pharmacologically and be transfected with genetic constructs to determine underlying mechanisms such as those regulating aberrant development of granule cell dendrites (Danzer et al., 2002). The key advantage to our system is the use of such cultures with fluorescent protein expressing mice, specifically Thy1-Yellow fluorescent protein

(YFP) expressing mice (Feng et al., 2000). Thy1 is a glycosylphosphatidylinositol-linked cell surface protein expressed in various tissues and cell-types, such as thymus and the nervous system; this particular promoter was modified so that expression was limited to the neurons

(Caroni, 1997). This allows for the observation of dentate granule cell morphology and underlying cellular processes of aberrant morphology development using time-lapse confocal microscopy.

The second experiment in Chapter 2 as well as the experiment in Chapter 3 uses in vivo administration of kainic acid to chemically induce prolonged seizure activity called status epilepticus (SE). Kainic acid is a neurotoxin that was isolated from the red alga Digenea

14 simplex in Japan in 1953 (Moloney, 1998). It is a known gultamate analog, and is an agonist for the subtype of ionotropic glutamate receptors that are highly expressed within the hippocampus (Bettler and Mulle, 1995; Bureau et al., 1999; Vincent and Mulle, 2009).

Kainate receptors are also found within the entorhinal cortex and amygdala (Miller et al., 1990).

Due to these patterns of receptor expression, kainic acid-induced seizures produce cell loss in

CA1 and CA3 layers as well as interneuron cell loss in the hilus, which are reminiscent of hippocampal sclerosis (Ben-Ari, 1985; Nadler et al., 1978). Because of these effects, kainic acid administration has become a useful model of MTLE (Ben-Ari, 1985; Engel and ILAE, 2001). In Chapter 2, the intrahippocampal injection of kainic acid model of MTLE was used in Thy1-YFP expressing mice in order to assess ectopic migration and changes in granule cell morphology. In this model, kainic acid is injected unilaterally into the dorsal hippocampus to induce SE for 8-10 hours (Heinrich et al., 2006; Riban et al., 2002). During this period, behavioral manifestations of seizure activity develop, including mild clonic movements of the forelimbs, rotations and immobility, which are associated with seizure activity within the hippocampus and cortex (Riban et al., 2002). This model is widely used to induce dispersion of the granule cell layer (Bouilleret et al., 1999; Riban et al., 2002; Suzuki et al., 1995). In Chapter

3, kainic acid is administered systemically via subcutaneous injection in bi-transgenic Gli1-

CreERT2;Green fluorescent protein (GFP) reporter mice in order to investigate changes in neural stem/progenitor cell fate following early-life seizures. This leads to the development of SE, as well as behavioral manifestations of seizure activity including, forelimb clonus, hyperextension of the hindlimbs and ‘swimming’ behavior, which lasts up to six-to-seven hours (Ben-Ari et al.,

1984; Danzer et al., 2004; Stafstrom et al., 1992; Tremblay et al., 1984).

In Chapter 4, in vivo administration of pilocarpine to chemically induce SE is used to investigate changes in adult-generated granule cell fate and synaptic structure using bi- transgenic Gli1-CreERT2;GFP reporter mice. Pilocarpine is a muscarinic acetylcholine receptor

15 agonist, and these receptors are highly expressed in the hippocampus, striatum and cortex

(Kuhar and Yamamura, 1976; Levey et al., 1994). Hyperactivation of these receptors leads to automatisms, “wet dog shakes,” and clonic seizures that develop into SE usually observed as consistent head-bobbing. Additionally, EEG recordings following pilocarpine administration have found that fast spikes occur within the hippocampus, which then spread to the cortex

(Cavalheiro et al., 1987). In our laboratory, we suppress SE after three hours with the administration of diazepam, a derivative of benzodiazepine, which is a GABAA receptor agonist

(Riss et al., 2008). Following a latent period of several weeks, spontaneous recurrent seizures begin to occur (Curia et al., 2008; Leite et al., 2002). Additionally, the neuropathology within the hippocampus of this model is also reminiscent of hippocampal sclerosis, including the loss of

CA3 pyramidal cells and hilar interneurons (Turski et al., 1987; Turski et al., 1989; Zhang et al.,

2009), loss of dentate granule cells (Danzer, 2008; Muller et al., 2009) as well as changes in mossy fiber pre- and post-synaptic structures (Danzer and McNamara, 2004; Danzer et al.,

2010). Additionally, this model has reliably reproduced the formation of hilar basal dendrites

(Jessberger et al., 2007; Ribak et al., 2000; Shapiro et al., 2005; Walter et al., 2007), ectopic migration of granule cells (Parent et al., 1997; Parent et al., 2006), mossy fiber sprouting

(Jessberger et al., 2007; Okazaki et al., 1999) and also leds to learning and memory deficits

(Muller et al., 2009a; Muller et al., 2009b).

The studies in this dissertation focus on three main aims:

(1) To gain insight into the mechanisms underlying changes in granule cell dendritic structure, in particular the formation of recurrent basal dendrites, which are observed in resected tissue from epileptic patients as well as in models of MTLE. (Chapter 2)

(2) To elucidate whether or not granule cells of different ages (immature vs. mature) are more or less susceptible to seizure-induced changes in morphology. (Chapters 3 & 4 vs. Chapter 2)

16 (3) To determine whether seizure activity within the developing or adult brain affect granule cell neurogenesis, maturation and development in similar or distinct ways. (Chapters 3 & 4)

Results from the studies of this dissertation will aid in the development of future therapeutics targeting changes in hippocampal circuitry that are hypothesized to underlie the development of epilepsy and associated cognitive impairments.

17 Figure 1. The “trisynaptic circuit.” Neural input flows into the hippocampus from entorhinal cortex synapsing first on granule cell dendrites in the dentate gyrus (d; dashed arrow, 1), which is then transmitted to CA3 pyramidal cells (CA3; dash-dot arrow, 2) and finally onto CA1 pyramidal cells (CA1; dotted arrow, 3; Note: this is not actual pathway). H, hilus; sl, stratum lucidum. Scale bar, 200 m.

18 Figure 2. Examples of aberrant dentate granule cells within the epileptic brain. This figure is a photomontage of pseudo-colored GFP-expressing dentate granule cells with normal and abnormal morphology, Prox1-immunoreactivity in the granule cell layer (granule cell marker) and illustrations of other aberrant morphologies. (a) A typical differentiated granule cell (green) with apical dendrites projecting into the molecular layer and an axon extending from the basal portion of the cell body (white arrowhead). Mossy fiber sprouting into the granule cell and inner molecular layers is depicted in light purple, and a hilar projecting basal dendrite is illustrated in red. (b) Examples of differentiated granule cells localized in the hilus (light blue) and one with a recurrent basal dendrite (yellow) that has an axon originating off of the dendritic loop (white arrowhead). Also, note the dispersion of the granule cell layer relative to a. GCL, granule cell layer; ML, dentate molecular layers. Scale bar, 30 m.

19 Figure 3. Representative Thy1-YFP organotypic hippocampal explant culture. Confocal maximum projection of a living hippocampal explant made from a Thy1-YFP expressing mouse.

The image shown was taken after 14 days in vitro. YFP-imaging reveals brightly labeled cells, preservation of principle cell layers and preservation of major axon pathways. dg, dentate gyrus.

CA3, CA3 pyramidal cell layer. CA1, CA1 pyramidal cell layer. mf, granule cell mossy fiber pathway. sc, Schaeffer collateral pathway. Scale bar, 500 m.

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41 Chapter 2

Somatic translocation: a novel mechanism of granule cell dendritic dysmorphogenesis and dispersion*

*The majority of this work has been submitted to the Journal of Neuroscience as a Brief Communication.

42 Abstract

Pronounced neuronal remodeling is a hallmark of temporal lobe epilepsy. Animal models of the disease provide an opportunity to identify and study these neuroplastic processes, particularly, the appearance of granule cells with deformed dendritic trees. Here, we use real-time confocal imaging to demonstrate that these deformities can evolve on fully differentiated granule cells following translocation of the into an apical dendrite. Somatic translocation converts dendritic branches into primary dendrites and shifts adjacent apical dendrities to the basal pole of the cell. Moreover, somatic translocation contributes to the dispersion of the granule cell body layer in vitro, and when granule cell dispersion is induced in vivo, the dispersed cells exhibit virtually identical derangements of their dendritic structures.

Together, these findings identify a novel form of neuronal plasticity, which contributes to granule cell dysmorphogenesis in the epileptic brain.

43 Introduction

The epileptic brain is characterized by numerous alterations in hippocampal granule cell structure, including dispersion of the normally compact granule cell body layer (Houser, 1990), accumulation of cells with dendrites projecting in the wrong direction (Spigelman et al., 1998;

Ribak et al., 2000), formation of recurrent basal dendrites, which project inappropriately off the basal pole of the cell, but then turn and correctly target the dentate molecular layer (Ribak et al.,

2000), and a variety of distortions of the dendritic tree (Scheibel and Scheibel, 1973; von

Campe et al., 1997). Although many of these aberrant plastic changes were first described decades ago, the cellular processes by which they develop have only recently begun to be elucidated.

A significant advance followed the demonstration that granule cell neurogenesis continues throughout adulthood in mammals (Kaplan and Hinds, 1977; Eriksson et al., 1998).

While these new cells develop normally in healthy animals, in the epileptic brain many of these cells exhibit defects in migration and dendritic growth (Parent et al., 1997; Jessberger et al.,

2007; Walter et al., 2007). Developing neurons are extremely sensitive to a host of perturbations to which mature neurons are resistant. Disruption of cells actively engaged in axonal and dendritic elongation might induce them to grow abnormally, leading to speculation that newborn neurons might account for most, if not all, of the aberrant cells in the epileptic brain. This assumption may be true under certain conditions; however, the sheer number of affected cells in some epileptic animals makes it unlikely that all abnormal cells are adult- generated. Moreover, profoundly impaired neurogenesis in some epilepsy models (Heinrich et al., 2006, Nitta et al., 2008) and at later disease stages (Rao et. al., 2008; Hattiangady and

Shetty, 2010) suggests that mature neurons are also capable of substantial restructuring.

44 To determine whether mature granule cells are capable of dendritic restructuring, organotypic entorhinal-hippocampal cultures were made from Thy1-Yellow fluorescent protein

(YFP)-expressing mice (Feng et al., 2000). Granule cells in these explants become hyperexcitable and recapitulate neuroplastic changes observed in the intact epileptic brain

(Zimmer and Gahwiler, 1984; Stoppini et al., 1991; Coltman et al., 1995; Bender et al., 1998;

Gutierrez and Heinemann, 1999; Routbort et. al., 1999), likely due to trauma and denervation

(injuries which also induce epilepsy in vivo). Importantly, YFP is only expressed in fully differentiated granule cells in these cultures. By imaging these cells with real-time confocal microscopy, we sought to reveal how granule cell dysmorphogenesis occurs in this relatively mature cell population. Real-time imaging also reveals subtle changes in structure that would be undetectable with other methods. Using this approach, we discovered two novel forms of neuronal plasticity exhibited by fully differentiated granule cells. Furthermore, by examining the cellular steps leading to these changes, we were able to predict and confirm that a similar process may account for the appearance of dysmorphic granule cells in an in vivo model of epilepsy.

45 Materials and Methods

Animals

Male and female Thy1-YFP-expressing mice on a C57BL/6 background were used for the present study. These animals are from the “H” line, as described previously (Feng et al., 2000).

All procedures conformed to National Institutes of Health and institutional guidelines for the care and use of animals.

Organotypic entorhinal-hippocampal explant cultures

Organotypic entorhinal-hippocampal explant cultures (n=26) were prepared from male and female 5-8 day old mouse pups (n=20) using a modification of the method described by Stoppini

(Stoppini, Buchs and Muller, 1991, Danzer et al., 2002). Briefly, mice were anesthetized with ketamine (100mg/kg), decapitated and the brain removed. The brain was immediately cooled in ice-cold dissection buffer (Gey’s Balanced Salt Solution plus 6.5mg/ml glucose [all from Sigma-

Aldrich, St. Louis, MO], pH 7.2), blocked to remove the and frontal lobe and mounted to a tissue chuck. Four hundred micron thick slices through the cortex and hippocampus were cut at 20-30° off the horizontal plane using a motorized Vibraslice (model #

MA752; Campden Instruments, Lafayette, IN). Brain slices were further dissected in ice-cold dissection buffer to generate intact sections containing both entorhinal cortex and hippocampus.

Entorhinal-hippocampal explants were transferred to pre-warmed Millicell inserts (Millipore,

Bedford, MA) in 35 mm sterile Petri dishes (Fisher Scientific) containing 1.1 ml of Stoppini medium [50% minimal essential medium, 25% hanks buffered salt solution, 25% heat inactivated horse serum, 10mM HEPES, 0.5% GlutaMax (all from Invitrogen, Carlsbad, CA) and

o Glucose (0.65g/100ml; Sigma-Aldrich)]. Explants were incubated at 37 C in a 5% CO2/95% air mix at 100% humidity. Culture medium was changed every three to four days.

46 Neuron selection and confocal time-lapse imaging

After 6-7 days in vitro (DIV), 26 cultures were screened for the presence of YFP-expressing dentate granule cells. Poorly labeled granule cells, granule cells exhibiting degenerative changes and granule cells obscured by the processes of other cells were excluded from the study.

All time-lapse imaging was conducted with the investigator blinded to treatment conditions. While imaging, explants were maintained at 37oC using a Ludin chamber supplied with a humidified 5% CO2/95% air mix. YFP-expression was imaged using the 514-laser line, and emission wavelengths between 520 and 600 nm were collected. Single optical sections were captured at 1.5X optical zoom to reveal the overall structure of the dentate, and individual granule cells meeting selection criteria were imaged through their Z-depth at 4-5X optical zoom.

Z-stacks were collected at 3-5 m increments. All imaging sessions were completed within 15 minutes, after which explants were returned to the home incubator. The first imaging sessions for granule cells in this study were conducted on 6-7 DIV to capture baseline morphology. To assess changes in dendritic structure in vitro, cells were re-imaged 24 hours, 48 hours and between 5-7 days after the initial imaging session. No evidence of phototoxicity was observed.

Eleven days after the first imaging session (17-18 DIV), explants were fixed in 2.5% paraformaldehyde and 4% sucrose in PBS, pH 7.4 for 90 minutes at room temperature.

Survival and final morphology of imaged cells was assessed after fixation. Cells that were absent at this final time point (dead cells, which rapidly lose their YFP due to loss of membrane integrity) and cells exhibiting obvious degenerative changes (e.g. dendritic beading) were excluded from the study.

47 In vitro kainic acid treatment

On the day of neuron selection (6-7 DIV), explants containing one or more granule cells meeting the selection criteria were randomly assigned to either kainic acid treatment or vehicle control. For treatment, explants were transferred to new dishes containing either 5M kainic acid (Sigma Aldrich) dissolved in Stoppini medium, or vehicle (Stoppini medium alone) for 6 hours. After 6 hours, explants were returned to their original dishes containing conditioned

Stoppini medium, and were then transferred to fresh medium one hour later.

Analysis of confocal time series

All data collection was conducted with the investigator blind to treatment conditions.

The length of granule cell primary dendritic segments were quantified from confocal image stacks imported into Neurolucida software (version 7.50.4; MicroBrightField,Williston, VT).

Granule cell layer dispersion in explants was measured using Leica Application Suite software

(1.7.0 build 1240). Briefly, for the first and last images in a time series, a box was drawn with 3-

5 granule cells serving as “corners”. Corner granule cells were selected from both the inner and outer granule cell layer, so that any spreading of the granule cell layer would be evident as an increase in the box’s size. Percent change was defined as: (initial area – final area)/initial area.

Notably, this approach does not rely on the assumption that the position of any particular cell is constant over time, since there are no immutable reference points to measure from in these cultures. Dispersion analyses were conducted on all explants containing morphologically stable granule cells, defined as cells that did not exhibit any notable movement of dendrites or dendritic branches relative to the cell body, and on all explants exhibiting the most dramatic morphological changes, defined as formation of a recurrent basal dendrite or a novel primary dendrite. Explants that did not contain at least three “corner” granule cells, however, were excluded from this analysis.

48 To generate comparative data for assessing recurrent basal dendrite frequency in explants, four fourteen-day-old Thy1-YFP expressing mice were overdosed with pentobarbital (100 mg/kg) and perfused with 0.1 M PBS+1U/ml heparin followed by 2.5% paraformaldehyde and

4% sucrose in PBS, pH 7.4. Brains were removed and post-fixed for 12 hours, cryoprotected in sucrose (10, 20, 30%) and snap frozen in 2-methyl-pentane at -25oC. Coronal sections were cut on a cryostat at 60 m, slide mounted and cover-slipped using Gel/Mount (Biomeda, Foster

City, CA). Fourteen-day-old animals were selected because YFP-expressing granule cells in fourteen-day-old mice would be equivalent in age to YFP-expressing granule cells in explants made from 7-day-old mice and maintained for 7 DIV. Brain sections from the four control animals and from sixteen 7 DIV explants were examined under epifluorescent illumination (63X,

NA 1.4) and the percentage of YFP-expressing granule cells with recurrent basal dendrites was determined from totals of 152 (controls) and 136 (explants) randomly selected cells. Recurrent basal dendrites were defined as dendrites originating from the lower half of the granule cell body that initially project towards the hilus but then turn back towards the granule cell layer.

