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The role of the parabrachial/Kolliker Fuse respiratory complex in the control of respiration Boon, Joyce A. 2004

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THE R O L E OF THE P A R A B R A C H I A L / K O L L I K E R FUSE RESPIRATORY C O M P L E X IN THE CONTROL OF RESPIRATION by  JOYCE A. BOON B.Sc. Honors The University of Alberta, 1967 M.Sc. The University of British Columbia, 1970  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES ZOOLOGY  THE UNIVERSITY OF BRITISH C O L U M B I A D E C E M B E R 2004  ©Joyce A . Boon, 2004  Abstract: M y goal was to explore the role of the parabrachial/Kolliker Fuse region (PBrKF) of the pons in the production of "state-related" changes in breathing in rats. I hypothesized that the effects of changes in cortical activation state on breathing and respiratory sensitivity are relayed from the pontine reticular formation to the respiratory centres of the medulla via the PBrKF. I found that urethane anaesthetized Sprague Dawley rats spontaneously cycled between a cortically desynchronized state (State I) and a cortically synchronized state (State III), which were very similar to awake and slow wave sleep (SWS) states in unanaesthetized animals, based on E E G criteria. Urethane produced no significant respiratory depression or reduction in sensitivity to hypoxia or hypercapnia. However, breathing frequency (TR), tidal volume ( V ) and total ventilation ( V TOT) all increased on T  cortical activation, and changes in the relative sensitivity to hypoxia and hypercapnia with changes in state were similar to those seen in unanaesthetized rats. This indicated that the urethane model of sleep and wakefulness could be used to investigate the effects of cortical activation state on respiration. Since NMDA-type glutamate receptor mediated processes in the P B r K F are known to be important in respiratory control, I examined the role of the PBrKF as a relay site for state effects on respiration by blocking neurons with NMDA-type glutamate receptors with MK-801. I first used systemic blockade and found that it altered resting ventilation and modified the hypoxic, but not the hypercapnic ventilatory response, as has been reported in unanaesthetized animals.  Microinjection of MK-801 into the PBrKF confirmed that the 'wakefulness' stimulus for breathing involved glutamate activation of N M D A r on PBrKF neurons, but these neurons were not involved in the response to either continuous or intermittent hypoxia, nor did they change chemosensitivity on cortical activation. However, they indirectly modulated the H V R by altering cortical activation state as animals cycled between State I during hypoxia, and State III post hypoxia. N M D A r in the PBrKF also functioned to return tidal volume to normal following hypoxia and in so doing, prevented the development of long-term facilitation of breathing.  iv  Table o f Contents Abstract:  ii  Table of Contents  iv  List of Tables:  viii  List of Figures  ix  List of Abbreviations:  xiii  Acknowledgements:  xv  Chapter 1  1  General Introduction  1  Introduction 2 Central Organization of Neurons for Respiratory Rhythm and Pattern Generation 3 Respiratory Rhythm Generation 4 Respiratory Pattern Generation 7 Interconnections between Respiratory Related Neurons in the Brainstem 13 The Role of the Respiratory Related Neurons in the PBrKF 14 The Role of Glutamate 17 Location of the NMDA-type Glutamate Receptors 18 Blockade of N M D A Receptors 19 Systemic Injections of Glutamate-receptor Antagonists 19 P B r K F Injections of Glutamate-receptor Antagonists 21 Control of Inspiratory Timing: the Inspiratory Off-Switch (IOS) 21 Effects of Cortical Activation State on Breathing Pattern 22 Anaesthesia, Breathing and the Urethane-Anaesthesia Model of Sleep-Wake 24 Role of the Pons and NMDA-type Glutamate Receptor Mediated Processes in Hypoxic Ventilatory Response 27 Hypotheses 32 References: 34 Chapter 