dissertation (rev 2011-05-23)_final_pdf
TRANSCRIPT
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
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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
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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.
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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
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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
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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
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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 activity 107 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
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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
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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
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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.
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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.
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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 University of California, Los Angeles
2008-2009 Predoctoral Fellow UCLA-NIH Training Program in Neural Microcircuits Department of Neurobiology University of California, Los Angeles
2010-2011 Predoctoral Fellow Ruth L. Kirschstein National Research Service Award Department of Neurobiology University of California, Los Angeles
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.
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ABSTRACT OF THE DISSERTATION
Long-term Enhancement of Respiratory-Related Activity by
Increasing the AMPA Receptor-Mediated Excitability of
Hypoglossal Motoneurons In Vitro
by
Walter Edward Babiec
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
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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.
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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
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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).
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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,
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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).
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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
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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
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“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.
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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
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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.
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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.
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 [3H]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
[3H]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
53
tandem mass spectrometry indicated that the long-lasting time course of CIF was due to
residual presence of CTZ, rather than a novel form of plasticity.
These data show the importance of regulating AMPA receptor kinetics for normal
functioning of the neural circuit controlling breathing. In addition, they suggest the basis
for formulating pharmacological therapies to be used alone or in combination with
existing mechanical or surgical techniques to counteract the impaired excitability of MNs
in diseases like OSA.
54
3.2 Methods
3.2.1 Preparation
All animal procedures were performed according to National Institutes of Health
guidelines and approved by the Office for the Protection of Research Subjects, University
of California Research Committee. Neonatal (P0-P4) Sprague-Dawley rats (Charles River
Laboratories International Inc., Wilmington, MA, USA) were anesthetized in a small
chamber by inhalation of isoflurane (5 ml for ~15 min). A lack of pedal withdrawal reflex
assured that the level of anesthesia was sufficient. The anesthetized rat was placed ventral
side up and rapidly decerebrated with a scalpel. A second cut caudal to the cervical
backbone was made, separating the skull and vertebrae containing the brainstem and
cervical spinal cord from the rest of the body. This reduced preparation was pinned in a
dish and submerged in chilled (4°-8º C) artificial cerebral spinal fluid (ACSF, in mM,
120 NaCl, 3 KCl, 1.5 CaCl2, 1 MgSO4, 23.5 NaHCO3, 0.5 NaH2PO4, 30 D-glucose, pH
7.4) that was gassed with 95% O2 - 5% CO2. Via a ventral entry, the brainstem and
cervical spinal cord were exposed and removed with care to preserve the XII nerve
rootlets. The dura mater was stripped away, the individual XII rootlets teased apart, and
the cerebellum and choroid plexus removed, exposing the IVth ventricle. Finally, a scalpel
cut was made near the pontomedullary border.
Still in oxygenated ACSF, the resulting en bloc preparation was pinned ventral
surface up to a holder made of Sylgard® 184 (Dow Corning Corp., Midland, MI USA)
backed with rigid plastic and placed in the chuck of a Vibratome® 1000 (Vibratome,
55
Bannockburn, IL, USA). Several cuts were made from the rostral end of the preparation.
Once the facial nucleus was removed and the compact formation of the nucleus ambiguus
exposed, a 700 μm slice was cut. This slice retained a sufficient proportion of the
respiratory network to generate an inspiratory rhythm that could be measured in the
activity of the XII nerve rootlets (Smith et al. 1991). The slice was transferred to a 1.5 ml
recording chamber (Warner Instruments, Hamden, CT USA) and held in place with a
harp. The slice was superfused (≥5 ml/min) with ACSF containing elevated K+ (9 mM) to
sustain stable inspiratory activity (Smith et al. 1991). The slice was maintained at a
constant temperature of 28°C and allowed to recover for ~ 1 hour before beginning
experiments.
3.2.2 XII Nerve Recordings
XII nerve activity was recorded using a suction electrode and differential
amplifier (A-M Systems, Carlsborg, WA USA). The signal coming from the amplifier
was split into two channels, one for direct data acquisition and a second that was rectified
and integrated (Paynter filter, τ = 100 ms) using a custom-built integrator. Signals were
sampled at 10 – 20 kHz and stored using a Digidata® 1440A analog-to-digital converter
and pCLAMP® 10 software (Molecular Devices, Sunnyvale, CA, USA) running on a PC.
The rhythmic burst discharges of the XII nerve defined the inspiratory period.
3.2.3 Whole-cell Recordings
Inspiratory-active XII MNs were visualized with an infinity corrected 40x water
immersion objective using differential interference contrast microscopy on an Axioskop
56
FS1 microscope (Carl Zeiss MicroImaging, Thornwood, NY, USA). The image was
displayed on a monitor using a CCD72 camera (Dage-MTI, Michigan City, IN USA).
Whole-cell voltage-clamp recordings (Vh= -70 mV for inspiratory drive currents, Vh= -90
mV for mEPSCs) were made using borosilicate glass electrodes (8250 glass, 3 5 MΩ).
The internal solution contained, in mM, 135 K gluconate, 1.1 EGTA, 5 NaCl, 0.1 CaCl2,
10 HEPES, 2 ATP (Mg2+ salt), 0.3 GTP (Na+ salt), pH 7.3 adjusted using KOH. Cs
methanesulfonate and CsOH were substituted for K-gluconate and KOH, respectively, for
mEPSC recordings. Signals were recorded via and Axopatch1-D patch clamp amplifier
and CV-4 1/100 headstage (Molecular Devices). Signals were filtered in the patch clamp
amplifier with a low pass Bessel filter (-3dB at 5kHz) and sampled at 20 kHz via
Digidata® 1440A both of which were controlled through pClamp® software. Post hoc,
currents were filtered further in pClamp® using a low pass 8-pole Bessel filter (-3dB at 1
kHz). During recordings, access resistance was monitored for stability throughout the
experiment. Junction potentials between bath solution and electrode were corrected
before forming a gigaohm seal and whole-cell capacitance was compensated before
break-in. For recording mEPSCs, XII MNs having inspiratory activity were silenced with
TTX (1 µM) and non-NMDA glutamate receptor minis isolated using D-AP5 (50 µM),
picrotoxin (100 µM), and strychnine (10 µM). Because of the requirement for
inspiratory-modulated XII MNs, only one MN per slice was used.
3.2.4 Mass Spectrometry
A liquid chromatography - tandem mass spectrometry - multiple reaction
monitoring (LC-MSMS-MRM) assay was conducted to determine whether significant
57
amounts of CTZ remained in tissue at different times during washout. 700 µm non-
rhythmogenic brainstem slices (as many as 4 from a single animal) from neonatal (P0-P4)
Sprague-Dawley rats were exposed to ACSF-only, 1 hour of CTZ (90 µM), 1 hour of
CTZ plus 1 hour wash with ACSF, or 1 hour of CTZ plus 6 hour wash with ACSF.
Excess surface fluid was removed from the treated slices, which were then placed in pre-
weighed microcentrifuge tubes, weighed again, and frozen at -20°C for later processing.
During processing, 1 ml of methanol was added to the samples, which were then
disrupted with an ultrasonic cell disrupter. The samples were centrifuged (13,000g for 5
min) and the supernatant was removed into a clean tube and taken to dryness. To the
dried residue 100 µl of water/acetonitrile/formic acid (95/5/0.1) was added and the
samples were vigorously mixed, centrifuged again (13,000g for 5 min) and the
supernatant was transferred to LC injector vials. 20 µl of each sample was injected and
analyzed. The limit of detection was 40 fmol.
3.2.5 Drugs
6-Chloro-3,4-dihydro-3-(5-norbornen-2-yl)-2H-1,2,4-benzothiazidiazine-7-
sulfonamide-1,1-dioxide (CTZ), N-[2-[[3-(4-Bromophenyl)-2-propenyl]amino]ethyl]-5-
isoquinolinesulfonamide (H 89) dihydrochloride, 1,2-Dimethoxy-12-
methyl[1,3]benzodioxolo[5,6-c]phenanthridinium (chelerythrine) chloride, D-(-)-2-
Amino-5-phosphonopentanoic acid (D-AP5), 6-Cyano-7-nitroquinoxaline-2,3-dione
(CNQX), and tetrodotoxin citrate (TTX) were acquired from Tocris (Ellisville, MO,
USA). 1-(1,4-Benzodioxan-6-ylcarbonyl)piperidine (CX546), strychnine, and picrotoxin
were acquired from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO;
58
Sigma-Aldrich) was used to dissolve, CTZ, CX546, picrotoxin, and CNQX into 100 mM
stocks and chelerythrine into a 10 mM stock. Stocks were then diluted directly to their
final concentrations as reported.
3.2.6 Electrophysiological Data Analysis.
For systems recordings, ∫XII nerve bursts were identified using threshold
detection in pClamp®. The peak value for each burst was measured and averaged with the
peak values from the other bursts in 5-minute epochs. Averages were normalized to the
first 25 minutes of a 30-minute control period. The last 5 minutes of the control period,
also normalized to the first 25 minutes of the control period, served as the control
measurement. XII nerve burst rate was determined by taking the inverse of the average of
the individual inter-burst intervals occurring over the same periods used to measure
amplitude and normalized to the control rate.
For whole-cell recordings of XII MN inspiratory drive currents, charge transfer
was calculated for individual inspiratory periods using DataView (Version 5.2.2, WJ
Hitler, University of St. Andrews) and averaged over 5-minute periods. mEPSCs were
identified using template matching in pClamp®. 200 - 600 mEPSCs per cell were
recorded and used in analyzing amplitude and inter-event interval. The maximum
amplitude of each individual event was averaged to determine the average amplitude,
while individual inter-event intervals were averaged to determine the average interval. To
calculate decay time constants, all mEPSC waveforms recorded from a given MN were
averaged into a single average waveform from which the decay time constant was
59
computed by fitting a single decaying exponential curve using data from the minimum
amplitude of the average mEPSC back to baseline.
3.2.7 Statistics
Data are summarized as mean ± SEM unless otherwise reported. Differences
associated with p-values ≤0.05 were deemed significant. Although they were not
considered significant, values of 0.05<p≤0.10 are reported. Values for p>0.10 are
reported as n.s. (not significant).
For data containing more than two groups an ANOVA was first conducted. One-
way ANOVAs were used for dose-response and exposure-response analyses. Two-way
repeated measures ANOVA (RMANOVA) was used for analyzing longitudinal data from
different treatment groups. Then individual difference tests (see below) were run for
specific comparisons of interest. Individual p-values are reported, but Holm-Bonferroni
analysis for multiple comparisons ensured a familywise error rate ≤ 0.05 for all difference
comparisons made within a data set. The similarity of cumulative distributions for
mEPSC amplitudes and intervals for neurons exposed to different treatment conditions
was assessed by piecewise comparison mEPSC amplitude and interval distributions in
addition to group mean comparisons.
Non-normal, assessed using the Shapiro-Wilk test, and heteroscedastic, assessed
using Levene’s test, sample populations appeared in a number of the data sets reported. In
addition, in some cases ANOVAs were carried out on slightly unequal data populations
to take advantage of all available data. Since normality (t-test and ANOVA) and
60
homoscedasticity (pooled variance t-test and ANOVA) are basic assumptions of
parametric statistics (Cohen and Lea, 2004), we chose to use bootstrapping statistical
methods to analyze all data. Bootstrapping avoids making any underlying assumptions
about parent data distributions, instead working solely with existing data distributions.
Bootstrapping also allows the use of data values rather than ranks. Similar to regular t-
tests and ANOVA, the difference tests and ANOVAs assumed as a null hypothesis that
data from all treatment groups were part of the same distribution. This null hypothesis
was tested by sampling, with replacement, from the composite population to come up
with differences in means or F-ratios for comparison. This process was repeated 10,000
times to construct distributions of the mean differences or F-ratios based on the null
hypothesis. The actual two-sample difference in means or multi-sample F-ratio for the
treatment groups was compared to these distributions to assess the likelihood that they
would have occurred if the null hypothesis had been correct. In repeated measures (RM)
situations resampling was done at the level of the subject, maintaining within-subject
correlations across time. More detail on the rationale for and use of bootstrapping in
analysis of biological data may be found in Manly (2006).
Bootstrapping was also employed to compare the similarity of mEPSC amplitude
and interval distributions among treatment groups. The control distribution was binned
into histograms and resampled with replacement 10,000 times to develop confidence
intervals around the bin value for each original control data histogram. The number of
data points selected during each resample depended upon the number of samples
contained in the histogram for the treatment (either CTZ or CTZ plus 1 hour wash) being
61
compared to the control distribution. Then the non-control treatment distribution was
binned into the same bin sizes and compared against the control distribution confidence
intervals. If the bin count was outside of confidence intervals for more than 20% of the
bins consecutively, the distributions were deemed significantly different at the level of
the confidence intervals. If confidence intervals were violated sporadically, those
violations were evaluated instead at the Bonferroni level (α divided by the number of
bins) level of confidence to determine significance.
3.2.8 Regressions
Single-variable linear regressions were performed using StatPlus® (AnalystSoft
Inc., Vancouver, BC, Canada). Only regressions having an F-test with p≤0.05 and
normally distributed residuals were considered significant.
62
3.3 Results
3.3.1 CIF
Our previous investigation effects of CTZ on inspiratory activity in the medullary
slice employed a cumulative dose-response protocol. Slices were exposed to 5 increasing
concentrations of CTZ in the range from 10 - 100 μM. At each level 7 minutes was given
for equilibration before 3 minutes of data were recorded (Funk et al., 1995). Here, our
preliminary experiments using 100 μM CTZ indicated that the amplitude of ∫XII nerve
activity continued to increase for the entire duration of treatment (~2 hours), although the
rate of rise slowed considerably within the first hour (Figure 3.1A). The increase in the
amplitude of ∫XII nerve activity, however, appeared to be much larger than previously
reported. Also, we saw sporadic increases in tonic activity after 1 hour (Figure 3.1A), as
previously reported (Funk et al., 1995).
To avoid these sporadic periods of increased nerve tonicity, we limited our
maximum CTZ concentration to 90 μM and treatment duration to 1 hour (Figure 3.1B-C).
∫XII nerve amplitude increased to 236%±21% of (pre-treatment) control at the conclusion
of bath application and peaked at 262%±23% 1 hour post-treatment, i.e., after start of
washout. Given our interest in the persistent effects of CTZ, we followed activity for 12
hours post-treatment (Figure 3.1C, Figure 3.2A). ∫XII amplitude remained significantly
elevated relative to pre-treatment for the entire duration of the experiment (Table 3.1).
The effect of CTZ on the rate of inspiratory burst activity (Figure 3.2B) was
significant but more modest. Respiratory rate elevated to 147%±14% at the conclusion of
63
treatment and rose to 151%±12% 1 hour post-treatment (Table 3.1). By 6 hours, the rate
returned to baseline 108%±15% remaining there at 12 hours (Table 3.1).
DMSO, the solvent for CTZ, has excitatory effects on neurons of the lamprey
locomotor CPG (Tsvyetlynska et al., 2005). To control for any effects DMSO might have
on neurons in this preparation, we exposed slices to 0.1% DMSO alone for 1 hour (Figure
3.1C, Figure 3.2A-B). (This concentration was slightly larger than the 0.09% that slices
were exposed to when we applied 90 µM CTZ). DMSO did not increase ∫XII amplitude
or rate, which was depressed at all time points relative to pre-treatment (Table 3.1).
The response to CTZ was compared to DMSO and CX546 (see below) in a two-
way RMANOVA to look for treatment effects as well as time effects followed by
difference tests at specific time points (reported in Table 3.1 and Figure 3.2). For ∫XII
nerve burst amplitude, the effects of treatment and the interaction of time and treatment
were both very highly significant, while the effect of time was not (Table 3.1). The
amplitude of ∫XII nerve activity for CTZ-treated slices was significantly larger than for
DMSO-treated slices at all time points (Figure 3.2C). Treatment and time effects on the
rate of inspiratory activity showed highly significant effects for treatment and the
interaction of time and treatment as well as a significant effect for time (Table 3.1).
Similar to amplitude, the rate of ∫XII nerve activity of CTZ-treated slices was higher than
in DMSO slices (Figure 3.2D).
The absolute amplitude of inspiratory ∫XII nerve bursts can vary by more than an
order of magnitude from slice to slice. Similarly, the rate of inspiratory activity can vary
64
by a factor of 2-3 from slice to slice. For this reason, normalized values are useful in
evaluating data taken from groups of slices. Analyzing normalized values, however, left
the possibility that, for example, the effects of CTZ on respiratory rate might be greater in
slices whose pre-treatment respiratory rates were at the lower end of the expected range.
On the other hand, the effects of CTZ on a slice with burst amplitudes at the high end of
the amplitude range during the pre-treatment period might be suppressed, because the
level of nerve activity could already be saturated. To assess whether the pre-treatment
raw values of amplitude or rate of inspiratory XII nerve activity predicted the response to
CTZ, we regressed the percentage effect on ∫XII amplitude or burst rate at 1 hour post-
treatment against the raw amplitude or rate of activity for that slice during the pre-
treatment period. Pre-treatment amplitude ranged from 0.3-13.1 a.u., and pre-treatment
control rate ranged from 6.3 to 20.4 bursts per minute. We saw no significant relationship
between amplitude effect size and raw pre-treatment amplitude or rate effect size and raw
pre-treatment burst rate (Figure 3.2E-F).
Finally, to see if the long-lasting effects of CTZ on amplitude and rate were
typical of agents that affect AMPA receptor desensitization and deactivation, we tested
CX546 (90 µM) using the same protocol (Figure 3.1C). In slices treated with CX546,
∫XII amplitude was increased immediately post-treatment, declined greatly but was still
elevated for 1 hour post-treatment, returned to baseline by 2 hours post-treatment and
remained there for the rest of the experiment (Figure 3.2A, Table 3.1). Relative to
DMSO, ∫XII amplitude was significantly different only immediately post-treatment
(Figure 3.2C).
65
Only immediately post-treatment, the rate of inspiratory ∫XII nerve bursts was
significantly elevated in CX546-treated slices relative to pre-treatment control (Figure
3.2B, Table 3.1). Similar to slices treated with DMSO to which they were statistically
similar (Figure 3.2D), slices treated with CX546 had a lower respiratory rate relative to
pre-treatment (Table 3.1). Therefore, in the presence of CX546, inspiratory activity in the
medullary slice responds in a way similar to CTZ, but the effects of CX546 abate during
washout.
3.3.2 Dose-Response
Next we characterized the effect of CTZ dose and duration of exposure on slice
inspiratory activity. We either applied 3, 9, or 30 µM to separate groups of slices for 1
hour or we applied 90 µM CTZ to separate groups of slices for 10 or 30 minutes. These
data were then grouped with the data for slices exposed to 90 µM for 1 hour in dose-
response (Figure 3.3A) or exposure response (Figure 3.3B) curves. The effect of CTZ on
∫XII nerve burst amplitude to be significant at 1 hour post-treatment for both dose and
exposure time (Figure 3.3). CTZ dose but not exposure time had a significant effect on
the respiratory rate measured 1 hour post-treatment (Figure 3.3), indicating the possibility
that the effect of CTZ on the amplitude and rate of inspiratory activity were separable.