Assessment of YFP-expressing granule cell postmitotic age

In order to assess the postmitotic age of YFP-expressing granule cells at the time imaging sessions were begun, a subset of cultures (n=17) made from seven-day old Thy1-YFP expressing (n=7) mouse pups were fixed in 2.5% paraformaldehyde and 4% sucrose in PBS, pH 7.4 for 90 minutes at room temperature after 7 days in vitro (DIV). Cultures were immunostained while still attached to the Millicell insert with calretinin (n=5), calbindin (n=6) or neuronal nuclei (NeuN; n=6). Briefly, cultures were incubated for 1 hour in blocking solution

[5% normal donkey serum (Millipore, S30-100ML) plus 1%% Igepal (Sigma-Aldrich, I3021-

500ML) in 0.1 M PBS] at room temperature, and then overnight at 4oC in primary antibody followed by a four-hour incubation at room temperature in 1:750 donkey anti-mouse AlexaFluor

647. Primary antibodies used were: 1:1000 mouse anti-Calretinin (Millipore, MAB1568), 1:500

49 mouse anti-Calbindin (Sigma-Aldrich, C9848) and 1:200 mouse anti-NeuN (Millipore, MAB377).

A section of the Millicell insert containing the culture(s) was then cut out and mounted on sliane- coated slides (Lab Scientific, 7801), which were cover-slipped using Gel/Mount (Biomeda, M01) and sealed using clear nail polish.

Confocal z-stacks were acquired for each culture using a Leica SP5 confocal system set up on a DMI 6000 inverted microscope equipped with a 10X (NA 0.3) and a 63X (NA1.4) objective. Endogenous YFP was excited using the 514-laser line (25% power), and emission wavelengths between 516 and 560 nm were collected. AlexaFluor 647 secondary antibody was excited using the 633-laser line, and emission wavelengths between 645 and 700 nm were collected. Images were acquired through the z-depth of each granule cell layer at 3 m steps using the 10X objective with 1.5X optical zoom. The image stacks were then imported into

ImageJ (version 1.43) for quantification. Only cultures with at least five YFP-expressing granule cells were used for this analysis, and all YFP-expressing cells within the granule cell layer of those cultures were quantified. A total number of YFP-expressing cells in the granule cell layer of each culture was obtained as well as the number of YFP-expressing cells that coexpressed calretinin, calbindin or NeuN.

Intrahippocampal kainic acid injection to induce granule cell dispersion in vivo

Ten-week-old male (25-30g) mice (n=12) were used for intrahippocampal kainic acid

(IHpKA) injection experiments (Heinrich et al., 2006, Suzuki et al., 1995, Bouilleret et al., 1999,

Riban et al., 2002). Mice were maintained under an inhaled anesthesia mixture containing

68.5% N2O, 30% O2 and 1.5% Isoflurane during IHpKA administration. Briefly, mice were placed in a stereotaxic frame and a single anterior-posterior cut was made along the scalp to expose the surface of the skull. A 1 mm diameter hole was drilled through the skull at the following coordinates, using bregma as a reference: 1.6 mm posterior and 1.6 mm lateral.

50 Next, a single 0.5 ml Hamilton syringe with a 25-guage needle was positioned 2.0 mm below dura using the stereotax holder, and 60-70 nl of a 20 mM solution of kainic acid in 0.9% sterile

NaCl (1.2-1.4 nmol kainic acid) was injected into the right dorsal hippocampus over 1 minute.

After the injection, the needle was left in the hippocampus for an additional 5 minutes to avoid reflux along the needle track. Following the injection, the needle was removed and the scalp re- sealed with TissueMend II (Veterinary Products Laboratories, Phoenix, AZ). Upon recovering from anesthesia, mice were monitored for 8-10 hours for behavioral seizure activity (Heinrich et al., 2006, Riban et al., 2002). During this time, animals experienced status epilepticus characterized by mild clonic movements of the forelimbs, rotations and immobility as previously described (Riban et al., 2002). At the end of the 8-10 hour observation period, mice that developed status epilepticus were weighed and given sufficient lactated Ringers+5% dextrose

(Hospira Inc, NDC No. 0409-7929-09) subcutaneously to offset any weight lost during surgery and status epilepticus. Animals recovered in a 31.5oC incubator with food and water ad libitum for two days. Hydration was given, as needed, to maintain pre-treatment body weight, with subcutaneous injections of lactated Ringers+5% dextrose.

One (n=4) and two weeks (n=8) after IHpKA injection (eleven and twelve weeks of age), mice were perfused and brains were prepared as described for 14-day-old animals. Uninjected eleven-week-old (n=4) and twelve-week-old (n=6) mice were perfused in an identical fashion to serve as naïve controls (n=6). Control animals were not surgically manipulated to avoid inadvertently disrupting granule cells by trauma alone.

Prox1 Immunohistochemistry

To assess granule cell layer dispersion following IHpKA injection, slides with up to four brain sections between bregma –1.22 mm and bregma –2.46 mm (Paxinos and Franklin, 2001) were immunostained with the granule cell marker Prox1. Briefly, slides were incubated for 1

51 hour in blocking solution [5% normal donkey serum (Millipore, S30-100ML) plus 0.5% Igepal

(Sigma-Aldrich, I3021-500ML) in 0.1 M PBS] at room temperature, and then overnight at 4oC in

1:2000 rabbit anti-Prox1 antibody (Chemicon, AB5475) in blocking solution followed by a four hour incubation at room temperature in 1:750 donkey anti-rabbit AlexaFluor 647. Slides were cover-slipped using Gel/Mount (Biomeda, M01) and sealed using clear nail polish.

Quantification of granule cell layer area

Granule cell layer dispersion was quantified from confocal image stacks of Prox1 immunoreactivity. Image stacks were captured at 3 m steps through the z-depth of the tissue using a 10X objective. The borders of the granule cell layer, defined by Prox1 immunolabeling of granule cells, were digitally encoded from the image files using Leica Application Suite software (2.0.0 build 1934) to generate area measurements. A total of 22 Prox1 immunostained dentate gyri were examined: four sections from one week naïve mice (1wk- naïve; n=4; one hemisphere in one brain section), four from one week kainic acid injected mice

(1wk-IHpKA; n=4; the hemisphere ipsilateral to the injection site in one brain section), six from two week naïve mice (2wk-naïve; n=6; one hemisphere in one brain section) and eight from two week kainic acid injected mice (2wk-IHpKA, n=8; the hemisphere ipsilateral to the injection site in one brain section).

Quantification of ectopic cells

Ectopic YFP-expressing granule cells – defined as granule cells with their cell bodies located at least one cell body within the dentate molecular layer – were quantified in twenty-two experimental mice. Counts were collected in one dentate per animal, the right hemispheres in naïve controls and the hemisphere ipsilateral to kainic acid injection in IHpKA mice.

Prox1 immunostaining was used to confirm the granule cell identity of all YFP-labeled ectopic cells. Ectopic cells were quantified by an investigator blinded to treatment group using a

52 DMI6000 inverted microscope equipped with a 63X objective (NA1.4) under epifluorescent illumination.

Identified YFP-expressing ectopic granule cells and Prox1 immunoreactivity were further assessed by confocal microscopy to determine whether they possessed recurrent basal dendrites. Endogenous YFP was excited using the 514-laser line (25% power), and emission wavelengths between 516 and 560 nm were collected. Prox1-immunoreactivity was excited using the 633-laser line, and emission wavelengths between 645 and 700 nm were collected.

Images were acquired through the z-depth of the granule cell of interest at 0.5 m steps using the 63X oil immersion objective with 2-3X optical zoom.

Statistics and data analysis

For all analyses, statistical significance was determined using Sigma Stat software

(version 2.03) using an  of 0.05. Parametric tests were used for data that met assumptions for normality and equal varience. Data that violated assumptions of normality or equal varience were either normalized prior to running a parametric test, or a non-parametric version of the test was run. Specific tests were used as noted in the results. Data are presented as either means±standard error of the mean, or as median (range). Statistical significance was accepted for P<0.05. Graphs were made using GraphPad Prism (version 2.01).

Figure Preparation

Unless otherwise stated, all images are maximum projections exported as TIFF files and imported into Adobe Photoshop. Some images were adjusted using Leica morphological erosion filter (radius=3; iterations=1) or deconvolution software, as noted. Brightness and

53 contrast of digital images were adjusted to optimize cellular detail using Adobe Photoshop.

Identical adjustments were made to all images meant for comparison.

Results

YFP is expressed in morphologically and phenotypically mature dentate granule cells.

The present study utilized a Thy1-YFP expressing mouse line to visualize hippocampal dentate granule cells (Feng et al., 2000). In order to determine which stage of neuronal development YFP is expressed in granule cells, we completed immunohistochemical analyses of calretinin, a calcium binding protein expressed during the early postmitotic stage of neuronal development (Brandt et al., 2003); calbindin, a marker of fully differentiated neurons (Sloviter,

1989; Kempermann et al., 1997; Vuksic et al., 2008) and NeuN, a postmitotic neuronal marker

(Mullen et al., 1992). A total of 5 out of 138 (3.62%) YFP cells in five cultures colocalized with calretinin, 182 out of 202 (90.09%) YFP cells in six cultures colocalized with calbindin and 127 out of 130 (97.69%) of YFP cells in six cultures colocalized with NeuN, Thus, YFP-expressing granule cells co-expressed NeuN and calbindin, but almost never colocalized with calretinin one week after culture preparation (Fig. 1). These immmunohistochemical data indicate that postnatal YFP-expressing granule cells phenotypically correspond to stage six of neuronal development in adult mammals (for review see Kempermann et al., 2004).

Additionally, all YFP-labeled cells in the time-lapse imaging experiment exhibited fully differentiated granule cell morphologies, including spine-coated dendrites projecting to the hippocampal fissure, somata located throughout the granule cell layer, and axons projecting into the hilus and stratum lucidum. Hilar basal dendrites were absent, as is typical for fully differentiated, but not immature, granule cells (Jones et al., 2003; Shapiro and Ribak, 2005).

Granule cells of this age and morphology are considered to be functionally mature (Jones et al.,

2003, Markakis and Gage, 1999, Liu et al., 2000, Ambrogini et al., 2004, Esposito et al., 2005,

54 Overstreet-Wadiche, Bensen and Westbrook, 2006, Zhao et al., 2006); however, we do recognize that these cells may be newly differentiated mature neurons as it takes four-to-seven weeks before newly differentiated cells become functionally indistinguishable from older granule cells (van Praag et al., 2002; Jessberger et al., 2003). Together, these findings indicate that the novel morphological plasticities described in this study occurred among fully differentiated, rather than immature granule cells.

Formation of recurrent basal dendrites by relocation of existing apical dendrites

Granule cells with recurrent basal dendrites are common in the epileptic brain (Ribak et al., 2000, Yan et al., 2001, Dashtipour et al., 2002), and one week after culture preparation (7

DIV), a dramatic increase in the percentage of granule cells with recurrent basal dendrites was observed in cultures (20.263.66%) relative to age-equivalent (14-day-old) intact control Thy1-

YFP expressing mice (2.782.78%; P=0.033, z-test). This finding demonstrates that culture preparation is sufficient to induce pathologies evocative of the epileptic brain.

We next sought to reveal the cellular processes underlying recurrent basal dendrite formation by serial confocal imaging. To ensure that only stable cultures were used, imaging was begun at 6-7 DIV. A total of 43 granule cells from 26 cultures were imaged. Approximately half of these cultures were also treated with the excitotoxin kainic acid, but as this produced no additional increase in recurrent basal dendrite formation over culture preparation alone (see

Supplementary Table 1), data from all cultures was pooled.

Eight of the 43 (18.6%) granule cells examined developed recurrent basal dendrites during the imaging period, revealing for the first time the cellular processes by which this pathology develops. Surprisingly, recurrent basal dendrites were not formed by growth of new

55 dendritic processes from the basal pole of the cell (in fact, no examples of dendritic growth were observed). Rather, existing apical dendrites were observed to shift positions from the apical to the basal pole of the cell (Fig.2, a-h), indicating – quite unexpectedly – that even though these cells are fully differentiated, dendritic origins are not stable relative to the cell body.

Conversion of dendritic branches into primary dendrites

A second, entirely novel, form of neuronal plasticity observed was the conversion of dendritic branches into primary dendrites. Specifically, secondary dendritic branches moved onto the cell body, such that they now became, by definition, primary dendrites (Fig. 3). In the most extreme example observed, a secondary dendritic branch moved its origin to the cell body and existed as a primary dendrite for several days (Fig. 4, a-d). Following swelling and contraction of the cell body, this dendritic branch – now primary dendrite – moved back into position as a dendritic branch, but on a different dendrite (Fig. 4, e and f). Branch-to-dendrite conversion was observed on 10 out of 43 (23.3%) granule cells in explants (two of these cells also developed recurrent basal dendrites).

Somatic translocation contributes to dendritic dysmorphogenesis

Careful examination of serial images revealed that, as an apical dendrite shifted to the basal pole of the cell, the primary dendritic segment (the segment between the soma and the first branch point) of an adjacent apical dendrite frequently decreased in length (Fig. 2, a-h).

Somal translocation into an apical dendrite would account both for the development of recurrent basal dendrites (as movement of the soma into one dendrite would shift neighboring dendrites towards the basal pole) and for the disappearance of dendritic branch points (as the soma encountered and absorbed these structures as part of its upward movement). To quantify this effect, changes in segment length were determined for the 16 cells exhibiting basal dendrite formation, branch point absorption or both. Eleven of these cells showed clear decreases in

56 segment length, supporting the conclusion that somatic translocation accounts for the observed dendritic plasticities. Indeed, in some cases, corresponding increases in primary segment length were observed for dendrites on the opposing side of the cell (as the soma moved towards a branch point in one dendrite, it moved away from branch points in other dendrites).

Based on these measurements, we were able to determine the total movement of the soma over the observation period, the average speed of movement and the maximum speed.

The eleven cells exhibiting clear movement shifted their somata on average 11.04±1.47 m towards the molecular layer, with the maximum movement observed being 20.45 m. Average speed of movement for these cells was 2.20±0.24 m/day, although somatic movement tended to occur in spurts, with a maximum speed observed of 9.6m/day. It is unclear how dendritic changes developed in the five cells that did not show obvious somatic translocation (including the cell depicted in Fig. 4). In some cases, the soma appeared to move first towards one dendrite, and then another, complicating interpretation; but the possibility that mechanisms apart from somatic translocation account for some plasticities should not be discounted.

Granule cell dysmorphogenesis is correlated with cell layer dispersion

Evidence of somatic translocation in the present study raises the possibility that this phenomenon may contribute to granule cell layer dispersion. Dispersion of cell body layers is typical of organotypic hippocampal explants, as cultures thin and spread during the incubation period. To determine whether dendritic dysmorphogenesis was correlated with granule cell dispersion, we conducted a post hoc analysis of the serial image files.

Granule cell dispersion was assessed by determining the area encompassed between three to five YFP-labeled granule cells in the initial and final images for each time series, with

57 greater area indicating movement of granule cells away from each other. Explants containing the most stable granule cells (n=10; see Fig. 5 for examples of stable cells) were compared to explants containing cells that exhibited the most dramatic changes (n=11). Explants exhibiting dendritic plasticities during the observation period displayed significantly more granule cell dispersion over this same period relative to explants containing stable cells (percent change for explants with disrupted cells, 27.0±16.3%; stable cells -3.9±2.6%; P=0.007, Mann-Whitney

RST), a finding which corresponds nicely with single cell evidence of somatic translocation in these same explants (see figure 2, e-l for an example of simultaneous dendritic dysmorphogenesis and granule cell dispersion). Together, these findings suggest that somatic translocation contributes, at least in part, to granule cell layer dispersion.

Granule cell dysmorphogenesis does not reflect acute cellular degeneration

Cultures were maintained for at least three days after the last live-imaging session to ensure that the plastic changes described here did not reflect terminal degenerative changes.

Serial-imaging data revealed a characteristic pattern of granule cell death. Dying cells first exhibited dendritic beading and a diminution of the YFP signal, followed by disintegration of the cell into a series of disconnected YFP-containing compartments (Supplemental Fig. 1). This process occurred rapidly, often leaving no evidence of the cell within 24 hours. Granule cells appearing to be in any stage of this process at the end of the experiment (3-5 days after the last imaging session) were excluded from the study.

