STATE-DEPENDENT VERSUS CENTRAL MOTOR EFFECTS OF ETHANOL ON BREATHING

by

Laura Marie Vecchio

A thesis submitted in conformity with the requirements for the degree of Master’s of Science

Department of Physiology University of Toronto

© Copyright by Laura Marie Vecchio (2009)

ii

STATE-DEPENDENT VERSUS CENTRAL MOTOR EFFECTS

OF ETHANOL ON BREATHING

Laura Marie Vecchio

Master’s of Science

Department of Physiology University of Toronto

2009

Abstract

This thesis tested the hypothesis that ethanol suppresses respiratory muscle activity by

effects at the central motor pool and/or by state-dependent regulation of motor activity via

influences on sleep/arousal processes. Ten rats were implanted with electroencephalogram and

neck electrodes to record sleep-wake states, and genioglossus and diaphragm electrodes for

respiratory recordings. Studies were performed following intraperitoneal injection of ethanol

(1.25g.kg-1) or vehicle. The effects on genioglossus activity of ethanol (0.025-1M) or vehicle

applied directly to the hypoglossal motor nucleus were also determined in sixteen isoflurane-

anaesthetized rats. The results of these studies suggest that ethanol at physiologically relevant

concentrations promoted sleep, and altered electroencephalogram and postural motor activities

indicative of a sedating effect. The lack of effect on genioglossus activity with ethanol applied

directly to the hypoglossal motor pool suggests that the suppression observed with systemic

administration may be mediated via effects on state-dependent processes rather than direct

effects at the motor pool per se.

iii

Acknowledgments

I’d like to acknowledge the contributions of Mr. Stephen Harding and Dr. Anh Lê (Department

of Neuroscience, Centre for Addiction and Mental Health, Toronto), who performed the blood

ethanol analyses for these studies. I’d like to thank Dr. Hattie Liu, for teaching surgical

techniques and assisting during surgeries. I’d also like to thank Mr. Kevin Grace for assisting

with injections, blood withdrawals, animal handling and scientific discussions. Thank you to Dr.

Horner and all members of the Horner lab.

iv

Table of Contents

Abstract ii

Acknowledgements iii

List of Tables ix

List of Figures x

Abbreviations xi

CHAPTER ONE: Introduction p. 1

1. Sleep-Disordered Breathing 2

2. Wakefulness and Sleep 4

3. The Neural Correlates of Sleep/Wake States: A Brief Introduction 8

3.1 Wakefulness 8

3.2 NREM Sleep 10

3.3 REM Sleep 16

3.3.1 Aminergic-Cholingeric Model of REM Sleep Generation 16

3.3.2 GABAergic-Glutamatergic Model of REM Sleep Generation 17

v

4. Neural Correlates of Breathing 19

5. Breathing During Sleep 24

6. Introducing GABAA Receptors 27

7. The Effects of GABAA Modulators on Sleep and Breathing 31

7.1 Effects of GABA Receptor Modulation on Sleep 31

7.2 Effects of GABA Receptor Modulation on Breathing 32

8. Ethanol in the Body 34

9. Ethanol: Where does it act? 40

9.1 NMDA and non-NMDA Glutamate Receptors 40

9.2 Serotonin Receptors 41

9.3 Glycine Receptors 41

9.4 GABA Receptors 44

10. Effects of Ethanol on Sleep and Breathing 48

11. Summary and Rational for the Study of State-Dependent Versus Central Motor Effects of Ethanol on Breathing 50

vi

CHAPTER TWO: Methods p. 53

Study 1: Blood Ethanol Levels in Conscious Rats 54

Study 2: Effects of Ethanol on Sleep and Respiratory Motor Activity in Freely Behaving Rats 54

Anaesthesia and Surgical Procedures 55

Recording Procedures 58

Protocol 58

Data Analysis 59

Study 3: Ethanol at the Hypoglossal Motor Pool and Effects on Genioglossus Activity 61

Anaesthesia and Surgical Procedures 61

Microdialysis Perfusion and Recordings 61

Protocol and Data Analyses 62

Tests of Function of Hypoglossal Motor Nucleus and 63 Histology

Statistical Analysis 63

CHAPTER THREE: Results p. 64

vii

Study 1: Blood Ethanol Levels In Conscious Rats 65

Study 2: Effects of Ethanol on Sleep and Respiratory Motor Activity in Freely Behaving Rats 66

Effects of Ethanol on Sleep-Wake Regulation 66

Effects of Ethanol on Postural Motor Tone and EEG Activity 67

Effects of Ethanol on Respiratory Motor Activity 68

Study 3: Ethanol at the Hypoglossal Motor Pool and Effects on Genioglossus Activity 70

Sites of Microdialysis 70

Effects of Ethanol at the Hypoglossal Motor Pool 71

CHAPTER FOUR: Discussion p. 80

General Discussion 81

Interpretation of Findings 83

Systemic Administration of Ethanol 83

Local Application of Ethanol at the Hypoglossal Motor Nucleus 84

viii

CHAPTER FIVE: Future Directions and Final Conclusions p. 90

Future Directions 91

Relevance of Animal Preparation and Final Conclusions 95

REFERENCES 98

ix

List of Tables

CHAPTER ONE: Introduction

Table 1.1 p. 37

x

List of Figures

CHAPTER ONE: Introduction

Figure 1.1 A p. 14

Figure 1.1 B 15

Figure 1.2 21

Figure 1.3 30

Figure 1.4 39

Figure 1.5 47

CHAPTER TWO: Methods

Figure 2.1 57

CHAPTER THREE: Results

Figure 3.1 72

Figure 3.2 73

Figure 3.3 75

Figure 3.4 76

Figure 3.5 78

Figure 3.6 79

xi

List of Abbreviations

ANOVA analysis of variance

CO2 carbon dioxide

DRG dorsal respiratory group

DRN dorsal raphé nuclei

EEG electroencephalogram

EMG electromyogram

eVLPO extended ventrolateral preoptic area

GABA gamma-aminobutyric acid

GiV gigantocellular nucleus

LC locus coeruleus

LDT laterodorsal tegmental nuclei

LORR loss of righting reflex

LPT lateral pontine tegmentum

NAD nicotinamide-adenine-dinucleotide

NREM non-rapid eye-movement sleep

OSA obstructive sleep apnoea

PBC pre-Bötzinger complex

PnO nucleus pontis oralis

PPT pedunculopontine

PRG pontine respiratory group

REM rapid eye-movement sleep

RTN retrotrapezoid nucleus

SLD sublaterodorsal nucleus

xii

TMN tubermammillary nucleus

vlPAG ventrolateral periaqueductal grey matter

VLPO ventrolateral preoptic region

VRG ventral respiratory groups

1

CHAPTER 1 INTRODUCTION

2

CHAPTER ONE

Introduction

1 Sleep-Disordered Breathing

Sleep-disordered breathing takes a variety of forms and can be caused by a number of

pathophysiological mechanisms. In some sleep disorders, the neural mechanisms monitoring

carbon dioxide (CO2) react sluggishly and in others, too vigorously, which can result in

inappropriate ventilatory responses, producing periods of hypercapnia at times and at other

times, instances of hyperpnoea and hypocapnia. These latter periods can then result in the

complete cessation of breathing, as during periods of very low CO2, no signal to breathing will

be transduced during sleep; during periods when breathing has stopped, known as apnoeas, CO2

will rise and oxygen saturation may fall to dangerous levels. This pattern of irregular breathing

due to the inefficient monitoring of CO2 is known as Cheyne-Stokes respiration [1, 2]. Such

problems contribute to disorders such as central sleep apnoea and sudden infant death syndrome

[2-4]. Other disorders, such as snoring and obstructive sleep apnoea, do not affect the

neurological signal to breath but the ability to maintain open airways for effective ventilation

during sleep [5]. Snoring is one common form of disordered breathing which involves periods of

loud snorting noises caused by narrowing airways during sleep [6], and is also often

accompanied by poor quality of sleep [7-9]. It is caused by a decrease in muscle tone in the

upper airway muscles, reducing the patency of the air passages, and is believed to share

pathological mechanisms with obstructive sleep apnoea (OSA), a more serious breathing

disorder [5].

Affecting up to 4 % of the male and 2% of the female population [10], OSA is characterized by

episodes of hypopnoeas and airway closures that lead to the cessation of breathing during sleep,

also accompanied by daytime sleepiness [5, 11]. Repeated periods of hypopnoea caused by

3

ineffective respiratory efforts can result in significant arterial oxygen desaturation (greater than 3

%) [5, 11]. Obstructive apnoeas are defined as the cessation of airflow into the lungs during

sleep for at least 10 seconds, in spite of any respiratory efforts made by primary pump muscles;

this is demonstrated by muscle recordings indicating the movement of the ribcage and the

abdomen are in opposite directions, resulting from attempts to breathe against a closed airway.

When breathing efforts occur with an unobstructed airway, ribcage movements and abdomen

movements are synchronous [12]. Airway closures are due, at least in part, to the decreased tone

of pharyngeal muscles that is typical of the onset of sleep, particularly the genioglossus muscle

of the tongue [13, 14]. Thus a person with already narrow airways may be predisposed to

disordered-breathing with the onset of sleep. Patients can experience hundreds of apnoeic events

a night that are usually terminated by recurrent arousals from sleep, temporarily restoring

effective ventilation. However, recurrent disruptions of sleep can result in excessive daytime

sleepiness, increased anxiety, and an increase in work and motor vehicle accidents [15, 16].

Furthermore, a correlational link has been made between OSA and heart disease, diabetes and

obesity [17-21].

It is well known that factors such as sleeping position, or posture, can increase the severity of

sleep-disordered breathing [11, 22]. Clinical observations have also shown that pharmacological

substances can also worsen the severity of OSA and other respiratory disorders such as benign

snoring, with alcohol purported to be one such substance [23-28]. Ingestion of ethanol impairs

breathing during sleep in otherwise normal individuals, and significantly worsens breathing

during sleep in individuals with pre-existing sleep-disordered breathing [25-31]. For example,

patients who normally exhibit mild snoring during sleep can experience airway obstructions with

ethanol; ethanol can also increase the duration and frequencies of apnoeic episodes in patients

with OSA [28]. Although impairment of breathing, particularly during sleep, has been attributed

to the depressant effects of ethanol on the respiratory control system, the mechanisms by which it

affects breathing are not clear. Ethanol is a central nervous system depressant, having

widespread effects on a variety of ion receptors and transporters [32-39]. Ethanol may worsen

OSA severity by increasing the time spent in sleep or delaying arousal from sleep during an

apnoeic event [28, 29]. Ethanol may also act to suppress respiratory muscle tone including

augmenting inhibition of upper airway muscles, effects that would also contribute to worsening

4

the severity of sleep-related breathing disorders. This thesis addresses the mechanisms by which

ethanol influences sleep mechanisms and upper airway motor control using a rodent model.

2 Wakefulness and Sleep

Sleep is believed to be important for many aspects of daily functions, including general physical

and emotional health, but also memory, endocrine and metabolic function [21, 40, 41].

Neurotransmitters in the hypothalamus and brainstem are involved in complex circuits believed

to be responsible for generating and regulating sleep and wakeful states [42-45].

There are three general but distinct states across the sleep-wake cycle: wakefulness, non-rapid

eye-movement sleep (NREM) and rapid eye-movement sleep (REM). Not only are behavioural

differences obvious across these states, but a change in sleep-wake state also evokes global

neurochemical changes in the brain [46]. Generally, both behavioural and electrophysiological

criteria are used to identify sleep-wake states, applied in both a clinical and research setting [47-

49]. Common standards used to assess sleep-wake state in humans were laid out by

Rechtschaffen and Kales (1968) in A manual for standardized terminology, techniques, and

scoring system for sleep stages of human subjects [47].

In human patients, polysomnograms are recordings measuring electroencephalic activity, ocular

movements, and postural muscle activity and are used to assess sleep-wake activity and to aid,

along with other recordings, in the diagnosis sleep-related disorders [47-49]. The

electroencephalogram (EEG) provides an index of cortical activity. In humans, recordings are

made by affixing electrodes to the surface of the scalp at more than one location, which detect

electrical signals generated by ionic currents between cells of large neuronal aggregates whose

summated activity can then be recorded with gross techniques [46, 50]. When experimenting

5

with animals such as rats, EEG electrodes are put in direct contact with the brain’s surface by

drilling small screws into the skull over the cortex. The electrodes then detect and record electric

activity. In both cases, the EEG reflects summated synaptic events, including both excitatory

and inhibitory postsynaptic potentials, of underlying neurons [46].

The EEG reflects summations of the concurrent activity of millions of neurons that have similar

orientations and thalamocortical pathways [46, 50, 51]. Changes in EEG frequency are induced

by changes in the activity of neurons located in the brainstem, posterior hypothalamus, and basal

forebrain, which project to the thalamus, neocortex and hippocampus [46]. Various frequencies

can be detected from less than 1 Hz to more than 60 Hz and reflect oscillations caused by

rhythmic bursts of cortical activity. Frequencies are measured as cycles per second, with each

cycle being a measured number of potential changes detected by the electrodes [48]. The

brainstem and basal forebrain exert modulatory control over activity of the thalamus and cortex,

which alter activity when excitatory inputs from the brainstem and forebrain are withdrawn. In

NREM sleep, the withdrawal of excitatory inputs from cell groups in brainstem results in

decreased thalamocortical bursting frequency (Please see Chapter 1, Section 3 of this thesis)

which is reflected in the EEG [46].

There are a number of cellular mechanisms and neural networks that underlie the various

frequency patterns typical of NREM sleep (to be discussed within this section). For example,

pacemaker properties of thalamic reticular neurons contribute to the formation of sleep spindles,

a common feature of NREM sleep. The appearance of slow oscillatory waves typical of NREM

sleep (delta waves) results from the hyperpolarization of thalamocortical neurons to membrane

potentials below – 70 mV [46]. While the generation of many of these oscillatory patterns have

different sources, these changes are generally the result of the changes in input from the

brainstem that coincides with the onset of NREM sleep. Similarly, high frequency oscillatory

patterns typical of REM sleep result from an increase in thalamocortical excitability produced by

excitatory inputs from forebrain and brainstem structures believed to be involved in the

generation and modulation of REM sleep (Please see Chapter 1, Section 3 of this thesis).

Conventionally, EEG “synchronization” refers to EEG frequencies typical of NREM sleep but

6

this is, in fact, an oversimplification since synchronized rhythms can also be observed in

thalamocortical neurons during some (albeit not all) periods of REM sleep and wakefulness (i.e.

high-frequency theta waves); synchronization occurs with the co-activation of large groups of

neurons whose summated synaptic activity is concurrently recorded [46]. Typically, frequencies

associated with REM sleep and alert wakefulness are desynchronized and irregular, not showing

a clear and consistent oscillatory pattern [46]. The frequency and amplitude of cortical activity

can be used to assist in analyzing the behavioural state of the person or animal. Spectral analysis

is used to quantify the EEG frequencies in any given time period to evaluate the frequency

content. The EEG signal consists of many components that are summated (i.e. many

frequencies), and the power spectrum reflects the relative proportions, or the distribution, of

signal power over various frequency bands [52]. More specifically, it is the amplitude of the

EEG signal that lies within a frequency band. In addition to the EEG, the activity in postural

muscles such as the neck is used to help assess behavioural states [46]. Electromyogram (EMG)

electrodes are affixed to the surface of the skin (in humans) or muscle (in an animal) to assess

activity.

During wakefulness, a person or animal is alert and engaging with the environment around them.

There is generally high, and often irregular, postural muscle tone. Moreover, actively engaging

with the environment requires high cortical activity and functions, resulting in a higher frequency

EEG. During active wakefulness, an animal may be engaged in eating, drinking, or grooming

behaviours or may simply be exploring the chamber in which it is placed. All of these will result

in a higher tonic tone as well as burst of high amplitude signals in the recordings of muscular

activity. During wakefulness, there is also an increase in other parameters such as both tonic and

phasic (respiratory-related) activity in respiratory muscles [47-49] and an increase in ocular

movements [48, 49]. The EEG typical of wakefulness consists mainly of alpha (8 – 13 Hz) and

beta waves (13 – 30 Hz) [47-49]. Desynchronized, irregular beta waves are common during

periods of attentiveness and alertness, and alpha waves are more dominant during relaxed

wakefulness [46]. Quiet wakefulness differs in that there is less postural muscle tone,

respiratory-related muscle activity becomes more regular and the EEG become progressively

slower in frequency during quiet wakefulness [46, 53].

7

NREM sleep is associated with a progressive decline in postural muscle activity as well as a

slowing of EEG frequency, which consists of synchronized low-frequency, high-amplitude

waves, as compared to wakefulness [46-49]. In humans, there are slow, rolling ocular

movements associated with NREM sleep. While there are several ways to score NREM sleep

and not one standard definition, a commonly-used method of scoring in humans identifies four

stages of NREM sleep. According to this method, during stage one NREM sleep, there is a

decrease in postural muscle activity and an increase in the power of theta-wave activity (4 – 8

Hz). Also known as “light sleep”, stage two sleep is characterized by an increase in the

proportion of slow oscillating waves in the EEG but with short bursts of high frequency waves

between 10 – 14 Hz (called sleep spindles) and the presence of K-complexes (high-amplitude

bipolar waves of 75 μV and also lasting >0.5 seconds). Stage two is usually the most dominant

stage of sleep during the night. Stage three and four sleep typically occupies 20 – 25 % of sleep

time in humans, and consists of very high amplitude EEG frequencies known as delta waves (δ2,

0.5 – 2 Hz; δ1, 2 – 4 Hz). The EMG of postural muscles is lower in stage three and four than in

other stages of NREM sleep. A common practise used while sleep-scoring is to score NREM

sleep as periods of uninterrupted sleep that last 10 seconds or longer [46-49]. Typically, the

“depth” of sleep correlates to the proportion of very low frequency waves, namely delta waves,

in the EEG; the arousal threshold to stimuli such as acoustic sounds increases during periods

when lower EEG frequencies are dominant [52, 54]. Because dividing NREM sleep into stages

is often not useful or readily applicable when studying laboratory animals (such as rats), a

common practise is to score periods of slow oscillatory EEG waves and low postural muscle

activity as simply NREM sleep.

REM sleep is differentiated from NREM sleep by rapid eye movements, from which it gets its

name, muscle atonia and high frequency EEG activity. The EEG is desynchronized and there is

a high proportion of electroencephalic activity in the theta frequency range. Despite the high

cortical activity, there is complete atonia of postural muscles with only occasional twitches. For

these reasons, it is sometimes referred to as “paradoxical sleep”. Towards the end of the normal

sleeping period, the frequency of REM episodes experienced increases due to the accumulation

8

of REM-sleep propensity, which is under the control by homeostatic and circadian mechanisms

[55]. Such as in NREM sleep, one common and readily-applicable practise is to score REM

sleep as a period of 10 seconds or longer. While different standards can be employed, one

regularly used identification of arousals from both NREM and REM sleep considers an arousal

to be a sudden change in EEG and EMG activity that lasts at least 3 seconds, also associated with

an increase in postural muscle tone if it occurs during REM [46-49].

3 The Neural Correlates of Sleep/Wake States: A Brief Introduction

Wakefulness, NREM sleep, and REM sleep are three unique states characterized by changes in

both muscle activity and neuronal activity in the brain, which can be identified using EMG and

EEG, respectively. The regulation of both NREM and REM sleep has long been studied, and

which neuronal nuclei are essential for the generation of sleep has been the concentrated focus of

research for many years [44, 56, 57] Various methodological approaches have been utilized to

identify the core brain regions responsible for generation and maintaining sleep-wake states,

including transection experiments, lesioning studies, discrete electrical and chemical stimulation,

discrete chemical inhibition, tracing studies, imaging, and in vitro and in vivo recordings.

3.1 Wakefulness The ascending arousal system that contributes to cortical activation involves a heterogeneous

population of neurons that begins in the rostral pons and the medulla, and runs through the

midbrain reticular formation [45, 58], a region spanning from the brainstem to the thalamus. It is

comprised of excitatory projections to the cortex, which influence electrocortical activity via

glutamatergic inputs. There are two major branches of this arousal system (see Figure 1.1 A).

The first branch (the dorsal pathway) originates in the cholinergic pedunculopontine and

9

laterodorsal tegmental nuclei (PPT and LDT) [45, 59], comprising heterogeneous neuronal

groups with cells showing activity during periods of cortical activity including both wakefulness

and REM sleep [60]. The PPT and LDT project to the thalamus and in turn to the cortex,

stimulating cortical activity [61, 62]. The ventral excitatory pathway of the ascending arousal

system is comprised of several types of monoaminergic projections; monoaminergic cells are

those that produce transmitters with an amino group that is connected to an aromatic ring by a

two-carbon chain (-CH2-CH2-) such as serotonin, histamine, dopamine, and noradrenaline,

amongst others. Histaminergic projections from the tubermammillary nucleus (TMN) and

orexinergic projections from the posterior hypothalamus contribute to cortical arousal via

excitatory inputs [44, 63-66]. The locus coeruleus (LC) and dorsal raphé nuclei (DRN) also

contribute to the ascending arousal system, with extensive noradrenergic and serotonergic

projections to the cortex, thalamus, hypothalamus and basal forebrain [67, 68]. Activity in

noradrenergic and serotonergic cells is highest during wakefulness, decreases during NREM

sleep and is minimal during REM sleep [46].

Monoaminergic and cholinergic cells involved in arousal send excitatory projections to other

areas involved in arousal, “reinforcing” wakefulness [46, 69, 70], but are also responsible for

inhibitory inputs to neurons believed to be responsible for the promotion of NREM sleep [71,

72]. For example, excitatory orexinergic neurons that project from the hypothalamus innervate

the PPT and LDT, LC, DRN, and TMN, further stabilizing these cells during periods when they

are normally highly active (i.e. wakefulness). Wakefulness-promoting neuromodulators such as

serotonin, noradrenaline, and acetylcholine then inhibit the ventrolateral preoptic region, which

is believed to be a sleep-promoting region [44, 69] (see Figure 1.1 B). The reciprocal inhibitory

relationship between monoaminergic arousal centres and sleep-promoting centres is known as

the “flip-flop” switch; this will be discussed in further detail in the next section (NREM Sleep).

When sleep-promoting neurons fire, they inhibit monoaminergic cells groups which, in turn,

disinhibits and reinforces their own firing; in the same way, when monoaminergic neurons fire

during wakefulness, they inhibit the sleep-promoting neurons which would normally regulate

their firing, thus reinforcing their own activity [44, 69].

10

3.2 NREM Sleep It was during the First World War, when a pandemic of encephalitis lethargica broke out, that

scientists began to investigate the cause of the prolonged state of sleepiness. Shortly thereafter, a

scientist by the name of Baron Constantin von Economo reported that injuries to the posterior

hypothalamus and rostral midbrain produced prolonged sleepiness, leading to the conclusion that

neurons promoting the state of wakefulness were located within these regions (as discussed in

Saper, 2001) [44]. Lesions of the the preoptic area and basal forebrain, however, had the

opposite effect on patients and induced periods of insomnia; it was hypothesized that these

regions, then, contained sleep-promoting cells. Yet it was not until after the Second World War

that scientists began to model an ascending pathway of arousal, regulating activity in the

forebrain and cortex, and investigate how sleep-promoting cells interact with those that promote

arousal [44].

A discrete group of neurons in the hypothalamus, known as the ventrolateral preoptic region

(VLPO), has been implicated in the generation of NREM sleep [44, 73-77]. This region was

characterized by Saper and his colleagues (1996) in a study that showed that the VLPO

contained neurons that are selectively active at sleep onset, and that the activity of these cells is

proportional to the depth of sleep [73]. The study examined cell activity using c-Fos

immunostaining techniques and the findings were later corroborated by electrophysiological

studies that demonstrated that, in freely-behaving animals, neurons within the VLPO increased

activity significantly during recovery from sleep deprivation [57, 78]. Moreover, lesioning

neurons within the VLPO significantly reduced the time spent in NREM sleep as well as the

delta power in the EEG signal [79]. The results agree with those of lesioning experiments done

in animals as many as 70 years ago, as well as microdialysis studies done 20 years ago,

suggesting that wakefulness is promoted by neurons in the posterior lateral hypothalamus and

sleep in the preoptic area [74-77].

The studies referred to above, in addition to many more, have demonstrated that the VLPO plays

a crucial, if not necessary, role in the generation and maintenance of sleep. Still, early studies

11

failed to elucidate the large networks involved in sleep generation. Changes in activity occur in

a wide variety of neuronal groups across the brain during sleep [57], and the interactions between

many of these neuronal groups are responsible for said changes. Approximately two-thirds of

neurons in the VLPO are active during sleep [78]. In an in vitro study, Gallopin (2000) and

colleagues hypothesized that neurons thought to be responsible for sleep onset in the

hypothalamus represent a homogeneous population of cells; moreover, it was argued that these

cells must be inhibited by neurons responsible for arousal during wakefulness [80]. In that study,

two-thirds of neurons within the VLPO were characterized by a low threshold spike (a

proportion that matches the number of “sleep-active” cells in the VLPO) and, using single-cell

reverse transcriptase, these cells were shown to include gamma-aminobutyric acid (GABA) –

containing neurons. GABA is one of the most common inhibitory molecules in the central

nervous system [81], so given the results of that experiment, it was concluded that the VLPO

likely plays a role in promoting sleep. VLPO neurons send inhibitory GABAergic projections to

the major neuronal groups thought to be responsible for mediating arousal in the hypothalamus,

basal forebrain and brainstem [73, 79, 80, 82, 83]. VLPO neurons also send widespread

inhibitory projections to the cortex [69]. Importantly, Gallopin (2000) also showed that these

same VLPO cells are inhibited by neurotransmitters implicated in arousal, namely noradrenaline

and acetylcholine, and some also by serotonin [80]. This observation led to the hypothesis that

reciprocal inhibitory interactions between GABAergic VLPO neurons and the monoaminergic

and cholinergic groups involved in arousal are functionally important in the generation of sleep

[73, 78, 82, 84-87]; maintaining the state of NREM sleep is an active process of inhibiting

neuronal groups responsible for arousal.

Early attempts to discern the neuronal regions involved in arousal began with transection studies

demonstrating that lesioning the brainstem at the rostral pons-caudal midbrain junction, at a

midcollicular level, caused a significant loss of wakefulness [44, 58]. Projections from this area

were traced through the paramedian midbrain reticular formation to the diencephalon. From

there, two pathways were found: one innervated the thalamus and the other, the hypothalamus

[44, 58]. As previously mentioned, this anatomical trajectory fits with the ascending arousal

system and involves a number of brainstem and posterior hypothalamic structures that send

projections throughout the forebrain and mediate arousal. Neuronal groups involved in this

12

arousal system include the cholinergic LDT and PPT nuclei, the histaminergic TMN, the

serotonergic DRN, and the noradrenergic LC. Key to this anatomical framework are interactions

with the sleep-promoting cells of the aforementioned VLPO, containing GABA as well as

another inhibitory neurotransmitter, galanin [44]. It is proposed that sleep-active neurons of the

VLPO receive inhibitory input from cell groups responsible for brain arousal as these latter cells

are actively promoting arousal [80, 88, 89]. During sleep, VLPO neurons both actively inhibit

these same arousal neurons and also disinhibit their own activity. Therefore, during periods of

wakefulness or NREM sleep, activity within the wake-promoting and sleep-promoting cells

(respectively) inhibit cells promoting the other state and at the same time stabilize their own

activity. The reciprocal, inhibitory relationship between cells in the VLPO and monoaminergic

cells is part of a system referred to as the “flip-flop” or “sleep switch” [44, 57, 69]. In such a

system, there is mutual inhibition between both states, with the system stable in each state.

While it is clear that this reciprocal relationship exists, it is not well established what causes the

rapid transition from wakefulness to sleep (and vice versa) and what allows sleep-promoting

cells to suddenly overcome inhibition from wake-promoting cells and in turn, begin to inhibit the

latter group with their own activity (and vice versa). The homeostatic drive for sleep (“Process

S”) and the circadian systems (“Process C”) are thought to bias the balance towards sleep or

wakefulness [90-95], and the mutual inhibition between the arousal and sleep-promoting

systems, promoting rapid transitions with the system stable in each state. For example, sleep

deprivation studies can illustrate the role of the homeostatic drive in promoting the switch of

state by demonstrating intensification of sleep and increasing durations of sleep during recovery.

During recovery from sleep deprivation, there is intensification of the power spectra typical of

NREM and REM sleep in the recovery phase, increasing slow-wave activity in NREM (although

with a progressive decline), and increasing theta activity in REM and wakefulness [93, 96-99].

There is also an increase in the total proportion of REM sleep and decrease in NREM sleep.

These results support the hypothesis of homeostatic mechanisms regulating a build-up in sleep

propensity [93, 96-99].

13

Along with an involvement in the ascending arousal system, orexin neurons are thought to play a

role in stabilizing the sleep switch [100-104]. During sleep, there is GABAergic inhibition to

orexin neurons in the perifornical region of the hypothalamus [105], with the latter neurons

having excitatory projections to aminergic neurons in the brainstem and hypothalamus,

particularly locus coeruleus neurons [104, 106-108]. Thus there is a decrease in excitation to the

monoaminergic “arousal” neurons, and this is thought to stabilize sleep [109]. Disruption of the

neural or chemical mechanisms thought to regulate the sleep-wake switch can therefore result in

the development of disrupted sleep and sleep-related disorders such as insomnia, lethargy and

narcolepsy. For example, there is reasonably strong evidence that the loss of orexin neurons is a

cause of narcolepsy, a disorder which involves the sudden and inappropriate transfer from

wakefulness into a cataplexic, REM-like state [45, 108, 110] .

