Dissertation (rev 2011-05-23)_final_pdf

UNIVERSITY OF CALIFORNIA
Los Angeles
Long-term Enhancement of Respiratory-Related Activity by
Increasing the AMPA Receptor-Mediated Excitability of
Hypoglossal Motoneurons In Vitro
A dissertation submitted in partial satisfaction of the
requirements for the degree Doctor of Philosophy
in Neurobiology
by
Walter Edward Babiec
2011

© Copyright by
2011

ii
The dissertation of Walter Edward Babiec is approved.
___________________________________
Nicholas C Brecha
____________________________________
Thomas J O’Dell
____________________________________
Thomas Stephen Otis
____________________________________
Jack L Feldman, Committee Chair
University of California, Los Angeles
2011

iii
DEDICATION
To my parents, for giving me the greatest gift any two people can give another: life.
To my sister and my brother, for being shining examples.
To my wife, for believing in me more than I could ever believe in myself.
To my sons, in the hope that this is some small example of what might be achieved with
patience, persistence, and commitment to following your dreams.

iv
TABLE OF CONTENTS
1 Introduction 1
1.1 Obstructive sleep apnea (OSA) 3
1.2 Why do upper airway obstructions form during sleep? 5
1.3 Strategies for treating OSA 6
1.3.1 Treating the symptoms of OSA 6
1.3.2 Preventing loss of tone during sleep 8
1.3.3 Overcoming sleep-related loss of muscle tone 9
1.4 Dissertation purpose and organization 10
1.5 Rhythmic slice preparation 11
2 The Role of Ionotropic Glutamate Receptors in the Transmission of
Respiratory Drive 14
2.1 iGluR structural overview 16
2.1.1 Common attributes of iGluRs 17
2.1.2 iGluR stoichiometry 19
2.1.3 RNA editing and alternative splicing 20
2.1.4 iGluR accessory proteins 21
2.2 Evidence for iGluRs in XII and phrenic MNs 23
2.2.1 AMPA and kainate receptors in XII and phrenic MNs 24
2.2.2 NMDA receptors in XII and phrenic MNs 26
2.3 Role of iGluRs in the transmission of respiratory drive 29
2.3.1 In vitro and anesthetized in vivo studies 30
2.3.2 Experiments in freely behaving animals 33
2.3.3 Non-NMDA receptors: AMPA v. kainate 34
2.4 Modulation and plasticity of iGluR currents in the transmission of respiratory-
related drive to MNs 35
2.4.1 Modulation of iGluR-mediated respiratory drive 36
2.4.2 iGluR-mediated synaptic plasticity of respiratory MNs 40
2.5 Discussion 45
3 Cyclothiazide-induced Persistent Increase in Respiratory-Related Activity
in vitro 51
3.1 Introduction 51
3.2 Methods 54
3.2.1 Preparation 54
3.2.2 XII Nerve Recordings 55
3.2.3 Whole-cell Recordings 55
3.2.4 Mass Spectrometry 56
3.2.5 Drugs 57
3.2.6 Electrophysiological Data Analysis. 58
3.2.7 Statistics 59
3.2.8 Regressions 61
3.3 Results 62

v
3.3.1 CIF 62
3.3.2 Dose-Response 65
3.3.3 Long-Term Effects of CTZ on XII MN Drive 66
3.3.4 Investigation of Intracellular Signaling as the Mechanism Underlying CIF 66
3.3.5 Does CTZ Washout? 69
3.4 Discussion 72
3.4.1 Mechanism of Action 73
3.4.2 Physiological Significance 74
3.4.3 Implications for Therapeutic Design 77
4 PKG-Dependent Mechanisms Modulate Hypoglossal Motoneuronal
Excitability and Long-Term Facilitation 89
4.1 Introduction 89
4.2 Methods 91
4.2.1 Slice preparation and ethical approval 91
4.2.2 XII nerve recording 92
4.2.3 Voltage-clamp recording 92
4.2.4 Data analysis 93
4.2.5 Drugs and drug application 94
4.3 Results 96
4.3.1 8-Br-cGMP depresses inspiratory drive currents. 96
4.3.2 8-Br-cGMP depresses exogenous AMPA-induced currents 96
4.3.3 Potentiation of endogenous excitatory drive by inhibition of PKG activity 97
4.3.4 PKG-dependent mechanisms directly depress AMPA receptor currents 97
4.3.5 Stimulation of PKG-dependent mechanisms facilitates ivLTF 98
4.4 Discussion 100
5 Critically Spaced Episodic Stimulation Enhances But Is Not Necessary For
in vitro Long-term facilitation 110
5.1 Introduction 110
5.2 Methods 112
5.2.1 Slice preparation and systems electrophysiology 112
5.2.2 Protocol and parameter space 113
5.2.3 Data analysis 116
5.2.4 Drugs and solutions 120
5.2.5 Statistical definitions 120
5.3 Results 125
5.3.1 ivLTF is parameter sensitive 125
5.3.2 Episodic stimulation is not required for ivLTF 126
5.3.3 Interdrug interval influences ivLTF 126
5.3.4 Is there a set of optimal parameter values? 127
5.3.5 The parameters explaining ivLTF variability are stable over time 129
5.4 Discussion 130
6 Summary of the Dissertation 141

vii
LIST OF TABLES
Table 2.1 Ionotropic glutamate receptor subunits 47
Table 2.2 AMPA and kainate receptor subunit localization studies in XII and phrenic
motor nuclei 48
Table 2.3 NMDA receptor subunit localization studies in XII and phrenic motor
nuclei 49
Table 3.1 Summary of statistical comparisons for medullary slices treated for 1 hour with
CTZ (90 µM), DMSO (0.1%), or CX546 (90 µM) 79
Table 5.1 Experimental parameter values 134
Table 5.2 Valid models fit for full data set 135
Table 5.3 Valid models fit for multiple episode data set 135
Table 5.4 Variation in model fit for ∫XIIn at 60 minutes post protocol with and without
inclusion of control data 136
Table 5.5 Variation of model parameters with time 136

viii
LIST OF FIGURES
Figure 1.1 Transverse medullary (rhythmic) slice 13
Figure 2.1 Similarities in signaling pathways for AIH-LTF and ivLTF 50
Figure 3.1 Bath application of CTZ leads to long-lasting facilitation of endogenous
inspiratory XII nerve activity in the neonatal rat medullary slice 80
Figure 3.2 CTZ, but not CX546 or DMSO, leads to long-lasting facilitation of
endogenous inspiratory ∫XII nerve activity 81
Figure 3.3 Dose-response and exposure-response effects of CTZ on ∫XII nerve burst
amplitude and rate 1 hour post-treatment 82
Figure 3.4 Bath application of CTZ induces long-lasting increases in endogenous
inspiratory drive to XII MNs 83
Figure 3.5 CIF does not depend upon activation of AMPA or NMDA receptors during
treatment with CTZ 84
Figure 3.6 CIF is not PKA or PKC dependent 85
Figure 3.7 CTZ treatment of medullary slices leads to long-lasting increases XII MN
non-NMDA mEPSC amplitude and decay 86
Figure 3.8 Comparison of mEPSC distributions shows further differences among
treatment groups 87
Figure 3.9 Large quantities of CTZ remain trapped in medullary slice following
wash with ACSF 88
Figure 4.1 Focal application of 8-Br-cGMP depresses inspiratory drive currents 105
Figure 4.2 Postsynaptic exogenous AMPA-induced currents are depressed by
8-Br-cGMP 106
Figure 4.3 Potentiation of endogenous excitatory drive by inhibition of PKG activity107
Figure 4.4 PKG-dependent mechanisms directly depress AMPA receptor currents 108
Figure 4.5 Activation of PKG facilitates induction of ivLTF 109
Figure 5.1 Summary of experimental data 137
Figure 5.2 Thicker slices show less facilitation 138
Figure 5.3 A single episode of PE can induce ivLTF 139
Figure 5.4 Changing interval duration relative to episode duration influenced ivLTF 140

ix
LIST OF ABBREVIATIONS
5-HT Serotonin
ACSF Artificial cerebrospinal fluid
AHI Apnea-hypopnea index
AIH Acute intermittent hypoxia
ALS Amyotrophic lateral sclerosis
AMPA 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid
AMPAR AMPA receptor
ANOVA Analysis of variance
ATD Amino-terminal domain
BBB Blood-brain barrier
cAMP Cyclic adenosine monophosphate
cGMP Cyclic guanosine monophosphate
CPG Central pattern generator
CPP Crossed phrenic phenomenon
CTD Carboxyl-terminal domain
CTZ Cyclothiazide
DMSO Dimethyl sulfoxide
EPSC Excitatory postsynaptic current
GABA γ-Aminobutyric acid
GG Genioglossus
iGluR Ionotropic glutamate receptor
ivLTF in vitro long-term facilitation
IR-DIC Infrared differential interference contrast
IUPHAR International Union of Basic and Clinical Pharmacology
LBD Ligand-binding domain
LTD Long-term depression
LTF Long-term facilitation
LTP Long-term potentiation

x
mEPSC miniature excitatory postsynaptic current
MLR Multiple linear regression
MN Motoneuron
MSA Multiple systems atrophy
NMDA N-Methyl-D-aspartate
NMDAR NMDA receptor
OSA Obstructive sleep apnea
PAP Positive airway pressure
PE Phenylephrine
PKA Protein kinase A
PKC Protein kinase C
PKG Protein kinase G
preBötC preBötzinger Complex
ROS Reactive oxygen species
RMANOVA Repeated measures analysis of variance
RSM Response surface methodology
RT-PCR Real-time polymerase chain reaction
RTN/pFRG Retrotrapezoid nucleus/parafacial respiratory group
SDB Sleep disordered breathing
TARP Transmembrane AMPA receptor regulatory protein
TMD Transmembrane domain
WSCS Wisconsin Sleep Cohort Study
XII Hypoglossal
∫XIIn Integrated hypoglossal nerve

xi
ACKNOWLEDGEMENTS
Thanks to:
• Jack Feldman, my mentor, for giving me the opportunity to work in his lab and
transition to the world of neuroscience.
• Feldman Lab members past and present for their camaraderie and scientific support.
• My committee (Nick Brecha, Reggie Edgerton, Tom O’Dell, and Tom Otis) for their
willingness, patience, ideas, and support in seeing me through this process.
• Thanks to my old neighbor Alan Garfinkel for encouraging me to pursue a career
change to neuroscience so many years ago.
• Thanks to the larger UCLA neuroscience community for showing me the excitement
and possibilities associated with a life committed to science.
With the following exceptions the work that follows is mine in collaboration with
Dr. Jack Feldman.
Chapter 4 is a version of Saywell SA, Babiec WE, Neverova NV, Feldman JL
(2010) Protein kinase G-dependent mechanisms modulate hypoglossal motoneuronal
excitability and long-term facilitation. J Physiol 588:4431-4439. A version of the
material associated with Figure 4.1-Figure 4.4 is also a part of Neverova N (2007)
Intracellular signaling pathways underlying respiratory plasticity in vitro. Dissertation.
University of California, Los Angeles. Natalia Neverova and Shane Saywell performed
the experiments associated with these figures. I performed the ANOVA for their data.

xii
Also, I performed the experiments and analyzed the data for Figure 4.5. I was also
responsible for the major rewrite of the paper as presented here and in press, including
the postulated connection between respiratory/ivLTF and ischemic preconditioning.
Rather than my portion, the entirety of the work is presented to provide greater context.
I am grateful for the assistance of Kym Faull of UCLA’s Pasarow Mass
Spectrometry Laboratory. He performed the mass spectrometry analysis in Chapter 3. I
am also grateful to Alan Garfinkel for collaborating with me on the development of the
statistical methods applied in this chapter and to Tom Otis for working with me on the
development of the minis experiment as a marker for cyclothiazide.
This work has been supported by a Ruth L. Kirschstein National Research Service
Award predoctoral fellowship (NS067933), UCLA-NIH Training Program in Neural
Microcircuits (NS058280), and NIH Grant NS24742.

