CENTRAL CHEMORECEPTION IN THE NEONATAL RAT TRANSVERSE MEDULLARY SLICE

PREPARATION

Sasha Aleksandar Necakov

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

University of Toronto

O Copyright bp Sasba Aleksandar Necakov 2001

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ABSTRACT

Central Chemoreception in the Neonatal Rat Transverse

Medullary Slice Preparation

University of Toronto, Department of Physiology Sasha Aleksandar Necakov Master of Science (200 1 )

This study investigates several aspects of central chemoreception in the neonatal rat. transverse

rnedullary siice preparation 1 found that the frequency of bursting recorded fiom hypoglossal

nerves changed significantly with step changes in pH produced by varying the CO2 of the

bathing solution of the slice. 1 also found that application of 1 mM acetazolarnide dissolved in

DMSO to the slice preparation produced no significant alterations in these responses to CO2.

However. acetazolarnide application did provoke a significant and prolonged decrease in

hypoglossal nerve burst duration. Although the pH sensitive fluorescent dye. BCECF-AM.

perfonned satisfactorily in bench tests. when it was used to label transverse medullary slice

preparations it exhibited a faster rate of decay in fluorescence ernission. Furthemore. 1 O bserved

no change in fluorescence emission when pH was varied in the slice. either globally or locally

using a COz diffusion pipette. 1 concluded that 1 ) the transverse rnedullary slice does contain

central chemoreceptors. which elicit a respiratory response: 7) the central chemoreceptor

response to pWCOz variations is not dependent upon carbonic anhydrase. and 3) the pH sensitive

fluorescent dye BCECF-AM is not suitable for assessing regional changes of pH in the

transverse medullary siice preparation.

ACKNO WLEDGEMENTS

1 have been blessed with the support of several individuals who have provided me with

the courage and drive to complete my thesis.

Firstly. 1 would like to thank the departialent of physiology for providing me with the

fiddler award. and the respiratory research group for their input.

I would like to thank John Peever for having the patience and kindness in helping me to

complete my Master's work. John provided me w*th a strong example of what is necessary to

become a capable researcher. For his efforts 1 sincerely thank him. I would like to thank S a k

Mahamed for his undying patience and help in providing me with an understanding of the

workings of our laboratory software. His help was instrumental in rny completion of this thesis.

and I am pleased to have had the opportunity to share in his insights on research and what lies

beyond. Thanks to my parents - you were there for me on those endless nights when the goal

seemed so distant. and when I thought I could go on no longer. You have nunured rny spirits and

have licked my wounds and I am deeply grateful for al1 that you have done. Thanks to Lillian.

Eli. Andrew. Aria and Miles - y u have always been proud and supportive of me. and 1 am

happy to share this achievement with you. To Alison. you have stood beside me along the way

and have lightened my load more than you will ever know. You have given me a reason for my

struggle and are the one with whom I wish to share my path.

Lastly. 1 would like to express my heartfit gratitude to Dr. James Dufin. Dr. Duffin.

you have never strayed fiom my side even in times when it would have been much easier for y u

to 50. You have been my fountain of inspiration and support. You have stuck with me to the end

and for that 1 am deeply grateful.

TABLE OF CONTENTS

Section

1 . 1

1.2

1.2.1

1.22

1 2 . 3

1.2.4

1.2.5

1.2.6

1.3

2.1

3.2

2.2.1

2.2.2

2.2.3

2.2.4

2.3

2.3-1

Content

.A bsmc t

Acknowledgements

Table of Contents

List of Abbreviations

Table of Figures

lNTRODUCTION (Chapter 1)

Ovcrview

Background

Genenl

C lassical Experiments

Recent Developments

The Central Chemoreceptor Stimulus

The Involvement of Carbonic Anhydrase

Preparations

Hypotheses

METHODS (Cha~ter 21 Experimentd Protocols

The Preparations

aCSF Preparation

The Brainstem-Spinal Cord Preparation

The Transverse Medullary SI ice Preparation

Nerve Recordings

Data Analysis

Nerve recordings

Page

Section Content

Statistical Analysis

Cross-Correlation

Acetazo lami de

BCECF-AM

Preparation

Fluorescence Image Analysis

BCECF Control Testing

BCECF-AM Labelled Transverse Medullary Slice Experirnents

COz Difision Pipette

RESULTS (Chapter 3)

Generai Resul ts

Cross-Correlation Experiments

LeH and Righi Phrenic Nerves

Ipsilateral Phrenic Nerve Rootlets

CO2 Sensitivity in the Neonatal Rat Transverse Medullary Slice

Acetazolamide

2mM Acetazolamide

5x 1 O* M Acetazolarnide

1 mM Acetazolamide in DMSO

Rate of pH Equilibration

BCECF-AM Quality Control Experiments

Uncleaved BCEC F-AM Sensitivity to pH

Cleaved BCECF-AM Sensitivity to pH

Cleaved BCECF-AM Fluorescence Decay Curve

Cleaved BCECF-AM Dye Concentration Test

The BCECF-AM Labelled Transverse Medullary Slice Preparation

pH Response in the BCECF-AM Labelled Transverse Medullarv Slice Pre~aration

Page

28

39

29

32

32

33

34

35

36

38

39

39

40

4 1

44

45

47

5 2

56

57

57

58

59

6 1

62

62

Section Content Page

3.6.2 COz Diffusion Pipette Application in the BCECF-AM Labeled Transverse Medullary Slice Preparation

3.6.3 Photobleaching and Differential Fluorescence Emission in the BCECF-AM Labeled Transverse Medullary Slice Preparation

DISCUSSION (Chapter 4)

4.1 General

4.2 Overview

4.3 Common Activation Between Lefi and Right Phrenic Nerve Rootlets in the Neonate

4.4 CO2 Sensitivity in the Neonatai Rat Transverse Medullary Slice Preparation

4.5 Carbonic Anhydmse

4.6 Fluorescence Microscopy and a pH Sensitive Dye

4.7 Conc t usions

4.8 Future Research

REFERENCES

APPENDICES

A. 1 Appendix 1 - Cross-Correlations

A. 1.1 ContraIateral Cross-Correlation

A. 1.2 Ipsilateral Cross-Correlation

A.2 Appendix 2 - CO2 Sensitivity

A.?. 1 COz sensitivity experiments

A.2.1.1 One way RM ANOVA data for the burst Frequency vs. pH in the COr sensitivity experirnent

A.2.1.2 One way RM ANOVA data for the burst amplitude vs. pH in the CO2 sensitivity experirnent

A.2.1.3 One way RM ANOVA data for the burst duration vs. pH in the CO2 sensitivity experiment

Section

A.3

A.3.l

A.3.1.1

A.3.1.2

A.3.2

A.3 .S. 1

A.3 2 . 2

A.3.3

A.3.3.1

A.3.3.2

'4.3.3.3

.4.3.3*4

A.3.3.5

A.3.4

A.4

A.4.1

A.4.2

A.4.3

A.4.4

A S

Content

Appendix 3- Acetazolamide

2mM AC2

One way repeated measures ANOVA between frequency of bursting and %CO2 for the 2 m M acetazolamide experiment

One way repeated measures ANOVA between amplitude of bunting and %COz for the 2 m M acetazolamide experiment

5 x 1 0 " ~ AC2

Two way repeated measures ANOVA on two factors - 5 x 1 O*M acetazolamide and pW%COz with burst frequency as the dependent variable

One way repeated measures ANOVA on one factor - pW% CO2 with burst frequency as the dependent variable

1 m M AC2 in DMSO

Two way repeated measures ANOVA on two Factors - 1 mM acetazolamide and pW%COz with bunt frequency as the dependent variable

One way repeated measures ANOVA on one factor - pH/% CO? with burst frequency as the dependent variable

Two way repeated measures ANOVA on two factors - 1 mM acetazolarnide and pW%COz with burst amplitude as the dependent variable

Two way repeated measures ANOVA on two factors - I mM acetazolarnide and pW%COz with burst duration as the dependent variable

One Way Repeated Measures ANOVA on one Factor - pWYo COz with Burst Duration as the Dependent Variable Rate of pH Equilibration

Appendix 4 - BCECF-AM Quality Control Experiments Uncleaved BCECF-AM Sensitivity to pH

Cleaved BCECF-AM Sensitivity to pH

Cleaved BCECF-AM Fluorescence Decay

Cleaved BCECF-AM Dye Concentration Test

Appendix 5 - The BCECF-AM Labelled Transverse Medullary S l ice Pre~aration

Page

1 O7

1 O7

1 O7

1 O7

108

1 O8

1 O9

1 1 1

113

113

114

115

117

118

119

119

119

120

121

121

- vii -

Content Page

A.5.l pH Response in the BCECF-AM Labelled Transverse Medullary 121 Slice Preparation

LIST OF ABBREVIATIONS

aCSF

AC2

ANOVA

BCECF-AM

CaCI?