2 2.1. Introduction 2.2. Methods 2.2.1. Animal Care 2.2.2. Surgical Preparation 2.2.3. Experimental Protocol 2.2.4. Data Analysis 2.3. Results 2.3.1. E E G and Respiratory Traces 2.3.2. Effects of cortical activation 2.3.3. Effects of hypoxia and hypercapnia 2.4. Discussion  46 47 49 49 50 50 52 53 53 56 61 66  2.4.1. State Distribution 2.4.2. Effects of changes in state on breathing 2.4.3. Effects of hypoxia/hypercapnia on breathing 2.5. Conclusions 2.6. References:  66 68 69 71 72  Chapter 3  75  3.1. Introduction 76 3.2. Methods 79 3.2.1. Animal Care..... 79 3.2.2. Experimental Protocol 80 3.2.2.1. Surgical Preparation 80 3.2.2.2. Recordings in Animals Breathing Air, 10%O in N and 5% C 0 81 3.2.2.3. Injection of Saline and MK-801 82 3.2.3. Data Analysis -. 82 3.3. Results 84 3.3.1. Respiratory and E E G Traces for Rats Breathing Air 84 3.3.2. Time in State 87 3.3.3. Effects of MK-801 on the E E G 90 3.3.4. Effects of MK-801 on Breathing Pattern 92 3.3.5. Effects of MK-801 on Cortical Activation 96 3.3.6. Effects of MK-801 on the Hypoxic Ventilatory Response 99 3.3.7. The Effect of MK-801 on the Time Domains of the H V R 101 3.3.8. The Effects of MK-801 on the Hypercapnic Ventilatory Response 104 3.3.9. The response of Heart Rate to State Changes and MK-801 106 3.4. Discussion 107 3.4.1. Role of NMDA-type glutamate receptors in determining state 107 3.4.2. Role of NMDA-type glutamate receptors in determining the effects of changes in state on breathing 109 3.4.3. The effects of changes in state on the H V R 110 3.4.4. Role of NMDA-type glutamate receptors in determining the hypoxic ventilatory response Ill 3.4.5. Role of NMDA-type glutamate receptors in determining the hypercapnic ventilatory response and the effects of changes in state on the hypercapnic ventilatory response 113 3.5. Conclusions 114 3.6. References 116 2  2  Chapter 4 4.1. Introduction: 4.2. Methods 4.2.1. Animal Care 4.2.2. Experimental Protocol: 4.2.2.1. Surgical preparation 4.2.2.2. Monitoring breathing pattern in air, hypoxia and hypercapnia  2  120 121 124 124 124 124 125  vi  4.2.2.3. Injection of Saline and MK-801 126 4.2.3. Data Analysis 127 4.2.4. Statistical Analysis: 128 4.3. Results 129 4.3.1. Respiratory and E E G traces 129 4.3.2. Time in State 133 4.3.3. Placement of Injections 135 4.3.4. Effect of MK-801 in rats breathing air 137 4.3.5. Effect of MK-801 on changes in breathing with changes in state 137 4.3.6. Effect of MK-801 in hypoxic rats 141 4.3.7. Effect of MK-801 on the timing of the hypoxic ventilatory response 143 4.3.8. Effect of MK-801 in hypercapnic rats 145 4.4. Discussion: 147 4.4.1. Role of N M D A r in Establishing State 147 4.4.2. Role of N M D A r in the PBrKF in producing changes in breathing with changes in state 148 4.4.3. Role of N M D A r in the PBrKF under resting conditions 149 4.4.4. Role of N M D A r in the PBrKF in the hypoxic ventilatory response and changes in hypoxic sensitivity with changes in state 150 4.4.5. Role of N M D A r in the PBrKF in the hypercapnic ventilatory response and changes in hypercapnic sensitivity with changes in state 152 4.5. Conclusion 153 4.6. References 155 Chapter 5  158  5.1. Introduction: 159 5.2. Methods 162 5.2.1. Animal Care: 162 5.2.2. Experimental Protocol: 162 5.2.2.1. Surgical preparation 162 5.2.2.2. Injection of Saline and MK-801 163 5.2.2.3. Monitoring breathing pattern in air and hypoxia 164 5.2.3. Data Analysis 165 5.2.4. Statistical Analysis: 166 5.3. Results 166 5.3.1. The effect of intermittent hypoxia on cortical activation state and breathing pattern in control rats 166 5.3.2. The effect of intermittent hypoxia on cortical activation state and breathing in MK-801 treated rats 169 5.3.3. The placement of MK-801 injections 169 5.3.4. The time domains of the exposure to intermittent hypoxia with air intervals. ; 173 5.3.5. A n examination of the possibility of long-term facilitation of breathing.... 175 5.3.6. The effect of MK-801 injection on the response to intermittent hypoxia.... 