xiii
VITA
1972 Born, Providence, Rhode Island
1994 S.B., Mechanical Engineering
Massachusetts Institute of Technology
Cambridge, Massachusetts
1995 S.M., Mechanical Engineering
Massachusetts Institute of Technology
Cambridge, Massachusetts
1995-2000 Hughes Space and Communications, Inc.
2000-2005 The Boeing Company
2005-2007 Research Assistant
Department of Neurobiology
2008-2009 Predoctoral Fellow
UCLA-NIH Training Program in Neural Microcircuits
2010-2011 Predoctoral Fellow
Ruth L. Kirschstein National Research Service Award
PUBLICATIONS AND PRESENTATIONS
Archer SF, Babiec WE, Atkins WJ (1996) Leveraging commercial technology for
SATCOM 2000. Space Programs and Technologies Conference AIAA-1996-4237.
Babiec WE, Feldman JL (2008) A parametric investigation of the induction of ivLTF and
hints about participating neural circuitry. 2008 Neuroscience Meeting Planner, Program
No. 340.6. Society for Neuroscience, Washington, D.C. Online.

xiv
Babiec WE, Saywell SA, Feldman JL (2010) Induction of long-lasting changes in
motoneuronal excitability. Motoneuron Meeting 2010 (Paris) Poster F2. Online.
Babiec WE, Saywell SA, Feldman JL, Janczewski (2010) Therapeutic uses of AMPA
receptor modulators for treatment of motor dysfunction. World Intellectual Property
Office PCT International Patent Application WO/2010/054336.
Feldman JL, Saywell SA, Babiec WE (2009) Control of respiratory motor outflow during
wakefulness and Sleep. Proc Physiol Soc 15:SA1.
Roper DH, Babiec WE, Hannan DD (2003) WGS phased arrays support next generation
DOD SATCOM capability. Proc Mil Comm (MILCOM) 2003 IEEE Conf 82-87.
Saywell SA, Babiec WE, Neverova NV, Feldman JL (2010) Protein kinase G-dependent
mechanisms modulate hypoglossal motoneuronal excitability and long-term facilitation. J
Physiol 588:4431-9.

xv
ABSTRACT OF THE DISSERTATION
Long-term Enhancement of Respiratory-Related Activity by
Increasing the AMPA Receptor-Mediated Excitability of
Hypoglossal Motoneurons In Vitro
by
Doctor of Philosophy in Neurobiology
University of California, Los Angeles, 2011
Professor Jack L Feldman, Chair
Breathing is an essential behavior required to meet metabolic needs. Even short
pauses in breathing may be enough to permanently impair or kill a mammal. Breathing is
also a complex behavior, requiring the precise coordination of pools of motoneurons
(MNs) throughout the brainstem and spinal cord that control upper airway and pump
muscles. Breathing is highly adaptive, accommodating changes in mammal size, O2
demands, posture, and sleep-wake state as well as challenges caused by low atmospheric
O2, birth, aging, illness, and injury.
Due to a variety of factors including genetic mutation, developmental insult,
aging, illness, or injury, breathing may be degraded or disrupted. Sleep is a time when

xvi
breathing is especially vulnerable to disruption. Obstructive sleep apnea (OSA) is a
disease of upper airway collapse during sleep, which leads to repetitive cycles of
hypoxemic hypoxia and compensatory sympathetic facilitation. These repetitive cycles
lead in the short-term to disrupted sleep, neurocognitive impairment, and increased risk
for automobile and workplace accidents. In the long-term untreated OSA raises the risk
of hypertension, cardiovascular disease, type 2 diabetes, and stroke by 2x – 5x depending
upon severity. Current treatments for OSA are cumbersome, suffering as a result from
low compliance, or they are highly invasive, requiring surgery.
I hypothesized that enhancing respiratory drive at the premotor-MN synapse of
upper airway MNs, which is mediated by fast glutamatergic signaling, to overcome sleep-
related loss of upper airway muscle tone offers an effective treatment for OSA.
Therefore, I pursued three studies of methods for enhancing AMPA receptor-mediated
respiratory drive at hypoglossal (XII) MNs. (XII MNs innervate all muscles of the
tongue, including the genioglossus muscle that plays an especially important role in
maintaining airway patency).
The first study used the diuretic, anti-hypertension, and AMPA receptor anti-
desensitization drug cyclothiazide (CTZ) to enhance the amplitude of respiratory-related
discharge from XII MNs for > 12 hours post-treatment by enhancing AMPA-receptor-
mediated drive to XII MNs. The maintenance of CTZ-induced facilitation of XII MN
activity depends upon the slow wash off kinetics of CTZ.

xvii
The second and third studies explored methods for enhancing in vitro long-term
facilitation (ivLTF), a plasticity phenomenon in XII MNs discovered by predecessors in
my mentor’s lab. ivLTF is of considerable interest, because it likely relates to acute-
intermittent hypoxia (AIH) induced long-term facilitation of ventilation in vivo, which
may be a naturally occurring mechanism for overcoming and avoiding apneas that fails in
sufferers of OSA. First, I show that stimulation of protein kinase G activity during
induction of ivLTF enhances respiratory-related XII nerve discharge. In Chapter 5, I
show that the magnitude of ivLTF is protocol dependent. Specifically, the duration of the
episodes of phenylephrine application and the length of the pauses between episodes of
stimulation as well as their ratio predict the level of ivLTF. All three studies were
performed in the transverse medullary (rhythmic) slice of neonatal rats, which maintains
endogenous respiratory rhythm while greatly simplifying the respiratory circuit.
In conclusion, I provide a summary of the dissertation. Limitations of my studies
are discussed along with ideas on future directions that the research described here might
take.

1
1 INTRODUCTION
Breathing is an essential behavior in mammals. Necessary to support metabolism,
breathing must persist from birth to death with only the shortest pauses (at most a few
minutes) before severe and irreversible damage to the brain and other organs results.
~500 million respiratory cycles occur in the average human lifetime (Feldman and Del
Negro, 2006).
Breathing is also complex, requiring the precise coordination of muscles in the
head, neck, chest, and abdomen to move air efficiently. During resting breathing
(eupnea), immediately prior to inspiration, upper airway muscles, e.g., the genioglossus
muscle of the tongue that is innervated by hypoglossal (XII) motoneurons (MNs),
activate to widen and stiffen the upper airway, reducing resistance to air flow. Then pump
muscles in the chest and diaphragm, the latter of which is innervated by phrenic MNs,
activate to increase the volume of the thoracic cavity, creating subatmospheric pressure
that draws air into the lungs. For breathing when active, depending upon O2 requirements
and posture, abdominal muscles may activate to help force O2–poor/CO2-rich air out of
the lungs to reduce the time required before the next inspiration.
Despite the distributed nature of muscle activation during breathing, one might
imagine a fairly simple control system of a square-wave or sinusoidal rhythm generator
transmitting drive through paths of varying delay to MNs located in the brainstem
(controlling the upper airway), the cervical spinal cord (controlling the diaphragm), the
thoracic spinal cord (controlling the intercostals), and the lumbar spinal cord (controlling

2
the abdominals). The problem that the neural circuits controlling breathing solve,
however, is much more complicated than the maintenance of a constant volume and rate
of breathing. First, the demand for O2 can vary by more than an order of magnitude as
result of changes in level of activity, e.g., exercise (Feldman and McCrimmon, 2003).
Second, the control system must adapt patterns of muscle activation to changes in
posture, organism size during development, and O2 levels in the surrounding air, as well
as impediments brought about by aging, illness, and injury. Therefore, the neural circuits
controlling breathing must be able to adapt over a variety of timeframes ranging from a
single breath to many decades, i.e., a range of ~10 orders of magnitude, to meet
metabolic needs over a lifetime.
For this purpose, humans and other mammals have evolved a distributed and
complex network of afferents, reflexes, and pattern generators, that are proposed to be
driven by a dual oscillator rhythm generator, to mediate adequate ventilation (Feldman
and McCrimmon, 2003; Feldman and Del Negro, 2006). These networks may be
modulated into higher or lower levels of activation by an array of neuro-transmitters, -
modulators, and -peptides that lead to changes on the timescale of synaptic release, or
more long lasting changes due to plasticity. Plasticity occurs throughout respiratory
control circuits, but, most recently, synaptic plasticity at respiratory MNs, such as XII
and phrenic MNs, has been discovered and is thought to play an important role in
adaptation of breathing to, for example, repetitive hypoxic challenges as well as spinal
cord injury (Bocchiaro and Feldman, 2004; Neverova et al., 2007; Wilkerson et al., 2007;
Dale-Nagle et al., 2010).

3
This complex system for controlling breathing, however, is susceptible to
degradation or outright failure. The source may be genetic, for example, Rett’s Syndrome
or congenital central hypoventilation syndrome (CCHS) (Glaze, 2005; Grigg-Damberger,
2009). Developmental insults, e.g., prenatal nicotine or alcohol, or unknown
developmental mechanisms, e.g., sudden infant death syndrome, may also play a role
(Feldman and Del Negro, 2006; Fregosi and Pilarski, 2008; Kinney, 2009). High cervical
spinal cord injury, neurodegenerative diseases, e.g., amyotrophic lateral sclerosis (ALS)
or multiple systems atrophy (MSA), and cardiovascular disease may also degrade or
eliminate altogether essential breathing behavior (Feldman and Del Negro, 2006; Selim et
al., 2010).
1.1 Obstructive sleep apnea (OSA)
An especially challenging time for the maintenance of proper ventilation is during
sleep. Sleep disordered breathing (SDB) is highly prevalent among adults. The gold-
standard of SDB studies, the Wisconsin Sleep Cohort Study (WSCS), estimates the
prevalence for SDB among adults, defined as more than 5 apneas or hypopneas per hour
of sleep (AHI ≥ 5), to be 24% in men and 9% in women (Young et al., 1993). Since
habitual snoring (a precursor of OSA) is a significant predictor of SDB likelihood, most
SDB sufferers in this study were thought to have apneas and hypopneas of obstructive,
i.e., collapse of the upper airway with continued movement of respiratory pump muscles,
rather than central, i.e., failure of pump muscle movement, origin (Young, 2009). This
conclusion seems reasonable, since studies in the elderly and those under treatment for
opiate addictions have a 2-3x greater likelihood for OSA versus central sleep apnea,

4
despite being at greater risk than the general population for apneas of central origin
(Ancoli-Israel et al., 1987; Johansson et al., 2009; Sharkey et al., 2010).
OSA, itself, does not cause death, but the long-term health impacts seen in
sufferers of this disease are severe and may lead to premature death. If untreated, those
suffering from moderate OSA (AHI of 5-15) are twice as likely to develop hypertension
or depression within 4 years of first diagnosis of OSA, while sufferers of severe OSA
(AHI ≥ 15) are nearly 3x as likely to develop hypertension and more than 2.5x as likely
to develop depression for the same period. In addition, severe OSA sufferers are also 4.5x
as likely to suffer stroke, 5x as likely to suffer cardiovascular related death, and nearly 4x
as likely to suffer death from all causes within 14 years from first diagnosis of OSA
(Young, 2009). OSA is also an independent risk factor for the development of Type 2
diabetes with the risk increasing according to the severity of OSA (Selim et al., 2010).
The reason for increased risk of cardiovascular disease and stroke is likely related
to the response of the body to an apneic event. Apnea leads to hypoxemic hypoxia, low
arterial O2, due to the absence of airflow. There is a massive sympathetic response to the
hypoxia, which causes spikes in blood pressure as high as 240 mm Hg at apnea
termination when there is arousal from sleep (Selim et al., 2010). In sufferers of severe
OSA, this can happen hundreds of times a night or in the severest cases nearly 90 times
an hour often without patients being aware (Young et al., 1993). Because of these
continuous arousals, many but not all sufferers of OSA report increased daytime
sleepiness, which is sometimes used as a second criterion along with AHI for the clinical
diagnosis of OSA (Young et al., 1993; Young et al., 2002).

