purinergic regulation of breathing in cane toads bufo marinus · 2015-04-17 · ii purinergic...

Purinergic Regulation of Breathing in Cane toads (Bufo marinus)

by

Joseph Chau

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

Cell and Systems Biology

University of Toronto

© Copyright by Joseph Chau (2014)

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Purinergic Regulation of Breathing in Cane toads (Bufo marinus)

Joseph Chau

Master of Science

Graduate department of Cell and Systems Biology

University of Toronto

(2014)

Abstract

Previous studies have shown that descending inputs from the midbrain exert an inhibitory

modulation on fictive breathing frequency in the cane toad (Bufo marinus) and that these inputs

become strengthened during chronic hypoxia (CH). I hypothesize that adenosine (ADO) plays a

role behind this inhibitory modulation, due to the fact that (1) ADO is a naturally circulating

metabolite, (2) extracellular [ADO] increases significantly during CH, and (3) the adenosine 1

receptor (A1R) is the primary receptor for ADO in the brain. In this study breathing was

measured by recording motor output (fictive breathing) from respiratory nerves in an isolated

brainstem-spinal cord preparation superfused with artificial cerebral spinal fluid (aCSF). The

results indicate that ADO inhibited fictive breathing frequency (fR) and total fictive ventilation

(TFV) and that this inhibition was mediated primarily by the A1R. Transecting the midbrain

caused an increase in fictive fR and TFV at hypercapnic aCSF pH levels and reduced the

inhibitory modulation mediated by A1R such that A1R activation by endogenous ADO no longer

influenced breathing. Collectively, the study supports a role for ADO as a factor behind the

inhibitory modulation on breathing exerted by descending input from the midbrain.

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Table of Contents

List of Abbreviations .......................................................................................................................... vii

List of Figures ....................................................................................................................................... x

Chapter 1: General Introduction .......................................................................................................... 1

1.1 Breathing in Animals ............................................................................................................... 2

1.2 Breathing in Amphibians ......................................................................................................... 2

1.3 Breathing Patterns in Amphibians .......................................................................................... 4

1.4 Control of Breathing ................................................................................................................ 5

1.4.1 Central Processes ......................................................................................................... 5

1.4.2 Olfactory Chemoreceptors .......................................................................................... 6

1.4.3 Pulmonary Stretch Receptors ..................................................................................... 6

1.4.4 Peripheral O2/CO2/pH sensitive chemoreceptors ...................................................... 7

1.4.5 Central pH/CO2 sensitive chemoreceptors................................................................. 7

1.4.6 Neurotransmitters ........................................................................................................ 8

1.5 In vitro Brainstem – Spinal Cord Preparation ........................................................................ 8

1.6 Adenosine ................................................................................................................................. 9

1.7 Hypothesis and Objectives .................................................................................................... 10

Chapter 2: General Materials and Methods ...................................................................................... 12

2.1 Experimental Animals ........................................................................................................... 13

2.2 Artificial Cerebral Spinal Fluid (aCSF) Solution ................................................................ 13

2.3 The In vitro Brainstem-Spinal Cord Preparation ................................................................. 14

2.4 Experimental protocol: aCSF pH Changes and Treatment with Adenosine, CCPA or

DPCPX ................................................................................................................................... 15

2.5 Experimental protocol: Midbrain Transection ..................................................................... 17

2.6 Controls .................................................................................................................................. 18

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2.7 Data and statistical analyses .................................................................................................. 18

Chapter 3: The Effects of Adenosine, CCPA and DPCPX on intact Brainstem-Spinal Cord

Preparations ................................................................................................................................... 20

3.1 Introduction ............................................................................................................................ 21

3.2 Hypothesis & Objectives ....................................................................................................... 22

3.3 Materials and Methods .......................................................................................................... 24

3.4 Data Analysis ......................................................................................................................... 26

3.5 Results..................................................................................................................................... 27

3.5.1 Fictive Breathing Frequency ..................................................................................... 27

3.5.2 Fictive Episodes per Minute ..................................................................................... 29

3.5.3 Fictive Breaths per Episode ...................................................................................... 31

3.5.4 Integrated Area of Fictive Breaths ........................................................................... 33

3.5.5 Fictive Breath Duration ............................................................................................. 35

3.5.6 Total Fictive Ventilation ........................................................................................... 37

3.6 Discussion............................................................................................................................... 39

Chapter 4: The effects of a Midbrain Transection on Fictive Breathing ........................................ 41

4.1 Introduction ............................................................................................................................ 42

4.2 Hypothesis & Objectives ....................................................................................................... 43


4.4 Data Analysis ......................................................................................................................... 46

4.5 Results..................................................................................................................................... 47




4.5.4 Integrated area of fictive breaths .............................................................................. 51


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4.6 Discussion............................................................................................................................... 57

Chapter 5: Effects of Adenosine, CCPA and DPCPX on Fictive Breathing Following a

Midbrain Transection .................................................................................................................... 60

5.1 Introduction ............................................................................................................................ 61


5.3 Data Presentation and Analysis ............................................................................................. 64

5.4 Results..................................................................................................................................... 66




5.4.4 Integrated Area of Fictive Breaths ........................................................................... 76



5.5 Discussion............................................................................................................................... 86

5.5.1 Possible Sites of Adenosine Action.......................................................................... 86

5.5.2 The Effects on Adenosine, CCPA and DPCPX ...................................................... 87

Chapter 6: Summary, Conclusions and General Discussion ........................................................... 91

6.1 Summary of the Major Results of the Thesis ....................................................................... 92

6.2 The Major Conclusions of the Thesis ................................................................................... 93

6.3 The Experimental Approach and Manipulation of Adenosine Receptors .......................... 94

6.4 The Effects of pH and Adenosine on Respiratory-Related Motor Output ......................... 95

6.5 Chronic Hypoxia and Adenosine .......................................................................................... 96

6.6 Experimental Limitations & Future Suggestions ................................................................. 97

6.7 Conclusion ............................................................................................................................ 100

References ......................................................................................................................................... 101

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Appendix ........................................................................................................................................... 112

A1. Summary of the Experimental Protocol and an Explanation of the Data Contained

Within the Appendix ............................................................................................................ 113

A2. Data Analysis ....................................................................................................................... 116

A3. Results................................................................................................................................... 117

A3.1 Fictive Breathing Frequency ................................................................................... 117

A3.2 Fictive Episodes per Minute ................................................................................... 118

A3.3 Fictive Breaths per Episode .................................................................................... 119

A3.4 Total Fictive Ventilation Index............................................................................... 120

A3.5 Integrated Area of the Fictive Breaths ................................................................... 121

A3.6 Fictive Breath Duration ........................................................................................... 122

A3.7 Time.......................................................................................................................... 123

A3.8 Midbrain Transection .............................................................................................. 124

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List of Abbreviations

A1R: adenosine A1 receptor

A2AR: adenosine A2A receptor

A2BR: adenosine A2B receptor

A3R: adenosine A3 receptor

AC: adenylate cyclase

aCSF: artificial cerebral spinal fluid

ADO: adenosine

AMP: adenosine monophosphate

AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

ATP: adenosine tri-phosphate

Ca2+

: calcium

CaCl2: calcium clhloride

cAMP: cyclic adenosine mono-phosphate

CCPA: 2-chloro-N(6)-cyclopentyladenosine

CH: chronic hypoxia

CL-IB-MECA: 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide

CNS: central nervous system

CO2: carbon dioxide

CSF: cerebral spinal fluid

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CRG: central rhythm generator

DPCPX: 8-Cyclopentyl-1,3-dipropylxanthine

ECD: excitotoxic cell death

eng X: raw electroneurogram

fR: fictive breathing frequency

GABA: gamma-aminobutyric acid

Gi: inhibitory G proteins

GIRK: G protein-gated inwardly rectifying K+ channels

Gs: stimulatory G proteins

H+: hydrogen

I-neuron: inspiratory neuron

K+: potassium

KCl: potassium chloride

LC: locus coeruleus

MCT: multiple comparison test

MgCl2: magnesium chloride

MS222: 3-aminobenzoic acid ethyl ester

NaCl: sodium chloride

NaHCO3: sodium bicarbonate

NI: nucleus isthmus

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NMDA: N-methyl-D-aspartate

NO: nitric oxide

O2: oxygen

PCO2: partial pressure of carbon dioxide

pFRG: parafacial respiratory group

PKC: protein kinase C

PO2: partial pressure of oxygen

PreBötzC: Pre-Bötzinger complex

PSR: pulmonary stretch receptors

RT-PCR: reverse transcription polymerase chain reaction

S.E.M: Standard Error of the Mean

TFV: total fictive ventilation

Vth: trigeminal nerve root

VIIth: facial cranial nerve

IXth: glossopharyngeal cranial nerve

Xth: vagus nerve

∫eng X: integrated electroneurogram

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List of Figures

Figure 1.1: The respiratory cycle of the adult anuran amphibian.

Figure 2.1: Schematic diagram of the apparatus used to measure brain activity from isolated

cane toad brainstem preparations.

Figure 2.2: Flow chart illustrating the general steps taken within the following study.

Figure 2.3: A lateral view of the toad brain illustrating the transected areas (dotted lines) used in

the current study.

Figure 2.4: Pyramid illustrating the relationship between respiratory variables evaluated in the

current study.

Figure 3.1: Overview of the stages and general procedures taken within the experiment on intact

brainstem preparations.

Figure 3.2: The effects of ADO and A1R analogs on fictive breathing frequency in intact


Figure 3.3: The effects of ADO and A1R analogs on fictive episodes per minute in intact


Figure 3.4: The effects of ADO and A1R analogs on fictive breaths per episode in intact


Figure 3.5: The effects of ADO and A1R analogs on fictive breath area in intact brainstem

preparations.

Figure 3.6: The effects of ADO and A1R analogs on fictive breath duration in intact brainstem

preparations.

Figure 3.7: The effects of ADO and A1R analogs on the total fictive ventilation index in intact


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Figure 4.1: Overview of the stages and general procedures taken within the midbrain transection

experiment.

Figure 4.2: The effects of transecting the midbrain on fictive breathing frequency.

Figure 4.3: The effects of transecting the midbrain on fictive episodes per minute.

Figure 4.4: The effects of transecting the midbrain on fictive breaths per episode.

Figure 4.5: The effects of transecting the midbrain on fictive breath area.

Figure 4.6: The effects of transecting the midbrain on fictive breath duration.

Figure 4.7: The effects of transecting the midbrain on the total fictive ventilation index.

Figure 5.1: Overview of the stages and general procedures taken within the experiment on

midbrain transected preparations.

Figure 5.2: The effects of ADO and A1R analogs on fictive breathing frequency in midbrain

transected brainstem preparations.

Figure 5.3: The difference in effects of ADO and A1R analogs on fictive breathing frequency

between intact brainstem preparations and midbrain transected brainstem preparations during the

dose and pH treatment period.

Figure 5.4: The effects of ADO and A1R analogs on fictive episodes per minute in midbrain


Figure 5.5: The difference in effects of ADO and A1R analogs on fictive episodes per minute



Figure 5.6: The effects of ADO and A1R analogs on fictive breaths per episode in midbrain


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Figure 5.7: The difference in effects of ADO and A1R analogs on fictive breaths per episode



Figure 5.8: The effects of ADO and A1R analogs on fictive breath area in midbrain transected


Figure 5.9: The difference in effects of ADO and A1R analogs on fictive breaths are between

intact brainstem preparations and midbrain transected brainstem preparations during the dose and

pH treatment period.

Figure 5.10: The effects of ADO and A1R analogs on fictive breath duration in midbrain


Figure 5.11: The difference in effects of ADO and A1R analogs on fictive breath duration



Figure 5.12: The effects of ADO and A1R analogs on TFV in midbrain transected brainstem

preparations.

Figure 5.13: The difference in effects of ADO and A1R analogs on TFV between intact

brainstem preparations and midbrain transected brainstem preparations during the dose and pH

treatment period.

Figure A.1: Fictive breathing frequency (fictive breaths per minute) measured during the pre-

treatment phase of the experiment in all groups examined within the current study.

Figure A.2: Fictive episodes per minute measured during the pre-treatment phase of the

experiment in all groups examined within the current study.

Figure A.3: Fictive breaths per episode measured during the pre-treatment phase of the


Figure A.4: Total fictive ventilation index (V s/min) measured during the pre-treatment phase of

the experiment in all groups examined within the current study.

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Figure A.5: Integrated area of the fictive breaths measured during the pre-treatment phase of the


Figure A.6: The fictive breath duration measured during the pre-treatment phase of the


Figure A.7: The effect of time on respiratory variables in intact control in vitro brainstem

preparations.

Figure A.8: Fictive breathing (vagal motor output) traces from midbrain transaction

experiments.

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Chapter 1

General Introduction

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1.1 Breathing in Animals

In general, respiratory rhythmogenesis in all animals is produced centrally and can be modified

by central and afferent inputs. Respiratory centers and rhythm generating neurons that are found

in the medulla , such as the inspiratory (I) neurons located within the preBötzinger complex

(PreBötzC), are crucial to the constitution of the basic rhythm of breathing in mammals and other

vertebrates (Smith et al., 1991; Rekling and Feldman, 1998; Gray et al., 1999; Feldman et al.,

2003). The basic respiratory rhythm is subjected to modification by central CO2/pH

chemoreceptors and peripherial (arterial) chemoreceptors. The central CO2/pH chemoreceptors

respond to pH changes associated with changing CO2 levels within the cerebrospinal fluid and

the peripheral chemoreceptors monitor PCO2, pH and PO2 of the arterial blood. Pulmonary

stretch receptors (PSR) are mechanoreceptors found on the lung that monitor the degree of lung

inflation/deflation and can have significant modulatory effects on respiratory rhythm and

breathing pattern formation.

The morphology of the respiratory system differs amongst various vertebrate groups. The form

and function of the respiratory system is usually tailored to exploit the external environment such

that the respiratory system provides the organism with adequate oxygen, expels carbon dioxide

(CO2) to the external environment and maintains homeostasis of arterial O2 and CO2 levels

(Wasserman, 1978; Cohn, 1983; Kinkead, 1997).

1.2 Breathing in Amphibians

Breathing in amphibians is dynamic as the respiratory organs undergo morphological changes

throughout the life cycle. During pre-metamorphosis, the respiratory organs present in tadpoles

are the gills and the skin. Tadpoles are water breathers and utilize cutaneous respiration for

approximately 60% of gas exchange and the gills for 40% for both O2 uptake and CO2 excretion.

During metamorphosis, the amphibian becomes a facultative air-breather marked by the retention

of the gills and the progressive development of the lungs. The emergence of air breathing that

occurs during metamorphosis is believed to be triggered by an increase in circulating

corticosterone and concurrent changes to GABAergic neurotransmission (Fournier et al., 2012).

During post-metamorphosis, the adult amphibian switches from facultative air-breathing to

obligate air-breathing. At the adult stage, the gills have completely rescinded and the lungs

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become the primary respiratory organ for O2 uptake (approximately 90%) with the skin still

playing a role in CO2 excretion (Burggren and West, 1982; Pinder and Burggren, 1986; Wells,

2007).

Like other animals, anuran amphibians possess a central rhythm generator (CRG) for lung

ventilation. However, unlike other animals, amphibians also have a CRG that regulates the non-

ventilatory buccal oscillations that occur between breaths (Wilson et al., 2002). Unlike mammals

that utilize a negative pressure pump to inflate and deflate the lungs, amphibians utilize a buccal

force pump (positive pressure pump) to control lung inflation (Jones, 1982).

The pulmonary respiratory system of amphibians consists of the buccal cavity and the lungs.

Inbetween the buccal cavity and the lungs is the glottis, which controls the passage of air

between the two internal spaces. The step-by-step process behind amphibian breathing has been

described by several studies (West and Jones, 1975; Macintyre and Toews, 1976; Vitalis and

Shelton, 1990; Jones, 1982). First, air enters the buccal cavity through the nares by negative

pressure generated by the lowering of the floor of the buccal cavity. Air in the lungs remains

trapped within the lungs due to the closed glottis. When the glottis opens, the pre-existing air

held in the lungs from the previous breath is drawn into the upper region of the buccal cavity,

while the newly inspired air remains in the lower portion of the buccal cavity. The expired air

from the lungs then exits through the open nares into the atmosphere. The nares will close while

the glottis remains open, and the newly inspired air gets pushed into the lungs by positive

pressure that is generated by the upward contraction of the floor of the buccal cavity. The glottis

then closes, which completes a single lung breath. Since the surface of the buccal cavity lacks

vasculature, only air in the lungs is believed to be involved with gas exchange. Lung inflation

cycles, in which the lung is “pumped” up by successive breaths, are associated with elevated

respiratory drive, defense behaviours and preparation for vocalization. Deflation cycles follow

inflation cycles after a period of apnea (Gargaglioni and Milsom, 2007).

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Figure 1.1. The respiratory cycle of the adult anuran amphibian. The inhalation of air begins with the generation of

negative pressure in the buccal cavity (achieved by the depression of the floor in the buccal cavity) which draws in

atmospheric air through the open nares into the lower half of the buccal cavity (A). The pre-existing air in the lungs

is under high pressure and when the glottis opens the pre-existing air in the lungs gets pushed out in to the

atmosphere through the open nares (B). The nares then close and the air trapped in the lower half of the buccal

cavity get pumped into the lungs through the open glottis by the elevation of the floor of the buccal cavity (C). The

glottis closes and the nares re-open which allows the cycle to either begin again or allow buccal oscillations to occur

without lung ventilation.

