Genetic and Anatomical Dissection of Sleep and Arousal in Drosophila melanogaster

185 Pages·2015·28.08 MB·English

by  James Robinson

UC San Diego
UC San Diego Electronic Theses and Dissertations
Title
Genetic and Anatomical Dissection of Sleep and Arousal in Drosophila melanogaster
Permalink
https://escholarship.org/uc/item/0sr4628g
Author
Robinson, James Edward
Publication Date
2015

Peer reviewed|Thesis/dissertation
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University of California
UNIVERSITY OF CALIFORNIA, SAN DIEGO

Genetic and Anatomical Dissection of Sleep and Arousal in Drosophila
melanogaster

A dissertation submitted in partial satisfaction of the requirements of the
degree Doctor of Philosophy

in
Biomedical Sciences
by
James Edward Robinson

Committee in Charge:
Professor William Joiner, Chair
Professor Rolf Bodmer
Professor Joan Heller Brown
Professor Gabriel Haddad
Professor Jing Wang

2015
Copyright
James Edward Robinson, 2015
All rights reserved.
The dissertation of James Edward Robinson is approved, and it is acceptable
in quality and form for publication on microfilm and electronically:

Chair

University of California, San Diego
2015

iii
DEDICATION
In recognition of the support, assistance, love, and patience given to me, this
dissertation is dedicated to:

My parents, who have always fostered an environment of lifelong learning and
curiosity. Thank you for providing me with the experiences that have shaped
me into who I am today.

My siblings, who challenge and push each other toward betterment. Thank
you for the fun we have had and will have.

My daughter, Edith, who despite the brevity of her time with us so far, has
already shown me another fun-filled side of life. Thank you for the joy you
bring and will continue to bring.

My wife, who tolerates and supports my ambitions and interests while keeping
me focused on what is truly important. Thank you for your love and patience
as we journey through life together.

iv
EPIGRAPH

Always plan with the end result in mind… Be clear. Be specific.
Stephanie Goddard Davidson

v
TABLE OF CONTENTS
Signature Page ................................................................................................. iii

Dedication ......................................................................................................... iv

Epigraph ............................................................................................................ v

Table of Contents ............................................................................................. vi

List of Abbreviations ......................................................................................... xi

List of Figures ................................................................................................. xiv

List of Tables .................................................................................................. xvii

Acknowledgements ....................................................................................... xviii

Vita .................................................................................................................. xx

Abstract of the Dissertation ............................................................................ xxii

Chapter 1: Introduction .................................................................................. 1
1.1 Sleep as a historical question ........................................................... 1
1.2 Sleep in health and disease .............................................................. 2
1.3 Drosophila as a model organism for sleep research ........................ 4
1.4 References ....................................................................................... 6

Chapter 2: ADAR-mediated RNA editing suppresses sleep by acting as a
brake on glutamatergic synaptic plasticity ................................ 13
2.1 Summary ........................................................................................ 13
2.2 Introduction ..................................................................................... 13
2.3 Results ............................................................................................ 15
2.3.1 ADAR is required for wake maintenance .......................... 15
2.3.2 ADAR suppresses glutamatergic signaling ....................... 17
vi
2.3.3 Elevated DVGLUT and NMDA receptor activity are
required for increased sleep in Adar mutants ................... 19
2.3.4 Expansion of the reserve pool of synaptic vesicles is
required for increased sleep in Adar mutants ................... 21
2.4 Discussion ...................................................................................... 24
2.5 Methods .......................................................................................... 27
2.5.1 Fly stocks .......................................................................... 27
2.5.2 Sleep and locomotor measurements ................................ 28
2.5.3 Sleep deprivation and rebound ......................................... 28
2.5.4 Sleep reversibility measurements ..................................... 29
2.5.5 Climbing assays ................................................................ 29
2.5.6 Ethanol sensitivity and tolerance ...................................... 29
2.5.7 Immunohistochemistry ...................................................... 30
2.5.8 Western blot analysis ........................................................ 30
2.5.9 Electrophysiology .............................................................. 31
2.5.10 Data analysis and statistics ............................................. 32
2.6 Acknowledgements ......................................................................... 33
2.7 Figures ............................................................................................ 34
2.8 References ..................................................................................... 46