177 5.3.7. A n analysis of the breathing pattern at critical time points during the intermittent hypoxia exposure 180  vii  5.4. Discussion: 183 5.4.1. The Effects of Intermittent Hypoxia on Cortical Activation State 183 5.4.2. Effects of MK-801 on resting breathing pattern prior to hypoxic exposure. 184 5.4.3. Time domains of the H V R before and after blockade of N M D A r in the PBrKF : 185 5.4.3.1. The Acute Response (AR) 185 5.4.3.2. Short Term Potentiation (STP) 186 5.4.3.3. Short Term Depression (STD) 186 5.4.3.4. Progressive Augmentation (PA) 187 5.4.3.5. Long Term Facilitation (LTF) 187 5.5. Conclusions: 189 5.6 References: 190 Chapter 6 6.0 Introduction •. 6.1 Preliminary considerations: Urethane anaesthesia 6.2 The Role of the PBrKF as a relay for State-related information 6.3 The Role of the PBrKF in Chemosensitivity 6.4 The Role of the PBrKF in stabilizing the breathing pattern after hypoxia 6.5 The Role of the PBrKF in the Generation of Respiratory Rhythm 6.6. Summary 6.6 References  193 194 194 198 200 201 202 203 205  Vlll  L i s t o f Tables: Chapter 2 Table 2.1 Summary of times of Inspiration and Expiration in States I and III in rats breathing air, 10% 0 or 5% C 0 2  58  2  Table 2.2 A comparison of the absolute values of fR, V and V TOT from urethaneT  anaesthetized rats with those from unanaesthetized rats in a plethysmograph  60  Chapter 3 Table 3.1 Inspiratory and expiratory times before and after MK-801 injection  95  Table 3.2 Heart Rate measured before and after the administration of MK-801  106  Chapter 4 Table 4.1 Mean values ± S E M for Frequency of respiration (f ), Tidal volume (V ), R  T  Total ventilation ( V TOT), Inspiratory time (TO and Expiratory time (T ) for Sprague E  Dawley rats breathing air, 10% 0 in N or 5% C 0 in air 2  2  2  132  Chapter 5 Table 5.1 Mean values ± S E M for f , V and V TOT for Sprague Dawley rats before and R  T  after injection of MK-801 into the PBrKF  178  IX  L i s t o f Figures Chapter 1 Fig. 1.1 Pontine Respiratory Areas and V R G  9  Fig. 1.2 Schematic of the brain stem with the location of pontine and medullary respiratory areas superimposed  11  Fig. 1.3 Time domains of the hypoxic ventilatory response  29  Fig. 1.4 Long term facilitation  31  Chapter 2 Fig. 2.1 Recordings of the E E G and integrated differential pressure signal (respiration) during State I and State III in a urethane anaesthetized Sprague Dawley rat breathing air, 10% 0 in nitrogen and 5% C 0 in air 2  54  2  Fig. 2.2 Spectral Analysis of the E E G patterns in States I and III in urethane anaesthetized rats using a Fast Fourier Transform  55  Fig. 2.3 Distribution of time spent in arousal States I and III as a function of respiratory gas in urethane-anaesthetized Sprague-Dawley rats....  57  Fig. 2.4 The effect of cortical activation (the change from State III to State I) on frequency, tidal volume and total ventilation in urethane anaesthetized Sprague Dawley rats breathing air, 10% 0 in nitrogen and 5% C 0 in air 2  58  2  Fig. 2.5 Respiratory responses in urethane-anaesthetized rats breathing 10% 0 and 5% 2  C 0 expressed as the % increase in frequency, tidal volume and total ventilation from air, 2  in both State I and State III  63  Fig. 2.6 A n analysis of the breathing pattern in hypoxia in States I, II and III  64  Fig. 2.7 A Pointe Carre plot  65  Chapter 3  Fig. 3.1 Recordings of the E E G and the differential pressure signal (respiration) during States I, II and III in urethane anaesthetized rats breathing air before injection of MK-801 and in States m and IV after injection of MK-801  86  Fig. 3. 2 Distribution of time spent in each arousal state  89  Fig. 3.3 Spectral analysis of the E E G patterns in urethane anaesthetized rats  91  Fig. 3. 4 The pattern of breathing and the E E G trace for one rat A) breathing 12% oxygen in nitrogen, B) breathing air after the injection of MK-801, and C) breathing 12% oxygen in nitrogen after injection of MK-801  93  Fig. 3.5 The effects of MK-801 on breathing pattern of urethane anaesthetized rats breathing air in States I and III  94  Fig. 3. 