Since the effect of CTZ on burst amplitude was so much larger and longer lasting than its
effect on the rate of activity, we focused solely on the former in the rest of our
experiments.
66
3.3.3 Long-Term Effects of CTZ on XII MN Drive
Manipulation of the excitability of XII MNs affects the amplitude but not the rate
of inspiratory activity in the medullary slice (Funk et al., 1993). Drive to these MNs is
primarily AMPA-receptor dependent in vitro (Funk et al., 1993) and is enhanced by acute
CTZ exposure (Funk et al., 1995). To assess the long-term post-treatment effects of CTZ
on inspiratory drive to XII MNs, we continuously measured currents from XII MNs
before, during, and up to 1 hour post-treatment with 90 µM CTZ. CTZ was applied for
only 10 minutes in order to reliably maintain stable whole-cell recordings for the duration
of the experiment, while still getting reliable long-lasting facilitation of activity (Figure
3.3B, Figure 3.4A). At 1 hour post-treatment, endogenously generated drive to XII MNs
was facilitated (Figure 3.4A). Charge transfer was 153%±8.1% of pre-treatment while
∫XII nerve amplitude increased to 146%±19% (Figure 3.4B). Furthermore, the size of
increase of ∫XII nerve amplitude correlated well with the increase in charge transfer
(Figure 3.4C).
3.3.4 Investigation of Intracellular Signaling as the Mechanism Underlying CIF
Having established that CTZ leads to a profound and long-lasting facilitation of
inspiratory XII nerve discharge and AMPA receptor-mediated synaptic drive to XII MNs,
we focused on identifying the mechanism underlying the long-lasting component of CIF.
Increased internal Ca2+ is a mechanism critical for inducing a variety of long-term
plasticity mechanisms (Malenka and Bear, 2004; Vogt and Canepari, 2010). NMDA
receptors, which are highly permeable to Ca2+, require depolarization (to remove Mg2+
67
block) in addition to glutamate to open. NMDA receptors are colocalized with AMPA
receptors at excitatory synapses in XII MNs (O’Brien et al., 1997). Therefore, we
hypothesized that enhanced depolarization resulting from facilitated AMPA receptor-
mediated synaptic currents could raise the level of intracellular Ca2+ through a
commensurate increase in activation of NMDA receptors. To investigate this possibility
we blocked AMPA (10 µM CNQX) and NMDA (50 µM D-APV) receptors for 30
minutes before and continuing through 1 hour of CTZ application and 1 hour of washout.
By eliminating the main source of XII MN excitability in the slice, we would also likely
reduce activation of another major source of Ca2+, voltage-gated Ca2+ channels.
Blockade of AMPA and NMDA receptors abolished inspiratory activity. To guard
against the possibility than any facilitation we might see during recovery from CNQX
and D-APV was due to rebound excitation from silencing neurons rather than from the
effects of CTZ, we also exposed slices to the same protocol but without applying CTZ.
Treatment with CTZ in the presence of these glutamate receptor antagonists had a
significant effect on ∫XII nerve burst amplitude relative the antagonists alone (Figure
3.5B). Slices treated with CTZ in the presence of CNQX and D-APV saw their activity
start to return within 30 minutes of starting antagonist washout (85.6%±22% of pre-
treatment control ∫XII amplitude, n=6). ∫XII amplitude was significantly greater than pre-
treatment from 1 hour after the start of washout of CNQX and D-APV through the end of
the experiment 5 hours after start of washout of CNQX and D-APV (Figure 3.5B). In
contrast, only 1 of 5 slices treated with the antagonists alone saw activity return within 30
minutes of starting antagonist washout. Activity in all 5 slices had returned by 1 hour
68
after the start of antagonist washout (80.8%±16% of pre-treatment control ∫XII
amplitude, n=5), but on average ∫XII amplitude in these slices was neither facilitated nor
depressed relative to pre-treatment control through the rest of the experiment (Figure
3.5B). At both 1 and 5 hours post CNQX and D-APV, ∫XII nerve burst amplitude was
larger in slices treated with CTZ relative to those not treated with CTZ (Figure 3.5C). To
test whether a component of the facilitation seen when slices were treated with CTZ
without glutamate receptor antagonists was missing when treatments were done in the
presence of antagonists, we compared equivalent time points for CTZ-treated slices in
this experiment with CTZ-treated slices from our initial characterization (Figure 3.1).
There was no significant difference in amplitude of response between slices treated with
CTZ and slices treated with CTZ in the presence of CNQX and D-APV (F(1,9)=3.04,
n.s., two-way RMANOVA). Together these data indicated that it was unlikely CIF
depended, on whole or in part, on activation of AMPA or NMDA receptors during
treatment with CTZ.
Next, we considered whether CTZ affected the activity of PKA and PKC, both of
which have a role in increasing XII MN excitability (Bocchiaro et al., 2003, DuBord et
al., 2010, Neverova et al. 2007). We blocked PKA (10 µM H 89) and PKC (10 µM
chelerythrine) 30 minutes prior to applying CTZ for 1 hour. The kinase antagonists were
maintained throughout CTZ treatment and for 1 hour post-treatment. We controlled for
the long-term effects of H 89 and chelerythrine, alone, on the amplitude of ∫XII nerve
activity in the slice by applying these antagonists without applying CTZ. Long-term
exposure to H 89 and chelerythrine greatly reduced the amplitude of inspiratory activity
69
in slices, which only partially recovered after 30 minutes of washout (Figure 3.6A). Some
slices were allowed more than an hour of washout but never showed complete recovery
(data not shown). Despite the effect on activity of these kinase antagonists, CTZ still
facilitated the amplitude of ∫XII nerve activity, which was significant when compared to
slices treated only with H 89 and chelerythrine alone at 30 minutes following washout of
the kinase antagonists (Figure 3.6B). From these experiments, we concluded CTZ was
most likely acting in a manner independent of PKA and PKC.
3.3.5 Does CTZ Washout?
Having eliminated several of the mechanisms most likely to underlie the long-
lasting component of CIF, we considered an alternative hypothesis. While some studies
report that CTZ washes out from the preparation (Ballerini et al. 1995; Funk et al. 1995;
Qi et al. 2006), other studies maintain that CTZ lingers in the tissue, sequestered in the
lipid neuron membrane (Patneau et al., 1993; Larson et al., 1994) where it can continue to
affect AMPA receptor desensitization. We addressed this issue in two ways. First, we
used a functional assay for residual CTZ, whereby we could measure certain
electrophysiological parameters for any telltale signature of the continued presence of
CTZ. Thus we measured non-NMDA, presumably AMPA, mEPSCs. CTZ can increase
amplitude, decay, and frequency of non-NMDA mEPSCs (Diamond & Jahr, 1995). We
measured XII MNs from slices that were bathed in ACSF only, bathed in CTZ for 1 hour,
or bathed in CTZ for 1 hour and then washed for 1 hour. Following a given treatment
regimen, XII MNs were patched, verified to have inspiratory-related drive, and action
potentials (TTX 1 µM) and receptors for NMDA (50 µM D-APV), GABAA and glycine
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(100 µM picrotoxin and 10 µM strychnine) were blocked. Average mEPSC peak
amplitude was significantly larger in neurons treated with CTZ and washed for 1 hour
relative to neurons treated with CTZ for 1 hour and to neurons bathed in ACSF alone,
which was in line with results from comparing mEPSC peak amplitude distributions in a
piecewise manner (Figure 3.8A). mEPSC decay time was greater for neurons treated with
CTZ and washed for 1 hour relative to neurons bathed in ACSF alone, although less than
for neurons treated with CTZ but not washed (Figure 3.7C).
CTZ had at most a marginally significant effect on the average interval between
mEPSCs (Figure 3.7D). Use of the more sensitive piecewise comparison of interval
distributions, however, showed a very highly significant difference between the interval
distribution of CTZ-treated slices compared to both ACSF-treated neurons and CTZ-
treated neurons that were washed for 1 hour (Figure 3.8B). There was also a marginally
significant difference between the ACSF-treated and CTZ-treated with 1 hour wash
groups that indicated XII MNs treated with CTZ and then washed had fewer large (>1.25
s) intervals.
The mEPSC amplitude and decay data provided indirect evidence that CTZ
remained in the tissue. We pursued a second, more direct measure for the residual
presence of CTZ in treated but washed tissue, i.e., liquid chromatography tandem mass
spectrometry. We made 4 different treatment groups of slices: (1) exposed to ACSF only,
(2) treated with CTZ (90 µM) for 1 hour and removed immediately, (3) treated with CTZ
(90 µM) for 1 hour and then washed with ACSF for 1 hour before removal, (4) treated
with CTZ (90 µM) for 1 hour and then washed with ACSF for 6 hours before removal.
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ACSF-only slices showed no evidence of CTZ (sensitivity of ~40 fmol). All of the other
treatment groups showed large quantities of residual CTZ (10±2.8 pmol/mg for CTZ
treated slices, 5.3±0.9 pmol/mg for slices treated with CTZ and washed for 1 hour, and
5.7±0.6 pmol/mg for slices treated with CTZ and washed for 6 hours). Although there
was a trend towards more CTZ remaining in tissue immediately post-treatment as
opposed to after being washed for 1 or 6 hours, the difference was not significant,
suggesting most of the CTZ remained in the slice despite hours of washing (Figure 3.9).
Assuming the slices have the approximate density of water and that CTZ could go
anywhere in the slice, which is conservative since CTZ likely does not easily cross the
cell membrane (Patneau et al., 1993), our measurements indicate the residual
concentration of CTZ in the tissue following treatment and wash was on average at least
5 µM and not statistically significantly different from unwashed CTZ-treated slices.
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3.4 Discussion
We demonstrate that CTZ profoundly increases the amplitude of inspiratory ∫XII
nerve discharge in the medullary slice preparation, while increasing the rate of inspiratory
activity in a smaller but significant manner. The ~150% average increase in amplitude
observed is ~3x the maximum facilitation previously reported for similar concentrations
of CTZ (Funk et al., 1995), with much of this facilitation remaining for at least 12 hours
post-treatment/after start of drug washout. The level of amplitude facilitation, also, is ~3x
that of ivLTF, another form of long-lasting facilitation of inspiratory activity than can be
induced in the medullary slice (Bocchiaro and Feldman, 2004; Neverova et al., 2007).
The increase in inspiratory ∫XII nerve discharge is associated with a well-correlated
increase in AMPA-mediated drive currents to the XII MN and an increase in the
amplitude and rate of decay of AMPA mEPSCs measured in XII MNs.
Unlike with ivLTF, CIF most likely is not a plasticity phenomenon. CIF is not
dependent upon activation of AMPA or NMDA receptors at the time of CTZ treatment,
nor is it dependent upon coincident PKA or PKC activity, which underlie increases in
AMPA receptor-mediated excitability in XII MNs (Bocchiaro et al., 2003; Neverova et
al., 2007; DuBord et al., 2010). Rather, the effects of CTZ are maintained, because CTZ
fails to wash out of the slice for at least 6 hours post-treatment. We are unaware of other
data that present direct evidence of the washout characteristics of CTZ in in vitro
preparations, despite some previous speculation and interpretation of indirect evidence
(Patneau et al., 1993; Larson et al., 1994; Funk et al. 1995). Therefore, these data provide
additional perspective for interpreting results acquired when using CTZ in in vitro
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systems. Furthermore, reversible enhancement of AMPAR-mediated signaling with
CX546, in part by blockade of AMPAR desensitization, did not lead to long-lasting
increases in respiratory activity, consistent with the idea that the extended bioavailability
of CTZ is critical for the persistent effects we observed.
3.4.1 Mechanism of Action
As a neuromodulator, CTZ is best known for its ability to limit or abolish
desensitization in primarily flip slice variants of AMPA receptors (Yamada and Tang,
1993; Patneau et al., 1993; Partin et al., 1994). CTZ, however, has a variety of other
effects at AMPA receptors, including dramatically lowering agonist EC50 (Patneau et al.,
1993; Partin et al., 1994; Dzubay and Jahr, 1999; 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). When considering these data in combination
with our analysis of endogenous drive currents and mEPSCs, which are consistent with
other studies showing increased spontaneous and mEPSC peak currents and decay time
constants (Yamada and Tang, 1993; Diamond and Jahr, 1995), we surmise that all of the
effects summarized here probably contribute to the effects we see on inspiratory activity.
In addition to its effects on AMPA receptors, CTZ has a variety of effects not
involving AMPA receptors, some of which could also contribute to the enhanced
inspiratory activity that we observed. CTZ originally was developed as a diuretic in the
1960s (Martz et al., 1962), acting on the thiazide-sensitive Na+-Cl- cotransporter in the
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distal loop of the kidney (Martinez-Moldonado and Cordova, 1990). This is highly
unlikely to be a mechanism affecting the medullary respiratory control circuit, however,
since this transporter is not found in the mammalian brain (Gamba, 2005). CTZ also has
antagonistic effects on GABAA (Deng and Chen, 2003), GABAC (Xie et al., 2008), and
α2-subunit containing glycine (Zhang et al., 2008) receptors. Whereas inhibitory
neurotransmitters have little effect on respiratory rhythm generation in vitro, their
blockade can dramatically change the pattern of motor output, including the amplitude
and shape of XII nerve bursts (Feldman and Smith, 1989; Shao and Feldman, 1997;
Saywell and Feldman, 2004). CTZ also inhibits metabotropic glutamate type 1 (Sharp et
al., 1994; Surin et al., 2007) and α3- but not α7-containing acetylcholine receptors
(Nooney and Feltz, 1995). Most likely blockade of these receptors would be antagonistic
to XII MN excitability, since both the effects of acetylcholine and activation of
metabotropic glutamate receptor type 1 tend to be excitatory in the preBötzinger
Complex and XII motor nucleus in vitro (Shao et al., 2008; Shao and Feldman, 2005;
Chamberlain et al., 2002; Sharifullina et al., 2004).
3.4.2 Physiological Significance
Recent studies show the importance of AMPA receptor desensitization to
survival. Knock-in of a desensitization inhibiting L483Y mutation to the gene encoding
the GluA2 receptor subunit is homozygous lethal, with most heterozygous mice suffering
from runted development and seizures developing at around P16, before premature death,
usually in the third postnatal week. Interestingly, no respiratory distress is reported
(Christie et al., 2010). In addition, over the past decade a number of endogenous proteins
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for controlling the level of desensitization have been identified. They include TARPs,
cornichons, and CKAMP44, which affect desensitization, deactivation, and activation
kinetics of AMPA receptors as well as the actions of CNQX and receptor trafficking to
the membrane (Milstein and Nicoll, 208; Brockie and Maricq, 2010; Kato et al., 2010;
von Engelhardt et al., 2010). Finally, predators use dysregulation of desensitization as a
method for capturing prey. A newly discovered Conus snail toxin works by inducing
excitotoxicity through blockade of AMPA receptor desensitization (Walker et al., 2009).
Our data, therefore, add to this growing set of data regarding the importance of
endogenous regulation of AMPA receptor kinetics to proper functioning of neural
circuits.
Remarkably, our induction of large increases in the efficacy of AMPA receptor
signaling greatly enhanced the amplitude of inspiratory nerve discharge while not grossly
distorting rhythmogenic behavior. Only the highest concentrations of CTZ at the longest
durations of application caused bouts of tonic activity that distorted the respiratory
rhythm. This result demonstrates the enormous residual firing capacity in XII MNs
available for enhancing inspiratory motor output under in vitro conditions. This suggests
the possibility of using CTZ as a therapeutic for enhancing reduced motor output due to
disease or dysfunction in vivo. One such therapeutic application might be in the treatment
of OSA, where loss of tone in upper airway muscles during sleep is associated with
repetitive upper airway collapse leading to periods of apnea or hypopnea and long-term
pathological consequences (Young et al., 2002; Young et al. 2009).
76
Many challenges remain, however, in translating our observations to an effective
treatment. First, CTZ, likely, does not cross the blood-brain barrier (BBB; Black, 2005).
The XII nucleus, however, is in close proximity to area postrema, a circumventricular
organ. Circumventricular organs are relatively leaky portions of the BBB, and, therefore,
may allow some local penetration of drugs like CTZ. Paradoxically, this might be a
benefit of using CTZ, since being BBB-impermeability would not allow for wide
penetration into the nervous system. In fact, improvement in apneic symptoms may be
present but go unnoticed in those treated for hypertension with cyclothiazide, because of
the relatively high-rate of undiagnosed cases of OSA. Alternatively, there are BBB-
permeable derivatives of CTZ, such as IDRA-21 and S18986, which are being
investigated as cognition-enhancing therapies (Black, 2005; Malkova, 2010).
Second, there is the concern that CTZ might induce negative non-specific effects.
In fact, CTZ is used to induce permanent seizures to study epilepsy (Qi et al., 2006; Kong
et al., 2010). However, the concentrations injected directly into the cerebral ventricle, for
example, 5 M (Qi et al., 2006), are much higher than the highest concentration used in
this study and more than 100x the concentrations that enhance inspiratory activity.
Although much lower concentrations of CTZ (Qi et al., 2006) can induce seizure-like
activity in in vitro cell cultures, which tend to be highly excitable on their own, the length
of incubation was for extremely long periods, 48 hours (Qi et al., 2006). Also, not only
derivatives of CTZ but other AMPA receptor modulators as well are being investigated
for a variety of cognitive deficits (Black, 2005; Arai and Kessler, 2007) without
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observing serious detrimental effects on breathing (modest changes in breathing may
very well go unnoticed) (Black, 2005).
Finally, the role of AMPA-mediated signaling in transmission of respiratory drive
to XII MNs in behaving animals during NREM and REM sleep is unclear. Injection of
CNQX into the XII motor nucleus during NREM and REM sleep has little effect on
phasic respiratory or tonic genioglossus muscle activity. But, injection of dihydrokainate,
a glutamate uptake inhibitor, increases tonic genioglossus muscle activity during NREM
sleep (Steenland and Horner, 2008). These data indicate that, although, glutamatergic
signaling might not be robust enough to drive genioglossus muscle activity under normal
circumstances, glutamatergic signaling is present. Therefore, amplifying the effects of
this signaling through the use of AMPA receptor modulators, such as CTZ, could serve to
increase upper airway tone during, at least, NREM periods of sleep.
3.4.3 Implications for Therapeutic Design
We have shown that CTZ appears to occupy a rather unique location in the trade
space of AMPA receptor modulator design. Working through multiple mechanisms, it
has profound impact on AMPA receptor function and inspiratory activity in vitro.
Because of its hydrophobicity and lipid solubility, it is extremely long-lasting, which also
may make it difficult to introduce in vitro. This latter attribute, however, may be taken
advantage of, serving as a starting point to precisely control the duration, location, or
localization of activity in the nervous system that a more soluble drug might not offer.
Although ampakines have received much more attention of late as a proposed treatment
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for neurological disorders, we feel the unique attributes of CTZ warrant taking a second
look for the development of treatments for disorders such as OSA.
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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).