Carbogen Gas

CCD

COz

D-Glucose

DMSO

W'I H'

H2CO3

H20

HCI

HCO,'

KCI

bec.,

KHzPOI

KOH

MgSO j

NaCl

NaHC03

PH SE

VRG

P

Artificial Cerebro-Spinal Fluid

Acetazolarnide

Analysis of Variance

2' .7'-bis-(2-carboxyethyl)-5-(and-6)-carboxy-tluorescein

acetoxymethy l ester

Calcium Chlonde

A 95% Oxygen / 5% Carbon Dioxide Gas Mixture

Charge Coupled Device

Carbon Dioxide

Dextrarotatory Glucose

Dimethyl Sul foxide

Hydrogen lon Concentration

Hydrogen Ion

Bicarbonate

Water

Hydmchlonc Acid

Bicarbonate Ion

Potassium Chlotide

Decay Constant

Potassium Dihydrogen Onho Phosphate

Potassium Hydroxide

Magnesium Sulfate

Sodium Chloride

Sodium Bicarbonate

The Negative Log of Hydrogen Ion Concentration

Standard Error

Ventral Respiratory Group

Micro

TABLE OF FIGURES

Figure Page

Chernosensi tive Areas

Vibratome Preparation of the Transverse Medul lq Slice

Recording Setup

Recording Sstup t'or the Brainstem-Spinal Cord and Transverse Medullary S lice Preparations

Fluorescently Labelled Transverse Medullary Slice and Di fiusion Pipette

Cross Correlation Histogram of Left and Right Contralateral P hrenic Nerve Rootlets

Cross Correlation of Ipsilatenl Phrenic Nerve Rootlets

Mcaii Burst Frequency vs. % C02/pH in the Transverse Medullap Slice Preparation

Mean Burst Amplitude vs. % COl/pH in the Transverse Meduilary Slice Prepantion

Mean Burst Duntion vs. % C02/pH in the Transverse Medullaly SI ice Preparation

Burst Frequency vs. pH/% CO? Before. During. and Afier Application of 3 mM Acetazolarnide

Burst Amplitude vs. pW% CO, Before. Dunng. and A%er Application of 2 mM Acetazolarnide

Burst Frequency vs. pH/% CO? Before. During. and A%er Application of 5 .u 10" M Acetazolarnide

Burst Amplitude vs. pH/% COz Before. During. and Afier Application of 5 x 1 o4 M Acetazolarnide Acetazolamide Crystals on the Transverse Medullary S lice

Burst Frequency vs. pH/% CO2 Before. Dunng. and Afier Application of 1 mM Acetazolamide in DMSO

Burst Amplitude vs. pW% COz Before. During and After Application of 1 m M Acetazolamide in DMSO

Bunt Dumtion vs. pH/% CO? Before. During. and Afier Application of 1 miM Acetazolamide in DMSO

Rate of pH Equilibration

Figure Name Page

Fluorescence Emission vs. pH of NonCleaved BCECF-AM

Fluorescence Emission of Cleaved BCECF-AM vs. pH

Cleaved BCECF-AM Fluorescence vs. Tirne

Fluorescence Emission of Cleaved BCECF-AM vs. Concentration

Fluorescence Emission of a BCECF-AM Labelled SIice vs. pH

Application of the CO2 Diffusion Pipene to a BCECF-AM Label led Transverse Medullary S 1 ice Preparation

BC ECF-AM Labelled Transverse Mrdullary Slice

BC EC F-AM Labelled Transverse Medullary Slice

CHAPTER 1

INTRODUCTION

1. I ) Overview

Respiration is controlied automaticaily largely through feedback fiom central respiratory

chmorecepton. They are located close to the ventral surface of the medulla for the most part.

but other areas such as the raph may also act as chernosensors for [H']. 1 chose the neonatal rat

transverse meduilary slice preparation to examine several questions about these c hemoreceptors.

First. are they effective in the slice in that hypoglossal motor output is increased in response to

an increase in the Pcoz of the aCSF bathing the s lice? Second. is carbonic anhydrase essential

for the chemoreceptor tnction? Third. do regions of high [H'] correspond to the locations of

the central chemoreceptors?

Considering the technical challenges involved in preparing medullary slices. my tst goal

was to become proficient in rriaking viable slices that produced a respiratory rhythm fiom their

hypoglossal nerve rootlets. As a first step 1 leamed to rnake viable brainstem spinal cord

preparations (Smith et al.. 1991) Eorn which medullary stices can be prepared. by assisting John

Peever in an expetiment invo lving cross-correlation of both contralateral and ipsilateral phrenic

nerve rootlets. The hypothesis tested was that the respiratory bursting pattern Eoom lefi and nght

phrenic nerve rootlets in the neonatal rat brainstem-spinal cord preparation is synchronized as a

result of common activation. This experiment was successful and determined that lefi and right

phrenic nerve rootlets in the neonatal rat do not receive excitation f?om a common source.

contrary to the case in the adult rat (Peever et al.. 1999a).

Upon anainhg proficiency in producing transverse meduliary slices that exhibited a

hypoglossal motor output. 1 helped John Peever to test the sensitivity of the hypoglossal motor

output to global changes in C02/pH. Peevefs hypothesis was that an increase in the

concentration of COz (resulting in a decrease in pH) in the bathing medium of the slice

preparation will lead to an increase in the hypoglossal output and should thus indicate the

presence of central chemoreceptors within the preparation. The results of t his experiment were

successil in demonstrating that increases in the concentration of COz of the bathing solution

produced increases in the fiequency of respiratory bursting recorded kom the hypoglossal nerve

roo tlets of the transverse medullary slice preparation ( Peever et al.. 1 999b).

As a result of demonstrating the e.&tence of central chemorecepton within the transverse

medullary slice preparation 1 probed the role of the enzyme carbonic anhydrase in centrai

chemoreception based on the hypothesis of Torrance (Tonance. 1 993) that the mechanism of

centrai chemoreception relies upon the action O f carbonic anhydrase. I hypo thesized that the

inhibition of carbonic anhydrase within the slice preparation through the application of

acetazolamide to the bathing medium wouId prevent increases in hypoglossai motor output in

response to increases in the concentration of CO? in the bathing medium. The results of this

expriment did not support this hypo thesis.

To answer the third question locating the central chemoreceptors within the slice. 1 made

use of a fluorescent dye sensitive to pH (BCECF-AM). 1 planned to test a hypothesis. based on

the hdings of Ichikawa (Ichikawa et al.. 1989) in adult cats. that regions within the transverse

medullary slice exhibithg a greater decrease in pH in response to increasing CO2 in the bathing

solution indicate the location of centrai chemoreceptors. In testing the characteristics of the dye

to ascertain its appropriateness for the task. 1 found that whiie satisfactory performance was

observed for the dye alone. when used in the slice 1 found no detectable changes in the

fluorescence emission of the dye in response to variations in pH. Despite identdjmg several

regions that were differentiaily stained. the experiment was therefore unsuccessful in providing

visual information of alterations in pH levels within the slice preparation. 1 concluded fkom my

experirnents that BCECF-AM dye could not be used to test my hypothesis.

As an alternative approach to localizing central chemoreceptor regions within the slice 1

attempted to implement the C O diffusion pipette pioneered by Nattie (Li and Nattie. 1997a) that

could be used to acida focal regions of tissue within the slice preparation. 1 hypothesized that

focal acidifkation of certain areas within the siice preparation would produce an increase in the

hypoglossal motor output. similar to that seen with increases in the concentration of COz of the

bathhg solution and so locate the central chemoreceptors. However. 1 encountered technical

dificulties in applying the diffusion pipette to the slice. and was thus unable to demonstrate its

ability to acidify any regions of tissue or to prove my hypothesis.

1.2) Background

1.2. i ) Generul

It has been well established that the neural substrate for the automa .tic control of

breathing. whose purpose is to supply tissues of the body with oxygen and to rernove the

metabolic waste product carbon dioxide. is localized in the pons and medulla and that it involves

feedback fiom many sources (Bianchi et al.. 1 995). These include receptors in the ainvays that

provide information about lung volume and its rate of change. as well as chemoreceptors in the

carotid body and medulia.

The latter are an important aspect of the feedbac k control syst em of the mammalian

respiratory rhythm generator. and are categorised as peripheral and central chemoretlexes

(Du& 1990). Central chemoreception hvolves sensors for Pco2/ [H?l w i t b the brainstem

(Erlichman et al.. 1998). It should be noted that these central chernosensors are shielded from

alterations in the blood pH by the blood-brain barrier and its associated ion transport

mechanisms.

1.2.2) Classicai Erperiments

As yet. the exact location of the central chemoreceptors has not k e n determined. nor has

their mechanism of action been elucidated. this despite the fact that from the time of the initial

experirnents conducted by Leusen in the late 1950's probing central chemoreception mtil now.

much work has been completed in search of the mechanisms involved. Loeschcke (Loeschcke

and Gertz 19%) found that acid infusions into the founh cerebral ventricle produced a

substantial increase in ventilation. and established the hypothesis that there exist sites within the

brain sensitive to acidification of the cerebrospinal fluid.

Mitchell expanded upon Loeschcke's work by demonstrating that exposure of a localised

subarachnoid region of the ventro lateral medullary surface to acidic solutions produced a large

increase in ventilation (Mitchell et ai.. 1963). Through Loeschcke and Mitchell's e'xperiments. a

topographical map was made in which two areas were outlined as the putative sites of central

chemoreception (Figure 1). These two areas came to be known as the rostral chemosensitive

area (Mitchell's area). and the caudal chemosensitive area (Loeschcke's area). A third site on

the ventral meduilary surface lying between the rostral and caudal chemosensitive areas was

dcovered by Scbefke (Schiaefke et al.. 1970). This site was named the intermediate

chemosensitive area (Schlaefke's area figure 1 ) and was not thought to be chemosensitive but

was considered important because ventilation and chemosensitivity were depressed subsequent

to its cooling (Cherniack et al.. 1979; Millhom et al.. 1982). Figure 1 below shows the three

chemosensit ive areas of the medulla (rostrd. caudal and intemediate).

C hemosensitive Areas

Ventral Aspect

Figure 1 - The diagrarn above shows the anatomical location of the chemoreceptive sites on the ventral surface of the medulla. These three sites are represented by M, S, and L. M refers to Mitchell's are% S refers to Schlaefke's area, and L refers to M. Loeschcke's area. The p i a re s associated with each site are photographs of the researchers after whom the sites are named.