Migration of granule cells and recurrent basal dendrite formation occur in vivo

The present study suggests that granule cell dispersion occurs following migration of the soma up a leading apical dendrite, which in turn leads to the formation of recurrent basal dendrites. If correct, granule cell dispersion and recurrent basal dendrite formation should occur in synchrony. To test this prediction, and to explore the cellular mechanisms by which recurrent

58 basal dendrites form in vivo, we utilized the IHp-KA injection model of epilepsy. In this model, kainic acid is injected unilaterally into the hippocampus, leading to status epilepticus, epileptogenesis and granule cell dispersion.

In this experiment, animals were sacrificed one and two weeks following IHpKA in order to identify in vivo susceptibility time points of granule cell dispersion and recurrent basal dendrite formation. In all twelve Thy1-YFP-expressing IHpKA mice (four 1wk-IHpKA and eight IHpKA-

2wk) acute behavioral status epilepticus as well as ipsilateral granule cell dispersion (Fig. 6) were observed. There was no significant difference found in the areas of the granule cell layer between either naïve control group (P=0.883), so data from these animals was pooled for the remainder of the study. The remainder of the analyses for this experiment focused on cells located in the dentate molecular layer, as these cells presumably migrated the greatest distances to arrive at this location. Firstly, to confirm that granule cell migration had occurred, the number of granule cells located in the molecular layer was determined for control and IHpKA mice. The number of granule cells in the molecular layer was significantly increased one and two weeks after IHpKA (Fig. 7, a-c) relative to controls (control, 15.6±1.4 ectopic cells/dentate;

1wk-IHpKA, 22.5±2.4; 2wk-IHpKA, 34.5±4.6; P<0.001, ANOVA on ranked data). The most parsimonious explanation for the appearance of these additional cells is that they migrated from the granule cell body layer to the molecular layer after IHpKA. Secondly, we asked whether recurrent basal dendrites were more common on these YFP-expressing granule cells relative to similarly-positioned granule cells in control and SE-1wk animals. A total of 276 Thy1-YFP- expressing granule cells located in the dentate molecular layer in eight brain sections from 2wk-

IHpKA mice, 92 cells in four brain sections from 1wk-IHpKA and 175 cells from ten brain sections in naïve mice were examined. The incidence of granule cells with recurrent basal dendrites (Fig. 7, d-f) was increased one (p=0.025) and two (p=0.035) weeks after IHp-

KA relative to controls (control, 0.75±0.29 ectopic cells with RBD’s/dentate; 1wk-IHpKA,

59 3.25±0.85; 2wk-IHpKA, 3.25±1.07, ANOVA on ranked data, p<0.034). Interestingly, between one and two weeks, the number of cells with recurrent dendrites was stable, although whether this reflects different mechanisms of cell movement, partial recovery of neuronal structure (see figure 2, c-d), neuronal loss, or other factors is not clear. These data demonstrate a clear positive association between aberrant granule cell migration into the dentate molecular layer and formation of recurrent basal dendrites in the intact brain.

60 Discussion

Dysmorphic neurons are present in the dentate gyrus of epileptic animals and humans; however, the neuroplastic mechanisms by which these pathologies develop remain unclear.

Here, using real-time imaging of living granule cells in organotypic cultures, we demonstrate that fully differentiated granule cells with initially normal morphologies can contort over a period of days, producing dysmorphic neurons remarkably similar to those observed in patients with epilepsy. Surprisingly, the process by which these changes evolve revealed two novel forms of neuronal plasticity. Firstly, apical dendrites were observed to shift their origins to the basal pole of the cell. Secondly, absorption of dendritic branch points by the soma was observed, converting these dendritic branches into primary apical dendrites. Intriguingly, these plastic changes occurred in synchrony with a shortening of apical dendrite initial segments and dispersion of the granule cell layer, leading us to speculate that they were a consequence of somatic movement up a leading apical process. To test this prediction, and to explore the cellular mechanisms by which recurrent basal dendrites form in vivo, we utilized the intrahippocampal kainic acid injection model of epilepsy. Morphological analysis of dispersed cells, which moved into the dentate molecular layer, revealed that they exhibited the same types of dendritic anomalies observed evolving in vitro. This suggests that they also migrated to their new positions by somatic movement up a leading apical process. Together, these data reveal two novel forms of neuroplasticity and provide critical insights into the mechanism by which fully differentiated neurons are able to migrate to new positions in the epileptic brain.

Somatic translocation leads to granule cell dispersion and dendritic dysmorphogenesis

In the present study, apical dendrites shifted to the basal pole of the cell, dendritic branches became apical dendrites, and granule cell bodies moved radially away from each other. One phenomenon that could account for all these plastic changes is translocation of the granule cell soma into an apical dendrite. In the model depicted in figure 8, somatic

61 translocation would lead to the shortening of dendritic initial segments, and in extreme cases, branch point absorption. Continued movement of the soma up an apical dendrite would gradually shift adjacent dendrites that are “left behind” to the basal pole of the cell, while simultaneously displacing the soma into the dentate molecular layer. No other single cellular process can account for all of these plastic changes, leading us to conclude that some form of somatic translocation is occurring.

Somatic migration of immature neurons

Somatic translocation is a principal migratory mechanism for developing neurons (Rakic,

1971; Rakic, 1972; Hatten, 1999; Nadarajah et al., 2001; Nadarajah et al., 2003), and occurs when the nucleus and soma are displaced into an unbranched leading process so that the neuron migrates in an undisrupted, straight trajectory (Rakic, 1971; Rakic, 1972; Nadarajah et al., 2001; Gupta et al., 2003). Notably, somatic translocation is believed to be the means by which newborn granule cells move from the subgranular proliferative zone to the granule cell layer (Eckenhoff and Rakic, 1984; Morozov et. al., 2006). Neuronal precursors can also migrate by a related process, known as branched somatic migration (Nadarajah et al., 2003; Gupta et al., 2003). Branched migration is characterized by branching of the leading process (Nadarajah et al., 2003; Gupta et al., 2003), which allows neurons to migrate by advancing their soma from one branch point to the next in a radial direction with a ‘zig-zag’ trajectory. This ultimately leads to the shortening of the initial segment of the leading process and retraction of side branches

(Nadarajah et al., 2003; Gupta et al., 2003). In general, branched somatic migration is slower than migration through an unbranched process (Gupta et al., 2003; Ohshima et al., 2007).

Somatic migration of fully differentiated neurons

Somatic migration has not been previously described among mature granule cells. It is clear, however, that they can shift position in vivo. Previous studies using the IHp-KA injection

62 model of epilepsy, which produces pronounced granule cell dispersion (Heinrich et al., 2006;

Bouilleret et al.; 1999, Riban et al., 2002), have revealed that movement of fully differentiated granule cells underlies the phenomenon (Heinrich et al., 2006; Nitta et al., 2008). The present data suggest that migration occurs via a process reminiscent of somatic translocation observed among migrating populations of neuronal precursors, although whether the underlying mechanisms are similar is not known. Clearly, migration rates are much slower; 2.20±0.24

m/day in the present study vs. 60 m/hr in neuronal precursors (Nadarajah et al., 2001;

Nadarajah et al., 2003). This difference in migration rates, however, does not necessarily indicate that different mechanisms are active: Multiple leading processes appears to slow the movement of neuronal precursors (Gupta et al., 2003; Ohshima et al., 2007), and a similar inference might occur for fully differentiated cells with multiple apical dendrites.

If both mature granule cells and granule cell precursors undergo somatic migration, then why do dendritic abnormalities only appear following migration of the former? The answer may lie both in the extent of process development and in the degree of synaptic innervation of these processes. Unlike mature granule cells, granule cell precursors typically possess a single prominent radial process which terminates in the inner molecular layer, with occasional highly labile side processes, all of which receive minimal synaptic input (Jones et al., 2003; Schmidt-

Hieber et. al., 2004; Shapiro and Ribak, 2006). Movement of the soma within the main radial process would produce little distortion, and any side branches “left behind” by such migration could be retracted. By contrast, mature granule cells – particularly the oldest cells located along the molecular layer border – typically have multiple apical dendrites, such that there is no single radial path in which the soma could translocate. Moreover, the dendrites of these mature cells are extensively innervated. Synaptic innervation may act to fix the dendritic membrane in place through the activity of cell adhesion molecules, preventing process retraction. Movement of the soma into an apical process, therefore, would unavoidably distort the dendritic tree as

63 synaptically fixed regions are left behind. Indeed, a similar mechanism has been proposed to explain the formation of axonal loops among a unique population of hippocampal interneurons.

Although occurring in the normal brain, these interneurons parallel the current findings in that they continue to migrate by somatic translocation after forming synaptic contacts with target neurons (Morozov et. al., 2006). In the model proposed by the authors, the initially straight axon is contorted into a loop as the soma migrates past the point of synaptic innervation.

Preservation of these contacts requires that affected axon segments be left behind as a loop structure. In that light, the appearance of dendritic loops (Fig. 8, b) may not be coincidental.

Conclusions

Granule cells with features strikingly similar to those observed evolving here are frequently observed in human epilepsy (Scheibel and Scheibel, 1973; von Campe et. al., 1997; da Silva et al., 2006). While the functional consequences of these changes are unclear, it is difficult to imagine that they are beneficial; indeed, patients with epilepsy exhibit a high incidence of comorbid neurocognitive deficits. The present demonstration that a novel form of neuronal plasticity may contribute to this pathology is a first step towards elucidating the underlying mechanisms. These mechanisms, in turn, may ultimately prove to be fruitful targets for novel therapies.

64 Figure 1. Postmitotic age of YFP-expressing granule cells in vitro. (a-c) Representative confocal maximum projections of YFP-expressing dentate granule cells at seven days in vitro.

(a-i, a-ii) Optical sections through thae granule cell soma showing the lack of colocalization

(yellow asterisk) between Thy1-YFP-expression (a-i) and calretinin-immunoreactivity (a-ii yellow asterisk). (b-i, b-ii) Optical sections through a granule cell soma illustrating the colocalization of

Thy1-YFP-expression (b-i, cyan asterisk) with calbindin (b-ii, yellow asterisk). (c-i, c-ii) Optical sections through a granule cell soma demonstrating the colocalization of Thy1-YFP-expression

(c-i, red asterisk) with NeuN (c-ii, yellow asterisk). Scale bars, 30 m.

65 Figure 2. Recurrent basal dendrite formation and granule cell layer dispersion. Serial confocal maximum projections of YFP-expressing granule cells showing conversion of apical dendrites into recurrent basal dendrites and granule cell layer dispersion. (a-d) Images taken over one week showing the formation of a recurrent basal dendrite at 10 DIV, and subsequent partial recovery (white arrow). Blue circles denote the distance between the origin and the first branch point on adjacent dendrites (the primary dendritic segment). Note the contraction of this distance over the course of the experiment. (e-h) Images of an initially normal dentate granule cell that develops two recurrent basal dendrites at 13 DIV. Note the contraction of the initial segment of central dendrite (distance between the two blue dots), suggesting somatic movement towards the first branch point. (i-l) Images of granule cell layer containing the cell shown in e-h (purple arrow). Note the simultaneous appearance of recurrent basal dendrites in e-h and granule cell dispersion in i-l. Scale bar: (a-d) 10 m; (e-h), 50 m; (i-l) 250 m.

66 Figure 3. Branch to dendrite conversion. Serial confocal maximum projections of a YFP- expressing dentate granule cell exhibiting conversion of a dendritic branch to a primary dendrite.

(a-c) Images taken over six days exhibiting the translocation of a dendritic branch (blue arrow) to the cell body (blue arrow) at 9 DIV, thus converting it to a primary dendrite. Blue circles denote the proximal dendritic segment on an adjacent dendrite. The distance between these points shrinks over time, indicative of somatic translocation into this process. Scale bar, 50 m.

67 Figure 4. Extreme example of branch to dendrite conversion. Serial confocal maximum projections of deconvolved images of a YFP-expressing dentate granule cell exhibiting the conversion of a dendritic branch to a primary dendrite and back to a dendritic branch. The imaged cell exhibits a dendritic branch (a, blue arrow), which moves to the cell body after 8 DIV

(b, c). Following hypertrophy of the cell body on 12 DIV (d), the previous dendritic branch – now primary dendrite – precedes to move back into position as a dendritic branch, this time however, on a different dendrite (e, f). Scale bar, 25 m.

68 Figure 5. Granule cells with normal morphology. Serial confocal maximum projections of YFP- expressing granule cells exhibiting stable dendritic structure. Representative images of two

YFP-expressing granule cells (a-c and d-g, respectively) not exhibiting dendritic rearrangements or granule cell dispersion over the course of the imaging period. Scale bar, 50

m.

69 Figure 6. Granule cell dispersion induced by IHpKA. Representative confocal maximum projections of Prox1 immunoreactivity within the dentate granule cell layer from (a) the right hemisphere of a naïve mouse, (b) the ipsilateral (right) hemisphere of a 1wk-IHpKA mouse and

(c) the ipsilateral hemisphere of a 2wk-IHpKA mouse. Scale bar = 200 m. (d) Bar graph of the mean area of the dentate granule cell layer per treatment group, which was acquired by outlining Prox1 immunoreactivity, a marker of granule cells, in optical sections. The y-axis is the mean area (m2) of Prox1 immunoreactivity, and the x-axis plots the data by treatment group.

Error bars are SEM. *P<0.05, **P<0.01, ANOVA on ranked data.

70 Figure 7. Ectopic granule cells and recurrent basal dendrites occur in vivo. Confocal maximum projections illustrating the location of the granule cell layer (GCL), inner molecular layer (IML) and outer two-thirds of the molecular layer (MML+OML) in a (a) naïve mouse, (b) 1wk-IHpKA mouse and (c) 2wk-IHpKA mouse. Note the greater numbers of ectopic granule cells in the inner (white arrows) and outer two-thirds (light blue arrows) of the dentate molecular layers in the ipsilateral hemisphere of a SE-2wk mouse. (d-f) Examples of YFP-expressing dentate granule cells with recurrent basal dendrites (white arrowheads) from the ipsilateral hemisphere of KA-SE mice. Granule cells are located in the molecular layer of the dentate gyrus. The insets in (d-f) are optical sections through the granule cell soma showing the colocalization of

Thy1-YFP-expression (d-, e-, f-i) and Prox1-immunoreactivity (d-, e-, f-ii). Note, d and e are imaged at the same magnification. Scale bars: (a-c) 30 m; (e, e-ii, f, f-ii) 20 m.

71 Figure 8. Model depicting granule cell dysmorphogenesis following somatic translocation into an apical dendrite. (a) As the soma moves into the initial segment of the left apical dendrite (1), the right apical dendrite is “left behind”, gradually shifting its position from the apical to the basal pole of the cell (2). Continuation of this process leads to a shortening of the initial dendritic segment on the left (3), and conversion of the right apical dendrite into a recurrent basal dendrite (4). In extreme cases, further migration of the soma leads to the absorption of branch points, converting these structures into primary dendrites (5), and the formation of dendritic loops projecting towards the hilus (6). (b) An example of a granule cell exhibiting a recurrent basal dendrite with a pronounced dendritic loop and an unusually large number of apical dendrites; the putative end product of the processes outlined in a. This process was ongoing during the imaging period, evident in the increasing length of the recurrent basal dendrite between 8 and 9 DIV. Note that the axon (white arrowhead) projects off the base of the dendritic loop, suggesting that this was the original location of the cell body. Scale bar, 50 m.

72 Supplemental Table 1 Recurrent basal dendrite formation and branch-to-dendrite conversion in control and kainic acid treated explants.

Control Kainic acid Mann-Whitney rank sum test (n=23) (n=20) P value Mean percentage of granule cells with 13±7 25±10 0.51 recurrent basal dendritesSEM

Mean percentage of granule cells with 22±9 25±10 0.86 branch-to-dendrite conversionSEM

73 Supplemental Figure 1 Dying YFP granule cell. Serial confocal maximum projections of a

YFP-expressing granule cell exhibiting disintegration of YFP. (a) Confocal maximum projections of a YFP-expressing dentate granule cell after 6 DIV, and during exposure to kainic acid one day later (b). This cell died during exposure, as is evident by its disintegration (b).

Scale bar, 50 m.

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82 Acknowledgements:

This work was supported by Cincinnati Children’s Hospital Medical Center and the National

Institute of Neurological Disorders and Stroke (SCD, Award Numbers R01NS065020 and

R01NS062806). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the

National Institutes of Health. Thy1-YFP mice were generously provided by Dr. Guoping Feng

(Duke University, Durham, North Carolina). We would also like to thank Keri Kaeding, Dr. Chia-

Yi Kuan, Dr. Andreas Loepke and Stefanie Bronson for useful comments on earlier versions of this manuscript.