1.3 Breathing Patterns in Amphibians

The pattern of breathing displayed by the adult anuran is described as intermittent (i.e.

discontinuous) (Milsom, 1991). The intermittent breathing pattern can be further described as

single breaths and/or clusters of breaths into doublets/triplets (two or three breaths in succession)

that are randomly distributed and separated by a brief period of apnea. Indeed it is possible to see

almost any type of “random” distribution of breaths in discontinuously breathing animals. In

contrast to the intermittent breath pattern displayed by amphibians, euthermic mammals, birds

and water-breathing fish all have a breathing pattern that is continuous. The stark difference

between breathing patterns observed in these animals is likely due to metabolic demands, which

are significantly lower in amphibians compared to birds and mammals (Gargaglioni and Milsom,

2007). In addition, the maintenance of the episodic breath patterns in the adult anuran amphibian

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appears to be independent of fluctuations in blood-gas levels (West et al., 1987; Kinkead et al.,

1994, 1997; Milsom et al., 1999; Reid et al., 2000).

Under certain conditions, the breathing pattern in both mammals and amphibians can be

switched (i.e. mammals can breathe episodically and adult amphibians can breathe

continuously). In mammals, episodic breathing can occur during hibernation, when metabolic

demands are low (Kinkead et al., 1997). In amphibians, the breathing pattern can be modified by

the level of respiratory drive. During elevated respiratory drive, the frequency of breaths

increases and the apnea periods between each breath shortens. Elevated respiratory drive can

occur by external stressors, such as hypoxia (low O2 levels) or hypercapnia (high CO2 levels).

When respiratory drive becomes extremely high, especially during severe hypercapnia,

amphibians exhibit continuous breathing (Milsom, 1991; Gargaglioni and Milsom, 2007).

Evidence from past studies has suggested that the caudal half of the midbrain is involved in the

clustering of breaths into episodes (Reid et al., 2000a; McAneney and Reid, 2007).

1.4 Control of Breathing

Regulation of breathing in amphibians can be described as a complex network of interactions

between stimulatory inputs from central and arterial chemoreceptors, and inhibitory inputs from

olfactory chemoreceptors in conjunction with CO2-sensitive tonic and/or phasic pulmonary

stretch receptor (PSR) feedback (Kinkead and Milsom, 1996). The interaction between

peripheral and central chemoreceptors is seen during both the acute hypercapnic ventilatory

response and the acute hypoxic ventilatory response (West et al., 1987; Smatresk and Smits,

1991; Reid, 2006). As in other animals, central integration of the afferent feedback provided by

these receptors leads to modified motor output to the respiratory muscles.

1.4.1 Central Processes

In mammals, the basic rhythm of breathing is generated centrally in the preBötzC and the

parafacial respiratory group (pFRG), which are located within the rostro-ventral medulla

(Onimaru and Homma, 2003; Feldman and Del Negro, 2006; Wilson et al., 2006; Gargaglioni

and Milsom, 2007). Within the preBӧtzC are inspiratory (I) neurons, which fire during

inspiration (Smith et al., 1991; Rekling and Feldman, 1998; Gray et al., 1999; Feldman et al.,

2003). Within the pFRG are pre-I neurons that are coupled to the I-neurons of the preBӧtzC. The

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pre-I neurons are believed to trigger the onset of bursting in the preBӧtzC (Smith et al., 1991;

Mellen et al., 2003; Onimaru and Homma, 2003; Feldman and Del Negro, 2006).

Regions within the medulla of anuran amphibians generates respiratory rhythm (McLean et al.,

1995a; Reid and Milsom, 1998; Reid et al., 2000a; Torgerson et al., 2001). In bullfrogs, the

ventral medullary reticular formation contains two rythmogenic sites involved with the

generation of endogenous respiratory activity (Vasilakos et al., 2005). One of these rythmogenic

sites can be found between the VIIth (facial) and IXth (glossopharyngeal) cranial nerves, which

is critical for lung ventilation. The other rythmogenic site is found at the level of the Xth (vagus)

nerve root and is required for buccal oscillation regulation (McLean et al., 1995b; Wilson et al.,

2002). During anuran development, the location of rhythm generation changes; in tadpoles,

respiratory rhythm generation occurs caudal to cranial nerve X and during metamorphosis to the

adult form, rhythm generation moves to a more rostral brainstem site (Torgerson et al., 2001).

1.4.2 Olfactory Chemoreceptors

The nasal mucosa of anuran amphibian contains CO2-sensitive olfactory chemoreceptors. These

receptors send an afferent signal to the brain via the trigeminal and olfactory nerves when

stimulated by high levels of CO2 (Sakakibara, 1978). Past studies have demonstrated that when

stimulated by CO2 the olfactory chemoreceptors produce an inhibitory signal that reduces

breathing frequency (Sakakibara, 1978; Ballam and Coates, 1989; Coates, 2001; Kinkead and

Milsom, 1996; Milsom et al., 2004). The degree of inhibition on breathing frequency from the

olfactory chemoreceptors is dependent on the level of CO2 with higher levels of CO2 leading to

greater levels of breathing frequency inhibition.

1.4.3 Pulmonary Stretch Receptors

Pulmonary stretch receptors (PSR) are mechanoreceptors that are located in the walls of the

lungs. These receptors monitor changes in lung volume during inspiration, expiration, and apnea,

and send their feedback to the brain via the pulmonary vagus nerve. PSR feedback can either

promote or inhibit respiratory activity depending on whether the PSR feedback is tonic or phasic.

Phasic PSR feedback stimulates breathing frequency via increases in the number of breathing

episodes (Reid and West, 2004). Tonic PSR feedback is considered to be inhibitory despite the

fact that it stimulates breathing frequency. This is because tonic PSR feedback, unlike phasic

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PSR feedback, stimulates breathing frequency by promoting lung deflation breaths instead of

lung inflation breaths (Sanders and Milsom, 2001). As such, this form of PSR feedback leads to

an acceleration in lung deflation. Tonic PSR feedback can also inhibit peripheral chemoreceptor-

induced increases in breathing under conditions of minimal stimulation from central pH/CO2

chemoreceptors (Wang et al., 2004). In anuran amphibians, PSR activity is sensitive to the level

of CO2 becoming inhibited with increasing levels of CO2 (Milsom and Jones, 1977; Kuhlman

and Fedde, 1979). In addition, depending on the level of CO2, tonic PSR feedback can function

to alter breathing pattern and the form of the individual breaths (Sanders and Milsom, 2001).

PSR feedback can also interact with central pH/CO2 chemoreceptor activity and enhance the

effects of aCSF pH on respiratory frequency (Kinkead et al. 1994).

1.4.4 Peripheral O2/CO2/pH sensitive chemoreceptors

The peripheral chemoreceptors are found in the aortic bodies within the aortic arch and the

carotid bodies at the bifurcation of the common carotid arteries. These receptors monitor the

PCO2, pH and PO2 of the arterial blood and when stimulated (during hypoxia and hypercapnia),

will transmit an afferent signal through the vagus and glossopharyngeal nerves to the respiratory

centers to increase breathing (West et al., 1987; Smatresk and Smits, 1991). Although arterial

chemoreceptors in the carotid labyrinth and aortic arch are both O2 and CO2-sensitive, exposing

the peripheral chemoreceptors in the carotid labyrinth to elevated levels of O2 will reduce

discharge at any given level of CO2 (Van Vliet and West, 1992). A comparison of respiratory

recordings between intact animals and in vitro brainstem-spinal cord preparations from

amphibians indicates that without afferent feedback from peripheral chemoreceptors, less

breathing activity is observed (Kinkead et al., 1994; Reid et al., 2000b; Reid, 2006).

1.4.5 Central pH/CO2 sensitive chemoreceptors

The central chemoreceptors found on the ventrolateral surface of the medulla sense the pH

associated with the CO2 level within the cerebrospinal fluid (CSF) (Smatresk and Smits, 1991;

Milsom, 2002; Taylor et al., 2003a). These receptors directly synapse with the respiratory

centers and are stimulated by reductions in CSF pH or increases in CSF CO2 levels (Smatresk

and Smits, 1991; Milsom, 2002). Stimulation of these receptors leads to an increase in

respiratory drive.

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1.4.6 Neurotransmitters

Several studies have examined the effects of neurotransmitters on respiration in anuran

amphibians (McLean et al., 1995a; Hedrick et al., 1998; Hendrick et al., 2005; Broch et al.,

2002; Straus et al., 2000). Neurotransmitters such as GABA and glycine have been demonstrated

to exert an inhibitory modulation to breathing frequency in amphibians (McLean et al., 1995a;

Broch et al., 2002). In addition, the clustering of breaths into episodes observed in amphibian

breathing may be regulated by the GABAB pathway (Straus et al., 2000). The neurotransmitter

that has been demonstrated to exert a stimulatory effect on breathing frequency in amphibians is

glutamate (McLean et al., 1995a). Studies by Hendrick et al. (1998 & 2005) demonstrated that

preventing NO generation via NMDA receptor (a glutamate receptor) activation results in the

abolishment of respiratory rhythm in the bullfrog (Rana catesbeiana) illustrating the importance

of NMDA receptors in respiratory rhythm generation in amphibians. Collectively, the effects of

various neurotransmitters on central respiratory modulation in amphibians are similar to those

observed in mammals, which suggests that a common mechanism to central respiratory rhythm

generation exists between the two (Wang et al., 1999).

In current literature the effects of serotonin, dopamine and noradrenaline on breathing have been

documented in mammals but not in anuran amphibians. The neurotransmitter serotonin has been

demonstrated to be a breathing stimulant in the CNS, producing increases in respiration during

normoxia (Millhorn et al., 1980). The neurotransmitter noradrenaline has a complex effect on

breathing, acting at the carotid bodies as a stimulant (Eldridge and Gill-Kunar, 1980) and as a

depressant at the CNS (Champagnat et al., 1978). The neurotransmitter dopamine also has a

complex effect on breathing, however opposite to noradrenaline, acting on the carotid body as a

ventilatory depressor and acting as a stimulant in the CNS (Nakano et al., 2002).

1.5 In vitro Brainstem – Spinal Cord Preparation

An in vitro brainstem preparation consists of the spinal cord and brainstem (which includes a

functioning medulla) as well as the midbrain. Using the in vitro brainstem preparation, fictive

breathing (motor output from respiratory nerves) can be measured from the cranial nerves.

Respiratory-related cranial nerves include the trigeminal nerves and hypoglossal nerves, which

regulate the raising or lowering of the floor of the buccal cavity, or the vagus nerve, a branch of

which controls the opening and closing of the glottis (Sakakibara, 1948a,b). The experimental

9

significance of the in vitro brainstem preparation is that it allows research to be done solely on

the central control of breathing, without any regulation or effects from peripheral input (Reid and

Milsom, 1998). In addition, in vitro brainstem preparations from amphibians can remain viable

for longer periods of time compared to mammals (some for more than 20 hours) due to their

lower metabolic rates and enhanced hypoxia tolerance (Morralles and Hedrick, 2002; Reid and

Milsom, 1998).

1.6 Adenosine

ADO is a neuromodulator / neurotransmitter that is generated by all living cells during the

breakdown of adenosine monophosphate (AMP) by ecto-5’-nucleotidase or cytosolic-5’-

nucleotidase (Latini and Pedata, 2001). When the intracellular concentration of ADO becomes

greater than that of the extracellular fluid, ADO is transported out of the cell and into the

extracellular fluid via nucleoside transporters. In the extracellular space, ADO can interact with

four different subgroups of ADO receptors (A1, A2A, A2B and A3), each of which have been

classified based on ligand affinities, structure, function and molecule interaction (Dohrman et al.,

1997). With reference to mammalian models, the A1 receptor (A1R) has the highest affinity for

ADO and is also the most abundant ADO receptor in the brain with the highest expression in the

cortex, cerebellum, hippocampus, and dorsal horn of the spinal cord (Goodman and Snyder,

1982; Dixon et al., 1996; Ribeiro et al., 2003). Coupled to the A1R are the inhibitory G proteins

(Gi) that function to hyperpolarize cells and reduce neuronal excitability when activated.

The A2AR has the second highest affinity for ADO and is also the second most abundant ADO

receptor in the brain that is found highly concentrated in the striatum and olfactory bulbs. Unlike

the A1R, the A2AR is coupled to stimulatory G proteins (Gs) and when activated will promote

cellular depolarization. Much like the A2AR, the A2BR is also stimulatory, but is less abundant

and has a lower affinity for ADO. Lastly the A3R, which functions similarly to the A1R, is

considered to be the least expressed adenosinergic receptor in the CNS with the least affinity for

ADO amongst the adenosinergic receptor subtypes (Ciruela et al., 2010).

The A1Rs and the A2ARs are primarily responsible for the central effects of ADO (Dunwiddie,

and Masino, 2001). The primary mode of effect of the A1R and the A2AR is the mediation of the

secondary-messenger enzyme adenylate cyclase (AC). Stimulation of the A1R activates the Gi

coupled proteins, which inhibit the enzyme AC and subsequent production of cAMP (Klinger et

10

al., 2002). On the contrary, stimulation of the A2AR activates the Gs coupled proteins, which

stimulate the AC and subsequent production of cAMP. The activation of the A1R and A2AR also

causes changes to ion channels. Activation of presynaptic A1Rs directly inhibits voltage-

dependent calcium (Ca2+

) channels (Ciruela et al., 2010). In addition, A1R activation produces

hyperpolarisation in a manner independent of cAMP by inducing a potassium (K+) current via

GIRK (G protein-gated inwardly rectifying K+ channels) (Klinger et al., 2002). In contrast, the

activation of A2ARs results in a Ca2+

-dependent release of glutamate and acetylcholine, by means

of a mechanism that may involve P-type Ca2+

channels (Ciruela et al., 2010).

1.7 Hypothesis and Objectives

A previous study from this lab investigated the effects of chronic hypoxia (CH) on central

respiratory-related pH/CO2 chemosensitivity in cane toads (Bufo marinus) (McAneney and Reid,

2007). Using in vitro brainstem–spinal cord preparations from cane toads, McAneney and Reid

(2007) had demonstrated that under normal physiological conditions, descending inputs from

the midbrain function to inhibit or reduce fictive breathing frequency during elevated respiratory

drive (i.e., during exposure of the preparations to lower levels of aCSF pH). In addition to the

study, the authors demonstrated that when cane toads were exposed to CH the inhibitory

modulation from these descending inputs from the midbrain became strengthened (i.e. fictive

breathing frequency was reduced even further).

The aim of the current study was to examine the mechanisms exerted by these descending

midbrain inputs that contribute to the inhibitory modulation of breathing that was observed in the

previous study. Since ADO is a naturally circulating metabolite that has been demonstrated to

increase in the extracellular fluid during CH (Klinger et al., 2002; Pamenter et al., 2008; Latini

and Pedata, 2001), I hypothesized that these descending inputs from the midbrain suppresses

respiratory activity through purinergic regulation by ADO acting primarily through the A1R. In

current literature, the effect of extracellular ADO on in vitro respiratory activity of the cane toad

has not been documented. As such, my primary objectives include the examination of the acute

effects of extracellular ADO on fictive breathing measured from the isolated brainstem-spinal

cord preparation and the isolation of the primary ADO receptor that is acted upon by ADO to

exert these effects.

11

To test the hypothesis, I superfused ADO, the A1R antagonist 8-Cyclopentyl-1,3-

dipropylxanthine (DPCPX) and the A1R agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA),

onto in vitro brainstem-spinal cord preparations while recording fictive breathing. To isolate the

effects of ADO and the A1R analogs, on fictive breathing, from descending midbrain inputs, the

superfusion of ADO and A1R analogs was repeated on midbrain transected in vitro brainstem

preparations.

12

Chapter 2

General Materials and Methods

13

2.1 Experimental Animals

Cane toads were obtained from a commercial supplier (Boreal Scientific, St Catherine’s, Ontario,

Canada). They were housed in the aquatics facility at the University of Toronto Scarborough.

Toads were kept at room temperature (20-22ºC) and held in fibreglass tanks that contained

plastic tubes and trays filled with de-chlorinated water to mimic both terrestrial and aquatic

habitats. The photoperiod was maintained at 12 h light; 12 h dark. The toads were fed once a

week with either live meal worms or crickets. All procedures were approved by the University of

Toronto Animal Care Committee and conform to standards set by the Canadian Council for

Animal Care.

2.2 Artificial Cerebral Spinal Fluid (aCSF) Solution

The artificial cerebral spinal fluid (aCSF) used to superfuse the isolated brainstem-spinal cord

preparations contained 103.5 mM NaCl, 25 mM NaHCO3, 4 mM KCl, 10 mM D-glucose, 1.36

mM MgCl2 and 2.43 mM CaCl2(all chemicals from Sigma) (Taylor et al., 2003a,b; Gheshmy et

al., 2006). The aCSF used for the surgical removal of the brainstem-spinal cord was placed in an

ice bath and gassed with 100% O2. The aCSF used during the experimental protocol was gassed

with both CO2 and O2 to achieve an aCSF pH between 7.4 and 8.0. All gas flow levels were

recorded using a gas flow meter (Smart-Trak 2000, Sierra instruments INC.). The pH of the

aCSF was measured constantly with a pH meter (VWR) submerged in the aCSF reservoir. It

was calibrated using standard buffer solutions.

14

Figure 2.1: Schematic diagram of the apparatus used to measure fictive breathing (respiratory motor output) from

isolated cane toad brainstem-spinal cord preparations. (a) aCSF reservoir (b) pH meter (c) CO2 inflow (d) O2 inflow

(e) Peristaltic pumps to circulate the aCSF (f) Preparation chamber (g) Suction electrode (h) Amplifiers and

integrator (i) data acquisition system.