For rnany years, the central chemoreceptors were believed to reside within a few hundred

rnicrometers of the ventral meduary surface in these three areas. However. a growing body of

evidence supports the notion that central chemoreceptors are more widely distnbuted throughout

the medulla. For example. Lipscomb and Bo yarsky (Lipscomb and Bo yarsky. 1 972) suggested

that the acidic solutions applied to the ventral rnedullary sudce could be transported deep into

the brauistem thus stimulating chemoreceptors at sites other than those proposed by the work of

Loeschcke and Mitchell. As weii. severai radiolabel studies (Keeler et al.. 1984: Nattie et al..

1988: Yarnada et al.. 1984) have indicated that both large and smail radiolabelled molecules are

transported deep Uito the meduiia when applied to the ventral meduliary surface. further

supponing the idea that surface application rnay influence deeper structures. Thus. the

groundwork was laid for the possible existence of central chemorecepton at sites within the

medulla away from the ventral surface.

1.2.3) Recent Developments

The experiments of Nattie. summarised in his review (Nattie. 1999). provided a wealth of

evidence in support of a wide distniution of central chemoreceptors throughout the meduiia.

Nattie used microinjections of acetazolamide to show that medullary chemoreceptive sites are

widespread (Coates et al.. 1993) and include the VRG (Nattie and Li. 1996). the midline raphe

(Bernard et al.. 1996: Wang et al.. 1998; Wang and Richerson 1999). as weii as the

retrotrapezoid nucleus (Akilesh et al.. 1997; Li and Nattie. 1997a: Nattie and Li. 1994). More

recently Nanie's group have used a COz diffusion pipette (Li and Nattie. 1997a): it is capable of

produchg a quickly reversible focal acidification of neurod tissue through the diffusion of CO2

through the tip of the pipette. with the extent of acidi6ication varying with the concentration of

COz circulated through the pipette. These investigators found that in anaesthetized adult rats.

focal acidification of the retrotrapezoid nucleus increased phrenic nerve amplitude. They also

determined the extent of their acidification through the use of tissue pH microelectrodes. Nattie

argues that the high solubility of CO2 in tissue and blood allows for its widespread diffusion

throughout the rnedulla. where it will determine [W. This view is in contrast to the classical

assumption that the ventral surface. because it is the initial site of exposure to the blood supply

f?om the basilar artery, is the location rnost capable of providing a rapid response to CO?/[?-f].

Several studies have used quite a dSerent approach in determining the potential sites of

central chemoreception. These studies exposed intact animak to hypercapnia and examined the

brainstem for the expression of the imrnediate early gene product c-fos (Haxhiu et al.. 1996:

Miura et al.. 1996: Miura et al.. 1994: Sato et ai.. 1992: Tepperna et al.. 1997). They showed that

COz activates neurones in widespread sites throughout the medulla and confhed the

involvement of the ventral medullary surtce. but they do not specificaily identiQ

chemoreceptors.

The experiments of Ichikawa provided indirect support for the concept of widespread

locations of central chemoreception (Ichikawa et al.. 1989). He completed a fscinating

experiment. which involved measuring the extraceilular pH within the medulla in vivo during

infusion of a hypercapnic solution of saline via the intravertebral artery. The experiment

demonstrated that the tissue pH of the meduiIa fo Uowing injection of the solution was not

unifiody distniuted but heterogeneous and depended on location. He postulated that sites in

the medulla e?duiiting a Io w pH in response to COr would logicdy prove to be the locations of

central chemoreceptors. and indeed they corresponded to rnany of the sites discovered by Nattie.

More recently in vitro preparations have also been used to examine the question of

central chemoreception. Decreases in pH of the solution bathing these preparations stimulate

neural activity in bot h ventral and dorsal areas of t he medulla (Fukuda et al.. 1 980: Morin-Sum

et al.. 1995; Okada et al.. 1993b). and include the locus coeruleus (Ruiz-Ortega et ai.. 1995). the

caudal midline raph (Richerson 1995; Wang et ai.. 1998; Wang and Richerson 1999). and the

nucleus tractus solitarius (Dean et al.. 1989). For example. Dean (Dean et al.. 1989) used

medullary slices whose ventral portions were removed in order to eliminate synaptic input from

ventral medullary surface chemoreceptors to demonstrate that cells within the nucleus tractus

solitarius increase the firing in response to increases in Pco2. and that ceils in the nucleus

ambiguus do no t. Ho wever. none of these studies specifically ident ifL chemosensitive neurones

but only neurones whose activity increases as a result of local or global changes in C 0 2 / [ q .

Nevertheless. these in vitro preparations can be used to h d central chemoreceptors.

Severai studies using the neonatal rat brainstem-spinal cord preparation have demonstrated the

existence of central chemoreceptors at sites deep within the medulla (Issa and Remmers. 1992:

Kawai et al.. 1996) and within the pre-Botzinger complex (Johnson et al.. 1998). In addition.

both extracellular recordings (Jaro lirnek et al.. 1 990; Richersoa 1 995) and intracellula

recordings (Kawai et al.. 1996: Weber-Kienitz et al.. 1998) of intrinsically [q chemosensitive

neurones have been made in these in-vitro preparations.

SirnilarIy. in a recent experirnent Okada (Okada et al.. 2000) dernonstrated that voltage

sensitive fluorescent dyes applied to the transverse medullary slice of a neonatal rat could be

used to visually identifl regions in which ceiis become active as a consequence of hypercapnic

exposure. He showed that neurones near the ventral surface as weii as at deeper sites like the

ventral respiratory group. and nucleus raph paihdus and obscurus were activated. By blocking

svnaptic transmission Okada showed that neurones intrinsically sensitive to COr exist in the

surface layer of the ventral medulla and that theu excitation is transmitted to deeper areas

through a synaptic connection.

2.2.4) The Centrai Chemoreceptor Stimulus

Although the approximate anatomical locations of the central chemoreceptors are known.

it remains unclear whether the central chernosensitive mechanisrn is located intra- or

extraceUularly and whether the stimulus is CO? or [m. In solution COz is hydrated into carbonic acid which itself freely dissociates to give a proton and a bicarbonate ion. This reaction

is catalysed by the enzyme carbonic anhydrase. The reaction equation is shown below:

CO2 + H z 0 u &CO3 IV + HCO;

This equation demonstrates that increases in COz cause increases in the concentration of

H'ions in solution leading to a decrease in pH. It is assumed that COz. able to diffise through

aqueous membranes Iike the blood-brain barrier fkeely. acts to stimulate the central

chemoreceptors either directly through its action or indirectly through altering pH. The high

rate of diffusion and ease of solubility ofCOz therefore rnakes it difficult to disceni whether COz

or [q is the signal for the central chemoreceptors.

Nattie (Nattie. 1999) has argued that changes in COr would be sensed equaiIy as quickly

at either an intraceUu1ar or an extracellular site due to its high rate of diffusion. but changes in

pH would not be sensed as quickly intracellulady as it would be extracellularly. Moreover.

intraceilular pH is subject to tight regulation. Therefore. an e~acellular location for the

chemosensors appears to be a more logical choice. Nevertheless. it remains undetermined

whether the central chemosensors are located t either intracellular. extraceilular. or both sites.

and in addition whether either COz. [m, or a combination of both is the transduced signai used to provide information to the respiratory control centre.

For exarnple. Harada (Harada et al.. 1 M a ) has shown that responses to both COz and

[m exist in the isolated neonatal rat brainstem preparation. Rigano (Rigatto et al.. 1994) has shown an identical response in cultured neurones taken from the neonatal rat medulla to both

COr and [m. By contrat. Neubauer (Neubauer et al.. 199 1 ) has shown in a similar experiment that cultured neurones are responsive to changes in CO? but not to changes in [Hl produced

under isocapnic conditions. E xperiments using meduilary slice preparat ions have no t helped to

conclusively ident@ the roles of CO2 and [m either. Fukuda and Honda(Fukuda and Honda 1976) have shown that increasing [m under isocapnic conditions acts to stimulate neurones near the ventral meduliary surface. However. Miles (Miles. 1983) aithough verifjmg this result.

frther demonstrated that this isocapnic [ H 1 mediated stimulation occurs with other neurones in

the medulla away from the ventral surface. equally as weii. Further contradictions exist.

Tojima (Tojima et al.. 1991) has demonstrated that a rise in [KI while PC02 is held constant acts

to stimulate certain neurones in the medulla while Fukuda (Fukuda and Honda, 1983) has show

that decreased pH caused by an increase in Pcoz with bicarbonate held constant stimulates other

neurones.

1.2.5) The Involverneni of Carbonic Anhydrase

The enzyme carbonic anhydrase. present both intracelluiarly and enracellularly in

neuronal tissues. is a zinc metdoenzyme known to catdyze the reversible hydration ofC02

(Neubauer. 1991). Histochemistry bas shown that carbonic anhydrase is found in the medula of

the cat in an area close to the ventral meduliary surface. medial to the roots of the hypoglossal

nerve. in the cell membrane of some large neurones. in the capillary endothelium. and in glial

c e k (Ridderstrale and Hanson 1985). Ridderstrale has also demonstrated that many neuronal

ce11 bodies. dendrites and axons in the medulla contain carbonic anhydrase.

Torrance (Torrance. 1 993) has &O shown that carbonic anhydrase exists within the

medulla and that it hydrates COz near the central chemoreceptors. He postdates that carbonic

anhydrase could act at the central chemoreceptors either in the intracellular space or at the ce11

surface as a membrane bound enzyme. Carbonic anhydrase could be involved in central

chemoreception by assisting the transduction of CO2/[K] into a neuronal signal (Neubauer.

1991): the presence of intraceilular carbonic anhydrase would act to accelerate the hydration of

CO2 and thus the rapid decrease in intracellular Ievels of [HT. in the face of a slower

intracellular regdation of pH. In any case. it seems likely that by allowing the rapid transduction

of a rise in COz into a decrease in pH. carbonic anhydrase rnay affect the rate of response of the

central chemoreceptors to a CO-[HT stimuIus by accelerating COd[H'] equilibration.