∫XII Amp ∫XII Rate
RMANOVA CTZ DMSO CX546 Treatment F(2,16)=47.7*** F(2,16)=42.9*** Time F(13,208)=6.4 F(26,208)=8.9*** Treatment x Time F(26,208)=5.5*** F(13,208)=49.8* Tests v. Pre-Treatment Control CTZ 0 Hours Post-Treatment 236%±21%*** 147%±14%*** CTZ 1 Hour Post-Treatment 262%±23%*** 151%±12%*** CTZ 6 Hours Post-Treatment 205%±5%*** 108%±15% CTZ 12 Hours Post-Treatment 190%±9%*** 63%±19% DMSO 0 Hours Post-Treatment 99%±4% 83%±6%* DMSO 1 Hour Post-Treatment 90%±4%** 60%±5%*** DMSO 6 Hours Post-Treatment 92%±7% 36%±4%*** DMSO 12 Hours Post-Treatment 90%±13% 22%±5%*** CX546 0 Hours Post-Treatment 160%±15%*** 137%±7%*** CX546 1 Hour Post-Treatment 126%±11%*** 75%±7%*** CX546 2 Hours Post-Treatment 114%±8% n/a CX546 6 Hours Post-Treatment 118%±18% 41%±5%*** CX546 12 Hours Post-Treatment 119%±19% 30%±3%***
Values are normalized relative to pre-treatment control (see Methods) and reported as mean±SEM (n = 5 for CTZ treated slices, n = 7 for DMSO and CX546 treated slices). Two-way RMANOVA included all slices. Measurements that were taken pre-treatment, immediately (0 hours) and 1-12 hours (hourly) post-treatment (14 data points in all per slice) were used in RMANOVA. Tests v. pre-treatment control were RM difference tests where measurements of activity were compared at two time points: once immediately prior to treatment and once at the time point indicated. Family-wise error rate, which also includes statistical tests shown in Figure 3.2, was protected to p≤0.05 using Holm-Bonferroni method. * p<0.05, ** p<0.01, *** p< 0.001.
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Figure 3.1 Bath application of CTZ leads to long-lasting facilitation of endogenous inspiratory XII nerve activity in the neonatal rat medullary slice. (A) ∫XII nerve activity continues to increase in the presence of bath-applied CTZ for the entire duration (~2 hours) of application. Arrows show periods of increased tonicity. Asterisk denotes period of tonicity shown on expanded timescale (right of main trace). (B) Protocol for experiments in (C) and Figure 2. (C) Example traces showing the impacts of bath application of CTZ (top), CX546 (middle), and DMSO (bottom) on ∫XII activity. Traces on expanded timescales below main traces show samples of ∫XII nerve activity before and 1, 6, and 12 hours post-treatment. Traces to the right of main trace show average of ∫XII nerve bursts taken in 5-minute intervals before (black) and immediately (blue), 1 (red), 6 (purple), and 12 (green) hours post-treatment.
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Figure 3.2 CTZ, but not CX546 or DMSO, leads to long-lasting facilitation of endogenous inspiratory ∫XII nerve activity. (A-B) Longitudinal data for the effect of 1-hour application of CTZ (90 µM, n=5), CX546 (90 µM; n=7), or DMSO (0.1%; n=7) on normalized ∫XII nerve burst amplitude (A) and rate (B). Thick lines show group averages. Dotted lines show individual experiments. CTZ (Black, ■), CX546 (Red, ◆), DMSO (Blue, ▲). Black bar above data shows timing and duration of treatment. (C-D) Comparisons for the effects of CTZ (■) and CX546 (◆) v. DMSO control (▲) on normalized ∫XII nerve burst amplitude (C) and rate (D) at 1, 6, and 12 hours post-treatment. Significance, assessed using difference tests following two-way RMANOVA, is as indicated in the figures. (E-F) Regressions assessing whether the effect of CTZ (90 µM; n=11) on normalized ∫XII at 1 hour post-treatment v. raw control amplitude (E) and normalized ∫XII nerve burst rate at 1 hour post-treatment v. raw control rate (F) were significantly correlated.
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Figure 3.3 Dose-response and exposure-response effects of CTZ on ∫XII nerve burst amplitude and rate 1 hour post-treatment. (A) Dose-response for 1 hour bath application of 3 µM, 9 µM, 30 µM, or 90 µM CTZ (n=5 for each concentration) on ∫XII nerve burst amplitude and rate. Concentration had a significant effect on both amplitude (F(3,16)=6.38, p<0.01) and rate (F(3,16)=6.50, p<0.01) as determined by a one-way ANOVA. (B) Exposure-response curves for 90 µM CTZ applied for 10 minutes (n=6), 30 minutes (n=6), or 1 hour (n=5). Exposure time had a significant effect on amplitude (F(2,14)=7.68, p<0.01) but not the rate (F(2,14) = 2.31, n.s.) of ∫XII nerve bursts as assessed by a one-way ANOVA. In both panels, ■, amplitude responses, and ●, rate responses. Large symbols show group averages and small symbols individual experiments. All measurements were taken at 1 hour post-treatment.
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Figure 3.4 Bath application of CTZ induces long-lasting increases in endogenous inspiratory drive to XII MNs. (A) Top trace shows the effect of treating a medullary slice with CTZ (90 µM) for 10 minutes. Expanded traces below show sample ∫XII nerve bursts and accompanying XII MN drive currents immediately prior to (black) and 1 hour post-treatment (blue). Overlaid current traces to the right show an average of 25 consecutive drive currents for each of these time points. (B) Comparison of normalized charge transfer of inspiratory drive currents in XII MNs before and 1 hour post-treatment. Lines connect measurements from the same cell before and 1 hour post-treatment. Significance tested using RM difference test (n=5). (C) Regression showing high correlation between increases in XII MN drive currents and ∫XII nerve burst amplitude 1 hour post-treatment.
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Figure 3.5 CIF does not depend upon activation of AMPA or NMDA receptors during treatment with CTZ. (A) Sample traces showing the effects on ∫XII nerve activity after bath application of CNQX (10 µM) and APV (50 µM) (control slices, top trace) or CTZ (90 µM) in the presence of CNQX and APV (bottom trace). CNQX and APV were applied for 2.5 hours. When CTZ was applied, it was applied for 1 hour, 30 minutes after the start of CNQX and APV. This allowed CNQX and APV to take effect before CTZ and 1 hour for the slices to be washed after CTZ before removing CNQX and APV. Black bars above traces illustrate the timing and duration of application. Transients resulting from electrostatic discharge during the slice silent periods have been removed. (B) Longitudinal data for all experiments run according to the protocols in A. CTZ had a significant effect on (F(1,9)=12.8, RMANOVA) on the amplitude of inspiratory activity. In slices treated with CTZ (n=6) ∫XII amplitude was significantly greater than pre-treatment from 1 hour post CNQX and D-APV (182%±7%, p<0.001, RM difference test) through the end of the experiment 5 hours post CNQX and D-APV (180%±25%, p<0.001, RM difference test). ∫XII amplitude in slices not treated with CTZ (n=5) was neither facilitated nor depressed relative to pre-treatment (115%±21% 5 hours post CNQX and D-APV, n.s., RM difference test). (■, slices receiving treatment with CTZ, n=6; ▲, control slices, n=5). Thick traces represent group means. Dotted traces represent individual experiments. (C) Comparison of activity 1 hour and 5 hours post CNQX and D-APV in slices treated and not treated with CTZ. Significance was computed using difference tests that followed a two-way RMANOVA.
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Figure 3.6 CIF is not PKA or PKC dependent. (A) Sample trace showing the effects of bath application of chelerythrine (10 µM) and H 89 (10 µM) on inspiratory ∫XII nerve activity. The last half hour of the trace (during washout of H 89 and chelerythrine) demonstrate the failure of some slices not treated with CTZ to recover pre-treatment activity levels. (B) Comparison of ∫XII nerve burst amplitude (relative to pre-treatment control) between CTZ-treated (CTZ) and untreated (No CTZ) slices 30 minutes post washout of H89 and chelerythrine, which was 1 hour post the start of washout of CTZ, when CTZ was used. Symbols represent individual experiments and lines represent group averages. Significance assessed using difference test.
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Figure 3.7 CTZ treatment of medullary slices leads to long-lasting increases XII MN non-NMDA mEPSC amplitude and decay. (A) Sample mEPSCs from a control cell (top), a cell treated for 1 hour with CTZ (90 µM; middle), an a cell treated with CTZ that was then washed for 1 hour before recording (bottom). (B-D) Group data comparing average mEPSC peak amplitude (B), mEPSC decay time constant (C), and average mEPSC interval (D) for cells treated under 1 of the 3 conditions described in A (n=6 for control cells and n=7 for cells treated with CTZ and cells treated with CTZ and then washed for 1 hour). Inset in B shows the average of the average waveforms for each experiment under a given condition. Control (black), CTZ (red), CTZ + 1 hour wash (blue). Inset in C shows waveforms in B scaled to have the same peak value. Significance in B-D assessed using difference test. Individual symbols in B-D represent values for single experiments. Lines represent group averages.
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Figure 3.8 Comparison of mEPSC distributions shows further differences among treatment groups. Symbols represent histogram values for the (A) magnitudes and (B) intervals of mEPSCs. Control (black diamonds), CTZ-treated (red circles), CTZ-treated and washed for 1 hour (blue triangles). Gray bars in (A) represent 5% confidence intervals in top and bottom graphs and 0.1% confidence intervals in middle graph. For (B) gray bars represent 95% confidence intervals for middle graph and 0.1% confidence intervals for top and bottom graphs. In both (A) and (B) confidence intervals are for control distribution in top and middle graphs and CTZ-treated distribution in bottom graph.
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Figure 3.9 Large quantities of CTZ remain trapped in medullary slice following wash with ACSF. Measurements made using liquid chromatography-tandem mass spectrometry. Slices treated in 1 of 3 ways: treated with CTZ (90 µM) for 1 hour, treated with CTZ for 1 hour and washed for 1 hour, treated with CTZ for 1 hour and washed for 6 hours (n=5 for all groups). No significant difference was found among the three groups using one-way ANOVA or difference tests between groups. Control slices (bathed in ACSF only) showed no CTZ and were not included in the figure. Individual symbols represent individual experiments. Lines represent group averages.
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4 PKG-DEPENDENT MECHANISMS MODULATE HYPOGLOSSAL MOTONEURONAL EXCITABILITY AND LONG-TERM FACILITATION
4.1 Introduction
Adaptive changes in breathing are essential to maintain blood-gas homeostasis. A
compromised ability to make such adaptations may underlie conditions such as
obstructive sleep apnea (OSA), where flaccidity of upper airway muscles, including the
genioglossus muscle of the tongue, during non-REM and REM sleep leads to collapse
and obstruction of the airway precipitating nocturnal hypoxia (Horner and Bradley,
2008). OSA affects a substantial fraction of the adult population and has severe health
consequences including neurodegeneration and increased incidence of cardiac failure and
stroke (Shamsuzzaman et al., 2003). Failure of a form of adaptive motoneuronal
plasticity known as long-term facilitation (LTF) may underlie OSA (Mahamed and
Mitchell, 2008). LTF is characterized in vivo by increased respiratory motoneuronal
output in response to episodic but not continuous bouts of hypoxia in adult (Baker and
Mitchell, 2000; Fuller et al., 2000; Mitchell et al., 2001) and neonatal (McKay et al.,
2004) rats, as well as in adult humans during sleep (Babcock and Badr, 1998; Shkoukani
et al., 2002). In vitro LTF (ivLTF) in XII MNs, which innervate the genioglossus muscle,
can be induced by episodic application of α-methyl-5HT (Bocchiaro and Feldman, 2004)
or phenylephrine (Neverova et al., 2007), the latter response being protein kinase C
(PKC), but not protein kinase A (PKA) dependent.
XII MNs receive excitatory (Funk et al., 1993) and inhibitory (Saywell and
Feldman, 2004) inputs from premotor neurons in respiratory rhythmic slices from
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neonatal rodents. Protein kinase activity contributes to neuronal plasticity at
glutamatergic and GABAergic synapses in XII MNs (Bocchiaro et al., 2003; Saywell and
Feldman, 2004; Neverova et al., 2007). Given the manifold roles of protein kinases in
modulating the excitability of XII MNs and the abundance of PKG in XII MNs (de Vente
et al., 2001), we investigated the role of PKG in XII motoneuronal plasticity. Elsewhere
PKG is involved in synaptic plasticity. For example, cerebellar postsynaptic long-term
depression (LTD; Levenes et al., 1998) or long-term potentiation (LTP; Lev-Ram et al.,
2002) is induced by stimulation of a cGMP-dependent pathway. cGMP-dependent
pathways are active postsynaptically during induction of hippocampal LTD (Wu et al.,
1998) and presynaptically during induction of hippocampal LTP (Arancio et al., 1996).
Based on its role in other neurons and evidence in vivo for a role of PKG in control of
genioglossus activity (Aoki et al., 2006), we hypothesized that PKG could affect
motoneuronal excitability and impact ivLTF.
Here we demonstrate that stimulating the cGMP-dependant pathway in XII MNs
depresses inspiratory drive but has an opposite effect on ivLTF. Stimulation of the PKG
pathway increases ivLTF relative to that induced by phenylephrine (PE) alone, yet it is
not sufficient on its own to induce facilitation. These data further illuminate the very
different and important roles played by protein kinases in modulating short-term
excitability and long-term plasticity in XII MNs.
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4.2 Methods
4.2.1 Slice preparation and ethical approval
All animal procedures were performed according to National Institutes of Health
guidelines and approved by the Office for the Protection of Research Subjects, University
of California Research Committee. In addition experiments comply with the policies and
regulations documented in Drummond (2009), which the authors have read. Experiments
were performed on neonatal Sprague-Dawley rats (P0-P4; n = 48; Charles River
Laboratories International Inc., Wilmington, MA) anesthetized in initial experiments by
hypothermia for a minimum of 3 minutes or in latter experiments with isoflurane
inhalation (5 ml for 15 minutes). Surgical anesthesia was assessed by the absence of limb
withdrawal to noxious pinch. Rats were then rapidly decerebrated.
A medullary slice was prepared that retains a sufficient proportion of the respiratory
network to generate a respiratory-related rhythm (Smith et al., 1991). Briefly, the
brainstem and upper cervical cord were isolated and bathed in artificial cerebrospinal
fluid (ACSF) comprised of (in mM): NaCl 128.0, KCl 3.0, CaCl2 1.5, MgCl2 1.0,
NaHCO3 23.5, NaH2PO4 0.5, D-glucose 30.0, pH 7.4, gassed with 95% O2 - 5% CO2 pH
7.4 at room temperature. The dura mater, superficial blood vessels and the cerebellum
were removed. The remaining brainstem was mounted on a chuck and serial transverse
sections (200-300 μm) were cut with a Vibratome VT 100 (Vibratome, Bannockburn, IL
USA) until the identifiable landmarks of compact formation of the nucleus ambiguus and
the inferior olive could be seen. Then a transverse 700 μm slice including the
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preBötzinger Complex (preBötC) and the XII motor nucleus and rootlets was cut. The
slice was transferred to a recording chamber and superfused (≥5 ml min 1) with ACSF
containing elevated K+ (9 mM) to sustain a stable respiratory-related output. The slice
was maintained at a constant temperature of 28°C.
4.2.2 XII nerve recording
A suction electrode was applied to the cut ends of the XII nerve rootlets and discharges
from the XII nerve recorded, amplified 1000 - 5000x and filtered at 1 kHz using a
conventional amplifier. Population discharges of the XII nerve rootlets were then
rectified and integrated using a Paynter filter (τ=100 ms). Signals were digitized and
stored using Digidata™ analog-to-digital converters and pClamp™ software (Molecular
Devices, Sunnyvale, CA USA). The rhythmic burst discharges of the XII nerve defined
the inspiratory period.
4.2.3 Voltage-clamp recording
XII MNs (classified according to the criteria of Funk et al. 1993) were visualized using
IR-DIC microscopy. Whole-cell voltage-clamp recordings (holding potential Vh = –70
mV) were made from XII MNs using electrodes pulled from borosilicate glass on an
electrode puller (Model P-97, Sutter Instrument Comp., Novato, CA USA), and filled
with patch solution comprised of (in mM); 120 K-gluconate, 11 glycol-bis-(b-
aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), 5 NaCl, 1 CaCl2, 10 HEPES, 2
ATP (Mg2+ salt), pH 7.3 adjusted with KOH (resistance 4-8 MΩ). To help confirm the
neurons as MNs, Lucifer yellow (Molecular Probes, OR USA) was included in the patch
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solution to intracellularly label the neurons. Neurons were subsequently examined under
an epifluorescent microscope (Axioskop, Carl Zeiss MicroImaging, Thornwood, NY
USA) to confirm their location, examine their morphology and identify axons projecting
in the XII nerve tract.
The patch-clamp electrode was advanced toward neurons under positive pressure.
Once the electrode tip approached a neuron, positive pressure was released and a
gigaohm seal formed by application of negative pressure. Neurons were then ruptured by
an additional brief application of negative pressure. Access resistance was monitored and
was always < 30 MΩ. Cells with large or unstable access resistances were rejected.
Intracellular signals were acquired using an Axopatch 1D™ amplifier filtered using -3 dB
Bessel filter and digitized at 10 kHz via a Digidata 1200™ interface with a software filter
(bandpass: 2 Hz – 5 kHz) in pClamp™ software (Molecular Devices, Sunnyvale, CA
USA). Junction potentials between bath solution and electrode were corrected for and
whole-cell capacitance was compensated.
4.2.4 Data analysis
Averages of integrated XII nerve activity and respiratory-related membrane currents were
constructed using the rising phase of the integrated XII nerve activity to trigger
acquisition of a 5 second epoch of membrane current and integrated XII nerve discharge.
Averages of 10 consecutive respiratory cycles were constructed. Recordings were
analyzed off-line using Clampex™ software (Molecular Devices, Sunnyvale, CA USA),
Data View™ (W. Hitler, www.st-andrews.ac.uk/~wjh/dataview/) and exported to
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Origin™ (OriginLab Corp., Northampton, MA USA). For ivLTF measurements, peak
amplitudes of all integrated XII nerve bursts occurring in 5-minute windows centered at
15, 30, 45, or 60 minutes post-ivLTF protocol were averaged and normalized relative to
the 30-minute pre-protocol control period.
Results are given as means ± S.D. T-tests were used to determine statistical
significance between paired groups of observations. P ≤ 0.05 was termed significant. For
experiments where repeated measures were involved, repeated measures ANOVA (one-
way or two-way mixed) was conducted first to determine a significant influence of the
factor in question (p ≤ 0.05). Then protected repeated measures t-tests were conducted to
compare paired groups of observations (Cohen and Lea, 2004). A natural logarithmic
transformation was used on normalized data used for statistical tests.
Power analysis was conducted by measuring the cumulative non-central F-distribution for
a given effect size that fell below the critical value of the central F-distribution that
represented a 5% chance of falsely rejecting a valid null hypothesis. Degrees of freedom
and variance estimates for the power analysis were taken from the parent ANOVA. SAS
(SAS Institute, Cary, NC USA) was used to calculate non-central and central F-
distributions.