6 The effects of cortical activation (the change from State III to State I), on breathing pattern in rats breathing air, 12% 0 in nitrogen and 5% CO2 in air 2  98  Fig. 3.7 The effect of 12% oxygen in nitrogen on breathing pattern in urethane anaesthetized rats in both States I and HI before and after the injection of MK-801  100  Fig. 3.8 Changes in ventilation during exposure to hypoxia and during the post-hypoxic period before and after injection of MK-801  103  Fig. 3.9 The effect on breathing of exposure to 5% CO2 in air before and after MK-801 injection  .•  105  XI  Chapter 4  Fig. 4.1 Recordings of the E E G and the differential pressure signal (respiration) from one rat before and after the injection of MK-801 into the PBrKF  131  Fig. 4.2 The percent time spent in States I, II and III in rats breathing air, 10% oxygen in nitrogen and 5% CO2 in air before and after injection of MK-801 into the PBrKF region. 134 Fig. 4.3 The placement of injections shown superimposed on schematic diagrams of serial sections of the brain (Paxinos and Watson, 1986)  136  Fig. 4.4 The effects of MK-801 injected into the PBrKF are shown for rats breathing air in State I and State HI  139  Fig. 4.5 The effects of cortical activation, the change from State III to State I, shown for rats breathing air, 10% oxygen in nitrogen and 5% CO2 in air both before and after MK801 injection into the PBrKF  140  Fig. 4.6 The effect of breathing 10% oxygen in nitrogen on breathing pattern in urethane anaesthetized rats in both States I and IU before and after the injection of MK-801into the PBrKF  142  Fig. 4. 7 The time course of the hypoxic ventilatory response shown with changes in breathing in the post-hypoxic period before and after injection of MK-801 into the PBrKF  144  Fig. 4.8 The effect of breathing 5% CO2 in air on breathing pattern in States I and m, before and after injection of MK-801 into the PBrKF region  146  Xll  Chapter 5  Fig. 5.1. Respiratory and E E G traces from a control urethane-anesthetized Sprague Dawley Rats  168  Fig. 5.2. Respiratory and E E G traces from a urethane-anesthetized Sprague Dawley rat after an injection of MK-801 into the PBrKF region of the pons  170  Fig. 5.3. The position of injections shown superimposed on serial sections from "The Rat Brain in Stereotaxic Coordinates" Paxinos and Watson, 1986  172  Fig. 5.4. The effects of intermittent hypoxia and the post-hypoxia periods of air on breathing in the control rats  174  Fig. 5.5. The percent change for the frequency of respiration, tidal volume, and total ventilation for one representative rat in the post-hypoxic period  176  Fig. 5.6. A comparison of the pattern of the hypoxic and post-hypoxic response in control and MK-801 treated rats  179  Fig. 5. 7. Values for f , V , V TOT at various times during the H V R R  T  181  Fig. 5.8. A comparison of the post-hypoxic tidal volume between control rats and rats treated with MK-801....!  182  xiii  List of Abbreviations: 5n - trigeminal nerve A M P A - a amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid AP5 - 2-amino-5-phosphonopentanoic acid AP7 - 2-amino-7-phosphonoheptanoic acid B C - Botzinger complex C N - cranial nerve C S N - carotid sinus nerve c V R G - caudal Ventral respiratory group dl pons - dorsolateral pons D R G - Dorsal respiratory group E E G - electroencephalogram E K G - electrocardiogram fR - frequency of respiration G A B A - gamma amino butyric acid H V D - hypoxic ventilatory decline H V R - hypoxic ventilatory response IOS - inspiratory off-switch ITR - intertrigeminal region i V R G - intermediate Ventral respiratory group Ka - kainate K F - Kolliker Fuse LcPBr - lateral crescent parabrachial L T F - long-term facilitation Mo5 - motor nucleus of C N 5 NAc - compact part of Nucleus ambiguus nACh receptors - nicotinic acetylcholine N M D A -N-methyl-d-aspartate