14

Figure 1.1 A: Schematic drawing of the ascending arousal system. This system involves two excitatory pathways. Dorsal pathway: The relay and reticular nuclei of the thalamus (yellow) receive excitatory inputs from cholinergic neurons in the the pedunculopontine (PPT) and laterodorsal tegmental nuclei (LDT) of the pons, which in turn, stimulates thalamocortical transmission. Ventral pathway: Responsible for main activation of the cerebral cortex and facilitating the processing of inputs from the thalamus. The main components are monoamerinergic cells groups such as the tuberomammillary nucleus (TMN) containing histamine (His), the A10 cell group containing dopamine (DA), the dorsal and median raphe nuclei containing serotonin (5-HT), and the locus coeruleus (LC) containing noradrenaline (NA). Excitatory inputs also originate in the peptidergic neurons in the lateral hypothalamus (LHA), which contains orexin (ORX) and melanin-concentrating hormone (MCH), and from basal forebrain (BF) neurons, which contain gamma-aminobutyric acid (GABA) and ACh. From Saper et al. (2006). [43]

15

Figure 1.1 B: A schematic drawing depicting projections from the ventrolateral preoptic nucleus (VLPO) to main components of the ascending arousal system. VLPO neurons (purple) sends inhibitory projects to monoaminergic cells (red) such as tuberomammillary nucleus (TMN), the A10 cell group, the raphe cell groups and the locus coeruleus (LC). It also projects to cholinergic groups (yellow) such as the pedunculopontine (PPT) and laterodorsal tegmental nuclei (LDT) and neurons in the lateral hypothalamus (LHA; green), which includes periforinical (PeF) orexinergic neurons. Other abbreviations: 5-HT, serotonin; GABA, gamma-aminobutyric acid; gal, galanin; NA, noradrenaline; His, histamine. Cells of the VLPO inhibit the activity of cells in the ascending arousal system; also, they are themselves inhibited by these same groups of cells involved in arousal. From Saper et al. (2006). [43]

16

3.3 REM Sleep Sixty years ago, while studying sleep in cats, Michel Jouvet and François Michel described a

stage of sleep characterized by muscle atonia but with accompaniment of a “paradoxically”

active EEG and rapid eye movements [111]. REM, or paradoxical sleep, is differentiated from

NREM sleep by complete muscle atonia, high-frequency and low-amplitude cortical activity, and

rapid eye movements [111, 112]. The onset of REM sleep is thought to be generated by

neuronal structures distinct from those generating NREM (“slow-wave”) sleep. Transection

studies performed in animals, as well as electrical and chemical lesioning studies, demonstrated

that cells within the region of the nucleus pontis oralis (PnO) are responsible for the onset of the

neural events that comprise REM sleep [113-116]. There are some studies that have suggested

that cholinergic mechanisms played a significant role in the generation of REM sleep [60, 114].

Carbachol injections into a discrete region identified as the peri-locus coeruleus α in cats, and the

sublaterodorsal nucleus (SLD) in rats, induced the onset of REM-like sleep [117-119]. Neurons

that become active during REM sleep (“REM-on” neurons) located in this region were then

subdivided into two groups based on location within the nuclei, projections, and response to

chemical stimulation: those believed to be responsible for cortical activation during REM sleep,

and others, for the muscle atonia that accompanies it [113, 117, 120]. More recently the

importance of acetylcholine in REM sleep generation has been questioned, and the involvement

of GABA has been the focus of study. Thus, there are two competing models of REM sleep

generation: the aminergic-cholinergic model and the GABAergic-glutamatergic model of REM

sleep.

3.3.1 Aminergic-Cholingeric Model of REM Sleep Generation

The reciprocal aminergic-cholingeric model of REM sleep onset, based largely on studies

involving local application of agonists to discrete nuclei, proposes that the switch to the state of

REM sleep is a result of reciprocal interactions between cholinergic REM-on neurons and

monaminergic REM-off neurons [113, 121]. Noradrenergic neurons in the LC, serotonergic

neurons in the DRN and histaminergic neurons in the TMN all decrease their discharge rates

during NREM sleep and are virtually silent during REM sleep, and have therefore been

17

designated “REM-off” cells [122-124]. As previously mentioned, these cells are involved in

mutually inhibitory interactions with the VLPO, involved in switching from wakefulness to

NREM sleep. Pharmacologically inhibiting the uptake of noradrenaline and serotonin suppresses

REM sleep, suggesting a role for these transmitters in regulating REM sleep generation [125].

Evidence suggests that monoamines not only act on the SLD in regulating REM sleep generation

but also on other areas believed to be involved in REM sleep modulation and maintenance.

These areas include the cholinergic PPT and LDT nuclei, and the gigantocellular nucleus (GiV)

in the ventrolateral medullary reticular formation, an area believed to be involved in muscle

atonia during REM sleep [113, 126]. Large lesions of the PPT and LDT nuclei result in a

significant decrease in REM sleep propensity [127-130] and pharmacological studies have

shown that these cells are inhibited by aminergic inputs via α2-adrenergic and 1A-serotonin

receptors [113]. While PPT and LDT cells are inhibited by aminergic inputs during wakefulness,

they are disinhibited when those same amerigic neurons are inhibited by the VLPO during

NREM sleep. The reciprocal aminergic-cholingeric model suggests that cholinergic neuronal

activation is responsible for cortical activation and motor inhibition via descending pathways

[69, 131, 132].

3.3.2 GABAergic-Glutamatergic Model of REM Sleep Generation

Recent studies have pointed out shortcomings in the aminergic-cholingeric model based on

inconsistencies between rats and cats used in various studies, on the lack of studies using

chemical antagonists at specific nuclei involved in the model, and on the fact that lesioning

monoaminergic nuclei in the brainstem have relatively little effect on REM sleep generation [45,

113, 133]. In addition, lesions of the PPT are usually quite large in area. Recent evidence has

suggested crucial roles for GABA and galanin in the switch to REM sleep, and a new model has

emerged based on these findings [113, 134]. The inhibitory transmitter GABA and excitatory

transmitter glutamate modulate the onset of REM sleep in this model by acting on nuclei

previously identified to have key roles in REM sleep generation and modulation [113, 133, 135].

When bicuculline, a GABAA receptor antagonist, was administered locally to the SLD in freely

behaving rats, there was a significant increase in the quantity of REM sleep [136]; these results

18

agreed with those of previous studies using microiontophoretic application of bicuculline or

gabazine in head-restrained rats [137].

The extended part of the ventrolateral preoptic area (eVLPO) contains REM-on cells that contain

GABA and galanin, and it was thus hypothesized to be involved in inhibitory networks with

known REM-off neurons [133]. Using anterograde tracers, it was found that the eVLPO is

involved in a network with an arc of tissue that extends from the ventrolateral part of the

periaqueductal grey matter (vlPAG) to the lateral pontine tegmentum (LPT). A study showed

that lesioning the vlPAG or LPT more than doubled the amount of REM sleep in rats during dark

periods (during light periods, normal sleeping hours for rodents, lesioning the LPT only

increased the number of REM periods) [133]. The use of retrograde tracers, c-Fos protein

analysis, and messenger RNA analysis showed that vlPAG neurons projected to SLD neurons,

which also contained glutamate. Further analysis revealed that the REM-on cells of the SLD

were not only connected to the vlPAG but there were GABAergic projections between the two,

suggesting mutual inhibition and a flip-flop switch for REM generation [133]. Again, the

implication of a mutually inhibitory relationship necessitates that while one group inhibits the

other, it also disinhibits its own cells and promotes further activity. Lesioning the SLD

significantly reduced total REM sleep time but increased the number of transitions between

NREM and REM sleep; in fact, lesioning either the vlPAG or SLD increased transitions. This

further suggests an important interaction between the vlPAG and the SLD in the onset of REM

sleep [133]. Furthermore, lesioning experiments demonstrated that the vSLD may be critical in

the motor atonia that accompanies REM-sleep [133].

Reports on the importance of the involvement of regions such as the LC and DRN in this model

are mixed. The manipulation of GABAA receptors in the LC and DRN with the application of

receptor modulators and antagonists has demonstrated that GABA is critical in eliciting the

reduced activity observed in these cell groups (particularly the LC) during NREM sleep and

silencing the neurons during REM sleep [86, 124, 126, 138-141]. However more recent studies,

including lesioning experiments, suggest that lesioning LC neurons does not significantly affect

19

the time spent in REM sleep [133]. These results contradict others showing that chemical

manipulation of the LC does affect the proportion of time spent in REM sleep.

Considering the results of these studies, GABA seems to play a clear role in the flip-flop switch

of REM generation. REM-on cells believed to be responsible for REM sleep modulation (such

as those in the eVLPO) have been shown to send GABAergic projections to cells that are

considered to be REM-off, such as several monoaminergic groups involved in arousal [113,

133].

4 Neural Correlates of Breathing

At the turn of the nineteenth century, French scientist Julien-Jean-Cesár Legallois found that the

centre for respiration was localized in the medulla of the brainstem by methodically lesioning the

midbrain, pons and medulla [142, 143]. More than two hundred years later, it has been well

established that respiratory neurons that are required for rhythmic respiratory motor activity are

found in the medulla and pons. These discrete centres are known as the dorsal and ventral

respiratory groups in the medulla (DRG and VRG, respectively) and the pontine respiratory

group (PRG). Respiratory neurons have also been identified in the lateral tegmental field of the

reticular formation in the medulla (see Figure 1.2).

Neurons in the PRG are not essential for generating respiratory rhythm, but are responsible for

stabilizing respiratory rhythm, and exert a tonic control on the length of both inspiration and

expiration [144, 145]. The PRG contains the nucleus parabrachialis medialis and the Kölliker-

Fuse nucleus in the dorsal lateral pons and studies in cats suggest they receive projections from

medullary respiratory neurons in the DRG and VRG. Some neurons located in the PRG send

projections back to medullary respiratory groups, while many neuron located in the Kölliker-

20

Fuse nucleus send direct projections to the phrenic motoneurons controlling the diaphragm [144,

146, 147].

Both the DRG and the VRG are concentrated, bilaterally, in the medulla-oblongata. The DRG is

constituted of afferent relay neurons in the ventrolateral nucleus tractus solitarius [148, 149].

The neurons located in the DRG receive input from slowly-adapting pulmonary stretch receptors

as well as the carotid bodies that sense changes in pH and oxygen saturation [150, 151]. These

neurons then send projections to motoneurons innervating primary and secondary respiratory

muscles such as the diaphragm and intercostals, respectively [144, 148, 152, 153].

21

Figure 1.2: A schematic representation of the location of the main groups of respiratory neurons involved in rhythm generation of modulation. Illustration represents the mammalian brainstem (medulla and pons) and spinal cord. The transverse section is meant to approximate the level of the obex illustrated in the coronal section. Main respiratory groups are located in the VRG, DRG, and PRG. Abbreviations: nA, nucleus ambiguus; nVII, facial nucleus; nXII, hypoglossal nucleus; nTS, nucleus of the solitary tract; DRG, dorsal respiratory group; PRG, pontine respiratory group; RVLM, rostral ventrolateral medulla; pFRG, para-facial respiratory group; VRG, ventral respiratory group; and C4, corresponding segment of the spinal cord. From Duffin (2004). [144]

22

The VRG consists of a bilateral longitudinal column extending between the retrofacial nucleus

and the cervical spinal cord and includes important structures such as the Bötzinger and pre-

Bötzinger complexes, retrofacial nucleus, nucleus ambiguus and nucleus retroambiguus [147].

The VRG contains both inspiratory and expiratory neurons, located in the rostral and caudal

parts of the VRG, respectively. The inspiratory VRG neurons send projections primarily to the

motoneurons innervating the diaphragm and those controlling the external intercostal muscles.

Cells in the pre-Bötzinger complex (PBC) of the VRG have been shown to have intrinsic

rhythmicity in slice preparations from neonatal animals in vitro, and they exhibit pacemaker

activity when synaptic input is removed [154, 155]. Whether this pacemaker region is the source

of rhythm generation in the mature animal in vivo is the subject of some debate [153, 156-158].

There are two main competing models of respiratory rhythm generation: the “pacemaker” model

and the “network model”.

Evidence supporting the hypothesis that the PBC is involved in respiratory generation comes

primarily from studies using in vitro slice preparations and en bloc preparations, which include

an intact brain and spinal cord [159, 160]. In vitro slice studies have isolated a small area of the

ventrolateral medulla encompassing the PBC, and these cells generate a rhythm that can persist

with the pharmacological blockade of synaptic inputs from other medullary regions [154-157].

This respiratory rhythm is indistinguishable from respiratory rhythms generated in en bloc

preparations, a preparation involving both the brain and spinal-cord. However, this type of

study has been criticised on the basis that the rhythm generated may not accurately reflect what

is observed in intact animals. Supporters of these results could argue that the pattern differences

are likely due to the absence of peripheral and descending inputs in the slice preparations [156].

In an intact animal, near-complete ablation of a subclass of PBC neurons (those that contain

neurokinin 1 receptors) produces an extreme form of ataxic, disordered breathing that is

irreversible [158]. The deterioration in breathing occurs over a number of days and is first

evident in REM sleep, then NREM sleep, and finally wakefulness. While this seems to indicate

that the PBC does play an important role in maintaining a constant and predictable respiratory

23

rhythm (i.e. eupneic breathing) in vivo, it does not necessarily demonstrate that these are the only

cells involved in rhythm generation [158, 161].

A thin layer of respiratory neurons was found in the cat using retrograde tracers and labelled the

retrotrapezoid nucleus (RTN); this region may be anatomically the same as the region known as

the parafacial nucleus [159, 160]. The RTN lies between the facial nucleus and the ventral

surface of the medulla, and consists of glutamatergic neurons that are chemosensitive [160, 162-

164]. Cells in the RTN have been suggested to have rhythm generating properties [165], but

have been shown to discharge several hundred milliseconds prior to those that produce

inspiratory bursts in the PBC. For this reason, they are known as “pre-inspiratory” neurons,

although it can be argued that they would be better called “expiratory” rhythm generating cells.

The cells are active prior to inspiration, are actively inhibited during inspiration (when PBC cells

are active), and are again active for a short period post-inspiration/early expiration [165, 166].

The cells have been shown to have chemosensitive properties and project to central pattern

generators, leading to the suggestion that they provide tonic input that allows respiratory

generators (ie. respiratory oscillators) to function and also receive synaptic feedback from pattern

generating cells [162, 163, 167, 168]. One hypothesis is that the PBC and RTN are both

involved in rhythm generation and that these centres are coupled [169, 170]; in this model, the

RTN, like the PBC, is an oscillator involved in rhythm generation. According to this hypothesis,

these oscillators are coupled but responsible for different roles in respiratory rhythm generation

with the PBC mainly responsible for inspiration and the RTN for expiration [165, 169]. The

“Network Hypothesis” relies on mutual inhibition between inspiratory and expiratory neurons.

While it is unclear if synaptic inhibition is necessary for rythmnogenesis, glycinergic and

GABAergic inhibition is believed to be involved in modulating respiratory rhythm [153, 171-

173] . Despite a persistent rhythm generated by PBC cells in vitro, evidence from in vivo studies

seems to support network models. It is possible that the way respiratory rhythm is generated

changes across development, perhaps explaining discrepancies between in vitro and in vivo

studies. For instance, neonatal and juvenile animals (often used for in vitro slice preparations)

may rely more heavily on pacemaker neurons to generate a rhythm while adults may have a

network of cells generating the respiratory rhythm [156, 174].

24

5 Breathing During Sleep

Breathing is significantly affected during both NREM and REM sleep. During NREM sleep,

there is a decrease in sensitivity to respiratory stimuli and motor output to some respiratory

muscles, such as the upper airway muscles, is suppressed, with a further suppression occurring

during REM sleep [147, 175-178]. The brainstem neurons believed to be involved in respiratory

rhythm generation and modulation also receive input from areas involved in wakefulness. The

activity of medullary VRG and DRG neurons significantly decreases during NREM sleep [179,

180]. However, in comparison to NREM sleep, activity of medullary and pontine respiratory

neurons increases in REM sleep [175, 181-183].

The change in activity of respiratory neurons, produced by changes in input from arousal centres

and chemoreceptors, results in breathing that is characteristic of each vigilant state [13, 176, 184,

185]. As compared to wakefulness, NREM sleep is usually accompanied by a decrease in

respiratory tidal volume. Diaphragmatic activity is not significantly reduced during sleep so

while the frequency remains statistically unchanged, the resistance caused by a reduction in tone

in the airways results in reduced tidal volume, and mean ventilation. The respiratory pattern

during NREM sleep is defined by even and regular breaths, both in frequency and amplitude. On

the contrary, REM sleep is characterized by erratic breathing, comprising irregular frequency and

amplitude [175, 181-183, 185-187]. Hypoventilation during sleep has been suggested to be

tightly linked to neural mechanisms that control sleep itself [147]. The changes in respiratory

activity during REM sleep are accompanied by a further suppression in the patency of upper

airways as compared to wakefulness and NREM sleep. As a result, obstructive apnoeic events

can occur most readily during this stage of sleep [13].

25

In addition to input from respiratory generation centres and chemosensors, respiratory

motoneurons receive tonic excitatory input from arousal centres; this control over respiratory

motoneurons is known as the “wakefulness drive” to breathe [176, 188]. During NREM sleep,

this drive is removed and respiratory muscle activity decreases. However, not all respiratory

muscles are affected by the removal of this input in equal proportions: the phrenic motoneurons

driving the diaphragm are less affected by the loss of the wakefulness drive to breathe because

they are mostly driven by central respiratory inputs [185, 187-191]. In fact, diaphragmatic

activity in REM sleep increases compared to NREM sleep and resembles that seen in active

wakefulness [187]. The upper airway is surrounded by skeletal muscles and soft tissue, which

makes behaviours such as speaking, suckling and swallowing possible. However, these

properties also make the airway susceptible to collapse during periods of decreased patency (i.e.

during sleep) [5, 13, 192]. On average, activity in pharyngeal muscles such as the genioglossus

muscle of the tongue is known to decrease in NREM sleep and even more so during REM sleep

(apart from transient events), and this activity is influenced by a number of neuromodulators

across the sleep-wake cycle [71, 193, 194]. Major suppression of genioglossus muscles has been

demonstrated to occur during REM sleep in comparison to wakefulness and NREM sleep, even

with respiratory stimulation by CO2 [191]. Diaphragmic activity is minimally affected by sleep

and so continues to generate inspiratory efforts; thus, when airway size and patency is reduced, a

higher resistance to airflow is produced. Moreover, negative pressure can cause flaccid airways

to collapse due to suction and thus may predispose people with narrow airways to sleep-related

breathing disorders such as OSA. It is essential that this airway remain open for effective

ventilation, so a decrease in airway motor tone that does result in airway collapse is a causal

factor in some sleep-related breathing disorders [71].

The withdrawal of endogenous noradrenaline has been shown to suppress both tonic and

respiratory-related genioglossus activity, which may account at least in part for the suppression

of this airway muscle during NREM sleep. However, it alone is insufficient to explain the

suppression seen in REM sleep [195, 196]. Endogenous serotonin was also found to play a

minimal role in normal modulation of genioglossus activity [197]. Withdrawal of serotonin and

noradrenaline input does disinhibit cholinergic neurons which, in turn increases ACh in pontine

reticular formation, promoting REM sleep. Injection of the cholinergic agonist carbachol into

26

the pontine reticular formation is used to produce many of the electrophysiological signs of REM

sleep, but does not produce the entire range of cortical and respiratory events normally

associated with the state [71]. For example, respiratory rate is attenuated and more regular with

injections of carbachol as compared to natural REM sleep, which is highly variable and at times,

quite rapid [175]. The depressant effects of carbachol injections into the pons are more strongly

exerted on hypoglossal, vagal pharyngeal and external intercostal nerve activities than on phrenic

and internal intercostal nerve activities, which can be the result of the loss of tonic influences

that may have stronger effects on the former group of respiratory nerves than on the latter [175].

Cholinergic input to hypoglossal neurons may mediate a component of genioglossal suppression,

but the exact role and mechanisms by which cholinergic input induces suppression have not yet

been determined.

Studies utilizing carbachol models of REM sleep have not only provided a basis for examining

the role of noradrenaline and serotonin, but also of glycine and GABA in the inhibition of spinal

motoneurons in the generation of muscle atonia [194]. Microdialysis of GABA and glycine

agonists at the hypoglossal motor nucleus causes suppression of genioglossus activity which can

be significantly reversed by perfusion of receptor antagonists. This suggests some physiological

role for these inhibitory transmitters in the suppression of genioglossus activity but does not

identify their role to any state. Studies in freely-behaving animals indicate that the contributions

of glycine and GABA to motor control differ from NREM to REM sleep. Data suggest that the

hypoglossal and trigeminal motor pools are under tonic GABAergic and glycinergic inhibition

during wakefulness and NREM sleep, but that these inhibitory amino acids do not contribute to

the major suppression of hypoglossal and trigeminal motor activities during REM sleep [71, 194,

198-201]. Nevertheless, the results of experiments that antagonize GABA and glycline at the

hypoglossal motor nucleus do suggest a role for the inhibitory transmitters in regulating transient

motor events during REM [71, 194, 198-201].

GABA is of particular interest because it not only suppresses motor activity during wakefulness

and NREM sleep but as previously mentioned, is also proposed to play a critical role in the

generation of sleep (Please see Chapter 1, Section 3 of this thesis). Furthermore, GABA

27

receptors are the target of many sedative drugs such as benzodiazepines, alcohol, anaesthetics

and barbiturates [202-206].

6 Introducing GABAA Receptors

Gamma-aminobutyric acid is the chief inhibitory neurotransmitter in the mammalian nervous

system. GABA synthesis is promoted by L-glutamic acid decarboxylase, the rate limiting step,

and is stored in presynaptic vesticles, from where it is released by stimulation of GABAergic

neurons [81, 203, 207, 208]. After the release from presynaptic butons, the molecule then

diffuses into extracellular space where it acts on postsynaptic GABA receptors of adjoining

neurons, or on extrasynaptic receptors on near-by neurons [81, 207, 208]. Tonic levels of GABA

in the extracellular space, regulated by the balance between its release and uptake, are known to

regulate a variety of neurological states. Excessive GABA can lead to a variety of cognitive and

functional impairments including sedation, amnesia, and ataxia; low GABA levels may produce

anxiety, restlessness, insomnia, and exaggerated reactivity [81, 208, 209].

There are two main types of GABA receptors: fast-acting ionotropic receptors and slow-acting

metabotropic receptors. Fast-acting receptors, GABAA and GABAC receptors, are chloride (Cl-)

channels which mediate synaptic inhibition. These channels consist of five subunits arranged

around a channel pore, making them part of a superfamily of ligand-gated ion channels, which

also includes nicotinic acetylcholine, strychnine-sensitive glycine and serotonin-3 receptors [81,

210].

Slow-acting receptors, GABAB receptors, are G-protein-linked and coupled by intracellular

proteins to calcium (Ca2+) and potassium (K+) channels. When GABA binds to these receptors,

K+ conductance is increased and voltage-dependent Ca2+ currents are reduced, which produce a

28

chloride influx and neuronal hyperpolarization [81, 211, 212]. This hyperpolarization reduces

the likelihood that any further stimulation of the cell will be sufficient to increase the membrane

voltage enough to reach the threshold required for an action potential, thus reducing neuronal

activity and excitability [208, 212].

GABAA receptors are the most common type of GABA receptors, found within approximately

50% of all synapses [203, 208, 212, 213]. The primary binding site on these receptors

accommodates the GABA molecule, however they also contain additional allosteric binding sites

for ligands such as benzodiazepines, alcohol, volatile anaesthetics and barbiturates [203-205].

Allosteric modulators of the GABAA receptors induce a conformational change once bound in

the presence of GABA, thus increasing Cl- flux and enhancing the inhibitory effects of the

receptors. Which ligand binds allosterically depends on the subtypes present within the receptor.

These receptors have five subunits and thus several possible isoforms, found in different regions

of the brain: α1-6, β1-4, γ1-3, δ1-3, ε and σ [203, 208, 212, 213]. The most common

combination of these isoforms is α1/2β2γ, which contains the benzodiazepine binding site.

Benzodiazepines are GABAA receptor modulators that lower the concentration of GABA

required to open the channel and allow for an influx of Cl-; they bind at the α-γ junction, while

GABA binds at the α-β junction [206, 208]. Ethanol, while it will bind to benzodiazepine

binding-sites, has been shown to affect less-common isoforms α4/6β3δ even at very low

concentrations in a number of in vitro and in vivo studies [33, 34, 214-221] (see Figure 1.3). The

effect of ethanol on α4/6β3δ receptors is believed to play a role in a number of effects of ethanol,

including the sedating effects and motor impairment experienced with intoxication [219].

While the most common receptor subunits are widely distributed throughout the brain, neuronal

specificity does exist, particularly among the less common subunits. Immunohistochemical

studies have identified and localized subunit expression throughout the brain [210, 213, 222].

For example, the α4 and δ receptor subunits, often paired, are more highly concentrated in the

thalamus, hippocampus, olfactory tubercle and the basal ganglia. The α6 subunit, however, is

exclusively paired with δ and found only in the cerebellum [223-225]. Extrasynaptic α6βδ

GABA receptors in the cerebellum have been implicated in the impairment of motor function

29

caused by sedative-hypnotic drugs such as ethanol [206, 208, 224-226]. Importantly, genetic

studies allowing for the knock-in or knock-out of specific amino acids can establish an increased

affinity at receptor binding sites or can create non-functional receptor sites. This type of genetic

manipulation can help identify which subunits are affected by GABAA receptor modulators such

as benzodiazepines and ethanol [206, 219, 220].

30

Figure 1.3: A schematic diagram of a typical trans-membrane GABAA receptor. GABAA receptors consist of five subunits, made of various combinations of at least 19 different glycoprotein subunits. There is a primary binding site for the GABA molecule (A), and a secondary site for receptor modulators (M). Ethanol binds to receptors with high affinity to receptors that include δ subunits. Benzodiazepines bind to receptors that include the much more common γ2 subunit. When GABA molecules bind, a chloride channel is open, resulting in hyperpolarization of the cell.

31

7 The Effects of GABAA Modulators on Sleep and Breathing

7.1 Effects of GABA Receptor Modulation on Sleep Neuronal excitability in the central nervous system is known to be regulated by fast, transient

inhibitory postsynaptic currents via GABAA receptors [219, 227, 228]. In addition to inhibition

via post-synaptic transmission, several regions of the brain are under constant regulation by

persistent tonic inhibition, conducted via extrasynaptic receptors [206]. These include the

cerebellum [206], thalamocortical relay neurons of the ventral basal complex [226], neocortex

[229], as well as several types of hippocampal neurons and interneurons [230]. Tonic GABA

inhibition has been studied using patch-clamping techniques in vitro [231-235], as well as in in

vivo experiments [234, 236]. Tonic GABA conduction causes a change in resting membrane

voltage and also decreases the membrane time constant of neurons so there is less spatial and

temporal summation of the excitatory signals they might receive, all of which make it more

difficult for excitatory inputs to elicit action potentials from inhibited cells [237].

Neuromodulating agents can bind to GABAA receptors and increase their binding affinity to low

levels of ambient GABA in extracellular space [206]. Benzodiazepines [206], alcohol [33, 34,

219, 220, 238, 239], inhaled and intravenous general anaesthetics [240-243] , and neurosteroids

[244-246] have all been proposed to depress the activity of the central nervous system via

GABAA receptors [247].

As previously mentioned, GABAA receptors are dispersed widely throughout the brain and vary

in subunit constitution, so it is difficult to determine precisely how GABA agonists and

modulators may induce sleep. However, the importance of GABAergic input to the cerebral

cortex and thalamus have been emphasized as likely sources of action, as well as the indirect

activation of the VLPO [72, 202, 206, 248]. Sleep-promoting neurons in the VLPO are under

tonic inhibition during wakefulness by input from wakefulness promoting regions in the

hypothalamus and brainstem [45]. Studies suggest that GABA receptor agonists may, in part,

32

promote sleep by inhibiting wakefulness-related neurons, particularly in the TMN and LC [72].

This would disinhibit GABAergic neurons in the VLPO, having an overall result of

thalamocortical suppression [44, 72, 248]. Studies have demonstrated that administration of

sedative gaboxadol resulted in an increase in slow-wave EEG patterns and an increase in delta-

power [72]. Concurrent with this was an increase in c-fos expression in VLPO neurons and a

decrease in c-fos expression in the cortex [72].

Not all sedative-hypnotic drugs, however, promote sleep via the same pathways [206].

Different sedative agents interact with different subtypes on GABAA receptors, which, in turn are

concentrated in different areas of the brain including the hypothalamus, thalamus, and cortex.

The effects of benzodiazepines are mediated mainly through α1 subunits, which are widely

dispersed. They are abundantly found in the thalamus, cerebral cortex, and TMN, on which they

may act to disinhibit VLPO neurons [206]. Extrasynaptic receptors containing α4 and δ subunits

are sensitive to positive allosteric modulation by gaboxadol, neurosteroids and ethanol; these

receptors show much more regional specificity and are mainly located in the thalamus. These

drugs act mainly to increase tonic GABA conductance in areas involved in sleep processes [206].

Extrasynaptic receptors are now being strongly promoted as important targets for sedative-

hypnotic drugs [206, 248].