The reversible carbonic anhydrase inhibitor acetazolamide has k e n used in many studies

involving the de t eda t ion of the egects of carbonic anhydrase inhibition on respiration

(Torrance, 1993. Torrance. 1 996. Neubauer. 1 99 1. Natt ie. 1 999). Acetazolarnide is used in the

treatment of acute mountain sickness Ui humans because it causes COz retention and thereby

stimulates ventilation and leads to increased tissue o.xygenation (Laux and Raic hle. 1 978). With

respect to the investigation of central chemoreception. a number of studies have used

ace tazo lamide to inhi'bit car bonic anhydrase.

Adams found that application of acetazolamide to the brain tissue in the in vivo

anaesthetized adult rat preparation increases the rebreathing response to COz. implying that

central chemosensitivity provides a greater response to CO2 upon carbonic anhydrase inhibition

(Adams and Johnson. 1 990). By contnist. others found that inhibition of carbonic anhydrase

with acetazolamide produces a response of normal magnitude but of a slo wer thne course in

response to a step change in PC02 in the meduia (Coates et al.. 1993) and in the isolated carotid

body (Iturriaga et ai.. 1 99 1 ). Furthemore. it has ken shown that the respiratory fiequency

response to CO2 in the in vivo anaesthetized rat preparation is slowed upon the inhibition of

carbonic anhydrase with acetazolamide (Tojima et al.. 1 988).

As mentioned previously. Nattie (Coates et al.. I993: Coates et al.. 199 1 ) used focal

acetazolamide injections in the in vivo anaesthetized adult rat preparation to probe the location of

central chemoreceptors. The focal acetazolamide injections produced a local tissue acidosis that

resulted in increased phrenic nerve activity. indicating the presence of central chemoreceptors at

widespread locations within the medulla.

1.2.6) Preparations

In my experiments I utilized the in vitro brainstem-spinal cord preparation. and the

transverse meduiary slice preparation fiom Sprague-Dawley neonatal rats.

The brainstem-spinal cord preparation was pioneered by Sume ( 1 984) and provides a

mode1 for probing respiratory rhythrnogenesis in vitro (Feldman and Smith 1989). This isolated

preparation includes the meduiIa and the spinal cord to the C-7 level. is lacking afferent inputs

and vascular circulation and is maintained at Iower temperatures than in the intact animal yet it

is found to produce neuronal activity correspondhg to the central respiratory rhythm. Smith et

al. (Smith et al., 1991) demonstrated that this preparation contains the pre-Botzinger cornplex.

necessary for respiratory rhythm generation. by showing that t s elirnination leads to the

cessation of respiratory rhythmic bursting of phrenic and hypoplossal nerves. The preparation

produces a respiratory bursting pattern from its phrenic and hypoglossal nerve rootlets. thus

providing a measurable respiratory output indicative of the activity of the isolated respiratory

control centre.

The transverse meduilary slice preparation pioneered by Smith and Feldman (Smith et

ai.. 1991). is one in which the brainstem spinal cord preparation is sectioned to produce a

transverse slice which includes the Pre-Botzinger complex the neuronal krrnel of respiratory

rhythmogenesis (Koshiya and Smith. 1 999). as weil as several of the ro stral hypoglossal nerve

rootlets. The transverse medullary slice preparation produces a respiratory bursting signal fiom

its hypoglossal nerve rootlets similar to that seen with the brainstem-spinal cord preparation.

However. it has also k e n postulated that the brainstemspinal cord and transverse

rnedullary stice preparations are not vaiid models in whkh the mechanisrns driving eupneic

respiration cari be tested (St.John. 19%). These preparations m y be exhibit h g gasping rarher

than eupnoea and these may represent fundarnentaliy difFerent rhyhnic respiratory patterns

generated by dserent centres. Feldrnan's group (Funk et al.. 1993) postdate that the

dserences in respiratory burst ing pattem are the result of deafferentation of the preparation

(particularly of mec hanosensory afferents). the removal of the pons. the hypoxic condit ion of the

tissues. and ambient temperature dserences. They argue that these condit ions promote changes

in the respiratory bursting pattern fkom the ramp-like rises of eupnoea seen in in-vivo. to the

square-like pattern seen in gasping observed Ni-vitro. With respect to the hypothesis that

eupnoea and gasping are produced by separate meduUary centres. Ramirez's group (Lieske et al..

2000) has recently demonstrated that three separate si@ that resemble eupnea sighs. and

gasping c m ali be obtained in the neonatal mouse transverse medullary slice preparation. They

suggest that a reconf!iguration in the respiratory rhythm generator occurs when switching

between these distinct patterns of respiration. In this case the transverse medullary slice

preparation is a valid mode1 of respiratory rhythm generation: its different pattem of respiratory

activity is the result of ambient conditions rather than the operation of a difFerent neural rhythm

generat or.

Regardless of the rhythm generator question it is important to note that both

spontaneously breathing neonatal rats (Saetta and Mortola 1987) and decerebrate. vagotornized

neonatal rats (Zhou et ai.. 1996) respond to hypercapnia similady. by increasing the tidal volume

of respiration rather than fkquency. By contrat. the neonatal rat brainstem-spinal cord

preparation responds to increases in [m (elicited by an increase in the % CO2 of the perfusion solution) by increases in p h r e ~ c and hypoglossal nerve bursting frequency with variable changes

in amplitude (Harada et al.. 1 985 b: Sume. 1 984: Voipio and Ballanyi. 1 997). Sunilarly. the

neonatal rat transverse meduilary slice preparation increases hypoglossal nerve bursting

fkquency in response to decreases in pH (Johnson et al.. 1997: Johnson et al.. 1998).

The dflering responses between the in vivo and the in vitro preparations has k e n

attributed to the fact that the temperature at which the in vitro preparations are rnaintained is

much lower than that in the in vivo preparation (25-27' C in vitro as compared to 37' C in vivo).

and that the respiratory burst signal is transfonned fiom a decrementing pattem to a rarnp-ke

incrementing pattern when temperatures are increased in the in vifro preparations ( Peever et al..

1 999b).

With the caveats above I believe that the transverse brainstem slice is likely a suitable

preparation for the study of the central chemoreceptors: moreover it offers the opportunity to use

fluorescent dyes as weil as conventional electrophysiological techniques. One such dye is 2l.7'-

bis-(2-carboxyethy1)-5-(andd6)-c~boxyxyfluore~eUi acetoxymethyl ester (BCECF-AM.

Molecular Probes). It is sensitive to changes in pH: its fluorescence emission is dependent upon

the hydrogen ion concentration it is exposed to. BCECF-AM dye has been used in the transverse

meduilary siice preparation (Ritucci et al.. 1 996) to provide a measure of the pH within stained

neurones.

1.3) Hypotheses

1) In the adult rat leA and right phrenic motoneurones are driven by a common source

ftom medullary premotoneurones whose avons bifurcate in the medulla to descend both

sides of the spinal cord. Whether they also do so in the neonatal rat is unknown. 1

hypothesised that they do. I tested my hypothesis by cross-conelating the discharges

recorded fiom lefl and right phrenic nerve rootlets of the brabtem-spinal cord

preparation. If the lefi and nght phrenic motoneurones were driven by common

premotoneurones then I should observe a central peak in the cross-~orrelo~prn.

The transverse medullary slice preparation provides an isolated region of the medulla

that exhibits a "respiratory" bursting activity on its hypoglossal nerves that some

investigaton find responds to changes in COz. 1 therefore hypothesised that this

preparation is suitable for the study of central chemoreceptors. 1 tested my hypothesis

by Uicreasing the concentration of CO2 (resulting in a decrease in pH) in the bathing

medium of the slice preparation and recorded the discharge of the hypoglossal rootlets.

If central chemoreceptors are present. the increase in COz should produce an increase in

the hypog lossai ac t ivity .

Carbonic anhydrase may play a role in central chernoreception of CO^/[^. either as an

essential factor in chemoreception or to facilitate the speed of response. I hypothesised

that carbonic anhydrase is essential to the central chemorecept ion process.

Aitematively. I hypothesized that carbonic anhydrase acts to increase the rate of

response of the central chemoreceptors to COz. 1 tested my hypotheses by blocking

carbonic anhydrase in the transverse medullary slice with acetazo lamide and recording

the response of the hypoglossal rootlet discharge to increases in CO2 in the slice-

bathing medium If carbonic anhydrase is essential the response to CO2 when

acetazolamide is present should be absent. If carbonic anhydrase speeds the response

then the response to CO? when acetazolamide is present should be slowed.

The transverse brainstem slice lends itself to the use of fluorescent dyes. which cm be

viewed via fluorescent rnicroscopy. One such dye is the H* sensitive dye BCECF-AM.

I hypothesized t hat BCECF-AM could be used to show variations in the tissue [H'] in

response to increasing CO2 of the bathhg medium. 1 tested my hypothesis first by

performing several qualitative tests on the dye in order to determine the optimal

concentrations for its use: then 1 used fluorescence microscopy to observe its rate of

fluorescence decay. and its sensitivity to [m in the slice bathing medium solution. and finally in the slice tissue itself. If the dye was effective in locating regions of altered

pH then 1 should be able to observe dserences in fluorescence in the slice tissue.

The CO2 difhsion pipette has been shown capable of acidifig specific localised

regions of brain tissue in anaesthetised rats. I note that the transverse brainstem slice

lends itselfto the use of a focal acidification of specific regions of the slice with a COz

diffusion pipette to localise central chemoreceptors. 1 therefore hypothesized that the

CO2 diffusion pipette could be used to produce focal increases of [m within the tissue of transverse medullary siices. which would increase the respiratory hypoglossal motor

output of the preparation. I tested my hypothesis by applying local acidification via a

CO2 difision pipette to known central chemoreceptor locations like the ventral surface

and recording the discharge of the hypoglossal nerve. If the technique was effective

then 1 should be able to observe increases in hypoglossal activity when the CO2

difision pipette was applied.