N M D A r - NMDA-type glutamate receptor N M D A r 1 - N M D A receptor subunit 1 nonREM sleep - non- rapid eye movement sleep N P B L - Nucleus parabrachialis lateralis N P B M - Nucleus parabrachialis medialis N R E M - non- rapid eye movement sleep NTS - Nucleus Tractus Solitarius PbC - parabrachial complex PBC - pre-Botzinger complex PBr - parabrachial P B r K F - Parabrachial Kolliker Fuse pFRG -para-facial respiratory group PHFD - post-hypoxia frequency decline Post-I neurons - post-inspiratory neurons PPT - pedunculo pontile tegmentum Pre-I neurons - pre-inspiratory neurons PRF - pontine reticular formation  xiv  P R G - Pontine respiratory group PS - paradoxical sleep QA - quisqualate R E M sleep- rapid eye movement sleep R R G - Respiratory rhythm generator rTZ - retrotrapezoid r V R G - rostral Ventral respiratory group STD - short term depression STP - short term potentiation SWS - slow wave sleep T - expiratory time Tj - inspiratory time V - tidal volume V TOT - total ventilation V A H - ventilatory acclimatization to hypoxia vl pons - ventrolateral pons V R G -Ventral respiratory group E  T  XV  Acknowledgements:  The decision to embark on a Ph.D. at a later stage of life is not one that should be taken lightly. M y decision came as a result of changing life circumstances. Changes in family circumstances were accompanied by changes at Okanagan College, which became Okanagan University College. All of a sudden I found myself considered to be unqualified, by some people, for the work that I had been doing. The unfairness of this was very clear to me. Not completing a Ph.D. during my first grad school experience was not due to lack of ability, but because I was in a generation when the vast majority of professors at both U of A , where I did my honors B.Sc. in Biochemistry, and at U B C where I did my M . S c , were male. In fact the only women with Ph.D. degrees that I knew were research assistants, and I was not interested enough in doing research to want to spend my life like that. I had also discovered, through teaching student laboratories, that I really liked teaching and I seemed to have a knack for it.  M y decision to pursue a  teaching career was fortuitously accompanied by the birth of the college system in B.C. and positions were coming up all the time. So I took my M . S c , got married and embarked on a teaching career. I loved teaching at Okanagan College, and I enjoyed the challenge of keeping upto-date for my courses, and of learning all sorts of new things so that I can teach 'real' biology courses. When the changes at OUC started, I was involved with The Health Educators Group, a group of people who taught nursing students and other students in the allied health sciences. At a memorable meeting in Naramata I met B i l l Milsom. Over a few beers in the pub I told him about my frustrations with the situation at OUC and he told me that if I wanted to do a Ph.D. I could come and do it in his lab. I'm not sure he  xvi  would make the same offer again; it hasn't been easy for either of us to try to work together from a distance. Nevertheless, I am greatly indebted to him for staying the course with me, helping me through the many ups and downs of doing research for a Ph.D., and for giving me support and encouragement when I was unsure of how to or whether to proceed. There are other people who have been very instrumental in helping me complete this work. The OUC students who worked for me as research assistants during the summer not only helped with the research but also enriched my life by giving me the opportunity to get to know them and share in the success of their lives. I'd like to thank Dr. Janna Johnston Bentley, Dr. Jenna Toplak, Dr. Natasha Garnett, Susan Stewart, Frann Antignano, Margaret Harvey and Ulrieke Birner. I would like to thank my family for their support, understanding and unfailing belief that I could do this if I set my mind to it. Thank you to my husband Dennis, who