5
The annual health costs of OSA in the U.S. are thought to total in the billions of
dollars, resulting from an approximately two-fold increase in medical costs associated
with patients that are subsequently diagnosed with OSA when compared to non-OSA
patients (Kapur, 2010). Increased societal costs beyond the increased healthcare costs of
untreated OSA sufferers include the costs resulting from motor vehicle accidents related
to OSA, which one study estimates were $15.9 billion in 2000 (Sassani et al., 2004). The
alarmingly rapid increase in obesity in the U.S. and the fact that obesity is a risk factor
for OSA, mean the prevalence and costs associated with untreated OSA and treatment of
OSA will likely continue to rise in coming years Young, 2009).
1.2 Why do upper airway obstructions form during sleep?
Sleep, especially during the REM phase, causes dramatic decreases in muscle
tone, including the tone of upper airway muscles. The upper airway of humans is
especially prone to collapse. Human evolution of speech was supported by anatomical
changes to the upper airway, including shortening of the maxillary, ethmoid, palatal and
mandibular bones, acute oral cavity-skull base angulation, pharyngeal collapse with
anterior migration of the foramen magnum, posterior migration of the tongue into the
pharynx, descent of the larynx and shortening of the soft palate with loss of the
epiglottic–soft palate lock-up, and the development of a “floating” hyoid bone (Davidson,
2003; Horner 2008). The hyoid bone, which supports the root of the tongue, therefore, is
not articulated to another bone and is unique among bones in the human body for this
reason. As a result of these changes, the human upper airway is much narrower and more
compliant, making it prone to collapse (Davidson, 2003; Horner, 2008). Even in healthy

6
adults, loss of tone during sleep narrows the upper airway, which increases airway
resistance that leads to hypoventilation and an increase of 3-5 mm Hg in the pressure of
arterial CO2 (Horner, 2008). For sufferers of SDB, suppression of activity during sleep in
the genioglossus muscle (Remmers et al., 1978) as well as other muscles of the tongue
(Horner, 2008), which are all innervated by the XII MNs, as well as possibly muscles of
the soft palate (Horner 2008), which are innervated by trigeminal MNs, leads to apneic
events.
Studies over the last decade in freely behaving rats indicate that the source of
drive supporting upper airway tone during wakefulness that abates during sleep is
noradrenaline with a much smaller component arising from 5-HT (Horner, 2008).
Noradrenergic efferents arising from the sub-coeruleus and possibly A5 or A7 are likely
the source of the noradrenaline (Horner, 2008). Whether these drives or the
responsiveness of MNs to them is different between non-sufferers and sufferers of OSA
is not known.
1.3 Strategies for treating OSA
Three strategies have evolved over time to treat OSA: (1) treat the symptoms;
(2) restore the wakefulness drive to upper airway MNs during sleep, and; (3) overcome
the reduction in upper airway muscle tone with enhanced respiratory drive.
1.3.1 Treating the symptoms of OSA
Addressing the symptoms of OSA is the predominant method for treating OSA.
The most common form of OSA treatment is the use of positive airway pressure (PAP) to

7
“splint” open the upper airway during sleep. A pump forces air into the nose through a
facemask continuously or in phase with inspiration, while the individual sleeps. PAP is
effective in many but not all cases, but its main drawback is compliance. Patients often
cite mask discomfort, pressure intolerance, and airway irritation as reasons for non-
compliance but ethnic and socio-economic issues play a role as well (Campbell et al.,
2010; Randerath et al., 2011).
A second strategy for treating symptoms is surgery, where a variety of procedures
including uvulopalatopharyngoplasty, tongue radiofrequency midline glossectomy,
genioglossus advancement or genioplasty, tongue stabilization, hyoid suspension, and
maxomandibular advancement are used to remove or relocate tissue likely to cause
constriction of the upper airway during sleep (Kezirian et al., 2010; Randerath et al.,
2011). Surgical approaches have the obvious drawback of being highly invasive, and,
although many procedures now occur on an outpatient basis, ~20% of procedures in the
U.S. in 2006 required inpatient surgery (Kezirian et al., 2010). Only maxomandibular
advancement, one of the most invasive of these procedures, yields improvements in
symptoms at a level similar to PAP, while uvulopalatopharyngoplasty works in specific
cases of obstruction limited to the oropharyngeal area. Other surgical procedures either
have been disproven or lack evidence supporting their efficacy (Randerath et al., 2011).
The final approach to the treating OSA symptoms is through the use of oral
appliances. The oral appliances are of two types: mandibular advancement devices and
tongue restraining devices. Only mandibular advancement devices improve OSA. While
being worn, they reposition the lower jaw forwards and downwards opening the airway.

8
Daytime sleepiness in patients improves the same amount with these devices when
compared to PAP, but snoring does not improve as much. Although compliance is better
than with PAP, approximately a quarter of patients discontinue use within the first year,
and one third of patients discontinue use by the end of 4 years (Randerath et al., 2011).
1.3.2 Preventing loss of tone during sleep
The approach to preventing loss of tone has been pharmaceutical based and, to
this point, largely ineffective. That being said, the development of such treatments is
immature, since the basic science underlying their development is still evolving.
Strategies have focused on the use of 5-HT and, to a lesser extent, noradrenaline reuptake
inhibitors with no or limited improvements in AHI or daytime drowsiness (Randerath et
al., 2011). This is likely the case, because the efferents providing wakefulness drive to
MNs are depressed during sleep, leaving little residual 5-HT and noradrenaline for uptake
inhibitors to preserve (Horner, 2008). Agonists for these receptors may be more helpful,
but care must be taken with noradrenergic stimulants, because of the potential for
cardiovascular effects. Furthermore, the focus on 5-HT rather than noradrenaline, based
on studies of respiratory drive in anesthetized rather than freely behaving animals, has led
to emphasis on the less important of the sources of wakefulness drive until relatively
recently (Horner, 2008).
In addition, adenosine receptor antagonists and cholinergic receptor agonists have
been studied. Adenosine receptor agonists increased sleep disruption, worsening daytime
sleepiness. Cholinergic agonists had some success but have had limited study and to this

9
point have required intravenous administration, making it unclear if an oral treatment
would be efficacious (Randerath et al., 2011).
1.3.3 Overcoming sleep-related loss of muscle tone
Methods to enhance respiratory drive and studies of their effectiveness in
overcoming sleep-related loss of muscle tone are relatively unstudied. Whyte et al. (1988)
studied the use of acetazolamide to treat OSA in 10 patients. Acetazolamide inhibits
carbonic anhydrase, producing a metabolic acidosis that increases respiratory drive.
Treatment for one week improved AHI, but there was no improvement of daytime
drowsiness, while longer treatment could not be tolerated.
Setting aside concern over side effects, acetazolamide likely might be a more
optimal agent for treating central apneas, because it enhances drive to the rhythm
generator by activation of chemosensitive afferents signaling excess arterial CO2. In more
severe cases of OSA or in the cases where apneas are of mixed origin, this enhanced
central drive could stimulate greater contractions in the diaphragm and intercostals,
resulting in increased pressure differentials that could lead to more instances of airway
collapse or longer duration obstructions when airway collapse occurs (Sharp et al, 1985).
However, direct enhancement of existing respiratory drive at the synapses of upper
airway MNs is an untested but promising method to treat OSA, because its specific
location of action might avoid side effects induced by more indirect methods of
enhancing respiratory drive.

10
1.4 Dissertation purpose and organization
This dissertation focuses on developing methods for enhancing respiratory drive
to MNs of the upper airway with the long-term goal of overcoming the sleep-related loss
of upper MN excitability. Chapter 2 provides a review of the evidence that fast
glutamatergic signaling, via AMPA, NMDA, and possibly kainate receptors, mediates
transmission of respiratory drive from the preBötC to upper airway and pump MNs alike.
This chapter also discusses a variety of mechanism for modifying the strength of fast
glutamatergic synapses via modulation with endogenous or exogenous agents as well as
by inducing long-lasting plastic changes at fast glutamatergic synapses onto respiratory
MNs.
Chapters 3-5 document experimental studies of methods that I and my colleagues
hypothesized would lead to long-lasting (> 1 hour) enhancements to AMPA-mediated
respiratory drive to XII MNs. Chapter 3 describes use of the diuretic, anti-hypertension,
and AMPA receptor anti-desensitization drug cyclothiazide to enhance the amplitude of
respiratory-related discharge from XII MNs for > 12 hours in vitro, by enhancing AMPA
receptor-mediated drive to XII MNs. This is an example of modulation of synaptic
efficacy rather than plasticity, since the phenomenon appears to rely on continued
presence of cyclothiazide to maintain its effects.
Chapters 4 and 5 are studies of in vitro long-term facilitation (ivLTF), a plasticity
phenomenon in XII MNs discovered by predecessors in my mentor’s lab. Episodic
application (3, 3-minute episodes spaced at 5-minutes) of α-Me-5HT, a 5-HT2 receptor
agonist, or phenylephrine, an α1-adrenergic agonist increases AMPA receptor-mediated

11
excitability postsynaptically in XII MNs in an activity-independent manner (Bocchiaro
and Feldman, 2004; Neverova et al., 2007). This increase in excitability results in an
increase in the respiratory-related discharge of XII MNs and lasts for >1 hour following
induction. ivLTF is of considerable interest as a phenomenon, because it likely relates to
the in vivo phenomenon of acute-intermittent hypoxia (AIH) induced long-term
facilitation of ventilation, which may be a naturally occurring mechanism for overcoming
and avoiding apneas that fails in sufferers of OSA (Mahamed and Mitchell, 2007).
In Chapter 4, I show that stimulation of protein kinase G activity during induction
of ivLTF enhances respiratory-related nerve discharge in vitro. In Chapter 5, I show that
the magnitude of ivLTF is protocol dependent. Specifically, the duration of the episodes
of phenylephrine application and the length of the pauses between episodes of stimulation
as well as their ratio predict the level of ivLTF. In conclusion, Chapter 6 provides a
summary of the dissertation. Limitations of the current studies are discussed along with
ideas on future directions that the research described here might take.
1.5 Rhythmic slice preparation
All studies described in this dissertation were performed in the transverse
medullary (rhythmic) slice taken from neonatal rats. Developed in my mentor’s
laboratory (Smith et al., 1991), the slice is an ~700 µm thick medullary slice with its
rostral boundary at the compact formation of nucleus ambiguus and its caudal boundary
at area postrema (Figure 1.1). The rhythmic slice is unique among in vitro slice
preparations for studying mammalian motor behavior, because it contains all the

12
necessary circuitry to generate and transmit motor, i.e., respiratory, drive endogenously,
i.e., without the addition of 5-HT, NMDA, or dopamine receptor agonists, which are
required for locomotor preparations.
The rhythmic slice contains the preBötC, the source of inspiratory rhythm and one
of two centers that interact to form the presumed dual oscillator underlying breathing
behavior (Feldman and Del Negro, 2006), as well as the XII nucleus and intervening
premotor network that transmits drive from preBötC to the XII nucleus. XII MNs
innervate muscles of the tongue, including the genioglossus muscle, whose loss of tone is
central to the development of airway obstructions in OSA. The rhythmic slice provides
direct, visualizable access to important constituent respiratory circuit elements, e.g., XII
MNs, so that direct, localized intracellular measurements and manipulations may be
made. In addition, most of the components of respiratory control that modulate basic
respiratory rhythmogenesis and transmission of drive have been removed, simplifying the
interpretation of experiments. The simplifications offered by the rhythmic slice enhance
our ability to perform basic studies of respiratory behavior like those described in this
dissertation.

