Early Life Iron Deficiency Changes the Ratio Between Interneuron Subtypes and Alters Excitability

112 Pages·2017·3.83 MB·English

Early Life Iron Deficiency Changes the Ratio Between Interneuron Subtypes and Alters
Excitability in the Adult Brain
by
Michael John Rudy
Submitted in Partial Fulfillment of the
Requirements for the Degree
Doctor of Philosophy
Supervised by Professor Margot Mayer-Pröschel
Toxicology
School of Medicine and Dentistry
University of Rochester
Rochester, New York
2017
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Table of Contents
Biographical Sketch....................................................................................................... vii
Acknowledgements ....................................................................................................... viii
Abstract .......................................................................................................................... ix
Contributors and Funding Sources .................................................................................. x
List of Tables ................................................................................................................. xii
List of Figures................................................................................................................. xi
List of Abbreviations ..................................................................................................... xiv
Chapter 1 Introduction ..................................................................................................... 1
1.1 Prevalence and stages of iron deficiency ............................................................... 2
1.2 Iron homeostasis within the body ........................................................................... 3
1.3 Consequences of early life iron deficiency ............................................................. 5
1.4 Iron deficiency and the excitatory/inhibitory balance of the brain ............................ 5
Chapter 2 Acute and gestational ID have different effects on seizure susceptibility ......... 7
2.1 Introduction ............................................................................................................ 8
2.2 Materials and Methods ........................................................................................... 8
2.2.1 Animal care ..................................................................................................... 8
2.2.2 Diet groups ...................................................................................................... 9
2.2.3 Pentylenetetrazole seizure testing ................................................................... 9
2.2.4 Calculation of “seizure response” and A-E values for PTZ dosed animal ....... 10
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2.2.5 Hypoxia seizure testing ................................................................................. 13
2.2.6 Hematocrit measurements: ........................................................................... 13
2.2.7 Western blot analysis: ................................................................................... 13
2.2.8 Statistical Analysis: ........................................................................................ 14
2.3 Results................................................................................................................. 15
2.3.1 Establishment of iron deficiency throughout gestational and postnatal life using
a nutritional model: ................................................................................................. 15
2.3.2 Gestational ID and acute ID have different effects on Pentylenetetrazole (PTZ)
induced seizures .................................................................................................... 19
2.3.3 Inverse relationship between seizure Class I and Class V ............................. 24
2.3.4 Gestationally ID animals are less sensitive to hypoxia induced seizures ....... 26
2.4 Discussion ........................................................................................................... 28
2.4.1 Suppressive interaction between Class I and Class V seizures in aID group . 28
2.4.2 Greater inhibitory tone in gID animals ............................................................ 28
2.4.3 Opposite effects of aID and gID on seizure response and conflict in the
literature ................................................................................................................. 29
2.4.4 Sex differences in seizure susceptibility ........................................................ 31
2.4.5 Gestational ID decreases excitation or increases inhibition in the brain ......... 31
Chapter 3 Gestational ID disrupts interneuron ratios in postnatal cortex........................ 33
3.1 Introduction .......................................................................................................... 34
3.2 Materials and methods ......................................................................................... 35
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3.2.1 Preparation of postnatal brains for staining.................................................... 35
3.2.2 IHC staining ................................................................................................... 35
3.2.3 DAPI counts using imageJ ............................................................................ 36
3.2.4 IHC counts for somatostatin, parvalbumin, and calretinin .............................. 37
3.2.5 Statistical analysis ......................................................................................... 37
3.3 Results................................................................................................................. 38
3.3.1 Resolution of ID following supplementation ................................................... 38
3.3.2 Interneuron ratios are significantly altered under gestational ID ..................... 38
3.3.3 Loss of PV expression at P14 is layer specific ............................................... 42
3.3.4 Growth of ID and control animals after iron supplementation ......................... 45
3.4 Discussion ........................................................................................................... 47
3.4.1 Specific loss of layer 2/3 parvalbumin expressing cells .................................. 47
3.4.2 Increase in SST positive interneurons following gID ...................................... 48
3.4.3 Somatostatin and growth ............................................................................... 48
3.4.4 ASD and early life ID ..................................................................................... 49
3.4.5 PV expressing interneurons are no longer significantly reduced at P100 ....... 50
Chapter 4 Iron deficiency disrupts early patterning of the medial ganglionic eminence .. 52
4.1 Introduction .......................................................................................................... 53
4.2 Materials and methods ......................................................................................... 55
4.2.1 Timed matings for embryonic ages ................................................................ 55
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4.2.2 Orienting and matching embryonic sections .................................................. 56
4.2.3 IHC staining ................................................................................................... 58
4.2.4 Real time quantitative PCR ........................................................................... 59
4.2.5 Gli1/LacZ reporter mouse .............................................................................. 59
4.2.6 Calculation of Ki67 and Nkx2.1 overlap for proliferation measurements ........ 60
4.2.7 Calculation of CoupTF2+ cells per mm in P0 cortex ...................................... 60
4.2.8 Calculation of percent GABA+ cells in P0 cortex ........................................... 61
4.2.9 Statistical analysis ......................................................................................... 62
4.3 Results................................................................................................................. 62
4.3.1 Gli1 and Nkx2.1 signaling increased at E12 under ID .................................... 62
4.3.3 Expansion of Nkx2.1 and increased proliferation at E14 under ID ................. 66
4.3.4 CoupTF2 unchanged at E12 and P0 ............................................................. 70
4.3.5 Pax6 unchanged at E14 ................................................................................ 73
4.3.6 GABA expressing cells increased at P0 ........................................................ 75
4.3.7 Oxysterols increased at E14 under ID ........................................................... 77
4.4 Discussion ........................................................................................................... 79
4.4.1 Not all brains show expansion of Nkx2.1 ....................................................... 79
4.4.2 Nkx2.1 and Gli1 do not overlap despite both being SHH targets ................... 80
4.4.3 Why iron deficiency may specifically target Sonic Hedgehog......................... 81
4.4.4 Regions surrounding the MGE are not affected ............................................. 82
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4.4.5 Increase in GABAergic cells of P0 cortex shows successful migration from
MGE ....................................................................................................................... 82
4.4.6 Programmed cell death between P0 and P14 ................................................ 83
Chapter 5 Conclusion .................................................................................................... 84
5.1 Summary of work ................................................................................................. 85
5.2 Future directions .................................................................................................. 87
5.2.1 Staining for markers of early and late born interneuron subtypes .................. 87
5.2.2 Changes in interneuron ratio within striatum and hippocampus ..................... 87
5.2.3 Confirmation of increased oxysterols under ID .............................................. 87
5.2.4 Increased somatostatin and changes in pituitary hormone release ................ 88
5.2.5 Tracking Nkx2.1 derived cells into adulthood ................................................. 88
Bibliography .................................................................................................................. 90
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Biographical Sketch
Michael Rudy was born in California, but lived in many places including Texas, Guatemala, and
Colorado. In 2002, he began taking courses at Pikes Peak Community College where his love of
knowledge drove him to take nearly every science class they offered. In 2005, he transferred to
the University of Colorado at Colorado Springs and continued his scholastic journey, taking many
additional biology and chemistry courses simply because he wanted to understand the topics.