2.3 The In vitro Brainstem-Spinal Cord Preparation

Toads were anaesthetised by submersion in a solution containing 500 ml of cold water mixed

with 0.5g of 3-aminobenzoic acid ethyl ester (MS222, 1.0g 1-1

; Sigma) and 1.0g of sodium

bicarbonate to buffer the solution to pH 7.0 (Reid and Milsom,1998; Reid et al., 2000a; Reid et

al., 2000b; Gheshmy et al., 2006). Once in the anaesthetic, toads were monitored for cessation of

buccal movements as well as eye-blink and toe-pinch reflexes. When these reflexes were

eliminated, the surgical removal of the brainstem-spinal cord commenced. An incision was

made in the skull rostral to the optic lobes and along the sides of the spinal cord. Using the bone

shears, the spinal cord was severed at the level of the third spinal nerve. The cranial case as well

as the associated dermal tissues and muscles surrounding the spinal cord were removed with

rongeurs and bone shears. Using Westcott scissors, the olfactory bulb was removed (Gheshmy et

al., 2007) and the cranial nerves were cut close to their exit from the skull. The brain preparation

was then isolated from the brain case and transferred to and immobilized within a Sylgard-coated

dissecting dish. With the aid of a light microscope, the dura mater surrounding the brain was

removed with tweezers to free the cranial nerve roots. Throughout the surgical procedure, a

15

constant supply of cold, oxygenated aCSF was applied to keep the isolated brain preparation

viable. The brainstem preparation was then transferred to the preparation recording chamber

(Fig. 2.1) and immobilized with insect pins. The trigeminal nerve root was aspirated into a

suction electrode and the preparation was kept in the recording chamber and superfused with the

aCSF solution (pH of 7.7-7.8) until respiratory motor output was stabile (approximately one

hour).

2.4 Experimental protocol: aCSF pH Changes and Treatment

with Adenosine, CCPA or DPCPX

Once the motor output from the isolated brainstem-spinal cord preparation had stabilised (i.e.

rhythmic recording became consistently visible), respiratory motor activity was recorded for data

analysis purposes for the final 10 minutes of that period (i.e., the stabilisation period). Following

the stabilisation period, the pH of the aCSF was changed to either 7.4, 7.6, 7.8 or 8.0. For each

isolated brain preparation the sequence of pH changes was randomized. The aCSF was

maintained at a given pH level for 15 minutes at which point the pH was changed again such that

the preparation was exposed to all four different aCSF pH levels. The motor output (fictive

breathing) during the last 10 minutes of exposure to each pH level was recorded for data analysis

purposes. After exposing the preparation to the various aCSF pH changes, the pH was readjusted

back to 7.8. At this point the aCSF was changed such that the new aCSF superfusing the

preparation contained either 1) 1 μM adenosine (n = 9), 2) 10 μM adenosine (n = 9), 3) 10 µM

of the A1R antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; n = 9) 4) 10 µM of the A1R

agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; n = 9) or 5) a control solution of normal

aCSF (n = 8). The new aCSF solution with either adenosine, DPCPA or CCPA was gassed with

O2 and CO2 to achieve a pH level between 7.7-7.8 and the preparation was superfused for a 40

minute stabilisation period. The last 10 minutes of the stabilisation period was used for data

analysis. The current study analysed the data after 30 minutes of drug exposure to ensure

maximum dose efficiency (Pamenter et al., 2008). After the 40 minute stabilisation period, the

aCSF pH was changed to either to 7.4, 7.8, 7.6 and 8.0 as described above in the continued

presence of adenosine, CCPA or DPCPX (aka dose & pH treatment period). Each pH change

lasted 15 minutes and motor output was recorded in the last 10 minutes of each pH change for

data analysis. Figure 2.2 summarizes the experimental procedure used in this type of

16

experiments. The data for this specific series of experiments (pH changes followed by drug

treatment followed by pH changes) is reported in chapter 3. Modifications to this protocol are

reported in subsequent chapters, as appropriate.

Figure 2.2: Flow chart illustrating the general stages of the experimental protocol used in the various series of

experiments outlined in this thesis. The first step was the stabilisation period, where the brainstem preparation was

placed into a bath of circulating aCSF at constant pH 7.8 until breathing becomes rhythmic for at least 15 minutes.

The second step is the pH treatment period, where the brainstem preparation was subjected to random alterations of

aCSF pH (7.4, 7.6, 7.8 and 8.0). The third step was the experimental manipulation period in which a dose of

adenosine, CCPA or DPCPX was introduced into the aCSF superfusing the brainstem preparations that were either

kept intact or transected at the midbrain. The fourth step was the “dose and pH treatment period”, where the

brainstem preparation was exposed to random alterations in aCSF in the continued presence of adenosine, CCPA or

DPCPX.

17

2.5 Experimental protocol: Midbrain Transection

These experiments used a similar procedure as described above. However, in addition to the

aCSF solution containing adenosine or an A1R agonist or antagonist, the midbrain was removed

from the isolated brainstem-spinal cord preparations. A transection through the optic lobes on the

dorsal surface to the caudal end of the acuate periventricular nucleus on the ventral surface was

made to remove the midbrain. Based on the Hoffmann (1973) atlas of the toad brain, this

transection site would correspond to 3.5 mm posterior to the zero mark (Fig. 2.3). The

transection is rostral to the nucleus isthmi (see Figs. 29 and 30 from Hoffmann, 1973). Further

details on the midbrain transection protocols are provided in chapters 4 and 5.

Figure 2.3: A lateral view of the toad brain illustrating the transected areas (dotted lines) used in the current study.

The rostral- and caudal-most transections were used to generate the in vitro brainstem preparation. The mid-brain

transection through the optic lobes was an experimental manipulation outlined in chapters 4 and 5. NI indicates the

location of the nucleus isthmi. V, trigeminal nerve root; VII, facial nerve root; IX, glossopharyngeal nerve root; X,

vagus nerve root.

18

2.6 Controls

In experiments conducted on intact preparations, control groups were intact brainstem

preparations exposed to aCSF that did not contain adenosine, CCPA or DPCPX. Similarly,

intact brainstem preparations exposed only to aCSF (no pharmacological treatment) were used as

controls for experiments examining the effects of the midbrain transection only (prior to drug

treatment). In experiments conducted on midbrain transected preparations, control groups were

midbrain transected preparations that were exposed to virgin aCSF during the dose stabilisation

and the dose & pH treatment period of the experiment. The pH level of 7.8 was used as the

control pH level during both pH treatment periods in the pre-treatment and post-treatment phase

of the experiment.

2.7 Data and statistical analyses

AcqKnowledge 3.7.3 (Biopac Systems) software was used to acquire and store the nerve

recordings from the in vitro brainstem preparations. The data recorded from the final 10 minutes

of each experimental period and/or aCSF pH level was used for analysis. Using the criteria

described by Reid and Milsom (1998), the brain traces were analysed to determine fictive

breathing frequency (fictive breaths/min), the number of fictive breathing episodes per min, the

number of fictive breaths per episode, fictive breath duration, and the integrated area of the

fictive breaths. Fictive breaths in a given episode were defined as occurring within 2 seconds of

each other according to general practices in the literature (Kinkead et al., 1994; Kinkead et al.,

1997; Reid et al., 2000a; Gheshmy et al., 2006). The product of integrated area of fictive breaths

and fictive breathing frequency yields the total fictive ventilation (mV•sec/min). The

relationships between the respiratory variables are illustrated in Fig. 2.3. To maintain analytical

consistency amongst all experimental groups, only neural respiratory recordings that had a mean

average in pre-treatment values that were approximately equal to that observed in controls were

used in the experiment (see Appendix). All statistical analyses were performed using commercial

software (SigmaStat 3.0, SPSS). See chapters 3-5 for further details on data and statistical

analysis appropriate to the given experiments.

19

Figure 2.4: Pyramid illustrating the relationship between respiratory variables evaluated in the current study. The

total fictive ventilation index is the product of fictive breathing frequency and breath area. Fictive breathing

frequency is the product of fictive episodes per minute and fictive breaths per episode. Fictive breath duration and

amplitude are components that make up fictive breath area.

20

Chapter 3

The Effects of Adenosine, CCPA and DPCPX on intact Brainstem-Spinal Cord Preparations

21

3.1 Introduction

In general, anuran amphibians are vulnerable to dehydration and lethal heat due to the high

permeability of their skin. However, despite this vulnerability, many amphibians can be found in

diverse habitats some of which have extreme climates, such as the dry and extreme heat of

deserts or the frigid cold temperatures of northern temperate climes (Boutilier et al., 1997). To

survive such extreme environmental conditions, amphibians utilize avoidance strategies such as

burrowing underground or overwintering under water. However, inhabiting underground

burrows or submergence under water introduces another problem for amphibians as both habitats

lack sufficient oxygen and may also, at least underground, have elevated levels of carbon dioxide

(Boggs et al., 1984; Schaefer and Sadleir, 1979). However, it has been documented that

amphibians can remain within these environments for several weeks or months (Breckenridge

and Tester, 1961; Pinder et al., 1992). Furthermore, in comparison to mammals, amphibians

have a greater tolerance to hypoxia (Boutilier, 2001) and can survive conditions of persistent

hypoxia by undergoing physiological adjustments to respiratory gas exchange and metabolism.

Inside underground burrows or underwater, amphibians may enter a state of torpor (i.e.,

aestivation). In this state, there is a reduction in basal metabolic rate and metabolic demand

which is vital to surviving prolonged periods in hypoxic environments. A reduction in metabolic

activity is usually observed in amphibians during exposure to high temperatures and low water

availability (Pinder et al., 1992) and as well as during exposure to cold temperatures (Lemckert,

2004; Yu and Guo, 2010; Bickler and Buck, 2007). The advantages of metabolic depression are

that it decreases the consumption of limited resources, such as oxygen and water (Boutilier,

2001), it allows for low levels of oxidative phosphorylation to meet ATP demand (Bickler and

Buck, 2007) and it lessens the impact of ATP demand on endogenous energy reserves

(Hochachka and Guppy, 1987; Storey and Storey, 1990; Flanigan et al.1991; Guppy et al.1994).

In addition to metabolic changes, respiratory activity in amphibians changes during hypoxia.

During exposure to moderate levels of acute hypoxia (10-15% O2), amphibians generally

increase their breathing in order to maintain oxygen homeostasis (Burggren and Doyle, 1987;

Kruhøffer et al., 1987; Smatresk and Smits, 1991). However, when hypoxic exposure becomes

chronic, some amphibians have been shown to exhibit a depression in respiratory activity

(McAneney et al., 2006; McAneney and Reid, 2007). The reduction in respiratory activity

22

observed during chronic hypoxia (CH) may serve as a means of conserving energy, since the

reduction in neuronal activity regulating breathing would help to reduce brain ATP demand

(Buck and Bickler, 2007) and motor activity of respiratory muscles would be reduced.

During exposure to hypoxia, brain ATP levels decline progressively (Winmill et al., 2005), while

the extracellular levels of potassium (K+) (Winmill et al., 2005) and adenosine (ADO) increase

progressively (Klinger et al., 2002; Pamenter et al., 2008; Latini and Pedata, 2001). Multiple

hypoxic/anoxic studies suggest that adenosine enhances cytoprotective mechanisms, including

the down-regulation of NMDA (N-methyl-D-aspartate) receptor activity which, in the absence of

down-regulation, is associated with excitotoxic cell death (ECD) during hypoxic and anoxic

insults (Bickler, 2004; Buck, 2004; Downey et al., 2007; Pagonopoulou et al., 2006). Other

benefits that adenosine has during hypoxia include (a) the stimulation of glycogenolysis which

provides the substrate for anaerobic glycolysis, (b) the stimulation of anaerobic glycolysis which

increases ATP production to meet demand and (c) reduction of neuronal energy requirements by

decreasing neuronal excitability (postsynaptic inhibition) as well as neurotransmitter release

(presynaptic inhibiton) (Bickler and Buck, 2007). Collectively, I suspect that adenosine maybe

the metabolite that induces the ventilatory depression exhibited by the cane toad during chronic

hypoxia (CH).

3.2 Hypothesis & Objectives

Cane toads exhibit ventilatory depression during CH (when extracellular adenosine levels are

significantly greater than during normoxic periods). As such, I hypothesized that superfusing

adenosine onto the in vitro brainstem preparation of the cane toad would cause a depression in

respiratory motor output (i.e. fictive breathing, the neural equivalent of breathing). I also

hypothesized that adenosine would work primarily via the A1R since the A1R has been

documented to be the most abundant and widely-distributed adenosinergic receptor in the brain

and that it has the highest affinity of all purinergic receptors for adenosine. In addition, A1R

stimulation leads to the release of K+ from neurons into the extracellular space which, in part,

leads to the hyperpolarization of that neuron. To determine whether a reduction in fictive

breathing is caused by adenosine and A1R stimulation, I superfused the in vitro brainstem cord

preparation with 1 μM adenosine (n = 9), 10 μM adenosine (n = 9), the A1R antagonist, 8-

Cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 µM; n = 9) or the A1R agonist, 2-chloro-N(6)-

23

cyclopentyladenosine (CCPA; 10 µM; n = 9). These pharmacological treatments were done at

various aCSF pH levels. I predicted that adenosine and CCPA treatment would reduce fictive

breathing and that DPCPX treatment would enhance fictive breathing. Furthermore, based on

previous studies I also predicted that the effects would be more pronounced at lower aCSF pH

levels.

24

3.3 Materials and Methods

See chapter 2 for details on the generation of the in vitro brainstem-spinal cord preparation and

general details of the experimental protocol for altering aCSF pH and superfusing the various

pharmacological agents onto the preparation.

Figure 3.1 illustrates the experimental protocol used in the experiments outlined in this chapter.

Briefly, the brainstem-spinal cord preparation (with the midbrain attached) was removed from

the animal and allowed to stabilise for approximately 1 hour at an aCSF pH of 7.8 (pre-treatment

stabilisation period). After this period, the aCSF pH was changed to pH levels of 8.0, 7.8, 7.6 and

7.4 (selected randomly; “pH treatment” portion of the protocol). Following these pH changes, the

preparation was superfused with aCSF containing either adenosine (1 µM; n = 9), adenosine (10

µM; n = 9), CCPA (A1R agonist; 10 µM; n = 9), DPCPX (A1R antagonist; 10 µM; n = 9), or

normal aCSF (controls; n = 8). After a stabilisation period of 40 min (“dose stabilisation” portion

of the protocol), the aCSF pH changes were repeated (“dose and pH treatment” portion of the

protocol).

25

Figure 3.1: Overview of the stages and general procedures performed within the experiments on brainstem-spinal

cord preparations reported in this chapter. The experiment was one continual process consisting of two phases; (1)

the pre-treatment phase in which the brainstem preparations were exposed to normal aCSF (no pharmacological

agents) of varying pH levels and (2) the post-treatment phase in which the brainstem preparations were exposed to a

pharmacological agent (adenosine; CCPA or DPCPX) within the aCSF as well as varying aCSF pH levels. The pre-

treatment phase of the experiment was divided into two parts; (1) the stabilisation period in which the preparation

was superfused with aCSF at a constant pH of 7.8 and (2) the pH treatment period in which the brainstem

preparation was subjected to random alterations in pH (15 minute duration for each pH change) in the aCSF. In all

cases the data were analysed in the final 10 minutes of each pH change period. The post-treatment phase of the

experiment was also split into two parts; (1) the dose stabilisation period in which the preparation was exposed to a

new aCSF reservoir containing a dose of adenosine, CCPA or DPCPX (kept at constant pH 7.8) for 40 minutes with

the data analyzed in the final 10 minutes and (2) the dose and pH treatment period in which the dose of the

pharmacological agent was still present and the brainstem preparation was subjected to random alterations to pH (15

minute duration for each pH change) in the aCSF with the data analyzed in the final 10 minutes of each pH change.

26

3.4 Data Analysis

A one-way repeated measures analysis of variance followed by a Holm-Sidak multiple

comparison test (MCT) was used to analyze the effect of altering aCSF pH levels within each

treatment group (i.e., control, adenosine, CCPA or DPCPX); the values obtained for each

respiratory variable at pH 7.4, 7.6 and 8.0 were compared to the value of the respective

respiratory variable obtained at the initial pH of 7.8 (during the stabilisation period). A one-way

analysis of variance followed by a Holm-Sidak MCT was also used to evaluate the effects of the

pharmacological agents (adenosine, CCPA or DPCPX) within the first forty minutes at constant

pH 7.8 (dose stabilisation period) by comparing the value of the respective variables between

the control group (no dose) to a specific pharmacological treatment group. To evaluate the effect

of the pharmacological treatment and aCSF pH, a two-way ANOVA with a Holm-Sidak MCT

was used. This two-way ANOVA compared the values of the respiratory variable obtained at pH

7.4, 7.6, 7.8 and 8.0 in control groups to the values of the respective respiratory variable and

relative pH set in a specific treatment group. Only post-treatment data is reported in this chapter.

The pre-treatment data are reported in the appendix. The limit of significance was 5% (p < 0.05).

The data are expressed as the mean ± one standard error of the mean (S.E.M).

27

3.5 Results

3.5.1 Fictive Breathing Frequency

Figure 3.2: The effects of adenosine (1 and 10 µM), an A1R agonist (CCPA, 10 µM) and an A1R antagonist

(DPCPX) on fictive breathing frequency measured from intact brainstem-spinal cord preparations. Panel A

illustrates data recorded from the 40 minute stabilisation period at an aCSF pH of 7.8 following the introduction of

the pharmacological agents into the aCSF. Panel B illustrates the data recorded following the various pH changes in

the presence of the various pharmacological agents. In both panels a number sign (#) indicates a significant

difference between the control value (aCSF with no pharmacological agents) and the values recorded with the

various pharmacological manipulations, and an asterisk sign (*) indicates a significant difference between the value

at that particular pH (panel B only) to the value recorded at pH 7.8 in the stabilisation period (A).

28

During the last 10 minutes of the stabilisation period (aCSF pH of 7.8 following the application

of the pharmacological agents), 1 µM adenosine (p<0.001), 10 µM adenosine (p<0.001) and

CCPA (p=0.007) caused a reduction in fictive breathing frequency while DPCPX (p<0.001)

caused a significant increase in fictive breathing frequency (Figure 3.2A).