13
Figure 1.1 Transverse medullary (rhythmic) slice.

14
2 THE ROLE OF IONOTROPIC GLUTAMATE RECEPTORS IN THE TRANSMISSION OF
RESPIRATORY DRIVE
The role of signaling via ionotropic glutamate receptors (iGluRs), also referred to
as fast glutamatergic signaling, in mediating mechanisms underlying synaptic plasticity
and learning and memory in hippocampus, cortex, and cerebellum has captured the
imagination of myriad researchers for more than a quarter century. The roles of AMPA
receptors (AMPARs) as the workhorse of excitatory synaptic transmission and NMDA
receptors (NMDARs) as the coincidence detectors necessary for triggering plastic
changes in AMPAR number, subunit composition, and conductance, e.g., via
phosphorylation, have been worked out in exquisite detail as have many of the second
messengers underlying this process (Malenka and Bear, 2004; Kennedy et al., 2005;
Traynelis et al., 2010). Furthermore dozens of proteins interacting with these receptors in
the post-synaptic density have been identified and their roles in mediating iGluR activity
enumerated (Collingridge and Isaac, 2003; Collingridge et al., 2004; Kim and Sheng,
2004; Kennedy et al., 2005; Traynelis et al., 2010). Further, researchers have even come
to appreciate that some rules for synaptic activity and plasticity appear to be general,
while many others appear to be brain-area specific (Malenka and Bear, 2004).
During this same period the development of our knowledge about the neural
control of breathing has taken a very successful but much different path. The control of
breathing is highly distributed throughout the brainstem, spinal cord, and peripheral
nervous system, involving the complex interaction of rhythm and pattern generators,
reflexes, sensory feedback, and volitional commands. For this reason, tremendous

15
emphasis was placed on understanding how these elements of the system interact at a
highly intact level, i.e., whole animal. Also, during this period, there was a revolution in
our understanding of the genesis of respiratory rhythm spurred on by the development of
the reduced in vitro brainstem-spinal cord (Suzue, 1984) and rhythmic slice (Smith et al.,
1991) preparations, which fostered the landmark discoveries of the preBötC, the kernel of
inspiratory rhythm, and the retrotrapezoid nucleus/parafacial respiratory group
(RTN/pFRG), an area implicated as the source of active expiration (Feldman and Del
Negro, 2006).
As a result of concerted efforts in the highly integrated studies of breathing, the
relatively recent development of in vitro models of respiratory control, and the only
recent discovery of the rhythmogenic centers for breathing, far less progress has been
made in understanding the synaptic physiology of the connections within and between
respiratory centers that are critical to respiratory control. The field of respiratory control,
however, may be on the precipice of a new and vibrant period for furthering our
understanding of the synaptic physiology underlying the control of breathing. Recently,
long-lasting synaptic plasticity was discovered at MN synapses (Bocchiaro and Feldman,
2004). Also, we have recognized that our understanding of synaptic physiology in
breathing could aid in the treatment of disease and injury (Ren et al., 2006; Ogier et al.,
2007; Ren et al., 2009). Finally, there are many exciting improvements in the
electrophysiological, optical, and genetic techniques that are available for addressing
questions of synaptic physiology that were heretofore unassailable (Sakmann, 2006; Luo
et al., 2008).

16
Fast glutamatergic signaling plays an especially important role in both the
generation of respiratory rhythm and the transmission of that rhythm to MNs mediating
breathing movement (Liu et al., 1990; Greer et al., 1991; Funk et al., 1993). The goal of
this chapter is to review current knowledge about the role of iGluRs in the latter of these
functions. First, a brief overview of iGluR structure is provided. A discussion of the
types and relative amounts of iGluR subunits observed in respiratory MNs follows. Then
the evidence for the role of fast glutamatergic signaling as the primary path for
transmitting respiratory rhythm is discussed. Finally, mechanisms for modulating the
strength of excitatory synapses at respiratory MNs, either through the continued action of
endogenous substances and drugs or through the induction of lasting plastic changes
induced by specific events, are addressed. Throughout, areas where future work might be
helpful in clarifying issues or answering, as yet, unaddressed questions, are discussed.
2.1 iGluR structural overview
iGluRs form one of two main groups of glutamate receptors in the nervous
system, the other being metabotropic glutamate receptors. iGluRs are ligand-gated ion
channels, which, by homology and agonist specificity, can be divided into AMPA (2-
amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid), kainate, NMDA (N-Methyl-
D-aspartate), and the delta receptors. (Relatively little is known about the delta receptors
and they will not be discussed further.) Therefore, these receptors have many common
structural and functional elements. They also have unique variations that set the
subfamilies apart. This section briefly reviews the attributes of the iGluRs. Unless

17
specific references are given, the material in this section can be verified by reading the
extremely comprehensive review published by Traynelis et al. (2010).
2.1.1 Common attributes of iGluRs
Crystallographic studies of AMPARs show that iGluRs are comprised of 4
subunits that come together in a dimer-of-dimer structure (Sobolevsky et al., 2009). A
given receptor is formed only from subunits of one subfamily of iGluRs. The types of
subunits and the genes encoding them are summarized in (Table 2.1). Each subunit is
comprised of four discrete semiautonomous domains: the amino-terminal domain (ATD),
the ligand-binding domain (LBD), the transmembrane domain (TMD), and the carboxyl-
terminal domain (CTD).
The ATD influences receptor oligomerization and trafficking, but is not required,
however, for basic receptor functioning. Mutagenesis studies that remove the entire ATD
produce receptors that are functionally similar to wild-type. Changes to the ATD,
however, influence open probability, deactivation, desensitization, responses to certain
negative allosteric modulators, and regulation of subunit specific assembly. Also, an
amino acid sequence coding for a standard signal peptide at the very N-terminal end of
the ATD, which is common to all glutamate receptors, is required for membrane
insertion, after which it is then removed by proteolysis (Traynelis). Interestingly, the
ATD has putative binding sites for proteins such as N-cadherins, neuronal petraxins, and
ephrins and divalent cations such as Zn2+
and is subjected to glycosylation, suggesting

18
other roles for this region in trafficking, functional modulation, and proper synapse
formation.
The TMD is comprised of three transmembrane-spanning helices (M1, M3, M4)
with a re-entrant loop (M2) and with a short pre-M1 helix that is parallel to the plasma
membrane. M1-M3 form the ion channel core. M2 lines the inner cavity of the pore and
contains the QR mRNA editing site in GluA2, which regulates Ca2+
permeability in
GluA2-containing AMPARs. M3 lines the outer cavity of the pore and, likely, forms the
ion gate. The M1 helix is outside of the M2 and M3 helices. The M4 helix interacts with
M1-M3 helices of an adjacent subunit helping to maintain dimer interfaces in the
receptor.
The LBD is comprised of two extracellular stretches of amino acids: S1 and S2.
S1 is on the ATD side of M1, while S2 is between M3 and M4. S1 and S2 come together
to form a “clamshell” configuration that closes in the presence of agonists, thus imparting
conformational changes on the receptor that lead to pore opening. The interaction of S1
domains from different subunits provides the binding sites for agonists as well as
allosteric modulators of iGluRs. The S2 portion conveys the conformational changes
required for channel opening or desensitization, a state where agonist is bound but the ion
pore is closed.
The CTD is the most diverse of the domains of the iGluR subunit, varying greatly
in length and sequence of amino acids. Deletion of the CTD does not alter iGluR
function. Instead the CTD is thought to be involved with targeting, stabilization, post-

19
translational modifications, e.g., phosphorylation, and targeting for degradation. The
CTD interacts with dozens of proteins involved with receptor trafficking, synapse
formation, and second messaging.
2.1.2 iGluR stoichiometry
All iGluRs are tetramers of subunits from a single receptor subfamily, i.e.
AMPARs contain only GluA subunits, kainate receptors contain only GluK subunits, and
NMDARs contain only GluN receptors. At least for AMPARs, segregation of subunit
subfamilies is governed by the ATD. The details of which subunits can join within a
receptor subfamily differ between AMPA, NMDA, and kainate receptors, having
important functional consequences. The rules of association are least restrictive for
AMPARs, which appear to be able to associate in any combination, although mRNA
editing at sites in GluA2 and GluA4 subunits result in a tendency for these subunits to
favor heterodimerization.
In contrast, kainate receptors have a conditional set of stoichiometric
requirements. Like for AMPARs, GluK1 – GluK3 subunits can form functional
homomeric or heteromeric receptors of any combination. GluK4 and GluK5 subunits,
however, require the presence of GluK1 – GluK3 subunits to form functional receptors.
The need by NMDARs for both glutamate and glycine binding for activation is a
direct result of their stoichiometry. NMDARs require two GluN1 subunits in combination
with GluN2/GluN3 subunits. GluN1 and GluN3 subunits provide the glycine-binding
site, while GluN2 subunits provide the glutamate binding site. Interestingly, heterologous

20
expression of GluN1 and GluN3 subunits alone leads to the formation of glycine-gated
excitatory channels, although there are not data supporting the existence of such a
configuration in vivo. Electrophysiological evidence does exist, however, for naturally
occurring receptors that contain combinations of lower-conductance
GluN1/GluN2/GluN3 as well as the more common configuration of higher-
conductanceGluN1/GluN2.
2.1.3 RNA editing and alternative splicing
The role of mRNA editing and alternative splicing is probably best known in
AMPARs. Each GluA subunit comes as either a flip or flop splice variant. The alternative
splicing occurs in a 38 amino acid segment of the LBD (Sommer et al., 1990). As a
result, the splice variants have very different responses to allosteric modulators. For
example cyclothiazide, a drug that slows AMPAR desensitization and deactivation,
works preferentially on flip-containing receptors, but the ampakines, which also slow
desensitization and deactivation, prefer flop-containing receptors over flip-containing
receptors to varying degrees (Partin et al., 1994; Arai and Kessler, 2007). Similarly, ATD
splice variants of GluK1 also have different sensitivities to the influence of allosteric
modulators.
Substitution of arginine for glutamine at the QR mRNA editing site on the M2
segment of the GluA2 receptor significantly decreases both the rectification and Ca2+
permeability of GluA2-containing AMPARs. Editing at a similar site on GluK1 and
GluK2 subunits similarly affects the permeability properties of kainate receptors that

21
contain these subunits. Together with the RG editing site in the LBD of GluA2 and
GluA4 subunits, the QR site affects subunit pairing, conferring a preference for
heterodimerization over homodimerization. GluA2 also has two alternative splice
variants of the CTD, which influence receptor trafficking, synaptic plasticity, and several
receptor-protein interactions.
GluN1 and GluN2A both have alternative splice versions of their CTDs. There
are four alternates for GluN1 and two for GluN2A. Only the longest of the four GluN1
CTDs can be phosphorylated, while both of the splice variants of GluN2A allow for
phosphorylation. Also, alternative splicing of the GluN1 ATD allows for proton
inhibition of NMDARs, while alternative splicing of GluN1 and GluN2 influences
trafficking through the inclusion or exclusion of endoplasmic reticulum retention signals.
2.1.4 iGluR accessory proteins
The past decade has led to a growing awareness of and appreciation for a set of
proteins that are independent of iGluRs but dramatically affect their function, explaining,
for example, the differences in biophysical properties between heterologously expressed
recombinant and wild-type iGluRs. The best known of these are transmembrane AMPA
receptor regulatory proteins (TARPs). TARPs are found in the majority of AMPA
receptor complexes in the brain suggesting that they serve as auxiliary subunits to
naturally occurring AMPARs. They interact with extracellular, transmembrane and
intracellular regions of AMPARs and have the stoichiometry of 2-4 TARPs per AMPAR.
Functionally, TARPs increase AMPAR single channel conductance, open probability,

22
and activation rate, while slowing deactivation time course and reducing desensitization.
TARPs also play roles early in AMPAR synthesis and trafficking.
CINH proteins are additional AMPAR auxiliary proteins that are sometimes
referred to as cornichons, because they are homologous to the cornichon family of
proteins in flies and yeast. Relatively little is known about their role in AMPAR function,
but recent evidence points to a role in trafficking and possibly regulation of receptor
kinetics as well (Brockie and Maricq, 2010).
Neto1 is an NMDAR accessory protein that interacts with GluN2 by both the
extracellular domain and via interaction with PSD95 intracellularly. Without Neto 1,
GluN2A expression is completely abolished, but there is little effect on the expression of
GluN2B, implying that Neto1 may have a role to play in regulating learning and memory.
Its relative, Neto 2, affects the kinetics of GluK2-containing kainate receptors, increasing
peak amplitude and open probability, while slowing the decay time course of GluK2-
containing receptor-mediated mEPSCs but has no effect on trafficking.
Very little is known about the expression or function of iGluR accessory proteins
in the brainstem, and there are no published data looking at how these proteins might
influence respiratory control. But, their close relationship with and strong influence on
AMPARs of the hippocampus, cortex, and cerebellum as well as their heterogeneous
expression across brain areas (Montgomery et al., 2009; Jackson and Nicoll, 2011) makes
these accessory proteins of great interest for future study in respiratory control.

