UNIVERSITY OF CALIFORNIA

Los Angeles

Long-term Enhancement of Respiratory-Related Activity by

Increasing the AMPA Receptor-Mediated Excitability of

Hypoglossal Motoneurons In Vitro

A dissertation submitted in partial satisfaction of the

requirements for the degree Doctor of Philosophy

in Neurobiology

by

Walter Edward Babiec

2011

© Copyright by

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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7 References 146

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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.

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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.

13

Figure 1.1 Transverse medullary (rhythmic) slice.

14

2 THE ROLE OF IONOTROPIC GLUTAMATE RECEPTORS IN THE TRANSMISSION OF RESPIRATORY DRIVE

The role of signaling via ionotropic glutamate receptors (iGluRs), also referred to

as fast glutamatergic signaling, in mediating mechanisms underlying synaptic plasticity

and learning and memory in hippocampus, cortex, and cerebellum has captured the

imagination of myriad researchers for more than a quarter century. The roles of AMPA

receptors (AMPARs) as the workhorse of excitatory synaptic transmission and NMDA

receptors (NMDARs) as the coincidence detectors necessary for triggering plastic

changes in AMPAR number, subunit composition, and conductance, e.g., via

phosphorylation, have been worked out in exquisite detail as have many of the second

messengers underlying this process (Malenka and Bear, 2004; Kennedy et al., 2005;

Traynelis et al., 2010). Furthermore dozens of proteins interacting with these receptors in

the post-synaptic density have been identified and their roles in mediating iGluR activity

enumerated (Collingridge and Isaac, 2003; Collingridge et al., 2004; Kim and Sheng,

2004; Kennedy et al., 2005; Traynelis et al., 2010). Further, researchers have even come

to appreciate that some rules for synaptic activity and plasticity appear to be general,

while many others appear to be brain-area specific (Malenka and Bear, 2004).

During this same period the development of our knowledge about the neural

control of breathing has taken a very successful but much different path. The control of

breathing is highly distributed throughout the brainstem, spinal cord, and peripheral

nervous system, involving the complex interaction of rhythm and pattern generators,

reflexes, sensory feedback, and volitional commands. For this reason, tremendous

15

emphasis was placed on understanding how these elements of the system interact at a

highly intact level, i.e., whole animal. Also, during this period, there was a revolution in

our understanding of the genesis of respiratory rhythm spurred on by the development of

the reduced in vitro brainstem-spinal cord (Suzue, 1984) and rhythmic slice (Smith et al.,

1991) preparations, which fostered the landmark discoveries of the preBötC, the kernel of

inspiratory rhythm, and the retrotrapezoid nucleus/parafacial respiratory group

(RTN/pFRG), an area implicated as the source of active expiration (Feldman and Del

Negro, 2006).

As a result of concerted efforts in the highly integrated studies of breathing, the

relatively recent development of in vitro models of respiratory control, and the only

recent discovery of the rhythmogenic centers for breathing, far less progress has been

made in understanding the synaptic physiology of the connections within and between

respiratory centers that are critical to respiratory control. The field of respiratory control,

however, may be on the precipice of a new and vibrant period for furthering our

understanding of the synaptic physiology underlying the control of breathing. Recently,

long-lasting synaptic plasticity was discovered at MN synapses (Bocchiaro and Feldman,

2004). Also, we have recognized that our understanding of synaptic physiology in

breathing could aid in the treatment of disease and injury (Ren et al., 2006; Ogier et al.,

2007; Ren et al., 2009). Finally, there are many exciting improvements in the

electrophysiological, optical, and genetic techniques that are available for addressing

questions of synaptic physiology that were heretofore unassailable (Sakmann, 2006; Luo

et al., 2008).

16

Fast glutamatergic signaling plays an especially important role in both the

generation of respiratory rhythm and the transmission of that rhythm to MNs mediating

breathing movement (Liu et al., 1990; Greer et al., 1991; Funk et al., 1993). The goal of

this chapter is to review current knowledge about the role of iGluRs in the latter of these

functions. First, a brief overview of iGluR structure is provided. A discussion of the

types and relative amounts of iGluR subunits observed in respiratory MNs follows. Then

the evidence for the role of fast glutamatergic signaling as the primary path for

transmitting respiratory rhythm is discussed. Finally, mechanisms for modulating the

strength of excitatory synapses at respiratory MNs, either through the continued action of

endogenous substances and drugs or through the induction of lasting plastic changes

induced by specific events, are addressed. Throughout, areas where future work might be

helpful in clarifying issues or answering, as yet, unaddressed questions, are discussed.

