Astrocytes Role in Lipid Mediated Synaptic Activity By Nathan Anthony Smith Submitted in Partial ...

117 Pages·2013·4.95 MB·English

Astrocytes Role in Lipid Mediated Synaptic Activity

By

Nathan Anthony Smith

Submitted in Partial Fulfillment of the

Requirements for the Degree

Doctor of Philosophy

Supervised by Professor Maiken Nedergaard

Neuroscience
School of Medicine and Dentistry

University of Rochester
Rochester, New York

2013
ii

I dedicate this work to my family, especially my mother and grandmother because I
would not be where I am today if it were not for their love and support.
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Biographical Sketch

The author was born in Baton Rouge, Louisiana. He attended Xavier University of
Louisiana, and graduated with a Bachelor of Science Degree in Biology Pre-Med. He
began his doctoral studies in the Neuroscience at the University of Rochester in 2005. He
was award a National Institutes of Health Training Grant in 2007 and a National
Institutes of Health Training Grant in 2011. He was awarded Masters of Science degree
from the University of Rochester in 2010. He pursed his research in cellular
neuroscience under the direction of Professor Maiken Nedergaard.
The following publications were a result of work conducted during doctoral study:

Smith NA, Wang F, Xu Q, Fujita T, Baba A, Matsuda T, Takano T, Bekar L, Nedergaard
M (2012) Astrocytes modulate neural network activity by Ca(2)(+)-dependent uptake of
extracellular K(+). Science signaling 5:ra26.

Lovatt D, Xu Q, Liu W, Takano T, Smith NA, Schnermann J, Tieu K, Nedergaard M
(2012) Neuronal adenosine release, and not astrocytic ATP release, mediates feedback
inhibition of excitatory activity. Proc Natl Acad Sci U S A 109:6265-6270.

Fujita T, Williams EK, Jensen TK, Smith NA, Takano T, Tieu K, Nedergaard M (2012)
Cultured astrocytes do not release adenosine during hypoxic conditions. J. Cereb. Blood
Flow Metab 32:e1-7.

Kaback A, Soung DY, Naik A, Smith N, Schwarz EM, O’Keefe RJ, Drissi H (2008)
Osterix/Sp7 Regulates mesenchymal stem cell mediated endochodral ossification. Journal
of Cellular Physiology 214:173-82.

Wang YJ, Zhou CJ, Shi O, Smith N, Li TF (2007) Aging delays the regeneration process
following sciatic nerve injury in rats. Journal of Neurotrauma. 24(5):885-94.

Smith N, Dong Y, Lian JB, Prata J, Kingsley PD, Wijnen AJ, Stein JL, Schwatz EM,
O’Keefe RJ, Stein GS, Drissi H (2005) Overlapping Expression of Runx 1 (Cbfa2) and
Runx2 (Cbfa1) transcription factors support cooperative induction of skeletal
development. Journal of Cellular Physiology 203: 133-143.
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Acknowledgments

I would like to thank my advisor, Dr. Maiken Nedergaard, for her unwavering support
and constant guidance. I would also like to thank my committee members, Drs. Kerry
O’Banion, Jian Kang, and John Olschowka. An additional thanks to Drs. Kim Tieu,
Edward Vates, and Hicham Drissi for their strong letters of support.
I would also like to give a special thanks to Drs. Takahiro Takano and Lane Bekar for
their constant support and training throughout my graduate career. I also extend my
sincerest appreciation to all of laboratory colleagues in the Center for Translational
Neuromedicine.
My sincerest gratitude goes out to my funding sources from the National Institutes of
Health for National Service Research Award and Neuroinflammation and Glial Cell
Biology Training Grant.
I am very thankful for all the love and support I received from my family. Most
importantly, I am extremely grateful to both my mother and my grandmother who have
been a driving force in my life for years and for teaching me that education is the key to
achieve all of my dreams.
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Abstract