During his time at the University of Colorado he was given the opportunity to teach the lab portion
of their Anatomy and Physiology course. It was during his three years teaching this course that he
realized his desire to teach at a university. He graduated Cum Laude with a Bachelor of Science
degree in Biology in 2009.
He accepted an invitation to join the Toxicology Program in the Department of
Environmental Medicine at the University of Rochester and in 2012 joined the laboratory of
Margot Mayer-Proschel studying the effects of iron deficiency on neuronal development. He
successfully passed his qualifying exam in 2013 and received his Master of Science degree in
Toxicology. Over the next three years he presented his work at national conferences, served as a
teaching assistant for Biochemical Toxicology where he also had the opportunity to teach three
guest lectures on toxicodynamics and receptor theory, received the Weiss Toxicology Scholar
award for leadership and excellence in neurotoxicology research, served as chair for the monthly
toxicology meeting, and was awarded a New York Stem Cell Training Grant.
The following paper has been submitted for review: “Iron deficiency affects seizure
susceptibility in a time and sex specific manner”. Michael J Rudy, Margot Mayer-Proschel. 2017
journal of ASNneuro.
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Acknowledgements
I especially want to thank my wife, Leah, for all of the work she has done to support me during
this time. She has made meals, cleaned, unpacked, repacked, and gone to social events solo
without complaint; all the while giving up our date nights and weekends because I was busy
dissecting, or doing cell counts, or analyzing data. She encouraged me through the times when I
was burned out and forced me to remember that today’s difficulties will pay off in our future. I
couldn’t have done this without her.
I also want to thank Margot for all of her encouragement when experiments didn’t turn out
the way I expected, and for always having an open door to discuss the project (or anything else
that was on my mind). She provided a perfect balance of support during the difficult times and a
kick-in-the-butt when I could be working harder. I have immensely enjoyed the intellectual
challenge she provided in this project and the freedom she granted me in figuring out where we
should take the project.
I want to thank my mom and dad for encouraging me to think, both critically and
creatively, about all of the unknowns in this life. This skill has benefited me tremendously through
my schooling and in my life as a whole. I also want to thank them for all of the support they
provided through my many years of schooling.
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Abstract
Iron deficiency (ID) affects billions of people worldwide, making it the most common micronutrient
deficiency. Of greater concern, it disproportionately affects pregnancy and early development,
which are precisely when ID has the most detrimental effects. Many of the mental disorders,
which are associated with early life ID, are also associated with an imbalance between excitation
and inhibition in the brain. To examine the excitatory/inhibitory (E/I) balance of the ID brain, we
used seizurogenic insults to probe the seizure threshold under different nutritional models of iron
deficiency. We found that gestationally ID mice had an increased seizure threshold even after
iron was replete, suggesting that early life ID disrupts the E/I balance of the brain. When we
examined the composition of a gestationally ID brain, we found a perturbation in GABAergic
interneuron subtypes, with a permanent increase in the somatostatin subtype and an early
decrease in the parvalbumin subtype. Further examination of the embryonic regions that give rise
to GABAergic interneurons, revealed that ID brains had expanded expression domains for Nkx2.1
and Gli1: two transcription factors which are important for appropriate specification of GABAergic
interneurons, specifically the somatostatin and parvalbumin subtypes. Taken together, our data
suggests that embryonic ID disrupts the E/I balance of the adult brain by disrupting transcription
factors in the region that specifies inhibitory GABAergic interneurons during development.
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Contributors and Funding Sources
This work was supported by a dissertational committee consisting of Professor Margot Mayer-
Proschel (advisor) of the Department of Biomedical Genetics, and Professor Michael O’Banion of
the Neuroscience Department, and Professor Ania Majewska of the Neuroscience Department,
and Professor James Palis of the Peds M&D Hematology/ Oncology Department. The data
depicted in Chapter 2 was submitted for publication in 2017 in an article listed in the Biographical
Sketch. All work conducted for the dissertation was conducted by student independently.
Graduate study was supported by the Toxicology training grant (5T32ES007026-37) and the New
York Stem Cell Training Grant (GR525565).