2.1 iGluR structural overview

iGluRs form one of two main groups of glutamate receptors in the nervous

system, the other being metabotropic glutamate receptors. iGluRs are ligand-gated ion

channels, which, by homology and agonist specificity, can be divided into AMPA (2-

amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid), kainate, NMDA (N-Methyl-

D-aspartate), and the delta receptors. (Relatively little is known about the delta receptors

and they will not be discussed further.) Therefore, these receptors have many common

structural and functional elements. They also have unique variations that set the

subfamilies apart. This section briefly reviews the attributes of the iGluRs. Unless

17

specific references are given, the material in this section can be verified by reading the

extremely comprehensive review published by Traynelis et al. (2010).

2.1.1 Common attributes of iGluRs

Crystallographic studies of AMPARs show that iGluRs are comprised of 4

subunits that come together in a dimer-of-dimer structure (Sobolevsky et al., 2009). A

given receptor is formed only from subunits of one subfamily of iGluRs. The types of

subunits and the genes encoding them are summarized in (Table 2.1). Each subunit is

comprised of four discrete semiautonomous domains: the amino-terminal domain (ATD),

the ligand-binding domain (LBD), the transmembrane domain (TMD), and the carboxyl-

terminal domain (CTD).

The ATD influences receptor oligomerization and trafficking, but is not required,

however, for basic receptor functioning. Mutagenesis studies that remove the entire ATD

produce receptors that are functionally similar to wild-type. Changes to the ATD,

however, influence open probability, deactivation, desensitization, responses to certain

negative allosteric modulators, and regulation of subunit specific assembly. Also, an

amino acid sequence coding for a standard signal peptide at the very N-terminal end of

the ATD, which is common to all glutamate receptors, is required for membrane

insertion, after which it is then removed by proteolysis (Traynelis). Interestingly, the

ATD has putative binding sites for proteins such as N-cadherins, neuronal petraxins, and

ephrins and divalent cations such as Zn2+ and is subjected to glycosylation, suggesting

18

other roles for this region in trafficking, functional modulation, and proper synapse

formation.

The TMD is comprised of three transmembrane-spanning helices (M1, M3, M4)

with a re-entrant loop (M2) and with a short pre-M1 helix that is parallel to the plasma

membrane. M1-M3 form the ion channel core. M2 lines the inner cavity of the pore and

contains the QR mRNA editing site in GluA2, which regulates Ca2+ permeability in

GluA2-containing AMPARs. M3 lines the outer cavity of the pore and, likely, forms the

ion gate. The M1 helix is outside of the M2 and M3 helices. The M4 helix interacts with

M1-M3 helices of an adjacent subunit helping to maintain dimer interfaces in the

receptor.

The LBD is comprised of two extracellular stretches of amino acids: S1 and S2.

S1 is on the ATD side of M1, while S2 is between M3 and M4. S1 and S2 come together

to form a “clamshell” configuration that closes in the presence of agonists, thus imparting

conformational changes on the receptor that lead to pore opening. The interaction of S1

domains from different subunits provides the binding sites for agonists as well as

allosteric modulators of iGluRs. The S2 portion conveys the conformational changes

required for channel opening or desensitization, a state where agonist is bound but the ion

pore is closed.

The CTD is the most diverse of the domains of the iGluR subunit, varying greatly

in length and sequence of amino acids. Deletion of the CTD does not alter iGluR

function. Instead the CTD is thought to be involved with targeting, stabilization, post-

19

translational modifications, e.g., phosphorylation, and targeting for degradation. The

CTD interacts with dozens of proteins involved with receptor trafficking, synapse

formation, and second messaging.

2.1.2 iGluR stoichiometry

All iGluRs are tetramers of subunits from a single receptor subfamily, i.e.

AMPARs contain only GluA subunits, kainate receptors contain only GluK subunits, and

NMDARs contain only GluN receptors. At least for AMPARs, segregation of subunit

subfamilies is governed by the ATD. The details of which subunits can join within a

receptor subfamily differ between AMPA, NMDA, and kainate receptors, having

important functional consequences. The rules of association are least restrictive for

AMPARs, which appear to be able to associate in any combination, although mRNA

editing at sites in GluA2 and GluA4 subunits result in a tendency for these subunits to

favor heterodimerization.

In contrast, kainate receptors have a conditional set of stoichiometric

requirements. Like for AMPARs, GluK1 – GluK3 subunits can form functional

homomeric or heteromeric receptors of any combination. GluK4 and GluK5 subunits,

however, require the presence of GluK1 – GluK3 subunits to form functional receptors.