83 Chapter 3

Reduced neurogenesis of hippocampal subventricular zone progenitor cells exposed to neonatal status epilepticus

84 Abstract

The developing brain exhibits substantially reduced seizure thresholds relative to adults, and correspondingly, seizures are common in young animals and children. While the immature brain appears to be comparatively resistant to seizure-induced cell death, early-life seizures are associated with cognitive deficits and poorer prognosis in both animals and humans. Dentate granule cells may be particularly vulnerable to this insult, as granule cell neurogenesis continues through the postnatal period. To explore the impact of early-life seizures on granule cell development, we have developed a genetic fate-mapping strategy to characterize the types of cells generated in the dentate gyrus following SE. Gli1-CreERT2 mice expressing a conditional, inducible form of cre-recombinase were crossed to a GFP reporter line (Nakamura et al., 2006). Bi-transgenic offspring were treated with tamoxifen at five days-of-age, leading to the persistent expression of GFP in subgranular zone putative stem cells and their progeny.

Two days later, status epilepticus was induced with 1 mg/kg kainic acid (control animals received saline). Mice were sacrificed two months after treatment. Recombination was efficient and specific, leading to bright GFP expression among SGZ stem cells and their progeny, the latter including putative progenitor cells, immature granule cells, mature granule cells and glial cells. Other than immature and mature granule cells, no other neuronal types were observed in the hippocampus. This was true both for control and kainic acid-treated bi-transgenic mice.

Strikingly, however, fewer GFP-expressing progenitor cells, immature granule cells and mature granule cells were present in the hippocampi of animals examined two months after early-life

SE relative to saline-treated mice. This finding suggests either that GFP-expressing stem/progenitor cells exposed to kainic-acid become inactive (or die) shortly after treatment, or that the progeny of labeled stem cells active weeks after treatment fail to survive. In either case, the net effect appears to be a reduction in the number of granule cells produced by the cohort of exposed stem/progenitor cells; a deficit that could contribute to learning impairments associated with early-life seizures in this model.

85 Introduction

Seizures are more common during the early-life period compared to other periods of life

(Camfield et al., 1996; Lanska et al., 1995), and peak in frequency within the first two years of life (Hauser, 1994). This suggests that the developing brain is more susceptible to the development of epilepsy, which is indeed the case since the risk of subsequent unprovoked seizures is between 25-40% within the first two years following a first-ever unprovoked seizure episode (Maytal et al., 1989; Eriksson and Koivikko, 1997). Even though the developing brain is more susceptible to seizure activity than its mature counterpart, animal models of early-life seizures propose that the immature brain is fairly resistant to seizure-induced cell loss when compared to epilepsy models in adult rodents (Nitecka et al., 1984; Holmes et al., 1998; Toth et al., 1998; Bender et al., 2003; Dube et al., 2001). This is not to imply that early-life seizures are benign, especially since many studies indicate that prolonged or frequent seizures in patients can lead to cognitive deficits later in life (Farwell et al., 1985; Hauser, 1990; Hauser, 1995;

Austin and Dunn, 2002; Cormack et al., 2007). Correspondingly, behavioral results from previous studies using animal models of early-life seizures parallel these clinical observations

(Lynch et al., 2000, Sogawa et al., 2001, de Rogalski Landrot et al., 2001, Sayin et al., 2004;

Cornejo et al., 2007).

Recently, several laboratories have shown an increase in neurogenesis following seizures in adult models of epilepsy (Bengzon et al., 1997; Parent et al., 1997; Parent et al.,

1998; Scott et al., 1998; Gray and Sundstrom, 1998). This finding raises concern regarding the consequences of early-life seizures on neurogenesis since the peak of dentate neurogenesis occurs during early postnatal life (Schlessinger et al., 1975; Bayer, 1980; Altman and Bayer,

1990). However, studies using various models of early-life seizures remain inconclusive due to reports of both increases and decreases in neurogenesis following prolonged or frequent

86 seizures, which may be due to differences in animal age, frequency of seizures evoked and the model used (Holmes et al., 1998; Bender et al., 2003; Wasterlain, 1976; McCabe et al., 2001;

Dong et al., 2003). Additionally, recent studies from our laboratory (Walter et al., 2007) as well as others have demonstrated that seizures in adult animals disrupt the integration and structure of adult-generated granule cells. Specifically, these new neurons were found to exhibit hilar basal dendrites (Walter et al., 2007; Shapiro et al., 2005; Jessberger et al., 2007; Kron et al.,

2010) and become ectopically localized to the hilus (Walter et al., 2007; Jessberger et al., 2005;

Parent et al., 2006; Kron et al., 2010). Together, these findings indicate that developing dentate granule cells in the adult brain are extremely sensitive to seizure-induced disruption, which raises concern regarding the integration of these neurons when exposed to early-life seizures.

Few studies have focused on the fate of SGZ stem/progenitor cell progeny and integration of granule cells following early-life seizures. Here, we examined the impact of early- life SE on neurogenesis and granule cell integration using a granule cell-specific fate-mapping strategy in a mouse model system. By crossing conditional, inducible Gli1-CreERT2 mice to a

GFP reporter line, we selectively induced the expression of GFP in SGZ putative stem cells.

This allowed us to assess the effect of early-life SE on the fate of exposed stem/progenitor cells’ progeny as well as the subsequent development of postnatally generated dentate granule cells.

87 Materials and Methods

All procedures conformed to National Institutes of Health and institutional guidelines for the care and use of animals.

Animals

To label SGZ neural stem cells and their progeny with GFP, a transgenic mouse line expressing tamoxifen-inducible cre-recombinase under control of the Gli1 promoter (GLI-

Kruppel family member), which drives expression in sonic hedgehog responding stem cells (Ahn and Joyner, 2004; Ahn and Joyner, 2005), was crossed to an enhanced GFP-reporter line with an upstream “floxed” stop codon (CAG-CAT-EGFP reporter mice (Nakamura, Colbert and

Robbins, 2006). Mice were donated courtesy of Dr. Alexandra Joyner (Gli1-CreERT2; Sloan-

Kettering Institute) and Dr. Jeffrey Robbins (CAG-CAT-EGFP reporter mice; Cincinnati

Children’s Research Foundation). Mice were maintained on a C57BL/6 background. Six bi- transgenic male and female mice hemizygous for the Gli1-CreERT2 transgene and heterozygous for the GFP reporter transgene (control: 2 male, 1 female; kainic acid treatment: 2 male, 1 female) were used for experiments.

Polymerase chain reaction analysis of genomic DNA

DNA from bi-transgenic offspring of Gli1-CreERT2 hemizygous and GFP reporter homozygous breeder pair parents was extracted using the DNeasy blood and tissue kit (Qiagen,

69506) and genotyped for cre recombinase using the following primer sequences: 5’,

CGTTTTCTGAGCATACCTGGA and 3’, ATTCTCCCACCGTCAGTACG, which generated a 471 base pair DNA fragment (Supplemental Fig. 1). Polymerase chain reaction (PCR) was completed using GoTaq® Green Master Mix (Promega Corporation, Madison, WI, M7123) and the following thermal cycling parameters: initialization at 95˚C for 3 minutes, denaturation at

95˚C for 45 seconds, annealing at 60˚C for 45 seconds, extension at 72˚C for 1 minute, final

88 elongation at 72˚C for 1 minute and a final hold at 4˚C (denaturation, annealing and extension steps were repeated for 38 cycles).

Tamoxifen and kainic acid treatments

Bi-transgenic offspring were given a subcutaneous (s. c.) injection of 140 mg/kg tamoxifen (Sigma, T5648) dissolved in corn oil at five days-of-age to induce GFP expression.

Two days later, at postnatal day seven, mice were given s. c. injections of Lactated Ringer’s

(Hospira Inc, NDC No. 0409-7953-09) or 1 mg/kg kainic acid dissolved in Lactated Ringer’s to induce SE. SE in this model is characterized by behavioral convulsions, such as forelimb clonus, hyperextension of the hindlimbs and ‘swimming’ behavior, which usually began 20-30 min after administration of kainic acid and lasted six-to-seven hours in length (Stafstrom et. al.,

1992). Pups were separated from dam during this period, but immediately returned to their home cage following the cessation of SE. Two months after treatment, mice were overdosed with 100 mg/kg pentobarbital, and transcardially perfused with PBS+1U/ml heparin followed by

2.5% paraformaldehyde and 4% sucrose in 0.1 M PBS, pH 7.4. Brains were removed, post- fixed, cryoprotected in sucrose and snap frozen in 2-methyl-pentane at -25oC. Coronal sections were cut on a cryostat at 60 m, slide mounted and stored at –80oC until use.

GFP Immunohistochemistry

Slides with up to four brain sections, between bregma –1.46 and –2.46 (Paxinos and

Franklin, 2001), were thawed in 0.1 M PBS, pH 7.4, and then incubated in 0.1% H2O2 for 30 min to quench endogenous peroxidase activity. Slides were then incubated for 1 hour in blocking solution [5% normal goat serum (Invitrogen, 16210-072) plus 0.5% Igepal (Sigma-Aldrich,

I3021-500ML) in 0.1 M PBS] at room termperature, and then overnight at 4oC in 1:100 rabbit anti-GFP antibody (Chemicon, AB3080) in blocking solution. Slides were then rinsed in blocker.

A tyramide signal amplification (TSA) kit (Molecular Probes, T20922) was used to detect the

89 GFP. Briefly, brain sections were incubated for 2 hours in 1:100 horseradish peroxidase (HRP)- conjugated goat anti-rabbit secondary antibody in blocking solution, rinsed in PBS and then incubated for 30 minutes with 1:100 Alexa-Fluor 488-labeled tyramide in amplification buffer/0.0015% H2O2 in order to enzymatically amplify the Alexa Fluor 488 signal. Slides were then rinsed in PBS, dipped in distilled water, dehydrated in alcohol series (50%, 70%, 95%,

100%, 100%), cleared in xylenes, and mounted with Krystalon (Harleco). Lastly, there was no difference between bregma levels used in the analyses of this study (control, -2.180.07; SE, -

1.900.22; P=0.296, t-test).

Confocal microscopy

All data collection was conducted with the investigator blind to treatment conditions.

Images along the length and through the z-depth of the dentate gyrus were acquired of GFP- expressing cells at 1X optical zoom using a Leica SP5 confocal microscope equipped with 10X

(0.3 NA) and 63X (1.4 NA) objectives in 5 m and 1 m steps, respectively. GFP expression was imaged using the 488-laser line, and emission wavelengths between 495 and 556 nm were collected.

Characterization of GFP-expressing cell-type

GFP-expressing cells were characterized by cell type and quantified from confocal image stacks of GFP immunostained brain sections. Image stacks were acquired through the z- depth along the entire dentate gyrus using a 63X objective. These image stacks were then imported into ImageJ (version 1.43) for quantification of (1) the total number of GFP-expressing cells, and (2) the number of GFP-expressing cells of each cell type. The number of GFP- expressing cells/image stack in each dentate was quantified using a variation of the optical dissector method (Peterson, 1999). GFP-expressing cells within optical sections from the first

90 and last 5 m of the image stack were exluded from the analysis. Within each image stack along the dentate, GFP-expressing cells were also excluded from the analysis if they touched the bottom or right portions of the ImageJ counting frame. The number of GFP-expressing cells/dentate gyrus was quantified in one brain section (two dentate gyri) from each animal, and numbers averaged per animal. Cell type was identified by cellular morphology, as described in the results. Quantification of cells by cell type is expressed as absolute number as well as by proportion of total GFP-expressing cell number, due to group differences in overall number of cells labeled. Caveat: since only one brain section per animal was included in this analysis, the data and conclusions presented here are only relevant to the dorsal hippocampus between bregma levels of –1.46 and –2.46, and may differ in other regions of the hippocampus.

GFP-expressing cell death in corpus callosum

In order to ascertain whether the population of stem/progenitor cells in the hippocampal subventricular zone, which populate the corpus callosum (Navarro-Quiroga et al., 2006), were affected following SE, the number of GFP-expressing in the corpus callosum was assessed under epifluorescence with the 10X objective (0.3 NA) in brain sections stained with GFP. Cell death or loss of GFP-expressing cells, was scored using a semi-quantitative scale: (1) no obvious cell loss; (2) <25% cell loss; (3) ~50% cell loss and (4) >90% cell loss.

One brain section (two dentate gyri) was examined from each animal, and the scores were averaged per animal.

Implantation and EEG recording

In order to ensure that treatment with kainic acid on postnatal day seven lead to SE, a subset of Gli1-CreERT2-/-;GFP+/- mice (n=4) were cortically implanted with single stainless steel electrodes. The electrodes were made out of bent 25 gauge needles that were soldered to stainless steel wires, which had connectors at the end of the wire. These connecters were

91 plugged into an amplifier so that electroencephalography (EEG) activity could be recorded during kainic acid treatment. Mice were briefly anesthetized with 3.5% isoflurane in oxygen, and then maintained at 1.5% isoflurane throughout the implantation procedure. Sterile techniques were used for this procedure, and the scalp was sterilized with propidium-iodide. A single anterior-posterior cut was made along the scalp to expose the surface of the skull. Next, two small holes, about 0.5 mm, were drilled on the left and right side of the skull approximately 0.5 mm lateral to the midline, in line with bregma. The electrode was implanted just slightly into the right cortex. On the left side of the skull, a screw soldered to transmitter wire acted as a reference lead, which also had a connector on its end to plug into the amplifier. The electrode and screw were secured with dental acrylic to the skull, which also acted to close the incision site. Upon recovering from anesthesia, 1 hour of baseline EEG was recorded. Following this baseline, mice were administered 1 mg/kg kainic acid dissolved in Lactated Ringer’s (n=3;

Hospira Inc, NDC No. 0409-7953-09) or Lactated Ringer’s (n=1), which acted as vehicle control.

Mice were monitored for epileptiform activity recorded by EEG as well as graded for behavioral manifestations of seizures (Ben-Ari et al., 1984; Tremblay et al., 1984; Stafstrom et al., 1992;

Danzer et al., 2004), which lasted between six-to-seven hours.

Statistics and data analysis

Data are presented as mean±standard error. All statistical tests were performed using

Sigma Stat (version 2.03). Parametric tests were run since the data passed assumptions for normality and equal variance. Specific tests used are noted in the results. Statistical significance was accepted for P<0.05.

Figure Preparation

Unless otherwise stated, all images are maximum projections exported as TIFF files and imported into Adobe Photoshop. All images were adjusted using Leica morphological erosion

92 filter (radius=3; iterations=1) to minimize background artifact. Additionally, images were pseudo-colored when structures belonging to other cells obscured the cell of interest in the two- dimensional format presented here. Brightness and contrast of digital images were adjusted using Photoshop (version 7.0) to maximize detail. In all cases, identical adjustments were made to images meant for comparison.

93 Results

Kainic acid treatment leads to the development of status epilepticus

In order to ensure that treatment with kainic acid on postnatal day seven lead to SE, a subset of mice underwent simultaneous EEG recording and grading for behavioral manifestations of seizures during a pilot experiment using 1mg/kg kainic acid (n=3), a dose used in the literature to induce epileptiform activity as well as behavioral status epilepticus (Ben-

Ari et al., 1984, Tremblay et al., 1984, Stafstrom et al., 1992; Danzer et al., 2004) or vehicle control (n=1). Animals treated with kainic acid in this pilot experiment exhibited epileptiform activity (Fig. 1) as well behavioral seizures characterized by forelimb clonus, hyperextension of the hindlimbs and ‘swimming’ behavior, consistent with previous behavioral observations (Ben-

Ari et al., 1984, Tremblay et al., 1984, Stafstrom et al., 1992; Danzer et al., 2004). No seizure activity was observed in the implanted control animal.

Cre-mediated recombination is efficacious and specific

A second subset of mice (n=4) was generated in order to assess Cre-mediated recombination in this experimental paradigm. The conditions in which recombination was tested are as follows: (1) bi-transgenic mouse administered tamoxifen at postnatal day five and vehicle on postnatal day seven; (2) bi-transgenic mouse not administered tamoxifen, but given vehicle on postnatal day seven; (3) bi-transgenic mouse not administered tamoxifen, but given kainic acid to induce SE on postnatal day seven and lastly, (4) a heterozygous CAG-CAT-EGFP reporter mouse given tamoxifen on postnatal day five. All of these mice were sacrificed on postnatal day twelve. Euthanization and tissue processing was completed similarly to experimental mice in this study. Postnatal day twelve was chosen so that cre-mediated recombination could be assessed in a timely manner prior to starting the major experiment of this study. From this pilot experiment, it was determined that recombination was specific, and led to bright GFP expression among large numbers of cells in the dentate gyrus as well as along

94 the corpus callosum/alvus in tamoxifen-treated bi-transgenic mouse (Fig. 2a, white asterisks).

No endogenous GFP expression was found in the mice that were not exposed to tamoxifen

(Fig. 2b, c). Additionally, we did not observe GFP expression in the tamoxifen-treated heterozygous CAG-CAT-EGFP reporter mouse (data not shown).