Following the stabilisation period, the aCSF pH was changed from 7.8 to 7.4, 7.6, 7.8 or 8.0. In

the control group (Fig. 3.2B), fictive breathing frequency was increased at aCSF pH 7.4 (p =

0.001) and reduced at aCSF pH 8.0 (p = 0.004) compared to the values recorded at pH 7.8

(during the stabilisation period). In the 1 µM adenosine (p=0.018) and 10 µM adenosine (p <

0.001) groups, fictive breathing frequency increased at an aCSF pH of 7.4 compared to the value

at 7.8 during the stabilisation period. In the CCPA superfused group, fictive breathing frequency

was significantly different at pH 7.4 (p<0.001) and 7.6 (p<0.001) from the values recorded at pH

7.8. In the DPCPX superfused group, fictive breathing frequency was significantly different at

pH 8.0 (p = 0.049) from the values recorded at pH 7.8.

The reduction in fictive breathing frequency observed in response to adenosine (1 and 10 μM)

and CCPA, compared to the value in the control group, during the stabilisation period (Fig.

3.2A) was maintained during the aCSF pH changes at all pH levels with the exception of 8.0.

The increase in fictive breathing frequency observed in Fig. 3.2A with DPCPX treatment was

maintained at all aCSF pH levels (Fig. 3.2B).

29

3.5.2 Fictive Episodes per Minute


(DPCPX) on the number of fictive breathing episodes per minute measured from intact brainstem-spinal cord

preparations. Panel A illustrates data recorded from the 40 minute stabilisation period at an aCSF pH of 7.8

following the introduction of the pharmacological agents into the aCSF. Panel B illustrates the data recorded

following the various pH changes in the presence of the various pharmacological agents. In both panels, a number

sign (#) indicates a significant difference between the control value (aCSF with no pharmacological agents) and the

values recorded with the various pharmacological manipulations, and an asterisk sign (*) indicates a significant

difference between the value at that particular pH (panel B only) to the value recorded at pH 7.8 in the stabilisation

period (A).

30

During the stabilisation period at an aCSF pH of 7.8, the number of fictive breathing episodes

per minute was reduced, compared to the control group, in response to superfusion with 1 μM

adenosine (p = 0.005), 10 μM adenosine (p = 0.001) and CCPA (p = 0.003) while treatment with

DPCPX caused an increase in the number of fictive episodes per minute (p < 0.001) (Fig. 3.3A).

In the control group (Fig. 3.3B) lowering the aCSF pH to 7.4 (<0.001) and 7.6 (p = 0.036)

caused an increase in the number of fictive episodes per minute while raising pH to 8.0 caused a

reduction in episodes per minute (p = 0.001). In preparations superfused with adenosine (1 µM, p

= 0.006; 10 µM, p = 0.008) or CCPA (p = 0.040), reducing the aCSF pH to 7.4 caused an

increase in the number of fictive episodes per minute (Fig. 3.3B). There was no effect of altering

aCSF pH on the number of episodes per minute in the group superfused with DPCPX (p >

0.221).

During the aCSF-altering phase of the experiment (Fig. 3.3B), treatment with both doses of

adenosine and the single dose of CCPA caused a reduction in the number of fictive breathing

episodes per minute at pH levels of 7.4, 7.6 and 7.8 but not 8.0. Treatment with DPCPX caused

an increase in the number of fictive episodes per minute at pH levels of 7.4 and 7.6.

31

3.5.3 Fictive Breaths per Episode


(DPCPX) on the number of fictive breaths per episode measured from intact brainstem-spinal cord preparations.

Panel A illustrates data recorded from the 40 minute stabilisation period at an aCSF pH of 7.8 following the

introduction of the pharmacological agents into the aCSF. Panel B illustrates the data recorded following the various

pH changes in the presence of the various pharmacological agents. In both panels, a number sign (#) indicates a

significant difference between the control value (aCSF with no pharmacological agents) and the values recorded

with the various pharmacological manipulations, and an asterisk sign (*) indicates a significant difference between

the value at that particular pH (panel B only) to the value recorded at pH 7.8 in the stabilisation period (A).

32

During the stabilisation period, the number of fictive breaths per episode was increased,

compared to the control group, in response to treatment with 10 µM adenosine (Fig. 3.4A; p =

0.025). The other treatments had no effect.

When compared to the values recorded at pH 7.8 in the stabilisation period, altering aCSF pH

had no effect on the number of fictive breaths per episode with the exception of the CCPA

superfused groups at pH 7.6 (Fig. 3.4B; p = 0.016).

Superfusion with 10 µM adenosine and CCPA caused an increase in the number of fictive

breaths per episode during the aCSF pH-altering phase of the experiment at aCSF pH levels of

7.4, 7.6 and 7.8 but not 8.0. Superfusion with 1 µM adenosine and DPCPX had no effect on the

number of fictive breaths per episode at any aCSF pH level.

33

3.5.4 Integrated Area of Fictive Breaths


(DPCPX) on fictive breath area (mV•sec) measured from intact brainstem-spinal cord preparations. Panel A



the presence of the various pharmacological agents. A number sign (#) indicates a significant difference between the

control value (aCSF with no pharmacological agents) and the values recorded with the various pharmacological

manipulations, and an asterisk sign (*) indicates a significant difference between the value at that particular pH

(panel B only) to the value recorded at pH 7.8 in the stabilisation period (A).

34

During the stabilisation period at an aCSF pH of 7.8, treatment with DPCPX caused a reduction

in fictive breath area compared to the control values (Fig. 3.5A; p = 0.004). However, this

difference did not manifest during the aCSF-altering phase of the experiment (Fig. 3.5B). Neither

changing aCSF pH nor treatment with adenosine or CCPA had any effect on fictive breath area

during this phase of the experiment (Fig. 3.5B) although there were statistically significant

increases in area with DPCPX compared to the value recorded at pH 7.8 in the stabilisation

period.

35

3.5.5 Fictive Breath Duration


(DPCPX) on fictive breath duration (sec) measured from intact brainstem-spinal cord preparations. Panel A



the presence of the various pharmacological agents. In both panels a number sign (#) indicates a significant




36

During the stabilisation period at an aCSF pH of 7.8 (Fig. 3.6A), 10 µM adenosine caused a

small decrease in the duration of the fictive breaths compared to the control values (p = 0.007).

However, this decrease (at pH 7.8) was not observed during the aCSF-altering phase of the

experiment (Fig. 3.6B; p = 0.426). During the stabilisation period at an aCSF pH of 7.8 (Fig.

3.6A), there was no effect of DPCPX on fictive breath duration (p = 0.335). However, during the

pH-altering phase of the experiment there was a reduction in fictive breath duration at all aCSF

pH levels (Fig. 3.6B) compared to the control group. Neither 1 µM adenosine nor CCPA affected

fictive breath duration during any stage of the experiment.

37

3.5.6 Total Fictive Ventilation


(DPCPX) on total fictive ventilation (mV•sec/min) measured from intact brainstem-spinal cord preparations. Panel

A illustrates data recorded from the 40 minute stabilisation period at an aCSF pH of 7.8 following the introduction

of the pharmacological agents into the aCSF. Panel B illustrates the data recorded following the various pH changes

in the presence of the various pharmacological agents. In both panels a number sign (#) indicates a significant




38

Total fictive ventilation index (TFV) is the product of the integrated fictive breath area and

fictive breathing frequency and can be considered to be the primary index of fictive breathing.

During the stabilisation period (Fig. 3.7A), treatment with 1 µM adenosine (p<0.001), 10 µM

adenosine (p<0.001) and CCPA (p=0.018) caused a reduction in total fictive ventilation. There

was no effect of DPCPX (p=0.586)

During the aCSF pH-altering phase of the experiment, in the control group there was an increase

in TFV at an aCSF pH of 7.4 (p = 0.002) and a significant decrease at an aCSF pH of 8.0 (p =

0.001) compared to the values observed at pH 7.8 during the stabilisation period. In the group

treated with 10 µM adenosine (p = 0.002) or DPCPX (p = 0.032), there was an increase in TFV

at pH 7.4 compared to the value at 7.8 while in the CCPA-treated group there were increases at

pH 7.4 and 7.6 (p<0.001) compared to the values at 7.8 (Fig. 3.7B). However there were no

significant differences observed in the TFV value between the stabilisation period and the aCSF-

altering phase in the group superfused with adenosine 1 µM (Fig. 3.7B).

During the aCSF-altering phase of the experiment, TFV was reduced at aCSF pH levels of 7.4,

7.6 and 7.8 in the adenosine (1 and 10 µM) treated groups and the CCPA-treated group; there

was no effect at pH 8.0. In contrast, TFV was augmented at aCSF pH level 8.0 in the DPCPX-

treated group but remained unaffected at aCSF pH levels of 7.4, 7.6 and 7.8 (Fig. 3.7B).

39

3.6 Discussion

The major results of the experiments outlined in this chapter are: 1) Adenosine (both doses) and

the adenosine A1R agonist, CCPA caused a reduction in total fictive ventilation (TFV) during the

stabilisation period immediately prior to changing the aCSF pH while the adenosine A1R

antagonist, DPCPX had no effect. 2) Adenosine and CCPA caused a reduction in TFV during the

aCSF-altering phase of the experiment such that TFV was reduced at pH levels 7.4 to 7.8 but not

8.0; DPCPX had no effect. 3) The reducing effects of adenosine and CCPA on TFV were

mediated by changes in fictive breathing frequency. 4) DPCPX caused an increase in fictive

breathing frequency but, given simultaneous reductions, or no changes in, fictive breath area, this

increase in fictive breathing frequency did not translate into an increase in TFV. 5) The changes

in fictive breathing frequency caused by adenosine, CCPA and DPCPX were caused by changes

in the number of fictive breathing episodes per minute. 6) The changes in the number of fictive

breaths per episode were more ambiguous and did not appear to contribute to the reduction in

fictive breathing frequency.

Given the results of this chapter, the data suggest that adenosine acting via an A1R receptor,

leads to a decrease in fictive breathing mediated predominately by changes in fictive breathing

frequency. As such, the data suggest that increases in extracellular adenosine levels in the brain

during periods of chronic hypoxia may contribute to the reduction in breathing observed in

response to chronic exposure to low inspired oxygen levels.

The activation of the A1R stimulates the opening of K+ channels and the inhibition of calcium

(Ca2+

) channels on pre-synaptic neurons, such as those that are innervated by central pH/CO2

chemoreceptor cells (Dohrman et al, 1997). Under typical conditions, CO2 in the cerebrospinal

fluid (CSF) enters into the chemoreceptor cells and become hydrated to form a proton and a

bicarbonate ion by the enzyme carbonic anhydrase. The proton and bicarbonate ions are involved

with a series of ion exchange processes, which lead to an increase in Ca2+

uptake into the cell

causing cellular depolarization and subsequent release of neurotransmitters from pre-synaptic

neurons (Lahiri and Forster, 2003). However, upon subsequent activation of A1Rs by ADO,

neuronal K+ channels open via phosphorylation by protein kinase C (PKC). In addition, A1R

activation induces a K+ current via GIRK (Klinger et al., 2002). As a result, K

+ leaves the cell

which hyperpolarizes the cell membrane. In addition, Ca2+

uptake in neuronal cells is prevented

40

by A1R mediated inhibition of neuronal Ca2+

channels via the inhibition of adenylyl cyclase

(AC), making the post-synaptic nerve less excitable and pre-synaptic nerves less likely to release

neurotransmitters. Hence an overall attenuation of breathing was observed when the brainstem-

spinal cord preparations were exposed to ADO and CCPA treatment.

The activity of the A1R also appears to be dependent on pH, as the most significant effects on

pH-sensitive fictive breathing (breathing frequency, TFV and episodes per minute) of ADO and

CCPA treatment were observed from normocapnic to hypercapnic pH levels (aCSF pH 7.8 to

7.4) and not from hypocapnic pH levels (pH 8.0). This observation is consistent with the pH-

sensitive nature of the effects of CH observed from the previous study by McAneney and Reid

(2007), suggesting that both CH and ADO do not alter pH-sensitive fictive breathing under

hypocapnic conditions but do affect pH-sensitive fictive breathing during the in vitro equivalent

of normocapnia and during an elevation in the CO2-mediated drive to breathe. Also consistent

with the study by McAneney and Reid (2007) is the fact that ADO attenuated TFV, fictive

breathing frequency and fictive episodes per minute, but had no affect on fictive breath duration

and area as observed in CH exposed toads. Collectively, the similarities between the effects of

ADO observed in this study and the effects of CH observed in the previous study suggests that

ADO does play an important role in the modulation of respiratory responses in the cane toad

during CH.

41

Chapter 4

The effects of a Midbrain Transection on Fictive Breathing

42

4.1 Introduction

The medulla oblongata is the site of endogenous respiratory rhythm generation in all vertebrates

including amphibians and mammals (McLean et al. 1995a; Reid et al. 2000a; Torgerson et al.,

1998; Wilson et al., 2002). However, breathing patterns can differ substantially amongst

different groups of vertebrates and can differ within one class of vertebrates under different

conditions. In mammals, birds and water-breathing fish, breathing is almost always rhythmic and

continuous although there are conditions such as hibernation and torpor in which breathing may

become discontinuous (Milsom, 1991). In contrast, breathing in adult amphibians is frequently

discontinuous and intermittent although it may become continuous under certain conditions

(Milsom, 1991). While discontinuous breathing in mammals, under non-pathological conditions,

is due to differential and mismatched input to the brain from oxygen and carbon dioxide

chemoreceptors, the intermittent pattern of breathing in adult amphibians can be independent of

arterial blood-gas levels. Under these conditions, discontinuous breathing is an intrinsic property

of the central respiratory control system (Kinkead, 1997).

Central respiratory groups involved in the control of mammalian breathing have been well

studied. On the other hand, fewer studies have identified specific sites in the brain that are, or

may be, involved with the control of breathing in amphibians. Some of these sites include the

nucleus isthmi (NI) (Kinkead et al., 1997; Gargaglioni and Branco, 2004; Milsom et al., 2004),

the locus coeruleus (LC) (Noronhade-Souza et al., 2006) and the Kölliker Fuse (Adli et al.,

1999). Two of these specific sites (i.e. the locus coeruleus and the nucleus isthmi) are found in

the midbrain, which has been implicated by several studies as an important region of the brain

that regulates the clustering of breaths into episodes (Oka, 1958a,b; Reid et al., 2000a;

McAneney and Reid, 2007; Milsom et al., 1999).

In anuran amphibians the locus coeruleus is described as a group of cells that innervate the spinal

cord, cerebellum and telencephalon (Parent, 1975; González and Smeets, 1991; 1993). These

cells contain noradrenaline (González and Smeets, 1991, 1993) and can be found in the isthmus

region located at the rostral end of the hindbrain (González and Smeets, 1991). Due to their

position, noradrenergic content and projections to both the telencephalon and spinal cord, the

locus coeruleus of the anuran amphibian is believed to be homologous to the locus coeruleus

found in mammals (Marin et al., 1996). The homology is supported by the study by Noronhade-

43

Souza et al. (2006) which demonstrated that just like the locus coeruleus in mammals, the locus

coerulues neurons in amphibians acts as a chemosensitive site in the CNS that modulates central

chemoreceptor information. As such, the locus coeruleus of the midbrain in the amphibian is

believed to contain CO2/H+-sensitive neurons involved primarily with central chemosensitivity

rather than the generation of the respiratory rhythm (Gargaglioni and Branco, 2009; Gargaglioni

et al., 2010).

Studies by Oka (1958a, b) demonstrated that transecting the brain behind the optic lobes (i.e.,

behind the midbrain) just in front of the cerebellum eliminated the episodic breathing pattern in

the Japanese bullfrog. Further studies on American bullfrogs have isolated the production of

episodic breathing patterns to the areas located in the caudal half of the midbrain (Milsom et al.,

1999; Reid et al., 2000a; Gargaglioni et al., 2007). The nucleus isthmi (NI) is a mesencephalic

structure found within the caudal half of the midbrain (between the roof of the midbrain and the

cerebellum). Although it was hypothesised that the NI is responsible for the generation of

episodic breathing in anuran amphibians (Kinkead et al., 1997), subsequent studies that involved

the manipulation of the NI, either by lesion and drug microinjections, disproved the notion that

the NI is directly responsible for turning episodic breathing on and off (Kinkead et al. 1997;

Gargaglioni and Branco 2000, 2001, 2003; Gargaglioni et al., 2002). Thus the specific area

responsible for episodic breath generation in amphibians remains unknown.

4.2 Hypothesis & Objectives

Previous studies (Reid et al., 2000; McAneney and Reid. 2007) have demonstrated that

transection of the midbrain of isolated brainstem-spinal cord preparations from bullfrogs and

cane toads reduces the episodic nature of fictive breathing and causes an increase in fictive

breathing frequency. The implication of these results is that central descending inputs from the

midbrain to the medulla function to cluster breaths into episodes and to reduce breathing

frequency. Furthermore, it has been suggested that the clustering of breaths into episodes

separated by periods of apnea involves alternating excitatory and inhibitory inputs from the

midbrain.

The experiments in this current chapter (chapter 4) and the next chapter (chapter 5) address the

hypothesis that the adenosine-mediated reduction in fictive breathing reported in chapter 3 is a

result of adenosine-mediated inputs from the midbrain into the medulla. In other words, the

44

adenosine-mediated reduction in breathing occurs due to physiological processes originating in

the midbrain. In the current chapter (chapter 4), the results of midbrain transection alone are

presented while in the next chapter (chapter 5) the effects of midbrain transection coupled with

pharmacological manipulation of adenosine A1R receptors are reported. In the current chapter, I

predict that a midbrain transection will reduce the episodic nature of fictive breathing (under

hypercapnic conditions where episodic breathing is expressed) and cause an increase in fictive

breathing frequency.

45


The general methods (surgery; brainstem-spinal cord preparation) are described in chapter 2.

Figure 4.1 illustrates the experimental protocol used in this chapter. Briefly, after a stabilisation

period, the preparation was exposed to various aCSF pH levels. Following this, the brainstem

was transected slightly caudal to the optic chiasma and the preparation was allowed to stabilise

for a 40 minute period. The transected preparation was then exposed to aCSF levels of varying

pH.