7.2 Effects of GABA Receptor Modulation on Breathing Systemic administration of sedative agents have been shown to suppress motor drive to the

genioglossus muscle more than the diaphragm in both humans and animals [30, 249, 250],

potentially, by directly inhibiting motoneurons located in the hypoglossal motor nucleus. In

vitro experiments have shown that GABA, as well as glycine, exerts inhibitory effects on

hypoglossal motoneurons [251, 252]. Similar effects have been observed by microdialysis of

GABAA receptor agonists in both anaesthetized and freely behaving animals in vivo, clearly

demonstrating some level of inhibitory control on motoneurons located directly in the

hypoglossal motor nucleus [198, 199, 253, 254]. However, while both GABA and glycine

receptors are present at the hypoglossal motor nucleus and while GABAA receptor modulators do

33

affect motor activity, in vitro studies suggest that the majority of inhibition at the hypoglossal

motor nucleus is mediated by glycinergic inputs [255, 256]. Furthermore, the degree to which a

GABAA receptor agonist or modulator affects hypoglossal motor activity likely depends on the

identity and relative specificity of GABAA receptors subunits distributed within the motor pool.

Importantly, the experiments mentioned above look at the effect of GABAA receptor modulating

agents at a discrete location, namely, the hypoglossal motor nucleus. Anaesthetics and strong

sedatives, such as propofol, pentobarbital, and high concentrations of benzodiapezines, used

during surgical procedures have been known to cause respiratory depression in humans and

animals, which must be closely monitored [257-259]. The overall effects that more commonly-

used sedatives have on airway muscles have been studied with mixed results. Diazepam, a long-

acting benzodiazepine, has been shown to cause a selective reduction in genioglossus activity

compared to diaphragm activity, as well as a reduction in the ventilatory response to hypoxia in

healthy humans, and to hypercapnia in older human adults [260, 261]. In vagotomised

decerebrate, and artificially ventilated cats, diazepam selectively decreased hypoglossal nerve

activity and genioglossus muscle tone, having no effect on diaphragmatic activity [250], with

similar results found using sodium pentobarbital [249] and ethanol [249]. Other studies have

reported that obstructive apnoeas can be induced or exacerbated in human patients with OSA by

the administration of diazepam, flurazepam, and other benzodiazepines [262-265]. These studies

suggest that GABAA receptor modulating agents may worsen or exacerbate sleep-disordered

breathing, particularly in patients already with severe OSA.

However, more recent studies in patients with less severe OSA have indicated that this may not

always be the case. Studies of individuals with mild or moderate sleep apnoea showed oxygen

desaturation did not significantly change with sedative-hypnotic drugs, and many showed there

was no change in the frequency of apnoeas [266, 267]. Those studies that did show a minimal

increase in apnoea frequency also showed that these apnoeas were not accompanied by a

decrease in oxygen saturation [267-270]. Other studies in humans suggested that contrary to

widely-held clinical dogma, arousals from sleep may not be detrimental in all patients; in fact,

while arousals open the airway during an obstruction, they have been shown in some patients to

34

promote airway instability and recurrent arousals [271, 272]. Thus it was hypothesized that

some sedative may help stabilize breathing in certain patients selected for low thresholds for

arousals from sleep. Arousals are not absolutely necessary for airway re-opening following an

airway obstruction [271], and so sedatives may stabilize breathing and improve genioglossus

motor tone by allowing muscles to mount an appropriate compensatory response. A recent study

in rats demonstrated opposing effects on genioglossus muscle activity with systemic

administration of common hypnotics as opposed to local application directly to the hypoglossal

motor nucleus. In that study, an increase in arousal threshold during increasing levels of CO2

was accompanied by an increase in genioglossus activity compared to baseline with systemic

administration of GABAA agonists [254]. Local application at the hypoglossal motor nucleus of

the same agonists caused inhibition of genioglossus activity. Results showed that delayed

arousal from sleep following systemic administration of sedative GABAA receptor-modulating

agents can allow accumulating respiratory drive produced by hypercapnia to increase

genioglossus activity, and overcome the local inhibitory effects of GABAA receptor inhibition at

the hypoglossal motor pool [254].

Still, it is worth noting that not all GABAA receptor agonists or modulators have similar neuronal

targets or effects. Systemically administered alcohol, which also has sedative effects, has been

shown to selectively reduce genioglossus activity in both humans and animals [30, 249].

8 Ethanol in the Body

Ethanol - the type of alcohol commonly found in beverages - is one of the most widely used legal

drugs in society [273]. Alcohol intake in moderation typically has few serious consequences and

low doses of alcohol have even been suggested to have some beneficial cardiovascular effects

[274]. However, at higher doses, the consequences associated with alcohol ingestion can range

from mild cognitive and temporary motor impairment to liver damage and cardiovascular

35

problems seen in chronic abusers [275-277]. Intoxication is generally biphasic in nature, with a

potent sedation following a brief period of stimulation [278, 279]. However, increasing doses of

ethanol cause more significant motor and cognitive impairment which, anecdotally, is easily

recognizable and which has been studied mechanistically in both humans and animals [279].

Different alcoholic beverages contain different quantities of alcohol: beers contain approximately

3 – 8% alcohol by volume, while wines typically contain 8 – 15%, and liqueurs and spirits may

contain between 20 – 60% [279]. Ethanol is a hydrophilic compound that is readily absorbed

through the gastrointestinal tract and while some may be excreted through urine or evaporated on

breath, approximately 90% of ethanol ingested is oxidized in the liver [280, 281]. Accurate

blood alcohol levels can be analyzed from biological fluids, such as urine or blood, in a number

of ways. The methods of analysis are largely similar, but for the purposes of this discussion, I

will focus on blood alcohol analysis as this method was performed in this thesis. Most methods

are based on the principle of alcohol oxidation. Alcohol dehydrogenase is an enzyme that reacts

with alcohols, such as ethanol, and transfers a hydrogen atom to nicotinamide-adenine-

dinucleotide (NAD), producing acetaldehyde and NADH. The acetaldehyde is then further

oxidized to acetic acid, and finally to CO2 and water [279, 280]. Fluorescence

spectrophotometry, an older method, measures the product species of a known reaction at a

known band of UV light; in this case, NADH is measured, using light at a frequency of 340 nm.

Gas chromatography has also been used, a technique that separates and measures compounds,

such as ethanol, from biological fluids [279]. More recently developed techniques also rely on

principles of alcohol oxidation. However, such methods use a known reagent such as alcohol

oxidase and measure the rate of oxygen uptake, which should be proportional to the

concentration of ethanol in the blood. Once determined, blood alcohol concentrations can be

expressed in a number of units including millimolar (mM), milligrams per decilitre of blood or

plasma (mg/dL), or percentage (%). As an example, the maximum amount of blood ethanol

legally allowed in the blood stream while driving is widely taken as 0.08%, but this value also

corresponds to an approximate concentration of 17.4 mM [ethanol] and 80 mg/dl (see Table 1.1).

36

The elimination of drugs in the blood is normally characterized by the “half-life”, the time it

takes for the concentration of a drug or substance in blood plasma to be reduced to half its

starting concentration. Unlike many other drugs, the decay or elimination of ethanol in the blood

is relatively constant [279, 281]. A blood alcohol curve can be plotted based on blood ethanol

levels taken at several time intervals. Once ethanol equilibrates within the body, the rate of fall

in blood ethanol levels is, for the most part, taken as the slope of the linear part of the blood

alcohol curve. The “β-value” represents the rate of ethanol elimination from the blood (see

Figure 1.4). For humans, the average rate of elimination of alcohol in the blood is approximately

100 mg/kg/hr or 0.015 %; for rats, it is approximately 300 mg/kg/hr or 0.045 % [279, 282].

Metabolism of ethanol does depend on a number of factors including, but not limited to, the sex

of the consumer, weight, the rate at which alcohol is consumed, and the food content within the

stomach. As previously described, Alcohol is mainly metabolized by enzymes, alcohol

dehydrogenase, which convert ethanol to acetaldehyde, then to the non-toxic Acetyl-COA by

acetaldehyde dehydrogenase [279, 280, 283].

37

Table 1.1: Blood ethanol levels and associated behavioural effects. Taken from [276, 277, 279,

283-289]

Percent mg/dL mM Behavioural Effects

0.02 – 0.03 20 – 30 mg/dL 4.3 – 6.5 mM - Few obvious effects.

- No loss of motor coordination.

- Slight elevation of mood.

0.05 – 0.06 50 – 60 mg/dL 10.9 – 13.0 mM - Feeling of well-being and relaxation.

- Sensation of warmth.

- Relaxed inhibitions and minor impairment of caution, reasoning and memory.

- Emotions intensified and behaviours slightly exaggerated.

0.07 – 0.09 70 – 90 mg/dL 15.2 – 19.5 mM - Euphoria.

- Slight impairment of speech, vision, balance, hearing and reaction time.

- Increased confidence.

Note: 0.08 %, or 17.4 mM, is the legal blood alcohol driving limit in most jurisdictions.

0.10 – 0.14 100– 140 mg/Dl 21.7 – 30.4 mM - Euphoria.

- Significant impairment of motor coordination.

- Impaired reasoning and judgement.

- Impaired speech (may be slurred), balance, vision, hearing and reaction time.

38

0.15 – 0.19 150 – 190 mg/dL

32.6 – 41.2 mM - Euphoria reduced and dysphoric symptoms appear (i.e. feelings of anxiety, depression, and restlessness).

- Gross impairment of cognitive and motor control, impaired reaction time.

- Blurred vision, incoherent speech extremely difficult, lack of balance.

- Above 0.16 %, dysphoria predominates and nausea appears.

0. 20 200 mg/dL 43.4 mM - Confusion, disorientation.

- Trouble standing.

- Nausea and vomiting.

- Risk of asphyxiation and choking on vomit (gag reflexes impaired).

- Extreme cognitive, motor and memory impairment.

- “Blackouts” may occur.

0.30 300 mg/dL 65.1 mM - Stupor.

- Little comprehension of whereabouts.

- Loss of consciousness possible.

- Respiratory depression.

0.40 400 mg/dL 86.8 mM - Coma possible.

- Level of surgical anaesthesia.

0.3 – 0.6 300 – 600 mg/dL

65.1 – 130.2 mM

- Death possible (due to respiratory arrest).

39

Figure 1.4: Ideal blood alcohol curve. There are four phases to a blood-alcohol concentration curve: A, absorption phase; B, plateau; C, diffusion/equilibrium phase; D, elimination phase. The “β” factor characterizes the rate of elimination of alcohol in the blood, and is computed on the assumption that alcohol elimination proceeds at a uniform rate after alcohol has been equilibrated. Thus the fall of blood-alcohol concentration is an expression of the slope of the linear elimination phase of the curve. Based on Wallgren (1970). [279]

40

9 Ethanol: Where does it act?

Ethanol has been shown to act on a number of common receptors [290], inhibiting NMDA [36,

291, 292] and non-NMDA [293] glutamate receptors, and potentiating serotonin receptors [294-

296]. It has been shown to potentiate the inhibitory effects of glycine receptors and GABAA

receptors [38, 252, 297]. Ethanol has also been shown to affect G protein-coupled inward

rectifying K+ channels [39, 298]. The effect of ethanol on some of these common channels will

be discussed below.

9.1 NMDA and non-NMDA Glutamate Receptors Inhibition of excitatory NMDA channels has been suggested to contribute to both the neural and

cognitive impairment produced by ethanol ingestion [36]. For example, ethanol (5 – 50 mM) at

voltage clamped hippocampal cells inhibits ionic currents produced by the glutamate receptor

agonist NMDA in a dose-dependent manner. Studies employing electrophysiological and patch-

clamping techniques have demonstrated that ethanol acts by altering the gating of NMDA

channels rather than affecting conductance or permeance [36]. Other studies have shown that

concentrations of ethanol that produce anaesthetic effects (> 50 mM) are correlated with a

concentration-dependent inhibition of currents produced by glutamate receptor agonists kainate,

quisqualate and NMDA [291, 292]. These results suggest that this NMDA-receptor inhibition

produced by ethanol may contribute to its general anaesthetic effects. However results also

indicate that ethanol acts on other glutamate channels, namely kainate- and quisqualate-mediated

channels [291, 292]. In hippocampal interneurons, alcohol has been shown to strongly inhibit

the excitatory drive of kainate glutamate receptors [293]. Kainate receptors play an important

role in modulating the excitability of the hippocampus and ethanol has been shown to inhibit

these channels at physiologically relevant concentrations (5 – 10 mM). Studies suggest that,

mechanistically, ethanol increases excitability of hippocampal cells indirectly by inhibiting the

kainate-dependent drive of GABAergic interneurons [293, 299, 300]. In hippcampal slices,

ethanol inhibited synaptic currents of CA3 pyramidal neurons at concentrations as low 5 – 10

41

mM, concentrations that can be achieved in the blood after the ingestion of only 1 – 2 drinks.

CA1 interneurons have also been shown to be affected by ethanol, at concentrations of 20 – 80

mM [293]. Results seem to indicate that inhibition of kainate-mediated glutamate channels may

be involved in the intoxicating effects of ethanol.

9.2 Serotonin Receptors Ethanol also potentiates the action of serotonin on serotonin type 3 receptors and is supposedly

involved in ethanol intake and reward mechanisms, as well as symptoms of tolerance and

withdrawal [301]. The serotonin-3 receptor, found within brainstem, cortical and limbic

structures, is linked to cation channels conducting mainly Na+ and K+ and has been demonstrated

to influence dopamine transmission [302, 303]. In vivo experiments indicated that serotonin-3

receptor antagonists block the ethanol-potentiated increase in dopamine in the nucleus

accumbens [304]. Following the results of these studies, whole-cell patch clamp recordings

corroborated that ethanol can potentiate currents mediated by serotonin-3 receptors in a

concentration-dependent manner; given the location of these receptors on dopaminergic neurons

in reward-centres of the brain, authors suggest these mechanisms may be involved in the

“positive reinforcement”-effect of ethanol consumption [301, 303, 305] .

9.3 Glycine Receptors Glycine receptors have been purported to be involved in the effects of ethanol on everything

from perceived reward caused by intoxication to depressed breathing; these receptors have been

examined in regions from the amygdala to the hypoglossal motor nucleus. Glycine receptors are

located throughout the brain and spinal cord, found in the prefrontal cortex, hippocampus,

amaydala, hypothalamus, cerebellum, nucleus accumbens, ventral tegmental area, substantia

nigra, and many other areas [290, 306]. Single channel recordings have shown that ethanol

potentiates the effects of glycine receptors by increasing burst durations of channel conductance,

characterized by an increase in the length of time glycine channels remained open, and by

increasing the number of channels that open per burst; these effects were hypothesized to be

42

produced by a decrease in the rate of glycine unbinding. Electrophysiological studies also show

that 100 mM ethanol significantly decreases the EC50 value of glycine [307, 308].

It has also been suggested that glycine receptors in the nucleus accumbens are involved in the

effect that ethanol has on reward-behaviours by modulating dopamine circuitry. Dopamine

increases in the nucleus accumbens after ethanol ingestion. In one set of experiments, bilateral

infusion of glycine and strychnine into the nucleus accumbens had reciprocal effects on

dopamine in the nucleus accumbens, with strychnine preventing systemically administered

ethanol from activating dopaminergic pathways. [309] Bilateral infusion of strychnine

significantly decreased dopamine output despite an increased ethanol intake. Data from this

study further suggest that direct glycine infusion into the nucleus accumbens increases dopamine

output and reduces ethanol consumption [309]. The glycine re-uptake inhibitor, Org 25935,

produces a pronounced dose-dependent decrease in ethanol consumption in rats that further

suggests that glycine plays a role in the perceived “reward” resulting from ethanol consumption

[309-312].

Glycine is also believed to contribute to the general depressive effects of ethanol, and is believed

to contribute to such consequences of consumption as sedation [290, 313]. Studies have

demonstrated that at least some of the sedative effects of ethanol can be attributed to

manipulation of glycine receptors [306, 313, 314]. In one study, intraperitoneal injections of

ethanol (3.0 g/kg) induced a loss of righting reflex (LORR) in 100% of adult rats studied. This

measure of sedation was attenuated by subsequent subcutaneous injections of strychnine (up to

1.25 mg/kg) [306]. Furthermore, results indicated that strychnine shifted the dose-response

curve of ethanol-induced LORR to the right, both delaying the onset and decreasing the duration

of the period of sedation. Researchers do not suggest that glycine alone (i.e. without GABA) is

responsible for the sedative effects of ethanol, but rather that the results provide evidence that

glycine may be involved in the depression of brainstem neurons that is responsible for sedation

[306]. Similar results were obtain in experiments using mice [314].

43

Inhibitory amino acids have been shown to play an important role in the generation and

maintenance of the respiratory rhythm in mature organisms studied in vivo [171, 315, 316]. In

vitro studies have demonstrated a role for glycine receptors in ethanol’s attenuation of

hypoglossal nerve activity [252, 317], which may be clinically important when considering the

depressive effect of ethanol on breathing. Inhibitory amino acids GABA and glycine provide

significant tonic modulation of the hypoglossal motor nucleus in freely-behaving animals across

sleep-wake states as judged by the increase in genioglossus activity with the application of

GABAA and glycine receptor agonists [198, 199], likewise for the trigeminal motor pool [200,

318]. In vivo studies produced a graded suppression of genioglossus muscle activity with the

microdialysis of glycine that was reversed by the application of strychnine. This effect was

demonstrated both with and without respiratory stimulation by CO2 [253].

The effects of ethanol on respiratory-related hypoglossal nerve activity has also been studied in

vitro using neonatal brainstem slices [252] and reduced brainstem-spinal cord preparations [251].

In vitro studies indicate that there is glycine and GABAA-mediated inhibition of hypoglossal

nerve activity with bath applications of ethanol and this effect is antagonized with applications of

strychnine and bicuculline [256, 319-321]. Ethanol inhibits the amplitude and frequency of

respiratory-related hypoglossal nerve activity in a dose-dependent fashion, which has been

measured from the cut ends of hypoglossal rootlets in an in vitro transverse brainstem slice

preparation [252]. Applications of strychnine or bicuculline partially, but not significantly,

reversed the inhibition of respiratory-related activity; however, application of strychnine in

addition to bicuculline significantly blocked the inhibitory actions of ethanol. Likewise,

preincubation of both strychnine and bicuculline significantly blocked ethanol-induced inhibition

of respiratory-related hypoglossal activity [252]. This suggests that ethanol does modulate both

of these inhibitory receptors, at least in vitro, and that this is potentially a mechanism by which

ethanol depresses hypoglossal motoneurons in vivo. In in vitro experiments, it is possible that

ethanol directly enhances glycinergic and GABAergic inhibition of hypoglossal motor activity.

However PBC neurons, also contained in the slices, may also be a target [252]. While neither

glycine nor GABA play a major role in rhythm generation at perinatal ages, both are believed

play important modulatory roles. PBC neurons that are active in the late-inspiratory phase are

44

subject to inhibition during the inspiratory phase and so ethanol may cause respiratory

depression if it enhances glycinergic and GABAergic inhibition of these cells [252].

9.4 GABA Receptors Ethanol may alter the function of many membrane receptors and channels (see above

discussion); however, many in vitro experiments use bath concentrations upward of 100 mM,

which is a concentration that is fatal in humans. To put this observation in perspective, 50 mM

ethanol is an anaesthetic concentration, and the legal blood alcohol limit in most jurisdictions

corresponds to about 17.4 mM (≈ 0.08%) [34]. Virtually all cys-looped ligand-gated ion channel

receptors are modulated by >100 mM ethanol, which enhances the function of serotonin, glycine,

and GABAA receptors, and inhibits the function of nicotinic acetylcholine receptors [34, 36, 38,

294, 322, 323]. However, despite the widespread targets and diverse effects of ethanol in the

brain, GABAA receptors have been of particular interest in past years because they have been

shown to be affected by physiologically relevant concentrations of ethanol both in vitro [251,

252] and in vivo [33, 34, 214-221, 324]. In particular, the isoforms α4/6β3δ have been shown to

have a particular sensitivity to ethanol at low concentrations [33, 34, 214-221]. In fact, these

receptors are inhibited at such low concentrations that they have been termed ‘one glass of wine

receptors.’ The receptors have been found in the cerebellum, which may explain the adverse

effects of ethanol on motor control, and in the thalamus, which may contribute to the drug’s

sedating effects [206] .

Ethanol is thought to modulate the activity of the GABAA receptors by allosterically binding to

the receptor and increasing Cl- flux into cells even at low concentrations, causing neuronal

inhibition [34, 325, 326]. The role of GABA in the sedating and intoxicating effects of ethanol

was demonstrated by early experiments showing that GABAA receptor agonist muscimol

potentiated the sedating effect and motor-impairment caused by ethanol; administration of

GABAA receptor antagonists, bicuculline and picrotoxin, augmented these effects [327, 328].

Behavioural characteristics of alcohol-tolerant and alcohol non-tolerant rat lines have been

attributed to differences in GABAergic neurotransmission because not only do alcohol non-

45

tolerant rats show an increased sensitivity to ethanol, but also to other GABAA agonists

lorazepam and sodium pentobarbital [204, 324, 329].

As previously mentioned, the isoforms α4/6β3δ have been closely studied in recent years [216,

219, 220, 330]. Recombinant studies showed that the activity of GABAA receptors containing α4

or 6, β3 and δ were enhanced by ethanol concentrations as low as 10 mM, which would

correspond to a blood alcohol concentration below the legal driving limit (17.4 mM) [219].

Behavioural and electrophysiological studies in rats have shown that GABAA receptors

containing α6 and δ are critical targets of low-dose ethanol intoxication, and a single nucleotide

change in α6 from arginine to glutamine induces a marked increase in benzodiazepine

impairment of motor coordination. This α6 polymorphism enhances ethanol sensitivity to as low

as 0.75 g/kg (which would induce blood alcohol concentrations below the legal limit) and causes

more severe motor impairment as measured by cerebellar behavioural tests (i.e. rotarod test),

but not by non-cerebellar tests (i.e. measurements of the LORR) [219]. Knock-out mice lacking

a GABAA receptor δ subunit resulted in multiple deficits in the behavioural response to ethanol,

including reduced consumption, attenuation of withdrawal symptoms and reduced protection

from bicuculline-induced seizures [330]. In vitro studies, such as those conducted in

hippocampal dendate gyrus granule cells have also demonstrated the critical role of δ subunits by

showing that low ethanol concentrations known to affect humans selectively augment GABA-

mediated tonic inhibition by acting on δ subunit-containing receptors [33, 331]. Ethanol is

hypothesized to cause motor impairment by acting via α4/6β3δ receptors located extracellularly

on cerebellar granule cells, thus increasing tonic GABA conductance [219]; extracellular

receptors have also been implicated in mediating the sedative effects of ethanol, by enhancing

tonic GABA transmission [206].

The interaction of ethanol with GABAA-specific antagonists provides further evidence for the

critical role of GABAA in mediating behavioural effects induced by ethanol ingestion [34, 238,

332]. The GABAA receptor-active benzodiazepine Ro15-4513 specifically antagonizes the

effects of ethanol in vitro [238, 239] and reduces sedative, motor-impairing and amnestic effects

caused by ethanol in vivo [215, 238, 332-338]. Ro15-4513 significantly reduces motor

46

impairment and ethanol-induced sleep times, and attenuates ethanol-induced sedation measured

by LORR; however, Ro15-4513 does not significantly antagonize ethanol-induced hypothermia

[135]. These results indicate that GABAA may not be involved in mediating all pharmacological

effects of ethanol. This is consistent with the known distribution of highly sensitive α4/6β3δ

receptors, commonly found in the thalamus and cerebellum [206, 339, 340] (see Figure 1.5).

Based on this distribution, it is reasonable to hypothesize that GABAA receptors are most critical

in mediating ethanol’s effects on sedation and motor impairment at physiological relevant

concentrations.

47

Thalamus

Cerebellum

Thalamus Cerebellum

Figure 1.5: Immunocytochemical distribution of GABAA subunits that constitute receptors particularly sensitive to low doses of ethanol. Behavioural and genetic studies have suggested GABA receptors containing α4 or α6, and δ to be uniquely sensitively to low doses of ethanol. Those isoforms containing α4 are found highest in expression in the thalamus, and those with α6 are solely found in the cerebellum. From Pirker et al. (2000). [224].

48

10 Effects of Ethanol on Sleep and Breathing

The sedative effects of ethanol have long been recognized. Ethanol has been shown to not only

promote sleep by shortening sleep-onset latency, but increase measures of “sleepiness” during

the day [341-343]. Studies examining ethanol ingested at night-time showed, based on standard

measures of sleepiness/alertness used in human subjects, an increased tendency towards sleep

and reduced alertness at times of day when patients would normally feel alert [344]. Studies in

humans [342, 343] and rats [345, 346] have shown that ethanol alters sleep architecture,

increasing time spent in NREM sleep while decreasing time spent in REM sleep and

wakefulness. Studies in laboratory rats clearly demonstrate an overall increase in time spent in

sleep versus wakefulness, while also showing that the proportion of NREM to REM sleep is

increased with ethanol [345, 346]. Ethanol has also been reported to affect the EEG spectra by

increasing power in a number of low frequency bands (0 - 4 Hz, 2-4 Hz, and 4 – 6 Hz) in the

parietal cortex, characteristic of NREM sleep, in adult rats; ethanol was also reported to decrease

power in high frequency bands ( 16 – 32, 32- 50 Hz), markers of cognitive activity and arousal,

in the parietal and frontal cortices [347]. These changes may be indicative of a “deeper” sleep.

Changes in EEG spectra were accompanied by increased motor impairment [347, 348]. Ethanol

has clearly been shown to increase motor impairment and sedation as indexed by LORR [204,

219, 329, 347].

Clinical observations have also shown that ethanol ingestion exacerbates sleep-related breathing

disorders. Ingestion of ethanol increased the duration and frequency of apnoea episodes in

patients with OSA, also profoundly increasing the degree of oxygen desaturation in arterial blood

in the first two hours after ingestion. It has also been shown to induce airway obstructions in

people who normally experience only benign snoring in the same time period [28, 31]. Another

study reports that after having ingested alcohol, patients with OSA experienced a shorter latency

to severe obstructive events after sleep onset, an increase in the number and severity of hypoxic

events, and a decrease in the number of arousals [31]. Severity of the sleep-disordered breathing

has also been positively correlated to the amount of alcohol consumed [28]. Because the

49

frequency and duration of obstructions are increased, it has been postulated that both airway

motor tone is reduced and the arousal response to asphyxia is depressed under the influence of

ethanol [14].

The prolonged duration of apnoeic episodes induced by ethanol is a leading cause of the

increased and potentially dangerous oxygen desaturation that accompanies them. In healthy

adult humans, moderate doses of ethanol have been shown to increase the threshold of

inspiratory effort needed to elicit an arousal in response to airway occlusion; the rate of increase

in the magnitude of inspiratory effort is also reduced by ethanol. While the delay to arousal was

increased in all stages of NREM sleep, the effects of ethanol were more pronounced in stages 3

and 4 of NREM sleep (REM sleep was not studied) [29]. In addition, ethanol has been shown to

increase inspiratory resistance in both snoring and non-snoring men. Combined with increased

inspiratory efforts, it is possible that an ethanol-induced increased in airway resistance could

increase the propensity towards airway instability and collapse [349]. Together, these studies in

humans further suggest that both effects of ethanol on airway stability, as well as on sleep and

the arousal response, may contribute to the exacerbation of disordered-breathing during sleep.

Moderate doses of ethanol have also been shown to exert selective reduction of genioglossus

muscle activity in both healthy human subjects [30] and unanaesthetized, decerebrate cats [249].

Genioglossus activity was significantly reduced in both room air and CO2-stimulated breathing

in normal adults during quiet wakefulness. In decerebrate cats, intravenous ethanol caused a

decreased in hypoglossal and recurrent laryngeal activity while phrenic nerve activity remained

unchanged; intact, awake cats showed similar results, with ethanol depressing genioglossus and

posterior cricoarytenoid muscle activity but not diaphragmatic activity. Because the study

involved a group of decerebrate cats as well as cats with the carotid sinus nerve and vagus nerve

sectioned, results suggest that the suppression is not the complete result of higher cortical

structures, vagal afferents, or chemoreception [249]. Studies in both awake humans and cats

suggest that at moderate doses, ethanol more strongly inhibits motor activity in upper airways

compared to respiratory pump muscles such as the diaphragm [30, 249].

50

In both the clinical and research settings, ethanol has been shown to worsen sleep-related

breathing disorders and to suppress genioglossus activity. Yet mechanistically, the source of

genioglossus suppression is unknown. One possibility is that ethanol predominately acts directly

on motoneurons in the hypoglossal motor nucleus to suppress genioglossus activity. Another

possibility is that genioglossus activity is suppressed by state-dependent influences operating via

sleep/arousal processes (i.e. the same processes that promote sleep), possibly by inhibition within

reticular activating system [30, 249]. These two possibilities have not yet been differentiated.

Through its sedative properties, it is possible that altering sleep time, patterns, or depth may in

turn influence genioglossus activity by means of the normal process of suppression that sleep

exerts upon upper airways. This may occur in addition to or, or in fact instead of, suppression

that occurs directly on motoneurons if the drug acts on inhibitory receptors located on

hypoglossal motoneurons. To better understand the mechanisms by which ethanol worsens

sleep-related breathing disorders, it is necessary to differentiate between suppressive effects

acting via central motor inhibition and state-dependent influences. The current study is meant to

address these issues by examining the effects of both systemically administered ethanol and

ethanol applied directly to the hypoglossal motor nucleus in a rat model.