encouraged me when I was down and was very generous with his time and money to help me; to my children, Kristen, Kyle and Adrienne, all of whom offered love and listened graciously when I complained; to my mother Olive who would have been so proud to see me get my Ph.D., but who unfortunately passed away in 2002; to my mother-in-law Ethel, who was always kind, caring, encouraging and understanding and who passed away in 2003; and to my second father, Harold Baldwin, who would have been as happy for me as if I were his own child, but who passed away in 1998. I would like to acknowledge the friendship and support of Mary Ellen Holland of the OUC English department. Not only did she help with figures and editing, but also phoned me every day during the last two months of the struggle to write this thesis to  xvii  offer words of wisdom and encouragement and to convince me that I really could do this and that I wasn't losing my mind. Thank you Mary Ellen. I would also like to thank Rosemary Cappell, who wrote the program that enabled me to analyze all 10,000 respiration files in about l/10 of the time it would have taken th  without her help; Bruce Campbell who gave generously of his time to help me finally figure out how to do the stats and how to interpret them; Bruce Mathieson who helped me with the brain sectioning; and Mary Forrest for doing her best to give me some workload relief when she could. I wish that I had had the chance to work in the lab at U B C more and to get to know the other graduate students better. However, I am grateful for the time I had with Mike Harris, Steve Reid, Beth Zimmer, Glenn Tattersall, Lisa Skinner and the other grad students who always welcomed me when I came to U B C . Crystal Brauner was a godsend when it came to doing final figures, and I appreciate her help in printing and distribution of the thesis. I would not suggest that anyone try to do a Ph.D. the way that I did. I feel that while I learned a lot about physiology and myself, I missed out on the best parts of grad studies by not being there to learn from and interact with the other students.  1  Chapter 1  General I n t r o d u c t i o n  The Role of the Parabrachial/Kolliker Fuse Respiratory  Complex of the Pons in the Control of Respiration  2  Introduction The goal of this research was to explore the role of NMDA-type glutamate receptor-mediated processes in the Parabrachial/Kolliker Fuse region (PBrKF) of the pons in the control of "state-related" changes in breathing in rats using the urethaneanaesthesia model of'sleep' and 'wakefulness' (Hunter and Milsom, 1998; Hunter et al., 1998). The first goal of the study was to establish that the urethane model was applicable to Sprague Dawley rats; these results are presented in Chapter 2. Then, since 1) glutamate has been shown to be the main excitatory neurotransmitter involved in the control of respiration (see Bianchi et al., 1995 for review), 2) NMDA-type glutamate receptor -mediated processes in the pons have been shown to be important in respiratory control, and 3) the pontine reticular formation has been shown to be involved in sleep state regulation, we wanted to characterize the response of urethane-anaesthetized rats to the blockade of NMDA-type glutamate receptor-mediated processes with MK-801 (dizocilpine maleate - an N M D A receptor antagonist) administered systemically (Chapter 3) and then injected directly into the parabrachial/Kolliker Fuse region (PBrKF) of the pons (Chapter 4). In the course of these experiments we considered the role of N M D A type glutamate receptor-mediated processes in the changes in breathing that occur on the transition between cortical activation states (Chapters 3, 4), and in the responses to hypoxia (delivered either continuously (Chapters 3, 4) or intermittently (Chapter 5)) and hypercapnia (Chapters 3, 4), as a function of state. The rationale for this series of studies follows.  