Figure 4.1: Overview of the stages and general procedures in the midbrain transection experiment. The experiment

is continuous and consists of two phases; (1) the pre-treatment phase in which the brainstem preparations are intact

aCSF and (2) the post-treatment phase following the midbrain transection. The pre-treatment phase of the

experiment is divided into two parts; (1) the stabilisation period during which the brainstem preparation is

superfused with aCSF at constant pH 7.8 and (2) the aCSF pH altering period during which the preparation is

exposed to random alterations of aCSF pH (15 minute duration for each pH change). The post-treatment phase of the

experiment is also divided into two parts; (1) the transection stabilisation period following the midbrain transection

during which the preparation is superfused with aCSF at constant pH of 7.8 for 40 minutes and (2) the transection

and aCSF pH altering period in which the transected brainstem preparation is exposed to aCSF of different pH levels

(15 minute duration for each pH change).

46

4.4 Data Analysis

The effects of altering the aCSF pH in the pre-transection and post-transection periods were

evaluated using a one-way repeated measures analysis of variance followed by a Holm-Sidak

multiple comparison test (MCT) The values during the post-transection stabilisation period (at

the constant aCSF pH of 7.8) were compared to the values during the pre-transection

stabilisation period using a paired t-test. A two-way ANOVA followed by a Holm-Sidak MCT

was used to evaluate the combined effect of aCSF pH and pre-transection / post-transection. The

limit of significance was 5% (p < 0.05), and all data are expressed as the mean ± one standard

error of the mean (S.E.M).

47

4.5 Results


During the post-treatment stabilisation period (Fig. 4.2A), transecting the midbrain caused an

augmentation of fictive breathing frequency compared to that in preparations that had not had the

brainstem transected (p = 0.046).

Following the stabilisation period, the aCSF pH was changed from 7.8 to either 7.4, 7.6, 7.8 or

8.0. In the control group (Fig. 4.2B), fictive breathing frequency was increased at aCSF pH 7.4

(p = 0.001) and reduced at aCSF pH 8.0 (p = 0.004) compared to the values recorded at pH 7.8

(during the stabilisation period). In groups with the midbrain transected, fictive breathing

frequency decreased at an aCSF pH of 8.0 compared to the value at 7.8 during the stabilisation

period (Fig. 4.2B; p = 0.005). More importantly however, during the aCSF-altering phase of the

experiment (Fig. 4.2B), transecting the midbrain augmented fictive breathing frequency at aCSF

pH levels of 7.4 (p = 0.006) and 7.6 (p = 0.011); there was no effect at pH 7.8 and 8.0.

48

Figure 4.2: The effect of transecting the midbrain on fictive breathing frequency. (A) Fictive breathing frequency

during the stabilisation period (at a constant aCSF pH of 7.8). (B) Fictive breathing frequency at various aCSF pH

levels during the pH-altering phase of the experiment following the stabilisation period. A number sign (#) indicates

a significant difference between the treatment and control values. The data are reported as the mean ± S.E.M. An

asterisk (*) indicates a significant difference within a group at any given pH level compared to that at pH 7.8.

49


During the stabilisation period at an aCSF pH of 7.8, the number of fictive breathing episodes

per minute was increased, compared to the control group (no brainstem transection), in response

to the midbrain transection (Fig. 4.3A; p = 0.043).

In the control group (Fig. 4.3B) lowering the aCSF pH to 7.4 (<0.001) and 7.6 (p = 0.036)

caused an increase in the number of fictive episodes per minute while raising pH to 8.0 caused a

reduction in episodes per minute (p = 0.001). In groups with the midbrain transected, there were

no significant differences in the number of fictive episodes per minute at any aCSF pH level

during the aCSF-altering phase when compared to a pH level of 7.8 during the stabilisation

period. More importantly however, during the aCSF-altering phase of the experiment (Fig.

4.3B), transecting the midbrain caused an augmentation in the number of fictive episodes per

minute when compared to controls at pH levels of 7.4 (p = 0.030) and 7.6 (p = 0.028) but not 7.8

and 8.0.

50

Figure 4.3: The effect of transecting the midbrain on the number of fictive breathing episodes per minute. (A) The

number of fictive episodes per minute during the stabilisation period (at a constant aCSF pH of 7.8). (B) The number

of fictive episodes per minute at various aCSF pH levels during the pH-altering phase of the experiment following

the stabilisation period. A number sign (#) indicates a significant difference between the treatment and control

values. An asterisk (*) indicates a significant difference within a group at any given pH level compared to that at pH

7.8 (during the stabilisation period). The data are reported as the mean ± S.E.M.

51


There was no effect of transecting the brainstem on the number of fictive breaths per episode

either during the stabilisation period (Fig. 4.4A; p = 0.754) or during the pH-altering period (Fig.

4.4B; p = 0.689). There was no effect of altering aCSF pH on the number of fictive breaths per

episode in either the control group (Fig. 4.4B; p = 0.383) or the transected group (Fig. 4.4B; p =

p = 0.510).

4.5.4 Integrated area of fictive breaths

There was no effect of transecting the brainstem on fictive breath area either during the

stabilisation period (Fig. 4.5A; p = 0.953) or during the pH-altering period (Fig. 4.4B; p =

0.882). There was no effect of altering aCSF pH on fictive breath area in either the control group

(Fig. 4.5B; p = 0.491) or the transected group (Fig. 4.5B; p = 0.821).


There was no effect of transecting the brainstem on fictive breath duration either during the

stabilisation period (Fig. 4.6A; p = 0.118) or during the pH-altering period (Fig. 4.6B; p =

0.451). There was no effect of altering aCSF pH on the number of fictive breath duration in the

transected group (Fig. 4.6B; p =0.868). However, the values of fictive breath duration during the

aCSF pH-altering phase were significantly larger in control groups (Fig 4.6B) at pH 7.4 (p =

0.004), 7.8 (p = 0.012) and 8.0 (p = 0.012) in comparison to the value observed during the

stabilisation period.

52

Figure 4.4: The effects of transecting the midbrain on the number of fictive breaths per episode. (A) The number of

fictive breaths per episode during the stabilisation period (at a constant aCSF pH of 7.8). (B) The number of fictive

breaths per episode at various aCSF pH levels during the pH-altering phase of the experiment following the

stabilisation period. The data are reported as the mean ± S.E.M.

53

Figure 4.5: The effects of transecting the midbrain on fictive breath area. (A) Fictive breath area during the

stabilisation period (at a constant aCSF pH of 7.8). (B) Fictive breath area at various aCSF pH levels during the pH-

altering phase of the experiment following the stabilisation period. The data are reported as the mean ± S.E.M.

54

Figure 4.6: The effects of transecting the midbrain on fictive breath duration. (A) Fictive breath duration during the

stabilisation period (at a constant aCSF pH of 7.8). (B) Fictive breath duration at various aCSF pH levels during the

pH-altering phase of the experiment following the stabilisation period. The data are reported as the mean ± S.E.M.

An asterisk (*) indicates a significant difference within a group at any given pH level compared to that at pH 7.8

(during the stabilisation period). The data are reported as the mean ± S.E.M.

55


During the stabilisation period, at a constant aCSF pH of 7.8, following brainstem transection,

total fictive ventilation was not different in the transected preparations than in the control non-

transected preparations (Fig. 4.7A; p = 0.165). In the subsequent pH altering phase of the

experiment (Fig. 4.7B), total fictive ventilation was greater in the transected preparations,

compared to the controls, at aCSF pH levels of 7.4 (p = 0.041) and 7.6 (p = 0.022). Total fictive

ventilation increased as aCSF pH was lowered in the control group at pH levels 7.4 (p = 0.002)

and decreased as aCSF pH was raised to pH level 8.0 (p =0.001) compared to the value at 7.8. In

groups with the midbrain transected, TFV decreased at an aCSF pH of 8.0 compared to the value

at 7.8 during the stabilisation period (Fig. 4.7B; p<0.001).

56

Figure 4.7: The effects of transecting the midbrain on total fictive ventilation. (A) Total fictive ventilation during

the stabilisation period (at a constant aCSF pH of 7.8). (B) Total fictive ventilation at various aCSF pH levels during

the pH-altering phase of the experiment following the stabilisation period. A number sign (#) indicates a significant

difference between the treatment and control values. An asterisk (*) indicates a significant difference within a group

at any given pH level compared to that at pH 7.8. The data are reported as the mean ± S.E.M.

57

4.6 Discussion

The current study was able to demonstrate that transecting at the level of the midbrain resulted in

an augmentation of total fictive ventilation (TFV) which was mediated by an augmentation of

fictive breathing frequency which, in turn, was caused by an elevation in the number of episodes

per minute. The augmentation in fictive breathing frequency and the number of episodes per

minute was manifest during both the stabilisation period at pH 7.8 as well as during the aCSF-

altering period at pH 7.4 and 7.6. The augmentation of TFV was only manifest during the pH-

altering period at the hypercapnic pH levels of 7.4 and 7.6 but not at the normocapnic or

hypocapnic pH levels of 7.8 and 8.0, respectively, while not identical, these are more-or-less

consistent with the study by McAneney and Reid (2007), which suggest that descending inputs

from the midbrain do attenuate respiratory motor output and that this attenuation is more likely

to occur during elevated respiratory drive.

Of relevance to this finding, although contradictory to the current results, is the study by

Gargaglioni et al. (2002) which demonstrated that chemical lesions to the NI in the toad

enhanced the ventilatory response to hypercapnia (3% inspired CO2). However, a chemical

lesion targeting a single brain centre is not analogous to the entire mid-brain transection

performed in this current study. In a subsequent study, Gargaglioni and Branco (2003) were able

to demonstrate that L-glutamate and NO within the NI have no role in respiration under resting

conditions and that L-glutamate and NO exert an inhibitory modulation on the hypoxic and

hypercapnic drives to breathe. This is consistent with a past study by McAneney et al. (2006),

which demonstrated that NMDA receptor-mediated mechanisms normally inhibit the increase in

breathing frequency associated with acute hypoxia. Collectively, these findings suggest that the

transection in the current study may have compromised L-glutamate and NO mediated

mechanisms which in turn lead to increases in breathing at hypercapnic pH levels in transected

preparations.

Both the current study and the study by McAneney and Reid (2007) did not transect the NI,

rather the transection was made in an area more rostral to the NI. In addition, the transection in

the current study as mentioned in chapter 2 (Fig. 2.3) was made slightly more rostral to the

transection site described by McAneney and Reid (2007). Regardless, both studies observed an

increase in fictive breathing frequency in response to the transection. This could be explained by

58

the fact that respiratory neurons of the brainstem can receive modulatory synaptic input from

non-respiratory regions such as the motor cortex, pontine and medullary reticular formations,

cerebellum, hypothalamus, other limbic and cardiovascular regions of the brainstem as well as

from extrapyramidal motor areas (Lalley, 2008), which function to adapt breathing rhythm and

pattern for effective cardio-respiratory interactions and activities (Feldman and McCrimmon,

2003). Hence, the findings in the current study suggest that the inhibitory inputs to ventilation

from the midbrain to the medulla are not limited to the caudal half of the midbrain.

One discrepancy between the study by McAneney and Reid (2007) and the current study was

that the midbrain transection did not switch the breathing pattern permanently, i.e. from

characteristic episodic breathing to continuous breathing. In the current study, continuous

breathing did occur but only immediately after transecting the midbrain, reverting back to

episodic breathing usually within the first forty minutes of transection (refer to the appendix, Fig.

A.8).Since the transection in the current study was done via an incision with a pair of Westcott

scissors, the cells along the incision were most likely damaged. Within the cytosol that is

released from damaged cells are various chemical triggers, such as ATP and glutamate, which

can cause action potential firing (Cook and McClesky, 2002). Since breath pattern in the

amphibian can become continuous during extremely high levels of respiratory drive (Milsom,

1991; Gargaglioni and Milsom, 2007), it is possible that the brief change to a continuous breath

pattern may have been caused by the excitatory triggers within the cytosol released from

damaged cells (both of which are caused by high levels of excitation).

In current literature, the only known fact with regards to the site of episodic breath generation is

that it is found within the caudal half of the midbrain (Milsom et al. 1999; Reid et al. 2000;

Gargaglioni et al. 2007; McAneney and Reid, 2007). The current study supports these findings,

as the episodic breath pattern was still observable in the midbrain transected preparations that

was transected approximately through the middle of the midbrain rather than through the caudal

half as described in the study by McAneney and Reid (2007). The NI, which is a mesencephalic

structure that is found between the roof of the midbrain and the cerebellum, was proposed to be

that specific site of episodic breath generation (Kinkead et al., 1997). However a study by

Gargaglioni and Branco (2000), demonstrated that an electrolytic lesion of the NI failed to

eliminate the episodic breath pattern. Hence, the specific site of episodic breath generation

within the caudal half of the amphibian midbrain still remains unknown.

59

Regardless, from the point-of-view of this thesis, the important result is that the midbrain

transection caused an increase in fictive breathing at hypercapnic aCSF pH levels (7.4 and 7.6).

This indicates that descending inputs from the midbrain to the medulla normally inhibit

breathing under these conditions of elevated respiratory drive. As such, the next step is to

determine if these inhibitory inputs are adenosinergic (adenosine-mediated) in nature. This is the

focus of the next chapter in this thesis.

60

Chapter 5

Effects of Adenosine, CCPA and DPCPX on Fictive Breathing Following a Midbrain Transection

61

5.1 Introduction

It has been suggested that respiratory-related midbrain input to the medulla in anuran amphibians

is generally inhibitory (Gargaglioni and Branco, 2003; McAneney and Reid, 2007). However,

the nature of those midbrain factor(s) that exert inhibitory influences on breathing remains

unknown. Gargaglioni and Branco (2003) suggested that the putative mediators within the

nucleus isthmi (NI), L-glutamate and NO, exert inhibitory modulation on breathing during

hypoxia and hypercapnia.

During chronic hypoxia (CH), the inhibition of breathing arising from these midbrain descending

inputs is enhanced (McAneney and Reid, 2007). However, other studies have shown that NMDA

receptor function becomes reduced but not eliminated in hypoxia-tolerant animals during CH

(Bickler, 2004; Buck, 2004; Downey et al., 2007;McAneney et al., 2006; Pagonopoulou et al.,

2006) and that Ca2+

influx via activation of NMDA receptors is a key trigger for NO production

(Garthwaite et al., 1988; 1989). Collectively, the findings suggest that L-glutamate and NO,

which may play a role in the inhibitory nature of the midbrain during elevated respiratory drive,

may not have a role in the inhibitory nature of the midbrain during CH. Given this, I speculated

that a more universal factor, adenosine, that is both present and functional during elevated

respiratory drive and CH, is responsible for the inhibitory modulation of breathing exerted by the

descending inputs from the midbrain.

Since the descending inputs from the midbrain exert inhibitory influences during conditions of

elevated respiratory drive, which becomes further enhanced during CH, it is reasonable to

assume that the possible factor(s) behind the inhibitory nature of the midbrain must (1) inhibit

respiratory activity, (2) be present during a variety of levels of respiratory drive and (3) be

modified in such a manner that its inhibitory effects are enhanced during CH. One such factor is

the metabolite adenosine (ADO). Chapter 3 of this thesis determined that adenosine and an

adenosine A1R agonist (CCPA) reduced fictive breathing whilst an adenosine A1R antagonist

(DPCPX) augmented fictive breathing. Chapter 4 of this thesis indicated that transection through

the midbrain caused an increase in fictive breathing at hypercapnic aCSF pH levels indicating

that descending inputs from midbrain to the medulla normally inhibit breathing under conditions

of elevated respiratory drive. In this current chapter, I hypothesize that the midbrain input to the

medulla is inhibitory due to ADO-mediated mechanisms and subsequent A1R activation within

62

the midbrain. If this is the case, then I predict that transecting the midbrain would reduce the

inhibition to breathing caused by ADO treatment, at least in comparison to the effects of ADO

on intact brainstem-cord preparations. This hypothesis was tested by comparing the effects of

ADO, CCPA and DPCPX during both a stabilisation period and a pH-altering period on fictive

breathing recorded from brainstem-spinal cord preparations with and without the midbrain

transected at the same level as described in chapter 4.

63


The brainstem-spinal cord preparations were obtained as described in chapter 2. Figure 5.1

illustrates the protocol used in these experiments.

Figure 5.1: Overview of the stages and general procedures used within the experiment on midbrain transected

preparations. The experiment was one continual process made up of two phases; (1) the pre-treatment phase in

which brainstem preparations were kept intact and exposed only to aCSF of varying pH levels. (2) the post-

treatment phase in which the brainstem preparation with the midbrain transected was exposed to a dose of ADO,

CCPA or DPCPX along with varying aCSF pH levels. The pre-treatment phase of the experiment was split into two

parts; (1) the stabilisation period in which the brainstem preparation was exposed to aCSF at a constant pH 7.8 and

(2) the pH treatment period in which the brainstem preparation was exposed to alterations in aCSF pH (15 minute

duration for each pH change). The post-treatment phase of the experiment was also split into two parts; (1) the

transection & dose stabilisation period during which the midbrain of the brainstem preparation was transected and

exposed to a new aCSF reservoir containing a dose of ADO or the A1R agonist, CCPA or the A1R antagonist,

DPCPX (kept at constant pH 7.8) for 40 minutes and (2) the transection (with ADO or CCPA or DPCPX ) and pH

treatment period during which the ADO or CCPA or DPCPX was still present and the transected brainstem

preparation was exposed to alterations in aCSF pH (15 minute duration for each pH change).

64

5.3 Data Presentation and Analysis

In figures 5.2, 5.4, 5.6, 5.8, 5.10 and 5.12, panels A and B are the same figures (data) as

presented in chapter 3. They illustrate the effects of adenosine, CCPA and DPCPX on

preparations with the midbrain intact (no transection).