Gli1-CreERT2;GFP expression in the adult brain

In both control and kainic acid-treated animals, GFP-labeled cells were found within the two neuroproliferative zones that are active in the postnatal brain, the subventricular zone (not shown) and the hippocampal SGZ (Fig. 3). Additionally, scattered GFP-labeled cells were present within the corpus callosum, alveus, hippocampus and cortex (Fig. 3). The morphology and localization of these GFP-labeled glial cells is consistent with previous cell-fate studies, which indicate that the cells in the corpus callosum are oligodendroytes (Fig 3., white arrowheads), whereas those in the hippocampus and cortex are astrocytes (Fig. 3, blue arrowheads; Ahn and Joyner, 2005; Navarro-Quiroga et al., 2006).

Gli1-CreERT2;GFP expression in the adult dentate gyrus

Five different cell types expressed GFP within the dentate gyrus of both control and kainic acid-treated mice. Since GFP was expressed throughout the entire cell, and not localized to any one part of the cell, we were able to characterize the various cell types present within the denate gyrus based on cellular morphology as described below.

1) Putative neural stem cells: these cells are also known as the type-1 cell (Kempermann et al., 2004) or B cell (Seri et al., 2001), and they were located in the subgranular zone of the dentate gyrus (Fig. 4a). This is the thin layer between the hilus and granule cell layer. These cells appeared very similar to those found using the Nestin-GFP mouse (Mignone et al., 2004).

In these mice, neural stem cells were described as possessing a single radial projection that

95 branched into two or three processes within the granular cell layer and terminated in the molecular layer with arbor-like clusters of short processes (Mignone et al., 2004). These GFP- expressing putative stem cells are thought to give rise to the remainder of the cell-types labeled with this genetic cell-fating strategy.

2) Putative progenitor cells: these cells, also known as type-2 and type-3 transient amplifying cells (Kempermann et al., 2004) or early subtypes of D cells (D1 and 2) (Seri et al.,

2001), were characterized by a cell body located within the SGZ (Fig. 4b). Occasionally, these cells were found at the SGZ/hilar border (Fig 4b), and were also observed to possess horizontally projecting aspiny processes parallel to the granule cell layer (not shown) (Esposito et al., 2005).

3) Glial cells: these cells were characterized by a small cell body located anywhere in the dentate and numerous thin, aspiny processes projecting outwards in a stellate fashion from the cell body (Fig. 4c), similar to previously described protoplasmic astrocytes (Levison et al., 1999).

4) Immature granule cells: these cells exhibited somata within the granule cell layer and aspiny dendrites projecting into the molecular layer (Fig. 4d) (Esposito et al., 2005; Jones et al.,

2003; Overstreet et al., 2004; Overstreet-Wadiche et al., 2006). More specifically, these cells correspond to stage 5 of neuronal development (Kempermann et al., 2004) or later class D cells

(D3) (Seri et al., 2001).

5) Fully differentiated granule cells: these cells were also found with cell bodies located in the granule cell layer. They also possessed spine-coated, arborized dendritic trees that projected to the hippocampal fissure (Fig. 4e; Esposito et al., 2005; Jones et al., 2003). Granule cells of this morphology are considered to be functionally mature (Jones et al., 2003; Esposito et

96 al., 2005; Overstreet-Wadiche et al., 2006; Markakis and Gage, 1999; Liu et al., 2000;

Ambrogini et al., 2004; Zhao et al., 2006), correspond to stage 6 of neuronal differentiation

(Kempermann et al., 2004), and yet may be younger mature cells. For the study, these cells are referred to as mature cells.

Early-life status epilepticus leads to reduced numbers of GFP-labeled cells

Using the above morphological criteria, the number of progeny generated from sonic hedgehog responsive putative stem cells under control conditions as well as those exposed to early-life status epilepticus was quantified. From this analysis, we found the total number of

GFP-expressing cells generated following early SE was reduced by 35.5% (Fig. 3; total GFP- labeled cells per brain section; control, 293.3325.65; SE, 104.0011.53; P<0.01, t-test).

Early-life status epilepticus disrupts GFP-labeled stem cell progeny cell-fate

By quantifying the five cell types within the dentate, we did not find significant changes in the ratios of GFP-expressing cell types generated following early-life SE (Table 1). We next analyzed the absolute numbers of GFP-labeled cell-types. By doing so, we found there to be significantly fewer putative progenitor cells, immature granule cells and mature granule cells in the dentate gyri of SE mice (Table 2; Fig. 5).

Diminished cell number in the corpus callosum and dentate gyrus after SE are correlated

A possible explanation for the presence of GFP-expressing cells in the dentate gyrus and the corpus callosum is that they originated from the same neural stem/progenitor cell population. Previous cell-fate studies suggest that this is indeed the case (Navarro-Quiroga et al., 2006). Specifically, the authors demonstrated that nestin-expressing stem/prognitor cells in the hippocampal subventricular zone generated oligodendrocytes and dentate granule cells, which populated the corpus callosum and dentate gyrus, respectively. Therefore, we

97 hypothesized that there would be a reduction in the number of surviving oligodendrocytes following early-life SE. In mice exposed to SE, a trend of fewer surviving GFP-expressing oligodendrocytes in the corpus callosum was found (Fig. 3; control, 2.000.58; SE, 3.670.33;

P=0.067, t-test). Next, we used this information to investigate whether or not there was a relationship between the number of surviving oligodendrocytes in the corpus callosum and cells within the dentate gyrus. From this analysis, we found that as the semi-quantitative score for

GFP-labeled cell loss in the corpus callosum increased (number of surviving GFP-expressing cells decreased), the number of GFP-labeled cells within the dentate gyrus decreased

(correlation coefficient, -0.92; P=0.01, Pearson correlation). This relationship was also significant in the SE group, as expected (Fig. 6; control: correlation coefficient, -0.934; P=0.23;

SE: correlation coefficient, -0.997; P<0.05, Pearson correlation). This suggests that sonic hedgehog responsive neural stem/progenitor cells labeled with GFP at P5 within the hippocampal subventricular zone are selectively vulnerable to insults early in life, and may underlie the diminished cell numbers within the corpus callosum and dentate gyrus later in life.

Integration of mature granule cells into dentate gyrus following SE

In adult models of epilepsy, the appropriate development and incorporation of mature granule cells into the dentate gyrus is assessed by scoring these cells for the presence of hilar projecting basal dendrites or ectopic localizaton within the hilus (Shapiro, et. al., 2005; Parent et al., 2006; Walter et al., 2007; Jessberger et al., 2007). Mature dentate granule cells with basal dendrites were characterized by possessing all the features of category 5 neurons above plus a spiny basal dendrite projecting into the dentate hilus. Mature dentate granule cells that had their cell bodies in the hilus and possessed spiny dendrites projecting into the granule cell layer were categorized as hilar ectopic cells. Previous studies using models of early-life seizures have not characterized mature granule cells in this manner, and even though significance was not reached in this study (Supplemental Table 1), it is interesting to note that neither of these

98 morphologies were found under control conditions, but both were present in dentate of mice exposed to early-life SE (Fig. 7).

99 Discussion

This study demonstrates the utility of the bi-transgenic Gli1-CreERT2;GFP fate-mapping approach to examine the impact of early-life seizures on brain development. Strikingly, this approach revealed that early-life SE leads to a reduction in the number of surviving cells within the dentate gyrus two months following a single episode of SE. The reduction in total GFP- labeled cell numbers is perplexing given that the percentage of GFP-labeled cell type ratios remained unchanged. Initially, ratios of cell-types to total GFP cells were compared in order to control for GFP labeling by tamoxifen. Due to the overall reduction in cell number, these data suggest that by normalizing the cell-types against total GFP-labeled cells, an underlying explanation for the change in total GFP cell numbers may have been missed. Therefore, we next analyzed the absolute numbers of GFP-labeled cell-types. By doing so, we found there to be significantly fewer putative progenitor cells, immature granule cells and mature granule cells in the dentate gyri of SE mice, which has not been previously shown. These data suggest several potential mechanisms that may underlie the observed decrease in GFP-labeled progeny. Firstly, the putative stem cell population may have become inactive, which would ultimately lead to the cessation of progeny generated. Alternatively, the putative stem cell population remained active, but their putative progenitor cell progeny died, which would also lead to a cessation of progeny generated. Lastly, all the GFP-labeled progeny generated shortly after SE failed to survive, which would result in reduced cell numbers later in life.

We propose that the diminished GFP-expressing cell number is mediated by alterations within the neural stem cell/progenitor cell population in the hippocampal subventricular zone.

This is conceivable since on postnatal day five, when tamoxifen was administered to induce

GFP expression among sonic hedgehog responsive neural stem/progenitor cells (Ahn and

Joyner, 2005), Gli1 mRNA is present within two populations of cells (Dahmane et al., 2001) – those located in the hippocampal subventricular zone, the secondary dentate germinal matrix

100 (Navarro-Quiroga et al., 2006), as well as those within the hilus/SGZ, the tertiary dentate germinal matrix (Pleasure et al., 2000). Interestingly, these two populations exist prior to postnatal day five, as Ahn and colleagues (2005) have shown that sonic hedgehog responsive neural stem cells are present in subgranular zone as early as embryonic day 18. Our data suggests that these populations may respond differently to early-life SE such that GFP- expressing neural stem/progenitor cells from the hippocampal subventricular zone en route to colonize the hilus (Navarro-Quiroga et al., 2006) either died or became inactive shortly after kainic acid-induced SE. Future studies are necessary in order to conclude which of these two scenarios might be occurring. However, a study by Dong et al. (2003) suggests the latter of the two situations. The authors used bilateral intrahippocampal injection of a sub-convulsive dose of kainic acid at P7 and did not find apoptotic cells within the dentate gyrus at several time points following kainic acid administration. Alternatively, the progeny of hippocampal subventricular zone neural stem/progenitor cells generated weeks following early-life SE failed to survive into adulthood.

Whether these changes lead to a lasting deficit in granule cells numbers remains to be determined; however, it is intriguing to note that early-life seizures using this model produce lasting deficits in hippocampal-dependent learning tasks in rodents (Sayin et al., 2004). Since granule cells are critical for hippocampal function (Snyder et al., 2005; Ge et al., 2007; Deng et al., 2009), and the fact that neonatally born granule cells dominate the dentate gyrus of adult mice (Muramatsu et al., 2007), impaired production of these neurons during development could contribute to these learning deficits.

Additionally, there are several limitations to this study. Firstly, significance may not have been reached with some parameters of the study due to the low numbers of mice used for the study. Future studies will address this, and should provide sufficient ‘n’ for all analyses.

101 Secondly, although morphology is used widely for identifying neurons and glia (Cajal, 1911) as well as distinguishing immature from mature neurons (Jones et al., 2003; Esposito et al., 2005), future studies should be completed that utilize cell-type markers to confirm the findings of the current study, and further investigate the three progenitor cell populations. Studies focused on these subclasses of progenitor populations will aid in determining which cell type is most vulnerable to early-life SE.

In summary, we conclude that early-life SE leads to a suppression of granule neurogenesis in a population of progenitor cells active two days before the insult. Several of our findings contribute to this conclusion. Firstly, we found significantly fewer GFP-expressing cells in the dentate gyrus of mice that experienced early-life SE. Secondly, this reduced cell number is illustrated by fewer numbers of GFP-expressing putative stem/progenitor cells, immature granule cells and mature granule two months after early-life SE. Thirdly, the reduced number of surviving GFP-expressing cells in the dentate was correlated with the amount of cell loss within the corpus callosum. This correlation suggests that hippocampal subventricular zone GFP- expressing progenitor cells exposed to kainic-acid induced SE become inactive (or die) shortly after treatment, or that the progeny of labeled stem cells active weeks after treatment fail to survive.

102 Figure 1. Cortical EEG recording from a seven day old Gli1-CreERT2 -/-;GFP+/- mouse. Images show (a) baseline EEG activity prior to kainic acid injection, and recordings of SE (b) 3 and (c) 6 hours after the injection. SE lasted for a minimum of six hours in all kainic acid-treated animals.

103 Figure 2. Cre-mediated recombination is efficacious and specific. (a) GFP-expressing cells are present in the dentate gyrus and corpus callosum/alveus of a bi-transgenic Gli1-CreERT2 +/-

;GFP+/- mouse given tamoxifen on postnatal day five and sacrificed on postnatal day twelve. (b, c) GFP-expressing cells are absent from bi-transgenic Gli1-CreERT2 +/-;GFP+/- mice not exposed to tamoxifen regardless of treatmenconditions (vehicle or kainic acid-induced SE) two days later on postnatal day seven. dg, dentate gyrus; cc/A, corpus callosum/alveus. Scale bar = 150 m.

104 Figure 3. GFP-expression in the adult mouse brain. Bi-transgenic Gli1-CreERT2 +/-;GFP+/- mice were treated with tamoxifen on postnatal day five, kainic acid (or Lactated Ringer’s) on postnatal day seven and sacrificed two months later. Confocal maximum projections of endogenous GFP staining exhibit GFP-expressing cells within the dentate gyrus in (a) control mice as well as (b)

SE mice. In addition, oligodendrocyres were present along the corpus callosum/alveus (white arroheads), and astrocytes were present in the hippocampus and surrounding cortex (blue arrowheads). There were significantly less GFP cells found within the dentate gyrus as well as a trend toward fewer GFP-labeled oligodendrocytes present in the corpus callosum following kainic acid-induced SE. CA1, CA1 pyramidal cell layer; CA3, CA3 pyramidal cell layer; dg, dentate gyrus; cc, corpus callosum and alveus. Scale bar = 300 m.

105 Figure 4. GFP-expression in five cell types within the adult dentate gyrus. Confocal projections of endogenous GFP staining in Gli1-CreERT2;GFP mice. GFP expression is present in (a) putative neural stem cells, (b) putative progenitor cells, (c) glial cells, (d) immature neurons and

(e) mature neurons (white arrowheads indicate cells of interest). The white lines in a, b, d, e represent the border(s) between the hilus (H) and subgranular zone (SGZ) as well as between the SGZ and the granule cell layer (GCL). Scale bar = 20 m.

106 Table 1. Mean percentage of GFP-labeled cell types per dentate gyrus Immature Mature Putative Putative Treatment Glia granule granule stem cells progenitors cells cells Control 0.680.68 22.331.82 0.970.97 38.626.30 37.4115.69 (n=3)

SE 2.441.05 18.443.78 4.171.21 27.591.20 47.374.66 (n=3) Values are meansSEM

107 Table 2. Mean number of GFP-labeled cell types per dentate gyrus Putative Immature Mature Treat- Total GFP Putative stem Glia granule granule ment cells progenitors cells cells cells Control 293.3325.6 2.332. 3.333.3 113.0019.1 110.0018. 64.672.60 (n=3) 5 33 3 4 56 104.0011.5 18.332.33* 50.3310.8 SE 2.330. 4.331.2 28.673.28* 3** ** 4* (n=3) 88 0 P=0.012 P=0.003 P<0.001 P=0.05 Values are meansSEM; *P<0.05, t-test; **P<0.01, t-test; ***P=<0.001, t-test. Significant values are noted in bold.

108 Figure 5. Early-life SE disrupts putative neural stem/progenitor cell fate. Representative confocal maximum projections of endogenous GFP staining in Gli1-CreERT2;GFP (a) control and (b) kainic acid-induced SE mice illustrating the presence of GFP-labeled putative progenitor cells (white arrowheads), glial cells (blue asterisks), immature neurons (pink arrowheads) and mature neurons (yellow arrowheads) within the dentate gyrii of these mice. Putative progenitor cells, immature neurons and mature neurons were fewer in number following SE. Additionally, the decrease in total number of GFP-labeled cells following SE can be observed. In a, two immature neurons and three progenitor cells were pseudo-colored cyan so that they could be better visualized among the milieu of other GFP-labeled cells. Scale bar = 20 m.

109 Figure 6. Scatter plot of Pearson correlation results. This plot illustrates an inverse relationship between GFP-expressing cell loss score in the corpus callosum and total GFP-expressing cell number in the dentate gyrus in control and SE conditions.

110 Figure 7. Examples of mature dentate granule cells with abnormal integraton following early- life SE. Confocal maximum projections of endogenous GFP staining illustrating that mature dentate granule cells were found to (a) possess spiny hilar projecting basal dendrites (white arrohead) as well as (b) become ectopically localized to the hilus (white arrowhead) following early-life SE. Glial cells were also present within the hilus in both control and SE animals (blue asterisks). The white line in a represents the border between the hilus (H) and the granule cell layer. Scale bars = 20 m.

111 Supplemental Figure 1. Cre-recombinase polymerase chain reaction. Representative genotyping results from PCR for Cre-recombinase. Lane 1 = DNA ladder, 500 base pair marker is noted (white arrowhead). Lane 2 = Promega master mix + distilled water, which acted as a negative control. Lane 3 = Promega master mix + DNA from a C57BL/6 mouse, which acted as an additional negative control. Lane 4 = PCR product from Promega master mix + DNA from a Gli1-CreERT2+/-;GFP+/- mouse, which resulted in a 417 base pair sequence of DNA corresponding to Cre-recombinase, and also served as a positive control for the remaining PCR reactions.