4.2.5 Drugs and drug application
Drugs were dissolved in ACSF for bath application: 8-bromoguanosine-3’,5’-
cyclomonophosphate sodium salt (8-Br-cGMP; 100 µM; Sigma-Aldrich, St. Louis, MO
USA or Tocris Bioscience, Ellisville, MO USA), tetrodotoxin (TTX; 1 μM; Sigma),
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phenylephrine hydrochloride (PE; 10 µM; Sigma). For intracellular dialysis, the
membrane impermeable inhibitory peptide of PKG (PKGI; 100 μM; Sigma) was placed
in the patch solution. For application into the whole XII motor nucleus, 8-Br-cGMP (100
µM) was injected from pressure-ejection pipettes (5 psi, 5-6 µm tip diameter, ejection
duration 10 seconds) over XII MNs. Similarly, AMPA (10 μM; Sigma) was applied via
pressure-ejection pipettes (15-20 psi, tip 1-2 μm diameter, injection duration 100 ms,
interval of 10 s) positioned within 5 μm of the motoneuronal soma.
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4.3 Results
4.3.1 8-Br-cGMP depresses inspiratory drive currents.
To determine if activation of the cGMP-dependent pathway affected endogenous
inspiratory drive currents, we recorded from XII MNs in whole-cell patch-clamp mode.
We ejected 8-Br-cGMP (100 μM) focally over the MN. There was a significant effect of
the treatment (p<0.01; n = 6, one-way repeated measures ANOVA). In particular, 8-Br-
cGMP decreased inspiratory drive currents almost immediately to 66±11% of control
(p<0.01; n=6; repeated measures protected t-test comparing treatment with 8-Br-cGMP to
control). Currents returned to their control value 94±9% (n=6; ns; repeated measures
protected t-test comparing post-treatment to control) within 5 minutes after terminating
ejection (Figure 4.1). The return of the currents to baseline within 5 minutes indicated
that the effects were not long-lasting, and post-8-Br-cGMP currents were not monitored
for subsequent intracellular experiments.
4.3.2 8-Br-cGMP depresses exogenous AMPA-induced currents
To determine whether 8-Br-cGMP acted postsynaptically to depress inspiratory
drive currents, XII MNs were synaptically isolated by bath application of TTX (1 µM).
Excitatory inspiratory drive to XII MNs is mediated almost exclusively via AMPA
receptors in neonatal rodent in vitro preparations (Funk et al., 1993). Pressure ejection of
AMPA (10 µM) to excite postsynaptic AMPA receptors induced an inward current.
Currents induced by successive (100 ms) AMPA ejections at 10-second intervals (to
reduce the possibility of receptor desensitization) were constant for at least 60 minutes
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(see example control trace Figure 4.3B). Bath application of 8-Br-cGMP (100 μM)
decreased the amplitude of AMPA currents within minutes to 77±10% of control (n=7;
p<0.01; paired t-test; Figure 4.2).
4.3.3 Potentiation of endogenous excitatory drive by inhibition of PKG activity
The above results suggest that stimulation of PKG depresses inspiratory drive
currents. To determine whether PKG is endogenously active in XII MNs, XII MNs were
intracellularly dialyzed via the patch pipette with an inhibitory peptide for PKG (PKGI)
and Lucifer yellow (to determine post hoc if the MN had been successfully dialyzed).
After patch formation, the amplitude of the endogenous inspiratory currents progressively
increased to 144±17% relative to values preceding break-in (n=5; p<0.01; paired t-test);
this value peaked within 10-30 minutes (Figure 4.3), at which time the amplitude
remained stable for over an hour.
4.3.4 PKG-dependent mechanisms directly depress AMPA receptor currents
To exclude the possibility that 8-Br-cGMP directly affected AMPA receptors (Lei
et al., 2000), MNs were dialyzed with PKGI after bath application of TTX, and AMPA
was focally ejected. Once a steady state was reached and the currents fully potentiated, 8-
Br-cGMP was bath applied (100 μM). If 8-Br-cGMP exerted any direct (or indirect not
via PKG) effects on AMPA receptors we would have expected to see a further change in
the AMPA currents; no such effect was observed (Figure 4.4) as the currents remained
stable at 99±5% of control (n=4; not significant).
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4.3.5 Stimulation of PKG-dependent mechanisms facilitates ivLTF
Episodic application of PE induces long-lasting increases in the amplitude of XII
nerve activity and AMPA-induced motoneuronal currents. These increases are dependent
on PKC but not PKA (Neverova, et al., 2007). As stimulation of PKG pathways
decreased motoneuronal excitability, we hypothesized that stimulation with 8-Br-cGMP
during induction would decrease ivLTF magnitude.
To test this hypothesis, we superfused slices with 3 3-minute episodes of PE (10
µM) or PE and 8-Br-cGMP (100 µM) at 5-minute intervals, to determine if ivLTF was
affected. During the application of 8-Br-cGMP with PE, the level of tonicity appeared to
be greater than for PE alone (Figure 4.5A). For long-term effects, a two-way repeated
measures ANOVA with time as the repeated measure (4 time points: 15, 30, 45, and 60
minutes post-treatment) and treatment (PE v. PE and 8-Br-cGMP) as the second
independent variable, showed the effects of treatment (F(1,14) = 4.678, p < 0.05, n = 8
slices for each treatment) and time (F(3,42) = 22.81, p<0.001, n = 8 slices for each
treatment) were significant, while the interaction between time and treatment (F(3,42) =
0.126, p > 0.05, n = 8 slices for each treatment ) was not significant, revealing that 8-Br-
cGMP significantly affected facilitation of XII nerve activity.
The difference in facilitation at 60 minutes post-treatment between slices treated
with PE (120±15%) or PE with 8-Br-cGMP (136±25%) could have been due to an
independent effect of 8-Br-cGMP that linearly added to the effect of PE alone or an
interaction between the two drugs that led to facilitation that was greater than a linear
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sum of their independent effects. We applied 8-Br-cGMP, alone, to slices in the same
episodic protocol as before, 3 3-minute episodes with 5-minute intervals, with no
observable acute effect (Figure 4.5A). To consider the long-term effects of 8-Br-cGMP
independent of those of PE, we would have expected to see an effect size equal to the
difference between episodic application of PE or PE with 8-Br-cGMP, i.e., ~ 16% on
average. However, a one-way repeated measures ANOVA showed that there was a much
smaller, non-significant effect resulting from episodic application of 8-Br-cGMP (105 ±
12%; n.s.; n = 9 slices; repeated measures ANOVA; Figure 4.5C). We, therefore,
analyzed the power of our study, i.e., the probability of detecting an ~ 16% difference
given the number of slices (n=9) and the error variance. The power was > 95%, meaning
that there was a < 5% chance that this study missed an effect size large enough to account
for the difference in levels of facilitation seen between PE and PE with 8-Br-cGMP
(Figure 4.5D). Therefore, the more likely explanation was that an interaction between the
effects of these two drugs accounted for the difference in facilitation. Together these data
suggest that 8-Br-cGMP serves to enhance facilitation of XII nerve activity brought on by
episodic application of PE rather than acting independently.
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4.4 Discussion
This study demonstrates diverse roles for the cGMP/PKG signaling pathway in
controlling motoneuronal excitability and long-term plasticity. In the case of
motoneuronal excitability, focal application of 8-Br-cGMP, a PKG activator,
significantly decreased excitatory inspiratory drive currents. There was a significant
postsynaptic component to these effects that was shown by patching MNs that were
synaptically isolated with TTX and then focally applying AMPA to mimic endogenous
currents (Bocchiaro et al., 2003; Bocchiaro and Feldman, 2004; Neverova et al., 2007).
Activation of PKG induced a significant depression of AMPA receptor-mediated currents
in these neurons. Non-specific actions of PKG that might occur under current-clamp
conditions can be excluded, e.g., effects upon ion channels affecting changes in
membrane potential, because recordings were made under voltage-clamp conditions.
Furthermore, the role played by PKG in motoneuronal excitability is constitutive since
intracellular dialysis of PKGI, a membrane impermeable peptide that inhibits PKG
activity, potentiated inspiratory drive currents. Considered together, these data indicate
that PKG is constitutively active in rhythmically firing XII MNs, dampening their
excitability.
Intracellular cGMP can depress AMPA receptor currents and inhibit excitatory
postsynaptic currents in hippocampal neurons through a phosphorylation-independent
mechanism (Lei, et al. 2000). Thus, cGMP modulation of excitatory transmission may
involve a direct coupling to AMPA receptors. However, our data show that this action, if
present in XII MNs, does not contribute significantly to regulation of motoneuronal
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excitability. Specifically, bath application of 8-Br-cGMP had no effect on the amplitude
of AMPA currents in MNs dialyzed with intracellular PKGI (Figure 4.4). We should
have seen a reduction in these currents if cGMP played a phosphorylation-independent
role in depressing AMPA currents in our experiments, but we did not. Thus, we conclude
that the main action of 8-Br-cGMP is via PKG activation, which, in turn, reduces AMPA
receptor-mediated currents.
XII MNs, like neurons in many parts of the brain, exhibit both protein kinase-
dependent synaptic plasticity and excitability (Bocchiaro et al., 2003; Saywell and
Feldman, 2004; Neverova et al., 2007). For example, PKC activity is necessary for PE-
induced ivLTF but does not constitutively modulate AMPA receptor-mediated currents
(Neverova et al., 2007). In contrast, while PKA constitutively modulates excitatory and
inhibitory currents (Saywell and Feldman, 2004; Bocchiaro et al., 2003), it is not
necessary for ivLTF (Neverova et al., 2007).
PKG is unique compared to PKA and PKC, since its activity regulates both
motoneuronal excitability and long-term plasticity. While depressing excitatory currents,
it augments long-term motoneuronal facilitation when activated during induction of PE-
induced ivLTF. Specifically, simultaneous episodic application of 8-Br-cGMP and PE
enhanced ivLTF 60 minutes after induction relative to the use of PE alone.
A possible explanation for increased facilitation is the observation that the there is
an effect of PKG presynaptic to the MN that is in addition to any postsynaptic effects
within the MN. For example, PKG pathway stimulation may lower excitability of
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inhibitory neurons that synapse onto XII MNs (Saywell and Feldman, 2003), thereby
lowering endogenous inhibition resulting in increased MN activity. We do not favor this
explanation, because episodic 8-Br-cGMP application alone did not induce ivLTF.
We favor the explanation that PKG activation within XII MNs converges on and
modulates intracellular pathways leading to ivLTF, possibly similar to the interactions
observed between PKG and PKC during induction of ischemic preconditioning (Costa et
al., 2008). Ischemic preconditioning, first discovered in myocardium (Murry et al., 1986),
is a phenomenon whereby low doses of noxious insults like ischemia or hypoxia protect
cells from future, more severe insults and appears to be a general phenomenon, occurring
in the brain, lungs, liver, intestine, and kidney as well as the heart (Shpargel et al., 2008).
Ischemic preconditioning, like ivLTF, was first induced using short, episodic
events (5-minute episodes of ischemia separated by 5-minute intervals (Murry et al.,
1986)). Also, like ivLTF, ischemic preconditioning in the heart and the brain is PKC-
dependent (Ytrehus et al., 1994; Ping et al., 1997). During myocardial ischemic
preconditioning, one mechanism of PKC activation is PKG-dependent. Specifically,
activation of myocardial cell membrane bradykinin and opioid receptors triggers a
phosphatidylinositol 3-kinase/Akt/extracellular-signal regulated kinase/nitric oxide
synthase pathway that creates nitric oxide, which in turn activates guanylyl cyclase
producing cGMP and activating PKG. PKG then causes the opening of mitochondrial
ATP-sensitive K+ channels, inducing reactive oxygen species (ROS) formation that
activates PKC via redox signaling (Costa et al., 2008).
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Similar to ischemic preconditioning, pharmacological scavenging of ROS blocks
phrenic and XII respiratory long-term facilitation in the anesthetized, paralyzed, and
ventilated adult rats (MacFarlane and Mitchell, 2008). Acute intermittent hypoxia (AIH)
induced LTF may be related to ivLTF and shares many of the same signaling components
(Feldman et al., 2005). We speculate that 8-Br-cGMP causes the production of additional
ROS that augments PKC activity via PKG activation and opening of mitochondrial ATP-
sensitive K+ channels. This in turn increases ivLTF relative to that induced by PE alone.
The increased tonicity of XII nerve activity when 8-Br-cGMP was present in
addition to PE during the induction of ivLTF may be some indication of this interaction;
however, studies correlating levels of tonicity during induction and the amount of
facilitation long-term do not exist for ivLTF. For AIH-LTF in vivo, most recent meta-
analysis (Baker-Herman and Mitchell, 2008) indicates that the amplitude of phrenic
bursts during the hypoxic ventilatory response is a significant predictor of LTF. The
authors postulate two potential reasons for this correlation: (1) a stronger hypoxic
response leading to greater release of serotonin in the vicinity of phrenic motor neurons
or (2) a limited dynamic range of phrenic motor output which limits an increase in
phrenic burst amplitude during hypoxia may similarly limit phrenic increases during
LTF. The authors allow, however, that there may be no causal relationship between
phrenic burst amplitude during the hypoxic ventilatory response and phrenic amplitude at
long-term time points. Whether an increase in amplitude of phrenic nerve bursts seen
during induction of AIH-LTF in vivo is related to enhanced tonicity in XII nerve activity
in vitro is unknown. Similarly, whether the increased tonicity we observed during
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application of PE plus 8-Br-cGMP v. PE alone was a part of inducing the observed
increase in ivLTF or a separate short-term phenomenon remains unstudied.
In total, our study provides additional clarity to a complex picture of the roles played by
protein kinases in the control of motoneuronal excitability and long-term plasticity. We
propose that at the time of activation PKA and PKG exert constitutive antagonistic
effects upon inspiratory drive currents, modulating motoneuronal excitability on a state-
dependent, cycle-by-cycle timeframe. Additional PKG activation with 8-Br-cGMP,
occurring during ivLTF induction, augments long-term facilitation by increasing PKC
activity via ROS production and redox signaling mechanisms.
Interestingly, both ischemic preconditioning and LTF take advantage of what appear to
be a common set of intracellular signaling mechanisms to promote survival against
hypoxic/anoxic/ischemic events, which makes both phenomena of therapeutic as well
etiological interest. For example, LTF is a proposed compensatory mechanism for
obstructive sleep apnea (Mahamed and Mitchell, 2008). Thus, exploiting the cGMP/PKG
pathway is potentially of interest therapeutically in augmenting LTF responses in OSA
patients. Alternatively, pathophysiological changes in the cGMP/PKG pathway could
underlie decreased XII MN excitability seen in OSA, again, offering a point for
therapeutic intervention.
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Figure 4.1 Focal application of 8-Br-cGMP depresses inspiratory drive currents. (A) Endogenous glutamatergic inspiratory drive currents in a XII MN before (black) and ~1 min after (red) focal application of 100 μM 8-Br-cGMP. Traces are averages of 10 individual currents. (B) Peak endogenous current amplitude decreased following the focal application of 8-Br-cGMP (n=6; p<0.01 (**); repeated measures t-test) returning to control within 5 minutes.
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Figure 4.2 Postsynaptic exogenous AMPA-induced currents are depressed by 8-Br-cGMP. (A) Continuous recording showing effect of 100 μM 8-Br-cGMP bath application on exogenous AMPA-induced currents. Whole-cell currents were generated by focal application of 10 μM AMPA after bath application of 1 μM TTX. (B) AMPA-induced current before (black) and several minutes after (red) initiation of bath application of 8-Br-cGMP. AMPA ejection at arrow. (C) Peak AMPA-induced current amplitude following focal application of 8-Br-cGMP (n=7; p<0.01 (**); paired t-test).
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Figure 4.3 Potentiation of endogenous excitatory drive by inhibition of PKG activity. (A) Endogenous glutamatergic inspiratory drive current right after (black) and 30 minutes after (red) establishing whole-cell patch on a XII MN with electrode filled with 100 μM PKGI. (B) Time course of effect of dialysis with PKGI on endogenous motoneuronal currents. Current increased to its maximal value and stabilized within 15 minutes after break-in. Example control trace (green) demonstrates the stability typical of endogenous motoneuronal currents in untreated cells following break-in. (C) Increase in endogenous peak current amplitude 30 minutes after establishing whole-cell patch conditions (n=5; p<0.01 (**); paired t-test).
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Figure 4.4 PKG-dependent mechanisms directly depress AMPA receptor currents. (A) Single AMPA-induced currents in a MN dialyzed with PKGI for at least 30 minutes before (black) and several minutes after (red) 100 μM 8-Br-cGMP bath application. (B) No change in the peak AMPA-induced current amplitude following bath application of 8-Br-cGMP. The current amplitude was 99±5% (n=4; not significant).
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Figure 4.5 Activation of PKG facilitates induction of ivLTF (A) Integrated XII (∫XII) nerve activity in response to episodic PE application (10 µM; upper trace), 8-Br-cGMP (100 µM; middle trace) or PE and 8-Br-cGMP together (10 µM and 100 µM respectively, lower trace). (B) Co-application of 8-Br-cGMP with PE significantly increased ivLTF relative to PE alone (p<0.05 (*); two-way repeated measures ANOVA). ●PE alone, ■ co-application PE and 8-Br-cGMP. Horizontal bars show group mean responses. (C) Episodic application of 8-Br-cGMP alone did not significantly increase ∫XII nerve activity (105±12% at 60 minutes; n=9 slices; not significant; one-way repeated measures ANOVA). (D) Power analysis for the probability of failing to detect a long-term effect of episodically applied 8-Br-cGMP on XII nerve activity plotted as a function of the size of the effect.
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5 CRITICALLY SPACED EPISODIC STIMULATION ENHANCES BUT IS NOT NECESSARY FOR IN VITRO LONG-TERM FACILITATION
5.1 Introduction
Plasticity in intact animals or in vitro preparations depends on the specific
pattern of induction stimulus. In hippocampal slices, long-term potentiation (LTP) can
be induced by high frequency, 100 Hz tetanic stimulation (Bliss and Lømo, 1973), 100
Hz bursts at theta frequency, i.e. ~6-10 Hz (Larson et al., 1986), or primed burst
stimulation (Bliss and Collingridge, 1993). In contrast, continuous low frequency, i.e.,
1-5 Hz, stimulation leads to long-term depression (LTD) (Massey and Bashir, 2007). In
Aplysia, the duration of memory for sensitization of siphon withdrawal to tail shock is
dependent on the number and temporal spacing of tail-shock stimuli (Sutton et al.,
2002). Respiratory long-term facilitation (LTF), a progressive increase in respiratory
motor output in intact mammals in the minutes and hours following exposure to
hypoxia, requires that the stimulus be episodic rather than continuous (Baker and
Mitchell, 2000).