3  Central Organization of Neurons for Respiratory Rhythm and Pattern Generation Breathing in mammals is generated by neurons in the central nervous system that function to set a basic rhythm of respiration. Other neurons modify this rhythm to produce a pattern of breathing that meets the needs of the animal for delivery of oxygen, and removal of carbon dioxide (Ramirez et al., 2002; Ballanyi et al. 1999; Richter et al., 1997; Bianchi et al. 1995, for review). The latter depends on many things, including levels of O2, and C02/pH; stretch receptor feedback from the lungs, intercostals and diaphragm; sleep/wake state, body temperature, locomotion, voluntary activity and emotions/stress (Feldman et al., 1990; Richter et al., 1997; Ballanyi et al. 1999). To what extent do the neurons that set the rhythm of respiration operate separately from the neurons that generate pattern? This is not a simple question since the organization and function of the basic rhythm generator or 'kernel' may be independent of the inputs from lung afferents, chemoreceptors, muscle afferents and forebrain structures (the reductionist hypothesis) (Feldman et al., 1990); or there may be a reorganization or transformation of this 'kernel' by the addition of inputs such as sleepwake state and the peripheral inputs that shape the pattern (the so-called transformational hypothesis) (Feldman et al., 1990). Such a transformation makes it difficult to determine which elements are essential components of the rhythm generator and which elements are only modulatory and may be part of a respiratory pattern generator. Such considerations are significant in determining the role of the parabrachial/Kolliker fuse respiratory complex of the pons in respiratory control.  4  Respiratory Rhythm Generation M a n y m o d e l s have been d e v e l o p e d to e x p l a i n the o r g a n i z a t i o n a n d c o m p o s i t i o n o f the respiratory r h y t h m generator. T h i s is perhaps not s u r p r i s i n g g i v e n that the various possible m o d e l s have been based o n experiments p e r f o r m e d o n a n i m a l s o f different ages, f r o m neonatal to adult, w i t h a variety o f and anaesthetized animals, a n d i n  in vivo  preparations, i n c l u d i n g unanaesthetized  in vitro preparations  such as the ' w o r k i n g heart-  brainstem preparation' (Paton, 1996), the isolated brainstem-spinal c o r d preparation (Suzue, T . , 1984) a n d the m e d u l l a r y slice preparation ( S m i t h et a l . , 1991). A m o n g s t the m a n y m o d e l s put f o r w a r d , one m o d e l is a m e d u l l a r y p a c e m a k e r model  ( S m i t h et a l . , 1991) another is a m e d u l l a r y n e t w o r k m o d e l ( R i c h t e r a n d S p y e r ,  2001, R a m i r e z et a l . , 1997), a third is a h y b r i d m o d e l , where a m e d u l l a r y p a c e m a k e r r e g i o n is e m b e d d e d w i t h i n a network o f neurons ( S m i t h et al.2000), a n d a fourth is a network m o d e l i n v o l v i n g a p o n t o m e d u l l a r y circuit (St. J o h n , 1998). T h e first m o d e l posits that there is a 'medullary pacemaker' that acts as the central respiratory r h y t h m generator ( C R G ) . T h e r e are two contenders for the site o f the m e d u l l a r y p a c e m a k e r . T h e first is a network o f pre- inspiratory neurons (Pre-I neurons that fire just before the onset o f inspiration) that are f o u n d ventrolateral to the facial nucleus a n d close to the ventral surface o f the m e d u l l a ( O n i m a r u a n d H o m m a ; 1987, 2003). T h i s g r o u p o f neurons has been termed the p a r a - f a c i a l respiratory group ( p F R G ) . T h e s e c o n d is a g r o u p o f inspiratory neurons i n a r e g i o n o f the ventral respiratory group ( V R G ) c a l l e d the p r e - B 6 t z i n g e r c o m p l e x ( S m i t h et a l . , 1991). W h i l e it has b e e n s h o w n that the neurons i n the p F R G are active - 5 0 0 m s e c before inspiratory activity is detected in the C 4 root o f the p h