112 Supplemental Table 1. Mean percentage of GFP-expressing dentate granule cells possessing basal dendrites (DGC+BD) or ectopically localized in the hilus (HEC) per dentate gyrus.

Treatment DGC+BD HEC

Control 0.000.00 0.000.00 (n=3)

SE 0.180.18 1.630.83 (n=3) Values are meansSEM; Significant values are noted in bold.

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122 Acknowledgements

This work was supported by Cincinnati Children’s Hospital Medical Center and the National

Institute of Neurological Disorders and Stroke (1R03NS064378-01A1). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National

Institute of Neurological Disorders and Stroke or the National Institutes of Health. Gli1-CreERT2 mice were generously provided by Dr. Alexandra Joyner (Memorial Sloan-Kettering Cancer

Center, New York, New York). Green fluorescent protein reporter mice (CAG-CAT-EGFP) were generously provided by Dr. Jeffrey Robbins (Cincinnati Children’s Hospital Medical Center,

Cincinnati, Ohio). We would like to thank Jane Allendorder and Stefanie Bronson for useful comments on earlier versions of this manuscript.

123 Chapter 4

Genetic fate-mapping of adult-generated hippocampal dentate granule cells in the epileptic mouse brain

124 Abstract

The development of temporal lobe epilepsy is associated with altered proliferation and impaired integration of adult-generated hippocampal granule cells. These changes could contribute to the development of epilepsy and/or to associated co-morbidities of epilepsy, such as memory deficits. To further elucidate the role of adult-generated cells in epilepsy, we have developed a genetic fate-mapping approach to label newborn cells in the epileptic brain. Specifically, Gli1-

CreERT2 mice, which express a conditional, tamoxifen-inducible form of Cre-recombinase under control of the Gli1 promoter, were crossed to a GFP reporter line (Nakamura et al., 2006). Bi- transgenic offspring were treated with pilocarpine at four weeks of age to induce acute SE and epileptogenesis. Two and four days following treatment, mice were administered 700 mg/kg doses of tamoxifen, leading to the persistent expression of GFP in Gli1-expressing granule cell progenitors and all their progeny. Mice were sacrificed five or twelve weeks following pilocarpine treatment to assess the fate of labeled stem/progenitor cells and their progeny.

Within the hippocampus, GFP expression was detected in all cell types typically produced by

SGZ stem cells, including progenitor cells, immature granule cells, mature granule cells and glia-like cells. Interestingly, the relative proportions of these cell types were significantly altered in epileptic animals. The labeled cell population in epileptic animals consisted primarily of mature granule cells, with immature granule cells and progenitor cell types being largely absent.

By contrast, significant numbers of mature and immature cell types were present in control animals, implicating an accelerated maturation of newborn granule cells or decreased proliferation of prognitor cells in epileptic animals. Furthermore, labeled mature granule cells in epileptic animals exhibited structural abnormalities including ectopic somata, basal dendrites and mossy fiber axon sprouting that were absent from control animals.

125 Introduction

Patients with chronic epilepsy exhibit cognitive deficits (Helmstaedter, 2002) and changes in mood (Kanner, 2005) later in life. Associated with these recurring seizures are several neuropathological changes in the hippocampus (Babb et al., 1984, 1991; Franck et al.,

1995; Houser et al., 1990; Isokawa et al., 1993; Represa et al., 1989; Scheibel and Scheibel,

1973; von Campe et al., 1997). It is thought that these neuropathological changes to hippocampal circuitry, specifically in granule cell development, may underlie the changes in mood and cognition attributed to chronic seizures (Jessberger et al., 2007).

Dentate granule cells within the hippocampus are some of the last neuronal cell types generated in the brain (Altman and Bayer, 1990a, 1990b; Bayer, 1980) and are critical for learning and memory (Jessberger et al., 2009; Snyder et al., 2005). Furthermore, granule cell neurogenesis continues throughout life in both humans and animals (Altman and Bayer, 1990a;

Altman and Bayer, 1990b; Bayer, 1980; Eriksson et al., 1998; Knoth et al., 2010; Schlessinger et al., 1975; Seress et al., 2001) but is aberrant in epilepsy. Adult animal models of epilepsy have demonstrated that granule cell neurogenesis significantly increases beginning three days after a seizure and continuing for several weeks (Bengzon et al., 1997; Gray and Sundstrom,

1998; Parent et al., 1997; Parent et al., 1998; Scott et al., 1998). Our laboratory as well as several others have shown that these newly generated granule cells are more susceptible to generating aberrant connections, such as the formation of hilar basal dendrites (Jessberger et al., 2007; Shapiro et al., 2005; Walter et al., 2007) and ectopic localization to the hilus

(Jessberger et al., 2005; Parent et al., 2006; Walter et al., 2007). Recent studies have also shown that the differentiation (Hattiangady and Shetty, 2010; Huttmann et al., 2003; Jessberger et al., 2005) and survival of newborn granule cells is altered in the epileptic brain (Ekdahl et al.,

2001). However, these studies are controversial with regard to the effects that seizures have on the differentiation of these newly generated cells. This is likely due to differences in the model

126 used, severity of the seizures as well as the time point after seizure induction in which these changes were examined (Hattiangady and Shetty, 2010; Huttmann et al., 2003; Jessberger et al., 2005).

Previous studies have used cell-labeling techniques such as transgenic reporter mice and the expression of several transcription factors and proteins to examine the various cell-type populations during stages of neuronal differentiation within the subgranular zone (SGZ) following seizure activity. These studies were crucial in providing a glimpse of how seizure activity within the hippocampus affects neuronal differentiation, but did not answer the question of what happens to progeny produced by a specific pool of neural stem/progenitor cells. Given that the timing of granule cell neurogenesis corresponds to a critical time period for the functional integration of these new neurons into hippocampal circuitry (Snyder et al., 2005), it is important to understand the consequences that an insult to the brain, such as status epilepticus

(SE), might have on the differentiation of these newborn granule cells. In this study, we investigated the effect of seizure activity on the generation and differentiation of progeny from stem/progenitor cells that gave rise to new cells within the first four days following a prolonged seizure episode, also known as SE. We used the Cre-loxP genetic strategy to investigate the cell fate of neural stem and progenitor cells within the SGZ. Specifically, Gli1-CreERT2 mice were crossed to green fluorescent protein (GFP) reporter mice so that SGZ neural stem and progenitor cells that are responsive to sonic hedgehog (Shh) and their progeny would be labeled with GFP for their lifetime.

127 Methods

All procedures conformed to NIH and institutional guidelines for the care and use of animals.

Animals

Gli1-CreERT2 mice were provided by Dr. Alexandra Joyner (Sloan-Kettering Institute).

These animals express tamoxifen-inducible Cre recombinase under the control of the Gli1 promoter (GLI-Kruppel family member), which drives expression in Shh-responding progenitor cells (Ahn and Joyner, 2004; Ahn and Joyner, 2005). GFP reporter mice containing a loxP- flanked stop codon (CAG-CAT-EGFP, (Nakamura et al., 2006)) were provided by Dr. Jeffrey

Robbins (Cincinnati Children’s Research Foundation). Mice were maintained on a C57BL/6 background. Bi-transgenic male and female mice hemizygous for the Gli1-CreERT2 transgene and heterozygous for the GFP reporter transgene were used for experiments.

Polymerase chain reaction analysis of genomic DNA

DNA from offspring of Gli1-CreERT2 hemizygous and GFP reporter homozygous breeder pair parents was extracted using the DNeasy blood and tissue kit (Qiagen, 69506) and genotyped for Cre recombinase using the following primer sequences: 5’,

GCGGTCTGGCAGTAAAAACTATC and 3’, GTGAAACAGCATTGCTGTCACTT, which generated a 102 base pair DNA fragment (Supplemental Fig. 1). Polymerase chain reaction was completed using GoTaq® Green Master Mix (Promega Corporation, Madison, WI, M7123) and the following thermal cycling parameters: initialization at 95˚C for 5 minutes, denaturation at

95˚C for 30 seconds, annealing at 53˚C for 45 seconds, extension at 72˚C for 45 seconds, final elongation at 72˚C for 5 minutes and a final hold at 4˚C (denaturation, annealing and extension steps were repeated for 34 cycles).

128 Pilocarpine and tamoxifen treatments

Bi-transgenic littermates were treated either with pilocarpine to induce SE or served as saline-treated controls, in accordance with published protocols (Danzer and McNamara, 2004;

Walter et al., 2007; Danzer et al., 2010). Briefly, four-week-old mice were injected with 1mg/kg methyl scopolamine nitrate s.c. in saline solution, followed fifteen minutes later by 400 mg/kg pilocarpine in saline to induce SE. Three hours after the onset of SE, mice were given two doses of 10 mg/kg diazepam at 15 minute intervals to suppress SE. Mice that failed to develop

SE were excluded from the study. Two and four days after pilocarpine treatment, mice were given 700 mg/kg doses of tamoxifen (Sigma-Aldrich, St. Louis, MO, T5648) dissolved in corn oil s.c. (one dose per day). Mice were overdosed with pentobarbital (100 mg/kg) either five or twelve weeks after pilocarpine treatment and were perfused with PBS+1 U/ml heparin, followed by 2.5% paraformaldehyde and 4% sucrose in PBS (pH 7.4). Brains were removed and post- fixed for 12 hours, cryoprotected in sucrose (10%, 20%, 30%), and 60 m coronal sections were cut on a cryostat. Slide-mounted sections were stored at –80oC until use. A total of 18 animals were used (5 wk control, n=5; 5 wk SE, n=4; 12 wk control, n=5; 12 wk SE, n=4).

Immunohistochemistry

Immunolabeling for the zinc transporter ZnT-3 and enhancement of the endogenous GFP was conducted on slide-mounted sections (McAuliffe et al., 2010). Sections were thawed in 0.1 M PBS, pH 7.4, blocked for one hour at room temperature in blocking solution [5% normal goat serum + 0.75% Glycine + 0.5% Triton X-100 in 0.1 M PBS] and incubated overnight at 4º C in 1:500 chicken anti-GFP (Abcam, Cambridge, MA, ab13970) and

1:3,000 polyclonal rabbit anti-ZnT3 (Synaptic Systems, Göttingen, Germany, 197 002) primary antibodies diluted in blocking solution. Sections were then rinsed in blocking solution and incubated for four hours at room temperature in 1:750 Alexa Fluor 488 goat anti-chicken and

1:750 Alexa Fluor 594 goat anti-rabbit secondary antibodies (Molecular Probes, Carlsbad, CA,

129 A-11039 and A-11012) in blocking solution, followed by rinses in 0.1 M PBS. Lastly, sections were dehydrated in alcohols (50%, 70%, 95%, 100%, 100%) and cleared in xylenes before mounting with Krystalon (Harleco). Importantly, no significant differences were found between bregma levels used for the five-week analysis (control-5wk, n=5, -2.040.08; SE-5wk, n=4, -

1.950.19; P=0.659, t-test) or the twelve-week analysis (control-12wk, n=5, -1.820.07; SE-

12wk, n=4, -1.950.23; P=0.560, t-test).

Characterization of GFP-expressing progeny in the dentate gyrus

Brain sections from dorsal hippocampus (Bregma -1.46 to -2.30) immunostained for

GFP were examined under epifluorescent illumination using the 63X (NA 1.4) objective by an observer blind to treatment group. One brain section (two dentate gyri) was examined from each animal. GFP-expressing cells were categorized by cell type and maturational stage using morphological criteria previously described (see Chapter 4; Fig. 3). Briefly, seven categories were scored, as follows: (1) Mature granule cells, defined as cells with their somata located in the granule cell body layer and spine-coated dendrites projecting to the hippocampal fissure.

(2) Mature granule cells with basal dendrites, which possess all the features of category 1 plus a spiny basal dendrite projecting into the dentate hilus (Dashtipour et al., 2003). (3) Mature hilar ectopic granule cells, which have their somata located in the hilus and possess spiny dendrites projecting through the hilus and frequently into the molecular layer. Ectopic location was defined as greater than or equal to two cell body widths outside the granule cell layer (Kron et al., 2010). (4) Immature granule cells, with somata in the granule cell body layer and aspiny apical dendrites terminating prior to reaching the hippocampal fissure; these cells occasionally possess short, aspiny basal dendrites. (5) Putative progenitor cells, also known as transient amplifying cells, which have somata located in the SGZ; these cells also occasionally possessed short, horizontally projecting, aspiny processes. (6) Glial cells, characterized by a

130 small cell body located anywhere in the dentate and numerous thin, aspiny processes projecting outwards in a stellate fashion from the cell body. (7) Putative neural stem cells, with a small cell body located in the SGZ and a single, radially projecting process that terminates in the inner molecular layer. Caveat: since only one brain section per animal was included in this analysis, the data and conclusions presented here are only relevant to the dorsal hippocampus between bregma levels of –1.46 and –2.30, and may differ in other regions of the hippocampus.

Mossy fiber sprouting

Brain sections from dorsal hippocampus (Bregma -1.46 to -2.30) immunostained for

ZnT-3 were examined for mossy fiber sprouting into the dentate inner molecular layer under epifluorescent illumination using the 10X (NA 0.3) objective by an observer blind to treatment group. Mossy fiber sprouting (ZnT-3 immunoreactivity) was scored in one hippocampus of each twelve-week animal using a semi-quantitative scale: (1) no obvious ZnT-3 immunoreactivity, (2) sparse ZnT-3 immunoreactivity, and (3) dense ZnT-3 immunoreactivity within the inner molecular layer.

Statistics

Data are presented as mean±standard error. All statistical tests were performed using

Sigma Stat (version 11.0). Parametric or nonparametric tests were run, based on whether the data met assumptions for normality and equal variance; specific tests used are noted in the results. Statistical significance was accepted for P<0.05.

Figure preparation

Images presented in the figures are confocal maximum projections and were prepared using Leica’s LAS-AF Confocal software (2.0.0 build 1934). These images were processed using an erosion filter run for one iteration with a three-pixel radius (Leica software) to reduce

131 background artifact, and some were pseudo-colored to highlight the cell(s) of interest among neighboring GFP-expressing cells. Brightness and contrast of digital images were adjusted using Adobe Photoshop (version 7.0) to maximize detail. In all cases, identical adjustments were made to images meant for comparison.

132 Results

Specificity of induction and cell labeling in Gli1-CreERT2;GFP reporter mice

Previous studies in our laboratory have found that tamoxifen treatment of bi-transgenic

Gli1-CreERT2;GFP reporter mice leads to GFP-labeled cells in the dentate gyrus (Fig. 1). GFP- labeled cells were also found in the subventricular zone as well as within the cerebellum, which likely corresponded to stem/progenitor cells and Bergman glia, respectively (Fig. 1). Although continued neurogenesis occurs in the (Zhao et al., 2008), GFP-labeling in the olfactory bulb was not observed when tamoxifen administrated on P17/18 (Fig. 1). Lastly, no

GFP-expressing cells were found in Gli1-CreERT2-/-;GFP+/- mice treated with tamoxifen (Fig. 2) or in bi-transgenic mice not treated with tamoxifen (data not shown).

Granule cell birth and differentiation in control and epileptic bi-transgenic mice

In order to determine whether SE disrupts progenitor cell activity, Gli1-CreERT2;GFP bi- transgenic mice were given tamoxifen two and four days following SE to induce GFP-expression in Shh-responsive SGZ stem/progenitor cells and their progeny. The numbers of GFP-labeled putative stem cells (Fig. 3, a), putative progenitor cells (Fig. 3, b), immature granule cells (Fig. 3, c) and mature granule cells (Fig. 3, d) were quantified at two time points following SE, five weeks and twelve weeks, to assess the fate of surviving GFP-expressing cells acutely as well as chronically. There were significantly fewer GFP-expressing cells found within the dentate gryri of mice five weeks following SE (control-5wk, n=5, 53.2025.67; SE-5wk, n=4, 3.751.34;

P=0.016, Mann-Whitney Rank Sum Test [MWRST]). Correspondingly, the proportion of GFP- expressing cells categorized as putative progenitor cells in SE mice was 25% less than the proportion of GFP-labeled cells in control mice (P<0.05, t-test; Table 1*). The most striking difference in cell-types generated between the two treatment conditions was in mature granule

* Supplemental Table 1 reports the mean number of GFP-expressing cells as requested for data completeness

133 cells which made up more than 50% of the total GFP-expressing cells generated at this time point in epileptic mice compared to less than 10% of the total GFP-labeled cell population in control animals (P<0.05, MWRST; Table 1). Since it takes approximately four weeks for granule cells to be considered fully differentiated or mature (Ambrogini et al., 2004; Esposito et al.,

2005; Jones et al., 2003; Liu et al., 2000; Markakis and Gage, 1999; Overstreet-Wadiche et al.,

2006a; Zhao et al., 2006), these cells were most likely some of the first to be generated following SE.