In vitro long-term facilitation (ivLTF) is an increase in XII motoneuronal
excitability in the respiratory rhythmically-active medullary slice preparation of
neonatal rats (Feldman et al., 2005) associated with an increase in XII motor nerve
output as well as AMPA-mediated motoneuronal currents that lasts longer than one
hour. The induction of ivLTF appears to be sensitive to the pattern of induction
stimulus. Three 3-minute episodes of slice superfusion with either 10 µM phenylephrine
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(PE), an α1-adrenoreceptor agonist, or 10 µM α-methyl-5-hydroxytryptamine (α-Me-
5HT), a 5-HT2 receptor agonist, spaced at 5-minute intervals induce this phenomenon,
whereas a single 9-minute episode of either drug does not (Bocchiaro and Feldman,
2004; Neverova et al., 2007). As with many protocols for inducing plasticity, the
parameters in these earlier studies of ivLTF were rather arbitrary. Once they were found
to work, whether they were optimal was not considered.
As the induction of ivLTF appears to require episodic stimuli, the underlying
signal transduction pathways must have dynamic components that are affected by the
stimulus pattern. Consequently, we systematically tested the protocol sensitivity of
ivLTF, determining which protocol parameters influenced the amount of facilitation and
investigated whether episodic stimulation is an absolute requirement for induction. We
also investigated if a combination of parameters exists that yields depression, instead of
facilitation.
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5.2 Methods
5.2.1 Slice preparation and systems electrophysiology
Details of this preparation and the administration of ivLTF experiments have
been previously described (Bocchiaro and Feldman, 2005; Neverova et al., 2007). All
experiments were performed in the standard medullary slice preparation that generates
endogenous respiratory activity (Smith et al., 1991). Animal procedures were performed
according to National Institute of Health guidelines and approved by the Office for the
Protection of Research Subjects, University of California Research Committee.
Neonatal Sprague-Dawley rats, postnatal days 0-4, were deeply anesthetized with
isoflurane. The level of anesthesia was assessed as sufficient by absence of a
withdrawal reflex to a noxious pinch of the hind paw, after which the rat was rapidly
decerebrated. The brainstem and cervical spinal cord were removed from the skull and
vertebrae with the aid of a dissection microscope, preserving the XII nerve rootlets, and
pinned ventral side up. The tissue was placed in the specimen vice of a Vibratome 1000
(Vibratome, St. Louis, MO) for sectioning. Using primarily the facial nucleus and
compact formation of the nucleus ambiguus as landmarks, one transverse slice (700-
1000 μm thick) containing the preBötzinger Complex, the XII motor nucleus and nerve
rootlets were cut.
Following cutting, the slice was transferred to a recording chamber (Warner
Instruments, LLC, Hamden, CT), where it was superfused at ≥ 3 ml/min with
oxygenated ACSF containing 9 mM K+ and maintained at 28°C. The slice was allowed
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to recover for at least 30 minutes before beginning the experimental control period.
Nerve activity was recorded from the cut ends of the XII nerve rootlets using a glass
suction electrode (A-M Systems, Sequim, WA), amplified 1,000 times, bandpass
filtered (1 Hz – 1 kHz), rectified, and integrated (Paynter Filter, τ = 100 ms). Raw and
integrated signals were sampled at 20 kHz using a Digidata (Molecular Devices,
Sunnyvale, CA) and recorded and stored on a computer hard drive (Clampex, Molecular
Devices, Sunnyvale, CA) for off-line processing (Clampfit, Molecular Devices,
Sunnyvale, CA).
5.2.2 Protocol and parameter space
For experiments described throughout, PE was bath applied according the
concentrations, number of episodes, and durations of episodes and intervals shown in
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. Each combination is referred to as a protocol. XII activity was recorded ≥ 65 minutes
following the end of each protocol to allow for a 5-minute window of averaged activity
at 60 minutes and compared to the activity during the 30 minutes immediately prior to
starting the protocol, referred to as the control period. A protocol was not started unless
XII activity had stable, less than 10% variation in amplitude in a non-ramping manner,
during the control period.
The approach for designing the parameter space was adapted from response
surface methodology (RSM), a chemical and manufacturing process optimization
technique (Myers and Montgomery, 2002). RSM combines parameter-space design
rules for varying experimental parameters with multiple linear regression (MLR)
analysis of the resulting experimental data to fit a mathematical model, known as a
response surface, which describes how variations in parameter values affect the
outcome being studied. This approach has two advantages. First, it reduces the number
of required repetitions of a unique set of parameter values, even in appropriate
circumstances, n=1 for a given protocol, and, second, it allows for the assessment of
interactions between parameters (Myers and Montgomery, 2002).
Each protocol was comprised of a unique set of four parameters: drug
concentration, number of episodes of drug application, drug-application episode
duration, and interdrug interval duration (Figure 5.1A). The protocol of Neverova et al.
(2007), referred to as the control protocol, served as a departure point for choosing
experimental values of each parameter, which were set to be linearly equidistant from
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the values used in the control protocol. The following rationales were used for picking
the limits of these four parameters. Number of episodes was chosen so that the
minimum would be one. In this way, we could further investigate the requirement for
episodicity, using different episode durations than had been used in the previous studies.
In order for the control protocol parameter value of 3 to remain linearly centered, we
chose the maximum number of episodes to be 5. In choosing the episode duration, we
used experience from our previous studies (Bocchiaro and Feldman, 2004; Neverova et
al., 2007) that a 9-minute stimulation did not yield facilitation. We hypothesized that the
length of the individual episode of drug application, 3 minutes in previous studies,
might be more important than keeping the total duration of stimulation constant as had
been done in the previous studies, i.e., 3 by 3 minutes and 1 by 9 minutes both provide 9
minutes of total stimulation. We chose the minimum duration of drug application that
provided a reliable slice response, 1:45, and set 4:15 to be equidistant to the other side
of 3 minutes, providing a range of ± 40%. A ± 40% range was also chosen for the drug
concentration, since concentrations higher than 14 µM yielded excessive amounts of
tonicity that made distinguishing phasic respiratory activity impossible and caused
concern that we might induce excitotoxicity.
A total of 12 unique parameter combinations resulted (Table 5.1). Experiments
with a single drug-application episode did not, by definition, have interdrug intervals.
The order in which the experiments were run was randomized to prevent bias. Each
slice was exposed only to a single protocol. In addition, experiments that used the
control protocol were scattered amongst the other experiments to assure that ivLTF
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induction occurred for this protocol as expected. The data from the control protocol
experiments were also used to aid in evaluating the parameter space for non-linear
relationships between the level of ivLTF and the parameters considered. Rat age and
medullary slice thickness were not varied systematically, but these parameters were
recorded and included in our analysis, as stage of development and anatomical
variations can affect biological phenomena, especially in neonates.
For this study, the level of facilitation measured in a given post-protocol
timeframe, e.g., facilitation 60 minutes post protocol, was the outcome we considered.
MLR combined with analysis of variance (ANOVA) determined which parameters had
a significant effect on ivLTF.
5.2.3 Data analysis
5.2.3.1 Raw data reduction
Raw data files of integrated nerve burst activity were decimated to improve
processing speed. For each integrated XII nerve burst (∫XIIn), the peak value was
measured. For each experiment, an average of the peaks was taken over 5-minute
windows from 5 – 60 minutes following completion of the selected PE protocol and
normalized to the average of the peaks during the 30-minute control period.
5.2.3.2 MLR
Multiple linear regression was used to assess how multiple parameters
simultaneously affected the outcome of the experiment. Specifically, coefficients were
fit to equations whose variables were selected subsets of the parameters of interest in
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our study. Each equation contained a unique combination of parameters and was
referred to as a model. Parameter coefficients were fit using method of least squares,
which minimized the sum square of the errors between measured data and model
predictions. The R2 value, which measures how much of the variance in data was
explained by the model, was used as the primary metric for grading how well the model
fit the data, where 0≤ R2≤ 1.
ANOVA determined whether whole models, or their individual parameters,
contributed in a statistically significant way to fitting the data. A combination of
parameters was determined to have produced a valid description of the data if p≤0.05
for the global F-test, which determines whether the parameters used in a regression
provide a curve that is significantly different from a horizontal straight line or surface
that is equal the average value of the entire data set.
Finding a combination of parameters that provided a valid model did not mean
that all of the parameters were useful in explaining the data. Therefore, unnecessary
parameters, i.e., ones not contributing significantly to the fit, were eliminated using a
two-stage process. First, if the individual F-test for that parameter produced a p≤0.05,
the parameter was retained. An individual F-test determined whether the R2 produced by
a model containing that parameter was significantly larger than the fit produced by the
same model without that parameter. Second, variables with p>0.05 for their individual
F-test were grouped into all possible combinations of two or more parameters. Partial F-
tests determined whether the R2 of the model after removal of a given combination of
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parameters was significantly (p≤0.05) lower than before their removal. If a combination
of parameters that failed at the individual F-test criterion did, together, significantly
contribute to the R2 as a group, they were retained in the final model description. For
models with even a moderate number of parameters, e.g., 3 or 4, the entire process
could require many iterations of these individual and partial F-tests to arrive at a final
set of valid parameters. If there were multiple valid models for describing ivLTF, they
were ranked on the basis of best fit, i.e., the statistically valid model with largest R2
value was chosen.
5.2.3.3 “Lack-of-fit” test
When multiple experiments were conducted for a given combination of
parameter values, a lack-of-fit test was run (Myers and Montgomery, 2002). This test
measures whether the mean square error due to the lack of fit of the regression to the
data is equal to the model-independent estimate of the variance of the experimental
noise resulting from measurement error and experimental and preparation variability.
Failure of this test indicates an overlooked variable or curvature of the response surface
in the parameter-space. Specifically, this test computes an F-statistic of the ratio of the
mean square errors of the lack-of-fit and pure errors. Computation of the pure error
serves as an estimate of the model-independent measure of the experimental error; it
was determined by summing the squares of the difference between the level of
facilitation for each experimental replication for a given protocol and the average of all
the trials for that replicated protocol over all replicated protocols divided by the
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appropriate degrees of freedom. While the lack of fit error is a weighted sum of squares
of the difference between the value predicted by the regression for that combination of
protocol parameters and the true average of the experiments for that particular set of
parameter values divided by the appropriate degrees of freedom, where the weighting is
by the number of experiments run at a given combination of parameters. If the F-
statistic is significantly greater than the critical value determined by the confidence
interval (p<0.05), then the regression is considered to have failed the lack-of-fit test.
For a detailed explanation of regression techniques, including fitting curves with least
squares method and testing model validity with global, individual, and partial F-tests,
and lack-of-fit test, see Kutner et al. (2005). Mathematical definitions for these
statistical methods are at the end of this section.
5.2.3.4 Repeated measures ANOVA
For investigations of whether a single combination of values for the protocol
parameters yielded statistically significant ivLTF, repeated measures ANOVA with
Dunnett’s analysis for multiple comparison was performed. Specifically, ∫XIIn at points
10, 20, 30, 40, 50, and 60 minutes post protocol were compared to the average of the
30-minute pre-protocol period.
5.2.3.5 Data analysis software
∫XIIn was analyzed for peak values and binned according to time in Clampfit
(Molecular Devices, Sunnyvale, CA). Averages and their normalization to control
period were performed using Excel (Microsoft, Redmond, WA). Repeated measures
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ANOVA and regression analyses were performed using SAS (SAS Institute, Inc., Cary,
NC).
5.2.4 Drugs and solutions
ACSF for bathing slices contained the following chemical concentrations (in mM):
NaCl 128, KCl 9, CaCl2 1.5, MgCl2 1, NaHCO3 23.5, NaH2PO4 0.5, D-glucose 30,
pH 7.4, gassed with 95% O2 - 5% CO2. Phenylephrine hydrochloride (PE) (Sigma, St.
Louis, MO) was dissolved in ACSF in a 1000x stock solution and frozen until use in
experiments, where it was bath applied in concentrations varying from 6 – 14 μM.
5.2.5 Statistical definitions
5.2.5.1 Variables & subscripts
yij is the experimental measurement for the ith time that the jth combination of
parameters, i.e., the jth protocol, was run.
ŷij is the regression estimate for the ith time that the jth protocol was run
ŷi,j = ŷj = b0 + b1X1j + b2X2j+ … + bp-1Xp-1,j (5.1),
where p is number of regression coefficients, bk is the kth regression coefficient
associated with the Xkth parameter. i,j ≥ 1. p ≥ 0.
n is the total number of experiments
n = Σcnj (5.2),
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where nj is the number of times that the jth protocol was run, and c is the total number of
unique protocols.
μY is the average of all experimental measurements for all replications of all
protocols
μY = (ΣiΣjyij) / n = Σjnjμyj (5.3)
μyj= (Σiyij)/nj (5.4).
5.2.5.2 Partitioning of sum square errors
SSTO = SSR + SSE (5.5),
where SSTO is the total sum of squares, SSR is the regression sum of squares, and SSE
is the error sum of squares.
SSTO = ΣiΣj(yij - μY)2 (5.6)
SSR = ΣiΣj(ŷij - μY)2 (5.7)
SSE = ΣiΣj(yij - ŷij)2 (5.8)
SSE = SSLF + SSPE (5.9),
where SSLF is the lack-of-fit sum of squares and SSPE pure error sum of squares
SSLF = ΣiΣj(μyj - ŷij)2 (5.10)
SSPE = ΣiΣj(yij - μyj)2 (5.11).
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5.2.5.3 Mean squares
MSR = SSR / (p - 1) (5.12)
MSE = SSE / (n - p) (5.13)
MSLF = SSLF / (c – p) (5.14)
MSPE = SSPE / (n – c) (5.15),
where MSR is the regression mean square, MSE is the error mean square, MSLF is the
lack-of-fit mean square, and MSPE is the pure error mean square.
5.2.5.4 Coefficient of multiple determination
R2 = SSR / SSTO (5.16)
5.2.5.5 Definition of F-tests
5.2.5.5.1 Global F-test
Null Hypothesis: b1 = b2 = … = bp-1 = 0
Alternative: At least one coefficient of the regression is not zero
F* = MSR / MSE (5.17),
where F* ~ F(0.95, p - 1, n - p), and F(confidence interval, ν1, ν2) is the F distribution
with ν1, ν2 degrees of freedom and confidence interval of 0.95. If F* ≥ F(0.95, p - 1, n -
p), p ≤ 0.05 that there are no valid parameters within the proposed regression.
5.2.5.5.2 Individual Parameter F-test
Null Hypothesis: bk = 0, where 1 ≤ k ≤ p
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Alternative: bk ≠ 0
F* = MSR(Xk | X1, X2, … ,Xk-1, Xk+1 … Xp-1) / MSE (5.18),
where F* ~ F(0.95, 1, n-p), and MSR (Xk-1 | … ) is the regression mean square for the
regression including all parameters except the kth parameter. If F* ≥ F(0.95, 1, n – p), p
≤ 0.05 that kth parameter is not valid.
5.2.5.5.3 Partial (group of parameters) F-test
Null Hypothesis: the set of coefficients for m parameters, {b}m, = 0, where the
coefficients are any m coefficients from the coefficients b1, … ,bp-1 associated with the
parameters.
Alternative: At least 1 parameter of {b}m ≠ 0.
F* = MSR ({X}m | X1, …, Xp-1) / MSE (5.19),
where F*~ (0.95, m, n-p), and MSR ({X}m | … ) is the regression mean square for the
regression including all parameters except the excluded parameters in the m-parameter
set. If F* ≤ F(0.95, m, n-p), p ≤ 0.05 that none of the parameters are valid.
5.2.5.5.4 “Lack-of-fit” F-test
Null Hypothesis: E(MSLF) = E(MSPE)
Alternative: E(MSLF) > E(MSPE)
F* = MSLF/MSPE (5.20),
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where F* ~ (0.95, c – p, n – c), and E(MSLF) is the expected value of the lack-of-fit
mean square. If F* ≥ F(0.95, c – p, n – c), p ≤ 0.05 that the model is not linear and the
mean square of the lack-of-fit error is not equivalent to the mean square of the pure
error.
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5.3 Results
5.3.1 ivLTF is parameter sensitive
The group average for peak ∫XIIn amplitude at 60 minutes post protocol taken
from all experiments (n=28) for the entire range of protocols used (n=13) was 112% ±
21% of control (range 73%-154%; n=28; Figure 5.1B). Based on the criterion that ∫XIIn
was neither facilitated nor depressed if 95%<∫XIIn<105%, 68% of slices facilitated
(122% ± 16%, n=19) and 18% depressed (83% ± 8%, n=5).
To determine, then, which of the parameters under control of the experimental
regimen (Table 5.1) were responsible for the measured variations in facilitation, ∫XIIn at
60 minutes post protocol was regressed against all 1-, 2-, 3-, 4-, and 5-parameter
combinations of the following parameters: number of episodes of drug-application, drug
concentration, drug application episode duration, animal age, and slice thickness.
Variation in age had no significant effect (p > 0.05), but slice thickness did (Figure 5.2),
explaining 30% of the variation (R2 = 0.30, p<0.01, n = 24), including the depressed
cases. The addition of the duration of drug application, alone, or in combination with
drug concentration in the regression improved the fit (Table 5.2). In both cases,
however, individual F-tests and partial F-tests eliminating both episode duration and
drug concentration had p>0.05, indicating that these parameters did not aid significantly
in explaining the variations in ∫XIIn.
The lack-of-fit test for the slice thickness model indicated, however, that either
certain variables had been missed or there was curvature to the response surface
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(p<0.01, n=24). This outcome was expected, considering that none of the parameters
that had been varied for each of the experimental protocols was included in the model.
We hypothesized that this might be due to our inclusion of multiple episode and single
episode cases within the same parameter space. We thus looked at single episode and
multiple episode cases separately.
5.3.2 Episodic stimulation is not required for ivLTF
When we looked at the single episode cases more closely, we observed several
cases of single-episode ivLTF, indicating that multiple episodes of stimulation were not
essential for inducing ivLTF. This is in contrast to the conclusion from our previous
studies (Bocchiaro and Feldman, 2004; Neverova et al., 2007). The single-episode case
that yielded the most facilitation was a single 14 µM PE application that was 4:15 in
duration (125%±14%, p<0.05, n=4; Figure 5.3A-B). Although episodicity was not a
requirement for ivLTF, we still wanted to known whether multiple episodes of drug
application could lead to greater facilitation. Regressing ∫XIIn at 60 minutes post
protocol for cases that showed facilitation against the number of drug-application
episodes demonstrated that more episodes predicted greater facilitation (R2 = 0.31,
p<0.05, n=19; Figure 5.3C).
5.3.3 Interdrug interval influences ivLTF
Since episodicity positively influenced facilitation, we considered whether the
duration of the interval between drug episodes affected ivLTF. For the experimental
cases using 5 episodes of drug application (protocols 1-8 from Table 5.1, n = 16), drug-
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application episode duration and interdrug interval explained 36% of the variation
(R2=0.36, p=0.05, n=16; Table 5.3). Was the optimal time interval between drug
applications the same for all durations of drug application or was the optimal interdrug
interval dependent on the duration of the drug application? To test this, we computed a
new parameter, the ratio of the drug episode duration to the interdrug interval duration.