CHAPTER 2

METHODS

2. I ) Experiimenta Prolocols

Ail experiments were performed on Sprague Dawley neonatal rats between the ages of 2.

and 8 days. Their mean age and standard error were 3.8 days old 7- 0.37. Surgery was

performed to iso late the brainstem-spinal cord of each rat as described in detail belo W. These

isolated brainstem spinal cord preparations were used in either the cross-correlation experirnents.

or to produce transverse medullary slice preparations as described in detail below. In each of the

experiments electrophysiological recordings were made of the act ivity of the phrenic (brainstem-

spinal cord preparation) or hypoglossal nerve rootlets (transverse medullaq slice preparation)

using suction electrodes. Details of the experirnents are provided below but bnefly the protocols

used were as follows:

a) To detect the presence of comrnon short-the scale synchronisation of le ft and right phrenic

nerves in the brainstem spinal cord preparation I cross-correlated the discharge recorded

tiom left and right phrenic rootlets and examined them for broad peaks at tirne zero in siu

preparations. I also cross-correlated the discharge between ipsilateral phrenic nerve rootlets

in one preparation.

b) To demonstrate the CO2 sensitivity of the transverse medullary slice preparation I recorded

the response of the rhythmic hypoglossal rootlet discharge to changes in the pH of the

bathing aCSF as weU. as the time course of the pH changes in six preparations. I used a pH

of 7.42 as a baseline and then decreased pH in steps to 7.70 and then 7.00. allowing at least 5

minutes between changes for equiliiration.

c) To test whether carbonic anhydrase is involved in the central chemoreceptive process. I

compared the response of the hypog lossal disc harge of the transverse medullary siice

preparation to pH variations in the aCSF. with and without the presence of acetazolamide (a

carbonic anhydrase inhibitor) in the aCSF bathing solution in thirteen preparations.

d) To test the applicability of the pH sensitive dye fluorescent dye BCECF-AM for visualizing

focal areas of acidificat ion wit hin the transverse rneduhry slice preparat ion I carried out a

series of experiments as follows. First. 1 measured the pH sensitivity of both the cleaved and

un-cleaved t o m of BCECF-AM. the rate of decay of fluorescence emission for cleaved

BCECF-AM. and the optimal concentration ofcleaved BCECF-AM for maximal

fluorescence emission. Then, 1 measured the changes in fluorescence of one BCECF-AM

labeiied transverse meduiiary slice preparation in response to changes in the COr content of

the bat hing aCS F.

e) To test the feasibility of ushg a CO2 diffusion pipette to focally acidw regions of tissue to

probe the location of central c hemorecept ive sites within the transverse medullary slice

preparation. 1 appiied it to various areas while recording the hypogIossai nerve rootlet

discharge in nine preparations.

2*2) The Preparations

2* 2- 1) aCSF Preparation

In order to deliver o'xygen to neurones within the brainstem-spinal cord and transverse

meduiiary siice preparations. experiments were conducted in a recording chamber (2.5 ml)

through which a solution termed 'art ificial Cerebro-S pinal Fluid' (aCSF) constant ly flowed. The

solution is named for its similarity in composition to that of the cerebro-spinal fluid to which the

neonatal rat brain is exposed in the intact anunal.

The aCSF composition was as follows: 125 mM sodium chloride (NaCi). 25 mM sodium

bicarbonate (NaHCOs). 30 mM dextrorotatory-Glucose. 3 m M potassium chloride ( K I ) . I mM

potassium dihydrogen ortho-phosphate (KH2P04). 2 rnM calcium chloride (CaCI?). and 1 mM

magnesiurn sulphate (MgSO4) in distiiled H20. Once the components were combined the aCSF

was well mived and lefi to cool in a retiigerator to a temperature of 18 OC. From this mixture ice

cubes. used to cool the aCSF bathing the preparations. were made in a standard ice tray ( 12 ice

cubes with a total volume of 300 ml).

I controiied the pH of the aCSF bathing solution by bubbling it with speck gas

mixtures. The baseline pH of the bathing solution was maintained at 7.42 by bubbling it with

carbogen (5% C02/95% Oz). To decrease the pH to approxirnately 7.20 and 7.00.1 increased

the percentage of CO? bubbled into the aCSF by 5% and 10%. respectively.

2.2.2) The Brainstem-Spinai Curd Preparation

The brainstem-spinal cord preparation implemented in the experiments was prepared as

outlined by (S unie. 1 984). Sprague-Dawley neonatal rats were anaesthetised using 3 %

Halothane pas (Halocarbon laboratones Inc.) in oxygen and were sectioned caudal to their fiont

legs. The head and t o m ofthe rat were isolated and placed into a dissection charnber filled with

cold aCSF (-12 OC) bubbled with carbogen (95% O?/ 5%C02) at a pH of 7.42 (pH meter. Hanna

Corporation). The skin covering the skuil. along with the fiontal and occipital bones. were

removed using surgical scissors. The portion of skuil covering the cerebeiium was removed.

dong with the dorsal portion of the vertebrae. taking extreme care not to rupture any spinal or

cranial nerves in the process.

The entire brain and spinai cord (cerebellum cerebnim midbrain and spinal cord) were

then excised fiom the rat by cutting al1 connecting nerves as close to their distal end as possible.

Following this excision the c e r e b m was removed through a transection at the intercollicular

leveL and the cerebeilum was removed as weil leaving the brainstem-spinal cord free for

experimentation. It is important to note t hat shorter times of complet ion provide more viable

preparations in t e m of their ability to produce a rhythrnic respiratory output. The time taken in

producing each brainstem-spinal cord preparation was approximately 8 minutes. and was

ni t t ic idy short for maintenance of viability.

Following its removal. the brainstem-spinal cord preparatim was placed in the recording

chamber on top of a nylon mesh and perfused at a rate of 20 dminu te with aCSF at a

temperature of 25.0 OC and bubbled with carbogen gas to achieve a pH of 7.42. In order to

maintain the temperature of the aCSF bathing solution within the recordkg charnber constant. an

automatic temperature controiier (mode1 TC-324B. Warner Instrumentation Corp.) was used. To

stabilize the preparation two smaii tungaen rods (diameter = 70 prn length = 1 cm) were

inserted through its caudal and rostral ends into Sylgard ( 184 Silicone Elastorner. Dow Coming)

Lining the bonom of the recording chamber.

Care was taken not to dismpt the spinal cord above the level of the 7" spinal nerve. and

the medulia caudal to the level of the pons. Once stabilized with regards to position.

temperature. and pH. the dura covering the hypoglossal and spinal nerve rootlets of the

preparation was removed using micro forceps (Dumoxel non-magnetic microforceps #5.

A.Durnont & Fils Co.) with the aid of a 40x dissection microscope (Wild Heerbrug Co.). The

spinal nerve rootlets were then carefully separated fkom one another using micro forceps in order

to clear them for electrophysiological recording. It is important to note that extreme care was

taken in order to ensure that the rootlets were not damaged.

2.2.3) The Transverse Medu f fury S k e Prepuration

The transverse meduliary s lice preparat ion used for the central chemosensitivity

experiments was that pioneered by (Smith et al.. 199 1 ). The initial steps in obtaining the slice

preparation were the same as those outlined for the brainstem-spinal cord preparation in section

2.2.2.

In preparation for making a slice. 50 ml of distiued water was heated to a boil in a 230 r d

pyrex beaker and to it was added 3.5 gram of agar (Bactom Agar. Becton Dickinson & Co.).

This agar solution was affowed to cool until hard. and was then cut to produce a small rectangle

(approxhately 10 mm x 8 mm x 4 mm). which was glued to a plastic vibratome mount using

cyanoacrylate glue (Krazy glue. Elmer's Co. ).

Once the brainstern-spinal cord preparation was obtained. t was removed kom the

dissection tray flled with bubbied aCSF using a d spatula dried carefuily with filter paper.

and its dorsal end was glued to the mounting bIock/agar rectangle with the rostral end down (see

figure 2.2.3 on the folIowing page) using the cyanoacrylate glue. The mounting block was

Vibratome Preparation of the Transverse MeduIIary SIice

Agar Block

II

- - - Inrtia! Cut --

- - A -

Vibratome Am

Figure 2.23 - The diagram above shows the brainstem-spinal cord preparation mounted ont0 the vibratome block in preparation for sectioning to produce a transverse medullary slice.

placed into the tray of the vibratome (mode1 752M. Vibroslice. Campden Instruments). which

was Nled with cold (-12 OC), carbogen gas bubbled (pH = 7.42) aCSF. The vibratome iight was

adjusted in order to illuminate the ventral surface of the brainstem-spinal cord so that the dura

and the hypoglossal nerve rootlets underlying it were h l y visible.

An initial cut was made using the vibratome. removing the section of tissue caudal to the

spinal medullary border in order to expose the hypoglossal nerve rootlets of the preparation to

full view. Using two sets of micro forceps (Dumoxel non-rnagnetic microforceps. A.Dumont &

Fils). the dura was removed ffom the ventral surface of the preparation in order to fkther expose

the hypoglossal rootlets to plain view. Then the blade of the vibratome was positioned

approxirnately 10 pm rostral to the rostrai-most hypoglossal nerve rootlet. and the blade was

moved upwards (caudal) to the desired thickness of the slice. The slice thickness in al1

experirnents varied between 400 and 1 200 prn with a mean thic kness and error of 930 7- 24

microns.