In figures 5.2, 5.4, 5.6, 5.8, 5.10 and 5.12, panels C and D represent the effects of the

pharmacological agents (adenosine, CCPA and DPCPX) on preparations in which the midbrain

was transected (i.e., previously un-reported data). Panel C shows the data from the stabilisation

period and panel D shows the effects of changing the aCSF pH following the stabilisation period

(i.e., during the pH-treatment/varying period). Note, in these transection experiments, the 1µM

adenosine dose was not administered as the effects of both 1 and 10 µM adenosine with the

midbrain intact were identical. This was done to shorten the experimental protocol and reduce

the number of animals used. In panel C of these figures, “midbrain” refers to the stabilisation

period following midbrain transection with no pharmacological agent treatment. In panel D,

“midbrain transection” refers to preparations that had the midbrain transected but were not

treated with any pharmacological agents during the pH-altering period. In other words, it is

analogous to the “control” data in panel A.


comparison test was used to compare the effects of controls (i.e., normal aCSF), adenosine,

CCPA and DPCPX during the 40 minute stabilisation period either with the midbrain intact or

the midbrain transected [i.e., the effects illustrated in panels A (midbrain intact) and C (midbrain

transected) of figures 5.2, 5.4, 5.6, 5.8, 5.10 and 5.12].

A two-way ANOVA followed by a Holm-Sidak test was used to compare the effects of treatment

with the pharmacological agents and aCSF pH changes during the “pH-altering phase” of the

experiment [i.e., the effects illustrated in panels B (midbrain intact) and D (midbrain transected)

of figures 5.2, 5.4, 5.6, 5.8, 5.10 and 5.12].

Figures 5.3, 5.5, 5.7, 5.9, 5.11 and 5.13 re-plot the data in panels B (midbrain intact) and D

(midbrain transected) of figures 5.2, 5.4, 5.6, 5.8, 5.10 and 5.12 in order to more readily observe

the effects of the different pharmacological agents with the midbrain intact and the midbrain

transected. As such, these figures show the effects of midbrain transection on fictive breathing

65

under control conditions (panel A) and in response to treatment with 10 µM ADO (panel B),

CCPA (panel C) or DPCPX (panel D).

A two-way ANOVA followed by a Holm-Sidak test was used to compare the effects of midbrain

transection and aCSF pH changes under control conditions and in response to treatment with

ADO, CCPA or DPCPX (i.e., the data in figures 5.3, 5.5, 5.7, 5.9, 5.11 and 5.13).

The limit of significance was 5% (p < 0.05), and all data are expressed as the mean ± one

standard error of the mean (S.E.M).

66

5.4 Results


Note, panels A (stabilisation period) and B (aCSF pH-altering period) are the data collected from

preparations with the midbrain intact. They are the same data as reported in chapter 3. As such,

there is no written description of these figures in the subsequent text which focuses on those

preparations in which the midbrain was transected (i.e., the data in panels C and D).

During the dose stabilisation period of the experiment in which the midbrain was transected (Fig.

5.2C), perfusion of ADO (p = 0.140), or CCPA (p = 0.130) or DPCPX (p = 0.980) had no

significant effect on fictive breathing frequency.

During the pH-altering phase of the experiment on preparations with the midbrain transected

(Fig. 5.2D), superfusion with 10 µM ADO caused a decrease in fictive breathing frequency,

compared to the controls (no pharmacological agents) at aCSF pH levels of 7.4 (p < 0.001), 7.6

(p = 0.003) and 7.8 (p = 0.038). Superfusion with CCPA caused a reduction in fictive breathing

frequency at aCSF pH levels of 7.4 (p= 0.014) and 7.6 (p = 0.013). Superfusion with DPCPX

had no effect on fictive breathing frequency, compared to the controls, at any aCSF pH level (pH

7.4, p = 0.644; pH 7.6, p = 0.951; pH 7.8, p = 0.430; pH 8.0, p = 0.214).

Altering the aCSF pH had minor effects on fictive breathing in the various groups (see the

asterisks on Fig. 5.2D).

67

Figure 5.2: The effects of adenosine (1 and 10 µM or10 µM only), an A1R agonist (CCPA, 10 µM) and an A1R

antagonist (DPCPX; 10 µM) on fictive breathing frequency measured from brainstem-spinal cord preparations with

the midbrain intact (A and B) and those with the midbrain transected (C and D). Panels A and B are the same figures

as reported in chapter 3. Panels A and C represent data recorded during the 40 minute stabilisation period either with

the midbrain intact (panel A) or transected (panel C). Panels B and D represent the data recorded during the pH-

altering phase of the experiment that followed the stabilisation period with the midbrain intact (panel B) or the

midbrain transected (panel D). In all panels a number sign (#) indicates a significant difference between the value

recorded with no pharmacological agent in the aCSF (i.e., “control” in panels A and B; “midbrain” in panel C and

“midbrain transection” in panel D) and the values recorded with the various pharmacological manipulations. An

asterisk (*) indicates a significant difference between the value of a respective group at a particular pH during the

pH manipulation phase compared to the value of the same respective group recorded at pH 7.8 during the

stabilisation period .

68

Figure 5.3 illustrates the effects of midbrain transection on the responses to ADO, CCPA,

DPCPX, as well as under control conditions, at various aCSF pH levels. Under control

conditions (Fig. 5.3A), fictive breathing frequency was elevated following a midbrain transection

at aCSF pH levels of 7.4 (p = 0.006) and 7.6 (p = 0.011). Fig. 5.3B illustrates that midbrain

transection increased fictive breathing frequency at aCSF pH levels of 7.4 (p < 0 .001) and 7.6 (p

= 0.0222) in response to ADO. Midbrain transection increased fictive breathing frequency at pH

levels 7.4 (p < 0.001), 7.6(p = 0.026) and 7.8 (p = 0.008) in response to CCPA (Fig. 5.3C).

Midbrain transection had no effect on the response to DPCPX treatment (Fig. 5.3D; p = 0.970).

69

Figure 5.3: Fictive breathing frequency recorded from brainstem-spinal cord preparations with the midbrain intact

(closed symbols) and the midbrain transected (open symbols) under control conditions (panel A) and following

treatment with 10 µM adenosine (panel B), 10 µM CCPA (panel C) or 10 µM DPCPX (D). In all panels a number

sign (#) indicates a significant difference between preparations with the midbrain intact and the preparations with

the midbrain transected.

70


During the dose stabilisation period of the experiment, perfusion with ADO caused a significant

reduction in the number of fictive episodes per minute in midbrain transected preparations (Fig.

5.4C; p = 0.015). Unlike ADO, treatment with CCPA (p = 0.107) or DPCPX (p = 0.948) on

midbrain transected preparations did not have a significant effect on the number of fictive

episodes per minute during the dose stabilisation period (Fig. 5.4C).

During the pH-altering phase of the experiment (Fig. 5.4D), superfusion with ADO caused a

reduction in the number of fictive episodes per minute at all aCSF pH levels (pH, 7.4, p < 0.001;

pH 7.6, p < 0.001; pH 7.8, p = 0.041; pH 8.0, p = 0.022). Treatment with CCPA caused

reductions in the number of episodes per minute at pH levels of 7.4 (p = 0.003), 7.6 (p = 0.003)

and 8.0 (p = 0.047). In DPCPX-treated midbrain-transected brainstem preparations, no

significant differences in the number of fictive episodes per minute were observed at any of the

pH levels (p = 0.339). With the exception of pH 8.0 in the CCPA-treated group, alterations in

aCSF pH had no effect on the number of fictive episodes per minute.

Figure 5.5 illustrates that midbrain transection caused an increase in the number of fictive

episodes per minute under control conditions (Fig. 5.5A) at aCSF pH levels of 7.4 (p = 0.030)

and 7.6 (p = 0.028). The number of episodes per minute was also elevated in response to CCPA

treatment (Fig 5.5C) at aCSF pH levels of 7.4 (p = 0.006), 7.6 (p = 0.030) and 7.8 (p = 0.013).

Midbrain transection had no effect on the number of fictive breathing episodes during treatment

with ADO (Fig. 5.5B) or DPCPX (Fig. 5.5D).

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antagonist (DPCPX; 10 µM) on fictive episodes per minute measured from brainstem-spinal cord preparations with











72

Figure 5.5: The number of fictive breathing episodes per minute recorded from brainstem-spinal cord preparations

with the midbrain intact (closed symbols) and the midbrain transected (open symbols) under control conditions

(panel A) and following treatment with 10 µM adenosine (panel B), 10 µM CCPA (panel C) or 10 µM DPCPX (D).

In all panels a number sign (#) indicates a significant difference between preparations with the midbrain intact and

the preparations with the midbrain transected.

73


During the dose stabilisation period of the experiment (Fig. 5.6C), perfusion of ADO (p =

0.428), CCPA (p = 0.160) or DPCPX (p = 0.953) did not affect the number of fictive breaths per

episode in brainstem preparations with the midbrain transected.

During the pH-altering phase of the experiment on midbrain-transected preparations (Fig. 5.6D),

treatment with ADO caused an increase in the number of fictive breaths per episode compared to

the controls at aCSF pH levels of 7.4 (p = 0.009) and 7.6 (p = 0.034). CCPA caused an increase

in the breaths per episode at pH 8.0 (p = 0.005).

Altering aCSF pH had no effect on the number of fictive breaths per episode in any of the groups

(Fig. 5.6D).

Figure 5.7 illustrates that midbrain transection had no effect on the number of breaths per

episode under control conditions (p = 0.689) or in response to treatment with ADO (p = 0.577),

CCPA (p = 0.207) or DPCPX (p = 0.184).

74


antagonist (DPCPX; 10 µM) on fictive breaths per episode measured from brainstem-spinal cord preparations with











75

Figure 5.7: The number of fictive breaths per episode recorded from brainstem-spinal cord preparations with the

midbrain intact (closed symbols) and the midbrain transected (open symbols) under control conditions (panel A) and

following treatment with 10 µM adenosine (panel B), 10 µM CCPA (panel C) or 10 µM DPCPX (D). In all panels a

number sign (#) indicates a significant difference between preparations with the midbrain intact and the preparations

with the midbrain transected.

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5.4.4 Integrated Area of Fictive Breaths

During the dose stabilisation period of the experiment (Fig. 5.8C), fictive breath area in the

midbrain-transected brainstem preparations was not affected by treatment with ADO (p = 0.669),

CCPA (p = 0.094) or DPCPX (p = 0.748). Similarly, during the pH-altering phase of the

experiment there was no effect of ADO (p = 0.938), CCPA (p = 0.304) or DPCPX (p = 0.186).

Fictive breath area was not affected by the alteration of aCSF pH levels in the control, adenosine

and DPCPX treated groups (Fig. 5.8D). However, the group treated with CCPA had a

significantly lower fictive breath area during the aCSF pH-altering phase at all pH levels than it

was during the stabilisation period (Fig. 5.8D; pH 7.4, p = 0.003; pH 7.6, p = 0.011; pH 7.8, p =

0.007; pH 8.0, p = 0.003; compare all values in Fig. 5.8D with the value in Fig. 5.8C).

Transection of the midbrain did not affect fictive breath area under control conditions (Fig. 5.9A;

p = 0.953) nor in response to CCPA (Fig. 5.9C; p = 0.302) or DPCPX (Fig. 5.9D; p = 0.142). In

response to ADO (Fig. 5.9B), midbrain transection caused an increase at pH 7.8 (p = 0.003) but

not at the other aCSF pH levels (pH 7.4, p = 0.061; pH 7.6, p = 0.075; pH 8.0, p = 0.305).

77


antagonist (DPCPX; 10 µM) on fictive breath area measured from brainstem-spinal cord preparations with the

midbrain intact (A and B) and those with the midbrain transected (C and D). Panels A and B are the same figures as

reported in chapter 3. Panels A and C represent data recorded during the 40 minute stabilisation period either with









78

Figure 5.9: Fictive breath area recorded from brainstem-spinal cord preparations with the midbrain intact (closed

symbols) and the midbrain transected (open symbols) under control conditions (panel A) and following treatment

with 10 µM adenosine (panel B), 10 µM CCPA (panel C) or 10 µM DPCPX (D). In all panels a number sign (#)

indicates a significant difference between preparations with the midbrain intact and the preparations with the

midbrain transected.

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Fictive breath duration observed in midbrain-transected preparations was not affected during the

stabilisation period (Fig 5.10C) during treatment with ADO (p = 0.826) or CCPA (p = 0.075) or

DPCPX (p = 0.050).

During the pH-altering phase of the experiment (Fig. 5.10D), treatment with ADO caused an

increase in fictive breath duration, compared to the control values at aCSF pH levels of 7.4 (p <

0.001) and 7.8 (p = 0.006). In CCPA treatments, fictive breath duration in midbrain transected

preparations was significantly augmented at all aCSF pH levels (Fig. 5.10D; pH 7.4, p = 0.004;

pH 7.6, p = 0.006; pH 7.8, p < 0.001; pH 8.0, p = 0.001). On the other hand, DPCPX treatment

did not significantly affect fictive breath duration at any aCSF pH level (Fig. 5.10D; pH 7.4, p =

0.092; pH 7.6, p = 0.066; pH 7.8, p = 0.216; pH 8.0, p = 0.211).

When compared to the values recorded at pH 7.8 in the stabilisation period (Fig. 5.10B), fictive

breath duration in response to ADO (Fig. 5.10D) was significantly larger during the aCSF pH-

altering phase at all pH levels (pH 7.4, p = 0.001; pH 7.6, p = 0.012; pH 7.8, p < 0.001; pH 8.0, p

= 0.007). In response to CCPA treatment, (Fig 5.10D), the value of fictive breath duration was

larger when compared to that observed in the stabilisation period at pH 7.8 (p = 0.044).

80


antagonist (DPCPX; 10 µM) on fictive breath duration measured from brainstem-spinal cord preparations with the











81

In the control group (Fig. 5.11A), there was no effect of transecting the midbrain on fictive

breath duration (p = 0.451). However, midbrain transection led to significant increases in fictive

breath duration in response to ADO (Fig 5.11B), CCPA (Fig 5.11C) and DPCPX (Fig. 5.11D)

treatment. Following ADO treatment (Fig 5.11B), fictive breath duration in midbrain transected

brainstem preparations was significantly greater than in intact preparations at aCSF pH levels of

7.4 (p = 0.019), 7.6 (p = 0.028) and 7.8 (p = 0.004). Similarly, superfusion with CCPA (Fig.

5.11C) significantly increased fictive breath duration in midbrain transected brainstem

preparations compared to intact brainstem preparations at all aCSF pH levels (pH 7.4, p < 0.001;

pH 7.6, p < 0.001; pH 7.8, p < 0.001; pH 8.0, p < 0.001). In DPCPX-treated transected brainstem

preparations (Fig. 5.11D), fictive breath duration was significantly elevated compared to intact

brainstem preparations at pH 7.6 (p = 0.014) and 8.0 (p = 0.012).

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Figure 5.11: Fictive breath duration recorded from brainstem-spinal cord preparations with the midbrain intact





83


Total fictive ventilation (TFV) was not affected in midbrain-transected brainstem preparations in

response to treatment with ADO (p = 0.348), CCPA (p = 0.395) or DPCPX (p = 0.727) during

the dose stabilisation period of the experiment (Fig. 5.12C).

During the pH-altering phase of the experiment (Fig. 5.12D), ADO treatment caused a

significant reduction in TFV at pH 7.4 (p = 0.022) and 7.6 (p = 0.033) while CCPA treatment

caused a significant reduction at pH 7.4 (p = 0.024). DPCPX had no effect (p = 0.879).

In the CCPA group, TFV during the pH-altering phase of the experiment (Fig. 5.12D) was less

than the value observed at pH 7.8 during the stabilisation phase at all aCSF pH levels (pH 7.4, p

= 0.004; pH 7.6, p = 0.003; pH 7.8, p = 0.001; pH 8.0, p < 0.001). There was also a significant

decrease in the control group at pH 8.0 (p = 0.017). There was no effect of DPCPX on the

response to altered pH levels.

Figure 5.13 illustrates the effects, on TFV, of transecting the midbrain under control conditions

and in response to the various pharmacological agents. Under control conditions (Fig. 5.13A),

transection led to increases in TFV at pH levels of 7.4 (p=0.041) and 7.6 (p = 0.022). In

midbrain-transected preparations that were treated with ADO (Fig. 5.13B) or CCPA (Fig.

5.13C), TFV was significantly greater in midbrain-transected brainstem preparations compared

to intact preparations at aCSF pH levels 7.4 (ADO, p = 0.008; CCPA, p = 0.023), 7.6 (ADO, p =

0.016; CCPA, p = 0.037) and 7.8 (ADO, p = 0.014; CCPA, p = 0.029). Conversely, transecting

the midbrain did not affect TFV in response to DPCPX at any pH level (Fig. 5.13D; p = 0.401).

84


antagonist (DPCPX; 10 µM) on total fictive ventilation measured from brainstem-spinal cord preparations with the











85

Figure 5.13: Total fictive ventilation recorded from brainstem-spinal cord preparations with the midbrain intact





86

5.5 Discussion

The experiments in chapter 3 illustrated that treatment with ADO and the A1R agonist, CCPA

caused a decrease in total fictive ventilation (TFV) mediated by a decrease in fictive breathing

frequency. Treatment with the A1R antagonist DPCPX caused an increase in fictive breathing

frequency which led to a non-statistically significant increase in TFV due to a concomitant

decrease in fictive breath area; TFV being the product of frequency and area. The conclusion

from that chapter is that adenosine-mediated mechanisms cause a reduction in fictive breathing

in the isolated brainstem-spinal cord preparations. Logic suggests that similar mechanisms would

lead to a decrease in breathing in the intact animal.