Next, we queried whether or not the changes in GFP-expressing cell number and proportions of cell-types labeled with GFP differed between groups at twelve weeks after SE.

The total number of GFP-expressing cells found within the dentate gryri of mice 12 weeks following SE was still less but not significantly different than the control mice (control-12wk, n=5,

38.6010.76; SE-12wk, n=4, 20.7510.73; P=0.285, t-test). Consistent with this finding, there was also a 10% increase in the proportion of GFP-expressing cells categorized as mature granule cells from five weeks to twelve weeks following SE (Tables 1 and 2*). We also found a

26% increase in the proportions of GFP-expressing mature granule cells within the granule cell layer twelve weeks after SE compared to control mice (P<0.05, MWRST; Table 2). However, the diminished proportion of GFP-labeled putative progenitor cells observed five weeks after SE remained seven weeks later, and 25% fewer immature granule cells were found in the epileptic hippocampus at this time compared to control mice (P<0.05, t-test; Table 2).

Abnormal integration of GFP expressing granule cells in epileptic bi-transgenic mice

Previous studies from our laboratory as well as others have established that seizure- induced adult-generated dentate granule cells dentate granule cells migrate ectopically into the

* Supplemental Table 1 reports the mean number of GFP-expressing cells as requested for data completeness

134 hilus (Jessberger et al., 2007; Jung et al., 2004; Kron et al., 2010; Parent et al., 1997; Parent et al., 2006; Scharfman et al., 2000; Scharfman et al., 2003; Walter et al., 2007) as well as form hilar projecting basal dendrites (Jessberger et al., 2007; Kron et al., 2010; Shapiro et al., 2005;

Walter et al., 2007). Therefore, we assessed whether or not these changes in the development and integration of newborn granule cells following SE were detected using our genetic fate- mapping strategy. Hilar ectopic granule cells were observed in both SE groups. When we quantified the percentage of GFP-labeled hilar ectopic granule cells, we found 4.421.98%

(mean number per dentate: SE, 0.250.14) and 5.251.98% (mean number per dentate: SE,

1.500.89) five and twelve weeks after SE, respectively (Fig. 3, d). No hilar ectopic cells were found in age-matched controls; however, this difference did not reach significance for either time point analyzed (5-wk, P=0.094, t-test; 12-wk, P=0.122, t-test). In some cases, dendrites of hilar ectopic cells extended to the hippocampal fissure (Fig. 3, d and e). Lastly, we found hilar projecting basal dendrites on mature granule cells twelve weeks after SE (Fig. 3, f), which made up 18.153.92% of GFP-expressing cells (mean number per dentate: SE, 3.061.05) compared to 0% in control mice (P=0.001, t-test). We did not observe any of the GFP-labeled mature granule cells five week after SE to possess hilar basal dendrites.

Labeling of mossy fiber axons in stratum lucidum

In order to determine that seizure-induced newborn neurons became functional, GFP- labeled mossy fibers terminals were examined for the presence of presynaptic terminals.

Firstly, we found that GFP-labeled mossy fibers projected into the stratum lucidum (Fig. 1). At higher magnification, we observed the presence of GFP-labeled mossy fiber presynaptic terminals. Specifically, we found the presence of mossy fiber boutons (Fig. 4) as well as filopodia (Fig. 4). Mossy fiber boutons form synapses with CA3 dendrites while filopodia form synapses with local inhibitory interneurons (Acsady et al., 1998; Amaral and Dent, 1981).

135 Therefore, the presence of these two types of presynaptic terminals on GFP-expressing granule cell mossy fibers suggests that seizure-induced, adult-generated granule cells have the potential to become functionally integrated into the hippocampal circuitry.

Mossy fiber sprouting in epileptic mice

Several laboratories have recently shown that immature granule cells exposed to SE and granule cells generated following SE contribute to mossy fiber sprouting (Danzer, 2008;

Kron et al., 2010; Parent et al., 1997). In order to determine whether our genetic strategy to label newly generated granule cells is useful in assessing such changes in granule cell circuitry, we examined sections from the twelve week group that were immunostained for GFP and ZnT3, a zinc transporter that is localized to mossy fiber terminals (McAuliffe et al., 2009; Palmiter et al., 1996; Wenzel et al., 1997). A semi-quantitative scale was used to score the extent of mossy fiber sprouting into the dentate inner molecular layer, which was based on ZnT3 immunoreacitivity. From this analysis, we found that mossy fiber sprouting was very dense twelve weeks after SE (control, 1.00; SE, 3.00 [2.00-3.00]; P=0.016, MWRST). In fact, we observed GFP-labeled mossy fibers within the dentate inner molecular layer, indicating that seizure-induced, newborn granule cells contribute to mossy fiber sprouting (Fig. 5). Next, we assessed whether the proportions of GFP-expressing cell types found twelve weeks after SE were related to the extent of mossy fiber sprouting. We found that as the density of mossy fiber sprouting increased, the proportion of GFP-expressing cells categorized as putative stem, progenitor and immature granule cells decreased (Table 3). We also found that the proportion of GFP-expressing cells exhibiting hilar basal dendrites was positively associated with the extent of mossy fiber sprouting (Table 3).

136 Discussion

In this study, we examined the short and long-term impact of SE on dentate granule cell neurogenesis from a specific pool of SGZ stem and progenitor cells. In order to accomplish this task, we utilized the Cre-loxP system to selectively induce GFP expression in SGZ stem and progenitor cells as well as their progeny. SGZ stem and progenitor cells were initially labeled with GFP two and four days following SE. Recent studies demonstrate that several types of dividing cells exist within the SGZ of the adult hippocampus (Fukuda et al., 2003; Kempermann et al., 2004; Kronenberg et al., 2003; Mignone et al., 2004). One of these dividing cell types are

Type-1 cells that have stem cell-like characteristics, and we did not find a change in the proportion of these cells labeled with GFP at five or twelve weeks following SE. This is in contrast with data presented by Huttmann and colleagues (2003) who showed an increase in

Type-1 cells 3 days after SE. Instead, our data support the thought that Type-1 putative stem cells are not responsible for the increase in neurogenesis following SE (Jessberger et al., 2005).

With regard to the second category of dividing cells, transient amplifying progenitor cells, we found there to be 25% fewer cells of this type labeled with GFP five weeks after SE compared to control animals, which was similar twelve weeks after SE (27%). This finding is different from other studies that demonstrated an increase in these progenitor cells following SE (Jessberger et al., 2005; Steiner et al., 2008), which may be due to differences in the time points examined after SE, nine days versus five and twelve weeks in this study. Furthermore, our study investigated a more specific pool of these progenitor cells using the Cre-loxP system compared to Jessberger and colleagues (2005) who utilized cell-type markers. Our data suggest that five weeks after SE, which is approximately the time when spontaneous recurrent seizures begin in this model of MTLE (Curia et al., 2008; Leite et al., 2002), a decrease in neurogenesis for the labeled progenitor pool is apparent, which is still somewhat present twelve weeks after SE.

137 In the present study, we observed a larger proportion of mature GFP-expressing cells five weeks and twelve weeks following SE relative to controls. This increase in mature granule cells is consistent with the finding that SGZ neurogenesis is increased for several weeks after SE

(Parent et al., 1997). However, this may have only been the case for the first week following SE in the present study. In addition to the finding of fewer GFP-labeled progenitor cells five and twelve weeks after SE, several other data in this study indicate that neurogenesis of the particular pool of progenitor cells is in fact suppressed. Surprisingly, we found significantly fewer GFP-labeled immature granule cells twelve weeks after SE, which actually suggests that the increase in mature GFP-expressing cells twelve weeks after SE was not due to increased neurogenesis, but maturation of immature granule cells that were present five weeks after SE.

The percentage of immature granule cells twelve weeks after SE can be attributed to suppression of neurogenesis in this progenitor pool, decreased survival of newly generated neurons following SE (Mohapel et al., 2004) or accelerated maturation of these neurons into differentiated neurons (Overstreet et al., 2006b). We propose that the former and later phenomena are occurring here. The former explanation is consistent with the finding of fewer

GFP-labeled progenitor cells at five and twelve weeks after SE. Additionally, the proportions of

GFP-labeled mature granule cells in control mice increased more than six-fold between five weeks and twelve weeks. This implies that if an increase in neurogenesis is occurring in the

GFP-labeled pool of progenitor cells following SE, a greater or equal change in the proportions of mature granule cells generated would be expected. However, this is not the case since the percentage of GFP-labeled mature granule cells only increased by 10% in SE mice from five weeks to twelve weeks. Therefore, we can conclude that neurogenesis is suppressed in the

GFP-labeled pool of progenitor cells following SE. We also observed a partial recovery in the number of GFP-expressing cells per dentate from five to twelve weeks in SE mice. The most plausible explanation for this recovery as well as the continued increase in proportion of GFP- expressing mature granule cells after SE is that GFP-expressing immature granule cells

138 matured faster five and twelve weeks after SE. This is evidenced by the fact that there was no difference in the proportion of GFP-expressing cells categorized as immature granule cells between control and SE mice five weeks after SE (P=0.100; Table 1). Examination of the ratio of immature to mature GFP-expressing cells five weeks after SE indicates a disproportionately high percentage of mature cells five weeks following SE, which suggests an accelerated maturation of immature neurons born after SE. Similarly, this ratio of immature to mature GFP- expressing cells is evident twelve weeks. Thus, accelerated immature granule cell maturation continues up to twelve weeks after SE. It is plausible that this acceleration of immature granule cell maturation may underlie the formation of hilar basal dendrites and ectopic granule cells if key steps in neuronal migration were to become altered following SE. Correspondingly, such changes in the rate of differentiation may underlie cognitive deficits associated with seizures due to the pivitol role of newly generated granule cells in learning amd memory (Jessberger et al., 2008).

In the present study, we found that the density of mossy fiber sprouting twelve weeks after SE was negatively correlated with the proportions of GFP-expressing cells identified as stem, progenitor and immature granule cells. This means that as mossy fiber sprouting became denser, the proportion of GFP-labeled stem, progenitor and immature granule cells decreased.

Mossy fiber sprouting is a hallmark characteristic of MTLE (Chang and Lowenstein, 2003), and the extent of mossy fiber sprouting into the inner molecular layer is highly correlated with the frequencies of recurrent seizures as well as the severity of seizures in animal models (Nadler,

2003; Sutula, 2002). If we use the extent of mossy fiber sprouting as a measure of the severity and frequency of seizures, we can conclude that mice with more frequent or severe seizures are more likely to exhibit alterations in cell-fate decisions. A similar case can be made for the formation of granule cells with hilar basal dendrites.

139 Together, this study indicates that the pool of stem/progenitor cells in the SGZ generating new granule cells during the first five days following SE is susceptible to disruption of cell-fate decisions and the rate of granule cell maturation later in life. Changes such as these may have severe consequences to hippocampal circuitry given their proposed importance of newborn granule cells in learning and memory (Deng et al., 2009; Jessberger et al., 2009; Kee et al., 2007; Shors et al., 2001; Snyder et al., 2005; Trouche et al., 2009), and may underlie cognitive deficits (Helmstaedter, 2002) and changes in mood (Kanner, 2005) associated with chronic epilepsy.

140 Figure 1: Endogenous GFP expression in the adult brain. Confocal maximum projections of

GFP-labeled cells in a Gli1-CreERT2;GFP reporter mouse treated with tamoxifen on P17/P18 and sacrificed on P25. One week after tamoxifen treatment, GFP-labeled putative progenitor cells were present in the dentate gyrus and the sub-ventricular zone (SVZ). Bergmann glial cells are also present in the cerebellum. Labeled cells were not observed in cortex or olfactory bulb at this time point. Scale bar = 300 µm.

141 Figure 2: Specificity of induction and cell labeling in Gli1-CreERT2;GFP reporter mice. (left)

Confocal maximum projection of GFP-labeled cells in a Gli1-CreERT2+/-;GFP reporter mouse treated with pilocarpine on P30, with tamoxifen on P32 and P34 and sacrificed on P120. GFP- labeled cells were present in the dentate gyrus. (right) Confocal maximum projection of a Gli1-

CreERT2-/-;GFP reporter mouse treated with pilocarpine on P30, with tamoxifen on P32 and P34 and sacrificed on P120. Note the lack of GFP-labeled cells in the dentate gyrus.

Scale bar = 300 µm.

142 Figure 3: GFP-expression in cells of the SGZ, dentate gyrus and aberrantly integrated dentate granule cells. Confocal maximum projections on endogenous GFP staining in the Gli1-

CreERT2+/-;GFP mice. GFP expression is present in (a) putative neural stem cells, (b) putative progenitor cells, (c) immature neurons and (d) mature neurons (white arrows). GFP-labeled granule cells localized to the hilus (d, white arrowhead) with dendrites projecting to the hippocampal fissure (e, white box in d) and granule cells with spiny hilar basal dendrites (f) were also present. Scale bars: (c is same for a and b), 20 mm; (d), 100 mm; (e, f) 10 mm.

143 Table 1. Mean percentage of GFP-expressing cells per dentate gyrus five weeks following SE Immature Mature Putative Putative Group Treatment Glia granule granule stem cells progenitors cells cells Control 12.587.31 35.437.37 2.221.62 42.953.93 6.822.37 (n=5) 57.9814.54* SE 10.776.25* 5 weeks 1.921.92 3.171.91 21.7311.70 P=0.016 (n=4) P=0.043 MWRST Values are meansSEM; *P<0.05, t-test or Mann-Whitney Rank Sum test (MWRST).

Significant values are noted in bold.

144 Table 2. Mean percentage of GFP-expressing cells per dentate gyrus twelve weeks after SE Immature Mature Putative Putative Group Treatment Glia granule granule stem cells progenitors cells cells Control 2.890.95 27.835.61 7.004.38 26.454.96 42.371.58 (n=5)    68.17 8.30* SE  0.71 0.71**  0.71 0.71** 12 weeks 0.00 0.00 0.45 0.30 P=0.016 (n=4) P=0.004 P=0.003 MWRST Values are meansSEM; *P<0.05, MWRST; **P<0.01, t-test. Significant values are noted in bold.

145 Figure 4: Labeling of mossy fiber axons in stratum lucidum. Confocal maximum projections mossy fiber pre-synaptic terminals. morphologies in both control and SE mice. Giant mossy fiber boutons, or typical mossy fiber boutons, were defined for this study as axonal expansions with an area greater than 4 µm2. Complex boutons consist of a bouton attached to another terminal bouton by a thin process. Filopodia were defined as finger-like projections from the bouton longer than 1 µm and no wider than 1 µm. MFB = mossy fiber bouton, ax = axon, arrowhead = filopodia. Scale bar = 5 µm.

146 Figure 5: Mossy fiber sprouting in epileptic mice. Confocal maximum projection illustrating the sprouting of mossy fibers through the granule cell layer (pseudo-colored red). Scale bar, 20 µm.

147 Table 3. Pearson correlation results: Are the changes in the cell-fate of GFP-expressing cells related to mossy fiber sprouting 12 weeks following SE?

Putative Putative Immature Mature dentate granule stem cells progenitor cells granule cells cells + basal dendrites

Correlation -0.675 -0.800 -0.815 0.939 coefficient

P value 0.0459 0.0096 0.0075 0.0002 Mossy fiber sprouting score

Significant values are noted in bold.

148 Supplemental Figure 1. Cre-recombinase polymerase chain reaction. Representative genotyping results from PCR for Cre-recombinase. Lane 1 = DNA ladder, 100 base pair marker is noted (white arrowhead). Lane 2 = Promega master mix + distilled water, which acted as a negative control. Lane 3 = Promega master mix + DNA from a C57BL/6 mouse, which acted as an additional negative control. Lane 4 = PCR product from Promega master mix + DNA from a Gli1-CreERT2-/-;GFP+/- mouse, which acted as an additional negative control. Lane 5 = PCR product from Promega master mix + DNA from a Gli1-CreERT2+/-;GFP+/- mouse, which resulted in a 102 base pair sequence of DNA corresponding to Cre-recombinase, and also served as a positive control for the remaining PCR reactions.

149 Supplemental Table 1. Mean number of GFP-expressing cells per dentate gyrus five and twelve weeks after SE Immature Mature Total GFP Putative stem Putative Group Treatment Glia granule granule cells cells progenitors cells cells Control 53.2025.67 3.501.53† 24.0014.07† 0.600.40 21.609.66† 3.501.30 (n=5) 1.770.54* SE  

5 weeks 0.06 0.06†* 0.94 0.41†* P=0.016 0.630.38† 0.190.12 1.690.58 (n=4) P=0.036 P=0.019 MWRST Control 38.6010.76 1.300.60 10.903.48 0.200.12† 9.402.18† 16.805.01 (n=5)

SE 0.130.13* 0.680.28†* 12 weeks 20.7510.73 0.000.00 0.130.13†*** 15.259.16 (n=4) P=0.029 P=0.037 Values are meansSEM; *P<0.05, t-test; ***P=<0.001, t-test. †Denotes data normalized by square root transformation. Significant values are noted in bold.