Once the blade was positioned at the desired level. al1 hypoglossal rootlets caudal to the

location of t he intended cut were placed rostral to the edge of the blade in order to avoid their

king sectioned. and the vibratome was set in motion at its lowest speed and highest vibration

rate. As weii. the light was turned off in order to maintain a low temperature w i t h the

preparation. The blade was retracted following completion of the cut and the iight was t m e d on

in order to illumuiate the slice. The ronral portion of tissue produced was removed. and the

blade was repositioned to the previously deteniiined location that king 10 pm rostral to the

rostral-rnost hypoglossai roo tlets. Once the location of the cut was verified. the light was turned

off again and a cut was made exactly as descnid previously. The transverse meduilary siice

produced was removed fkom the vibratome chamber and placed into a 250 ml pyrex beaker fled

with cold (-12 OC) carbogen bubbled (pH = 7.42) aCSF. and aiiowed to sit for 20 minutes in

order for neuronal function to recuperate (Paton et al.. 1994).

Following the 20 minute recuperation period. the slice was placed into the recording

c h b e r as outlined in section la and s w e d by inserthg two srnail tungsten rods through the

attached agar backing and into Sylgard gel (Sylgard 1&4 elastorner. Dow Corning Co.) c o v e ~ g

the bottom of the recording chamber. he slice was positioned in the chamber such that the agar

backing was oriented towards the hcoming flow in order to ensure mechanical stability of the

slice and perfusion of both its sides. Once secured. the KCL concentration within the aCSF

bathing solution was increased to 8 mM by slowly adding 2.5 ml of 1 M KCI to the 500 ml of

aCSF in the reservoir over a period of 20 minutes in order to depolarise the neurones within the

prepmion and thus facilitate their firing. it was important to add KCI to the aCSF reservoir

only after the slice was placed in the recording chamber in order to provide a slow. graded

increase in [KCI] as it was my experience that rapid step increases in [KCL] are detrimental to

the viability of the slice. As weli. the temperature of the bathing solution was slowly (- 0.5

"C/minute) increased using the temperature controiier to a temperature of 25.0 O C . The

hypoglossal nerve rootlets were prepared for electrophysiological recording exactly as described

for the phrenic nerve rootlets of the brainstem spinal cord preparation (see section 2.2.3).

2.2.4) Nerve Recordings

The activity of newe rootlets was recorded using glas suction electrodes prepared by

pulling borosilicate glass capillary tubes (Kwik-FilTU capillary tubes. mode1 TW 100F-6. World

Precision instruments) using a rnicroelectrode puller (Mode1 750. David Knopf Instniments).

The tip diameters of the microelectrodes were varied by breaking the tip. thereby obtaining an

opening to fit the nerve rootlet and allow it to be secured through suction (approxhately 200

pm).

The suction microelectrode was inserted into a holder attached to a microelectrode pre-

amplifier (HS-ZA Headstage pre-amplifier. Axon instruments Inc.) mounted on a

micrornanipulator (mode1 10606. Narshige instruments) clamped to the base of the fluorescence

microscope (BXSOWI Fived Stage upright microscope. Olympus optical Co.). thus aliowing for

precise manipulation of the suction electrode tip. A 10 ml W g e attached to the microelectrode

holder was used to provide the suction necessary to pull a single phrenic nerve rootlet hto the tip

of the suction electrode. Once the nerve rootlet was secured within the suction electrode. and the

rootlet demonstrated evidence of respiratory bursting (discernible bursts seen on the

oscilioscope), it was possible to begin recording the respiratory bursting pattern of t he

preparat ion.

The recorded signal was arnpiified using a pre-amplifier (HS-IA Headstage pre-

amplifier. Axon instruments Inc.) and an amplifier (Neurolog. NL 104). The amplified signal

was then iltered (bandpass 0.12-8 kHz) and Uitegrated ( t h e constant = 50 rns). The resulting

signals were displayed on oscilioscopes (Tektronics. Nicolet) and monitored using loudspeakers.

A thermal anay chart recorder (Graphtec. WR3600) provided a permanent record of the signals

and a digitised videotape recording (Vetter) was also made for archival purposes. Figure 2.24

on the foilowing page illustrates the recording setup.

Recording Setup

Figure 2.2.4 - The diagrarn above shows the organization of the recording equipment used in both the brainstem-spinal cord and transverse medullary slice preparations.

2.3) Data Analysk

2* 3.1) Nerve recordings

To allow for stabiiization of the preparat ion. the rhythmic respiratory output of the s lice

was recorded for a period of approxirnately 40 respiratory bursts (Because of variation in the

bursting kquency between slice preparations a period involving a set nurnber of bursts was

chosen rather than a set period of tirne). For example. using t his criterion after making changes

to the pH of the bathing aCSF. at least 5 minutes passed before measurements were made. The

chart recordings of the rhythrnic respiratory output of al1 siice preparations were analysed to

obtain mean values for burst frequency. ampiitude. and duration as foiiows:

Mean bursr frequency was measured as the inverse of the mean t h e (seconds) taken for a

siice to complete 20 respiratory bursts (Hz).

Mean burst amplirude was measured as the mean height of the peak of the integrated

signal for 20 bursts.

Mean bursr hration was measured as the mean duration of 20 bursts.

2- 3. 2) Statisticu f Ana &sis

One-way repeated measures ahalysis of variance (ANOVA) tests of significance were

used to test (Sigmastat 1 .O. Jandel Corp.) whether the dserences in respiratory burst fiequency.

amplitude and duration of the slice at diffierent pH values were statistically significant. Because

of ditferences in recording characteristics between slices comparisons were only made within the

sarne stice preparations. In cases where natisticaily sigruficant differences were found. the

S tudent Neumann-Keuls test was irnplemented for comparisons.

2.4) Cross-Correiution

I used cross-correlation (Kirkwood. 1979) to detect short t h e scale synchronisation in

the respiratory buras recorded fiom lefi and right phrenic nerve rootlets (Cl-C5). For

cornputhg cross-correlation histograms. nerve action potentials were irst amplitude gated (Bak

Electronics Inc.). Then. cross-correlation histograms were computed on-line and occasionally

f?om the digital tape recordings. I used both a hard-wired cross-correlator (Anderson and Duffin

1976) as well as a cornputer, discriminated pulses were input via an A/D i n t d c e (AT-MIO-

16XE- 10. National Instruments). where specially written software (National Instruments.

LabVI E W). simultaneously cross-correlated neurone activity to phrenic and hypoglossal

disc harpe.

Features observed in the cross-correlation and post-stimulus h iaogam were quantified

using the k-ratio (Sears and Stagg. 1 976). For peaks. the k-ratio was calculated by dividing the

peak bin count by the mean bin count: for troughs. the sum of the mean bin count and the

difEerence between the mean bin count and the nadir for the trough divided by the mean bin

count determined the k-ratio. The mean bin count was measured away fiom any features present

in the histogram. Peaks and troughs were tested for statistical significance at P < 0.0 1 level

(Graham and Du& 198 1 ): only features shown to be statistically significant are reported.

Features were dso described by the latency to the start. and the half-amplitude width of the

feature: values are eqressed as mean I standard deviation.

2.5) Acetazolamide

To apply acetazolarnide (Sigma chernicd company) to the transverse medullary slice

preparation I used two separate reservoirs containing 500 ml aCSF and bubbled with carkgen

gas to a pH of 7-42 (See Figure 2.5 on the following page).

The first reservoir contained no acetazolamide and thus acted as the control solution. A

kno wn arnount of acetazolamide (2 mM ACZ in the fist 3 preparations. 5 x I o4 M AC2 U1 the

second 4 preparations. and I rnM ACZ in the third 6 preparations) was added to the second

reservoir. It is important to note that KCl was applied to both reservoirs to increase its

concentration to 8 mM. as detailed in section 2.2.3.

The 2 rnM and 5 x 10" M solutions of acetazolamide were prepared by mixing the

acetazolamide directly with the aCSF and dissolved by n k g vigorously for 20 minutes using a

magnetic stirrer and a stir bar. However. in the case of the 1 mM AC2 solution to enhance its

solubility it wa dissolved in DMSO (dimethyl sulfoxide) pnor to its administration to the aCSF

(0.1 1 1 1 gram of acetazolamide in 1 ml of DMSO added to a reservoir containing 500 ml of

aCSF. provided a bathing solution with a 1 mM concentration of acetazolamide). The solution

was then stirred vigorously with a rnagnetic stirrer and stir bar for 70 minutes prior to use in

order to evenly distribute the acetazolamide. In order to maintah consistency between the

control solution and the solution containing the acetazolamide. 1 ml of DMSO was also added to

the control reservoir containing 500 ml aCSF.

Three-way valves were used to direct the flow into the recording chamber fkom either

of the two reservoirs so that I could quickly alter the bathing medium of the slice between the

control aCSF and the acetazolamide containhg aCSF solution. Nevertheless. d e r any switch

the slice was leA to equilibrate for a period of 20 minutes in order to ailow t h e for wash in or

washout of the acetazolamide and for binding or unbinding to the carbonic anhydnse present

within the siice.

Recording Setup for the BrainsternSpinal Cord and Transverse Medullary Slice Preparations

Recording Chamber ---- -- Ternperatwe Robe Note - - = direction of K S F flow

Figure 2.5 - The diagram above shows the recording setup for the transverse rnedullary slice preparation. The setup above was used for the application of acetazolamide. The same setup was used for the brainstem spinal cord preparation. the only difference being that only one aCSF reservoir was used.

2.6) BCECF-AM

2. 6. I ) hepararion

BCECF-AM ( 2 ' , 7 ' - b i s - ( 2 - c a r b o x y e t h y l ) - 5 - ( a n d - 6 ) - c a acetoxymethyl

ester) is a membrane permeable fluorescent dye that. once within a ceL has its AM group

cleaved off by intraceilular esterases to produce its highiy active form BCECF. To prepare the

dye for experimentation 50 pg of BCECF-AM dye was hs t dissolved in 50 pl of Dimethyl

Sulfoxide (DMSO) and then sonicated in an ultrasonic cleaner (Cole Palmer) for 3 minutes. Low

Light conditions were maintained in al1 steps involving the dye in order to decrease the effects of

photobleaching. The resulting solution was termed the ' BCECF-.-1 M sruck solution ' and

provided a I pg/pl concentration of the dye.