The results of chapter 4 indicate that transection of the midbrain leads to an increase in fictive

breathing. The interpretation of these results is that input from the midbrain to the respiratory

centres in the medulla oblongata normally result in a reduction in breathing since their removal

by transection leads to increases in breathing. The current chapter addressed the hypothesis that

these descending influences are, at least in part, mediated by adenosine-mediated mechanisms

(i.e., neurotransmission or neuromodulation).

5.5.1 Possible Sites of Adenosine Action

Superfusion of adenosine or CCPA onto the brainstem-spinal cord preparation with the midbrain

intact could lead to decreases in fictive breathing via several pathways. First, ADO or CCPA

may stimulate adenosine receptors (all types in the case of ADO and A1R in the case of CCPA)

within respiratory centres in the medulla. These respiratory centres may or may not be innervated

by adenosinergic (purinergic) neurons originating from the midbrain although my hypothesis

suggests that they are. Second, ADO or CCPA may stimulate neurons in the midbrain which, in

turn, activate other neurons that are non-purinergic in nature that then descend and innervate

respiratory centres within the medulla. Treatment with the A1R antagonist DPCPX would be

expected to antagonise the effects of endogenous adenosine regardless of the anatomical location

of the receptors.

In current literature, the distribution of ADO receptors in the brain has been documented in a

variety of mammals (Goodman and Snyder, 1982; Fastbom et al., 1987; Dixon et al., 1996;

Naganawa et al., 2006) but not in anuran amphibians. The distribution of the A1Rs have been

87

found throughout the mammalian brain in areas such as the cortex, amygdala, striatum, olfactory

bulb, nucleus accumbens, hippocampus, hypothalamus, thalamus and cerebellum.

In this current chapter I hypothesized that the midbrain input to the medulla is inhibitory due to

ADO-mediated mechanisms and subsequent A1R activation within the midbrain. If this is the

case then I predict that transecting the midbrain would reduce the inhibition to breathing caused

by ADO/CCPA treatment compared to the effects of ADO/CCPA on intact brainstem-cord

preparations.

5.5.2 The Effects on Adenosine, CCPA and DPCPX

Total fictive ventilation (TFV) is the global measure of breathing recorded from the in vitro

brainstem-spinal cord preparations. A comparison of the effects of ADO, CCPA and DPCPX

with the midbrain intact (Fig. 5.12A) and the midbrain transected (Fig. 5.12C) during the dose

stabilisation period at an aCSF pH of 7.8 indicates that the ADO- and CCPA-mediated decreases

in breathing that occur with the midbrain intact are abolished when the midbrain is transected.

The interpretation of this result is that the adenosine-mediated mechanisms that cause a reduction

in breathing originate from the midbrain albeit at a normocapnic pH of 7.8. This result is

corroborated during the pH-altering phase of the experiment (compare Figs. 5.12C and D) as the

ADO- and CCPA-mediated decreases in TFV with the midbrain intact were abolished when the

midbrain was transected; again at a normocapnic pH of 7.8. As such, the data leads to the

conclusion that adenosine-mediated mechanisms acting through the A1 purinergic receptor

somewhere in the rostral midbrain cause decreases in breathing under normocapnic conditions.

However, removal of the midbrain influences via transection did not prevent the ADO- or

CCPA-mediated decreases in TFV at hypercapnic pH levels. At pH 7.4 and 7.6, ADO treatment

still led to a decrease in TFV following midbrain transection whereas at pH 7.4 CCPA treatment

still led to a decrease in TFV following midbrain transection. These results suggest that the

inhibition of breathing by adenosine-mediated mechanisms under hypercapnic conditions occurs

somewhere other than in the midbrain; presumably within respiratory centres in the medulla.

With the exception of the data at pH 8.0 with the midbrain intact, blocking the actions of

endogenously produced adenosine by superfusing the brainstem-spinal cord preparations with

DPCPX had no effect on total fictive ventilation either with the midbrain intact (Fig. 5.12B) or

88

following a midbrain transection (Fig. 5.12C). However, treatment with DPCPX did cause a

significant increase in fictive breathing frequency (Fig. 5.2A) and a significant decrease in fictive

breath area (Fig. 5.8A) during the stabilisation phase of the experiment (pH 7.8) with the

midbrain intact. The interpretation of these results is that under normocapnic conditions (pH 7.8),

adenosine-mediated mechanisms normally function to reduce breathing frequency and increase

breath amplitude (an index of breath volume; the equivalent of mammalian tidal volume). A

similar effect was observed at pH 7.8 (and indeed at all aCSF levels) during the pH-altering

phase of the experiment for breathing frequency (Fig. 5.2B) but not for breath area/amplitude

(Fig. 5.8B).

During the pH-altering phase with the midbrain intact (Fig. 5.2B), DPCPX caused an increase in

fictive breathing frequency under hypercapnic (7.4; 7.6), normocapnic (7.8) and hypocapnic

(8.0) pH levels indicating that endogenous adenosine-mediated mechanism working via A1

receptors function to reduce breathing frequency regardless of the pH/CO2-induced drive to

breathe. These effects of DPCPX were abolished following the midbrain transection (compare

the effects of DPCPX in Figs. 5.2B and D). However, the abolition of the DPCPX-induced

increase in breathing frequency following midbrain transection is due to the fact that the

midbrain transection itself elevated fictive breathing frequency in the control group but had no

effect in the DPCPX group (see Fig. 5.3A and D). The simplest explanation is that the midbrain

transection has removed the A1 receptors that were being influenced by DPCPX with the

midbrain intact. In other words, there are A1 receptors in the midbrain that normally cause a

reduction in breathing. Their effect can be removed either by treating with DPCPX with the

midbrain intact or by removing the rostral portion of the midbrain. With the rostral portion of the

midbrain gone, DPCPX had no further effect.

Alternatively, the augmentation to fictive breathing frequency following DPCPX treatment could

have been caused by the endogenous activation of stimulatory ADO receptors (A2A and A2B

receptors). Assuming the A1Rs are fully antagonized by DPCPX, the A2A receptors followed by

the A2B receptors have the next highest affinity for ADO. If this were the case, then one

possibility pertaining to the abolished response to DPCPX in midbrain-transected preparations is

that transecting the midbrain removed input from the striatum, olfactory tubercle, hypothalamus

and nucleus accumbens, which are areas that are highly concentrated with stimulatory A2A

receptors in the mammalian brain (Dixon et al., 1996; Mishina et al., 2007). Repeating the

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experimental procedure described in the current study but with A2A, A2B or A3 agonist/antagonist

treatment of midbrain-transected preparations could provide further insight as to whether

breathing is influenced by other types of adenosine receptors and whether the effects are altered

by removal of midbrain influences.

In the current study, DPCPX (A1R antagonist) and CCPA (A1R agonist) treatment demonstrated

that the transection of the midbrain does not eliminate adenosine-mediated modulation through

the A1R. Since the transected preparations remain responsive to CCPA, this suggests that A1R

are as diversely distributed in the brain of amphibians as they are in mammals. If A1Rs are

diversely distributed in the brain of cane toad, then any decrease in A1R efficiency by the

midbrain transection could be caused by the elimination of A1R regulation originating from the

midbrain and rostral brain sites, which would support the hypothesis that the descending inputs

from the level of the midbrain suppresses respiratory activity through purinergic regulation of

ADO primarily through the A1R. Indeed preliminary data (Peters and Reid, in preparation) from

RT-PCR experiments indicate that the A1R is, as expected, present in whole brain extracts from

the cane toad. Experiments are on-going to isolate the presence of these receptors to regions of

the midbrain and medulla. In addition, in vitro light microscopic autoradiographic methods

(Goodman and Snyder, 1982) could help verify the relative densities of ADO receptors in the

brain of cane toad.

The regulation of fictive breath duration in response to adenosine-mediated mechanisms appears

to be an intrinsic function of the midbrain. In intact preparations, 10 μM ADO caused a reduction

in duration at pH 7.8 during the stabilisation period but had no effect during the pH-altering

period while CCPA failed to alter breath duration. Antagonizing the A1R with DPCPX on intact

preparations caused an overall decrease in fictive breath duration during the pH-altering period.

The data therefore appear contradictory. The interpretation of the DPCPX data alone is that A1R-

mediated mechanisms normally cause an increase in breath duration (because it decreases when

the receptors are blocked with DPCPX). However, if A1R activation normally increases breath

duration then one might expect that the ADO and CCPA treatment would have caused increases

in breath duration compared to the controls. The fact that this did not occur suggests that the

duration of the fictive breaths under control conditions is already at a maximal level and that

further stimulation of A1R mediated mechanisms had no further effect (putting aside the effect

observed in Fig. 5.10A).

90

After transecting the midbrain, fictive breath duration increased significantly in response to ADO

and CCPA (see Fig. 5,11B and C). In other words, ADO and CCPA had no effect on fictive

breath duration (aside from the single anomalous result in Fig. 5.10A) with the midbrain intact

but caused increases when the midbrain was transected. Collectively, the results suggest that the

A1R activity functions to augment breath duration but is confined to a certain degree that is

governed by the input from midbrain, the nature of which cannot be surmised from the results of

this study.

91

Chapter 6

Summary, Conclusions and General Discussion

92

6.1 Summary of the Major Results of the Thesis

The major results of chapter 3 are as follows: 1) With the midbrain intact, treatment with

adenosine (1 and 10 µM) and an adenosine A1 receptor (A1R) agonist (10 µM CCPA) caused a

reduction in total fictive ventilation (TFV: chapter 3, figure 3.7) 2) The effects of ADO and

CCPA on TFV were due to reductions in fictive breathing frequency (chapter 3, figure 3.2)

rather than a reduction in fictive breath area (chapter 3, figure 3.5). 3) The effects of ADO and

CCPA on fictive breathing frequency were due to reductions in the number of fictive episodes

per minute (chapter 3, figure 3.3) rather than the number of fictive breaths per episode (chapter

3, figure 3.4). 4) With the midbrain intact, treatment with an A1R antagonist (10 µM DPCPX)

had no effect on TFV (chapter 3, figure 3.7). However, DPCPX did cause an increase in fictive

breathing frequency (chapter 3, figure 3.2) mediated by an increase in the number of fictive

breathing episodes per minute (chapter 3, figure 3.3) and a decrease in fictive breath area

(chapter 3, figure 3.5). 5) With the midbrain intact, the effects of ADO, CCPA and DPCPX were

manifest predominately at normocapnic (7.8) and hypercapnic (7.4 and 7.4) aCSF pH levels.

The major results of chapter 4 are as follows: 1) Transection of the midbrain to remove the

rostral regions caused an increase in TFV at hypercapnic aCSF pH levels (7.4 and 7.6; chapter 4,

figure 4.7) that was mediated by an increase in fictive breathing frequency (chapter 4, figure 4.2)

which in turn was mediated by an increase in the number of fictive episodes per minute (chapter

4, figure 4.3). 2) Despite causing an increase in TFV, the midbrain transection did not abolish the

discontinuous (episodic) nature of the fictive breathing pattern.

The major results of chapter 5 are as follows: 1) Following the midbrain transection, the effects

of ADO and CCPA on TFV under normocapnic conditions (pH 7.8) were abolished (chapter 5,

figure 5.12). 2) Following midbrain transection, the effects of ADO and CCPA on TFV under

hypercapnic conditions (pH 7.4 and 7.6) remain (chapter 5, figure 5.12). 3) Following the

midbrain transection, the effects of ADO and CCPA on fictive breathing frequency are retained

under hypercapnic conditions (pH 7.4 and 7.6: chapter 5, figure 5.2D) while under normocapnic

conditions (pH 7.8) the effects of CCPA were abolished while the effects of ADO remained but

only during the pH-altering phase of the experiment. 4) Following the midbrain transection the

effect of DPCPX on fictive breathing frequency was abolished due to an increase in the control

level of fictive breathing frequency figures 5.2B and D). 5) Following midbrain transection,

93

ADO and CCPA caused increases in fictive breath duration which did not occur prior to the

transection (chapter 5, figure 5.10).

6.2 The Major Conclusions of the Thesis

1) Given that treatment with ADO and CCPA caused decreases in TFV (with the midbrain

intact), the data indicate that activation of A1R causes a decrease in breathing. This is consistent

with the fact that the A1R is an inhibitory receptor. Given that A2 receptors are stimulatory, and

that the A3 receptors are the least expressed sub-type receptor with the lowest affinity for ADO,

it is unlikely that the effects of ADO were mediated by activating receptor sub-types other than

the A1R.

2) Activation of the A1R led to decreases in fictive breathing frequency and may also lead to

increases in fictive integrated breath area although the overall effect is a reduction in TFV. This

is supported by the fact that ADO and CCPA caused a reduction in fictive breathing frequency

while DPCPX caused an increase in fictive breathing frequency but a decrease in fictive

integrated breath area (albeit the effects on area were only observed at pH 7.8 during the

stabilisation period).

3) Activation of the A1 receptor causes a reduction in breathing frequency by reducing the

number of breathing episodes per minute even though A1 receptor stimulation appears to cause a

simultaneous increase in the number of fictive breaths per episode.

4) For the most part, the effects of A1R receptor activation on breathing with the midbrain intact

occur under both normocapnic (pH 7.8) and hypercapnic (pH 7.4 and 7.6) conditions although

the occasional effect is observed at the hypocapnic pH level of 8.0.

5) Adenosine-mediated descending inputs from the rostral region of the midbrain are important

for regulating overall breathing under normocapnic conditions (pH 7.8) but not under

hypercapnic conditions (pH 7.4 and 7.6). This is based on the fact that the effects of ADO and

CCPA on TFV were abolished at pH 7.8 following midbrain transection whereas the effects at

pH 7.4 and 7.6 were not abolished. This does not exclude the possibility or the likelihood that

adenosine-mediated mechanisms in the medullary respiratory centres are also involved in

regulating breathing under normocapnic conditions.

94

6) As an extension of the previous conclusion, the data suggest that adenosine-mediated

mechanisms within respiratory centres in the medulla are important for regulating overall

breathing under hypercapnic conditions (pH 7.4 and 7.6) and likely also play a role under

normocapnic conditions. This later point is supported by the fact that the effects of ADO (but not

CCPA) on breathing frequency were retained following the midbrain transection.

7) Adenosine-mediated mechanisms within the medulla normally function to reduce breath

duration but these mechanisms or influences are normally inhibited or reduced by influences

from the midbrain although the nature of these influences is unclear.

Global Conclusion: Activation of A1R receptors causes a reduction in breathing. There are A1R-

mediated mechanisms in the brainstem respiratory centres that reduce breathing under all levels

of respiratory drive (i.e., aCSF pH). In addition, there are A1R-mediated mechanisms within the

midbrain that serve to reduce breathing under normocapnic conditions but not under hypercapnic

conditions.

6.3 The Experimental Approach and Manipulation of Adenosine

Receptors

ADO and A1R analogs (CCPA and DPCPX) were used on intact and midbrain-transected

brainstem preparations to see if the inhibitory modulation on breathing (predominately breathing

frequency) exerted by descending inputs from the midbrain and other rostral brain centers are

mediated by ADO and subsequent activation of the A1R. Administration of ADO and A1R

analogs via circulating aCSF affects all areas of the brainstem preparation. In the intact

brainstem preparations, these include the telencephalon, diencephalon, mesencephalon and

rhombencephalon, whereas in transected brainstem preparations, these include only the

rhombencephalon and the caudal half of the mesencephalon. Hence the difference in breathing in

response to ADO and A1R analogs between intact and midbrain-transected preparations,

described in chapter 5, illustrates how much of the response to ADO and A1R analogs is

accredited to the telencephalon, diencephalon and the rostral half of the mesencephalon.

The data in the current study illustrate that the response to ADO is primarily mediated by the

A1R and that the inhibitory effect of the A1R decreased as the result of the midbrain transection.

95

Since the transection removed A1Rs found in the rostral midbrain and rostral brain sites, the

reduction in inhibitory modulation on breathing exerted by the A1R may be due to a reduction in

the overall A1R density in the amphibian brain or simply the specific removal of A1Rs in the

rostral regions of the brain. This finding is consistent with the age-related decrease in the density

of A1R in the brain, which has been demonstrated to decrease the ability of A1Rs to inhibit

neuronal activity (Sebastão et al., 2000) in both mice (Pagonopoulou and Angelatou, 1992) and

humans (Meyer et al., 2007). The current study was also able to demonstrate that antagonizing

the A1R significantly augments fictive breathing frequency in intact brainstem preparations of

the toad, but did not have any effect on fictive breathing frequency and TFV in transected

brainstem preparations. Collectively, the findings suggest that modulation of breathing by ADO

through the A1R is reduced (not eliminated) by the transection, so much so that the activation of

A1R via endogenous ADO does not affect fictive breathing frequency and TFV significantly.

Hence, the hypothesis that the descending inputs from the midbrain and other rostral brain

centers exert an inhibitory modulation on breathing via ADO-mediated mechanisms and the

activation of A1R is supported by these findings.