In the revised model, we replaced the parameter for interdrug interval with this new
parameter. This made interdrug interval a variable that was dependent on the duration of
drug application rather than being an independent variable as it had been. Doing this
improved the fit (R2=0.40, p<0.05, n=16; Table 5.3), suggesting that the interval
duration should be varied in such a way as to keep its length proportional to the duration
of drug application. Figure 5.4 illustrates the interplay between episode duration and
interval duration. The best combination of parameters, then, for explaining the outcome
of protocols with more than one episode of drug application was comprised of episode
duration, the ratio of episode duration to the interval duration, and slice thickness. It
explained 65% of the total variation in ivLTF (R2=0.65, p<0.01, n=16; Table 5.3). This
model passed the lack-of-fit test (p=0.30), indicating that there were not any missing
variables or any significant curvature to the response surface.
5.3.4 Is there a set of optimal parameter values?
A set of parameter values within the range considered that yields an optimal
amount of ivLTF would be indicated by curvature in the response surface, e.g., an
upside down parabola-like shape. Even though, the lack-of-fit test for the model of the
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multi-episode data did not indicate curvature, additional steps were taken to confirm
this. Specifically, the control protocol experiments (3, 3-minute 10 µM PE applications
at 5-minute intervals; n=4) were included in the regression analysis. This provided a
third value for each of the parameters through which curved surfaces explicitly could be
fit. The impact of adding quadratic parameter terms on R2 was considered. However,
there were no quadratic terms that significantly improved R2. The best parameter
combinations for describing the entire data set and the multi-episode data subset with
and without including the control protocol data did not differ. Furthermore, there was
only a modest difference in the values of the parameter coefficients and R2 between the
regressions with and without the control data. In addition, a lack-of-fit test run on the
model for the multi-episode data set did not indicate any missed parameters or curvature
in the response surface (p=0.22, n=20). Because the fits did not change and passed the
lack-of-fit test, these results indicated the absence of a significant nonlinear relationship
between the amount of facilitation and any of the experimental parameters (Table 5.4).
Together these results suggest that there are no optimal values in the parameter space
studied here. In addition, these data support the conclusion that the failure of the lack-
of-fit test for the model containing single and multi-episode data was due to including
both types of data in the model, likely due to a failure to consider interdrug interval for
the multiple episode cases.
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5.3.5 The parameters explaining ivLTF variability are stable over time
Plasticity varies over multiple timeframes that are differentially sensitive to induction-
protocol pattern (Sutton et al., 2002). ∫XIIn at 15, 30, and 45 minutes post protocol was
regressed against the preferred parameter set for the 60-minute time point to see
whether ∫XIIn for different time points was described equally well using the same set of
parameters. For the full data set and multi-episode data subset, the best combination of
parameters remained the same: drug-application episode, ratio of the durations of drug-
application episode to interdrug interval, and slice thickness. Both the parameter
coefficients and R2 values showed little variation for 30-, 45-, 60-minute time points
(Table 5.5) with some modest degradation in the quality of fit for the 15-minute time
point (data not shown).
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5.4 Discussion
These results illustrate that ivLTF can occur for a broad range of induction
protocols. Within the parameter space studied, there was no optimal combination of
parameter values that maximized facilitation or caused depression. Maxima and minima
were found at the edges of the parameter space, while no curvature existed in the
preferred response surface. This indicates that if any optimum exists, it is outside the
space considered.
Similar results are seen in the phenomenon of respiratory LTF (Mahamed and
Mitchell, 2007). In anesthetized, vagotomized, paralyzed, and ventilated rats 3, 5-
minute episodes of hypoxia spaced at 5-minute intervals (Baker-Herman and Mitchell,
2002), 3, 3-minute episodes of hypoxia spaced at 5-minute intervals (Baker and
Mitchell, 2000), 3, 5-minute episodes of hypoxia spaced at 10-minute intervals
(Hayashi et al., 1993), and 3 or 6 ventilator-induced apneas of <25 seconds at 5-minute
intervals (Mahamed and Mitchell, 2008) all induce phrenic and, where tested, XII
respiratory LTF.
Our data show that no combination of parameters repeatably yielded depression
once slice thickness was taken into account. MNs likely have mechanisms for
depression as well as facilitation to prevent saturation of excitability, much like LTP
and LTD are counterparts in other areas of the nervous system. Nevertheless, parameter
values that consistently induced depression were not found. Possibly the protocols
leading to depression reside in an entirely different regime of the parameter space than
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studied here, or they may be state- or activity-dependent. Alternatively, protocols
leading to depression may rely on different neurotransmitter-mediated signal
transduction cascades than those controlling facilitation.
Perhaps of most interest is that multiple episodes of drug application are not
necessary for the induction of ivLTF. ivLTF lasting for ≥1 hour could be induced using
a single application of PE. This conclusion contrasts with results from previous studies
of PE- and α-Me-5HT-induced ivLTF (Bocchiaro and Feldman, 2004; Neverova et al.,
2007) in which 3, 3-minute episodes of drug application spaced at 5-minute interdrug
intervals yielded facilitation, but a single episode of 9-minute drug application did not.
Similarly, respiratory LTF requires episodic rather than continuous hypoxia (Baker and
Mitchell, 2000).
This contradiction, however, may be more apparent than real. First, the drug-
application episodes in this study were ≤4:15, less than half of the fairly long bolus (9
minutes) previously used to test for single-episode facilitation in the studies of
Bocchiaro and Feldman (2004) and Neverova et al. (2007); this lends credence to the
idea that episode duration is more important than the total amount of stimulation. As a
matter of conjecture, past a certain point, prolonged exposure may initiate intracellular
cascades that attenuate ivLTF or even induce excitotoxicity.
In addition, in the neonatal rat brainstem-spinal cord preparation that, while both
cervical and thoracic motor roots exhibited facilitation after episodic stimulation with 5-
HT, thoracic motor roots did not require episodicity of 5-HT application to exhibit
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facilitation (Lovett-Barr et al., 2006). Taking together these results along with those of
our study suggest that different motor pools exhibit facilitation when exposed to unique
stimuli patterns as well as to a common episodic stimulus.
Despite concluding that ivLTF does not require multiple episodes of drug
application, we found that more episodes yielded more facilitation. Additionally, the
durations of these drug-application episodes and interdrug intervals are key
determinants of resulting facilitation: too short an interdrug interval reduces the amount
of facilitation. Furthermore, the optimal interdrug interval is a function of episode
duration. Thus, critically spaced episodicity enhances the induction of ivLTF.
The observation that thicker slices yield less facilitation may be due to either
drug efficacy or medullary anatomy. Drugs will take more time to diffuse to the core of
thicker slices. Yet, concentration never proved to be a significant parameter, and no
other parameter correlated with concentration in such a way as to suggest that it played
a role through a surrogate parameter, for example, episode duration. In such a case, one
would expect that before adjusting for thickness, episode duration would yield a
significant description of the data and after adjusting for thickness it would not. This,
however, was not the case in the global model describing single and multiple episodes
ivLTF, where episode duration did not contribute to a significant model before or after
adjusting for thickness, and the opposite of what occurred in the multi-episode model,
where episode duration remained as part of the model even after adjusting for thickness,
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presumably because of its relationship with interval duration in predicting the amount of
facilitation.
Lastly, since the difference in ivLTF between the thickest slice and the thinnest
slice was approximately 40% and the difference in concentrations was more than double
between some protocols, based on a linear diffusion gradient, it seems that if a critical
threshold existed for the proper concentration at the center of the slice, the parameters
selected and analysis undertaken should have captured this effect.
Alternatively, thicker slices might lead to some variation in the signaling
mechanisms underlying ivLTF induction. The experiments exhibiting the greatest
depression were in the thickest slices, and a change in the amount of reactive oxygen
species (ROS), due to a reduction in oxygen concentration at the center of thicker slices,
might be responsible. ROS are a necessary component of respiratory LTF (MacFarlane
and Mitchell, 2008) and may also play a role in ivLTF (Chapter 4 of this dissertation).
ivLTF is postulated to be a neural correlate of respiratory LTF (Feldman et al.,
2005) possibly related to diseases like obstructive sleep apnea (OSA), which afflicts 2-
4% of the adult US population (Young et al., 1993). This study describes the sensitivity
of ivLTF to variation in induction-protocol parameters. Within the parameter space
studied, there was no optimal combination of parameter values that maximized
facilitation, providing evidence that ivLTF is broadly tuned, perhaps making it
responsive to a broad range of physiological challenges, as might occur in intact
mammals during sleep.
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Table 5.1 Experimental parameter values
Protocol Number
of Episodes
Episode Duration (min:sec)
Interval Duration (min:sec)
Concen-tration (µM)
1 5 1:45 3:00 6
2 5 1:45 3:00 14
3 5 1:45 7:00 6
4 5 1:45 7:00 14
5 5 4:15 3:00 6
6 5 4:15 3:00 14
7 5 4:15 7:00 6
8 5 4:15 7:00 14
9 1 1:45 - - 6
10 1 1:45 - - 14
11 1 4:15 - - 6
12 1 4:15 - - 14
Control 3 3:00 5:00 10
"Episode" refers to one application of PE. "Interval" refers to the time between two drug episodes. Experimental cases with one episode do not have interdrug intervals. "Concentration" refers to the concentration of applied PE during each episode.
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Table 5.2 Valid models fit for full data set
Model Parameter Coefficient Individual
F-test R2 Global F-test
Intercept 213.4% <0.01 1 Slice
Thickness -13.2% / 100µm 0.01
0.30 <0.01
Intercept 198.7% <0.01
Slice Thickness
-12.6% / 100µm 0.01 2
Episode Duration 3.2% / min 0.31
0.33 0.01
Intercept 204.3% <0.01
Slice Thickness
-12.7% / 100µm 0.01
Episode Duration 3.2% / min 0.33
3
Concentration -0.5% / µM 0.64
0.34 0.04
The full data set (n=24) includes all data except for control experiments. "Individual F-test" provides the p-value for the individual F-test of that parameter. Similarly, "global F-test" provides the p-value for the global F-test. Both tests are defined in the methods. Fits are to ∫XIIn 60 minutes post protocol. A model is considered valid if the global F-test gives a p≤0.05.
Table 5.3 Valid models fit for multiple episode data set
Model Parameter Coeffi-cient
Individual F-test R2
Global F-test
Intercept 65.2% <0.01
Episode Duration 7.6% / min 0.10 4
Interval Duration 5.5% / min 0.06
0.36 0.05
Intercept 92.7% <0.01
Episode Duration
16.8% / min 0.01 5
Ep. Dur / Int. Dur -38.7% 0.04
0.40 0.04
Intercept 228.4% <0.01
Episode Duration
16.0% / min <0.01
Ep. Dur / Int. Dur -41.1% 0.01
6
Slice Thickness
-17.8% / 100 µm 0.01
0.65 <0.01
The multiple episode data set (n=16) includes the data for all 5-episode experiments. "Individual F-test" provides the p-value for the individual F-test of that parameter. Similarly, "global F-test" provides the p-value for the global F-test. Both tests are defined in the methods. Fits are to ∫XIIn 60 minutes post protocol. A model is considered valid if the global F-test gives a p≤0.05.
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Table 5.4 Variation in model fit for ∫XIIn at 60 minutes post protocol with and without inclusion of control data
Model Parameter
Coefficient (w/ Control
Data)
Coefficient (no Control
Data) Coefficient Difference
R2 (w/Control
Data)
R2 (no Control Data)
Fit Difference
Intercept 206.1% 213.4% 3.4% Full Data Set
(n=28/24) Slice Thickness
-12.2% / 100 µm
-13.2% / 100 µm 8.6%
0.25 0.30 15.9%
Intercept 204.7% 228.4% 10.4%
Episode Duration 16.0% / min 16.0% / min 1.1%
Ep. Dur / Int. Dur -40.8% -41.1% 0.5%
Multi-episode Data Set
(n=20/16) Slice
Thickness -14.7% / 100 µm
-17.8% / 100 µm 21.3%
0.57 0.65 13.1%
The control data adds four data points to each data set (28 v. 24 and 20 v. 16). The difference computed as the magnitude of the percentage difference of the minimum in absolute value relative to the maximum in absolute value for coefficient and R2 values.
Table 5.5 Variation of model parameters with time
Model Parameter Minimum Coefficient
Maximum Coefficient
Coefficient Difference
Minimum R2
Maximum R2
Fit Difference
Intercept 206.1% 232.8% 11.5% Full Data Set (n=24) Slice
Thickness -14.9% / 100 µm
-12.2% / 100 µm 18.0%
0.25 0.31 18.7%
Intercept 228.4% 248.1% 7.9%
Episode Duration 14.7% / min 17.3% / min 14.8%
Ep. Dur / Int. Dur -41.1% -32.8% 20.0%
Multi-episode Data Set (n=16)
Slice Thickness
-20.1% / 100 µm
-17.8% / 100 µm 11.6%
0.63 0.66 4.3%
Minimum and maximum refer to the minimum and maximum values for a given coefficient or R2 value across the model of ∫XIIn facilitation fit for each of three time points 30, 45, and 60 minutes post protocol. The difference in coefficient or fit is calculated as the magnitude of the percentage difference of the minimum in absolute value relative to the maximum in absolute value. Data sets used do not include data from control experiments.
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Figure 5.1 Summary of experimental data. (A) ∫XIIn at the control set of parameter values (PE concentration: 10 µM; number of episodes: 3; episode duration: 3 minutes; interdrug interval: 5 minutes). (B) ∫XIIn at 60 minutes post protocol for each replication of the 13 experimental cases (Table 5.1). 95%<∫XIIn<105% (gray band) is considered to be neither facilitated nor depressed. 11 out of 12 cases (◆) were repeated twice (■), with one being repeated three times (▲). The control case was repeated four times (open circles).
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Figure 5.2 Thicker slices show less facilitation. Regression line fitting ∫XIIn at 60 minutes post protocol as a function of slice thickness for all data points including control experiments shown (n=28). Slices with ∫XIIn<0.95 were considered to be depressed (■).
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Figure 5.3 A single episode of PE can induce ivLTF. (A) Sample trace showing ∫XIIn facilitation after application of 14µM PE for 4:15. (B) Group data for multiple replications of drug protocol shown in A. Facilitation was125 ± 14% for ∫XIIn at 60 minutes post protocol (n = 4, p < 0.05 (*); repeated measures ANOVA with Dunnett’s test for multiple comparison). (C) Facilitation of ∫XIIn at 60 minutes is positively influenced by increasing number of drug-application episodes. Regression line and formula for the 19 data points that showed facilitation at 60 minutes.
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Figure 5.4 Changing interval duration relative to episode duration influenced ivLTF. (A) 5-episode experiment, where both the episode duration and interval duration were maximized in the parameter space, 4:15 and 7:00 respectively. (B) Ratio of the durations of drug-application episode to inter-drug interval increased from 0.61 to 1.42 (interval duration of 3:00 for episode duration of 4:15), but ∫XIIn at 60 minutes decreased from 140% to 105%. (C) Drug-application episode duration was decreased from 4:15 to 1:45, while the inter-drug interval was held constant at 3:00, ∫XIIn recovered to 113% (ratio of the durations of drug-application episode to inter-drug interval reduced from 1.42 in B to 0.58 in C. The concentration of PE was the same, 14µM, for experiments in A-C. (D) Regression of group data for 5-episode experiments (n=16) showing the residual difference in ∫XIIn at 60 minutes, after adjusting for duration of drug-application episode and slice thickness, as a function of the ratio of the durations of drug-application episode and inter-drug interval. Facilitation decreases as the length of the duration of drug application increases relative to the inter-drug interval.
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6 SUMMARY OF THE DISSERTATION
Adaptive changes in breathing are essential to maintain blood-gas homeostasis. A
compromised ability to make such adaptations may underlie conditions such as
obstructive sleep apnea (OSA), where flaccidity of upper airway muscles, including the
genioglossus muscle of the tongue, induced by loss of muscle tone during sleep leads to
collapse and obstruction. Such collapses precipitate repetitive cycles of hypoxia followed
by sympathetic nervous system activation, blood pressure surges, and finally arousal.
OSA affects a substantial fraction of the adult population and has severe health
consequences including daytime sleepiness, cognitive impairment, and long-term
increased risk of cardiovascular disease and stroke. Current treatment paradigms for the
disease, some of which are effective, suffer from low compliance, due to the need for use
of facemasks or dental appliances. Others are highly invasive, requiring surgical
procedures followed by recovery. Therefore, new treatments using pharmacological
therapies might enhance compliance as well as offer a new method for treating cases of
OSA that do not respond to current treatment paradigms.
Drive to respiratory MNs is mediated by fast glutamatergic signaling in vitro and
in anesthetized in vivo preparations. The mechanisms for transmission of respiratory
drive in freely behaving animals may be more complicated. I along with my mentor and
colleagues hypothesize that enhancing the respiratory drive at upper airway MNs, such as
XII MNs, may aid in overcoming the loss of catecholaminergic wakefulness drive, so that
upper airway MN function for breathing is maintained.
142
As a first step towards developing these treatments, I investigated three methods
for increasing respiratory drive to XII MNs in vitro. Two methods attempted to enhance
the amount of facilitation seen during ivLTF. ivLTF is an activity-independent form of
MN plasticity induced in vitro that specifically enhances AMPAR-mediated signaling.
ivLTF likely is related to the in vivo phenomenon referred to as respiratory LTF, which is
characterized by increased respiratory motoneuronal output, tidal volume, and sometimes
rate of ventilation in response to episodic but not continuous bouts of hypoxia in adult
and neonatal rats, as well as in adult humans during sleep.
One study looked at the sensitivity of ivLTF to variation in protocol, since many
forms of neuronal and behavioral plasticity are sensitive to the stimulus pattern used for
induction. Specifically, I assessed the sensitivity of ivLTF to variations in induction
pattern in an attempt to identify critical parameters for maximizing the response. I found
that the duration of drug application and its relationship to the duration of the intervals
between drug applications were key predictors of the amount of ivLTF. Multiple episodes
of drug application induced greater facilitation, but in contrast to previous studies, ivLTF
could be induced by a single drug application. While no optimum was found, these data
inform further our understanding of the dynamics of inducing ivLTF.
The second study for enhancing ivLTF looked at the effects of stimulating PKG
signaling during the induction of ivLTF. I found that ivLTF was enhanced when PKG
signaling was stimulated during the induction protocol. Episodic stimulation of PKG
activity on its own, however, had no long-term effect on the amplitude of XII respiratory
discharge. Interestingly, other parts of the study performed by my colleagues showed that
143
acute enhancement of PKG activity in XII MNs decreased AMPAR-mediated signaling.
Together these experiments provide evidence for a previously unappreciated level of
complexity in how kinases regulate fast glutamatergic transmission in upper airway MNs.
Failure of respiratory LTF may underlie OSA. Conversely, a greater
understanding of the mechanisms underlying LTF and other phenomena mediating
plasticity of fast glutamatergic signaling in respiratory MNs could serve as a launch point
for the development of novel therapies for treating diseases such as OSA.