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Acknowledgements

This work was supported by Cincinnati Children’s Hospital Medical Center, the Epilepsy

Foundation of America and the National Institute of Neurological Disorders and Stroke (SCD,

Award Numbers R01NS065020 and R01NS062806). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of

Neurological Disorders and Stroke or the National Institutes of Health. Gli1-CreERT2 mice were generously provided by Dr. Alexandra Joyner (Memorial Sloan-Kettering Cancer Center, New

York, New York). Green fluorescent protein reporter mice (CAG-CAT-EGFP) were generously provided by Dr. Jeffrey Robbins (Cincinnati Children’s Hospital Medical Center, Cincinnati,

Ohio). We would like to thank Jane Allendorfer and Stefanie Bronson for useful comments on earlier versions of this manuscript.

162 Chapter 5

Conclusions and future directions

163 Conclusions

Differential changes in neurogenesis following SE: developing vs. mature brain

In Chapters 3 and 4, a bi-transgenic fate-mapping strategy was used to GFP-label a specific pool of stem/progenitor cells and assess the affect of early-life and adult SE on the production of their subsequent progeny, which also expressed GFP. In Chapter 3, the pool of stem/progenitor cells was labeled with GFP two days prior to early-life SE. We found that later in life, specifically two months after SE, fewer GFP-labeled prognitor cells, immature and mature granule cells were present within the dentate gyrus. This suggests that SE impaired the proliferative capacity of this specific progenitor cell population. However, this does not seem to be the case following SE in the adult brain. In Chapter 4, adult mice were exposed to SE, and stem/prognitor cells were labeled with GFP two and four days following SE. We found there to be fewer putative stem cells and more mature cells five weeks after SE. Twelve weeks after

SE, this scenario changed in that fewer progenitor cells and immature granule cells were present within the dentate gyrus, but there were still significantly more mature granule cells.

There was also a trend towards fewer putative stem cells at this time point (p=0.063). These data suggest that the GFP-labeled pool of putative stem cells are impaired five weeks following

SE, and GFP-labeled progenitor cells become impaired twelve weeks following SE. However, this decrease in the number of proliferative cells did not result in an overall suppression of neurogenesis like in the developing brain since significantly more mature granule cells were labeled with GFP at both time points in the adult brain. We proposed that this was due to an accelerated maturation of GFP-labeled immature granule cells. Alternatively, GFP-labeled progenitor cells present five weeks after SE may have undergone more rounds of replication.

Together, these data indicate that SE differentially affects stem/progenitor pools in the developing and adult brain. However, the reduced generation of granule cells (Chapter 2 – early-life SE) and accelerated maturation of immature granule cells (Chapter 3 – adult SE) may underlie cognitive impairments associated with seizures in that the normal amount and rate of

164 neurogenesis is perturbed. In the future, studies utilizing BrdU injections immediately following early-life and adult SE will be help in identifying the changes in proliferative capacity presented here. Lastly, studies of electrophysiology on mature GFP-labeled granule cells in these paradigms will aid in determining whether passive and plasticity related electrical properties of these cells is altered, which would further support their role in cognitive impairment following seizures.

Neuronal age is a critical factor in the formation of aberrant dendrites

Continuous generation of dentate granule cells through adulthood produces a population of neurons that varies widely in age (Altman and Das, 1965; Kaplan and Hinds, 1977; van Praag et al., 2002). The present findings suggest that granule cells of different ages make distinct contributions to pathology in the epileptic brain. Specifically, granule cells with basal dendrites, which are not observed in the normal brain, occur in two forms in the epileptic brain: (1) recurrent basal dendrites which project back into the dentate molecular layer; and (2) hilar basal dendrites which continue their trajectory into the dentate hilus. Data from Chapter 4 as well as recent studies using in vivo SE models of epilepsy demonstrate that hilar basal dendrites originate almost exclusively from newly generated granule cells (Jessberger et al., 2007b;

Walter et al., 2007). Whereas in Chapter 3, a more severe insult to the brain, slice preparation and subsequent granule cell dispersion, led to a pronounced disruption of mature granule cell dendritic structure and the formation of recurrent basal dendrites. In this study, no instances of hilar basal dendrite formation were observed. The apparent inability of these neurons to form hilar basal dendrites likely reflects their more mature state, and suggests the age of the neuron at the time of insult plays a critical factor in the formation of dendritic abnormalities. Additionally, data from each chapter as well as previous studies from our laboratory (Walter et al., 2007) demonstrate that mature granule cells and immature granule cells can become ectopically localized after an epileptogenic stimulus. Taken together, these data suggest that both

165 immature and mature granule cells contribute to pathology in the epileptic brain. Granule cell dispersion and neuronal dysmorphogenesis are likely to contribute to neurocognitive deficits associated with epilepsy (Jessberger et al., 2007a). The current demonstration that these two pathologies are linked provides new insights that will be important for understanding and perhaps treating this disease by targeting therapeutics to a specific age granule cell.

Potential cellular mechanisms of basal dendrite formation and granule cell dispersion

The dentate gyrus is a highly laminated structure, and because of its postnatal development, investigators have been able to study the molecular underpinnings of neurogenesis, neuronal migration and axonal pathfinding (for review see Forster et al., 2006).

The laminar specificity of the dentate gyrus is thought to come from lamina-specific distribution of cellular targets and molecular cues of the extracellular matrix (Zhao et al., 2003). A key player in this process is the glycoprotein reelin, which is secreted from Cajal-Retzius cells in the dentate outer molecular layer (Deller et al., 1999). Specifically, in mice that lack reelin, referred to as reeler mice, granule cells were no longer organized in a compact fashion, and some of these granule cells exhibited hilar basal dendrites and recurrent basal dendrites (Drakew et al.,

2002; Stanfield and Cowan, 1979).

However, the underlying cellular mechanisms of granule cell dispersion and basal dendrite formation remain largely unexplored. Intriguingly, genetic manipulations that produce granule cell dispersion in mice also produce dysmorphic granule cells. Several of these mice are those with mutations in reelin receptors, very low density lipoprotein receptor and apolipoprotein E receptor 2 (Drakew et al., 2002; Stanfield and Cowan, 1979); p35 (Patel et al.,

2004; Wenzel et al., 2001; Wenzel et al., 2007), 1 integrins (Forster et al., 2002); Lis1 (Fleck et al., 2000) and disrupted-in- 1 (DISC1; Duan et al., 2007). Coincidentally, these proteins are involved in regulating neuronal migration, positioning and development within the

166 neocortex as well as the dentate gyrus.

Although brief, the following synopsis of neuronal migration illustrates several key steps, which, if misregulated, may underlie granule cell dispersion and basal dendrite formation in the epileptic hippocampus as suggested by previously referenced mutant mice. Migration of newborn neurons in the postnatal brain is a complex and tightly regulated process. As with the lamination of the hippocampus, proper guidance of immature neurons to the correct location requires interactions between the extracellular matrix, guidance signals, cell adhesion molecules and cell-surface tyrosine kinase or integrin signaling receptors. Much of what is known regarding the cellular and molecular control of migrating newborn neurons in the postnatal brain stems from studies of newborn neurons originating from the subventricular zone en route to the olfactory bulb along the rostral migratory stream (for review see Ghashghaei et al., 2007). In order to commence migration, neurons must first set-up a leading process followed by polarization of the nucleus, a sequence that is facilitated by LIS1 (Reiner, 2000; Tsai and Gleeson, 2005). Once en route, migrating neurons are guided by cues from extracellular matrix proteins via cell surface signaling receptors, such as 1 integrins (Belvindrah et al.,

2007). When a neuron reaches its destination, local extracellular matrix molecules, such as

Reelin (D'Arcangelo et al., 1995), direct the cell’s correct positioning by binding with cell-surface receptors. In the case for Reelin, these receptors are very low density lipoprotein receptor and apolipoprotein E receptor 2 (D'Arcangelo et al., 1999; Hiesberger et al., 1999). Once correctly located, neurons then begin to develop neurites, which become the cell’s dendrites and axon.

Cyclin dependent kinase 5 (Cdk5) and its co-activators, p35 and p39, are implicated in this process (Nguyen et al., 2002; Nikolic et al., 1998; Xiong et al., 1997; Zheng et al., 1998).

Additionally, DISC1 has been recently implicated in regulating several of these processes

(Meyer and Morris, 2009; Porteous and Millar, 2009). Specifically, it has been implicated in regulating neuronal migration through its interaction with Nuclear distribution factor E

167 homologue 1 (NDE1) and NDE1-like proteins (Bradshaw et al., 2009). DISC1 has also been shown to function in neurite outgrowth, cell-cell and cell-matrix adhesions by regulating the expression of b1 integrins (Hattori et al., 2010). Importantly, DISC1 is implicated in the integration of newborn neurons into the postnatal hippocampus (Duan et al., 2007).

Recent studies have found that several of these proteins, which were thought to function separately, in fact interact with each other. Most important to the subsequently proposed work is the interaction between reelin signaling and p35/Cdk5. In neurons entering their final destination, the granule cell layer for example, reelin signaling acts to give neurons positional cues so that they become correctly located within their final destination (Ghashghaei et al.,

2007). In order for this process to commence, the adapter protein Disabled 1 (Dab1) must be phosphorylated by Fyn tyrosine kinase (Arnaud et al., 2003). Recently, Ohshima and colleagues (2007) have shown that Cdk5 suppressed this action by phosphorylation of multiple serine/threonine sites on Dab1, perhaps signaling the cell to re-initiate migration. This mechanism is potentially interesting as it may explain increases in neuron migration when the reelin signal is lost. Incidentally, decreased expression of reelin in two animal models of epilepsy has shown that mature and immature granule cells become ectopically localized (Gong et al., 2007; Heinrich et al., 2006). Such interactions between several signaling pathways involved in neuronal migration may partly explain why only some of the granule cells in each of the aforementioned mutant mice exhibit ectopic localization and basal dendrites.

Future directions

Today, the number of tools to manipulate and study genetics and cellular pathways are vast, and several laboratories have recently begun exploring the impact of Cdk5 signaling in postnatal granule cell neurogenesis, subsequent neuronal differentiation and dendrite development within the hippocampus. Indeed, some of its substrates, nestin and doublecortin

168 (DCX), are required for neurogenesis (Sahlgren et al., 2003; Tanaka et al., 2004). The activity of Cdk5 is restricted to postmitotic neurons (Hellmich et al., 1992), and requires association with co-activators p35 and p39 (Lew et al., 1992; Tsai et al., 1994). Lagace and colleagues (2008) utilized Nestin-CreERT2/R26R-YFP mice crossed to floxed Cdk mice to generate conditional knockout out Cdk5 mice, which occurred in nestin-expressing neural stem/progenitor cells. This lead to a decrease in neurogenesis with significantly less NeuN immunoreactive, YFP double- labeled cells and no change in the number of DCX, YFP double-labeled cells. They also demonstrated a significant amount of SGZ cell death, and ultimately concluded that Cdk5 expression is required for the survival of adult-generated neurons. Additionally, conditional knockout of Cdk5 in mature granule cells using the CAMKII-Cre mouse resulted in significantly less DCX immunoreacitve neurons, and no change in the number of NeuN immunoreactive neurons. These data suggest that Cdk5 regulates adult-generated neuron maturation by influencing the environment of the neurogenic niche. Interestingly, p35 knock out mice also had significantly fewer DCX immunoreactive neurons, confirming their earlier finding. Jessberger and colleagues (2008) conducted a second study of interest, in which they unilaterally injected a reporter retrovirus overexpressing a dominant-negative form of Cdk5 into the dentate gyrus, thereby limiting expression to neural stem/progenitor cells. From this experiment they found that newborn neurons deficient in Cdk5 developed aberrant dendrites that extended into the hilus as well as along the granule cell layer, implicating the regulation of Cdk5 in the formation of hilar basal dendrites.

Future experiments

Together, the above literature suggests that Cdk5 and/or its co-activator p35 may underlie the decrease in neurogenesis observed in the cell-fate studies outlined in Chapter 3 and 4. Additionally, the interaction of this signaling pathway with other signaling pathways such as reelin may underlie the ectopic migration of adult-generated and mature granule cells as well

169 as basal dendrite formation observed in Chapters 2 and 4. Whether similar or distinct cellular regulatory mechanisms underlie these phenomena remains to be explored; however a possibility is that lack or disruption of Cdk5/p35 function may vary depending on the age of the neuron affected. I propose, as a first step in determining whether this signaling pathway is affected in the animal models of seizures and epilepsy used in this dissertation, that tissue from

Chapters 2-4 be immunostained against Cdk5, p35 and its downstream effector phospho-tau

(Ser202) and total tau (Tian et al., 2010). This will aid in determining the expression pattern of these proteins following seizures during early-life and adulthood, and aim to determine whether

Cdk5 signaling disruption underlies our observed changes in cell-fate and hilar basal dendrite formation following seizures. In order to semi-quantify the expression levels of these proteins after seizures, western blot should also be performed. Additionally, there are several mouse lines that might be of interest to begin investigating Cdk5/p35 signaling in relation to the ectopic migration of granule cells and basal dendrite formation. Given that these are hypothesized to occur due to migration defects, we could selectively target our experimental design so it is aimed at neurons in this stage of differentiation. Therefore, I propose we use the POMC-

CreERT2 (Jackson laboratories) and DCX-CreERT2 (Jablonska et al., 2010) mice to induce genetic manipulations in late progenitor cells (Type-3). Previous work with POMC-GFP mice have shown GFP co-expression with PSA-NCAM (Overstreet et al., 2004), and have a similar morphologically to horizontal D cells (Seri et al., 2004), which differentiate into immature granule cells. Similarly, DCX is expressed in migrating neurons. Either of these two mouse lines could be crossed to make triple transgenic knockout using our current GFP reporter mouse as well as

Cdk5tm2Kul , floxed Cdk5, mouse. Given the findings reported by Jessberger and colleagues

(2008), it is plausible that floxed p35 mice might be a good candidate since p35 it is required for

Cdk5 function; p35 is also highly expressed in adult mouse dentate (Zheng et al., 1998) (no studies in neonates are available). Since we have identified formation of recurrent basal dendrites on fully differentiated granule cells, we can cross the same floxed mice with CamKII-

170 Cre(T50) mice (Kuhn et al., 2005). Although Lagace and colleagues (2008) used this genetic strategy, granule cell morphology was not explored in these mice. Together, these are several experiments that would help position our laboratory at the forefront of granule cell dysmorphogenesis research.

The proposed future directions are innovative as well as an important first step in elucidating the underlying mechanisms of ectopic granule cell migration and basal dendrite formation. Interestingly, several recent studies have implicated that p35/Cdk5, reelin signaling as well as DISC1 may all play a part in the regulation of the phosphatidylinositol 3- kinase/AkT/mammalian target of rapamycin signaling pathway (Beffert et al., 2004), which exerts its effects on cellular proliferation, growth survival and mobility (Endersby and Baker,

2008). This signaling pathway is also implicated in several neurological disorders such as tuberous sclerosis complex disease, epilepsy, autism schizophrenia and brain tumors

(Buckmaster et al., 2009; de Vries, 2010; Endersby and Baker, 2008; Greer and Wynshaw-

Boris, 2006; Huang et al., 2010; Kwon et al., 2006; Porteous and Millar, 2009; Wong, 2010;

Zeng et al., 2009).

Implications for the future of epilepsy research

Following a seizure is a latent period during which maladaptive changes occur, culminating with the appearance of spontaneous seizures. Thus, during this latent period, when significant remodeling of neuronal networks is thought to occur, is when a preventative might be most useful. Possible targets for such therapeutic intervention are dentate granule cells since aberrant neurogenesis, development and integration following seizure activity are implicated in the progression of epilepsy and associated cognitive deficits. The results of the studies of this thesis provide support for investigating the regulation of neurogenesis under control and epilptogenic conditions. Recently, psycotropic drugs have been shown to alter granule cell

171 neurogenesis (for review see Duman, 2004). Implenting such drugs as fluoxetine (Santarelli et. al., 2003; Nasrallah et. al., 2010), clozapine (Halim et. al., 2004) and MK-801 (Halim et. al.,

2004) in the experimental paradigms of Chapters 3 and 4, will provide insight into whether or not such drugs will rescue the alterations in neurogenesis presented in the studies of thesis. In contrast to the field of neurogenesis, there is currently no drug targeting the development of dentate granule cell pathologies, which is due to the fact that the underlying cellular mechanisms by which granule cell pathologies develop remain unclear. Therefore, elucidating the mechanisms by which an epileptogenic insult leads to maladaptive changes in the structure and connectivity of hippocampal dentate granule cells is also of great interest. It is our hope that our description of a novel process by which recurrent basal dendrites form will spur new research of the cellular mechanisms underlying the formation of recurrent basal dendrites.

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