11 Summary and Rationale for the Study of State-Dependent Versus Central Motor Effects of Ethanol on Breathing

Sleep-disordered breathing, comprising repetitive obstructive apnoeas and hypopnoeas during

sleep, constitutes a common respiratory problem and significant public health burden [350, 351].

Ethanol is one of the most widely used drugs in western society [273], and one consequence of

ethanol ingestion in humans is impairment of breathing during sleep in otherwise normal

individuals, and significant worsening of breathing during sleep in those with pre-existing

hypopnoeas or obstructive sleep apnoea [25-27, 31, 352-355]. In these latter clinical studies,

ethanol typically increased both the frequency and duration of disordered breathing events [25-

51

27, 31, 355]. However, the physiological mechanisms underlying how ethanol can adversely

influence breathing during sleep are not well understood because combined measurements of

pharyngeal and respiratory pump muscle activities with indices of sleep/arousal regulation have

not been performed in human [25-27, 31, 352-355] or animal studies [345, 346, 356]. Also, the

influence of ethanol on respiratory motor activity when applied directly to a central respiratory

motor pool has also not been studied in vivo to differentiate between the potential central effects

of ethanol on respiratory motor activity versus state-dependent effects operating via influences

on sleep/arousal processes.

Ethanol exerts complex effects on the central nervous system, generally depressing neuronal

function, at least in part via interactions with the GABAA receptor and exerting a GABA-

mimetic profile [38, 357-359]. GABAergic neurons are importantly involved in the initiation and

maintenance of NREM sleep [42, 360, 361], and a major component of the sedative effect of

ethanol [29, 344] may be mediated via potentiating this sleep promoting (i.e., state-dependent)

system. Accordingly, one explanation for an increase in the incidence and duration of disordered

breathing events observed clinically after ethanol ingestion [25-27, 31, 355] may be due to

reduced arousal processes leading to increased time spent asleep and/or deeper sleep, so

contributing to hypopnoeas via primary state-dependent influences on the respiratory system

(i.e., independent of an influence of ethanol on the respiratory motor pool per se).

In addition to its GABAA receptor modulating properties, ethanol can also influence neuronal

activity via (among others) potentiation of inhibitory glycinergic neurotransmission and

suppression of excitatory glutamatergic receptor-mediated responses [36, 292, 293].

Accordingly, a second explanation for how ethanol can predispose to, and worsen, obstructed

breathing during sleep is that ethanol may directly suppress respiratory neuronal and

motoneuronal activity, leading to suppression of respiratory muscle tone, which for the

pharyngeal muscles would also increase the incidence and duration of obstructed breathing

events. Such a scenario contributing to hypopnoeas via a central effect of ethanol on respiratory

motor activity (i.e., independent of a state-dependent mechanism) is plausible because in vivo,

GABA, glycine and glutamate each exert tonic modulation effects at the hypoglossal motor pool

52

innervating the muscle of the tongue [362-367], and ethanol potentiates inhibitory GABA and

glycine receptor function at this motor pool when studied in vitro [308, 317, 368].

Overall, the study presented in this thesis tested the hypotheses that systemic administration of

ethanol, at a physiologically relevant dose, would promote sleep and produce other

electrophysiological signs consistent with sedation (Hypothesis 1), as well as alter breathing and

suppress genioglossus activity in a freely-behaving rat model (Hypothesis 2). In additional

experiments, we also tested the hypothesis that local application of ethanol to the hypoglossal

motor pool would suppress genioglossus activity (Hypothesis 3). To our knowledge this is the

first study to determine the effects of systemically administered ethanol on both sleep/arousal

processes and state-dependent respiratory motor activity, and also respiratory activity with local

application to a brainstem respiratory motor pool, to differentiate between potential state-

dependent and central motor effects of ethanol on breathing in vivo.

53

CHAPTER 2

METHODS

54

CHAPTER TWO

Methods

Procedures conformed to the recommendations of the Canadian Council on Animal Care and the

University of Toronto Animal Care Committee approved the protocols. Rats were housed

individually, maintained on a 12–12 hr light/dark cycle (lights on at 0700 hr), and had free access

to food and water.

Study 1: Blood Ethanol Levels in Conscious Rats

Blood ethanol levels were determined in a group of six male Wistar rats (mean body weight =

360g, range 297-425g) following intraperitoneal injection of ethanol (1.25 g.kg-1 in saline, 15%

volume for volume). For sampling, the inner thigh over the saphenous vein was shaved. Blood

samples (200µl) were drawn from the saphenous vein using a sterile 23¾ gauge needle, with the

rats being held and the hind limb extended. Samples were taken immediately before (i.e., time 0)

and after (30, 60, 90, 120, 180 and 240 minutes) ethanol injection. To obtain each sample, the

needle tip was inserted into the vein and then quickly removed, following which a sample of

blood was collected in heparinised vials and centrifuged at 2500 rpm. The plasma was then

collected, frozen and analysed for ethanol concentration (Analox Instruments USA, Lunenburg,

MA).

Study 2: Effects of Ethanol on Sleep and Respiratory Motor Activity in Freely Behaving Rats

55

Anaesthesia and Surgical Procedures Experiments were performed on ten male Wistar rats (mean body weight = 262g, range 234-

285g). Sterile surgery was performed under anaesthesia induced and maintained by inhalation of

2.5-3% isoflurane. Rats were also intraperitoneally injected with buprenorphine (0.03 mg·kg-1) to

minimize potential post-operative pain, atropine sulphate (1 mg.kg-1) to minimize airway

secretions, and saline (3 ml, 0.9 %) for fluid loading. An anaesthesia mask was placed over the

snout throughout surgery and the rats also breathed a 50:50 mixture of room air and oxygen.

Effective anaesthesia was judged by abolition of pedal withdrawal and corneal blink reflexes.

During surgery, body temperature was maintained with a water pump and heating pad (T/Pump-

Heat Therapy System, Gaymar, Orchard Park, NY, USA).

With the rats supine the ventral surface of the genioglossus was exposed via a submental incision

and dissection of the overlying geniohyoid and mylohyoid muscles. Two insulated, multi-

stranded stainless steel wires (AS631; Cooner Wire, Chatsworth, CA, USA) were implanted

bilaterally into the genioglossus and secured with sutures and tissue glue. Tongue movement in

response to electrical stimulation (0.4-0.8 V) was also used to confirm electrode placements. In

previous experiments, tongue muscle activity has been shown to be markedly decreased, and

almost abolished, after section of the medial branches of the hypoglossal nerve, indicating that

the electrode placement was such that recordings were predominantly from the genioglossus

muscle [365]. To record diaphragm EMG activity, two insulated, multi-stranded stainless steel

wires (AS636: Cooner Wire) were then sutured onto the costal diaphragm via an abdominal

approach. The size, configuration and placement of the genioglossus and diaphragm electrodes

were consistent across experiments. To further ensure adequate electrode placements during

surgery, both the genioglossus and diaphragm signals were monitored on loudspeaker (AM8

Audio Amplifier, Grass) to document respiratory-related activity. The genioglossus and

diaphragm wires were tunnelled subcutaneously to a small incision on the skull and the sub-

mental and abdominal incisions were closed with absorbable sutures.

The rats were then placed in a stereotaxic apparatus (Kopf Model 962, Tujunga, CA, USA) with

56

blunt ear bars. To record the electroencephalogram (EEG), two stainless steel screws (1.5 mm

diameter) attached to insulated wires (30 gauge) were positioned on the skull approximately 2

mm anterior and 2 mm to the right of bregma, and 3 mm posterior and 2 mm to the left of

bregma respectively [369, 370]. The reference electrode was placed approximately 5 mm

anterior and 3 mm to the left of bregma [369, 370]. Two insulated, multi-stranded stainless steel

wires were also sutured onto the dorsal neck muscles to record neck muscle activity [369, 370].

For schematic drawing of electrode placement, see Figure 2.1.

At the end of surgery, the electrodes were connected to pins inserted into a miniature plug (STC-

89PI-220ABS, Carleton University, Ottawa, Canada). The plug was then fixed to the skull with

dental acrylic and anchor screws. After surgery, the rats were transferred to a clean cage and kept

warm under a heating lamp until full recovery as judged by normal motor activity, drinking and

eating. The rats were given soft food for the first day after surgery. The rats were then housed

individually and recovered for a minimum of 5 days before the experiments were performed.

57

GG EMG

Diaphragm EMG

EEG Neck EMG

Figure 2.1: Schematic drawing of animal model. Rats were implanted with EEG and neck EMG electrodes, to determine sleep and awake states. Diaphragmatic and genioglossus (GG) electrodes were used to assess respiratory muscle activity.

58

Recording Procedures For recordings, a lightweight shielded cable was connected to the plug on the rat’s head. The

cable was attached to a counterbalanced swivel that permitted free movement. All rats were

studied in a recording chamber (PLY 3223, Buxco Electronics, Wilmington, NC, USA) located

inside a noise-attenuated cubicle free from any disturbance. The rats were supplied with fresh

bedding, food and water. For habituation, the rats were connected to the cable and electrical

swivel apparatus and placed in the recording chamber the day before the experiments.

The electrical signals were amplified and filtered (Super-Z head-stage amplifiers and BMA-400

amplifiers/filters, CWE Inc., Ardmore, PA, USA). The EEG was filtered between 1 and 100 Hz,

whereas the neck, genioglossus and diaphragm EMGs were filtered between 100 and 1000 Hz.

The electrocardiogram was removed from the diaphragm EMG using an oscilloscope and

electronic blanker (Model SB-1, CWE Inc.). The moving-time averages of the neck (time

constant = 50 ms), genioglossus and diaphragm EMGs (time constants = 100 ms) were also

obtained (Model MA 821, CWE Inc.). The raw EEG and genioglossus signals, and the moving-

time averages of the genioglossus, diaphragm and neck EMGs, were digitized and recorded on

computer (Spike 2 software, 1401 interface, CED Ltd, Cambridge, UK).

Protocol Experiments began at approximately 0930 hrs and were performed during the day when the rats

normally sleep. Prior to monitoring the rats were administered, by intraperitoneal injection,

either ethanol (15% volume for volume solution, in 0.9% saline) or the vehicle control

(corresponding to ~2.5 ml of 0.9% saline, the exact amount corresponding to the volume used for

the ethanol experiments which was determined by body weight). In vitro experiments are more

suited to compare the effects of ethanol to other forms of alcohol such as methanol and octanol;

for example, different types of alcohol might be used to determine the biochemical aspects of the

structure of the alcohol in mediating the observed effects [251]. However, this experiment only

investigated the effects of ethanol because this alcohol is the type found in commercial beverages

59

and is, therefore, relevant clinically. A dose of 1.25 g.kg-1 ethanol was chosen based on the

results past studies in both animals and humans. The justification for the chosen dose is two-

fold. First, doses of 0.75 - 1.25 g.kg-1 of systemically administered ethanol were shown to

produce significant impairment of motor function in behavioural testing in rats [219]. Ethanol

doses between 1.1 and 2.5 g.kg-1 caused a dose-dependent increase in total sleep time in rats

[345]. In humans, doses around 0.8 g.kg-1 have mild sedative effects, produce motor and

cognitive impairment, and increase the severity of sleep-disordered breathing [28-30, 276, 277,

371-374]. Secondly, a dose of 1.0 g.kg-1 produces a blood-alcohol concentration of

approximately 15 - 16 mM in rats [219]. This dose is physiologically relevant to the

concentration that is the legal driving limit in humans, 17.4 mM (Please see Chapter 1, Table

1.1). Thus, the justification for the chosen dose is based both on the effects that it has on motor

and cognitive functions in animals and humans, and on the approximate blood ethanol level that

it induces.

Each rat was studied with both ethanol and the vehicle control (i.e. a repeated measures design),

with the order of studies randomized and each study separated by at least 48 hours. Data

collection began 15 minutes after injection which, based on prior studies [282, 375, 376] and our

own measurements of blood ethanol (see Results), would correspond to approximately 10-15

minutes before the peak blood ethanol level given the dose administered. The rats were then

monitored for 4 hrs by the end of which the ethanol is largely cleared from the blood, based on

previous studies of the metabolism of alcohol in rats [282, 376] and our own measurements of

blood ethanol levels (see Results). After completion of the studies, the rats were re-anaesthetised

with isoflurane and tongue movement was tested again in response to stimulation of the

genioglossus electrodes prior to overdose of 5% isoflurane.

Data Analysis Each experiment was coded and the experimenter was blinded to the treatment (saline or ethanol)

during the analyses of sleep and respiratory motor activities. Sleep-wake states were identified

visually and classified using standard criteria [369]. Measurements of respiratory muscle

60

activities within the identified sleep-wake states were made during all periods of quiet

wakefulness (> 30 sec in duration, with these periods excluding any with body movements

including overt behaviours such as eating, drinking or grooming), NREM sleep (> 60 sec

duration) and REM sleep (> 30 sec duration). Data were included in the analyses of respiratory

activity only if they were obtained during such unequivocal and clearly defined states. Data

obtained during periods of active wakefulness (i.e., with movements and overt behaviours), and

transitional states (e.g., drowsiness, arousals from sleep and transitions from NREM to REM

sleep) were not included in the analysis of respiratory muscle activities. Periods of quiet

wakefulness without any body movements are typically shorter in duration than periods of

uninterrupted NREM sleep, and there are typically fewer periods of REM sleep compared to

these other states, which accounts for why periods > 30 sec were analyzed in quiet wakefulness

and REM sleep as opposed to > 60 sec in NREM sleep. Furthermore, the transition from

wakefulness to NREM sleep is typically a slower process than from NREM to REM sleep, which

is again why periods longer than 60 seconds were chosen [377, 378].

Quantification of respiratory muscle activities was performed as previously described [366, 369].

The EMG signals were analyzed from the respective moving-time average signals (above

electrical zero). The genioglossus signal was quantified as mean tonic activity (i.e. basal activity

in expiration) and respiratory-related activity (peak inspiratory activity - tonic activity). The

amplitude of diaphragm activity, respiratory rate and mean neck muscle activity were also

calculated. The tonic and respiratory-related genioglossus signals were quantified both in

arbitrary units and as the percent of maximum. The maximum genioglossus level in each rat was

determined from the evening of recording before the experiment the next day, i.e., after the

animals were hooked-up for the habituation period. This period corresponded to the normal time

of day with high behavioural activity.

The EEG was sampled by computer at 500 Hz then analyzed on overlapping segments of 1024

samples, windowed using a raised cosine (Hamming) function and subjected to a fast Fourier

transform to yield the power spectrum [366, 369]. The window was advanced in steps of 512

samples, and the mean power spectrum of the EEG signal for each 5 sec epoch was calculated. The

61

power contained within six frequency bands was recorded as absolute power and as a percentage of

the total power of the signal. The band limits were δ2 (0.5 - 2 Hz), δ1 (2 - 4 Hz), θ (4 - 7.5 Hz), α

(7.5 - 13.5 Hz), β1 (13.5 - 20 Hz) and β2 (20 - 30 Hz); the ratio of high (20-30 Hz) to low (0.5-2 Hz)

frequency activity was also calculated as a relative index of EEG activation [370].

Study 3: Ethanol at the Hypoglossal Motor Pool and Effects on Genioglossus Activity

Anaesthesia and Surgical Procedures Sixteen male Wistar rats (mean body weight = 293g, range 260-327g) were anesthetized with

isoflurane (2-3 %), and were given atropine (1 mg/kg) to minimize airway secretions. Following the

onset of surgical anaesthesia the rats were tracheotomized and the femoral artery and vein

cannulated. The rats spontaneously breathed a 50:50 mixture of room air and oxygen throughout the

experiments. Core body temperature was monitored with a rectal probe and maintained between 36-

38°C with a heating pad (TC-1000 Temperature Controller, CWE Inc., Ardmore, PA, USA). The

rats received continuous intravenous fluid (0.4 ml/hr) containing 7.6 ml saline, 2 ml 5% dextrose

and 0.4 ml of 1M NaHCO3. Bipolar electrodes were inserted into the genioglossus and costal

diaphragm for EMG recordings [365, 369]. The rats were then placed in a stereotaxic apparatus

(Kopf Model 962, Tujunga, CA) and two stainless steel screws attached to insulated wire were

implanted in the skull over the frontal-parietal cortex to record the cortical EEG as described above

for Study 2 [365, 369]. To ensure consistent positioning between rats the flat skull position was

achieved with an alignment tool (Kopf Model 944).

Microdialysis Perfusion and Recordings Microdialysis probes (CMA/11 14/01, CSC, St. Laurent, QC) were targeted into the hypoglossal

motor nucleus to infuse artificial cerebrospinal fluid (ACSF) followed by ethanol dissolved in

62

ACSF (n=10 rats), or continuous ACSF for the same time period (n=6 rats, time control

experiments). The probes were placed 13.7 ± 0.12 (SEM) mm posterior to bregma, 0.2 ± 0.04 mm

lateral to the midline and 10.1 ± 0.12 mm ventral to bregma. The rats stabilized for at least 30 min

before any interventions. The microdialysis probes were 240 μm in diameter with a 1 mm

cuprophane membrane. The probes were connected to FEP Teflon tubing (inside diameter =0.12

mm) that were in turn connected to 1.0 ml syringes via a zero dead space switch (Uniswitch, B.A.S.

West Lafayette, IN). The probes were continually flushed with fluid at a flow rate of 2.1 μl.min-1

using a syringe pump and controller (MD-1001 and MD-1020, B.A.S. West Lafayette, IN). The

composition of ACSF (mM) was NaCl (125), KCl (3), KH2PO4 (1), CaCl2 (2), MgSO4 (1),

NaHCO3 (25) and D-glucose (30). The electrical signals were amplified and filtered as for the

experiments in Study 2. Each signal, along with blood pressure (DT-XX transducer, Ohmeda,

Madison, WI and PM-1000 Amplifier, CWE Inc.) was also recorded on computer (as above).

Protocol and Data Analyses Interventions were performed during steady-state periods with predominantly high-voltage and low-

frequency EEG activity. In a group of ten rats, the microdialysis probes were perfused with ACSF

for at least 30 min followed by 25, 50, 100, 300 and 1000 mM ethanol, each for 30 min. To

examine if any of the potential changes in genioglossus activity were caused by effects of time per

se, i.e., independent of ethanol, further experiments were performed in a separate group of six rats in

which repeated switches to perfusion of ACSF into the hypoglossal motor nucleus were performed

(i.e., “sham interventions”) over the same time course as the drug interventions.

For ACSF or ethanol delivered to the hypoglossal motor nucleus, measurements were taken over 1

min periods at the end of each 30 min drug or sham intervention. Genioglossus and diaphragm

responses were measured from the moving average signals above electrical zero, as described in

previous experiments [365, 369] and for the above experiments in sleep (Study 2). In practice there

was no tonic genioglossus activity under anaesthesia, and so data are only reported for respiratory-

related activity.

63

Tests of Function of Hypoglossal Motor Nucleus and Histology

At the end of each experiment, 10mM serotonin (creatinine sulphate complex) was applied to the

hypoglossal motor nucleus as a positive control to confirm that it was still functional and able to

respond to manipulation of neurotransmission as judged by the expected increase in genioglossus

activity [379]. At the end of each study the rats were overdosed with isoflurane. The rats then were

perfused intracardially with 40 ml of 0.9% saline followed by 40 ml of 10% formalin, following

which the brain was removed and fixed in 10% formalin. Medullary regions containing the

hypoglossal motor nucleus were blocked and transferred to a 30% sucrose solution for

cryoprotection. The tissue was then cut in 50 µm sections using a cryostat (CM1850, Leica,

Nussloch, Germany). Sections were mounted and stained with neutral red, and the lesion sites left

by the microdialysis probes were recorded on a corresponding standard cross-section using a

stereotaxic atlas of the rat brain [380].

Statistical Analysis

The statistical test used for each analysis is indicated in the text where appropriate. For all

comparisons, differences were considered significant if the null hypothesis was rejected at P <

0.05. Where post-hoc comparisons were performed after analysis of variance with repeated

measures (ANOVA-RM), the Bonferroni corrected P value was used to infer statistical

significance. Data were tested for normality using the Kolmogorov-Smirnov test, and if not

normally distributed then analyses were performed on the log transformed data. Log

transformations are appropriate for positively–skewed data to produce a more normal

distribution [381]. Where data were significantly skewed, transformations were performed

before the statistical tests. Analyses were performed using SigmaStat (SPSS Inc., Chicago, IL).

Data are presented as means ± SEM. unless otherwise indicated.

64

CHAPTER 3

RESULTS

65

CHAPTER THREE

Results

Study 1: Blood Ethanol Levels In Conscious Rats

Figure 3.1 shows the changes in blood ethanol levels over time following intraperitoneal

injection of ethanol. A blood sample was taken immediately prior to injection, and levels

measured at this time were considered baseline levels. Blood ethanol levels peaked

approximately 30 min post-injection, increasing to a recorded maximum of 125.5 ± 15.8 mg.dL-

1. Statistical analysis confirmed the significant effect of time on blood ethanol concentration

(F6,30 = 50.68, P < 0.001, 1-way ANOVA-RM). Post-hoc analyses showed that blood ethanol

concentration remained significantly elevated above baseline levels at all time points up to 2 hrs

post-injection (all t5 > 6.40, P < 0.001, Bonferroni t-test comparisons with the pre-injection control,

Figure 3.1). For this reason, all subsequent analyses of sleep and respiratory activity were limited to

the first 2 hrs post-injection. Blood ethanol levels were not significantly different than baseline at

3 hrs (t5 = 6.40, P = 0.475) or 4 hrs (t5 = 1.82, P = 1.000) post-injection.

Although no formal behavioural motor tests were conducted in this experiment, the rats appeared

to be sedated as judged by less exploration and slower movements around the home cage,

especially in the first hour. They also appeared less alert and less engaged with their

environment for the first hour after injection. The rats still showed signs of reduced alertness

and activity when handled for the blood samples at 60 and 90 minutes post-injection,

observations consistent with behavioural studies of sedation and motor impairment induced by

ethanol [219]. More detailed analysis of the effects of ethanol on sleep and EEG power spectra,

in addition to postural and respiratory motor activities, are included in results of Study 2.

66

Study 2: Effects of Ethanol on Sleep and Respiratory Motor Activity in Freely Behaving Rats

Effects of Ethanol on Sleep-Wake Regulation The effects of ethanol on sleep and wakefulness were determined from the data obtained in the

first 2 hrs post-injection, i.e., during the time when blood ethanol levels were significantly

elevated above baseline (Figure 3.1). A representative example of the effects of ethanol on sleep

architecture and the latency to sleep onset is shown in Figure 3.2. Figure 3.2 also shows group

data for the effects of ethanol on sleep-wake regulation, including total sleep time, as well as the

number and duration of NREM and REM sleep episodes. Ethanol significantly reduced sleep

latency, as judged by the time from the onset of injection to the time of the first sustained (60

sec) period of NREM sleep (23.25 ± 1.25 min for ethanol vs. 37.56 ± 3.88 min for saline (P =

0.005, paired t-test). Ethanol also had significant affects on sleep-wake states (F3,27 = 9.43, P <

0.001, 2-way ANOVA-RM) by significantly reducing the amounts of active wakefulness (i.e.,

periods with movements and overt behaviours, t9 = 4.46, P < 0.001, post-hoc Bonferroni paired t-

test, symbol ‘*’ in Figure 3.2 B), and increasing the amounts of NREM sleep (t9 = 4.21, P < 0.001,

symbol ‘*’ in Figure 3.2 B). Amounts of quiet wakefulness (i.e., periods without any body

movements) and REM sleep were unchanged by ethanol (both t9 < 0.18, P > 0.861, Figure 3.2 B).

The effects of ethanol on the proportion of time spent in each sleep-wake state were due to

significant influences on the duration of the sleep-wake episodes rather than the number of episodes

(F3,27 = 4.85, P = 0.008 for durations, and F3,27 = 1.13, P = 0.353 for the number of episodes, 2-

way ANOVA-RMs, Figure 3.2 C-D). Analyses showed that ethanol significantly reduced the

duration of the periods spent in active wakefulness, and increased the duration of NREM sleep

episodes (both t9 > 2.32, P < 0.026, post-hoc Bonferroni paired t-tests, symbol ‘*’ in Figure 3.2 D).

67

Effects of Ethanol on Postural Motor Tone and EEG Activity Neck EMG activity: There was a significant effect of sleep-wake state on neck EMG activity

(F2,18 = 20.43, P < 0.001, 2-way ANOVA-RM), with neck EMG being significantly decreased in

both NREM and REM sleep compared to wakefulness (both t9 > 5.21, P < 0.001, post-hoc

Bonferroni paired t-tests, symbol ‘#’ in Figure 3.3 A). Ethanol also had a significant effect on

neck EMG activity that depended upon sleep-wake state (F2,18 = 5.00, P = 0.019, 2-way

ANOVA-RM). Compared to the saline controls, ethanol significantly reduced neck EMG

activity in wakefulness, as shown by post-hoc analyses (t9 = 4.12, P < 0.001, Bonferroni paired t-

test, symbol ‘*’ in Figure 3.3 A). This effect was not observed in NREM or REM sleep (both t9 <

1.11, P > 0.278, Figure 3.3 A).

EEG Activity: As expected there was a significant effect of sleep-wake state on the ratio of

high (20-30 Hz) to low (0.5-2 Hz) frequency EEG power (i.e., the β2/δ1ratio, F2,18 = 117.53, P <

0.001, 2-way ANOVA-RM), with significant differences observed between all pairs of states (all

t9 > 7.52, P < 0.001, post-hoc Bonferroni paired t-tests, symbol ‘#’ in Figure 3.3 B). For example,

the β2/δ1 ratio was minimal in NREM sleep indicating significantly reduced EEG power in the

faster (β2) frequency range and/or increased power in the slower (δ1) frequency range (Figure 3.3

B). Moreover, further analyses showed that ethanol also significantly modulated the EEG signal

as judged by the β2/δ1ratio, with this effect being dependent upon the prevailing sleep-wake state

(F2,18 = 5.55, P = 0.013, 2-way ANOVA-RM). Compared to the saline control, ethanol

significantly reduced the β2/δ1ratio in NREM sleep, indicating reduced power in the faster

frequency bandwidths and/or increased power in the slower frequency bandwidths (t9 = 3.63, P =

0.001, post-hoc Bonferroni paired t-test, symbol ‘*’ in Figure 3.3 B). These changes are in a

direction expected with deeper sleep.

Given the above effects of ethanol on the ratio of β2/δ1activity in the EEG, additional analyses were

performed to further determine the effects of ethanol and sleep-wake states on the power of the EEG

signal in each of the different bandwidths, and these data are shown in Figure 3.3 C-H. In general,

the data in this figure show that compared to wakefulness and REM sleep, NREM sleep was

68

associated with significantly increased power in the δ2 and δ1 bandwidths (0.5-4 Hz) and

significantly reduced power in the θ (4-7.5 Hz) and β2 (20-30 Hz) bandwidths (see symbols ‘#’ in

Figure 3.3 C-H). Importantly, however, ethanol significantly increased δ2 and δ1 power in NREM

sleep and decreased β1 (13.5 - 20 Hz) power in NREM sleep and wakefulness (see symbols ‘*’ in

Figure 3.3 C-H), all changes consistent with a sedating effect.

Effects of Ethanol on Respiratory Motor Activity Like the effects on sleep-wake regulation, the effects of ethanol on respiratory motor activity

were also determined from the data obtained in the first 2 hrs post-injection (Figure 3.1).

Furthermore, data were included in these analyses only if they were obtained during unequivocal

and clearly defined states of quiet wakefulness, NREM sleep and REM sleep (see Methods). A

total of 11,972 5-sec epochs, equalling 16.63 hrs of data, were included in the analysis of

respiratory motor activity. Of these epochs, 1632 epochs were from periods of quiet

wakefulness, 8439 epochs were from NREM sleep, and 1901 epochs were from REM sleep.

Saline treatment accounted for 5123 of these epochs and ethanol, for 6849. Figure 3.4 shows

both a representative example from a single animal example, and group data, for the effects of

systemically administered ethanol or saline on respiratory motor activity.

Genioglossus Activity: Ethanol had significant effects on respiratory-related genioglossus

activity depending upon the prevailing sleep-wake state (F2,18 = 5.07, P = 0.018, 2-way ANOVA-

RM). Compared to the saline controls, ethanol significantly reduced respiratory-related

genioglossus activity in wakefulness (t9 = 2.32, P = 0.028, post-hoc Bonferroni paired t-test,

symbol ‘*’ in Figure 3.4 B), but this effect was not observed in NREM or REM sleep (both t9 <

1.38, P > 0.181, Figure 3.4 B). Since the vehicle control and ethanol were administered on

different days, it is possible that a difference in genioglossus activity may have been related to

differences in muscle-electrode coupling over time. This potential concern was addressed in two

ways. First, saline and ethanol doses were randomized such that any order effect was removed.