Neither of these approaches for enhancing AMPAR-mediated excitability
described above, however, was nearly as effective as the use of cyclothiazide (CTZ), a
diuretic, anti-hypertensive, and AMPA receptor modulator. I found that CTZ induces
profound and long-lasting increases in the amplitude of respiratory-related XII nerve
activity in rhythmically active neonatal rat medullary slices. The facilitation was nearly
3x that of the facilitation seen in my and other studies of ivLTF and lasted at least 12
hours. The amount of CTZ-induced facilitation was dependent upon both CTZ dose and
exposure time and was accompanied by a long-lasting increase in endogenous AMPAR-
mediated drive currents to XII MNs. The facilitation, however, is not a form of plasticity,
depending rather on continued presence of CTZ in slices.
In total, the results from the studies documented in this dissertation illustrate the
tremendous residual capacity that exists in AMPAR-mediated respiratory drive to XII
MNs. Thus, there is the potential for the development of pharmacological agents to
access this residual capacity for the treatment of upper airway motor deficits. Based on
144
my investigations, the most immediate promise seems to come from the use of CTZ. This
is because CTZ yielded the largest increases in XII MN activity of the methods I studied,
and it is an already approved drug in the clinic.
Even once a potentially viable target for pharmacotherapy has been identified,
however, a number of barriers to efficacy remain: (1) delivery, (2) specificity, (3)
variation in response across sleep-wake state, and (4) variability in how OSA affects
individuals (Eastwood et al., 2010). As I described in Chapter 3 there may be issues with
CTZ crossing the blood-brain barrier, and the doses required to enhance upper airway
tone may be larger than the ones approved for current clinical uses. Therefore, the next
logical step would be to study the effects of CTZ on breathing and behavior in freely
behaving animals. Such studies would give insight into issues related to BBB
permeability of CTZ, required dosing, variations in effects with changes in sleep-wake
state, as well as possible side effects. While such studies would be a good first step, rats,
even obese ones, do not suffer from OSA. In fact, there is no good model for OSA,
because modifications of the upper airway that allow us to walk upright and speak have
not been duplicated in other animals. Ultimately, trials in humans will be required.
Some of the data, however, may already exist. Millions of doses of cyclothiazide
have been prescribed. I wonder along with my mentor and colleagues whether primary
care physicians have mistaken improvements in symptoms of undocumented OSA as
general improvements in well-being brought on, for example, by reductions in
hypertension. A wealth of data ripe for retrospective analysis likely remains in the files of
these primary care physicians, through which the hidden benefits of this and other drugs
145
for treating not only OSA but a variety of other diseases may be found. Perhaps the
ongoing transition to electronic medical records might be exploited to do such
retrospective studies.
Furthermore, future treatment studies and meta-analyses should be designed with
the idea of capturing unexpected benefits of treating diseases other than the subject of the
study, i.e., “good” side effects. Such studies might tell us that we know far more about
treating a whole host of diseases with out current arsenal of pharmaceuticals than first
thought. More than 50 years have passed since the dawn of the information age, yet our
ability to amass data has far outstripped our ability to synthesize and make sense of it.
Our future success in treating diseases and injury, however, may rely more on how we
transform data gathered into information informing treatment strategies than in increasing
the number or types of studies we perform.
146
7 REFERENCES
Ancoli-Israel S, Kripke DF, Mason W (1987) Characteristics of obstructive and central sleep apnea in the elderly: an interim report. Biol Psychiatry 22:741-750.
Aoki CRA, Liu H, Downey GP, Mitchell J, Horner RL (2006) Cyclic nucleotides modulate genioglossus and hypoglossal responses to excitatory inputs in rats. Am J Respir Crit Care Med 173:555-565.
Arai AC, Kessler M (2007) Pharmacology of ampakine modulators: from AMPA receptors to synapses and behavior Curr Drug Targets 8:583-602.
Arancio O, Kiebler M, Lee CJ, Lev-Ram V, Tsien RY, Kandel ER, Hawkins RD (1996) Nitric oxide acts directly in the presynaptic neuron to produce long-term potentiation in cultured hippocampal neurons. Cell 87:1025-1035.
Babcock M, Badr M (1998) Long-term facilitation of ventilation in humans during NREM sleep. Sleep 21:709-716.
Bach KB, Mitchell GS (1996) Hypoxia-induced long-term facilitation of respiratory activity is serotonin dependent Respir Physiol 104:251-260.
Baker TL, Mitchell GS (2000) Episodic but not continuous hypoxia elicits long-term facilitation of phrenic motor output in rats. J Physiol 529:215-219.
Baker-Herman TL, Bavis RW, Dahlberg JM, Mitchell AZ, Wilkerson JE, Golder FJ, Macfarlane PM, Watters JJ, Behan M, Mitchell GS (2010) Differential expression of respiratory long-term facilitation among inbred rat strains. Resp Physiol Neurobiol 170:260-267.
Baker-Herman TL, Mitchell GS (2002) Phrenic long-term facilitation requires spinal serotonin receptor activation and protein synthesis. J Neurosci 22:6239-6246.
147
Baker-Herman TL, Mitchell GS (2008). Determinants of frequency long-term facilitation following acute intermittent hypoxia in vagotomized rats. Resp Physiol Neurobiol 162:8-17.
Ballerini L, Bracci E, Nistri A (1995) Desensitization of AMPA receptors limits the amplitude of EPSPs and the excitability of motoneurons of the rat isolated spinal cord. Eur J Neurosci 7:1229-1234.
Bavis RW, Mitchell GS (2003) Intermittent hypoxia induces phrenic long-term facilitation in carotid-denervated rats. J Appl Physiol 94:399-409.
Bellingham MC, Berger AJ (1996) Presynaptic depression of excitatory synaptic inputs to rat hypoglossal motoneurons by muscarinic M2 receptors. J Neurophysiol 76:3758-3770.
Berger AJ, Dieudonné S, Ascher P (1998) Glycine uptake governs glycine site occupancy at NMDA receptors of excitatory synapses. J Neurophysiol 80:3336-3340.
Bertolino M, Baraldi M, Parenti C, Braghiroli D, DiBella M, Vicini S, Costa E (1993) Modulation of AMPA/kainate receptors by analogues of diazoxide and cyclothiazide in thin slices of rat hippocampus. Receptors Channels 1:267-278.
Billard JM, Rouaud E (2007) Deficit of NMDA receptor activation in CA1 hippocampal area of aged rats is rescued by D-cycloserine. Eur J Neurosci 25:2260-2268.
Black MD (2005) Therapeutic potential of positive AMPA modulators and their relationship to AMPA receptor subunits. A review of preclinical data. Psychopharmacol 179:154-163.
Bliss TV, Collingridge GL (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361:31-39.
Bliss TV, Lφmo T (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol 232: 331-356.
148
Bocchiaro CM, Feldman JL (2004) Synaptic activity-independent persistent plasticity in endogenously active mammalian motoneurons. PNAS 101:4292-4295.
Bocchiaro CM, Saywell SA, Feldman JL (2003) Dynamic modulation of inspiratory drive currents by protein kinase A and protein phosphatases in functionally active motoneurons. J Neurosci 23:1099-1103.
Böhmer G, Schmid K, Schauer W (1991) Evidence for an involvement of NMDA and non-NMDA receptors in synaptic excitation of phrenic motoneurons in the rabbit. Neurosci Lett 130:271-274.
Bouryi VA, Lewis DI (2003) The modulation by 5-HT of glutamatergic inputs from the raphe pallidus to rat hypoglossal motoneurones, in vitro. J Physiol 553:1019-1031.
Bouryi VA, Lewis DI (2004) Enkephalinergic inhibition of raphe pallidus inputs to rat hypoglossal motoneurones in vitro. Neuroscience 129:55-64.
Brockie PJ, Maricq AV (2010) In a pickle: is cornichon just relish or part of the main dish? Neuron 68:1017-1019.
Campbell A, Neill A, Lory R (2010) Ethnicity and socio-economic status predict initial Continuous Positive Airway Pressure compliance in New Zealand adults with Obstructive Sleep Apnoea. Intern Med J doi: 10.1111/j.1445-5994.2010.02360.x.
Chamberlin NL, Bocchiaro CM, Greene RW, Feldman JL (2002) Nicotinic excitation of rat hypoglossal motoneurons. Neuroscience 115:861-870.
Chitravanshi VC, Sapru HN (1996) NMDA as well as non-NMDA receptors mediate the neurotransmission of inspiratory drive to phrenic motoneurons in the adult rat. Brain Res 715:104-112.
149
Christie LA, Russell TA, Xu J, Wood L, Shepherd GM, Contractor A (2010) AMPA receptor desensitization mutation results in severe developmental phenotypes and early postnatal lethality. Proc Natl Acad Sci 107:9412-9417.
Cohen BH, Lea RB (2004) Essentials of Statistics for the Social and Behavioral Sciences. John Wiley & Sons, Inc.: Hoboken.
Collingridge GL, Isaac JT (2003) Functional roles of protein interactions with AMPA and kainate receptors. Neurosci Res. 47:3-15.
Collingridge GL, Isaac JT, Wang YT (2004) Receptor trafficking and synaptic plasticity. Nat Rev Neurosci 5:952-962.
Costa ADT, Pierre SV, Cohen MV, Downey JM, Garlid KD (2008) cGMP signaling in pre- and post-conditioning: the role of mitochondria. Cardiovasc Res 77:344-352.
Dale-Nagle EA, Hoffman MS, MacFarlane PM, Satriotomo I, Lovett-Barr MR, Vinit S, Mitchell GS (2010) Spinal plasticity following intermittent hypoxia: implications for spinal injury. Ann N Y Acad Sci 1198:252-259.
Davidson TM (2003) The great leap forward: the anatomic basis for the acquisition of speech and obstructive sleep apnea. Sleep Med. 4:185-194.
Deng L, Chen G (2003) Cyclothiazide potently inhibits γ-aminobutyric acid type A receptors in addition to enhancing glutamate responses PNAS 100:13025–13029.
de Vente J, Asan E, Gambaryan S, Markerink-van Ittersum M, Axer H, Gallatz K, Lohmann SM, Palkovits M (2001) Localization of cGMP-dependent protein kinase type II in rat brain. Neurosci 108:27-49.
Diamond JS, Jahr CE (1995) Asynchronous release of synaptic vesicles determines the time course of the AMPA receptor-mediated EPSC. Neuron 15:1097-1107.
150
Drummond GB (2009) Reporting ethical matters in The Journal of Physiology: standards and advice. J Physiol 587:713-719.
DuBord MA, Liu H, Horner RL (2010) Protein kinase A activators produce a short-term, but not long-term, increase in respiratory-drive transmission at the hypoglossal motor nucleus in vivo. Neurosci Lett 486:14-18.
Dzubay JA, Jahr CE (1999) The concentration of synaptically released glutamate outside of the climbing fiber-Purkinje cell synaptic cleft. J Neurosci 19:5265-5274.
Eastwood PR, Malhotra A, Palmer LJ, Kezirian EJ, Horner RL, Ip MS, Thurnheer R, Antic NA, Hillman DR (2010) Obstructive Sleep Apnoea: From pathogenesis to treatment: Current controversies and future directions. Respirology. 15:587-595.
Ellenberger HH, Feldman JL (1988) Monosynaptic transmission of respiratory drive to phrenic motoneurons from brainstem bulbospinal neurons in rats. J Comp Neurol 269:47-57.
Essin K, Nistri A, Magazanik L (2002) Evaluation of GluR2 subunit involvement in AMPA receptor function of neonatal rat hypoglossal motoneurons. Eur J Neurosci 15:1899-1906.
Feldman JL, Del Negro CA (2006) Looking for inspiration: new perspectives on respiratory rhythm. Nat Rev Neurosci 7:232-242.
Feldman JL, McCrimmon (2003) Neural control of breathing. LR Squire, FE Bloom, SK McConnell, JL Roberts, NC Spitzer, MJ Zigmond (Eds.) Fundamental Neuroscience (2nd ed. pp. 967-990). Boston: Academic Press.
Feldman JL, Mitchell GS, Nattie EE (2003) Breathing: rhythmicity, plasticity, chemosensitivity. Annu Rev Neurosci 26:239-266.
Feldman JL, Neverova NV, Saywell SA (2005) Modulation of hypoglossal motoneuron excitability by intracellular signal transduction cascades. Resp Physiol & Neurobiol 147:131-143.
151
Feldman JL, Smith JC (1989) Cellular mechanisms underlying modulation of breathing pattern in mammals. Ann N Y Acad Sci 563:114-130.
Fregosi RF, Pilarski JQ (2008) Prenatal nicotine exposure and development of nicotinic and fast amino acid-mediated neurotransmission in the control of breathing. Resp Physiol Neurobiol 164:80-86.
Fucile S, Miledi R, Eusebi F (2006) Effects of cyclothiazide on GluR1/AMPA receptors. PNAS 103:2943-2947.
Fuller DD, Bach KB, Baker TL, Kinkead R, Mitchell GS (2000) Long term facilitation of phrenic motor output. Resp Physiol 121:135-146.
Funk GD, Smith JC, Feldman JL (1993) Generation and transmission of respiratory oscillations in medullary slices: role of excitatory amino acids. J Neurophysiol 70:1497-1515.
Funk GD, Smith JC, Feldman JL (1995) Modulation of neural network activity in vitro by cyclothiazide, a drug that blocks desensitization of AMPA receptors. J Neurosci 15:4046-4056.
Gamba G (2005) Molecular pathophysiology of electroneutral cation-chloride cotransporters. Physiol Rev 85:423–493.
García Del Caño G, Millán LM, Gerrikagoitia I, Sarasa M, Matute C (1999) Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat. J Neurocytol 28:455-468.
Ge Q, Feldman JL (1998) AMPA receptor activation and phosphatase inhibition affect neonatal rat respiratory rhythm generation. J Physiol 509:255-266.
Glaze DG (2005) Neurophysiology of Rett syndrome. J Child Neurol 20:740-6.
152
Goshgarian HG (2003) The crossed phrenic phenomenon: a model for plasticity in the respiratory pathways following spinal cord injury. J Appl Physiol 94:795-810.
Goshgarian HG (2009) The crossed phrenic phenomenon and recovery of function following spinal cord injury. Resp Physiol Neurobiol 169:85-93.
Greer JJ, Smith JC, Feldman JL (1991) Role of excitatory amino acids in the generation and transmission of respiratory drive in neonatal rat. J Physiol 437:727-749.
Grigg-Damberger M, Wells A (2009) Central congenital hypoventilation syndrome: changing face of a less mysterious but more complex genetic disorder. Semin Respir Crit Care Med 30:262-74.
Hayashi F, Coles SK, Bach KB, Mitchell GS, McCrimmon DR (1993) Time-dependent phrenic nerve responses to carotid afferent activation: intact vs. decerebellate rats. Am J Physiol 265:R811-R819.
Hoffman MS, Mitchell GS (2011) Spinal 5-HT7 receptor activation induces long-lasting phrenic motor facilitation. J Physiol 589:1397-1407.
Horner RL (2008) Neuromodulation of hypoglossal motoneurons during sleep. Resp Physiol Neurobiol 164:179-196.
Horner RL, Bradley TD (2008) Update in sleep and control of ventilation 2007. Am J Respir Crit Care Med 177:947-951.
Huang Y, Goshgarian HG (2009) Postnatal conversion of cross phrenic activity from an active to latent state. Exp Neurol 219:66-73.
Huang Y, Goshgarian HG (2009a) The potential role of phrenic nucleus glutamate receptor subunits in mediating spontaneous crossed phrenic activity in neonatal rat. Int J Dev Neurosci 27:477-483.
153
Ireland MF, Lenal FC, Lorier AR, Loomes DE, Adachi T, Alvares TS, Greer JJ, Funk GD (2008) Distinct receptors underlie glutamatergic signaling in inspiratory rhythm-generating networks and motor output pathways in neonatal rat. J Physiol 586:2357-2370.
Jackson AC, Nicoll RA (2011) The expanding social network of ionotropic glutamate receptors: TARPs and other transmembrane auxiliary subunits. Neuron 70:178-199.
Johansson P, Alehagen U, Svanborg E, Dahlström U, Broström A (2009) Sleep disordered breathing in an elderly community-living population: Relationship to cardiac function, insomnia symptoms and daytime sleepiness. Sleep Med 10:1005-1011.
Kapur VK (2010) Obstructive sleep apnea: diagnosis, epidemiology, and economics. Respir Care. 55:1155-1167.
Kato AS, Gill MB, Yu H, Nisenbaum ES, Bredt DS (2010) TARPs differentially decorate AMPA receptors to specify neuropharmacology. Trends Neurosci 33:241-248.
Kennedy MB, Beale HC, Carlisle HJ, Washburn LR (2005) Integration of biochemical signalling in spines. Nat Rev Neurosci 6:423-434.
Kezirian EJ, Maselli J, Vittinghoff E, Goldberg AN, Auerbach AD (2010) Obstructive sleep apnea surgery practice patterns in the United States: 2000 to 2006. Otolaryngol Head Neck Surg 143:441-447.
Kim E, Sheng M (2004) PDZ domain proteins of synapses. Nat Rev Neurosci 5:771-781.
Kinney HC (2009) Brainstem mechanisms underlying the sudden infant death syndrome: evidence from human pathologic studies. Dev Psychobiol 51:223-233.
Koizumi H, Wilson CG, Wong S, Yamanishi T, Koshiya N, Smith JC (2008) Functional imaging, spatial reconstruction, and biophysical analysis of a respiratory motor circuit isolated in vitro. J Neurosci 28:2353-2365.
154
Kong S, Qian B, Liu J, Fan M, Chen G, Wang Y (2010) Cyclothiazide induces seizure behavior in freely moving rats. Brain Res 1355:207-13.
Kono Y, Shigetomi E, Inoue K, Kato F (2007) Facilitation of spontaneous glycine release by anoxia potentiates NMDA receptor current in the hypoglossal motor neurons of the rat. Eur J Neurosci 25:1748-1756.
Kus L, Saxon D, Beitz AJ (1995) NMDA R1 mRNA distribution in motor and thalamic-projecting sensory neurons in the rat spinal cord and brain stem. Neurosci Lett 196:201-204.
Kutner MH, Nachtsheim CJ, Neter J, Li W (2005) Applied Linear Statistical Models (5th ed.) McGraw-Hill Irwin: New York.
Larson J, Le T-T, Hall RA, Lynch G (1994) Effects of cyclothiazide on synaptic responses in slices of adult and neonatal rat hippocampus. Neuroreport 5:389-92.
Larson J, Wong D, Lynch G (1986) Patterned stimulation at the theta frequency is optimal for the induction of hippocampal long-term potentiation. Brain Res 368:347-350.
Lei S, Jackson MF, Jia Z, Roder J, Bai D, Orser BA, MacDonald JF (2000) Cyclic GMP-dependent feedback inhibition of AMPA receptors is independent of PKG. Nat Neurosci 3:559-565.
Levenes C, Daniel H, Crepel F (1998). Long-term depression of synaptic transmission in the cerebellum: cellular and molecular mechanisms revisited. Prog Neurobiol 55:79–91.