Astrocytes are a major cell type in both the human and rodent central nervous systems.
Due to their inability to be electrically excited, astrocytes have been viewed as supportive
cells providing a suitable environment for neuronal signaling without participating in
information processing. This simplistic view has been overturned in the past few decades
by evidence showing that astrocytes can respond directly to local neuronal activity with
subsequent modification. Neurotransmitters such as glutamate can initiate intracellular
Ca2+ signaling in astrocytes that leads to astrocyte release of gliotransmitters, including
glutamate, D-serine, ATP or endocannabinoids, which can modulate nearby synaptic
strength. Moreover, astrocytes release Arachidonic Acid metabolites, such as PGE ,
2
modulating local blood flow to meet the energy demands of increased neuronal activity.
Observations in our lab have demonstrated that vasodilation is seemingly dissociable
from astrocytic calcium dynamics in many cases, thus prompting us to test whether
astrocytes, through lipid release, can signal on a faster signaling scale in a calcium-
independent manner. Thus far, virtually all studies looking at astrocytic signaling
mechanisms focus on intracellular Ca2+, which is on a signaling time scale of seconds.
However, astrocytes are capable of Ca2+ independent signaling that is potentially on a
time scale one to two orders of magnitude faster (msec). In line with this, astrocytes
possess Ca2+-independent PLA that can lead to the production of AA in the absence of
2
calcium. However, further investigation is needed to reveal the existence of Ca2+-
independent signaling from astrocytes and whether this can influence physiological
function. We report that upon Ca2+ chelation and receptor stimulation astrocytes can
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release lipid in a Ca2+ independent manner and that these lipids can indeed affect
neuronal signaling. Furthermore, using a model of transient Heterosynaptic Depression
(tHSD) in cortical slices, we report that astrocytic modulation of synaptic transmission
occurs independent of traditional gliotransmitters, such as ATP and glutamate, but
through astrocytic release of endocannabinoids. Taken together, these results provide
novel insight into the controversial role of astrocytes in synaptic regulation and thereby in
higher information processing.
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Contributors and Funding Sources
This work was supervised by a dissertation consisting of Professors Maiken Nedergaard
(advisor), Kerry O’Banion of the Department of Neurobiology and Anatomy, Jian Kang
of the Department of Cell Biology and Anatomy at New York Medical, and John
Olschowka of the Department of Neurobiology and Anatomy. The data analyzed in the
last figure of chapter 2 was provided by Dr. Fushun Wang. All other work conducted for
the dissertation was completed by the student independently. Graduate study was
supported by a National Institutes of Health Training Grant for Neuroinflammation and
Glial Cell Biology and a National Institutes of Health National Research Service Award.
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Table of Contents

Chapter 1: Introduction 1
1.1: Astrocytes are multifunctional cells 2
1.2: Astrocytic Gliotransmitters 2
1.3: Astrocytes role in Blood Flow 7
1.4: Astrocytic modulation of Synaptic Activity 10
1.5: Astrocytic Calcium Signaling 15
1.6: Astrocytic Receptor Mediated Lipid Release 16
1.7: Significance 18
Chapter 2: Calcium Independent Astrocytic Release of Lipid Modulation 20
Abstract 21
2.1: Introduction 22
2.2: Results 23
2.2.1: GPCR-mediated Ca2+-independent release of 3H-AA 23
and/or its metabolites from astrocytic cultures

2.2.2: iPLA activity is essential for Ca2+-independent 25
2
liberation
2.2.3: GPCR-mediated Ca2+-independent release of PGE 31
2
from astrocytic cultures
2.2.4: Connexin 43 does not mediate PGE2 release 35
2.2.5: Ca2+-independent astrocytic lipid release enhances 39
mEPSCs via Kv channels blockade
2.2.6: Discussion 44
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Chapter 3: Astrocytic Endocannabinoids Mediate Transient 48
Heterosynaptic Depression
Abstract 49
3.1: Introduction 50
3.2: Results 52
3.2.1: Astrocytes are necessary for tHSD 52
3.2.2: Group II mGluR are necessary for tHSD 56
3.2.3: Astrocytic vesicular release is not involved in 60
Group II mGluR-mediated tHSD
3.2.4: CB1R antagonists block Group II mGluR-mediated tHSD 66
3.2.5: Discussion 68
Chapter 4: General Discussion 71
Chapter 5: Experimental Procedures 82
5.1: Culture, Ca2+ imaging of cultured cells, and small interfering RNA 83
5.2: Isolation of human fetal astrocytes 84
5.3: Immunocytochemistry 85
5.4: Radiolabelling and Assessment of AA Release 85
5.5: PGE Release Assessment 86
2
5.6: Slice preparation and electrophysiology/Patch clamp 86
5.7: Animals 88
5.8: Slice preparation for tHSD 88
5.9: Electrophysiology Recording and Analysis for tHSD 88
6.0: Statistical Analysis 89
x

References 89

List of Figures
Figure Title Page
Figure 1.1. Mechanisms of Gliotransmitter Release 5
Figure 1.2. Astrocytic modulation of blood flow 8
Figure 1.3. Astrocytic modulation of synaptic activity 13
Figure 2.1. Agonist mediated Calcium Rises in Astrocytic Cultures 27
Figure 2.2. GPCR-mediated Ca2+ independent release of 3H-AA 29
and/or its metabolites from astrocytic cultures
Figure 2.3. GPCR-mediated Ca2+ independent release of PGE 33
2
from astrocytic cultures
Figure 2.4. Connexin 43 does not mediate PGE release 37
2
Figure 2.5. Ca2+-independent astrocytic lipid release enhances 42
mEPSCs via Kv channel blockade
Figure 3.1. Astrocytes are necessary for tHSD 54
Figure 3.2. Group II mGluR are necessary for tHSD 58
Figure 3.3. Astrocytic vesicular release is not involved in 62
Group II mGluR mediated tHSD
Figure 3.4. CB1R antagonist blocks Group II mediated 66
MGluR-mediated tHSD
Figure 4.1 Astrocytic Processes 73
Figure 4.2. Astrocytes role in lipid mediated synaptic activity 77