The need by NMDARs for both glutamate and glycine binding for activation is a

direct result of their stoichiometry. NMDARs require two GluN1 subunits in combination

with GluN2/GluN3 subunits. GluN1 and GluN3 subunits provide the glycine-binding

site, while GluN2 subunits provide the glutamate binding site. Interestingly, heterologous

20

expression of GluN1 and GluN3 subunits alone leads to the formation of glycine-gated

excitatory channels, although there are not data supporting the existence of such a

configuration in vivo. Electrophysiological evidence does exist, however, for naturally

occurring receptors that contain combinations of lower-conductance

GluN1/GluN2/GluN3 as well as the more common configuration of higher-

conductanceGluN1/GluN2.

2.1.3 RNA editing and alternative splicing

The role of mRNA editing and alternative splicing is probably best known in

AMPARs. Each GluA subunit comes as either a flip or flop splice variant. The alternative

splicing occurs in a 38 amino acid segment of the LBD (Sommer et al., 1990). As a

result, the splice variants have very different responses to allosteric modulators. For

example cyclothiazide, a drug that slows AMPAR desensitization and deactivation,

works preferentially on flip-containing receptors, but the ampakines, which also slow

desensitization and deactivation, prefer flop-containing receptors over flip-containing

receptors to varying degrees (Partin et al., 1994; Arai and Kessler, 2007). Similarly, ATD

splice variants of GluK1 also have different sensitivities to the influence of allosteric

modulators.

Substitution of arginine for glutamine at the QR mRNA editing site on the M2

segment of the GluA2 receptor significantly decreases both the rectification and Ca2+

permeability of GluA2-containing AMPARs. Editing at a similar site on GluK1 and

GluK2 subunits similarly affects the permeability properties of kainate receptors that

21

contain these subunits. Together with the RG editing site in the LBD of GluA2 and

GluA4 subunits, the QR site affects subunit pairing, conferring a preference for

heterodimerization over homodimerization. GluA2 also has two alternative splice

variants of the CTD, which influence receptor trafficking, synaptic plasticity, and several

receptor-protein interactions.

GluN1 and GluN2A both have alternative splice versions of their CTDs. There

are four alternates for GluN1 and two for GluN2A. Only the longest of the four GluN1

CTDs can be phosphorylated, while both of the splice variants of GluN2A allow for

phosphorylation. Also, alternative splicing of the GluN1 ATD allows for proton

inhibition of NMDARs, while alternative splicing of GluN1 and GluN2 influences

trafficking through the inclusion or exclusion of endoplasmic reticulum retention signals.

2.1.4 iGluR accessory proteins

The past decade has led to a growing awareness of and appreciation for a set of

proteins that are independent of iGluRs but dramatically affect their function, explaining,

for example, the differences in biophysical properties between heterologously expressed

recombinant and wild-type iGluRs. The best known of these are transmembrane AMPA

receptor regulatory proteins (TARPs). TARPs are found in the majority of AMPA

receptor complexes in the brain suggesting that they serve as auxiliary subunits to

naturally occurring AMPARs. They interact with extracellular, transmembrane and

intracellular regions of AMPARs and have the stoichiometry of 2-4 TARPs per AMPAR.

Functionally, TARPs increase AMPAR single channel conductance, open probability,

22

and activation rate, while slowing deactivation time course and reducing desensitization.

TARPs also play roles early in AMPAR synthesis and trafficking.

CINH proteins are additional AMPAR auxiliary proteins that are sometimes

referred to as cornichons, because they are homologous to the cornichon family of

proteins in flies and yeast. Relatively little is known about their role in AMPAR function,

but recent evidence points to a role in trafficking and possibly regulation of receptor

kinetics as well (Brockie and Maricq, 2010).

Neto1 is an NMDAR accessory protein that interacts with GluN2 by both the

extracellular domain and via interaction with PSD95 intracellularly. Without Neto 1,

GluN2A expression is completely abolished, but there is little effect on the expression of

GluN2B, implying that Neto1 may have a role to play in regulating learning and memory.

Its relative, Neto 2, affects the kinetics of GluK2-containing kainate receptors, increasing

peak amplitude and open probability, while slowing the decay time course of GluK2-

containing receptor-mediated mEPSCs but has no effect on trafficking.

Very little is known about the expression or function of iGluR accessory proteins

in the brainstem, and there are no published data looking at how these proteins might

influence respiratory control. But, their close relationship with and strong influence on

AMPARs of the hippocampus, cortex, and cerebellum as well as their heterogeneous

expression across brain areas (Montgomery et al., 2009; Jackson and Nicoll, 2011) makes

these accessory proteins of great interest for future study in respiratory control.

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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).

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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

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