Secondly, tonic and respiratory-related data were also quantified as a percentage of maximal

genioglossus activity. The maximum level was chosen during periods of high behavioural

69

activity from a recording taken the evening before the experiments were performed, after the

animals were hooked-up for the habituation period. This time typically corresponded to the end

of their sleep period and the beginning of their active period. This same state-specific suppression

of respiratory-related genioglossus activity by ethanol was also observed if the data were quantified

and analysed as a percent of maximum activity (F2,18 = 6.88, P = 0.006, 2-way ANOVA-RM),

with the suppression also observed in wakefulness only (t9 = 2.87, P = 0.008, post-hoc

Bonferroni paired t-test). In other words, the results and conclusions are the same regardless of the

measure of genioglossus activity. Figure 3.4 B also shows that with the vehicle control, respiratory-

related genioglossus activity was normally decreased from wakefulness to NREM and REM sleep

(both t9 > 3.70, P < 0.003, symbol ‘#’ in Figure 3.4 B). Due to the aforementioned suppression

effects of ethanol, however, this normal suppression of genioglossus activity from wakefulness to

NREM and REM sleep was not observed in the presence of ethanol (both t9 < 1.70, P > 0.297,

Figure 3.4 B).

The significant suppression of respiratory genioglossus activity by ethanol in wakefulness but not

sleep may have been because genioglossus activity was already low in sleep such that it was more

difficult to detect a change. Accordingly we sought to determine if the magnitude of suppression of

respiratory-related genioglossus activity observed with ethanol varied with the level of the

respiratory genioglossus signal during the vehicle control. Figure 3.4 C shows the significant

positive relationship between the level of respiratory genioglossus activity recorded with vehicle

within a rat and the magnitude of decrease observed in response to ethanol (r = 0.627, P = 0.0002,

Pearson Product Moment Correlation). This suggests that it is possible that no significant changes

were detected in NREM or REM sleep due to the already low levels of genioglossus activity.

There was also a significant effect of sleep-wake state on tonic genioglossus activity (F2,18 = 21.80,

P < 0.001, 2-way ANOVA-RM), with tonic activity also significantly decreasing from

wakefulness to NREM and REM sleep (both t9 > 5.09, P < 0.001, post-hoc Bonferroni paired t-

tests, symbol ‘#’ in Figure 3.4 D). However, for the group there was no independent effect of

ethanol on tonic genioglossus activity compared to the saline controls (F1,9 = 0.06, P = 0.815, 2-

way ANOVA-RM), and there were no effects of ethanol that were specific to any particular

70

sleep-wake state (F2,18 = 2.11, P = 0.150, 2-way ANOVA-RM). Nevertheless, there was a

statistically significant relationship between the level of baseline tonic genioglossus activity with

vehicle and the magnitude of decrease in response to ethanol (r = 0.701, P = 0.00002, Pearson

Product Moment Correlation, Figure 3.4 E).

Diaphragm Activity and Respiratory Rate: There were no effects of ethanol or sleep-

wake state on the amplitude of diaphragm activity at the given dose of 1.25 g.kg-1 (F1,9 = 0.94, P

= 0.357, and F2,18 = 2.53, P = 0.108 respectively, 2-way ANOVA-RMs, Figure 3.4 F), nor were

there any effects of ethanol that depended upon the prevailing sleep-wake state (F2,18 = 1.90, P =

0.178, 2-way ANOVA-RM). However, as expected, respiratory rate did vary with sleep-wake

state (F2,18 = 32.22, P < 0.001, 2-way ANOVA-RM). Respiratory rate in REM sleep was

significantly increased compared to both NREM sleep and wakefulness (both t9 > 6.93, P <

0.001, symbol ‘#’ in Figure 3.4 G). Nevertheless, there were no differences in respiratory rate

between the ethanol and saline conditions (F1,9 = 1.42, P = 0.264, 2-way ANOVA-RM), nor was

any effect of ethanol dependent upon the prevailing sleep-wake state (F2,18 = 0.55, P = 0.587, 2-

way ANOVA-RM, Figure 3.4 G). Overall, therefore, the significant effects of systemically-

administered ethanol on respiratory motor activity were confined to the genioglossus muscle, a

muscle with dual respiratory and non-respiratory (including postural) functions.

Study 3: Ethanol at the Hypoglossal Motor Pool and Effects on Genioglossus Activity

Sites of Microdialysis Figure 3.5 shows an example of a lesion site left by a microdialysis probe in the hypoglossal

motor nucleus. The distribution of microdialysis sites from all the experiments in Study 3 are

71

also shown in Figure 3.5, with the sites located within or immediately adjacent to the

hypoglossal motor nuclei in all animals.

Effects of Ethanol at the Hypoglossal Motor Pool In these anaesthetized rats, baseline respiratory rate averaged 49.9 ± 1.9 min-1, blood pressure

averaged 70.7 ± 2.0 mmHg, and respiratory-related genioglossus and diaphragm muscle

activities averaged 137.7 ± 22.8 and 370.7 ± 72.8 arbitrary units respectively. Group mean data

for the effects of ethanol at the hypoglossal motor pool on genioglossus activity and these other

variables are shown in Figure 3.6. There was a decline in genioglossus activity over the course of

the experiment (F5,70 = 7.83, P < 0.001, 2-way ANOVA-RM) that first became statistically

significant at a point corresponding to 100 mM ethanol and the same time-point (i.e., 120 min) in

the ACSF time-control experiment (t14 = 3.18, P = 0.033, post-hoc t-test, symbol ‘*’ in Figure

3.6 A). Statistical analyses showed, however, that there was no effect of drug treatment on the

change in genioglossus activity (F5,70 = 0.36, P = 0.876, 2-way ANOVA-RM); any effect on

genioglossus activity observed with direct application of ethanol to the hypoglossal motor pool

was statistically indistinguishable from the corresponding sham time-control experiment with

ACSF. Therefore, it is most reasonable to conclude that decline in genioglossus activity was a

product of time and not of experimental interventions.

There were also no effects of experimental protocol (i.e., ethanol or ACSF at the hypoglossal

motor pool) on the amplitude of diaphragm activity, respiratory rate or blood pressure during the

course of the experiments (all F5,70 < 1.17, P > 0.334, 2-way ANOVA-RM, Figure 3.6 B-D).

Likewise, there was no effects of delivery of ethanol or ACSF to the hypoglossal motor pool on

the ratio of high (β2, 20-30 Hz) to low (δ1, 2-4 Hz) frequency activity in the EEG, indicating no

relative change in EEG activation compared to the ACSF time-control experiments with these

localized interventions (F5,70 = 2.02, P = 0.086, 2-way ANOVA-RM).

72

Figure 3.1: Blood ethanol concentrations. Changes in blood ethanol levels as ethanol is metabolized over time following intraperitoneal injection of 1.25g.kg-1

ethanol. Data are shown as the mean (± SEM) from the group of six rats. Blood ethanol concentration was significantly elevated above baseline (shown as time 0) at 30, 60, 90 and 120 min post-injection (* indicates P < 0.05 compared to baseline).

73

Figure 3.2: Effects of ethanol on sleep-wake regulation. A single animal example, and group data, for the effects of ethanol on indices of sleep and wake regulation. (A) The distribution of sleep-wake states over time in a rat administered saline and the same rat administered ethanol. (B) Group data for the effects of ethanol on the percent (%) amounts of active wakefulness (i.e.,

74

periods with movements and overt behaviours), quiet wakefulness (i.e., periods without any body movements), NREM sleep and REM sleep. (C) The number of episodes of each sleep-wake state. (D) Average duration of each episode of each sleep-wake state. Values were obtained from the data collected in the first 2 hrs post-injection, i.e., during the time when blood ethanol levels were significantly elevated above baseline (Figure 3.1). Data are shown as mean + SEM (n = 10 rats). For the group data, the symbols indicate significant differences (P < 0.05) between ethanol and saline conditions (*).

75

Figure 3.3: Effects of ethanol on postural motor tone and electroencephalogram activity. The figure shows group data for the effects of systemically administered ethanol and vehicle (saline) on (A) neck muscle activity, (B) the ratio of high (20-30 Hz) to low (0.5-2 Hz) frequency activity in the electroencephalogram (i.e., the β2/δ1 ratio) and (C-H) the power of the EEG signal in the different frequency bands spanning 0.5 to 30 Hz. The symbols denote significant differences (P < 0.05) between ethanol and saline conditions (*) and the respective sleep-wake states (#). Data are shown as mean + SEM (n = 10 rats). The mean values for each individual rat were first calculated from the population of values that occurred for all the 5 sec epochs during each sleep-wake state in the first two hours following ethanol or saline administration. Data were averaged as described in Methods. In general, the changes in postural muscle tone and electroencephalogram activity are consistent with a sedating effect of ethanol.

76

Figure 3.4: Effects of ethanol on respiratory motor activity. The figure shows an example (A) and group data (B-G) for the effects of systemically administered ethanol and vehicle (saline) on respiratory motor activity across sleep-wake states. The traces show the electroencephalogram

77

(EEG) and neck electromyogram (EMG) signals. The genioglossus (GG) and diaphragm (DIA) signals are displayed as their moving-time averages (MTA) in arbitrary units (AU). The increasing levels of muscle activity during inspiration are indicated by the arrows. The baseline of the integrator (i.e. electrical zero) is shown for the GG MTA. For the group data, the symbols indicate significant differences (P < 0.05) between ethanol and saline conditions (*) and the respective sleep-wake states (#). Data are shown as mean + SEM (n = 10 rats). The mean values for each individual rat were first calculated from the population of values that occurred for all the 5 sec epochs during each sleep-wake state in the first two hours following ethanol or saline administration. The means from each individual rat in each condition were then averaged to yield the grand means for the group which are shown in the figures. For the correlations in panels C and E, the symbols ●, ▼, and ■ refer to wakefulness, NREM and REM sleep respectively (symbols show 3 states per animal for 10 animals). The correlations in C and E are indicated by the solid lines, with the 95% confidence intervals shown by the dashed lines. Some individual values for the reduction in GG activity with ethanol are negative because in these instances GG activity was increased compared to the saline controls.

78

Figure 3.5: Example and group data showing location of the microdialysis probes from all the experiments with ethanol at the hypoglossal motor pool and the artificial cerebrospinal fluid (ACSF) time controls. The top images show histological sections with a lesion site (indicated by the arrow) made by the microdialysis probe within the hypoglossal motor nucleus (HMN). Also shown are coronal diagrams from the rat medulla (Paxinos & Watson, 1998) illustrating the distribution of individual microdialysis sites from all rats administered ethanol and ACSF. The microdialysis probe locations are represented by grey cylinders which are drawn to scale. Overlap obscures some of the dialysis sites.

79

Figure 3.6: Effects of ethanol delivered to the hypoglossal motor pool. (A) Group data illustrating that there was a statistically significant decline in genioglossus activity over the course of the experiment with microdialysis perfusion of ethanol into the hypoglossal motor pool, that first became statistically significant (P < 0.05) at 100 mM ethanol (indicated by symbol *). Importantly, however, there was no significant difference in the effect of ethanol at the hypoglossal motor pool compared to the ACSF time-controls. This latter result indicated that compared to the ACSF time-controls, there was no ethanol-specific inhibitory effect operating at the hypoglossal motor pool and that any changes in GG activity in the ethanol experiments were due to the effects of time. There were also no effects on (B) the amplitude of diaphragm activity, (C) respiratory rate, or (D) blood pressure. See text for further details. All data are shown as mean + SEM (n = 10 and 6 rats with ethanol and ACSF time controls).

80

CHAPTER 4

DISCUSSION

81

CHAPTER FOUR

Discussion

Ethanol worsens, and in some cases can induce, sleep-disordered breathing [26, 28, 29]. Ethanol

decreases genioglossus muscle activity compared to diaphragmatic activity [30, 249], and is also

known to have sedating effects [219, 344-346]. This study is the first to determine the effects of

ethanol on both pharyngeal and respiratory pump (i.e. diaphragm) muscle activities combined

with indices of state-dependent arousal processes, and to also determine the effects of ethanol on

genioglossus activity when applied to the hypoglossal motor pool, the source of motor outflow to

the muscles of the tongue. The aim of this study was to distinguish between two major

mechanisms that could mediate ethanol-induced suppression of pharyngeal (i.e. genioglossus)

motor tone: one mechanism being a state-dependent influence on motor activity via the effect of

ethanol on sleep/arousal processes, the other being a motor suppression effect operating via an

inhibitory influence of ethanol directly at the hypoglossal motor pool. Overall, the results

support the former state-dependent mechanism, and not the latter mechanism involving direct

inhibitory effects at the motor pool, in explaining the influences of ethanol on breathing in the

intact organism in vivo.

Previously, the physiological mechanisms underlying how ethanol can adversely influence

breathing had not been adequately addressed because combined measurements of respiratory

muscle activities and indices of sleep/arousal regulation (i.e., beyond the identification of sleep-

wake states) had not been performed in either human [25-27, 31, 352-355] or animal studies

[345, 346, 356]. Moreover, the influence of ethanol on respiratory motor activity when applied

directly to a central respiratory motoneuronal pool had also not been studied in vivo. While the

effects of ethanol on hypoglossal nerve activity had been studied in in vitro brainstem slice

preparations of neonatal and juvenile rat medulla, these studies did not address the effects

specifically at the hypoglossal motor pool because ethanol was applied in a bath preparation

[251]. Other regions contained in the brainstem slices used in these preparations, such as the

PBC (which is involved in rhythm generation), may also be targets of ethanol. Thus in vitro

82

preparations used in the past have been unable to demonstrate the specific effect of ethanol on

the motor pool innervating the muscle of the tongue. The current study uses both systemically

administered ethanol and local application of ethanol to the hypoglossal motor nucleus in an

intact animal preparation to differentiate between the potential central effects of ethanol on

respiratory motor activity (ie. direct inhibition of motoneurons) versus state-dependent effects

operating via influences on sleep/arousal processes (as determined by changes in sleep-wake

states, EEG activity and postural motor tone).

This study presents evidence that the suppressant effects of ethanol on respiratory motor activity

are most likely operating via effects on state-dependent/arousal processes rather than directly at

the motor pool, by examining activity of the pharyngeal (genioglossus) muscles at

physiologically relevant blood ethanol concentrations. To put the results into perspective,

ethanol is one of the most widely used, self-administered, drugs in western society [273], and

one serious consequence of ethanol ingestion in humans is impairment of breathing during sleep

in otherwise normal individuals, as well as significant worsening of breathing during sleep in

individuals with pre-existing hypopnoeas or obstructive sleep apnoea [25-27, 31, 352-355].

Moreover, obstructive sleep apnoea is a common and serious disorder [350], with the prevalence

increasing with increasing levels of obesity and with most patients remaining undiagnosed [382,

383]. Accordingly, understanding the adverse impact of ethanol on breathing and recognizing

the important influence of state-dependent modulation has both physiological significance and

clinical relevance, in addition to broader implications for the impact of other sedative agents on

breathing.

83

Interpretation of Findings

Systemic Administration of Ethanol Ethanol was systemically administered at a concentration chosen to produce physiologically

relevant blood concentrations, reaching a maximum of 125.5 ± 15.8 mg.dL-1 that declined over

the subsequent 2-3 hrs (Figure 3.1). The justification for the dose administered was twofold: it is

a physiologically relevant dose that produces impairment of motor function in behavioural

testing in rats, and it corresponds to the blood alcohol levels that produce moderate intoxication

and impaired cognitive and motor behaviour in humans [219, 276, 277, 286]. Data were

analyzed for the first two hours post-injection, as statistical tests of blood ethanol levels showed

significantly elevated concentrations compared to baseline values during this time only. The

results showed that the chosen dose of ethanol decreased wakefulness and increased the

proportion of time spent in NREM sleep, and shortened sleep latency. In addition, it

significantly reduced postural (neck) muscle tone and shifted the component frequencies in the

EEG toward increased power in the lower frequency bands and decreased power in the higher

frequency bands. All of these changes are consistent with a sleep-promoting/sedating effect.

Ethanol may act at various nuclei within the brainstem’s arousal system to produce sedating

effects (Please see Chapter 1, Section 3). As previously mention, during sleep, the VLPO sends

GABAergic projections to arousal-promoting cells in the brainstem. Based on the results of this

study, it is unlikely that ethanol acts at the VLPO itself as inhibition of sleep-promoting cells of

the VLPO would result in a disinhibition of arousal-promoting cells, and an increase in

wakefulness would be expected (Please see Chapter 1, Figure 1.1 A and B). Instead, it is

possible that ethanol produces the sedating effects reported in this thesis by potentiating the

effects of endogenous GABA at arousal-promoting nuclei. One area that is potentially involved

in producing electrocortical signs of sedation is the thalamus, which is involved in the (dorsal)

ascending arousal system. The thalamus has been shown to contain a particular isoform of the

GABAA receptor believed to be uniquely sensitive to low and physiologically relevant

concentrations of ethanol [72, 206, 224]. Immunocytochemical studies of the distribution of 13

84

GABAA subunits in the adult rat brain showed that the α4 and δ subunits are highly expressed in

the thalamus [224], both of which are believed to constitute ethanol-sensitive GABAA receptors.

As well as producing markers of sleep-promotion/reduced arousal, ethanol caused a state-

dependent decrease in genioglossus activity but did not affect diaphragm activity or respiratory

rate across any sleep-wake state. The pattern of this state-dependent decrease in genioglossus

activity after ethanol injection was also similar to that produced in the non-respiratory, postural

(neck) muscle by ethanol (compare Figure 3.3 B and D with Figure 3.4 A). Furthermore, the

magnitude of decrease in respiratory-related and tonic genioglossus activities produced by

ethanol was related to baseline activity without ethanol; that is, the ethanol-induced decrease in

activity was larger when baseline activity was higher. This latter observation can explain why

the motor suppression produced by ethanol was most apparent in wakefulness when the levels of

activity are typically increased compared to those in sleep (3.4 B and D). Moreover,

wakefulness also corresponds to a time when arousal processes are most active, and therefore the

suppressant effects of ethanol on the components of motor activity supported by state-dependent

arousal processes would be most apparent in wakefulness. Nevertheless, the suppressant effects

of ethanol on state-dependent arousal processes were also observed in sleep; for instance,

sedation was observed after ethanol administration in NREM sleep, as judged by the reduced

ratio of high (20-30 Hz) to low (0.5-2 Hz) frequency EEG power (i.e., the β2/δ1ratio) (Figure 3.3).

Also in NREM sleep, ethanol led to increased power in the δ2 and δ1 frequency bands (spanning

0.5 - 4 Hz) and reduced power in the β1 (13.5 - 20 Hz) bandwidth (Figure 3.3). Given the already

low motor tone in sleep, however, any reduction in neck and genioglossus muscle activities

produced by ethanol were not significant for the group in states outside of wakefulness (Figures ,

and Figure 3.3 A, 3.4 B and D).

Local Application of Ethanol at the Hypoglossal Motor Nucleus

Despite state-dependent effects of ethanol on genioglossus motor activity following systemic

administration, local application of ethanol directly to the hypoglossal motor pool caused no

85

change in genioglossus activity in anaesthetized rats compared to the time-control (sham)

experiments. These additional sham experiments were performed because of the need to

determine if a component of the observed decrease in genioglossus activity that occurred in the

presence of the ethanol at the hypoglossal motor pool was due to the effects of ethanol per se or

the effects of time over which the experiments were performed. This distinction was achieved

by comparing the responses to ethanol with appropriate time-controls. From such comparisons,

there was no evidence supporting ethanol-induced suppression of hypoglossal motor output to

genioglossus muscle via local effects at this motor pool in intact animals, as any changes were

indistinguishable from the time-controls.

In notable contrast to ethanol, local application of the sedative hypnotic drugs lorazepam (a

benzodiazepine) and zolpidem (an imidazopyridine compound) to the hypoglossal motor pool

cause suppression of genioglossus activity in an anaesthetized preparation in vivo [254]. We

have also shown previously that GABA, glycine and glutamate each exert tonic modulation

effects at the hypoglossal motor pool innervating genioglossus muscle in this same anaesthetized

preparation [362-367]. This observation is relevant because these GABA, glycine and glutamate

receptor responses have the capacity to be significantly influenced by ethanol, influences that

could reduce genioglossus activity based on in vitro studies both at the hypoglossal motor pool

and other brain regions [36, 292, 293, 308, 317, 368, 384]. Nevertheless, there was no

physiological consequence of ethanol on genioglossus activity when locally applied to the

hypoglossal motor pool, over a wide range of applied doses (Figure 3.6).

Although ethanol is known to affect a variety of receptors at high concentrations, more recent in

vivo experiments have focused on ethanol’s interaction with GABAA receptors in eliciting

sedative effects as well as cognitive and motor impairment. In particular, recent evidence from

both in vitro and in vivo experiments have suggested that low and physiologically-relevant doses

of ethanol act on extrasynaptic receptors that contain α 4 or 6 subunits in addition to a δ subunit

[33, 34, 216, 217, 219, 220, 238, 239]. Recombinant studies showed that GABAA receptors

containing α4/6β3δ were enhanced by ethanol concentrations as low as 10 mM in vitro, and in

vivo behavioural studies show that rats with a single mutation in the gene coding for α6 induces a

86

marked increase in motor impairment in response to ethanol [219]. The importance of the

subunit δ [330, 331, 340] has also been emphasized in the tonic GABA conductance of

extracellular receptors, by which ethanol is believed to act [72, 202, 206]. However, α4 and α6

receptors are not distributed heterogeneously throughout the brain; α6 are located exclusively in

the cerebellum and α4 is found highly expressed mainly in the thalamus, but with expression also

in striatum and nucleus accumbens, the tuberculum olfactorium and the dentate gyrus [224]. In

rats, up to 30% of all GABAA receptors contain α4 subunits, 70% of those pairing with δ subunits

[385-387]. Tonic GABA inhibition, mediated by α4β2δ GABAA receptors has been demonstrated

in thalamocortical relay neurons which thereby affect cortical activity [385]. Inhibitory neurons

in the reticular thalamic nucleus and thalamocortical relay neurons are highly interconnected,

both of which use T-type calcium currents to generate a slow rhythmic output when

hyperpolarized which in turn, can entrain firing within the cortex producing slow-frequency δ

waves [385].

Based on this discussion, I hypothesize that motor impairment is caused by ethanol acting on

highly sensitive α6β3δ receptors located in the cerebellum [219], and the sedative effects of

ethanol affect tonic conductance of GABA in the thalamus by acting on α4/6β3δ receptors [206,

224]. It is possible that hypoglossal motor nucleus neurons do not contain the highly-sensitive

GABAA isoforms on which ethanol acts at physiologically relevant concentrations, and thus fail

to respond to discrete application in our preparation.

Overall, our data showing a lack of effect of ethanol at the hypoglossal motor pool on

genioglossus activity do not dispute the potentiating effects of ethanol on inhibitory GABA and

glycine receptor function and augmentation of inhibitory postsynaptic currents at hypoglossal

motoneurons when studied in neonatal and juvenile rodent brainstem slice preparations in vitro

[252, 308, 317, 368]. Rather, these data show that local application of ethanol to the hypoglossal

motor pool in the adult organism in vivo, over the same dose range, does not suppress total motor

outflow to the genioglossus muscle. This difference may relate to the older age of animals used

in the present study or may indicate that the influences observed at single motoneurons do not

necessarily reflect the influence on population motor outflow as subsequently recorded by

87

genioglossus muscle activity, particularly if the GABAA receptor subunit isoforms responsible

for mediating the inhibitory effects of ethanol are not present at the motor pool in the adult.

While the results of this study suggest that the genioglossus suppression seen in freely-behaving

rats was not produced by ethanol acting directly at the hypoglossal motor pool, it is possible that

ethanol modulates the activity of neurons at pre-motor nuclei. The lateral tegmental field is

known to contain respiratory pre-motor neurons projecting to the hypoglossal motor nucleus

(such as those contained within the Kölliker Fuse nucleus) [388, 389]. It is unclear if neurons at

these pre-motor nuclei themselves possess receptors that are sensitive to low doses of ethanol.

However, the lateral tegmental field is known to have state-dependent activity so ethanol may

modulate the activity of pre-motor neurons projecting to the hypoglossal motor pool by way of

effecting sleep-wake process. If ethanol affects neurons groups responsible for sleep-wake

states within the ascending activating system, it may in turn affect genioglossus activity by

influencing respiratory pre-motor neurons such as those in the state-dependent lateral tegmental

field.

There is potential criticism of performing the experiments with local application of ethanol to the

hypoglossal motor pool in anaesthetized (albeit otherwise intact) rats. The rationale to study

anaesthetized, rather than conscious, rats for this component of the investigation lay in the

impracticality of applying to the hypoglossal motor nucleus the multiple doses of ethanol that

were required to span the dose range previously used in vitro [308, 317, 368]. In addition,

controlled application of these multiple doses would not have been possible in conscious animals

because of the confounding influence of unpredictable changes in ongoing behavioral state and

spontaneous motor activities that typify the conscious state [366, 379]. A potential concern is

that under anaesthesia, genioglossus muscle activity is suppressed such that any further

suppression would not be reflected by statistical tests. However, isoflurane, the type of

anaesthetic used in this study has been shown to increase respiratory-related genioglossus

activity at low doses, in contrast to other anaesthetics such as propofol [259]. In fact, the same is

true for inhaled-anaesthetic halothane. Halothane (0.2 to 0.9%) has been shown in a previous

study to significantly increase respiratory-related genioglossus activity compared to levels

observed in the same rat during NREM sleep [366]. Certain types of inhaled anaesthetics such

as halothane and isoflurane are proposed to increase the magnitude of hypoglossal nerve

88

discharge in intact animals by increasing the activity of pre-motor neurons such as those found in

the Kölliker Fuse nuclei, as demonstrated by c-Fos staining [390]. In addition, during the course

of the studies presented in this thesis, respiratory-related genioglossus activity was observed to

be elevated even compared to respiratory-related activity during wakefulness in freely-behaving

animals, although this was not compared using statistical tests. Therefore, it is unlikely that the

effects of ethanol on genioglossus activity are masked by the effects of the chosen anaesthetic.

Despite these concerns of anaesthesia [259, 391], it remains reasonable to expect that if ethanol

was exerting a physiological action to suppress hypoglossal motor output to genioglossus muscle

via an effect at the hypoglossal motor pool, then we would still have expected to observe such a

decrease in genioglossus activity when locally applied even under anaesthesia, and this simply

did not occur.

In summary, the significant changes in sleep-wake states, sleep latencies, EEG frequencies and

postural and genioglossus motor tone produced by systemically administered ethanol are all

indicative of reduced arousal processes. Coupled with the lack of effect of ethanol on

genioglossus activity with discrete application to the hypoglossal motor pool, these data suggest

that the genioglossus motor suppression observed with systemic administration was mediated via

effects of ethanol on sleep/arousal (i.e., state-dependent) processes rather than via a

physiological effect of ethanol at the motor pool per se. Ethanol, along with other GABA-

modulating sedatives, therefore can induce sedation and influence respiratory motor activity by

acting on a number of structures involved in sleep and wakefulness processes, and so influence

breathing via an influence on central nervous system state or the “wakefulness stimulus” to

breathe [175, 176, 188]. Although not directly involved in the generation of NREM sleep, the

thalamus is involved in integrating inputs from brainstem structures and, in turn, influences

cortical activity; it is known to contain the particular GABAA receptor isoforms on which low

doses of ethanol are purported to act. As previously discussed, GABA has emerged as having a

significant role in sleep induction (Please see Chapter 1, Section 3 and Section 7 of this thesis),

and in the interactions between critical sleep-promoting and arousal nuclei. This primary

influence of ethanol on state-dependent inputs to the respiratory system would also explain why

genioglossus activity was suppressed by ethanol at physiologically-relevant concentrations, but

diaphragm activation and respiratory rate were unchanged. Several lines of evidence indicate

89

that hypoglossal motor output is more strongly influenced by state-dependent influences than

diaphragm activity [191, 392-396]. In the present study, alterations in state-dependent drives to

the hypoglossal motor pool produced by ethanol may result in reduced motor excitability and

genioglossus muscle activity, independent of a potential effect of ethanol at the motor pool per

se.

90

CHAPTER 5:

FUTURE DIRECTIONS AND FINAL

CONCLUSIONS

91

CHAPTER FIVE

Future Directions and Final Conclusion

Future Directions

Ethanol has been clinically shown to worsen sleep-related breathing disorders such as OSA, to

increase time spent in sleep, and to selectively decrease genioglossus motor activity as compared

to diaphragmatic activity at moderate doses [28, 30, 344]. The study presented in this thesis is

unique in that it combined measurements of respiratory muscle activity and indices of sleep-

wake regulation in a rodent model, and also tested the effects of ethanol at the hypoglossal motor

pool. To this end, the study presented in this thesis addresses the possible mechanisms by which

ethanol may suppress genioglossus activity (i.e. state-dependent influences or central motor

suppression via direct inhibition). The results of this study demonstrate that respiratory-related

genioglossus activity is reduced by the systemic administration of a physiologically relevant dose

of ethanol (1.25 g.kg-1); this same dose also decreases the power of high frequency β1 waves and

reduces neck EMG activity during wakefulness, suggestive of a sedative effect. Increased power in

β1 bandwidths is associated with increased alertness and focused attention, and generally associated

with high cortical activity [46]. Thus, a decrease in β1 power may indicate a reduction in alertness

and general arousal during wakefulness [46]. Past studies have also shown that periods of NREM

sleep accompanied by an increased in the proportion of delta waves are associated with periods of

decreased arousability and responsiveness to stimuli such as sound in both humans and rats [54,

397, 398]. In this study, there was an increase in power in lower frequency bands, such as δ1 and

δ2, as well as a decrease in β1 power during NREM sleep with ethanol compared to control. There

was also a state-dependent decrease in postural motor tone and observable effects on behaviour

(although these were not directly measured). Together, these results demonstrate the sedative effect

of ethanol. Ethanol caused a state-dependent decrease in respiratory-related genioglossus activity

but did not affect diaphragm activity, with the magnitude of genioglossus decrease related to

92

baseline activity. Ethanol did not alter genioglossus activity when applied directly to the

hypoglossal motor pool. Overall, these results suggest that ethanol at physiologically relevant

concentrations promoted sleep and altered electroencephalogram and postural motor activities

indicative of a sedating effect. The lack of effect on genioglossus activity with ethanol applied

directly to the hypoglossal motor pool suggests that the suppression observed with systemic

administration was mediated via effects on state-dependent processes rather than effects at the

motor pool per se.