Lev-Ram V, Wong ST, Storm DR, Tsien RY (2002) A new form of cerebellar long-term potentiation is postsynaptic and depends on nitric oxide but not cAMP. PNAS 99:8389–8393.
Liu G, Feldman JL, Smith JC (1990) Excitatory amino acid-mediated transmission of inspiratory drive to phrenic motoneurons. J Neurophysiol 64:423-436.
155
Liu Q, Wong-Riley MT (2005) Postnatal developmental expressions of neurotransmitters and receptors in various brain stem nuclei of rats. J Appl Physiol 98:1442-1457.
Liu Q, Wong-Riley MT (2010) Postnatal development of N-methyl-D-aspartate receptor subunits 2A, 2B, 2C, 2D, and 3B immunoreactivity in brain stem respiratory nuclei of the rat. Neuroscience 171:637-654.
Lorier AR, Funk GD, Greer JJ (2010) Opiate-induced suppression of rat hypoglossal motoneuron activity and its reversal by ampakine therapy. PLoS One 5:e8766.
Lovett-Barr MR, Mitchell GS, Satriotomo I, Johnson SM (2006) Serotonin-induced in vitro long-term facilitation exhibits differential pattern sensitivity in cervical and thoracic inspiratory motor output. Neuroscience 142:885-892.
Luo L, Callaway EM, Svoboda K (2008) Genetic dissection of neural circuits. Neuron 57:634-660.
MacFarlane PM, Mitchell GS (2008) Respiratory long-term facilitation following intermittent hypoxia requires reactive oxygen species formation. Neuroscience 152:189-197.
Mahamed S, Mitchell GS (2007) Is there a link between intermittent hypoxia-induced respiratory plasticity and obstructive sleep apnea? Exp Physiol 92:27-37.
Mahamed S, Mitchell GS (2008) Simulated apnoeas induce serotonin-dependent respiratory long-term facilitation in rats. J Physiol 586:2171-2181.
Malenka RC, Bear MF (2004) LTP and LTD: an embarrassment of riches. Neuron 44:5-21.
Malkova L, Kozikowski AP, Gale K (2010) The effects of huperzine A and IDRA 21 on visual recognition memory in young macaques. Neuropharmacol [Epub ahead of print]
156
Manly BFJ (2006) Randomization, Bootstrap and Monte Carlo Methods in Biology (3rd ed.) Chapman & Hall/CRC: Boca Raton.
Martinez-Maldonado M, Cordova HR (1990) Cellular and molecular aspects of the renal effects of diuretic agents. Kidney Int 38:632-641.
Martz BL, Steinmetz E, Kraner JC (1962) Studies of a new diuretic, cyclothiazide. J Indiana State Med Assoc 55:173-176.
Massey PV, Bashir ZI (2007) Long-term depression: multiple forms and implications for brain function. Trends Neurosci 30:176-184.
McCrimmon DR, Feldman JL, Speck DF (1986) Respiratory motoneuronal activity is altered by injections of picomoles of glutamate into cat brain stem. J Neurosci 6:2384-2392.
McCrimmon DR, Smith JC, Feldman JL (1989) Involvement of excitatory amino acids in neurotransmission of inspiratory drive to spinal respiratory motoneurons. J Neurosci 9:1910-1921.
McGuire M, Zhang Y, White DP, Ling L (2005) Phrenic long-term facilitation requires NMDA receptors in the phrenic motonucleus in rats. J Physiol 567:599-611.
McKay LC, Janczewski WA, Feldman JL (2004) Episodic hypoxia evokes long-term facilitation of genioglossus muscle activity in neonatal rats. J Physiol 557:13-18.
Milstein AD, Nicoll RA (2008) Regulation of AMPA receptor gating and pharmacology by TARP auxiliary subunits. Trends Pharmacol Sci 29:333-339.
Mitchell GS, Baker TL, Nanda SA, Fuller DD, Zabka AG, Hodgeman BA, Bavis RW, Mack KJ, Olson EBJ (2001) Invited review: Intermittent hypoxia and respiratory plasticity. J Appl Physiol 90:2466-2475.
157
Montgomery KE, Kessler M, Arai AC (2009) Modulation of agonist binding to AMPA receptors by 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546): differential effects across brain regions and GluA1-4/transmembrane AMPA receptor regulatory protein combinations. J Pharmacol Exp Ther 331:965-974.
Morgado-Valle C, Feldman JL (2007) NMDA receptors in preBotzinger complex neurons can drive respiratory rhythm independent of AMPA receptors. J Physiol 582:359-368.
Morris KF, Shannon R, Lindsey BG (2001) Changes in cat medullary neurone firing rates and synchrony following induction of respiratory long-term facilitation. J Physiol 532:483-497.
Murry C, Jennings R, Reimer K (1986) Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74:1124-1136.
Myers RH, Montgomery DC (2002) Response Surface Methodology: Process and Product Optimization Using Designed Experiments. John Wiley & Sons: New York.
Neverova N (2007) Intracellular signaling pathways underlying respiratory plasticity in vitro. Dissertation. University of California, Los Angeles.
Neverova NV, Saywell SA, Feldman JL (2002) The role of cGMP-mediated signaling pathway in modulation of hypoglossal (XII) motoneuron excitability. 2002 Neuroscience Meeting Planner, Program No. 856.5. Society for Neuroscience, Washington, D.C. Online.
Neverova NV, Saywell SA, Nashold LJ, Mitchell GS, Feldman JL (2007) Episodic stimulation of α1-adrenoreceptors induces protein kinase C-dependent persistent changes in motoneuronal excitability. J Neurosci 27: 4435-4442.
Nooney JM, Feltz A (1995) Inhibition by cyclothiazide of neuronal nicotinic responses in bovine chromaffin cells. Br J Pharmacol 114:648-655.
158
O'Brien JA, Isaacson JS, Berger AJ (1997) NMDA and non-NMDA receptors are co-localized at excitatory synapses of rat hypoglossal motoneurons. Neurosci Lett 227:5-8.
Ogier M, Wang H, Hong E, Wang Q, Greenberg ME, Katz DM (2007) Brain-derived neurotrophic factor expression and respiratory function improve after ampakine treatment in a mouse model of Rett syndrome. J Neurosci 27:10912-10917.
Oshima S, Fukaya M, Masabumi N, Shirakawa T, Oguchi H, Watanabe M (2002) Early onset of NMDA receptor GluR epsilon 1 (NR2A) expression and its abundant postsynaptic localization in developing motoneurons of the mouse hypoglossal nucleus. Neurosci Res 43:239-50.
Paarmann I, Frermann D, Keller BU, Hollmann M (2000) Expression of 15 glutamate receptor subunits and various splice variants in tissue slices and single neurons of brainstem nuclei and potential functional implications. J Neurochem 74:1335-45.
Partin KM, Patneau DK, Mayer ML (1994) Cyclothiazide differentially modulates desensitization of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor splice variants. Mol Pharmacol 46:129-138.
Partin KM, Patneau DK, Winters CA, Mayer ML, Buonanno A (1993) Selective modulation of desensitization at AMPA versus kainate receptors by cyclothiazide and concanavalin A. Neuron 11:1069-1082.
Patneau DK, Vyklicky L, Mayer ML, (1993) Hippocampal neurons exhibit cyclothiazide-sensitive rapidly desensitizing responses to kainate. J Neurosci 13:3496-3509.
Perrais D, Pinheiro PS, Jane DE, Mulle C (2009) Antagonism of recombinant and native GluK3-containing kainate receptors. Neuropharmacol 56:131-140.
Ping P, Zhang J, Qiu Y, Tang X-L, Manchikalapudi S, Cao X, Bolli R (1997) Ischemic preconditioning induces selective translocation of protein kinase C isoforms {epsilon} and {eta} in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ Res 81: 404-414.
159
Powell FL, Milsom WK, Mitchell GS (1998) Time domains of the hypoxic ventilatory response. Resp Physiol 112:123-134.
Qi J, Wang Y, Jiang M, Warren P, Chen G (2006) Cyclothiazide induces robust epileptiform activity in rat hippocampal neurons both in vitro and in vivo. J Physiol 571:605-618.
Quitadamo C, Fabbretti E, Lamanauskas N, Nistri A (2005) Activation and desensitization of neuronal nicotinic receptors modulate glutamatergic transmission on neonatal rat hypoglossal motoneurons. Eur J Neurosci 22:2723-2734.
Randerath WJ, Verbraecken J, Andreas S, Bettega G, Boudewyns A, Hamans E, Jalbert F, Paoli JR, Sanner B, Smith I, Stuck BA, Lacassagne L, Marklund M, Maurer JT, Pepin JL, Valipour A, Verse T, Fietze I (2011) Non-CPAP therapies in sleep apnoea. Eur Respir J 37:1000-1028.
Rekling JC, Funk GD, Bayliss DA, Dong XW, Feldman JL. (2000) Synaptic control of motoneuronal excitability Physiol Rev 80:767-852.
Remmers JE, deGroot WJ, Sauerland EK, Anch AM (1978) Pathogenesis of upper airway occlusion during sleep. J Appl Physiol 44:931-938.
Ren J, Ding X, Funk GD, Greer JJ (2009) Ampakine CX717 protects against fentanyl-induced respiratory depression and lethal apnea in rats. Anesthesiology 110:1364-1370.
Ren J, Poon BY, Tang Y, Funk GD, Greer JJ (2006) Ampakines alleviate respiratory depression in rats. Am J Respir Crit Care Med 174:1384-1391.
Robinson D, Ellenberger H (1997) Distribution of N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate receptor subunits on respiratory motor and premotor neurons in the rat. J Comp Neurol 389:94-116.
Sakmann B (2006) Patch pipettes are more useful than initially thought: simultaneous pre- and postsynaptic recording from mammalian CNS synapses in vitro and in vivo. Pflugers Arch 453:249-259.
160
Sassani A, Findley LJ, Kryger M, Goldlust E, George C, Davidson TM (2004) Reducing motor-vehicle collisions, costs, and fatalities by treating obstructive sleep apnea syndrome. Sleep 27:453-458.
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.
Saywell SA, Feldman JL (2003) Protein kinases differentially modulate inhibition in respiratory phased hypoglossal (XII) motoneurons. 2003 Neuroscience Meeting Planner, Program No. 499.16. Society for Neuroscience, Washington, DC. Online.
Saywell SA & Feldman JL (2004) Dynamic interactions of excitatory and inhibitory inputs in hypoglossal motoneurones: respiratory phasing and modulation by PKA. J Physiol 554:879-889.
Selim B, Won C, Yaggi HK (2010) Cardiovascular consequences of sleep apnea. Clin Chest Med 31:203-220.
Shamsuzzaman ASM, Gersh BJ, Somers VK (2003) Obstructive Sleep Apnea: Implications for Cardiac and Vascular Disease. JAMA 290:1906-1914.
Shao XM, Feldman JL (1997) Respiratory rhythm generation and synaptic inhibition of expiratory neurons in pre-Bötzinger complex: differential roles of glycinergic and GABAergic neural transmission. J Neurophysiol 77:1853-1860.
Shao XM, Feldman JL (2005) Cholinergic neurotransmission in the preBötzinger Complex modulates excitability of inspiratory neurons and regulates respiratory rhythm. Neuroscience 130:1069-1081.
Shao XM, Tan W, Xiu J, Puskar N, Fonck C, Lester HA, Feldman JL (2008) Alpha4* nicotinic receptors in preBotzinger complex mediate cholinergic/nicotinic modulation of respiratory rhythm. J Neurosci 28:519-528.
161
Sharifullina E, Ostroumov K, Nistri A (2004) Activation of group I metabotropic glutamate receptors enhances efficacy of glutamatergic inputs to neonatal rat hypoglossal motoneurons in vitro. Eur J Neurosci 20:1245-1254.
Sharkey KM, Kurth ME, Anderson BJ, Corso RP, Millman RP, Stein MD (2010) Obstructive sleep apnea is more common than central sleep apnea in methadone maintenance patients with subjective sleep complaints. Drug Alcohol Depend 108:77-83.
Sharp JT, Druz WS, D'Souza V, Diamond E (1985) Effect of metabolic acidosis upon sleep apnea. Chest 87:619-624.
Sharp RL, Mayne NG, Burnett JP (1994) Cyclothiazide differentially modulates human metabotropic glutamate receptors linked to phosphoinositide hydrolysis stimulation in oocytes. Eur J Pharmacol 269:R5-R7.
Shaw PJ, Ince PG, Johnson M, Perry EK, Candy J (1991) The quantitative autoradiographic distribution of [3H]MK-801 binding sites in the normal human spinal cord. Brain Res 539:164-168.
Shkoukani M, Babcock MA & Badr MS (2002) Effect of episodic hypoxia on upper airway mechanics in humans during NREM sleep. J Appl Physiol 92:2565-2570.
Shpargel KB, JalabiW, Jin Y, Dadabayev A, Penn MS & Trapp BD (2008) Preconditioning paradigms and pathways in the brain. Cleve Clin J Med 75:S77–S82.
Sibigtroth CM, Mitchell GS (2011) Carotid chemoafferent activity is not necessary for all phrenic long-term facilitation following acute intermittent hypoxia. Resp Physiol Neurobiol 176:73-79.
Singer JH, Bellingham MC, Berger AJ (1996) Presynaptic inhibition of glutamatergic synaptic transmission to rat motoneurons by serotonin. J Neurophysiol 76:799-807.
Smith JC, Ellenberger HH, Ballanyi K, Richter DW, Feldman JL (1991) Pre-Bötzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254:726-729.
162
Sobolevsky AI, Rosconi MP, Gouaux E (2009) X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature 462:745-756.
Sommer B, Keinänen K, Verdoorn TA, Wisden W, Burnashev N, Herb A, Köhler M, Takagi T, Sakmann B, Seeburg PH (1990) Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS. Science 249:1580-1585.
Steenland HW, Liu H, Horner RL (2008) Endogenous glutamatergic control of rhythmically active mammalian respiratory motoneurons in vivo. J Neurosci 28:6826-6835.
Steenland HW, Liu H, Sood S, Liu X, Horner RL (2006) Respiratory activation of the genioglossus muscle involves both non-NMDA and NMDA glutamate receptors at the hypoglossal motor nucleus in vivo. Neuroscience 138:1407-1424.
Surin A, Pshenichkin S, Grajkowska E, Surina E, Wroblewski JT (2007) Cyclothiazide selectively inhibits mGluR1 receptors interacting with a common allosteric site for non-competitive antagonists. Neuropharmacol 52:744-754.
Sutton MA, Ide J, Masters SE, Carew TJ (2002) Interaction between amount and pattern of training in the induction of intermediate- and long-term memory for sensitization in Aplysia. Learn Mem 9:29-40.
Suzue T (1984) Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat. J Physiol 354:173-183.
Tadjalli A, Duffin J, Peever J (2010) Identification of a novel form of noradrenergic-dependent respiratory motor plasticity triggered by vagal feedback. J Neurosci 30:16886-16895.
Teppema LJ, Dahan A (2010) The ventilatory response to hypoxia in mammals: mechanisms, measurement, and analysis. Physiol Rev 90:675-754.
163
Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev 62:405-496.
Tsvyetlynska NA, Hill RH, Grillner S (2005) Role of AMPA receptor desensitization and the side effects of a DMSO vehicle on reticulospinal EPSPs and locomotor activity J Neurophysiol 94:3951-3960.
Vogt KE, Canepari M (2010) On the induction of postsynaptic granule cell-Purkinje neuron LTP and LTD. Cerebellum 9:284-290.
von Engelhardt J, Mack V, Sprengel R, Kavenstock N, Li KW, Stern-Bach Y, Smit AB, Seeburg PH, Monyer H (2010) CKAMP44: a brain-specific protein attenuating short-term synaptic plasticity in the dentate gyrus. Science 327:1518-1522.
Walker CS, Jensen S, Ellison M, Matta JA, Lee WY, Imperial JS, Duclos N, Brockie PJ, Madsen DM, Isaac JTR, Olivera B, Maricq AV (2009) A novel Conus snail polypeptide causes excitotoxicity by blocking desensitization of AMPA receptors. Curr Biol 19:900–908.
Wang J, Irnaten M, Venkatesan P, Evans C, Baxi S, Mendelowitz D (2002) Synaptic activation of hypoglossal respiratory motorneurons during inspiration in rats. Neurosci Lett 332:195-199.
Whyte KF, Gould GA, Airlie MA, Shapiro CM, Douglas NJ (1988) Role of protriptyline and acetazolamide in the sleep apnea/hypopnea syndrome. Sleep. 11:463-472.
Wilkerson JE, Macfarlane PM, Hoffman MS, Mitchell GS (2007) Respiratory plasticity following intermittent hypoxia: roles of protein phosphatases and reactive oxygen species. Biochem Soc Trans 35:1269-1272.
Williams TL, Ince PG, Oakley AE, Shaw PJ (1996) An immunocytochemical study of the distribution of AMPA selective glutamate receptor subunits in the normal human motor system. Neuroscience 74:185-198.
164
Wu J, Wang Y, Rowan MJ, Anwyl R (1998) Evidence for involvement of the cGMP-protein kinase G signaling system in the induction of long-term depression, but on long-term potentiation, in the dentate gyrus in vitro. J Neurosci 18:3589–3596.
Xie A, Song X, Ripps H, Qian H (2008) Cyclothiazide: a subunit-specific inhibitor of GABAC receptors. J Physiol 586:2743–2752.
Yamada KA, Tang C-M, (1993) Benzothiadiazides inhibit rapid glutamate receptor desensitization and enhance glutamatergic synaptic currents. J Neurosci 13:3904–3915.
Yang Y, Ge W, Chen Y, Zhang Z, Shen W, Wu C, Poo M, Duan S (2003) Contribution of astrocytes to hippocampal long-term potentiation through release of D-serine. PNAS 100:15194-15199.
Young T (2009) Rationale, design and findings from the Wisconsin Sleep Cohort Study: Toward understanding the total societal burden of sleep disordered breathing. Sleep Med Clin 4:37-46.
Young T, Palta M, Dempsey J, Peppard PE, Nieto FJ, Hla KM (2009) Burden of sleep apnea: rationale, design, and major findings of the Wisconsin Sleep Cohort study. WMJ 108:246-249.
Young T, Palta M, Dempsey J, Skatrud J, Weber S, Badr S (1993) The occurrence of sleep-disordered breathing among middle-aged adults. N Engl J Med 328:1230-1235.
Young T, Peppard PE, Gottlieb DJ, (2002) Epidemiology of obstructive sleep apnea: a population health perspective. Am J Respir Crit Care Med 165:1217-1239.
Ytrehus K, Liu Y, Tsuchida A, Miura T, Liu GS, Yang XM, Herbert D, Cohen MV, Downey JM (1994) Rat and rabbit heart infarction: effects of anesthesia, perfusate, risk zone, and method of infarct sizing. Am J Physiol Heart Circ Physiol 267:H2383-2390.
Zhang XB, Sun GC, Liu LY, Yu F, Xu TL (2008) Alpha2 subunit specificity of cyclothiazide inhibition on glycine receptors. Mol Pharmacol 73:1195-1202.