23
2.2 Evidence for iGluRs in XII and phrenic MNs
Breathing involves muscles of the upper airway, rib cage, diaphragm, and, in the
case of active expiration, the abdomen through the coordinated activation of MNs from
the brainstem all the way down to the lumbar regions of the spinal cord. Most studies of
MNs in respiration have focused on those innervating the diaphragm (phrenic MNs) and
muscles of the tongue (XII MNs). Therefore, the study of MNs in these regions
predominate in this and subsequent sections.
More than ten studies of the subunit composition of iGluRs in XII and phrenic
MNs have been published, although most focus on a certain type of iGluR, rather than
comprehensively studying the full range. Based on these studies there seems to be some
linkage in the pattern of receptor subtype expression among respiratory MNs and other
respiratory areas, e.g., the preBötC, that is specific to breathing and is not shared in
common with other proximally located non-respiratory nuclei, possibly having
consequences for breathing instabilities during early postnatal periods (see Paarmann et
al., 2000; Oshima et al., 2002; Liu and Wong-Riley, 2005; Liu and Wong-Riley 2010 for
more details).
Studies of iGluR expression in phrenic and XII MNs most commonly use adult
rats, although some data for mice and humans do exist. Antibody-based methods
predominate, but data using other techniques including in situ hybridization, RT-PCR,
and radiolabeled antagonists are also found. Obvious disagreements among these studies
as to the types of subunits and their relative levels of expression mean, however, that

24
general conclusions about the iGluR subunit expression patterns must be treated with
caution.
2.2.1 AMPA and kainate receptors in XII and phrenic MNs
All types of AMPA receptor subunits, i.e., GluA1-4, appear in XII and phrenic
MNs of rats (Robinson and Ellenberger, 1997; Garcia del Caño et al., 1999) and XII MNs
of mice (Paarmann et al., 2000), as well as the XII and phrenic MNs of humans
(Williams et al., 1996) and tend to be located predominantly on the soma and proximal
dendrites with weak or no staining in the neuropil (Williams et al., 1996; Robinson and
Ellenberger, 1997). There is some disagreement among studies, however, over the
amount of GluA1 and GluA2 containing receptors that are present.
Using immunocytochemistry, Williams, et al. (1996) and Robinson and
Ellenberger (1997) report weak staining for GluA1 subunits in both XII and phrenic MNs
of humans and rats, respectively. Paarmann et al. (2000) report strong GluA1 expression
levels in XII MNs of neonatal mice using RT-PCR as the detection method. The
difference could be one associated with detection method or differences in species or
development. The study of Garcia del Caño, et al. (1999), however, offers another
explanation. This study details the expression of AMPAR subunits for each independent
subnucleus of the XII motor nucleus. The ventral, ventromedial, and rostral portion of the
dorsal subnuclei stain weakly for GluA1, while staining is moderate to intense in the
ventrolateral and caudal portion of the dorsal subnuclei. Therefore, the possibility exists
that Paarmann et al. (2000) may have selected the small sample of cells used for RT-PCR

25
in the ventrolateral subnucleus or caudal portion of the dorsal subnucleus. Interestingly,
the ventrolateral subnucleus contains most of the XII MNs involved in respiratory
activity (Garcia del Caño, et al., 1999), which would indicate a moderate to robust
presence of GluA1 subunits in XII MNs involved with breathing.
In the case of GluA2, some studies use antibodies that could not distinguish
between GluA2 and GluA3 (Williams et al., 1996; Robinson and Ellenberger, 1997).
Garcia del Caño et al. (1999) show that staining for their GluA2/3 antibody is strong
across all subnuclei of the XII, while staining with a separate GluA2 antibody is weak.
Since GluA2 confers Ca2+
impermeability on AMPA receptors, they conclude that high
Ca2+
entry into XII MNs is likely, because there should be high proportion of GluA2-less
AMPA receptors. In their opinion, this could explain the greater susceptibility of these
neurons to neurodegenerative diseases such as ALS. Paarmann et al. (2000), however,
indicate strong reaction products for GluR2 when RT-PCR is performed on a cell-by-cell
basis in XII MNs. Again, the difference could be one of differences in species or
development. To this point, Liu and Wong-Riley (2005) show a 50% decline in GluA2
immunoreactivity over the first three postnatal weeks in rats. Also, a pharmacological
study of rats in the first two postnatal weeks of life shows that the Ca2+
permeability of
AMPARs in XII MNs is somewhere in the middle of the range seen in other neurons of
the CNS (Essin et al., 2002). This study shows the ratio of Ca2+
to Na+
permeability in XII
MNs is 4x less than that of striatal and hippocampal interneurons, which are thought to be
relatively GluA2-less but 2.5x greater than that of hippocampal pyramidal cells, which
are thought to have a low quantity of GluA2-less AMPARs. Further, Essin et al. (2002)

26
support a hypothesis of graded Ca2+
permeability across AMPARs, depending upon how
many GluA2 receptors they contain rather than independent populations of Ca2+
-
permeable and Ca2+
-impermeable receptors.
Only the study from Paarmann et al. (2000) analyzes the relative level of
expression of flip and flop variants of AMPARs, using RT-PCR of aspirated patches of
the XII nucleus. There is a preference for flip over flop in GluA2 and GluA4 subunits and
for flop over flip in GluA3 subunits with no preference in GluA1 subunits. The
preferences, however, are not extreme. No such study exists for phrenic MNs.
In the case of kainate receptors, studies using antibodies that could not distinguish
between GluK1-GluK3 subunits find moderate to strong staining in the soma of phrenic
MNs (Robinson and Ellenberger, 1997) and in the soma and neuropil of XII MNs
(Robinson and Ellenberger, 1997; Garcia del Caño (1999)). RT-PCR for individual
kainate receptor subunits in the XII nucleus indicates that GluK2 is strongly expressed,
while GluK1 and GluK3 are weakly expressed or hardly expressed, respectively
(Paarmann et al., 2000). Therefore, GluK2 likely accounts for the strong
immunoreactivity of the non-specific antibodies. Additionally, GluK4 is strongly present,
while GluK5 is hardly detectable (Paarmann et al., 2000). Table 2.2 summarizes the
AMPA and kainate receptor subunit localization studies in XII and phrenic motor nuclei.
2.2.2 NMDA receptors in XII and phrenic MNs
NMDA receptors are in strong abundance in both XII (Shaw et al., 1991; Kus et
al., 1995; Robinson and Ellenberger, 1997; Garcia del Caño, 1999; Paarmann et al., 2000;

27
Oshima, 2002; Liu and Wong-Riley, 2010) and phrenic (Shaw et al., 1991; Kus et al.,
1995; Robinson and Ellenberger, 1997) MNs, localized mostly to neuronal somata in
humans, rats, and mice as well as in the neuropil of rats (Liu and Wong-Riley, 2010) and
mice (Oshima et al., 2002). Early studies, because of their use of the radiolabeled
antagonist [3
H]MK-801 or probes specific for GluN1-subunit mRNA or proteins, do not
provide specificity on the types of NR2 or NR3 subunits present.
Developmental studies of XII MNs shed light about the types of GluN2/3 subunits
that appear, but similar studies do not exist for phrenic MNs. One such study (Oshima et
al., 2002), using in situ hybridization in mice aged E13-P21, shows the GluN1 subunit is
expressed widely and strongly in neurons throughout the brainstem, including XII MNs
throughout the E13-P21 period. Similarly, high levels of GluN2A mRNA are seen in the
XII nucleus at E13, with mRNA further increasing and peaking in the first postnatal
week, before levels decrease gradually toward adult levels at P21. mRNA for GluN2B
and GluN2D is highly expressed at E13 and diminishes over the period of E15-E18,
indicating a specific developmental role for these subunits. Little expression of GluN2C
at any of the ages used in this study is reported.
Using immunohistochemistry in rats, another developmental study of NMDAR
subunit expression over the first three postnatal weeks shows a somewhat different
profile (Liu and Wong-Riley, 2010). Although largely in agreement on the postnatal
developmental profile of GluN2A with the mouse developmental study, this study shows
little agreement on the expression levels for the other GluN2 subunits. The study reports
GluN2A immunoreactivity in 65%-75% of neurons, which is present in cell bodies and

28
proximal processes as well as in the neuropil. GluN2A expression rises gradually from P2
to P11 with a significant dip at P12, slight rise at P13 and 14 and a gradual decline from
P17 to P21. GluN2C immunoreactivity is seen in 70%–85% of XII MNs in cell bodies
and some proximal processes, which is in glaring contrast to the study of Oshima et al.
(2002), where little evidence for GluN2C mRNA is reported for any age. Furthermore,
the study of Liu and Wong-Riley does not indicate a developmental role for GluN2B and
GluN2D in contrast to Oshima et al. (2002). This role might be obscured by looking only
at postnatal periods. GluN2B is in the cell bodies and some proximal processes of 70%–
90% of XII MNs with developmental expression relatively constant from P2 to P21,
although somewhat higher in expression at P5 and P7 than at P21. GluN2D
immunoreactivity is observed in about 60%–75% of XII MNs, distributed in cell bodies
and some proximal processes. Expression significantly decreases at P3 and P17, with a
small rise at P12. For GluN3B, immunoreactivity is present in 75%–85% of neurons that
generally increases with age. GluN3A is not considered by this study.
How much the differences in species v. that of technique contribute to the
differing data from these two developmental studies of NMDAR expression is unclear.
Unfortunately, this latter study failed to reference or comment on the earlier
developmental study, leaving it uncertain as to what the authors’ thoughts on the
differences might be. What role, if any, differences in NMDAR subunit stoichiometry
might make to respiratory function is unclear. Liu and Wong-Riley (2010) argue that
downregulation of GluN2 around P12 in MNs as well as in the preBötC may contribute
to a brief period where inhibition outweighs excitation in the respiratory control circuit.

29
Such an imbalance, they argue, could lead to reduced robustness against challenges to
stable breathing, thus resulting in pathologies like SIDS during the similar developmental
period in humans. Table 2.3 summarizes the NMDA receptor subunit localization studies
in XII and phrenic motor nuclei.
2.3 Role of iGluRs in the transmission of respiratory drive
Early studies of the role of glutamatergic signaling in the generation and
transmission of respiratory rhythm, e.g., McCrimmon, et al., 1986, show that injections of
small quantities of glutamate into brainstem centers involved in rhythm generation or into
motor nuclei controlling respiratory muscles increases the rate or amplitude of
respiratory-related activity, respectively. While showing that glutamatergic signaling
could influence respiratory behavior, studies like this one fail to answer the more
important question of whether glutamatergic signaling, in particular fast glutamatergic
signaling, is necessary for the generation and transmission of respiratory rhythm. Having
shown in the previous section evidence for the expression of AMPA, NMDA, and kainate
receptors in phrenic and XII MNs, this discussion summarizes critical studies using
antagonists of these receptors to demonstrate the necessity for fast glutamatergic
signaling in the transmission of respiratory drive to MNs in vitro and in anesthetized in
vivo preparations as. A recent study that will also be discussed calls into question the role
of fast glutamatergic signaling when the subjects are freely behaving.