Chapter 3: Development of a novel method for quantitative measurements
of arousal thresholds in Drosophila ............................................ 51
3.1 Summary ........................................................................................ 51
3.2 Introduction ..................................................................................... 51
3.3 Results ............................................................................................ 53
3.3.1 An apparatus for measuring arousal threshold ................. 53
3.3.2 Daytime and nighttime arousal are highly similar ............. 54
3.3.3 Arousal threshold measurements are reproducible across
days .................................................................................. 55
3.3.4 Interpulse interval length does not influence arousal ........ 56
3.3.5 Arousal threshold across time of day does not change .... 56
3.4 Discussion ...................................................................................... 57
3.5 Methods .......................................................................................... 59
vii
3.5.1 Fly stocks .......................................................................... 59
3.5.2 Sleep and locomotor measurements ................................ 59
3.5.3 Arousal threshold stimulations .......................................... 59
3.5.4 Arousal threshold analysis ................................................ 60
3.5.5 Statistics ............................................................................ 61
3.6 Acknowledgements ......................................................................... 62
3.7 Figures ............................................................................................ 63
3.8 References ..................................................................................... 68

Chapter 4: Identification of neurons with a privileged role in sleep
homeostasis in Drosophila melanogaster .................................. 70
4.1 Summary ........................................................................................ 70
4.2 Introduction ..................................................................................... 71
4.3 Results ............................................................................................ 73
4.3.1 Homeostatic recovery sleep can be uncoupled from prior
waking time ...................................................................... 73
4.3.2 Select arousal-promoting circuits suppress sleep
homeostasis ..................................................................... 77
4.3.3 Select groups of cholinergic neurons promote sleep
homeostasis ..................................................................... 79
4.3.4 Intersecting expression patterns of two promoters identifies
a restricted set of neurons that promote sleep homeostasis
.......................................................................................... 80
4.3.5 Split Gal4-labeled neurons are distinct from neural circuitry
that controls baseline sleep .............................................. 82
4.3.6 Sleep homeostasis is important for short-term memory ... 83
4.4 Discussion ...................................................................................... 85
4.5 Methods .......................................................................................... 89
4.5.1 Fly stocks and transgenic fly lines .................................... 89
4.5.2 Behavioral assays ............................................................. 89
4.5.3 Molecular biology .............................................................. 92
4.5.4 Immunohistochemistry ...................................................... 92
4.5.5 Statistics ............................................................................ 93
4.6 Acknowledgements ......................................................................... 93
viii
4.7 Figures ............................................................................................ 95
4.8 References ................................................................................... 108

Chapter 5: Nf1 is a sleep-promoting factor in Drosophila melanogaster ... 113
5.1 Summary ...................................................................................... 113
5.2 Introduction ................................................................................... 113
5.3 Results .......................................................................................... 115
5.3.1 Nf1 is required in cholinergic neurons for normal sleep .. 115
5.3.2 A Gal4 screen for neurons requiring Nf1 for normal sleep
behavior .......................................................................... 116
5.3.3 Generation of an endogenous epitope-tagged Nf1 allele
using CRISPR ................................................................ 119
5.3.4 NF1 is broadly expressed in the brain but is not expressed
in the mushroom bodies ................................................. 120
5.4 Discussion .................................................................................... 121
5.5 Methods ........................................................................................ 122
5.5.1 Fly stocks ........................................................................ 122
5.5.2 Sleep and locomotor measurements .............................. 123
5.5.3 Generation of epitope-tagged Nf1 allele ......................... 123
5.5.4 Generation of transgenic animals ................................... 124
5.5.5 Immunohistochemistry .................................................... 125
5.5.6 Western blot analysis ...................................................... 126
5.5.7 Statistics .......................................................................... 127
5.6 Acknowledgements ....................................................................... 127
5.7 Figures .......................................................................................... 128
5.8 References ................................................................................... 134

Chapter 6: SLEEPLESS is a bifunctional regulator of excitability and
cholinergic synaptic transmission ............................................ 139
6.1 Summary ...................................................................................... 139
6.2 Introduction ................................................................................... 140
6.3 Results .......................................................................................... 143
6.3.1 qvr/sss genetically interacts with nAChR subunits to control
sleep ............................................................................... 143
ix