6.4 The Effects of pH and Adenosine on Respiratory-Related

Motor Output

Neural respiratory-related activity from in vitro brainstem-spinal cord preparations is generated

by respiratory centers found in the medulla and regulated by central pH/CO2 chemoreceptors,

peripheral input from arterial chemoreceptors and lung mechanoreceptors as well as by central

influences from higher brain centers (Lahiri and Forster, 2003; Reid et al., 2000a; Reid, 2006). In

the current study, lowering the aCSF pH (8.0 to 7.4) caused an increase in TFV, fictive breathing

frequency and fictive episodes per minute. This phenomenon, also known as the acute

hypercapnic ventilatory response (or the neural equivalent thereof), is the result of central

pH/CO2 chemoreceptors responding to an increased content of hydrogen (H+) ions caused by the

increase in CO2 that was used to lower the pH of the aCSF (Reid, 2006). In intact animals, CO2

is readily diffusible from the blood and, as such, a rise in CO2 in the cerebrospinal fluid is an

indication of a rise in arterial PCO2 (Kinkead et al., 1994; Lahiri and Forster, 2003; Gheshmy et

al., 2006). Physiologically, hydrogen ions cannot pass through the blood brain barrier, so the

96

generation of H+ by the CO2 hydration reaction occurs in the CSF and intracellularly within the

chemoreceptor cells from CO2 as illustrated in the following reaction:

CO2 + H2O →H2CO3 → H+ + HCO3

-

The midbrain-transection in the current study not only maintained the acute hypercapnic

ventilatory response but augmented the sensitivity to elevated CO2 during hypercapnic pH levels

(see figure 5.13A). The maintenance of the acute hypercapnic ventilatory response was expected,

as the medulla and the locus coeruleus (Gargaglioni et al., 2010), which are both sites in the

anuran amphibian brain that contain central pH/CO2 chemoreceptors, were not removed by the

midbrain transection. The increased sensitivity to CO2 is most likely caused by the reduction of

inhibitory modulation through the A1R. In intact and transected brainstem preparations, the

inhibitory modulation of fictive breathing frequency by ADO or CCPA (A1R agonist) was

always observed at hypercapnic pH levels. In addition, treatment with DPCPX (A1R antagonist)

in intact brainstem preparations illustrates that endogenous ADO and subsequent activation of

A1R exert an inhibitory modulation to maintain typical breathing frequency. However,

transecting the brainstem at the level of the midbrain abolished the effect of DPCPX on

breathing frequency, which indicates that the subsequent activation of the A1R by endogenous

ADO no longer had a significant impact on fictive breathing frequency as a result of the

transection.

6.5 Chronic Hypoxia and Adenosine

During a hypoxic event, neurons can become damaged and killed by excitotoxic cell death

(ECD). The primary cause of ECD is the overactivation of NMDA and AMPA receptors which

cause an increase in release of the excitatory neurotransmitter glutamate. When glutamate

increases to excitotoxic levels, substantial amounts of Ca2+

enter into cells which can cause an

increase in cellular activity beyond the cells’ physiological “tolerance” causing the cells to die

(Choi, 1994). However, with an increase in cellular activity there is also an increase in

extracellular ADO due to the heightened use of metabolic by products (Klinger et al., 2002;

Pamenter et al., 2008; Latini and Pedata, 2001). During a hypoxic event, ADO can act as a

retaliatory metabolite by decreasing NMDA and AMPA receptor activity, which inhibits overall

nerve excitability and minimizes neuronal damage caused by ECD (Pamenter et al., 2008).

97

Another benefit of ADO during hypoxia is that it helps with the maintenance of brain ATP levels

which are essential to hypoxic tolerance. ADO does this by stimulating ATP production through

anaerobic glycolysis and reducing neuronal energy demands by decreasing neuronal excitability

as well as neurotransmitter release (Bickler and Buck, 2007).

Breathing in the anuran amphibian decreases during chronic hypoxia (McAneney et al., 2006;

McAneney and Reid, 2007). The findings in the current study are generally consistent with the

results observed in toads exposed to chronic hypoxia (CH) in the study by McAneney and Reid

(2007). The attenuation of fictive breathing frequency during CH or in response to perfusion of

ADO results from a decrease in the number of fictive episodes per minute rather than the number

of fictive breaths per episode. The similarity of the data suggests that the reduction in breathing

observed during exposure to CH could be caused by ADO-induced effects on breathing.

However, one notable discrepancy between the two studies does exist; application of 10 μM

ADO or CCPA augmented the number of fictive breaths per episode, an effect that was not

observed following CH. Interestingly, when 1 μM ADO was administered, the augmentation in

the number of fictive breaths per episode was not significant, which may suggest that the number

of breaths clustered into each episode is influenced by the A1R and is concentration dependent.

In future studies, performing microdialysis measurements ADO in the CSF of toads previously

exposed to CH could determine the specific concentration of extracellular ADO achieved in

toads during CH. In addition, administration of DPCPX via cannula to toads in vivo during CH

could determine whether the attenuation to breathing that occurs during CH is mediated by the

A1R.

6.6 Experimental Limitations & Future Suggestions

Westcott scissors were used in the current study to transect the midbrain; a clean cut from the

optic lobes on the dorsal surface to the caudal end of the acuate periventricular nucleus, which

severed the area between the spino-tectal and spino-mesencephalic connections and the dorsal

hypothalamus on the ventral surface (Fig. 2.3). The transection through the midbrain not only

severed modulation from the rostral half of the midbrain, but also from other rostral brain sites,

such as the thalamus, dorsal and ventral hypothalamus, septum, primordial hippocampus,

striatum, etc. In the study by Gargalioni and Branco (2000), electrolytic lesions were made

specifically to the NI located in the midbrain. The results from that study illustrated that the

98

lesion removed inhibitory modulation to breathing during elevated respiratory drive but not

under resting conditions, a result which is consistent with the data in the current transection

study. However, the lesions in the study by Gargalioni and Branco (2000) increased breathing

through changes in tidal volume (breath amplitude), but in this thesis the increase in breathing

was achieved through increased breathing frequency and not breath amplitude. The transection in

this thesis is therefore more consistent with the transection done in the study by McAneney and

Reid (2007), as the data from that study also shows an augmentation to breathing during elevated

respiratory drive via an augmentation of fictive breathing frequency rather than fictive breath

amplitude. The difference between the studies could be a reflection of the transection method

used, as both the current study and the study by McAneney and Reid (2007) used Westcott

scissors at a site rostral to the NI to transect the midbrain, whereas Gargaglioni and Branco

(2000) used electrolytic lesions to the NI. Collectively, the findings do not oppose the notion that

the NI may have an inhibitory input to respiratory sites by limiting breathing amplitude when

respiratory drive is elevated. However it still remains uncertain whether the inhibitory input to

respiratory sites that limits fictive breathing frequency during elevated respiratory drive,

demonstrated in the current study and the study by McAneney and Reid (2007), originated from

the midbrain or from other specific brain areas that are rostral to the midbrain. In future studies,

transection of more rostral sites such as the diencephalon or telencephalon would help to isolate

the origin of the inhibitory modulation to elevated respiratory drive.

In intact brainstem preparations, perfusion of ADO onto the brainstem preparation of the toad

had an inhibitory affect on fictive breathing frequency and TFV. The current study was able to

demonstrate that the amount of inhibition to breathing caused by a specific concentration of

ADO can be mirrored with the same concentration of CCPA (A1R agonist), which provides

supporting evidence that the effects of ADO observed in this study were mediated primarily by

the A1R. However, the current study did not test whether the effects of ADO can be mirrored by

stimulation of the A3R, which is also an inhibitory adenosine receptor. In future studies,

perfusion of the A3R agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (CL-

IB-MECA), at the same concentration as that of ADO perfusing the toad brainstem preparation,

could illustrate how much, if any, of the inhibitory effect of ADO on respiration can be

accredited to the A3R.

99

Perfusion of DPCPX (A1R antagonist) augmented fictive breathing frequency in intact brainstem

preparations, which illustrates the importance of A1R activation by endogenous ADO in

maintaining typical fictive breathing frequency in the toad. Whether the augmentation to fictive

breathing frequency as a result of DPCPX was caused by antagonizing the A1R alone, or in

combination with endogenous ADO activation of stimulatory A2A and/or A2B receptors, remains

uncertain. In future studies, to determine whether the augmentation of fictive breathing

frequency is mediated by antagonizing the A1R alone, the brainstem preparations could first be

superfused with DPCPX, followed by a wash-out phase and subsequent perfusion of an A2A

antagonist such as SCH-58621 or an A2B antagonist such as imiloxan. Alternatively, perfusion of

DPCPX to the brainstem preparation followed by perfusion of ADO could also determine

whether A1R are fully antagonized and whether the ADO would cause augmentation to breathing

by subsequent activation of stimulatory A2A and/or A2B receptors.

As mentioned previously, the augmentation of fictive breathing frequency seen in intact

preparations in response to DPCPX was not observed in transected brainstem preparations. The

current study demonstrated that A1R modulation was significantly reduced but not eliminated by

the transection. In studies that examined ADO receptor distribution, A2A receptors were found

highly concentrated in the nucleus accumbens, olfactory tubercle, hypothalamus and striatum in

mammals, which were rostral sites that were removed by the transection in the current study.

Hence, there are two possible reasons why the transected preparation no longer responded to

DPCPX: (1) the transection decreased both endogenous ADO sources and A1R modulation to the

point that activation by endogenous ADO no longer impacts fictive breathing, and/or (2) the

transection abolished stimulatory modulation from stimulatory adenosine receptors to fictive

breathing. In future studies, perfusion of A2 agonists (Guanabenz for A2A receptors and BAY

60–6583 for A2B receptor) to transected brainstem preparations should illustrate whether

stimulatory modulation through A2 receptors is still intact or removed by the transection.

Alternatively, utilizing RT-PCR experiments or an in vitro light microscopic autoradiographic

method (Goodman and Snyder, 1982) could help verify the relative densities of ADO receptors

in the brain of cane toad.

100

6.7 Conclusion

Activation of A1R receptors causes a reduction in breathing. There are A1R-mediated

mechanisms in the brainstem respiratory centres that reduce breathing under all levels of

respiratory drive (i.e., aCSF pH). In addition, there are A1R-mediated mechanisms within the

midbrain that serve to reduce breathing under normocapnic conditions but not under hypercapnic

conditions.

101

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Appendix

113

A1. Summary of the Experimental Protocol and an Explanation of the Data Contained Within the Appendix

Figure 3.1: Overview of the stages and general procedures performed within the experiments on intact brainstem-

spinal cord preparations.

Figure 5.1: Overview of the stages and general procedures used within the experiment on midbrain transected

preparations.

The two diagrams above (Figures 3.1 and 5.1) are reproduced from chapters 3 and 5,

respectively. Details can be found in the appropriate figure legends within these chapters. The

data reported within chapters 3, 4 and 5 of the thesis were all recorded during the “post-

treatment” phase of the experiments. All pre-treatment values (i.e. prior to experimental

manipulation – pharmaceutical agent administration and/or midbrain transection) are reported

within this appendix. The pre-treatment phase of the experiment was split into two parts; (1) the

stabilisation period: circulating virgin aCSF at constant pH of 7.8 and (2) the pH-treatment

(varying) period: alterations made to aCSF pH to achieve values of 7.4, 7.6, 7.8 and 8.0. During

114

the pre-treatment phase of the experiment, the in vitro brainstem-spinal cord preparations were

intact and were treated with circulating virgin aCSF only.

Data pertaining to the effects of time (i.e., during the time course of the entire experimental

protocol) are included within the appendix. To evaluate whether time had any effect on the

results, values from all the respiratory variables are reported from all four stages of the

experiment (Fig. 2.2; reproduced below) at a pH of 7.8 only in the intact control groups. These

data will show (see below) that all measured variables at pH 7.8 in control preparations with the

midbrain intact are identical throughout the four experimental stages of the protocol. This

indicates that “time alone” had no confounding effect on the results. Indeed this is consistent

with the long-held knowledge that the isolated brainstem-spinal cord preparation from

amphibians is robust and can function normally for many hours (and in some cases several days).

In addition, a series of recordings from midbrain transected preparations are included in this

appendix to illustrate that continuous fictive breathing did occur as a result of the transection,

albeit temporarily, reverting back to discontinuous (episodic) fictive breathing usually before the

end of the transection stabilisation period.

115

Figure 2.2. Flow chart illustrating the general stages of the experimental protocol used in the various series of

experiments outlined in this thesis. The first step was the stabilisation period, where the brainstem preparation was

placed into a bath of circulating aCSF at constant pH 7.8 until fictive breathing became rhythmic for at least 15

minutes. The second step was the pH treatment period, where the brainstem preparation was subjected to random

alterations of aCSF pH (7.4, 7.6, 7.8 and 8.0). The third step was the experimental manipulation period in which a

dose of adenosine, CCPA or DPCPX was introduced into the aCSF superfusing the brainstem preparations that were

either kept intact or transected at the midbrain. The fourth step was the “dose and pH treatment period”, where the

brainstem preparation was exposed to random alterations in aCSF in the continued presence of adenosine, CCPA or

DPCPX.

116

A2. Data Analysis


comparison test was used to analyze the effect of altering aCSF pH levels within each treatment

group; the value obtained for each respiratory variable at pH 7.4, 7.6 and 8.0 was compared to

the value of the respective respiratory variable obtained at the control pH of 7.8. A one-way

repeated measures analysis of variance was also used to evaluate fictive breathing during the

stabilisation period by comparing the value of the respective variable between the control groups

to a specific group designated for a specific treatment. To evaluate the effects of time, a one-way

repeated analysis of variance was used to compare respective respiratory variables obtained at

each stage of the experiment to one other. To evaluate the effect of both the dose and pH, a two-

way ANOVA with the Holm-Sidak method was used to compare the values of the respiratory

variable obtained at pH 7.4, 7.6, 7.8 and 8.0 in control groups to the values of the respective

respiratory variable and relative pH set in a group designated for a specific treatment. The limit

of significance was 5% (p < 0.05), and all data was expressed as the mean ± one standard error

of the mean (S.E.M).

117

A3. Results

A3.1 Fictive Breathing Frequency

Figure A.1: Fictive breathing frequency (fictive breaths per minute) measured during the pre-treatment phase of the

experiment in all groups examined within the current study. (A) The fictive breathing frequency values observed in

all groups was the same (no statistically-significant differences) during the stabilisation period of the pre-treatment

phase. (B) Fictive breathing frequency within all groups progressively decreased as aCSF pH was increased during

the pH treatment (pH-varying) period of the pre-treatment phase and the values in all groups were the same (no

statistically-significant differences). The grey area in panel A represents treatment groups that were subjected to a

midbrain transection later on in the study. The data are reported as the mean ± S.E.M.

118

A3.2 Fictive Episodes per Minute

Figure A.2: The number of fictive breathing episodes per minute measured during the pre-treatment phase of the

experiment in all groups examined within the current study. (A) The fictive breathing frequency values observed in

all groups was the same (no statistically-significant differences) during the stabilisation period of the pre-treatment

phase. (B) Fictive breathing frequency within all groups progressively decreased as aCSF pH was increased during

the pH treatment (pH-varying) period of the pre-treatment phase and the values in all groups were the same (no

statistically-significant differences). The grey area in panel A represents treatment groups that were subjected to a

midbrain transection later on in the study. The data are reported as the mean ± S.E.M.

119

A3.3 Fictive Breaths per Episode

Figure A.3: The number of fictive breaths per episode measured during the pre-treatment phase of the experiment in

all groups examined within the current study. (A) The fictive breathing frequency values observed in all groups was

the same (no statistically-significant differences) during the stabilisation period of the pre-treatment phase. (B)

Fictive breathing frequency within all groups progressively decreased as aCSF pH was increased during the pH

treatment (pH-varying) period of the pre-treatment phase and the values in all groups were the same (no statistically-

significant differences). The grey area in panel A represents treatment groups that were subjected to a midbrain

transection later on in the study. The data are reported as the mean ± S.E.M.

120

A3.4 Total Fictive Ventilation Index

Figure A.4: Total fictive ventilation measured during the pre-treatment phase of the experiment in all groups

examined within the current study. (A) The fictive breathing frequency values observed in all groups was the same

(no statistically-significant differences) during the stabilisation period of the pre-treatment phase. (B) Fictive

breathing frequency within all groups progressively decreased as aCSF pH was increased during the pH treatment

(pH-varying) period of the pre-treatment phase and the values in all groups were the same (no statistically-



121

A3.5 Integrated Area of the Fictive Breaths

Figure A.5: Fictive breath area measured during the pre-treatment phase of the experiment in all groups examined

within the current study. (A) The fictive breathing frequency values observed in all groups was the same (no

statistically-significant differences) during the stabilisation period of the pre-treatment phase. (B) Fictive breathing

frequency within all groups progressively decreased as aCSF pH was increased during the pH treatment (pH-

varying) period of the pre-treatment phase and the values in all groups were the same (no statistically-significant

differences). The grey area in panel A represents treatment groups that were subjected to a midbrain transection later

on in the study. The data are reported as the mean ± S.E.M.

122

A3.6 Fictive Breath Duration

Figure A.6: Fictive breath duration measured during the pre-treatment phase of the experiment in all groups

examined within the current study. (A) The fictive breathing frequency values observed in all groups was the same

(no statistically-significant differences) during the stabilisation period of the pre-treatment phase. (B) Fictive

breathing frequency within all groups progressively decreased as aCSF pH was increased during the pH treatment

(pH-varying) period of the pre-treatment phase and the values in all groups were the same (no statistically-



123

A3.7 Time

Figure A.7: The effect of time on respiratory variables recorded from intact control brainstem-spinal cord

preparations. (A) Fictive breathing frequency, (B) total fictive ventilation, (C) the number of fictive episodes per

minute, (D) fictive breath area, (E) the number of fictive breaths per episode and (F) fictive breath duration. Stage 1

represents the pre-treatment stabilisation phase; Stage 2 represents the pre-treatment pH treatment (pH-varying)

phase; Stage 3 represents the post-treatment stabilisation phase; Stage 4 represents the post-treatment dose & pH

treatment phase (pH-varying phase). All of the data were recorded at pH 7.8 at each stage. With the exception of

fictive breath duration in stage 3(#), the values for each variable recorded at pH 7.8 during the four experimental

stages were not statistically different. The data are reported as the mean ± S.E.M.

124

A3.8 Midbrain Transection

Figure A.8: Fictive breathing (trigeminal motor output) recorded at pH 7.8 before and after a midbrain transection.

(A) A recording made during the pre-treatment stabilisation phase (“pre-midbrain transection”). (B) A recording

during the post-treatment (i.e., post-transection) stabilisation period (“transection”). (C) A recording made during

the post-treatment (i.e., post-transection) pH-treatment (pH-varying) phase. Large peaks represent fictive lung

breaths and small peaks represent fictive buccal oscillations. In all cases the upper trace represents the raw

electroneurogram (eng X) recorded from the trigeminal nerve root while the lower trace (∫eng X) represents the

integrated trace.

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