30
2.3.1 In vitro and anesthetized in vivo studies
McCrimmon et al. (1989) showed the first evidence for the necessity of iGluRs in
the transmission of respiratory drive, using a split bath preparation of the rhythmically
active brainstem-spinal cord. At the spinomedullary junction, a fluid tight partition
allowed circulation of ionotropic glutamate antagonists to the spinal cord, while leaving
rhythmic activity in the brainstem unaffected. Phrenic and intercostal nerve activity was
sensitive to AP4, kynurenic acid, and DGG but largely insensitive to AP5 and DGT.
Similarly, in spontaneously breathing, anesthetized juvenile rats, when AP4 and
kynurenic acid were applied to the surface of the thoracic spinal cord, which provides
intercostal muscle innervation, reductions in MN activity in this region were seen.
The study by McCrimmon et al., however, did not demonstrate for certain that fast
glutamatergic signaling is required directly at synapses onto phrenic MNs. Liu et al.
(1990) directly addressed this question in the same preparation. Whole-cell patch clamp
recordings of phrenic MNs showed that inspiratory-related spiking and drive currents
were abolished by local application of the non-NMDA receptor antagonist CNQX to the
phrenic motor nucleus but largely insensitive to the similar local application of the
NMDA receptor antagonist MK-801.
Greer et al. (1991) demonstrated the necessity of non-NMDA receptor signaling
to rhythm generation when they saw a dose-sensitive slowing and finally abolition of
respiratory rhythm in cranial and spinal nerves after bath application of CNQX to the
medulla only. MK-801 had little effect on the respiratory rhythm or the amplitude of XII

31
nerve activity. The question remained, however, whether non-NMDA signaling was
obligatory for the transmission of drive to cranial nerves, e.g., the XII nerve. In addition,
the preBötC had not yet been discovered, making it unclear whether the importance of
non-NMDA receptor signaling in respiratory rhythmogenesis was localized to the
preBötC. Funk et al. (1993) answered both of these questions using the rhythmic slice.
Focal injection of CNQX unilaterally into the preBötC abolished activity in both the right
and left XII nerve rootlets, indicating the necessity for non-NMDA signaling in
respiratory rhythmogenesis. Furthermore, unilateral injection of CNQX into the XII
nucleus abolished activity in the ipsilateral but not contralateral XII nucleus, providing
evidence for the role of non-NMDA receptors in the transmission of respiratory drive to
cranial MNs.
Contemporaneous in vivo studies in anesthetized, vagotomized, and paralyzed
adult rabbits (Böhmer et al., 1991) and rats (Chitravanshi and Sapru, 1996), however
demonstrated an important role for NMDA receptors as well as non-NMDA receptors in
the transmission of respiratory drive. Microinjections of the non-NMDA receptor
antagonists DNQX, GAMS, and NBQX or the NMDA antagonists AP5 and AP7 into the
phrenic motor nucleus led to significant declines in activity. But only co-injection of non-
NMDA and NMDA receptor antagonists led to near abolition of phrenic nerve activity.
Because of these studies, Wang et al. (2002) revisited this issue of the relative roles of
non-NMDA and NMDA receptors in the transmission of respiratory drive in vitro. Using
the rhythmic slice under favorable conditions where Mg2+
was eliminated from the ACSF

32
bathing the slice and GABAA and glycine receptors were blocked, they measured that
only 14% of the inspiratory drive currents to XII MNs was NMDA-receptor dependent.
Morgado-Valle and Feldman (2007) looked at the problem a little differently,
however, shedding light on the issue. Similar to Wang et al., they eliminated Mg2+
in the
ACSF superfusing the rhythmic slice but they silenced non-NMDA receptors with
NBQX, leaving the NMDA receptors unaffected. Under these conditions, although
diminished in amplitude, inspiratory activity measured at the XII nerve rootlet continued
and was largely unaffected in rate. Only when MK-801 and NBQX were applied in
tandem was respiratory activity abolished. These data showed that NMDA receptors
alone, at least in 0 Mg2+
conditions, could support both respiratory rhythmogenesis and
transmission of respiratory drive to MNs, acting in an apparently parallel manner to non-
NMDA receptors. This agrees with the observation that the collocation of non-NMDA
and NMDA receptors at XII MN synapses is high (O’Brien et al., 1997). A reasonable
hypothesis arises from these data that, in vivo, various monoaminergic and peptidergic
drives that have been removed during the preparation of in vitro specimens likely provide
the extra depolarization required to remove Mg2+
block of NMDA receptors making them
more likely to carry current. Absent these drives in vitro, the respiratory control circuit
relies solely upon non-NMDA receptors to provide rhythmogenesis and transmission of
respiratory drive.

33
2.3.2 Experiments in freely behaving animals
Steenland et al. in a series of two studies (2006, 2008) explored the role of fast
glutamatergic signaling in transmission of respiratory drive to XII MNs, which innervate
the GG muscle of the tongue. Cannulae, allowing for microdialysis of agonists and
antagonists, were chronically implanted into the XII motor nucleus of adult rats along
with electrodes that were implanted into the genioglossus (GG) muscle of the tongue and
diaphragm to measure levels of respiratory and non-respiratory related activity. In their
2006 study, rats were anesthetized but not paralyzed. Independent microdialysis of high
enough concentrations of either CNQX (≥200 µM) or AP5 (≥1 mM) in the XII motor
nucleus was enough to abolish tonic and respiratory-related GG muscle activity. Applied
serially in either order, lower concentrations of AP5 and CNQX, together, could also
abolish GG activity. There was not a difference between vagotomized and non-
vagotomized animals. Under no circumstances was diaphragmatic activity affected,
indicating that the effects of the antagonists were local to the XII motor nucleus. These
results were in line with those described previously.
When, however, the same antagonists were applied by microdialysis to the XII
motor nucleus in freely behaving animals that exhibited periods of active wakefulness,
quiet wakefulness, non-REM, and REM sleep, as measured by EEG and neck EMG, only
subtle effects were observed (Steenland et al., 2008). AP5 significantly reduced but did
not abolish respiratory-related and tonic activity in GG muscles during active
wakefulness and significantly reduced but did not abolish respiratory-related activity in
non-REM sleep. Meanwhile, CNQX (as high as 5mM) did not have a significant effect

34
on tonic or phasic activity in any behavioral state. Microdialysis of DHK, a glutamate
uptake inhibitor, yielded an increase in tonic GG activity during periods of quiet
wakefulness and NREM sleep, providing evidence that glutamate was present. When
these rats were anesthetized, however, the results of the 2006 study were confirmed.
These data indicate that normal behavioral states introduce an added level of
complexity in understanding the role of iGluRs in the transmission of respiratory drive.
Unfortunately, under freely behaving conditions the authors did not simultaneously apply
CNQX and AP5 to rule out compensation by one set of iGluRs for another, i.e., NMDA
receptors for non-NMDA receptors or vice versa. Therefore, it remains unclear whether
iGluRs play a primary or backup role in transmission of respiratory drive during normal
behavior.
2.3.3 Non-NMDA receptors: AMPA v. kainate
The assumption in the field of respiratory control is that AMPA rather than
kainate receptors mediate non-NMDA receptor transmission of respiratory drive. But the
data speaking to this question are inadequate. The antagonists used in previous studies,
such as CNQX, NBQX, DNQX and kynurenic acid, do not distinguish between AMPA
and kainate receptors (Traynelis et al., 2010). GYKI 52466, which does distinguish
between the two receptor types, when applied focally to preBötC, abolishes respiratory
activity (Ge and Feldman, 1998). But these observations have not been extended to focal
application in respiratory motor nuclei. Therefore, only one study provides a partial
answer to the question of whether AMPA and kainate receptors both play a role in the

35
transmission of respiratory drive. Application of UBP-302, which selectively blocks
GluK1-containing receptors, to rhythmic slices does not affect either the rate or
amplitude of respiratory discharge in the XII nerve (Ireland et al., 2008). This result,
perhaps, is not surprising, since Paarmann et al. (2000) indicate GluK2 is the dominant
kainate receptor subunit expressed in XII MNs and neurons of the preBötC, and UBP-302
does not effectively block GluK2-containing recombinant or native receptors (Perrais et
al., 2009).
Cyclothiazide, which is selective for AMPA receptors relative to kainate receptors
(Partin et al., 1993), increases in the rate and amplitude of respiratory discharge when
bath applied in the rhythmic slice (Funk et al., 1995; Chapter 3 of this dissertation). This
result, however, does not preclude a role for kainate receptors. Similar data for kainate
receptor specific anti-desensitization agents, e.g., concanavalin A (Partin et al., 1993), are
absent from the literature. In addition, non-pharmacological methods, for example, EM
studies of kainate or AMPA receptor locations at synapses in motor nuclei or genetic
tools such as relevant knockouts, have not been applied to this problem. Thus, somewhat
surprisingly, this question remains unanswered.
2.4 Modulation and plasticity of iGluR currents in the transmission of
respiratory-related drive to MNs
The ability of an organism to adapt its breathing over timeframes ranging from a
single breath to a lifetime in response to changes in activity, posture, body size, sleep-
wake state, and disease and injury is essential to survival. The respiratory control circuit
changes both the rate and tidal volume (depth of breaths) to maintain the required levels

36
of minute ventilation (the volume of air moved per unit time) in the face of these
challenges. The locations and sources of this modulation are many and include changes to
iGluR-mediated respiratory drive to MNs (Feldman et al., 2003). These changes may
require the continued presence of the modulating signal (modulation), or they may last
beyond termination of the modulating signal (plasticity).
Much is known about neurotransmitters and neuropeptides that raise and lower
MN excitability, usually by modulating neuronal intrinsic properties (Rekling et al.,
2000). The focus of this section, however, relates to those neurotransmitters and second
messenger systems that specifically change iGluR-mediated currents at respiratory MN
synapses.
2.4.1 Modulation of iGluR-mediated respiratory drive
2.4.1.1 Presynaptic Modulation of iGluR signaling in XII MNs
5-HT, glutamate, enkephalin, and acetylcholine all influence presynaptic release
of glutamate in the XII MN. Probably the best studied of these transmitters is 5-HT,
which acts via 5-HT1A/B receptors to depress glutamatergic synapses presynaptically
(Singer et al., 1996; Bouryi and Lewis, 2003). Application of 5-HT (Singer et al., 1996;
Bouryi and Lewis, 2003), 5-HT1A agonist 8-OH-DPAT, and 5-HT1B agonist CP 93129
(Bouryi and Lewis, 2003) reduces the frequency but not the amplitude of mEPSCs
recorded in XII MNs in the presence of TTX. Also, EPSCs in XII MNs that are elicited
by stimulation of the reticular formation or raphe pallidus diminish in the presence of 5-
HT and the aforementioned subunit specific agonists. Although eEPSCs from the

37
reticular formation are only sensitive to 5-HT1B stimulation, indicating a more specific
subunit expression for presynaptic 5-HT receptors on axons originating in the reticular
formation (Singer et al., 1996).
Similar studies of mEPSCs as well as XII MNs EPSCs evoked by stimulation in
the reticular formation showed that nicotinic acetylcholine receptors, likely containing α4,
α7, and β2 subunits, facilitate presynaptic glutamate release (Quitadamo et al., 2005),
while activation of presynaptic M2 muscarinic receptors depresses presynaptic glutamate
release (Bellingham and Berger, 1996). In addition, enkephalin depresses glutamatergic
release from boutons on axons projecting from raphe pallidus, likely by acting on NK1
receptors (Bouryi and Lewis, 2004). Interestingly, while activation of presynaptic
mGluR1 receptors enhances glutamatergic release for spontaneous EPSCs in XII MNs, it
depresses XII MN EPSCs evoked by stimulation of the reticular formation lateral to the
XII nucleus in the presence of bicuculline and strychnine (to block effects on inhibition),
indicating the possibility of heterogeneity in the coupling of mGluRs to downstream
targets in different cell types sending their axons to the XII nucleus (Sharifullina et al.,
2005).
The previous data indicate that responses to the activation of a given receptor type
depends upon the origin of the specific axons. Little is known about the location or origin
of the axons providing respiratory drive to XII MNs (Koizumi et al., 2008), making it
impossible to know whether the observations described here hold for the presynaptic
elements carrying respiratory drive to MNs. In this context, then, it is difficult to say how
well the modulatory response of the glutamatergic synapses considered in these studies

38
represent the function of presynaptic boutons responsible for transmitting respiratory
drive to XII MNs.
2.4.1.2 Postsynaptic Modulation of iGluR signaling in XII MNs
Postsynaptic modulation of iGluR signaling can be accomplished by the action of
drugs and endogenous substances directly acting on AMPA and NMDA receptors as well
as by varying kinase activity.
Two classes of exogenous positive allosteric modulators of AMPARs,
benzothiadiazide diuretics and ampakines, increase respiratory drive currents measured in
XII MNs. Benzothiadiazide diuretics are best known for their ability to limit or abolish
desensitization in AMPARs (Yamada and Tang, 1993; Patneau et al., 1993) but also have
a variety of other effects at AMPARs, including dramatically lowering agonist EC50
(Patneau et al., 1993; Partin et al., 1994; Fucile et al., 2006), lengthening rate and length
of channel open time (Yamada and Tang, 1993; Fucile et al., 2006), increasing the
preference for larger conductance states (Fucile et al., 2006), and increasing deactivation
time (Patneau et al., 1993). Ampakines, derived from aniracetam, primarily work by
slowing AMPAR deactivation, although some formulations also inhibit desensitization as
well (Arai and Kessler, 2007; Traynelis, 2010).
The ampakines CX614 and CX717 increase respiratory drive to XII MNs (Lorier
et al., 2010). Similarly, cyclothiazide, the most potent of the benzothiadiazide diuretics
(Bertolino et al., 1993; Yamada and Tang, 1993), does the same also by acting
postsynaptically at AMPARs (Funk et al., 1995, Chapter 3 of this dissertation).