To prepare the cleaved dye 1 followed the directions provided by its manufacturer

(Molecular Probes Inc. product information sheet on acetoxymethyl (AM) asters and diacetates).

I dissolved 50 pg of BCECF-AM dye in 50 pl of DMSO. and subsequently added 50 pl of

methanol. Following this. 1 added 25 pl of 2 M KOH (this is the active reagent involved in

cleavage of the AM group). 1 sonicated the vial containing the solution for 3 minutes. and the

via1 was lefi to sit for 60 minutes for cleavage to take place. At the end of 60 minutes 1 adjusted

the pH in the vial back to approximately 7.00 by adding 25 pl of 2 M HCI. Care was taken to

prepare the entire solution under low Eght conditions in order to minimize the effects of

photobleaching on the dye. The resulting solution was tenned cleaved BCECF-.4 IV sfock

soluhm and had a volume of 150 pl and a BCECF concentration of l pg/3pl.

BCECF solutions were prepared for fluorescence measurements as foiiows. First

pluronic acid stock solution was prepared by dissolving 200 mg of pluronic acid in 1 ml of

DMSO. The pluronic acid stock solution acts as a detergent and thus aids in distnuting the

BCECF-AM dye evenly throughout the aCSF. Then, in a 250 ml beaker. 200 ml of aCSF at a

temperature of 25 OC was bubbled with carbogen gas (95% O?. 5% CO2). carbon dioxide. and

oxygen to obtain a desired pH. 300 pl of this aCSF was placed in a clear plastic via1 with a

volume capacity of 500 pl. dong with 1 pl of pluronic acid stock solution and 8 pl of the

BCECF-AM stock solution. Since the concentration of dye in the cleaved BCECF-AM stock

solution was one third that of the BCECF-AM aock solution 24 pl. as opposed to 8 pl of the

cleaved BCECF-AM stock solution was added to the viai containhg 300 pi aCSF and 1p1 of

piuronic acid stock solution. The solution in the vial was then mixed using a 2-20 pl

micropipettor (Wheaton Socorex) and placed under the fluorescence microscope and secured

with Plastik putty (Platignum inc.). Care was taken to quickly transfer the aCSF. pluronic acid,

and BCECF-AM stock solution into the vial (4 0 seconds) and capture an image immediately in

order to assure that the pH of the aCSF was rnaintained at the desired value.

2- 6- 2) Fluorescence Image Anuiysis

1 observed the fluorescence emission of the dye using a fluorescence microscope

(Olympus BXSO Wi fluorescent microscope with a Wi BA 530 nm ernission/excitation filter). and

too k pictures with a high performance CCD carnera (Sensicam) opera~ed by cornputer so ttware

(.&on Irnaging Workbench 2.2. Axon Instruments Inc.). To maintain consistency between the

vials. the microscope was focused just beneath the surface of the solution in the %st via1 viewed

and was not changed over the course of viewing the subsequent viak. As weil. the same

exposure t h e was maintained for each vial (The exposure was set to a value at which ali of the

pixels in each region of interest were w i t h the scale of measurement intensity).

1 used the histograms provided in Avon Irnaging Workbench Eorn each image captured

to provide a value for the emitred fluorescence of BCECF-AM dye. These provided an 8-bit

value of intensity (0-255) for each pixel in a region of interest. From the histogram 1 sumrned

the nurnber of pixels at that intensity level multip lied by their intensity to give an overall value of

ernitted fluorescence for the image that 1 temedfluorescence emission.

2- 6-3) BCECF Control Tesling

To determine the sensitivity of the cleaved and un-cleaved BCECF-AM dye to changes in

pH 1 measured the fluorescence emission at varying pH values (8.10. 7.42. 7.00. and 6.70). It

was important to ver% that the non-cleaved version of the dye did emit fluorescent light upon

excitation in order to determine whether the fluorescence emission of dye residing in the

extracellular space of the BCECF-AM labelled transverse medullary slice was sensitive to pH

variation.

To determine the effects of photobleaching on BCECF-AM. 1 measured the fluorescence

emission of a via1 containing 300 pl of aCSF (25 OC. pH = 8.00). 1 pl of pluronic acid stock

solution and 8 FI of the cleaved BCECF-AM stock solution each minute for 50 minutes. An

exponential regession iine was fitted to the data (Microsof Excel) to obtain a value for the

decay in fluorescence emission using an order and senes of 2.

To v e m the concentration ofcleaved BCECF-AM needed to provide an optimal value

of fluorescence emission (maximal fluorescence emission with minimal occurrence of self-

quenching). I measurinp the fluorescence emission of several vials. each containing 300 pl of

aCSF at a pH of 7.42 and a temperature of 25 OC. 1 pl of the pluronic acid stock solutioa and a

varying volume of the cleaved BCECF-AM stock solution ( 2 pl. 4 pl. 8 pl. 16 pi. and 32 pl).

These values encompassed the concentration of 8 pVpg recomrnended by the manufacturer

(Molecular Probes Inc.) for optimal fluorescence emission. A gaph of the tluorescence

emission venus the concentration of cleaved BCECF-AM was plotted for each via1 (Microsoti

ExceI).

2.6.4) BCECF-A M L abelled Transverse Medultary Stice Ekperiments

In one experiment 1 measured the tluorescence response of BCECF-AM to pH in the

transverse medullary slice preparation by recording the tluorescence emission at several pH

values (7.42. 8.10. 7.42. 7.00.6.70. 7.00. 7.42. and 8.10 in that order). A 500 prn thick

transverse rnedullary slice from a single 3 day old Sprague-Dawley rat was prepared and on

removal fiom the vibratome chamber was placed in a 500 pl plastic via1 containing 300 pi of

aCSF bubbled to a pH of 7.42. 1 pl of the pluronic acid stock solution. and 8 pl of the BCECF-

AM stock solution. and lefi to sit for 10 minutes in low light conditions before use. 1 maintained

the temperature of the recording charnber at 25.0" C. and ailowed a five minutes equilibration

period between each pH variation. 1 ploned the variation of fluorescence emission with pH on a

vaph (Microsoft Excel) to obtain a tluorescence ernission venus pH curve for the BCECF-AM z

labelled transverse medullary slice preparation. 1 did not record fiom the hypoglossal rootlets to

determine if the respiratory rhythm generator was operational. because my only goal in these

experiments was to demonstrate the fluorescence emission response of the dye-labelled slice:

tluorescence emission depends only on intracellular esterase activity for AM cleavage. An

rxponential regression line was fit to the tluorescence emission venus time gaph for the above

-35-

data (Microsoft Excel) to obtain a value for the decay in fluorescence emission with the

regression set to an order and series of 2.

1.6.5) CC2 Diffusion Pipette

1 tested the feasibility of using a CO2 di ffision pipette to probe the existence of focal sites

of central chemoreception withiri the transverse medullary slice preparation. labelled wirh

BCECF in nine experiments. With the diffusion pipette in place. a BCECF-AM labelled

transverse medullary slice was placed into the recording chamber. and the di fision pipette was

insened into the tissue of the slice in order to focally increase the concentration of COz. Images

of the slice were taken to visualize focal changes in pH.

The COz diffusion pipette was made from a glass suction microelectrode with a tip

diameter of 50 um and was c o ~ e c t e d to a preamplitier headstage micropipette holder with a

side port. and mounted on a micromanipulator to allow precise positioning as descnbed in

section 2. CO2 rich aCSF bubbled to a pH of 6.00 was circulated through the microelectrode in a

unique manner as follows (see figure 2.6.5 on the following page). A 50 ml syringe was tilled

with aCSF at a pH of 6.00 and placed ont0 an injector pump (World Precision instruments Inc).

The syinge outflow was connected to the inside of the microelectrode tip through the headstage

holder side port via P-50 Intrarnedic plastic tubing (Becton Dickinson and Company). The

syringe pump injected the aCSF at 1-1 0 mI/min so that the aCSF flowed into the microelectrode

tip. with the excess draining out through the side port of the headstage micropipette holder.

Fluorescently Labelled Transverse Medullary Slice & Diffusion Pipette

Co2 Rich ACSF

- s~nnge

Do rsa 1

Figure 2.6.5 - The diagram above shows the difision pipette setup implemented in my experiments.

CHAPTER 3

RESULTS

3.1) General Results

These statements btiefly surnmarize my main findings with respect to the hypotheses

proposed in the introduction: details for each project follow.

1 ) 1 found a significant central peak when cross-correlating the activities recordcd tiom

ipsilateral phrenic nerve rootlets. indicating that they receive excitation from a common source.

However. in experiments cross-correlating the respiratory bursting recorded from leti and nght

contralateral phrenic nerve rootlets of the neonatal rat brainstem spinal cord preparation. I found

no central peaks: indicating that left and right phrenic motoneurone pools do not receive a

common activation.

2 I found that significant increases in the burst frequency. recorded from hypoglossal

nerves of the neonatal transverse medullary slice preparation. occurred upon providing a step

increase in COz tiom 0% to 10%. and a step decrease in CO2 from 10% to 0% to Vary the pH of

the bathing solution of the slice preparation.