Importantly, a decrease in the patency of airways reduces the effectiveness and efficiency of

ventilation, which can result in periods of elevated CO2. There is a normal decrease in ventilatory

response to hypercapnia and hypoxia during sleep, although this is especially apparent in REM

rather than NREM sleep [177, 184, 190]. Central chemoreceptive structures are widely dispersed in

the brainstem [164, 399-402], and include regions that have also been implicated in the ascending

arousal system [400-402]. It is conceivable that ethanol, as well as other GABAA receptor

modulator with sedative effects, may act (directly or indirectly) on one or more of these regions thus

affecting sedation and respiratory chemoreflexes. Additional effects that a GABAA receptor

modulators may have on the effective CO2 detection, in addition to any sedative effects, would

result in a prolonged latency until arousal during periods of hypercapnia. In future experiments,

ethanol’s effect on the response to CO2 may be studied in the same freely-behaving animal

preparation as the one presented in this thesis by systemically administrating ethanol and, during

periods of unequivocal NREM and REM sleep, introducing a CO2 challenge. By slowly increasing

CO2 in a ramp-like fashion, it will be possible to test the effects of ethanol on both sleep and

breathing during periods of elevated CO2, such as the latency to arousal as well as the activity of

respiratory-related muscles.

In recent years, evidence has suggested an important role for extrasynaptic receptors and the

modulation of tonic GABA currents in mediating anaesthetic effects of GABA agents [242].

However, the regional targets of GABAA sedatives are also an area of great interest [348, 403].

One area of interest is the LC, a structure believed to be involved in ascending arousal system that

has also been suggested to have chemosensitive properties [400-402, 404-406]. The LC

93

demonstrates state-dependent activity, with activity highest during wakefulness, decreased activity

during NREM sleep and minimal activity during REM sleep. It has been established that

endogenous levels of GABA increase in the LC during NREM sleep and even more so during REM

sleep, indicating that noradrenergic neurons located in the LC are under GABAergic regulation

across sleep-wake states [86, 113, 138]. Studies suggest that a GABA-induced suppression of the

LC may play a role in sleep induction and modulation, although it may not be essential for the

generation of NREM nor REM sleep [84, 124, 126].

Electrical stimulation of the LC has been shown to reduce time spent in REM sleep. Bilateral

injections of picrotoxin, a GABAA receptor antagonist, also reduced REM sleep by decreasing the

duration of REM episodes but leaving the frequency of episode generation unaffected [140]. These

observations suggest that silencing of noradrenergic cells located in the LC may not be necessary

for the generation of REM sleep, but may be involved in the maintenance of the state. One major

source of GABA input to the LC is from the prepositus hypoglossi. Electrical stimulation of the

prepositus hypoglossi reduces REM sleep duration but has no effect when picrotoxin is

microinjected into the LC [407]. These results again suggest that GABAergic control of the LC

may modulate REM sleep by affecting its duration, rather than in the initiation or generation of the

state. However, the results of studies that investigate the role of the LC in both NREM and REM

sleep control are mixed. Other studies that involve lesioning and chemical inhibition of the LC have

demonstrated no effect on overall time spent in REM sleep [133, 403]. Muscimol, propofol, and

pentobarbital have been shown to increase activity in VLPO neurons and decrease activity in the

histaminergic TMN, an arousal centre which sends excitatory projections to the cortex (Please see

Chapter 1, Figure 1.1) [403]. Like the LC, the TMN is part of the ascending arousal system, and

has mutually inhibitory interactions with the sleep-promoting VLPO. Microinjections of muscimol

into the TMN have been shown to produce dose-dependent sedation, supporting the hypothesis that

it has a key role in the sedative response to GABAergic anaesthics. Although the LC also regulates

the activity of the VLPO, bilateral injections of muscimol into the LC alone does not induce a loss

of consciousness in rats [403]. Furthermore, although it has been suggested that the withdrawal of

noradrenergic input to widespread areas of the brainstem and cortex plays a role in the switch to

REM sleep, lesioning the LC does not result in a reduction in REM sleep [133, 403]. In light of

these mixed results, the role of the LC in the generation and/or maintenance of sleep remains

94

unclear. Nevertheless, because the LC plays a role in the ascending arousal system and because it

does have chemosensitive properties [400-402, 404-406], the effects that GABA agonists have on

wakefulness and sedation, as well as on breathing responses to chemical respiratory stimulation, are

worth examining.

Sleep-related breathing disorders such as obstructive sleep apnoea result in increasing levels of CO2

as a result of obstructive episodes. Arousals from sleep due to hypercapnia quickly open airways

and restore ventilation, and so agents that act to inhibit chemosensitivity and ventilatory

chemoreflexes may be dangerous. I have performed such a study, designed to investigate the effects

of microdialysis perfusion of the specific GABAA receptor agonist muscimol into the LC on both

the sleep and breathing response to elevated CO2 levels. To this end, I hypothesized that

potentiating GABAergic inhibition at the LC would decrease time spent in wakefulness and thus

increase time spent in sleep, and would also attenuate the ventilatory response to hypercapnia.

To test these hypotheses, I designed a four-part microdialysis experiment with drug intervention

at the LC (aCSF vs muscimol, 50 µM) during both room air and hypercapnia (7 % inspired CO2).

A level of 7 % inspired CO2 was chosen because it produces robust stimulation of diaphragm and

genioglossus muscle activities across sleep-wake states, and promotes wakefulness (while still

allowing measurable amounts of sleep) [191]. Each rat completed 90 minutes of each of the

following interventions, on the same day: room air and aCSF; CO2 and aCSF; room air and

muscimol; CO2 and muscimol. Both aCSF treatments were completed first, with the room air

and CO2 challenges being delivered in random order; the gas challenges were also completed in

random order within the muscimol treatment. The treatments of aCSF were delivered first

because of the length of time necessary for drug washout to be confident that no confounding

effects of residual drug would be incurred. As a four-part experiment, the length of the study

would present challenges in allowing for an adequate washout period and completion of the

study within a rat’s normal sleeping hours were muscimol to be delivered first. The time spent in

each sleep stage during each drug treatment and gas challenge will be evaluated, as well as the

respiratory response (as revealed by diaphragmatic activity) to CO2. To date, I have studied 14

rats (9 muscimol and 5 sham) but the results are in a preliminary stage of analysis at the time of

writing this thesis.

95

The effects of muscimol on sleep will be examined and compared to time control (sham) rats.

The additional control is necessary because as the muscimol interventions were always

completed after the aCSF interventions, they occurred later in the day. Sleep architecture

changes throughout the sleep-period, with REM sleep occurring more later in the day [48, 49,

408, 409]. Thus it is important to quantify sleep architecture (both with room air and CO2)

without any drug intervention in sham animals. The amount of NREM and REM sleep will be

compared between aCSF and muscimol animals during (1) room air periods and (2) continuous

7% CO2. Within each treatment (aCSF and muscimol), it is hypothesized that there will be more

sleep in room air than with CO2, and that CO2 will significantly reduce the amount of REM sleep

[253]. It is also hypothesized that muscimol at the LC will suppress sleep disruption caused by

hypercapnia. The number and duration of sleep periods will also be quantified, as well as the

number of arousals, with muscimol versus aCSF. The ventilatory response to CO2 will also be

compared between aCSF and muscimol treatments. The diaphragm amplitude and respiratory

frequency will increase with CO2 during aCSF perfusion [57]. The aim of this study is to test the

hypotheses that muscimol at the LC will affect sleep and suppress the ventilatory response to

CO2, in accordance with their hypothesized role as chemoreceptors.

Relevance of Animal Preparation and Final Conclusions

As discussed, previous studies addressing the effects of ethanol on breathing have not attempted

to separate, or distinguish between, the two major mechanisms that could mediate the

suppression of pharyngeal (genioglossus) motor tone, those being a state-dependent influence on

motor activity via an effect of ethanol on sleep/arousal processes and/or a motor suppression

effect operating via an inhibitory influence of ethanol directly at the hypoglossal motor pool.

This distinction is one of the major contributions of this thesis. Nevertheless, to determine

96

potential commonality of mechanisms and applicability to human studies it is necessary to

compare the results obtained in the present study to data obtained in other experiments in

animals and humans.

In the clinical literature it has been shown that night-time ethanol can increase total inspiratory

resistance in normal non-snoring subjects [352]. Although this effect was not localised to the

upper airway, as resistance was calculated from the pressure difference between the mouth and a

balloon in the mid-oesophagus, it is likely that suppression of pharyngeal motor tone was

responsible for the increased airway resistance observed in that study. In a separate study,

administration of ethanol to awake normal human subjects decreased genioglossus muscle

activity but did not alter minute ventilation [30], but components of arousal state and the effects

in sleep or were not studied. Nevertheless, the effects on genioglossus activity and minute

ventilation observed in those awake human subjects are in keeping with the suppression of

genioglossus activity and lack of effect on diaphragm activity and respiratory rate described in

this thesis. In a separate experiment in normal human subjects, ethanol also did not alter minute

ventilation in wakefulness or sleep in keeping with the results of this thesis, but the effects on

upper airway motor tone and components of arousal state or sedation were not investigated

[352]. The results of another study, again in normal human subjects, did suggest that ethanol’s

sedative effects may contribute to the worsening of disordered-breathing during sleep by

demonstrating that ethanol increased the latency to arousal during experimentally-induced

airway occlusions.

Previous studies performed using slices of neonatal rodent medulla or the isolated brainstem-

spinal cords of newborn rats in vitro showed suppression of hypoglossal nerve activity with

ethanol [251, 384]. However, whether that suppression occurred via effects at the motor nuclei

per se or via suppression of pre-motor inputs was not determined as the ethanol was bath

applied. Likewise, the physiological basis for the selective suppression of hypoglossal nerve

activity following systemic administration of ethanol was not determined in a previous study

using decerebrate or awake cats [356] as differentiation between the potential central effects of

97

ethanol on respiratory motor activity versus state-dependent effects operating via influences on

brainstem arousal neurons was not performed.

In conclusion, this study differentiates between the effects of ethanol at the hypoglossal motor

pool, and the effects on pharyngeal and respiratory pump muscle activities combined with

indices of sleep/arousal regulation. Together, the data support the novel concept of a primary

influence on state-dependent modulation of hypoglossal motor activity rather than an effect at

the motor pool per se. Moreover, the evidence that the magnitude of decrease in respiratory-

related and tonic genioglossus activities produced by ethanol was related to baseline activity

without ethanol (i.e., that the ethanol-induced decrease in activity was larger when baseline

activity was higher) has important clinical relevance. It has been postulated that individuals with

obstructive sleep apnoea patients require high levels of baseline genioglossus activity to keep the

upper airspace open and maintain adequate airflow [11]. The implication of the observations

presented in this thesis is that individuals with high baseline levels of genioglossus activity, such

as patients with obstructive sleep apnoea, would be most susceptible to suppression of activity

with ethanol, so explaining the worsening of sleep-disordered in such individuals [25-27, 31,

352-355].

98

REFERENCES

99

1. Bradley, T.D. and E.A. Phillipson, Central sleep apnea. Clin Chest Med, 1992. 13(3): p. 493-505.

2. Yumino, D. and T.D. Bradley, Central sleep apnea and Cheyne-Stokes respiration. Proc Am Thorac Soc, 2008. 5(2): p. 226-36.

3. Kinney, H.C., Brainstem mechanisms underlying the sudden infant death syndrome: evidence from human pathologic studies. Dev Psychobiol, 2009. 51(3): p. 223-33.

4. Kinney, H.C., et al., The brainstem and serotonin in the sudden infant death syndrome. Annu Rev Pathol, 2009. 4: p. 517-50.

5. Horner, R.L., Pathophysiology of obstructive sleep apnea. J Cardiopulm Rehabil Prev, 2008. 28(5): p. 289-98.

6. Pevernagie, D., R.M. Aarts, and M. De Meyer, The acoustics of snoring. Sleep Med Rev, 2009.

7. Sleep-related breathing disorders in adults: recommendations for syndrome definition and measurement techniques in clinical research. The Report of an American Academy of Sleep Medicine Task Force. Sleep, 1999. 22(5): p. 667-89.

8. Scott, S., et al., A comparison of physician and patient perception of the problems of habitual snoring. Clin Otolaryngol Allied Sci, 2003. 28(1): p. 18-21.

9. Kales, A., et al., Severe obstructive sleep apnea--I: Onset, clinical course, and characteristics. J Chronic Dis, 1985. 38(5): p. 419-25.

10. Young, T., et al., The occurrence of sleep-disordered breathing among middle-aged adults. N Engl J Med, 1993. 328(17): p. 1230-5.

11. Malhotra, A. and D.P. White, Obstructive sleep apnoea. Lancet, 2002. 360(9328): p. 237-45.

12. Strollo, P.J., Jr. and R.M. Rogers, Obstructive sleep apnea. N Engl J Med, 1996. 334(2): p. 99-104.

13. Remmers, J.E., et al., Pathogenesis of upper airway occlusion during sleep. J Appl Physiol, 1978. 44(6): p. 931-8.

14. Issa, F.G., et al., Genioglossus and breathing responses to airway occlusion: effect of sleep and route of occlusion. J Appl Physiol, 1988. 64(2): p. 543-9.

15. Findley, L.J. and P.M. Suratt, Serious motor vehicle crashes: the cost of untreated sleep apnoea. Thorax, 2001. 56(7): p. 505.

16. Dobbin, K.R. and P.J. Strollo, Jr., Obstructive sleep apnea: recognition and management considerations for the aged patient. AACN Clin Issues, 2002. 13(1): p. 103-13.

17. Bass, J. and F.W. Turek, Sleepless in America: a pathway to obesity and the metabolic syndrome? Arch Intern Med, 2005. 165(1): p. 15-6.

18. Laposky, A.D., et al., Sleep and circadian rhythms: key components in the regulation of energy metabolism. FEBS Lett, 2008. 582(1): p. 142-51.

100

19. Leung, R.S. and T.D. Bradley, Sleep apnea and cardiovascular disease. Am J Respir Crit Care Med, 2001. 164(12): p. 2147-65.

20. Parker, J.D., et al., Acute and chronic effects of airway obstruction on canine left ventricular performance. Am J Respir Crit Care Med, 1999. 160(6): p. 1888-96.

21. Spiegel, K., R. Leproult, and E. Van Cauter, Impact of sleep debt on metabolic and endocrine function. Lancet, 1999. 354(9188): p. 1435-9.

22. Cartwright, R.D., Effect of sleep position on sleep apnea severity. Sleep, 1984. 7(2): p. 110-4.

23. Punjabi, N.M., The epidemiology of adult obstructive sleep apnea. Proc Am Thorac Soc, 2008. 5(2): p. 136-43.

24. Young, T., P.E. Peppard, and D.J. Gottlieb, Epidemiology of obstructive sleep apnea: a population health perspective. Am J Respir Crit Care Med, 2002. 165(9): p. 1217-39.

25. Block, A.J. and D.W. Hellard, Ingestion of either scotch or vodka induces equal effects on sleep and breathing of asymptomatic subjects. Arch Intern Med, 1987. 147(6): p. 1145-7.

26. Mitler, M.M., et al., Bedtime ethanol increases resistance of upper airways and produces sleep apneas in asymptomatic snorers. Alcohol Clin Exp Res, 1988. 12(6): p. 801-5.

27. Scanlan, M.F., et al., Effect of moderate alcohol upon obstructive sleep apnoea. Eur Respir J, 2000. 16(5): p. 909-13.

28. Issa, F.G. and C.E. Sullivan, Alcohol, snoring and sleep apnea. J Neurol Neurosurg Psychiatry, 1982. 45(4): p. 353-9.

29. Berry, R.B., M.H. Bonnet, and R.W. Light, Effect of ethanol on the arousal response to airway occlusion during sleep in normal subjects. Am Rev Respir Dis, 1992. 145(2 Pt 1): p. 445-52.

30. Krol, R.C., S.L. Knuth, and D. Bartlett, Jr., Selective reduction of genioglossal muscle activity by alcohol in normal human subjects. Am Rev Respir Dis, 1984. 129(2): p. 247-50.

31. Scrima, L., et al., Increased severity of obstructive sleep apnea after bedtime alcohol ingestion: diagnostic potential and proposed mechanism of action. Sleep, 1982. 5(4): p. 318-28.

32. Cheng, G., et al., Ethanol reduces neuronal excitability and excitatory synaptic transmission in the developing rat spinal cord. Brain Res, 1999. 845(2): p. 224-31.

33. Wallner, M., H.J. Hanchar, and R.W. Olsen, Ethanol enhances alpha 4 beta 3 delta and alpha 6 beta 3 delta gamma-aminobutyric acid type A receptors at low concentrations known to affect humans. Proc Natl Acad Sci U S A, 2003. 100(25): p. 15218-23.

34. Wallner, M., H.J. Hanchar, and R.W. Olsen, Low dose acute alcohol effects on GABA A receptor subtypes. Pharmacol Ther, 2006. 112(2): p. 513-28.

35. Lovinger, D.M., G. White, and F.F. Weight, Ethanol inhibition of neuronal glutamate receptor function. Ann Med, 1990. 22(4): p. 247-52.

101

36. Lovinger, D.M., G. White, and F.F. Weight, Ethanol inhibits NMDA-activated ion current in hippocampal neurons. Science, 1989. 243(4899): p. 1721-4.

37. Roberto, M., et al., Actions of acute and chronic ethanol on presynaptic terminals. Alcohol Clin Exp Res, 2006. 30(2): p. 222-32.

38. Mihic, S.J., et al., Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors. Nature, 1997. 389(6649): p. 385-9.

39. Kobayashi, T., et al., Ethanol opens G-protein-activated inwardly rectifying K+ channels. Nat Neurosci, 1999. 2(12): p. 1091-7.

40. Hobson, J.A. and E.F. Pace-Schott, The cognitive neuroscience of sleep: neuronal systems, consciousness and learning. Nat Rev Neurosci, 2002. 3(9): p. 679-93.

41. Sterpenich, V., et al., Sleep promotes the neural reorganization of remote emotional memory. J Neurosci, 2009. 29(16): p. 5143-52.

42. Jones, B.E., Basic mechanisms of sleep-wake states, in Principles and Practice of Sleep Medicine, M.H. Kryger, T. Roth, and W.C. Dement, Editors. 2000, Saunders: Philadelphia. p. 134-154.

43. Saper, C.B., Staying awake for dinner: hypothalamic integration of sleep, feeding, and circadian rhythms. Prog Brain Res, 2006. 153: p. 243-52.

44. Saper, C.B., T.C. Chou, and T.E. Scammell, The sleep switch: hypothalamic control of sleep and wakefulness. Trends Neurosci, 2001. 24(12): p. 726-31.

45. Saper, C.B., T.E. Scammell, and J. Lu, Hypothalamic regulation of sleep and circadian rhythms. Nature, 2005. 437(7063): p. 1257-63.

46. Steriade, M., and McCarley, R.W., Brain Control of Wakefulness and Sleep. II ed. 2005, New York: Kluwer Academic/Plenum Publishers.

47. Rechtshaffen, A., and Kales, A., A.M.F.S.T., techniques, and scoring for sleep stages of human subjects., in A Manual of Standardized Terminology, Techniques and Scoring System for Sleep Stages of Human Subjects M. Kryger, Roth, T., Dement, W.C., Editor. 1968, Public Health Service, U.S. Government Printing Office Washington.

48. Carskadon, M.A., and Rechtschaffen, A., Monitoring and staging human sleep., in Principles and Practice of Sleep Medicine, M. Kryger, Roth, T., Dement, W.C., Editor. 2000, W B Saunders: Philadelphia. p. 1197.

49. Carskadon, M.A., and Dement, W.C., Normal human sleep: an overview., in Principles and Practice of Sleep Medicine, M. Kryger, Roth, T., Dement, W.C., Editor. 2000, W B Saunders: Philadelphia. p. 15-26.

50. Creutzfeldt, O.D., S. Watanabe, and H.D. Lux, Relations between EEG phenomena and potentials of single cortical cells. I. Evoked responses after thalamic and erpicortical stimulation. Electroencephalogr Clin Neurophysiol, 1966. 20(1): p. 1-18.

51. Nunez, P.L. and R. Srinivasan, A theoretical basis for standing and traveling brain waves measured with human EEG with implications for an integrated consciousness. Clin Neurophysiol, 2006. 117(11): p. 2424-35.

52. Dressler, O., et al., Awareness and the EEG power spectrum: analysis of frequencies. Br J Anaesth, 2004. 93(6): p. 806-9.

102

53. Yang, C.C., et al., Relationship between electroencephalogram slow-wave magnitude and heart rate variability during sleep in humans. Neurosci Lett, 2002. 329(2): p. 213-6.

54. Neckelmann, D. and R. Ursin, Sleep stages and EEG power spectrum in relation to acoustical stimulus arousal threshold in the rat. Sleep, 1993. 16(5): p. 467-77.

55. Benington, J.H. and H.C. Heller, REM-sleep timing is controlled homeostatically by accumulation of REM-sleep propensity in non-REM sleep. Am J Physiol, 1994. 266(6 Pt 2): p. R1992-2000.

56. von Economo, C., Sleep as a problem of localization. J. Nerv. Ment. Dis, 1930. 71: p. 249-259.

57. McGinty, D. and R. Szymusiak, The sleep-wake switch: A neuronal alarm clock. Nat Med, 2000. 6(5): p. 510-1.

58. Moruzzi, G. and H.W. Magoun, Brain stem reticular formation and activation of the EEG. Electroencephalogr Clin Neurophysiol, 1949. 1(4): p. 455-73.

59. Hallanger, A.E., et al., The origins of cholinergic and other subcortical afferents to the thalamus in the rat. J Comp Neurol, 1987. 262(1): p. 105-24.

60. Szymusiak, R., N. Alam, and D. McGinty, Discharge patterns of neurons in cholinergic regions of the basal forebrain during waking and sleep. Behav Brain Res, 2000. 115(2): p. 171-82.

61. Hallanger, A.E. and B.H. Wainer, Ascending projections from the pedunculopontine tegmental nucleus and the adjacent mesopontine tegmentum in the rat. J Comp Neurol, 1988. 274(4): p. 483-515.

62. Saper, C.B. and A.D. Loewy, Efferent connections of the parabrachial nucleus in the rat. Brain Res, 1980. 197(2): p. 291-317.

63. Li, Y., et al., Hypocretin/Orexin excites hypocretin neurons via a local glutamate neuron-A potential mechanism for orchestrating the hypothalamic arousal system. Neuron, 2002. 36(6): p. 1169-81.

64. Lin, J.S., et al., Histaminergic descending inputs to the mesopontine tegmentum and their role in the control of cortical activation and wakefulness in the cat. J Neurosci, 1996. 16(4): p. 1523-37.

65. Moore, R.Y., E.A. Abrahamson, and A. Van Den Pol, The hypocretin neuron system: an arousal system in the human brain. Arch Ital Biol, 2001. 139(3): p. 195-205.

66. Neuzeret, P.C., et al., Application of histamine or serotonin to the hypoglossal nucleus increases genioglossus muscle activity across the wake-sleep cycle. J Sleep Res, 2009. 18(1): p. 113-21.

67. Jacobs, B.L. and E.C. Azmitia, Structure and function of the brain serotonin system. Physiol Rev, 1992. 72(1): p. 165-229.

68. Jones, B.E. and R.Y. Moore, Ascending projections of the locus coeruleus in the rat. II. Autoradiographic study. Brain Res, 1977. 127(1): p. 25-53.

69. Pace-Schott, E.F. and J.A. Hobson, The neurobiology of sleep: genetics, cellular physiology and subcortical networks. Nat Rev Neurosci, 2002. 3(8): p. 591-605.

103

70. Mignot, E., S. Taheri, and S. Nishino, Sleeping with the hypothalamus: emerging therapeutic targets for sleep disorders. Nat Neurosci, 2002. 5 Suppl: p. 1071-5.

71. Horner, R.L., Neuromodulation of hypoglossal motoneurons during sleep. Respir Physiol Neurobiol, 2008. 164(1-2): p. 179-96.

72. Lu, J. and M.A. Greco, Sleep circuitry and the hypnotic mechanism of GABAA drugs. J Clin Sleep Med, 2006. 2(2): p. S19-26.

73. Sherin, J.E., et al., Activation of ventrolateral preoptic neurons during sleep. Science, 1996. 271(5246): p. 216-9.

74. Lin, J.S., et al., A critical role of the posterior hypothalamus in the mechanisms of wakefulness determined by microinjection of muscimol in freely moving cats. Brain Res, 1989. 479(2): p. 225-40.

75. McGinty, D.J. and M.B. Sterman, Sleep suppression after basal forebrain lesions in the cat. Science, 1968. 160(833): p. 1253-5.

76. Ranson, S.W., Some Functions of the Hypothalamus: Harvey Lecture, December 17, 1936. Bull N Y Acad Med, 1937. 13(5): p. 241-271.

77. Swett, C.P. and J.A. Hobson, The effects of posterior hypothalamic lesions on behavioral and electrographic manifestations of sleep and waking in cats. Arch Ital Biol, 1968. 106(3): p. 283-93.

78. Szymusiak, R., et al., Sleep-waking discharge patterns of ventrolateral preoptic/anterior hypothalamic neurons in rats. Brain Res, 1998. 803(1-2): p. 178-88.

79. Lu, J., et al., Effect of lesions of the ventrolateral preoptic nucleus on NREM and REM sleep. J Neurosci, 2000. 20(10): p. 3830-42.

80. Gallopin, T., et al., Identification of sleep-promoting neurons in vitro. Nature, 2000. 404(6781): p. 992-5.

81. Bormann, J., The 'ABC' of GABA receptors. Trends Pharmacol Sci, 2000. 21(1): p. 16-9.

82. Sherin, J.E., et al., Innervation of histaminergic tuberomammillary neurons by GABAergic and galaninergic neurons in the ventrolateral preoptic nucleus of the rat. J Neurosci, 1998. 18(12): p. 4705-21.

83. Szymusiak, R., et al., Preoptic area sleep-regulating mechanisms. Arch Ital Biol, 2001. 139(1-2): p. 77-92.

84. Osaka, T. and H. Matsumura, Noradrenergic inputs to sleep-related neurons in the preoptic area from the locus coeruleus and the ventrolateral medulla in the rat. Neurosci Res, 1994. 19(1): p. 39-50.

85. Osaka, T. and H. Matsumura, Noradrenaline inhibits preoptic sleep-active neurons through alpha 2-receptors in the rat. Neurosci Res, 1995. 21(4): p. 323-30.

86. Gervasoni, D., et al., Electrophysiological evidence that noradrenergic neurons of the rat locus coeruleus are tonically inhibited by GABA during sleep. Eur J Neurosci, 1998. 10(3): p. 964-70.

87. McCormick, D.A. and T. Bal, Sleep and arousal: thalamocortical mechanisms. Annu Rev Neurosci, 1997. 20: p. 185-215.

104

88. Chamberlin, N.L., et al., Effects of adenosine on gabaergic synaptic inputs to identified ventrolateral preoptic neurons. Neuroscience, 2003. 119(4): p. 913-8.

89. Chou, T.C., et al., Afferents to the ventrolateral preoptic nucleus. J Neurosci, 2002. 22(3): p. 977-90.

90. Fuller, P.M., J.J. Gooley, and C.B. Saper, Neurobiology of the sleep-wake cycle: sleep architecture, circadian regulation, and regulatory feedback. J Biol Rhythms, 2006. 21(6): p. 482-93.

91. Saper, C.B., G. Cano, and T.E. Scammell, Homeostatic, circadian, and emotional regulation of sleep. J Comp Neurol, 2005. 493(1): p. 92-8.

92. Sinton, C.M. and R.W. McCarley, Neurophysiological mechanisms of sleep and wakefulness: a question of balance. Semin Neurol, 2004. 24(3): p. 211-23.

93. Borbely, A.A. and P. Achermann, Sleep homeostasis and models of sleep regulation. J Biol Rhythms, 1999. 14(6): p. 557-68.

94. Daan, S., D.G. Beersma, and A.A. Borbely, Timing of human sleep: recovery process gated by a circadian pacemaker. Am J Physiol, 1984. 246(2 Pt 2): p. R161-83.

95. Borbely, A.A., A two process model of sleep regulation. Hum Neurobiol, 1982. 1(3): p. 195-204.

96. Trachsel, L., I. Tobler, and A.A. Borbely, Sleep regulation in rats: effects of sleep deprivation, light, and circadian phase. Am J Physiol, 1986. 251(6 Pt 2): p. R1037-44.

97. Franken, P., I. Tobler, and A.A. Borbely, Sleep homeostasis in the rat: simulation of the time course of EEG slow-wave activity. Neurosci Lett, 1991. 130(2): p. 141-4.

98. Endo, T., et al., Selective and total sleep deprivation: effect on the sleep EEG in the rat. Psychiatry Res, 1997. 66(2-3): p. 97-110.

