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

139 Pages·2015·7.77 MB·English

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

A dissertation submitted to the
Division of Research and Advanced Studies of the University of Cincinnati
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
In the Molecular and Developmental Biology Graduate Program
By
Shatrunjai P. Singh
M.Sc. Stem Cell Biology, Manipal University
September 15th 2015

Dissertation Committee:
Steve Danzer, Ph. D. (chair)
Mark Baccei, Ph. D.
Kenneth Campbell, Ph. D
Brian Gebelein, Ph. D.
Ronald Waclaw, Ph. D
ii

Abstract

Epilepsy is a common and devastating neurological disease with no real preventive or
cure. In most cases of acquired epilepsy, an initial precipitating injury to the brain is followed by
a silent period which eventually culminates into the development of spontaneous, recurrent
seizures. This interval between the primary insult and the first seizure is referred to as the latent
period of epileptogenesis and is characterized by abnormal morphological and physiological
changes in the hippocampus. In the studies described herein, I aim to elucidate changes in the
different phases of epileptogenesis with the end goal of deciphering critical epileptogenic
mechanisms.
To study the initial stages of epileptogenesis, I employed the early kindling model. In this
protocol, it is possible to administer a limited number of stimulations sufficient to produce a
lifelong enhanced sensitivity to stimulus evoked seizures without associated spontaneous
seizures. In these experiments, I characterized the morphology of GFP-expressing granule cells
from Thy-1 GFP mice either one day or one month after the last evoked seizure. I observed
several morphological changes at the one day time point, which all normalized to control levels
at the one month time point. Interestingly, I did not observe the presence of basal dendrites,
frequently observed in other models of epilepsy. These findings demonstrate that the early stages
of kindling epileptogenesis produces transient morphological changes but not the dramatic
pathological rearrangements of dentate granule cell structure seen in typical models associated
with spontaneous seizures.
To study epileptogenesis after the incidence of spontaneous, recurrent seizures, I used the
pilocarpine model of epilepsy. Our lab has previously used this model to show that adult
hippocampal neurogenesis is profoundly altered under epileptic conditions, leading to the
iii

production of morphologically abnormal dentate granule cells. Under epileptic conditions, these
adult generated cells migrate to ectopic locations and develop misoriented basal dendrites.
Although it has been established that these abnormal cells are newly-generated, it is not known
whether they arise ubiquitously throughout the progenitor cell pool or are derived from a smaller
number of bad actor progenitors. To explore this question, I describe clonal analysis
experiments conducted in epileptic mice expressing the brainbow fluorescent protein reporter
construct in dentate granule cell progenitors. Brain sections were rendered translucent so that
entire hippocampi could be reconstructed and all fluorescently-labeled cells identified. The
findings revealed that a small number of progenitors produced the majority of ectopic cells in
epileptic mice, indicating that either the affected progenitors or their local micro-environments
had become pathological. By contrast, granule cells with basal dendrites were equally
distributed among clonal groups. These findings strongly predict that distinct mechanisms
regulate different aspects of granule cell pathology in epilepsy.
The experiments described here utilize different models of epilepsy and employ cutting edge
technology to provide valuable insight into the process of epileptogenesis. The results and ideas
presented here are intended to advance our knowledge of epilepsy and eventually lead to better
antiepileptic therapies.
iv
v

Dedication

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

Acknowledgement

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

Completing a doctoral thesis, although long and sometimes demanding, was the best decision I
have ever made. My journey through graduate school has taught me a lot and has made me a
better, happier person. This would not have been possible without my mentors, family, friends
and colleagues, who have helped me at every step of my career. They have selflessly looked out
for my best interests and have tirelessly supported me in my pursuit of happiness.
I would like to thank Dr. Steve Danzer for showing me what an ideal boss should be like.
During the course of my business classes, I had enrolled in a leadership class and every lecture
made me realize that he had already showed me every technique they could possibly teach.
Steve’s scientific brilliance mixed with a high emotional quotient, empathy, perfectionism and
the ability to motivate people makes him the best mentor I could have asked for. I am indebted to
him for granting me the freedom to take business courses, do an internship and ultimately for
molding my career. Thank you.
I would also like to thank my dissertation committee for guiding me through the ups and downs
of graduate school. I am indebted to Dr. Brian Gebelein, for believing in me and writing endless
recommendation letters which helped me get grants and fellowships. I am grateful to Dr. Mark
Baccei for always being forthcoming with new ideas and making himself ever available for
advice and inspiration. I am obliged to Dr. Ronald Waclaw, for helping me develop the skill of
asking better scientific questions and for always reassuring me that I am always on track to
vii

graduate. I would like to thank Dr. Kenneth Campbell, for his constant guidance on the future
of my research and his unfaltering support in every committee meeting and at every poster
presentation. All of them have been an integral part of my graduate school experience and I will
forever be grateful for all your support.
I am thankful to my lab mates and colleagues, Mike Hester (by far the smartest guy I know),
Isaiah Rolle (for making me feel like family), Dr. Candi LaSarge (my favorite coffee
conversationalist and postdoc par excellence), Bethany Hosford (for showing me how to be an
excellent graduate student), Mary Dusing (for making all our lives livelier and more organized),
Dr. Shaadi Khademi (for patiently listening to all my ‘negative-result-again’ stories), Salwa
Ragab (for making the lab so much fun), Rylon Hofacer (my fellow NPR enthusiast), Dr.
Shane Rowley (for the much needed guy talk), Dongyi Tong (for the great conversations we’ve
had), Victor Santos (the Brazilian who taught me how to like Indian food more), Bernadin
Joseph (the greatest immunostaining genius that has ever lived), Amen An (undergraduate
rockstar whose also a Krav Maga expert) and Jules Rosen (for patiently putting up with all my
jokes). I would like to specially thank Dr. Ray Pun for teaching me electrophysiology, the virtue
of patience and the endless pharmacological support (coffee). I would also like to thank all my
other friends (you know who you are!) for making my life so lively and complete.
My family has always been my strongest quality. I am indebted to my mother, Aprajita Singh,
for her unconditional love, support and guidance at every step of my life. I thank my father,
Devendra Singh, for teaching me everything ranging from cricket to why charity makes you
happy. Dad, thanks for all the things you have done for me and Tutu; we are eternally grateful to
you. I would like to thank my younger brother, Dr. Vijendra Singh (Tutu), who has been my
strongest supporter for as long as I can remember. Tutu, we are all so proud of you (although
viii

mom says she’s more proud of me). I would also like to acknowledge my grandma, because her
voice makes everything better. I would like to thank my cousin, Dr. Sanjay Singh, who besides
being my role model, has always been a source of inspiration and a stronghold of support.
Finally, I would like to thank my extended family that has played such a critical role in my life.
Thank you all.
I would also like to acknowledge support from the University of Cincinnati, the Cincinnati
Children’s Medical Center, the Albert J. Ryan foundation, the American Heart Association
and the American Epilepsy Foundation, without which I could not have completed my studies.
Finally, I would also like to thank the MDB program for providing me the opportunity to
conduct my research at this excellent institute.

Thank you,
Shatrunjai Singh
ix

Table of contents

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

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

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