39
Interestingly, the effects of cyclothiazide last for at least 2 hours following application
(Funk et al., 1995). Whether the source of this prolonged enhancement is mediated by
plasticity phenomena is discussed in Chapter 3 of this dissertation. Both classes of drugs
also accelerate respiratory rate, making them of therapeutic interest in treating central
(Ren et al., 2006; Ogier et al., 1997; Ren et al., 2009) as well as obstructive (Chapter 3 of
this dissertation) apneas.
NMDA receptors require glycine binding at their GluN1 subunits as well as
glutamate binding to their GluN2/3 subunits to open. The glycine binding sites of XII
MN NMDARs are not fully saturated in vitro (Berger et al., 1998; Kono et al., 2007).
Therefore, under baseline conditions in slices, NMDA currents are submaximal. Addition
of D-serine (Berger et al., 1998) to the bathing medium or stimulation of glycinergic
synapses (Kono et al., 2007) facilitates currents resulting from subsequent NMDAR
activation. Whether regulation of glycine binding is a method for modulating NMDAR
currents in vivo in XII MNs is unknown, although there is evidence for it playing a role in
other brain areas, for example, in hippocampal function in vitro (Yang et al., 2003) and in
vivo (Billard and Rouaud, 2007).
The role of kinases and phosphatases in regulating the strength of iGluR synapses
has been widely studied in areas of the brain such as the hippocampus, cerebellum, and
cortex. Data in XII MNs also supports a role for phosphorylation in modulating AMPAR
synapses transmitting respiratory drive. In XII MNs in vitro, protein kinases A (PKA)
and G (PKG) play opposing roles in regulating the strength of AMPA receptor synapses.
Intracellular dialysis of the catalytic subunit of PKA into XII MNs in rhythmic slices

40
potentiates respiratory drive as well as currents elicited by exogenous application of
AMPA in the presence of TTX. Conversely, a peptide inhibitor of PKA inhibits
respiratory drive when intracelluarly dialyzed via patch pipette (Bocchiaro et al., 2003).
In vivo, microdialysis of the PKA activators 8-Br-cAMP and forskolin into the XII
nucleus increases GG activity, but microdialysis of the PKA inhibitor Rp-8-Cl-CAMPS
does not decrease GG activity, calling into question the constitutive role of PKA in
managing MN excitability (DuBord et al., 2010), although other compensating pre- or
post-synaptic effects of PKA activation could not be ruled out.
In contrast, in rhythmic slices, focal application of PKG activator 8-Br-cGMP to
XII MNs decreases respiratory drive and currents elicited by exogenous application of
AMPA in the presence of TTX. Intracellular dialysis with a PKG inhibitory peptide
increases respiratory drive and exogenous AMPA-induced currents in TTX (Saywell et
al., 2010). Finally, intracellular dialysis of XII MNs with microcystin, a phosphatase 1
and 2a inhibitor, increases respiratory drive and exogenous AMPA receptor-mediated
currents (Bocchiaro et al., 2003), arguing for the constitutive role of both phosphatases
and kinases in managing AMPAR-mediated excitability of XII MNs.
2.4.2 iGluR-mediated synaptic plasticity of respiratory MNs
The sensory neuron to MN synapse mediating siphon withdrawal in Aplysia
californica serves as a canonical model for studying synaptic plasticity. Despite this,
there has been relatively little study of synaptic plasticity in mammalian MNs.
Furthermore, most existing studies of synaptic plasticity involve some form of injury,

41
e.g., severing supraspinal inputs or axotomy, or disease, e.g., ALS, rather than exploring
synaptic plasticity under typical physiological conditions. On the other hand, there has
been considerable interest in respiratory plasticity, but it is unclear how many of these
plasticity phenomena involve plastic changes at MNs and if they do, whether those
changes are to excitatory synapses or intrinsic properties. This section considers several
respiratory plasticity phenomena that involve or are postulated to involve plastic changes
to iGluR synapses of MNs.
2.4.2.1 Acute-hypoxia induced long-term facilitation
Not surprisingly, the natural stimulus that induces many forms of respiratory
plasticity is hypoxia brought on by the lowering of the arterial pressure of O2, i.e.,
hypoxemic hypoxia (Powell et al., 1998; Teppema and Dahan, 2010). The response of the
respiratory control system greatly depends upon the depth (level of O2 desaturation),
duration (acute or chronic), and time course (single episode or intermittent) of hypoxia
and whether CO2 is held constant, as well as the age, sex, sleep-wake state, species, and,
even, strain of the animal (Powell et al., 1998; Baker-Herman et al., 2010; Teppema and
Dahan, 2010).
Long-term facilitation (LTF) of phrenic, intercostal, and XII motor activity
following acute intermittent hypoxia (AIH) is an example of hypoxia-induced plasticity
that is of interest for several reasons. First, LTF may be a naturally occurring response by
the body to respiratory challenges brought on by recurrent apneic episodes, e.g., during
sleep, and its failure may lead to diseases such as OSA (Mahamed and Mitchell, 2007).

42
Second, AIH-induced LTF has shown potential for treatment of motor deficits due to
diseases of ventilatory control (Wilkerson et al., 2007) and spinal cord injury (Dale-Nagle
et al., 2010). Third, there is an in vitro form of synaptic plasticity in MNs, ivLTF
(discussed below), that has similar induction protocols, shares many of the necessary
second messenger cascades, and results in postsynaptic increases in AMPAR-mediated
currents and respiratory drive at XII MNs.
AIH-LTF is induced by short episodic bouts of hypoxia, e.g., 3, 5-minute bouts of
isocapnic 10% O2 spaced at 5-minute intervals, although more apneic-like protocols also
prove effective for induction (Baker and Mitchell, 2000; Mahamed and Mitchell, 2008).
Most often AIH-LTF is studied in anesthetized, vagotomized and paralyzed adult rats but
can be induced in neonatal rats as well as a variety of other species as well as in freely
behaving animals, although the level of expression of facilitation is more variable under
these conditions (Feldman et al., 2003; McKay et al., 2004). AIH-LTF depends on the
action of 5-HT through 5-HT2 (Baker-Herman and Mitchell, 2002) and possibly 5-HT7
(Hoffman and Mitchell, 2011) receptors as well as noradrenaline via α1-adrenergic
receptors (Neverova et al., 2007). Protein kinase C, tyrosine receptor kinase B (TrkB),
brain-derived neurotrophic factor (BDNF), and reactive oxygen species (ROS) all play a
role in the signaling cascade required for its expression (Figure 2.1; Wilkerson et al.,
2007).
Denervation of the carotid bodies greatly reduces the level of AIH-LTF (Bavis
and Mitchell, 2003; Sibigtroth and Mitchell, 2011), and there is evidence that AIH-LTF
increases the excitability of bulbospinal neurons (Morris et al., 2001). Notwithstanding

43
these data, much of what is required to induce AIH-LTF is thought to takes place in the
respiratory motor nuclei and likely the MNs themselves. Localized injections of 5-HT
receptor antagonists into C4 attenuate AIH-LTF in phrenic but not XII nerve activity
(Wilkerson et al., 2007). Similarly, injection of MK-801 into the motor nuclei containing
phrenic MNs blocks induction of AIH-LTF, which also indicates a potential role for
iGluRs, specifically, NMDARs in inducing the phenomenon (McGuire et al., 2005).
Finally, a separate but potentially related phenomenon in XII MNs that is induced by
stimulation of vagal feedback requires activation of α1-adrenergic receptors in the XII
motor nucleus (Tadjalli et al., 2010).
2.4.2.2 In vitro long-term facilitation
Episodic application of α-Me-5HT (Bocchiaro and Feldman, 2004), a 5-HT2A
receptor agonist or phenylephrine (Neverova et al., 2007), an α1-adrenergic receptor
agonist, results in a long-lasting (≥1 hour) increase (~50%) in the amplitude of
respiratory activity in XII nerve of the rhythmic slice. The increased nerve discharge is
accompanied by a commensurate increase in non-NMDA mediated drive currents to XII
MNs. When the same protocol is run after silencing the rhythmic slice with TTX,
exogenous application of AMPA to the XII MN shows a similar increase in AMPAR-
mediated currents in the XII MN, indicating that this plasticity is postsynaptic, activity
independent, and dependent upon increases in synaptic AMPAR currents (Bocchiaro and
Feldman, 2004; Neverova et al., 2007). Similar to AIH-LTF, ivLTF is PKC, TrkB, ERK
dependent (Neverova et al., 2007; Neverova 2007). Chapter 4 of this dissertation
demonstrates that ivLTF can be enhanced via PKG signaling, likely involving ROS

44
activity. In addition, Chapter 5 of this dissertation shows that ivLTF is protocol sensitive
and may, in fact, not require episodic stimulation as first thought.
2.4.2.3 The crossed-phrenic phenomenon
Hemisection of the spinal cord rostral to C2 results in paralysis of the half
diaphragm ipsilateral to the hemisection. Over time (weeks to months, depending on the
species), the paralyzed part of the diaphragm recovers function spontaneously in a variety
of mammalian species. This is referred to as the crossed phrenic phenomenon (CPP;
Goshgarian, 2003). Recovery of activity is associated with pronounced restructuring of
axonal bulbospinal inputs to phrenic MNs as well as the dendritic arbors of the phrenic
MNs themselves. A variety of manipulations can hasten this recovery, including
damaging the contralateral phrenic nerve, enhancing cAMP activity, or treatment with
phosphodiesterase inhibitors, A1 adenosine receptors antagonists, or antagonists of
NMDA receptors (Goshgarian, 2003; Goshgarian, 2009). The last of these treatments
implicates a role for iGluR-mediated plasticity in CPP.
CPP is thought to take advantage of latent bulbospinal efferents to phrenic MNs
that are present and active in perinatal animals. In P2 rats, a portion of diaphragmatic
activity is maintained ipsilateral to the hemisection. The same is true for the just the
ventral portion of the diaphragm of rats aged ≤P28. By P35, all activity is lost (Huang
and Goshgarian, 2009). Associated with this loss of crossed-phrenic activity in perinatal
animals is a downregulation of GluN2A and GluA1 receptor subunits in phrenic MNs
(Huang and Goshgarian, 2009a). Finally, spontaneous recovery of activity in rats seen in

45
CPP is associated in time with, first, upregulation of GluN2A and subsequent
upregulation of GluA1 receptor subunits, strongly implicating a role for iGluRs in CPP
(Huang and Goshgarian, 2009a).
2.5 Discussion
Evidence from various studies over the last 20 years show not only the presence
of the panoply of iGluR subunits in respiratory MNs but also the potentially essential role
of iGluR signaling in the transmission of respiratory drive to MNs. Furthermore,
plasticity of these iGluR-mediated connections is implicated in a variety of plasticity
mechanisms resulting from normal and pathophysiological stimuli, i.e., hypoxia and
spinal cord injury.
Due to the relatively recent development of reduced models of breathing that offer
easy access to cellular components of respiratory rhythmogenesis and motor activity, our
understanding of both basic iGluR signaling as well as its modulation and plasticity in the
brainstem is in its early days. As described previously, more study of the types of iGluR
subunits, their stoichiometry, and intracellular location, i.e., synaptic, perisynaptic,
extrasynaptic, somatic as well proximal, dendritic, or localization to the neuropil, needs
to be understood. Furthermore, evidence is beginning to mount for the widespread role of
iGluR-mediated plasticity in this circuit.
Much more remains to be discovered regarding iGluR signaling in respiratory
control as whole, with the promise that therapeutics might be developed to take
advantage of these iGluR modulation and plasticity mechanisms in treating respiratory-

46
related disease and dysfunction. These are exciting times, indeed, for studying the
synaptic physiology of respiratory rhythmogenesis, pattern generation, and drive
transmission!