99. Borbely, A.A., I. Tobler, and M. Hanagasioglu, Effect of sleep deprivation on sleep and EEG power spectra in the rat. Behav Brain Res, 1984. 14(3): p. 171-82.

100. Brown, R.E., et al., Orexin A excites serotonergic neurons in the dorsal raphe nucleus of the rat. Neuropharmacology, 2001. 40(3): p. 457-9.

101. Brown, R.E., et al., Convergent excitation of dorsal raphe serotonin neurons by multiple arousal systems (orexin/hypocretin, histamine and noradrenaline). J Neurosci, 2002. 22(20): p. 8850-9.

102. Eriksson, K.S., et al., Orexin/hypocretin excites the histaminergic neurons of the tuberomammillary nucleus. J Neurosci, 2001. 21(23): p. 9273-9.

103. Korotkova, T.M., et al., Selective excitation of GABAergic neurons in the substantia nigra of the rat by orexin/hypocretin in vitro. Regul Pept, 2002. 104(1-3): p. 83-9.

104. Ohno, K. and T. Sakurai, Orexin neuronal circuitry: role in the regulation of sleep and wakefulness. Front Neuroendocrinol, 2008. 29(1): p. 70-87.

105. Nitz, D. and J.M. Siegel, GABA release in posterior hypothalamus across sleep-wake cycle. Am J Physiol, 1996. 271(6 Pt 2): p. R1707-12.

106. Espana, R.A., et al., Wake-promoting and sleep-suppressing actions of hypocretin (orexin): basal forebrain sites of action. Neuroscience, 2001. 106(4): p. 699-715.

105

107. Espana, R.A., et al., Organization of hypocretin/orexin efferents to locus coeruleus and basal forebrain arousal-related structures. J Comp Neurol, 2005. 481(2): p. 160-78.

108. Kilduff, T.S. and C. Peyron, The hypocretin/orexin ligand-receptor system: implications for sleep and sleep disorders. Trends Neurosci, 2000. 23(8): p. 359-65.

109. Yoshida, K., et al., Afferents to the orexin neurons of the rat brain. J Comp Neurol, 2006. 494(5): p. 845-61.

110. Thankachan, S., S. Kaur, and P.J. Shiromani, Activity of pontine neurons during sleep and cataplexy in hypocretin knock-out mice. J Neurosci, 2009. 29(5): p. 1580-5.

111. Jouvet, M. and F. Michel, [Electromyographic correlations of sleep in the chronic decorticate & mesencephalic cat.]. C R Seances Soc Biol Fil, 1959. 153(3): p. 422-5.

112. Aserinsky, E. and N. Kleitman, Regularly occurring periods of eye motility, and concomitant phenomena, during sleep. Science, 1953. 118(3062): p. 273-4.

113. Luppi, P.H., et al., Paradoxical (REM) sleep genesis: the switch from an aminergic-cholinergic to a GABAergic-glutamatergic hypothesis. J Physiol Paris, 2006. 100(5-6): p. 271-83.

114. Jouvet, M., [Research on the neural structures and responsible mechanisms in different phases of physiological sleep.]. Arch Ital Biol, 1962. 100: p. 125-206.

115. Jouvet, M., Paradoxical Sleep--a Study of Its Nature and Mechanisms. Prog Brain Res, 1965. 18: p. 20-62.

116. Jouvet, M., [The paradoxical phase of sleep]. Int J Neurol, 1965. 5(2): p. 131-50.

117. Sakai, K., et al., Tegmentoreticular projections with special reference to the muscular atonia during paradoxical sleep in the cat: an HRP study. Brain Res, 1979. 176(2): p. 233-54.

118. Garzon, M., I. De Andres, and F. Reinoso-Suarez, Sleep patterns after carbachol delivery in the ventral oral pontine tegmentum of the cat. Neuroscience, 1998. 83(4): p. 1137-44.

119. Lai, Y.Y. and J.M. Siegel, Cardiovascular and muscle tone changes produced by microinjection of cholinergic and glutamatergic agonists in dorsolateral pons and medial medulla. Brain Res, 1990. 514(1): p. 27-36.

120. Sakai, K. and Y. Koyama, Are there cholinergic and non-cholinergic paradoxical sleep-on neurones in the pons? Neuroreport, 1996. 7(15-17): p. 2449-53.

121. Hobson, J.A., R.W. McCarley, and P.W. Wyzinski, Sleep cycle oscillation: reciprocal discharge by two brainstem neuronal groups. Science, 1975. 189(4196): p. 55-8.

122. Aghajanian, G.K. and C.P. Vandermaelen, Intracellular identification of central noradrenergic and serotonergic neurons by a new double labeling procedure. J Neurosci, 1982. 2(12): p. 1786-92.

123. McGinty, D.J. and R.M. Harper, Dorsal raphe neurons: depression of firing during sleep in cats. Brain Res, 1976. 101(3): p. 569-75.

124. Jones, B.E., The role of noradrenergic locus coeruleus neurons and neighboring cholinergic neurons of the pontomesencephalic tegmentum in sleep-wake states. Prog Brain Res, 1991. 88: p. 533-43.

106

125. Gervasoni, D., et al., Effect of chronic treatment with milnacipran on sleep architecture in rats compared with paroxetine and imipramine. Pharmacol Biochem Behav, 2002. 73(3): p. 557-63.

126. Verret, L., et al., Localization of the neurons active during paradoxical (REM) sleep and projecting to the locus coeruleus noradrenergic neurons in the rat. J Comp Neurol, 2006. 495(5): p. 573-86.

127. Deurveilher, S. and E. Hennevin, Lesions of the pedunculopontine tegmental nucleus reduce paradoxical sleep (PS) propensity: evidence from a short-term PS deprivation study in rats. Eur J Neurosci, 2001. 13(10): p. 1963-76.

128. Shouse, M.N. and J.M. Siegel, Pontine regulation of REM sleep components in cats: integrity of the pedunculopontine tegmentum (PPT) is important for phasic events but unnecessary for atonia during REM sleep. Brain Res, 1992. 571(1): p. 50-63.

129. Jones, B.E., Paradoxical sleep and its chemical/structural substrates in the brain. Neuroscience, 1991. 40(3): p. 637-56.

130. Webster, H.H. and B.E. Jones, Neurotoxic lesions of the dorsolateral pontomesencephalic tegmentum-cholinergic cell area in the cat. II. Effects upon sleep-waking states. Brain Res, 1988. 458(2): p. 285-302.

131. Sakai, K., Sastre, J.P., Kanamori, N., Jouvet, M. , State-specific neurones in the ponto-medullary reticular formation with special reference to the postural atonia during paradoxical sleep in the cat., in Brain Mechanisms of Perceptual Awareness and Purposeful Behavior, O. Pompeiano, Aimone Marsan, C., Editor. 1981, Raven Press: New York. p. 405-429.

132. Kodama, T., Y. Takahashi, and Y. Honda, Enhancement of acetylcholine release during paradoxical sleep in the dorsal tegmental field of the cat brain stem. Neurosci Lett, 1990. 114(3): p. 277-82.

133. Lu, J., et al., A putative flip-flop switch for control of REM sleep. Nature, 2006. 441(7093): p. 589-94.

134. Fuller, P.M., C.B. Saper, and J. Lu, The pontine REM switch: past and present. J Physiol, 2007. 584(Pt 3): p. 735-41.

135. Sapin, E., et al., Localization of the brainstem GABAergic neurons controlling paradoxical (REM) sleep. PLoS One, 2009. 4(1): p. e4272.

136. Pollock, M.S. and R.E. Mistlberger, Rapid eye movement sleep induction by microinjection of the GABA-A antagonist bicuculline into the dorsal subcoeruleus area of the rat. Brain Res, 2003. 962(1-2): p. 68-77.

137. Boissard, R., et al., The rat ponto-medullary network responsible for paradoxical sleep onset and maintenance: a combined microinjection and functional neuroanatomical study. Eur J Neurosci, 2002. 16(10): p. 1959-73.

138. Nitz, D. and J.M. Siegel, GABA release in the locus coeruleus as a function of sleep/wake state. Neuroscience, 1997. 78(3): p. 795-801.

139. Leger, L., et al., Noradrenergic neurons expressing Fos during waking and paradoxical sleep deprivation in the rat. J Chem Neuroanat, 2009. 37(3): p. 149-57.

107

140. Kaur, S., R.N. Saxena, and B.N. Mallick, GABA in locus coeruleus regulates spontaneous rapid eye movement sleep by acting on GABAA receptors in freely moving rats. Neurosci Lett, 1997. 223(2): p. 105-8.

141. Mallick, B.N., S. Kaur, and R.N. Saxena, Interactions between cholinergic and GABAergic neurotransmitters in and around the locus coeruleus for the induction and maintenance of rapid eye movement sleep in rats. Neuroscience, 2001. 104(2): p. 467-85.

142. Finger, S., Origins of Neuroscience: A History of Explorations Into Brain Function. 1994, New York: Oxford University Press.

143. Spyer, K.M., To breathe or not to breathe? That is the question. Exp Physiol, 2009. 94(1): p. 1-10.

144. Duffin, J., Functional organization of respiratory neurones: a brief review of current questions and speculations. Exp Physiol, 2004. 89(5): p. 517-29.

145. Rybak, I.A., et al., Modeling the ponto-medullary respiratory network. Respir Physiol Neurobiol, 2004. 143(2-3): p. 307-19.

146. Dobbins, E.G. and J.L. Feldman, Brainstem network controlling descending drive to phrenic motoneurons in rat. J Comp Neurol, 1994. 347(1): p. 64-86.

147. Joseph, V., J.M. Pequignot, and O. Van Reeth, Neurochemical perspectives on the control of breathing during sleep. Respir Physiol Neurobiol, 2002. 130(3): p. 253-63.

148. Duffin, J. and J. Lipski, Monosynaptic excitation of thoracic motoneurones by inspiratory neurones of the nucleus tractus solitarius in the cat. J Physiol, 1987. 390: p. 415-31.

149. Hilaire, G., et al., Functional significance of the dorsal respiratory group in adult and newborn rats: in vivo and in vitro studies. Neurosci Lett, 1990. 111(1-2): p. 133-8.

150. Ezure, K. and I. Tanaka, Identification of deflation-sensitive inspiratory neurons in the dorsal respiratory group of the rat. Brain Res, 2000. 883(1): p. 22-30.

151. Mitchell, R.A., Site of termination of primary afferents from the carotid body chemoreceptors. Fed Proc, 1980. 39(9): p. 2657-61.

152. Hilaire, G. and R. Monteau, [Connections between inspiratory medullary neurons and phrenic or intercostal motoneurones (author's transl)]. J Physiol (Paris), 1976. 72(8): p. 987-1000.

153. Feldman, J.L., G.S. Mitchell, and E.E. Nattie, Breathing: rhythmicity, plasticity, chemosensitivity. Annu Rev Neurosci, 2003. 26: p. 239-66.

154. Del Negro, C.A., et al., Persistent sodium current, membrane properties and bursting behavior of pre-botzinger complex inspiratory neurons in vitro. J Neurophysiol, 2002. 88(5): p. 2242-50.

155. Johnson, S.M., N. Koshiya, and J.C. Smith, Isolation of the kernel for respiratory rhythm generation in a novel preparation: the pre-Botzinger complex "island". J Neurophysiol, 2001. 85(4): p. 1772-6.

156. Feldman, J.L. and C.A. Del Negro, Looking for inspiration: new perspectives on respiratory rhythm. Nat Rev Neurosci, 2006. 7(3): p. 232-42.

108

157. Smith, J.C., et al., Pre-Botzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science, 1991. 254(5032): p. 726-9.

158. McKay, L.C., W.A. Janczewski, and J.L. Feldman, Sleep-disordered breathing after targeted ablation of preBotzinger complex neurons. Nat Neurosci, 2005. 8(9): p. 1142-4.

159. Smith, J.C., et al., Brainstem projections to the major respiratory neuron populations in the medulla of the cat. J Comp Neurol, 1989. 281(1): p. 69-96.

160. Nattie, E.E., The retrotrapezoid nucleus and the 'drive' to breathe. J Physiol, 2006. 572(Pt 2): p. 311.

161. Paton, J.F., et al., Respiratory rhythm generation during gasping depends on persistent sodium current. Nat Neurosci, 2006. 9(3): p. 311-3.

162. Guyenet, P.G., R.L. Stornetta, and D.A. Bayliss, Retrotrapezoid nucleus and central chemoreception. J Physiol, 2008. 586(8): p. 2043-8.

163. Guyenet, P.G., et al., Retrotrapezoid nucleus: a litmus test for the identification of central chemoreceptors. Exp Physiol, 2005. 90(3): p. 247-53; discussion 253-7.

164. Nattie, E.E. and A. Li, Central chemoreception in the region of the ventral respiratory group in the rat. J Appl Physiol, 1996. 81(5): p. 1987-95.

165. Onimaru, H. and I. Homma, A novel functional neuron group for respiratory rhythm generation in the ventral medulla. J Neurosci, 2003. 23(4): p. 1478-86.

166. Onimaru, H., A. Arata, and I. Homma, Firing properties of respiratory rhythm generating neurons in the absence of synaptic transmission in rat medulla in vitro. Exp Brain Res, 1989. 76(3): p. 530-6.

167. Fortuna, M.G., et al., Botzinger expiratory-augmenting neurons and the parafacial respiratory group. J Neurosci, 2008. 28(10): p. 2506-15.

168. Guyenet, P.G., et al., Regulation of ventral surface chemoreceptors by the central respiratory pattern generator. J Neurosci, 2005. 25(39): p. 8938-47.

169. Smith, J.C., et al., Respiratory rhythm generation in neonatal and adult mammals: the hybrid pacemaker-network model. Respir Physiol, 2000. 122(2-3): p. 131-47.

170. Wittmeier, S., et al., Pacemakers handshake synchronization mechanism of mammalian respiratory rhythmogenesis. Proc Natl Acad Sci U S A, 2008. 105(46): p. 18000-5.

171. Pierrefiche, O., et al., Blockade of synaptic inhibition within the pre-Botzinger complex in the cat suppresses respiratory rhythm generation in vivo. J Physiol, 1998. 509 ( Pt 1): p. 245-54.

172. Shao, X.M. and J.L. Feldman, Respiratory rhythm generation and synaptic inhibition of expiratory neurons in pre-Botzinger complex: differential roles of glycinergic and GABAergic neural transmission. J Neurophysiol, 1997. 77(4): p. 1853-60.

173. Feldman, J.L. and J.C. Smith, Cellular mechanisms underlying modulation of breathing pattern in mammals. Ann N Y Acad Sci, 1989. 563: p. 114-30.

174. Onimaru, H. and I. Homma, Developmental changes in the spatio-temporal pattern of respiratory neuron activity in the medulla of late fetal rat. Neuroscience, 2005. 131(4): p. 969-77.

109

175. Orem, J., and Kubin, L., Respiratory Physiology: central neural control, in Principles and Practice of Sleep Medicine., M. Kryger, Roth, T., Dement, W.C., Editor. 2000, W B Saunders: Philadephia. p. 205-20.

176. Phillipson, E., and Bowes, G. , Control of breathing during sleep., in Handbook of Physiology Section 3: the Respiratory System. 1986, American Physiological Society: Baltimore. p. 649-90.

177. Phillipson, E.A., et al., Ventilatory and waking responses to CO2 in sleeping dogs. Am Rev Respir Dis, 1977. 115(2): p. 251-9.

178. Smith, C.A., et al., Ventilatory effects of specific carotid body hypocapnia in dogs during wakefulness and sleep. J Appl Physiol, 1995. 79(3): p. 689-99.

179. Orem, J., J. Montplaisir, and W.C. Dement, Changes in the activity of respiratory neurons during sleep. Brain Res, 1974. 82(2): p. 309-15.

180. Orem, J., et al., Activity of respiratory neurons during NREM sleep. J Neurophysiol, 1985. 54(5): p. 1144-56.

181. Lydic, R. and J. Orem, Respiratory neurons of the pneumotaxic center during sleep and wakefulness. Neurosci Lett, 1979. 15(2-3): p. 187-92.

182. Orem, J., Central respiratory activity in rapid eye movement sleep: augmenting and late inspiratory cells [corrected]. Sleep, 1994. 17(8): p. 665-73.

183. Orem, J., Augmenting expiratory neuronal activity in sleep and wakefulness and in relation to duration of expiration. J Appl Physiol, 1998. 85(4): p. 1260-6.

184. Krieger, J., Breathing in normal subjects, in Principles and Practice of Sleep Medicine, M. Kryger, Roth, T., Dement, W.C., Editor. 2000, W B Saunders: Philadelphia.

185. Phillipson, E.A., E. Murphy, and L.F. Kozar, Regulation of respiration in sleeping dogs. J Appl Physiol, 1976. 40(5): p. 688-93.

186. Orem, J., Medullary respiratory neuron activity: relationship to tonic and phasic REM sleep. J Appl Physiol, 1980. 48(1): p. 54-65.

187. Orem, J. and C.A. Anderson, Diaphragmatic activity during REM sleep in the adult cat. J Appl Physiol, 1996. 81(2): p. 751-60.

188. Lo, Y.L., et al., Influence of wakefulness on pharyngeal airway muscle activity. Thorax, 2007. 62(9): p. 799-805.

189. Orem, J. and T. Dick, Consistency and signal strength of respiratory neuronal activity. J Neurophysiol, 1983. 50(5): p. 1098-107.

190. Douglas, N.J., et al., Hypercapnic ventilatory response in sleeping adults. Am Rev Respir Dis, 1982. 126(5): p. 758-62.

191. Horner, R.L., et al., Effects of sleep-wake state on the genioglossus vs.diaphragm muscle response to CO(2) in rats. J Appl Physiol, 2002. 92(2): p. 878-87.

192. Horner, R.L., Respiratory motor activity: influence of neuromodulators and implications for sleep disordered breathing. Can J Physiol Pharmacol, 2007. 85(1): p. 155-65.

110

193. Horner, R.L., The neuropharmacology of upper airway motor control in the awake and asleep states: implications for obstructive sleep apnoea. Respir Res, 2001. 2(5): p. 286-94.

194. Horner, R.L., Control of genioglossus muscle by sleep state-dependent neuromodulators. Adv Exp Med Biol, 2008. 605: p. 262-7.

195. Fenik, V.B., R.O. Davies, and L. Kubin, REM sleep-like atonia of hypoglossal (XII) motoneurons is caused by loss of noradrenergic and serotonergic inputs. Am J Respir Crit Care Med, 2005. 172(10): p. 1322-30.

196. Chan, E., et al., Endogenous excitatory drive modulating respiratory muscle activity across sleep-wake states. Am J Respir Crit Care Med, 2006. 174(11): p. 1264-73.

197. Sood, S., et al., Role of endogenous serotonin in modulating genioglossus muscle activity in awake and sleeping rats. Am J Respir Crit Care Med, 2005. 172(10): p. 1338-47.

198. Morrison, J.L., et al., Role of inhibitory amino acids in control of hypoglossal motor outflow to genioglossus muscle in naturally sleeping rats. J Physiol, 2003. 552(Pt 3): p. 975-91.

199. Morrison, J.L., et al., GABAA receptor antagonism at the hypoglossal motor nucleus increases genioglossus muscle activity in NREM but not REM sleep. J Physiol, 2003. 548(Pt 2): p. 569-83.

200. Brooks, P.L. and J.H. Peever, Biochemical control of airway motor neurons during rapid eye movement sleep. Adv Exp Med Biol, 2008. 605: p. 437-41.

201. Brooks, P.L. and J.H. Peever, Glycinergic and GABA(A)-mediated inhibition of somatic motoneurons does not mediate rapid eye movement sleep motor atonia. J Neurosci, 2008. 28(14): p. 3535-45.

202. Walsh, J.K., Understanding GABA and its relation to insomnia and therapeutics. J Clin Sleep Med, 2006. 2(2): p. S5-6.

203. Mohler, H., GABA(A) receptor diversity and pharmacology. Cell Tissue Res, 2006. 326(2): p. 505-16.

204. Hellevuo, K., K. Kiianmaa, and E.R. Korpi, Effect of GABAergic drugs on motor impairment from ethanol, barbital and lorazepam in rat lines selected for differential sensitivity to ethanol. Pharmacol Biochem Behav, 1989. 34(2): p. 399-404.

205. Mihic, S.J. and R.A. Harris, GABA and the GABAA receptor. Alcohol Health Res World, 1997. 21(2): p. 127-31.

206. Orser, B.A., Extrasynaptic GABAA receptors are critical targets for sedative-hypnotic drugs. J Clin Sleep Med, 2006. 2(2): p. S12-8.

207. Jin, H., et al., Demonstration of functional coupling between gamma -aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles. Proc Natl Acad Sci U S A, 2003. 100(7): p. 4293-8.

208. Nutt, D., GABAA receptors: subtypes, regional distribution, and function. J Clin Sleep Med, 2006. 2(2): p. S7-11.

209. Mohler, H., GABAA receptors in central nervous system disease: anxiety, epilepsy, and insomnia. J Recept Signal Transduct Res, 2006. 26(5-6): p. 731-40.

111

210. Nutt, D.J. and A.L. Malizia, New insights into the role of the GABA(A)-benzodiazepine receptor in psychiatric disorder. Br J Psychiatry, 2001. 179: p. 390-6.

211. Chebib, M. and G.A. Johnston, The 'ABC' of GABA receptors: a brief review. Clin Exp Pharmacol Physiol, 1999. 26(11): p. 937-40.

212. Bowery, N.G., A.L. Hudson, and G.W. Price, GABAA and GABAB receptor site distribution in the rat central nervous system. Neuroscience, 1987. 20(2): p. 365-83.

213. Ong, J. and D.I. Kerr, Recent advances in GABAB receptors: from pharmacology to molecular biology. Acta Pharmacol Sin, 2000. 21(2): p. 111-23.

214. Radcliffe, R.A., et al., Behavioral characterization of alcohol-tolerant and alcohol-nontolerant rat lines and an f(2) generation. Behav Genet, 2004. 34(4): p. 453-63.

215. Wallner, M. and R.W. Olsen, Physiology and pharmacology of alcohol: the imidazobenzodiazepine alcohol antagonist site on subtypes of GABAA receptors as an opportunity for drug development? Br J Pharmacol, 2008. 154(2): p. 288-98.

216. Santhakumar, V., et al., Contributions of the GABAA receptor alpha6 subunit to phasic and tonic inhibition revealed by a naturally occurring polymorphism in the alpha6 gene. J Neurosci, 2006. 26(12): p. 3357-64.

217. Olsen, R.W., et al., GABAA receptor subtypes: the "one glass of wine" receptors. Alcohol, 2007. 41(3): p. 201-9.

218. Mody, I., J. Glykys, and W. Wei, A new meaning for "Gin & Tonic": tonic inhibition as the target for ethanol action in the brain. Alcohol, 2007. 41(3): p. 145-53.

219. Hanchar, H.J., et al., Alcohol-induced motor impairment caused by increased extrasynaptic GABA(A) receptor activity. Nat Neurosci, 2005. 8(3): p. 339-45.

220. Hanchar, H.J., M. Wallner, and R.W. Olsen, Alcohol effects on gamma-aminobutyric acid type A receptors: are extrasynaptic receptors the answer? Life Sci, 2004. 76(1): p. 1-8.

221. Glykys, J., et al., A new naturally occurring GABA(A) receptor subunit partnership with high sensitivity to ethanol. Nat Neurosci, 2007. 10(1): p. 40-8.

222. Smith, G.B. and R.W. Olsen, Functional domains of GABAA receptors. Trends Pharmacol Sci, 1995. 16(5): p. 162-8.

223. Korpi, E.R., G. Grunder, and H. Luddens, Drug interactions at GABA(A) receptors. Prog Neurobiol, 2002. 67(2): p. 113-59.

224. Pirker, S., et al., GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience, 2000. 101(4): p. 815-50.

225. Wisden, W., et al., The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J Neurosci, 1992. 12(3): p. 1040-62.

226. Brickley, S.G., S.G. Cull-Candy, and M. Farrant, Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors. J Physiol, 1996. 497 ( Pt 3): p. 753-9.

227. Low, K., et al., Molecular and neuronal substrate for the selective attenuation of anxiety. Science, 2000. 290(5489): p. 131-4.

112

228. Rudolph, U., et al., Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes. Nature, 1999. 401(6755): p. 796-800.

229. Porcello, D.M., et al., Intact synaptic GABAergic inhibition and altered neurosteroid modulation of thalamic relay neurons in mice lacking delta subunit. J Neurophysiol, 2003. 89(3): p. 1378-86.

230. Salin, P.A. and D.A. Prince, Spontaneous GABAA receptor-mediated inhibitory currents in adult rat somatosensory cortex. J Neurophysiol, 1996. 75(4): p. 1573-88.

231. Bai, D., et al., Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by gamma-aminobutyric acid(A) receptors in hippocampal neurons. Mol Pharmacol, 2001. 59(4): p. 814-24.

232. Nusser, Z. and I. Mody, Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells. J Neurophysiol, 2002. 87(5): p. 2624-8.

233. Semyanov, A., Cell type specificity of GABA(A) receptor mediated signaling in the hippocampus. Curr Drug Targets CNS Neurol Disord, 2003. 2(4): p. 240-7.

234. Semyanov, A., M.C. Walker, and D.M. Kullmann, GABA uptake regulates cortical excitability via cell type-specific tonic inhibition. Nat Neurosci, 2003. 6(5): p. 484-90.

235. Stell, B.M. and I. Mody, Receptors with different affinities mediate phasic and tonic GABA(A) conductances in hippocampal neurons. J Neurosci, 2002. 22(10): p. RC223.

236. Farrant, M. and Z. Nusser, Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors. Nat Rev Neurosci, 2005. 6(3): p. 215-29.

237. Chadderton, P., T.W. Margrie, and M. Hausser, Integration of quanta in cerebellar granule cells during sensory processing. Nature, 2004. 428(6985): p. 856-60.

238. Wallner, M., H.J. Hanchar, and R.W. Olsen, Low-dose alcohol actions on alpha4beta3delta GABAA receptors are reversed by the behavioral alcohol antagonist Ro15-4513. Proc Natl Acad Sci U S A, 2006. 103(22): p. 8540-5.

239. Hanchar, H.J., et al., Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to alpha4/6beta3delta GABAA receptors. Proc Natl Acad Sci U S A, 2006. 103(22): p. 8546-51.

240. Antkowiak, B., Different actions of general anesthetics on the firing patterns of neocortical neurons mediated by the GABA(A) receptor. Anesthesiology, 1999. 91(2): p. 500-11.

241. Bonin, R.P. and B.A. Orser, GABA(A) receptor subtypes underlying general anesthesia. Pharmacol Biochem Behav, 2008. 90(1): p. 105-12.

242. Orser, B.A., K.J. Canning, and J.F. Macdonald, Mechanisms of general anesthesia. Curr Opin Anaesthesiol, 2002. 15(4): p. 427-33.

243. Caraiscos, V.B., et al., Selective enhancement of tonic GABAergic inhibition in murine hippocampal neurons by low concentrations of the volatile anesthetic isoflurane. J Neurosci, 2004. 24(39): p. 8454-8.

244. Follesa, P., et al., Neurosteroids, GABAA receptors, and ethanol dependence. Psychopharmacology (Berl), 2006. 186(3): p. 267-80.

113

245. Maguire, J. and I. Mody, Steroid hormone fluctuations and GABA(A)R plasticity. Psychoneuroendocrinology, 2009.

246. Smith, S.S., et al., Neurosteroid regulation of GABA(A) receptors: Focus on the alpha4 and delta subunits. Pharmacol Ther, 2007. 116(1): p. 58-76.

247. Belelli, D. and J.J. Lambert, Neurosteroids: endogenous regulators of the GABA(A) receptor. Nat Rev Neurosci, 2005. 6(7): p. 565-75.

248. Harrison, N.L., Mechanisms of sleep induction by GABA(A) receptor agonists. J Clin Psychiatry, 2007. 68 Suppl 5: p. 6-12.

249. Bonora, M., et al., Selective depression by ethanol of upper airway respiratory motor activity in cats. Am Rev Respir Dis, 1984. 130(2): p. 156-61.

250. Bonora, M., W.M. St John, and T.A. Bledsoe, Differential elevation by protriptyline and depression by diazepam of upper airway respiratory motor activity. Am Rev Respir Dis, 1985. 131(1): p. 41-5.

251. Di Pasquale, E., et al., Effects of ethanol on respiratory activity in the neonatal rat brainstem-spinal cord preparation. Brain Res, 1995. 695(2): p. 271-4.

252. Gibson, I.C. and A.J. Berger, Effect of ethanol upon respiratory-related hypoglossal nerve output of neonatal rat brain stem slices. J Neurophysiol, 2000. 83(1): p. 333-42.

253. Morrison, J.L., et al., Glycine at hypoglossal motor nucleus: genioglossus activity, CO(2) responses, and the additive effects of GABA. J Appl Physiol, 2002. 93(5): p. 1786-96.

254. Park, E., et al., Systemic vs. central administration of common hypnotics reveals opposing effects on genioglossus muscle activity in rats. Sleep, 2008. 31(3): p. 355-65.

255. Donato, R. and A. Nistri, Relative contribution by GABA or glycine to Cl(-)-mediated synaptic transmission on rat hypoglossal motoneurons in vitro. J Neurophysiol, 2000. 84(6): p. 2715-24.

256. Donato, R. and A. Nistri, Differential short-term changes in GABAergic or glycinergic synaptic efficacy on rat hypoglossal motoneurons. J Neurophysiol, 2001. 86(2): p. 565-74.