3) The application of acetazolamide at a concentration of either 2 mM or 5 x 1 0 ~ M to the

neonatal rat transverse medullary slice preparation resulted in covering of the slice with

acetazolamide crystals. To prevent this crystal formation I dissolved acetazolamide in DMSO in

subsequent experiments. 1 subsequently found that application of 1 rnM acetazolamide dissolved

in DMSO to the transverse medullary slice preparation produced a significant change in

hypoglossal nerve burst duration. However. I found no sipificant changes in the sensitivity of

the slice to [H']/C02. with regards to burst frequency. amplitude or duration resulted fiom such

an application of acetazolarnide.

4) In quality control tests of the pH-sensitive fluorescent dye BCECF-AM. 1 determined that

both the cleaved and non-cleaved forms of BCECF-AM dye were sensitive to changes in pH.

that the fluorescence emission decay constant &,) of cleaved BCECF-AM is 0.01 02 min-'.

and that the concentration of cleaved BCECF-AM dye for optimal fluorescence emission is 8

pgi300ml. However. experiments involving staining of the transverse medullary slice

preparation with BCECF-AM dye demonstrated a faster rate of decay in tluorescence emission

than cleaved BCECF-AM dye alone. and no response in tluorescence emission to pH variation

was found.

5 ) While application of BCECF-AM dye to the transverse medullary slice preparation did

exhibit areas of differential tluorescence emission. nevenheless. the CO2 diffusion pipette failed

to produce a visually identitiable focal change in the tluorescence emission of the BCECF-AM

labeled transverse medullary slice preparation.

3.2) Cross-Correlation Experiments

3*2* I ) Left and Right Phrenic Nerves

I cross-correlated the rhythmic respiratory output tiom left and ri& phrenic nerve

rootlets in ~ neonatal rat brainstem-spinal cord preparations. The temperature of the bathing solutions varied from 25.0 to 26.0 OC. with a mean * SE of 25.3 * 0.01 2 OC. the pH varied fiom 7.34 to 7.42. with a mean * SE of 7.40 5 0.1 6. and the age of the rats varied tiom 2 to 5 days oid. with a mean L- SE of 3 m0.45 days old between preparations.

-3 9-

As figure 3.2.1 demonstrates the cross-correlation histopms did not display any central

peaks. The cross-cone1oga.m~ for al1 experiments are shown in appendix 1.1.

Cross-Correlation Histogram of Left and Rlght Contralateal Phmnic Nerve Rootlets

Figure 3.2.1 - Above is shown the cross-correlation his topm oftwo contralateral phrenic nerve rootlets tiom the brainstem-spinal cord preparation of a nvo day old nt. The gaph shows no indication of a central peak.

3.2.2) Ipsiloerai Ph renie Nerve Rootlets

1 cross-correlated the rhythmic respiratory output h m ipsilateral phrenic nerve rootlets

(C-4 to C-4) in one brainstem-spinal cord preparation. The temperature and pH were maintained

at 16.0 O C and 7.40 respectively throughout the experiment. and the rat used was 3 days old. nie

cross-correlation histopun for this experiment is shown in figure 3.2.2 below and in appendix

1.1. It displays a centrai peak. The mean bin count and the peak value of the histogram were

2000 and 25 10. respectively. Thus. the k-ratio was 1.255. and proved to be statistically

230Q --

\ l em Bin C'wnt = ?

Figure 3.2.2 - Above is show a cross-correlation histo-m o f ipsilateral phrenic nerve rootlets from the brainstem- spinal cord preparation of a three day old nt. The histograrn shows cvidence of a central peak.

3.3) CO2 Sensitivity in the Neonatai Rat Transverse Medullary Slice

1 probed & transverse meduliary slice preparations for their sensitivity to C02/[H-1. The

temperature of the slice bathing solutions varied from 25.0" C to 27.7' C with a mean * SE of

25.8 * 0.43' C. the age of the rats used varied from 2 to 8 days old with a mean * SE of 4 B0.93 days old. and the thickness of the slices varied from 700 to 1000 microns with a mean * SE of 900 -54 microns between preparations. The mean burst fiequency. amplitude. and duration

were cdculated (methods section 2.3.1 ) for each pH/% CO? value.

Mean Burst Fmquency vs. % CO, pH in the Tramverse Meduihry Slke Preparation

Figure 3.3.1 - Mean burst frequency = SE of six transverse medullary slice preparations vs. the pHiO'oCOL of their bathing solutions. Significant differences between coiumns are indicated by an asterisk. The n w data for the graph appears in appendix 2.1.

The burst tiequency vs. pH/% COz p p h (Figure 3.3.1 above) demonstrates that the

mean burst frequency of the slice preparations increases in response to a decrease in pH (as a

result of an increase in % CO2) of the bathing solution. A one-way repeated measures analysis

of variance ( ANOVA) test performed on the burst fiequency vs. pH data (appendix 2.1.1 )

indicated that significant differences in the burjt fiequencies of the preparations existed between

the initial pH of 7.42 and pH 7.00 (p=0.0046. power=0.9678). and between pH 7.00 and the final

pH of 7.42 ( p=O.O 1 86. powe~0.7528). The percentage increase in the frequency of bursting

tom pH 7.42 to 7.00 was 54.7% (mean burst frequency increased From 0.1 1 7 Hz to 0.18 1 Hz).

Conversely. the percentage decrease in the fiequency of bursting from pH 7.00 to 7.42 was

54.7% (mean burst frequency *SE decreased fiom 0.18 1 + 0.034 to 0.1 17 * 0.0 13 Hz). No

other statistically significant differences between burst tiequencies were found. Neither burst

amplitude (Figure 3.3.2) nor burst duration (Figure 3.3.3) changed significantly with pH/ %CO2.

Figure 3.3.2 - Mean burst amplitude = SE of six transverse meduIIary slice preparations vs. the pHl'O/oCO2 of their bathing solutions. No significant differences were found between columns. The raw data for the :-ph appears in appendix 2.1.

Figure 3.3.3 - Mean burst duration * SE of six transverse medufla- slice preparations vcrsus the pH/?/oC02 of their bathing solutions. No signrficant Merences were found beween columns. The raw data for the graph appears in appendk 2.1.

One-way repeated measures ANOVA tests indicated no significant differences (p>0.05)

between the burst amplitudes (append 2.1 2) or the burst durations (appendk 2.1 -3) when the

%C02/pH of the bathhg aCSF was varied.

3.4) Acetuzoiamide

The concentration of acetazolamide necessary to block al1 carbonic anhydrase enzyme

activity was unknown nor were any indirect effects on tissues of the transverse meduiiary slice.

1 first used 2m.M. a high concentration- thinking to ensure that aii carbonic anhydrase enzyme

activity would be blocked and tested to see if the response to COz was blocked. It was not.

However. the siice viabiiity was adverseiy aected. and so 1 used a lower concentration of 5 x

1 o4 M and tested to see if the response to CO2 was reduced. It was not. and slice viability was

still not good. I f o n d that the application of acetazolamide c a w d crystals to form on the

surface of the slices and thought that they rnight account for the reduced viability. To prevent

crystals forrning in subsequent applications of acetazo lamide I used dimethyl sulfo xide ( DMSO )

to assist in the solubilization of the acetazolamide. 1 then tested the slice response to COz with

and without L rnM acetazolamide present and the same concentration of DMSO (28.16 mM)

throughout. The detailed tndings for this set of experiments are provided below.

S. 4* 1) 2mM Acetnzolamide

1 probed fifieen transverse meduiiary slice preparations for changes in burst i-equency

and burst amplitude responses to variations in C02/[HT] of the bathing solution in response to the

application of 2 mM acetazolamide. Only three of these Meen slices. however. provided a

rhythmic respiratory output throughout the entire experiment. In these three slice preparations

the temperature of the bathing solutions vared fiom 26.2O C to 26.3 O C with a mean * SE of16.2 i 0.03"C. the slice thickness varied fiom 800 to 1000 microns. with a mean i SE of 900 * 58 microns. and the age of the rats varied fiom 3 to 8 days old with a mean * SE of5 1.5 days old.

The graph of bunt fiequency vs. pH is shown in figure 3 .4.1(a) below.

Bumt Fmquency M. pHACCO2 Before, During and Amr Application of 2 mM Acebizohmide

0% CO2

pH 7 42

Pm Aceiazdamide Contra

Figure 3.4.l(a) - Burst frequency = SE of three transverse rnedullary slice preparations in response to variations in pHI0'oCO2 of their bathin2 solutions during application of 2 mM acetazolamide. SipifIcant diff'rences between conditions are indicated by an asterisk. See appendix 3 . i for the MW data.

There was a 74.1 % rise in the mean burst frequency of the slice preparations upon

decreasing the pH of the bathing solution containine acetazolarnide tiom 7.43 to 7.00. A one-

way repeated measures ANOVA statistical analysis of the data (see appendix 3.1.1 ) indicated

that a statistically significant difference exists only between the 0% COz acetazolamide treatment

and the 1 0% CO2 acetazolarnide treatrnent (p=0.026. power=O. 835).

The _maph of burst amplitude vs. pH is shown in figure 3.4.l(b) below.

Burst Amplitude M. pHR6 CO2 Before, Durfng and A b r Application of 2 mM Acetmabmide

Figure

Pre Acetazdamide C m M

3.4.1(b) - Bunt amplitude = SE of three transverse medullary slice preparations in response to variations in pH/O/6CO2 of their bathing solutions durin application of 7 m M acetazolamide. No sigificant differences between conditions exist. See appendix 3. I for the n w data.

The results of the one way repeated mesures ANOVA analysis performed on the mean

amplitude of bursting data (appendix 3.1.2) did not provide evidence of sta~istically significant

differences in the burst amplitude of the slice preparations at varying pW0hC02 values.

3* 4.2) 5x1 06 M Acefazolamide

1 probed nine transverse medullary slice preparations for changes in burst frequency.

amplitude. and duration in response to variations in pH/%C02 of the bathing solution pior to.

during. and following the application of 5x1 O* M acetazolamide. Only four provided a rhythmic