47
Table 2.1 Ionotropic glutamate receptor subunits1
IUPHAR2
Name Common Name Gene Name3
AMPA Receptor Subunits
GluA1 GluR1, GluRA Gria1
GluA2 GluR2, GluRB Gria2
GluA3 GluR3, GluRC Gria3
GluA4 GluR4, GluRD Gria4
Kainate Receptor Subunits
GluK1 GluR5 Grik1
GluK2 GluR6 Grik2
GluK3 GluR7 Grik3
GluK4 KA1 Grik4
GluK5 KA2 Grik5
NMDA Receptor Subunits
GluN1 NMDAR1, NR1, GluRξ1 Grin1
GluN2A NMDAR2A, NR2A, GluRε1 Grin2a
GluN2B NMDAR2B, NR2B, GluRε2 Grin2b
GluN2C NMDAR2C, NR2C, GluRε3 Grin2c
GluN2D NMDAR2D, NR2D, GluRε4 Grin2d
GluN3A NR3A Grin3a
GluN3B NR3B Grin3b
Delta Receptor Subunits
GluD1 δ1, GluR delta-1 Grid1
GluD2 δ2, GluR delta-2 Grid2
1
Adapted from Traynelis, et al. (2010)
2
IUPHAR – International Union of Basic and Clinical Pharmacology
3
Human gene names would be capitalized (e.g., GRIA1)

48
Table 2.2 AMPA and kainate receptor subunit localization studies in XII and phrenic motor nuclei
AMPAR Subunit Kainate Receptor Subunit4
Study Method1
Model2
Age3
A1 A2 A3 A4 K1 K2 K3 K4 K5
Williams
et al.
(1996)5 IC H A + ++/+++6
++
Robinson &
Ellenberger
(1997)7 IC R A +/++8
+++6
+++ ++/+++9
Garcia del
Caño et al.
(1999)10 IH R A
+/++/
+++11 +++6,12,13
+++12
++/+++9,12
Paarmann
et al.
(2000)14,15
RT-
PCR
M
P4-
P7
4/4 3/416
3/2 2/4 2 4 1 3 0
Immunoreactivity: +, weak; ++, moderate; +++, strong
1
Immunocytochemistry (IC), Immunohistochemistry (IH), RT-PCR (Real-time polymerase chain reaction
2
Human (H), Rat (R), Mouse (M)
3
Adult (A), Postnatal day x (Px) where P0 is the day of birth
4
Blank column means presence of receptor subunit was not assessed.
5
Results for XII motor nucleus and ventral horn of cervical spinal column. All results similar between both locations.
6
Antibody could not distinguish between GluA2 and GluA3.
7
Results for XII MNs and phrenic MNs identified by fluoro-gold retrograde tracer applied to phrenic nerve.
8
+ for phrenic, ++ for XII
9
Antibody could not distinguish between GluK1/GluK2/GluK3.
10
Studied XII drawing distinctions between dorsal (D), ventral (V), ventromedial (VM), ventrolateral (VL) subnuclei.
11
+, rostral D, V, VM subnuclei; ++/+++, caudal D, VL subnuclei
12
Same intensity of immunoreactivity across subnuclei
13
Staining with separate GluA2 specific antibody was weak.
14
RT-PCR performed on aspirated areas of tissue that included neurons as well as glia
15
Shows # of positive samples out of 4 containing reaction products (x/x for flip/flop). Each sample from different animal.
16
Separate RT-PCR analysis in single XII MNs showed that 9/11 cells had detectable products for arginine edited
(Ca2+
-impermeable) mRNA. 0/11 showed products for glutamine containing mRNA (Ca2+
-permeable).

49
Table 2.3 NMDA receptor subunit localization studies in XII and phrenic motor nuclei
NMDA Receptor Subunit
Study Method1
Model2
Age3
N1 N2A N2B N2C N2D N3A N3B
Shaw et al.
(1991)4 [3
H]MK-
8015 H A
45-102 fmole/mg binding in ventral horn of spinal column
in generally increasing gradient from cervical to sacral
Kus et al.
(1995)6 ISH R A +++7
Robinson
&
Ellenberger
(1997)8
IC R A +++9
Garcia del
Caño et al.
(1999)10
IH R A +++11
Paarmann
et al.
(2000)12,13 RT-PCR M
P4-
P7
4 3 4 1 4 3
Oshima
et al.
(2002)14 ISH M
E13-
P21
+++
↓
+/++
+++
↓
+/++
+++
↓
+/-
-
+++
↓
+/-
Liu &
Wong-
Riley
(2010)15,16
IC R
P2-
P21
++/+++
↓
++
++/+++
↓
++
+++
++
↓
+
+/++
↓
++/+++
Immunoreactivity: -, none detected, +, weak; ++, moderate; +++, strong
1
Immunocytochemistry (IC), Immunohistochemistry (IH), RT-PCR (Real-time polymerase chain reaction
2
Human (H), Rat (R), Mouse (M)
3
Adult (A), Embryonic day x (Ex), Postnatal day x (Px) where P0 is the day of birth.
4
Binding analyzed in C3, C7, T1, T5, L1, L5, S2 levels of human spinal cord
5
Method does not distinguish between subunit types
6
XII and lumbar MNs studied
7
Staining similar in XII and lumbar MNs. Staining much higher than in sensory neurons.
8
Results for XII MNs and phrenic MNs identified by fluoro-gold retrograde tracer applied to phrenic nerve.
9
Immunoreactivity same for XII and phrenic MNs.
10
Studied XII drawing distinctions between dorsal (D), ventral (V), ventromedial (VM), ventrolateral (VL) subnuclei.
11
Same for all subnuclei
12
RT-PCR performed on aspirated areas of tissue that included neurons as well as glia.
13
Shows # of positive samples out of 4 containing reaction products. Each sample from different animal.
14
Developmental study of XII MNs. Days: E13, E15, E18, P1, P7, P14, P21.
15
Developmental study of XII MNs. Days: P2, P3, P4, P5, P7, P10, P11, P12, P13, P14, P17, P21.
16
GluN2A: 65%-75% MNs immunoreactive (IR), GluN2B: 70%-90% MNs IR, GluN2C: 70%-85% MNs IR,
GluN2D: 60%-75% MNs IR, GluN3B: 60%-80% MNs IR

50
Figure 2.1 Similarities in signaling pathways for AIH-LTF and ivLTF. (A) Proposed signaling pathways
on phrenic motor facilitation (PMF), a form of AIH-LTF (from Dale-Nagle et al., 2010). (B) Proposed
signaling pathways for induction of ivLTF (Adapted from Neverova, 2007). AMPAR, AMPA receptor;
BDNF, brain-derived neurotrophic factor; GC, guanylyl cyclase; MAPK, mitogen-activated protein kinase
(aka ERK); MEK, mitogen-activated protein kinase kinase; mGluR1, metabotropic glutamate receptor 1;
MIT, mitochondria; NOS, nitric oxide synthase; PKC, protein kinase C; PKG, protein kinase G; PMF,
phrenic motor facilitation; PP, protein phosphatase; Ras, rat sarcoma; ROS, reactive oxygen species; Trk,
tyrosine receptor kinase

51
3 CYCLOTHIAZIDE-INDUCED PERSISTENT INCREASE IN RESPIRATORY-RELATED
ACTIVITY IN VITRO
3.1 Introduction
Motor pools innervating muscles of the upper airway maintain upper airway
patency against subatmospheric pressures due to inspiratory airflow. Loss of upper
airway muscle tone resulting in restriction or closure of the airway can lead to hypopnea
or apnea. In obstructive sleep apnea (OSA) decrease or loss of MN activity innervating
genioglossus (tongue retractor) and other upper airway muscles during non-REM and
REM sleep leads to upper airway collapse, resulting in repeated apneic and hypopneic
events and (severe) disruption of sleep. Occurring in 15%-20% of people (Young et al.,
2002; Young et al., 2009), OSA leads to increased daytime drowsiness, risk of workplace
or car accidents and increased long-term risks of cardiovascular disease, stroke, and
hypertension (Young et al., 2002; Young et al., 2009). Therapies that specifically can
increase excitability of these MNs have the potential to ameliorate OSA.
XII MNs innervate the genioglossus muscle of the tongue, which is critical to
upper airway patency. In vitro (Funk et al., 1995; Greer et al. 1991) and under anesthesia
in vivo (Steenland et al. 2006; Steenland et al. 2008), phasic respiratory drive to these
MNs is mediated primarily by glutamatergic signaling through AMPA and NMDA (in
vivo) receptors suggesting that the excitability of XII MNs may be modulated by drugs
that change AMPA receptor kinetics. One class of drugs, which work at the AMPA
receptor by impeding deactivation and, to a lesser extent, desensitization is ampakines
(Arai and Kessler, 2007). Ampakines have therapeutic potential, successfully treating, in

52
rodents, central depression of breathing due to anesthetics (Ren et al., 2006; Ren et al.,
2009) or to the knock-out of the Rett’s syndrome related gene Mecp2 (Ogier et al., 2007).
They also facilitate respiratory-related activity in XII MNs in vitro (Lorier et al., 2010).
Another class of drugs that can upregulate AMPA receptor-mediated excitability is
benzothiadiazide diuretics (Bertolino et al., 1993; Arai and Kessler, 2007). Cyclothiazide
(CTZ) is the most potent of these (Bertolino et al., 1993; Yamada and Tang, 1993). CTZ
affects the amplitude and rate of respiratory-related activity measured on the XII nerve in
vitro (Funk et al., 1995). Interestingly, its effects last for at least 1-2 hours post-treatment
(Funk et al., 1995). What underlies this long-lasting facilitation is unclear, with the
possibility that a novel form of plasticity induced by CTZ may be the source (Funk et al.,
1995).
In this study, we explored the mechanisms underlying the persistence of CTZ-
induced facilitation (CIF) of respiratory-related (inspiratory) XII nerve activity. We
found that CTZ profoundly increased the amplitude of inspiratory activity, and the effects
lasted up to 12 hours post-treatment, i.e., from the start of washout. In contrast, the
effects of the ampakine CX546, though similar in character to those of CTZ during
treatment, dissipated following washout. The size of CIF was dose-dependent and
sensitive to the duration of treatment. CIF did not depend on AMPA or NMDA receptor
signaling at the time of CTZ treatment, nor did it depend on coincident protein kinase A
or C activity. Finally, investigation of the long-term effects of CTZ on non-NMDA,
presumably AMPA, miniature excitatory postsynaptic currents (mEPSCs) in XII MNs, as
well as analysis of untreated and treated tissue samples with liquid chromatography

Dissertation (rev 2011-05-23)_final_pdf

Dissertation (rev 2011-05-23)_final_pdf

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Viewers also liked

Viewers also liked (12)

Similar to Dissertation (rev 2011-05-23)_final_pdf

Similar to Dissertation (rev 2011-05-23)_final_pdf (20)

Dissertation (rev 2011-05-23)_final_pdf