257. Nieuwenhuijs, D., et al., Propofol for monitored anesthesia care: implications on hypoxic control of cardiorespiratory responses. Anesthesiology, 2000. 92(1): p. 46-54.

258. Eikermann, M., et al., Pentobarbital dose-dependently increases respiratory genioglossus muscle activity while impairing diaphragmatic function in anesthetized rats. Anesthesiology, 2009. 110(6): p. 1327-34.

259. Eikermann, M., et al., Differential effects of isoflurane and propofol on upper airway dilator muscle activity and breathing. Anesthesiology, 2008. 108(5): p. 897-906.

260. Leiter, J.C., et al., The effect of diazepam on genioglossal muscle activity in normal human subjects. Am Rev Respir Dis, 1985. 132(2): p. 216-9.

261. Lakshminarayan, S., et al., Effect of diazepam on ventilatory responses. Clin Pharmacol Ther, 1976. 20(2): p. 178-83.

114

262. Dolly, F.R. and A.J. Block, Effect of flurazepam on sleep-disordered breathing and nocturnal oxygen desaturation in asymptomatic subjects. Am J Med, 1982. 73(2): p. 239-43.

263. Guilleminault, C., et al., Aging and sleep apnea: action of benzodiazepine, acetazolamide, alcohol, and sleep deprivation in a healthy elderly group. J Gerontol, 1984. 39(6): p. 655-61.

264. Mendelson, W.B., et al., Effects of flurazepam on sleep, arousal threshold, and the perception of being asleep. Psychopharmacology (Berl), 1988. 95(2): p. 258-62.

265. Montravers, P., B. Dureuil, and J.M. Desmonts, Effects of i.v. midazolam on upper airway resistance. Br J Anaesth, 1992. 68(1): p. 27-31.

266. Berry, R.B., et al., Triazolam in patients with obstructive sleep apnea. Am J Respir Crit Care Med, 1995. 151(2 Pt 1): p. 450-4.

267. Camacho, M.E. and C.M. Morin, The effect of temazepam on respiration in elderly insomniacs with mild sleep apnea. Sleep, 1995. 18(8): p. 644-5.

268. Berry, R.B., C.R. McCasland, and R.W. Light, The effect of triazolam on the arousal response to airway occlusion during sleep in normal subjects. Am Rev Respir Dis, 1992. 146(5 Pt 1): p. 1256-60.

269. Cirignotta, F., et al., Zolpidem-polysomnographic study of the effect of a new hypnotic drug in sleep apnea syndrome. Pharmacol Biochem Behav, 1988. 29(4): p. 807-9.

270. McCann, C.C., et al., Effect of zolpidem during sleep on ventilation and cardiovascular variables in normal subjects. Fundam Clin Pharmacol, 1993. 7(6): p. 305-10.

271. Younes, M., Role of arousals in the pathogenesis of obstructive sleep apnea. Am J Respir Crit Care Med, 2004. 169(5): p. 623-33.

272. Douglas, N., Respiratory Physiology: Control of ventilation, in Principles and Practice of Sleep Medicine., M. Kryger, Roth, T., Dement, W.C., Editor. 2000, W B Saunders: Philadephia. p. 221-41.

273. Vallee, B.L., Alcohol in the western world. Sci Am, 1998. 278(6): p. 80-5.

274. Snow, W.M., et al., Alcohol use and cardiovascular health outcomes: a comparison across age and gender in the Winnipeg Health and Drinking Survey Cohort. Age Ageing, 2009.

275. Cargiulo, T., Understanding the health impact of alcohol dependence. Am J Health Syst Pharm, 2007. 64(5 Suppl 3): p. S5-11.

276. Schweizer, T.A. and M. Vogel-Sprott, Alcohol-impaired speed and accuracy of cognitive functions: a review of acute tolerance and recovery of cognitive performance. Exp Clin Psychopharmacol, 2008. 16(3): p. 240-50.

277. Schweizer, T.A., et al., Neuropsychological profile of acute alcohol intoxication during ascending and descending blood alcohol concentrations. Neuropsychopharmacology, 2006. 31(6): p. 1301-9.

278. Addicott, M.A., et al., The biphasic effects of alcohol: comparisons of subjective and objective measures of stimulation, sedation, and physical activity. Alcohol Clin Exp Res, 2007. 31(11): p. 1883-90.

115

279. Wallgren, H., and Barry III, H. , Actions of Alcohol. Vol. I. 1970, Amsterdam: Elsevier Publishing Co., LTD.

280. Lieber, C.S., Metabolism of Ethanol, in Metabolic Aspects of Alcoholism, C.S. Lieber, Editor. 1977, MTP Press Limited: Lancester.

281. Fox, A.F., Guzman, N.J., and Friedman, P.A., The Clinical Pharmacology of Alcohol, in Alcoholism: A Guide for the Primary Care Physician, H.N. Barnes, Aronson, M.D., Delbanco, T.L., Editor. 1987, Springer-Verlag New York Inc.: New York.

282. Owens, A.H., Jr. and E.K. Marshall, Jr., The metabolism of ethyl alcohol in the rat. J Pharmacol Exp Ther, 1955. 115(3): p. 360-70.

283. Wallgren, H., and Barry III, H. , Actions of Alcohol. Vol. II. 1970, Amsterdam: Elsevier Publishing Co., LTD.

284. Mitchell, M.C., Alcohol-induced impairment of central nervous system function: behavioral skills involved in driving. J Stud Alcohol Suppl, 1985. 10: p. 109-16.

285. Herrington, R.E., Alcohol Abuse and Alcohol Dependence: Treatment and Rehabilitation, in Alcohol and Drug Abuse Handbook, R.E. Herrington, Jacobson, G.R., Benzer, D.G., Editor. 1987, Warren H. Green, Inc.: St. Louis.

286. Meda, S.A., et al., Alcohol dose effects on brain circuits during simulated driving: An fMRI study. Hum Brain Mapp, 2008.

287. Miller, N.S., and Gold, M.S,, Alcohol. 1991, New York: Plenum Publishing Corporation.

288. Effects at specific B.A.C. levels. B.R.A.D: Be Responsible About Drinking. 2008 [cited; Available from: http://www.brad21.org/effects_at_specific_bac.html.

289. Understanding blood alcohol content (BAC). 2009 [cited; Available from: http://www.rochester.edu/uhs/healthtopics/Alcohol/bac.html.

290. Vengeliene, V., et al., Neuropharmacology of alcohol addiction. Br J Pharmacol, 2008. 154(2): p. 299-315.

291. Weight, F.F., D.M. Lovinger, and G. White, Alcohol inhibition of NMDA channel function. Alcohol Alcohol Suppl, 1991. 1: p. 163-9.

292. Weight, F.F., et al., Alcohol and anesthetic actions on excitatory amino acid-activated ion channels. Ann N Y Acad Sci, 1991. 625: p. 97-107.

293. Carta, M., et al., Alcohol potently inhibits the kainate receptor-dependent excitatory drive of hippocampal interneurons. Proc Natl Acad Sci U S A, 2003. 100(11): p. 6813-8.

294. Lovinger, D.M., Ethanol potentiation of 5-HT3 receptor-mediated ion current in NCB-20 neuroblastoma cells. Neurosci Lett, 1991. 122(1): p. 57-60.

295. Lovinger, D.M., Ethanol potentiates ion current mediated by 5-HT3 receptors on neuroblastoma cells and isolated neurons. Alcohol Alcohol Suppl, 1991. 1: p. 181-5.

296. Lovinger, D.M. and G. White, Ethanol potentiation of 5-hydroxytryptamine3 receptor-mediated ion current in neuroblastoma cells and isolated adult mammalian neurons. Mol Pharmacol, 1991. 40(2): p. 263-70.

116

297. Roberto, M., et al., Ethanol increases GABAergic transmission at both pre- and postsynaptic sites in rat central amygdala neurons. Proc Natl Acad Sci U S A, 2003. 100(4): p. 2053-8.

298. Lewohl, J.M., et al., G-protein-coupled inwardly rectifying potassium channels are targets of alcohol action. Nat Neurosci, 1999. 2(12): p. 1084-90.

299. Cossart, R., et al., GluR5 kainate receptor activation in interneurons increases tonic inhibition of pyramidal cells. Nat Neurosci, 1998. 1(6): p. 470-8.

300. Crowder, T.L., O.J. Ariwodola, and J.L. Weiner, Ethanol antagonizes kainate receptor-mediated inhibition of evoked GABA(A) inhibitory postsynaptic currents in the rat hippocampal CA1 region. J Pharmacol Exp Ther, 2002. 303(3): p. 937-44.

301. Kostowski, W., 5-HT3 receptors and central effects of ethanol. Pol J Pharmacol, 1996. 48(3): p. 243-54.

302. Derkach, V., A. Surprenant, and R.A. North, 5-HT3 receptors are membrane ion channels. Nature, 1989. 339(6227): p. 706-9.

303. Dyr, W. and W. Kostowski, Effects of 5-HT3 receptor agonists on voluntary ethanol intake in rats maintained on a limited access procedure. Alcohol Alcohol, 1997. 32(4): p. 455-62.

304. Carboni, E., et al., Differential inhibitory effects of a 5-HT3 antagonist on drug-induced stimulation of dopamine release. Eur J Pharmacol, 1989. 164(3): p. 515-9.

305. Lovinger, D.M. and Q. Zhou, Alcohols potentiate ion current mediated by recombinant 5-HT3RA receptors expressed in a mammalian cell line. Neuropharmacology, 1994. 33(12): p. 1567-72.

306. Ye, J.H., K.A. Sokol, and U. Bhavsar, Glycine receptors contribute to hypnosis induced by ethanol. Alcohol Clin Exp Res, 2009. 33(6): p. 1069-74.

307. Welsh, B.T., B.E. Goldstein, and S.J. Mihic, Single-channel analysis of ethanol enhancement of glycine receptor function. J Pharmacol Exp Ther, 2009. 330(1): p. 198-205.

308. Eggers, E.D. and A.J. Berger, Mechanisms for the modulation of native glycine receptor channels by ethanol. J Neurophysiol, 2004. 91(6): p. 2685-95.

309. Molander, A., et al., Involvement of accumbal glycine receptors in the regulation of voluntary ethanol intake in the rat. Alcohol Clin Exp Res, 2005. 29(1): p. 38-45.

310. Lido, H.H., et al., The glycine reuptake inhibitor org 25935 interacts with Basal and ethanol-induced dopamine release in rat nucleus accumbens. Alcohol Clin Exp Res, 2009. 33(7): p. 1151-7.

311. Molander, A. and B. Soderpalm, Accumbal strychnine-sensitive glycine receptors: an access point for ethanol to the brain reward system. Alcohol Clin Exp Res, 2005. 29(1): p. 27-37.

312. Molander, A. and B. Soderpalm, Glycine receptors regulate dopamine release in the rat nucleus accumbens. Alcohol Clin Exp Res, 2005. 29(1): p. 17-26.

117

313. Yevenes, G.E., et al., A selective G betagamma-linked intracellular mechanism for modulation of a ligand-gated ion channel by ethanol. Proc Natl Acad Sci U S A, 2008. 105(51): p. 20523-8.

314. Williams, K.L., et al., Glycine enhances the central depressant properties of ethanol in mice. Pharmacol Biochem Behav, 1995. 50(2): p. 199-205.

315. Hayashi, F. and J. Lipski, The role of inhibitory amino acids in control of respiratory motor output in an arterially perfused rat. Respir Physiol, 1992. 89(1): p. 47-63.

316. Schwarzacher, S.W., J.C. Smith, and D.W. Richter, Pre-Botzinger complex in the cat. J Neurophysiol, 1995. 73(4): p. 1452-61.

317. Sebe, J.Y., E.D. Eggers, and A.J. Berger, Differential effects of ethanol on GABA(A) and glycine receptor-mediated synaptic currents in brain stem motoneurons. J Neurophysiol, 2003. 90(2): p. 870-5.

318. Brooks, P.L. and J.H. Peever, Unraveling the mechanisms of REM sleep atonia. Sleep, 2008. 31(11): p. 1492-7.

319. Dobbins, E.G. and J.L. Feldman, Differential innervation of protruder and retractor muscles of the tongue in rat. J Comp Neurol, 1995. 357(3): p. 376-94.

320. O'Brien, J.A. and A.J. Berger, Cotransmission of GABA and glycine to brain stem motoneurons. J Neurophysiol, 1999. 82(3): p. 1638-41.

321. Oku, Y., et al., Modulation of glycinergic synaptic current kinetics by octanol in mouse hypoglossal motoneurons. Pflugers Arch, 1999. 438(5): p. 656-64.

322. Forman, S.A. and K.W. Miller, Molecular sites of anesthetic action in postsynaptic nicotinic membranes. Trends Pharmacol Sci, 1989. 10(11): p. 447-52.

323. Grobin, A.C., et al., The role of GABA(A) receptors in the acute and chronic effects of ethanol. Psychopharmacology (Berl), 1998. 139(1-2): p. 2-19.

324. Hellevuo, K. and K. Kiianmaa, Effects of ethanol, barbital, and lorazepam on brain monoamines in rat lines selectively outbred for differential sensitivity to ethanol. Pharmacol Biochem Behav, 1988. 29(1): p. 183-8.

325. Suzdak, P.D. and S.M. Paul, Ethanol stimulates GABA receptor-mediated Cl- ion flux in vitro: possible relationship to the anxiolytic and intoxicating actions of alcohol. Psychopharmacol Bull, 1987. 23(3): p. 445-51.

326. Suzdak, P.D., et al., Ethanol stimulates gamma-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes. Proc Natl Acad Sci U S A, 1986. 83(11): p. 4071-5.

327. Frye, G.D. and G.R. Breese, GABAergic modulation of ethanol-induced motor impairment. J Pharmacol Exp Ther, 1982. 223(3): p. 750-6.

328. Hakkinen, H.M. and E. Kulonen, Ethanol intoxication and gamma-aminobutyric acid. J Neurochem, 1976. 27(2): p. 631-3.

329. Hellevuo, K., et al., Intoxicating effects of lorazepam and barbital in rat lines selected for differential sensitivity to ethanol. Psychopharmacology (Berl), 1987. 91(3): p. 263-7.

118

330. Mihalek, R.M., et al., GABA(A)-receptor delta subunit knockout mice have multiple defects in behavioral responses to ethanol. Alcohol Clin Exp Res, 2001. 25(12): p. 1708-18.

331. Wei, W., L.C. Faria, and I. Mody, Low ethanol concentrations selectively augment the tonic inhibition mediated by delta subunit-containing GABAA receptors in hippocampal neurons. J Neurosci, 2004. 24(38): p. 8379-82.

332. Ticku, M.K. and S.K. Kulkarni, Molecular interactions of ethanol with GABAergic system and potential of RO15-4513 as an ethanol antagonist. Pharmacol Biochem Behav, 1988. 30(2): p. 501-10.

333. Suzdak, P.D., et al., A selective imidazobenzodiazepine antagonist of ethanol in the rat. Science, 1986. 234(4781): p. 1243-7.

334. June, H.L. and M.J. Lewis, Interactions of Ro15-4513, Ro15-1788 (flumazenil) and ethanol on measures of exploration and locomotion in rats. Psychopharmacology (Berl), 1994. 116(3): p. 309-16.

335. Hoffman, P.L., et al., Effect of an imidazobenzodiazepine, Ro15-4513, on the incoordination and hypothermia produced by ethanol and pentobarbital. Life Sci, 1987. 41(5): p. 611-9.

336. Rastogi, S.K. and M.K. Ticku, Anticonvulsant profile of drugs which facilitate GABAergic transmission on convulsions mediated by a GABAergic mechanism. Neuropharmacology, 1986. 25(2): p. 175-85.

337. Syapin, P.J., K.W. Gee, and R.L. Alkana, Ro15-4513 differentially affects ethanol-induced hypnosis and hypothermia. Brain Res Bull, 1987. 19(5): p. 603-5.

338. Dar, M.S., Antagonism by intracerebellar Ro15-4513 of acute ethanol-induced motor incoordination in mice. Pharmacol Biochem Behav, 1995. 52(1): p. 217-23.

339. Jia, F., P.A. Goldstein, and N.L. Harrison, The modulation of synaptic GABA(A) receptors in the thalamus by eszopiclone and zolpidem. J Pharmacol Exp Ther, 2009. 328(3): p. 1000-6.

340. Wafford, K.A., et al., Novel compounds selectively enhance delta subunit containing GABA A receptors and increase tonic currents in thalamus. Neuropharmacology, 2009. 56(1): p. 182-9.

341. Lumley, M., et al., Ethanol and caffeine effects on daytime sleepiness/alertness. Sleep, 1987. 10(4): p. 306-12.

342. MacLean, A.W. and J. Cairns, Dose-response effects of ethanol on the sleep of young men. J Stud Alcohol, 1982. 43(5): p. 434-44.

343. Williams, D.L., A.W. MacLean, and J. Cairns, Dose-response effects of ethanol on the sleep of young women. J Stud Alcohol, 1983. 44(3): p. 515-23.

344. Walsh, J.K., et al., Sedative effects of ethanol at night. J Stud Alcohol, 1991. 52(6): p. 597-600.

345. Mendelson, W.B. and S.Y. Hill, Effects of the acute administration of ethanol on the sleep of the rat: a dose-response study. Pharmacol Biochem Behav, 1978. 8(6): p. 723-6.

119

346. Hattan, D.G. and P.I. Eacho, Relationship of ethanol blood level to REM and non-REM sleep time and distribution in the rat. Life Sci, 1978. 22(10): p. 839-46.

347. Pian, J.P., et al., Differential effects of acute alcohol on EEG and sedative responses in adolescent and adult Wistar rats. Brain Res, 2008. 1194: p. 28-36.

348. Lu, J., et al., Role of endogenous sleep-wake and analgesic systems in anesthesia. J Comp Neurol, 2008. 508(4): p. 648-62.

349. Dawson, A., et al., Effect of bedtime alcohol on inspiratory resistance and respiratory drive in snoring and nonsnoring men. Alcohol Clin Exp Res, 1997. 21(2): p. 183-90.

350. Young, T., et al., The occurrence of sleep-disordered breathing among middle-aged adults. New England Journal of Medicine, 1993. 328(17): p. 1230-5.

351. Phillipson, E.A., Sleep apnea - a major public health problem. New England Journal of Medicine, 1993. 328(17): p. 1271-3.

352. Dawson, A., et al., Effect of bedtime ethanol on total inspiratory resistance and respiratory drive in normal nonsnoring men. Alcohol Clin Exp Res, 1993. 17(2): p. 256-62.

353. Scrima, L., P.G. Hartman, and F.C. Hiller, Effect of three alcohol doses on breathing during sleep in 30-49 year old nonobese snorers and nonsnorers. Alcohol Clin Exp Res, 1989. 13(3): p. 420-7.

354. Taasan, V.C., et al., Alcohol increases sleep apnea and oxygen desaturation in asymptomatic men. Am J Med, 1981. 71(2): p. 240-5.

355. Issa, F.G. and C.E. Sullivan, Alcohol, snoring and sleep apnea. Journal of Neurology, Neurosurgery and Psychiatry, 1982. 45(4): p. 353-9.

356. Bonora, M., et al., Selective depression by ethanol of upper airway respiratory motor activity in cats. American Review of Respiratory Disease, 1984. 130(2): p. 156-61.

357. Deitrich, R.A., et al., Mechanism of action of ethanol: initial central nervous system actions. Pharmacol Rev, 1989. 41(4): p. 489-537.

358. Harris, R.A., Ethanol actions on multiple ion channels: which are important? Alcohol Clin Exp Res, 1999. 23(10): p. 1563-70.

359. Breese, G.R., et al., Basis of the gabamimetic profile of ethanol. Alcohol Clin Exp Res, 2006. 30(4): p. 731-44.

360. Saper, C.B., T.C. Chou, and T.E. Scammell, The sleep switch: hypothalamic control of sleep and wakefulness. Trends in Neuroscience, 2001. 24(12): p. 726-31.

361. McGinty, D. and R. Szymusiak, The sleep-wake switch: A neuronal alarm clock. Nature Medicine, 2000. 6(5): p. 510-1.

362. Morrison, J.L., et al., GABA-A receptor antagonism at the hypoglossal motor nucleus increases genioglossus muscle activity in NREM but not REM sleep. Journal of Physiology, 2003. 548: p. 569-583.

363. Morrison, J.L., et al., Role of inhibitory amino acids in control of hypoglossal motor outflow to genioglossus muscle in naturally sleeping rats. Journal of Physiology, 2003. 552: p. 975-991.

120

364. Liu, X., et al., Suppression of genioglossus muscle tone and activity during reflex hypercapnic stimulation by GABA(A) mechanisms at the hypoglossal motor nucleus in vivo. Neuroscience, 2003. 116(1): p. 249-59.

365. Morrison, J.L., et al., Glycine at the hypoglossal motor nucleus: genioglossus activity, CO2 responses and the additive effects of GABA. Journal of Applied Physiology, 2002. 93: p. 1786-1796.

366. Steenland, H.W., H. Liu, and R.L. Horner, Endogenous glutamatergic control of rhythmically active mammalian respiratory motoneurons in vivo. Journal of Neuroscience, 2008. 28(27): p. 6826-35.

367. Steenland, H.W., et al., Respiratory activation of the genioglossus muscle involves both non-NMDA and NMDA glutamate receptors at the hypoglossal motor nucleus in-vivo. Neuroscience, 2006. 138: p. 1407-1424.

368. Eggers, E.D., J.A. O'Brien, and A.J. Berger, Developmental changes in the modulation of synaptic glycine receptors by ethanol. Journal of Neurophysiology, 2000. 84(5): p. 2409-16.

369. Horner, R.L., et al., Effects of sleep-wake state on the genioglossus vs. diaphragm muscle response to CO2 in rats. Journal of Applied Physiology, 2002. 92(2): p. 878-87.

370. Hamrahi, H., B. Chan, and R.L. Horner, On-line detection of sleep-wake states and application to produce intermittent hypoxia only in sleep in rats. Journal of Applied Physiology, 2001. 90(6): p. 2130-40.

371. Feige, B., et al., Effects of alcohol on polysomnographically recorded sleep in healthy subjects. Alcohol Clin Exp Res, 2006. 30(9): p. 1527-37.

372. Hernandez, O.H., et al., Acute dose of alcohol affects cognitive components of reaction time to an omitted stimulus: differences among sensory systems. Psychopharmacology (Berl), 2006. 184(1): p. 75-81.

373. Roehrs, T., et al., Residual sedating effects of ethanol. Alcohol Clin Exp Res, 1994. 18(4): p. 831-4.

374. Zwyghuizen-Doorenbos, A., et al., Individual differences in the sedating effects of ethanol. Alcohol Clin Exp Res, 1990. 14(3): p. 400-4.

375. Shram, M.J., et al., Motor impairing effects of ethanol and diazepam in rats selectively bred for high and low ethanol consumption in a limited-access paradigm. Alcohol Clin Exp Res, 2004. 28(12): p. 1814-21.

376. Tullis, K.V., et al., An animal model for the measurement of acute tolerance to ethanol. Life Sci, 1977. 20(5): p. 875-82.

377. Behn, C.G., et al., Mathematical model of network dynamics governing mouse sleep-wake behavior. J Neurophysiol, 2007. 97(6): p. 3828-40.

378. Borbély, A., and Achermann, P., Sleep homeostasis and models of sleep regulation, in Principles and Practice of Sleep Medicine, M. Kryger, Roth, T., Dement, W.C., Editor. 2000, W B Saunders: Philadelphia. p. 377-390.

121

379. Jelev, A., et al., Microdialysis perfusion of 5-HT into hypoglossal motor nucleus differentially modulates genioglossus activity across natural sleep-wake states in rats. Journal of Physiology, 2001. 532: p. 467-81.

380. Paxinos, G. and C. Watson, The rat brain in stereotaxic coordinates. 4th ed. 1998, San Diego: Academic Press.

381. Colton, T., Statistics in Medicine. 1974, Boston: Little, Brown and Co.

382. Young, T., P.E. Peppard, and D.J. Gottlieb, Epidemiology of obstructive sleep apnea: a population health perspective. American Journal of Respiratory and Critical Care Medicine, 2002. 165(9): p. 1217-39.

383. Young, T., P.E. Peppard, and S. Taheri, Excess weight and sleep-disordered breathing. Journal of Applied Physiology, 2005. 99(4): p. 1592-9.

384. Gibson, I.C. and A.J. Berger, Effect of ethanol upon respiratory-related hypoglossal nerve output of neonatal rat brain stem slices. Journal of Neurophysiology, 2000. 83(1): p. 333-42.

385. Jia, F., L. Pignataro, and N.L. Harrison, GABAA receptors in the thalamus: alpha4 subunit expression and alcohol sensitivity. Alcohol, 2007. 41(3): p. 177-85.

386. Khan, Z.U., et al., The alpha 4 subunit of the GABAA receptors from rat brain and retina. Neuropharmacology, 1996. 35(9-10): p. 1315-22.

387. Sur, C., et al., Preferential coassembly of alpha4 and delta subunits of the gamma-aminobutyric acidA receptor in rat thalamus. Mol Pharmacol, 1999. 56(1): p. 110-5.

388. Kuna, S.T. and J.E. Remmers, Premotor input to hypoglossal motoneurons from Kolliker-Fuse neurons in decerebrate cats. Respir Physiol, 1999. 117(2-3): p. 85-95.

389. Peever, J.H., L. Shen, and J. Duffin, Respiratory pre-motor control of hypoglossal motoneurons in the rat. Neuroscience, 2002. 110(4): p. 711-22.

390. Roda, F., et al., Effects of anesthetics on hypoglossal nerve discharge and c-Fos expression in brainstem hypoglossal premotor neurons. J Comp Neurol, 2004. 468(4): p. 571-86.

391. Eastwood, P.R., et al., Collapsibility of the upper airway during anesthesia with isoflurane. Anesthesiology, 2002. 97(4): p. 786-93.

392. Orem, J. and L. Kubin, Respiratory physiology: Central neural control, in Principles and Practice of Sleep Medicine, M.H. Kryger, T. Roth, and W.C. Dement, Editors. 2000, Saunders, W. B.: Philadelphia. p. 205-20.

393. Kubin, L., R.O. Davies, and L. Pack, Control of upper airway motoneurons during REM sleep. News in Physiological Sciences, 1998. 13: p. 637-656.

394. Horner, R.L., Emerging principles and neural substrates underlying tonic sleep state-dependent influences on respiratory motor activity. Philosophical Transactions of the Royal Society – Biology, 2000. In Press.

395. Krieger, J., Breathing in normal subjects, in Principles and Practice of Sleep Medicine, M.H. Kryger, T. Roth, and W.C. Dement, Editors. 2000, Saunders, W. B.: Philadelphia. p. 229-41.

122

396. Phillipson, E.A. and G. Bowes, Control of breathing during sleep, in Handbook of Physiology, section 3, The Respiratory System, Vol. II, Control of Breathing, part 2, N.S. Cherniack and J.G. Widdicombe, Editors. 1986, American Physiological Society: Bethesda, MD, USA. p. 649-689.

397. Rechtschaffen, A., P. Hauri, and M. Zeitlin, Auditory awakening thresholds in REM and NREM sleep stages. Percept Mot Skills, 1966. 22(3): p. 927-42.

398. Williams, H.L., et al., Responses to Auditory Stimulation, Sleep Loss and the Eeg Stages of Sleep. Electroencephalogr Clin Neurophysiol, 1964. 16: p. 269-79.

399. Guyenet, P.G., et al., The retrotrapezoid nucleus and central chemoreception. Adv Exp Med Biol, 2008. 605: p. 327-32.

400. Nattie, E., Multiple sites for central chemoreception: their roles in response sensitivity and in sleep and wakefulness. Respir Physiol, 2000. 122(2-3): p. 223-35.

401. Nattie, E. and A. Li, Central chemoreception 2005: a brief review. Auton Neurosci, 2006. 126-127: p. 332-8.

402. Nattie, E. and A. Li, Central chemoreception is a complex system function that involves multiple brain stem sites. J Appl Physiol, 2009. 106(4): p. 1464-6.

403. Nelson, L.E., et al., The sedative component of anesthesia is mediated by GABA(A) receptors in an endogenous sleep pathway. Nat Neurosci, 2002. 5(10): p. 979-84.

404. Biancardi, V., et al., Locus coeruleus noradrenergic neurons and CO2 drive to breathing. Pflugers Arch, 2008. 455(6): p. 1119-28.

405. Filosa, J.A., J.B. Dean, and R.W. Putnam, Role of intracellular and extracellular pH in the chemosensitive response of rat locus coeruleus neurones. J Physiol, 2002. 541(Pt 2): p. 493-509.

406. Ito, Y., et al., Optical mapping of pontine chemosensitive regions of neonatal rat. Neurosci Lett, 2004. 366(1): p. 103-6.

407. Kaur, S., R.N. Saxena, and B.N. Mallick, GABAergic neurons in prepositus hypoglossi regulate REM sleep by its action on locus coeruleus in freely moving rats. Synapse, 2001. 42(3): p. 141-50.

408. Shea, J.L., et al., Rapid eye movement (REM) sleep homeostatic regulatory processes in the rat: changes in the sleep-wake stages and electroencephalographic power spectra. Brain Res, 2008. 1213: p. 48-56.

409. Datta, S. and J.A. Hobson, The rat as an experimental model for sleep neurophysiology. Behav Neurosci, 2000. 114(6): p. 1239-44.