r e n i c nerve, a n d prior to activity i n the p r e - B o t z i n g e r r e g i o n  5  (Onimaru and Homma; 2003), neurons in the pre-Botzinger complex can maintain a respiratory rhythm in the absence of the pFRG (Smith et al., 1991). One possible interpretation of these results is that the pre-inspiratory pFRG neurons interact with the inspiratory neurons of the pre- Botzinger complex as an integral (but non-essential) component of a coupled oscillator (Feldman et al., 2003). In this context, it has been suggested that the pre-inspiratory neurons may drive expiratory activity (which is no longer essential in mammals), while the pre- Botzinger neurons drive inspiratory activity (Feldman et al., 2003). In the medullary network model, the rhythm generator is thought to be comprised of a network of neurons in the medulla that interact through inhibitory synaptic pathways involving the neurotransmitters glycine and G A B A (y amino butyric acid) (Ramirez et al.1997; Pierrefiche et al., 1998). Richter and Spyer (2001) suggest that the pacemaker activity seen in transverse slice preparations of neonatal rodents may represent an immaturity of expression and function of ion channels. They note that as animals mature, the pacemaker function becomes incorporated into a network where inhibitory synaptic processes become increasingly more effective, ultimately taking over the generation of rhythm. In this model there is a developmental sequence that occurs leading to the 'adult' or mature rhythm generator. Another model for respiratory rhythm generation is a hybrid model in which a set of medullary pacemaker cells that has an intrinsic oscillatory firing pattern interacts with a network of excitatory and inhibitory interneurons to generate the respiratory rhythm (Smith et al., 2000). The overlap of neuronal pathways in the network then gives a pacemaker/network hybrid where both components operate in parallel. This model is  6  consistent with the basic principles of rhythmic motor pattern generation as elaborated by Marder and Calabrese (1996). St. John and colleagues (St. John, 1998; St. John and Paton, 2000; St. John and Paton, 2003) argue that the pons is also essential for the generation of an eupneic (or normal) respiratory pattern. They envision a pontomedullary circuit or network that involves a rostral pontine pneumotaxic center, a caudal pontine apneustic center and the dorsal and ventral medullary respiratory nuclei. Removal of the rostral pons leads to apneustic breathing (a pattern of breathing in which inspiratory time is lengthened and can, in vagotomized animals, continue for many minutes) while removal of the caudal pons gives a gasping pattern of respiration (Lumsden, 1923). St. John and Paton (2003) argue that only with both pons and medulla intact is there an eupneic breathing pattern. They suggest that the neurons of the pFRG and the pre-Botzinger complex are part of a "gasping" center and not the pacemaker for eupneic breathing. They suggest that the in vitro brainstem slice preparation used to identify the pacemaker neurons is anoxic and therefore its activity is not representative of what would be seen in an in vivo preparation. In support of this, they found, in experiments with adult cats (St. John, 1999) and with juvenile rats (St. John and Paton, 2003), neuronal activity in the region of the preBotzinger complex, which under hypoxic conditions, would 'switch' from an inspiratory or phase spanning pattern to a pre-inspiratory pattern, typical of gasping. Many other neurons ceased to discharge at all. Rybak et al. (2004), with a computational model supported by experimental work, argue that neurons in the rostral (dorsolateral (dl) and ventrolateral (vl)) pons, together with input from the vagus, modulate the activity of ventral medullary neurons, especially post-inspiratory neurons, and thus control the  7  lengths of both inspiration and expiration. They hypothesize that neurons in the pons provide a tonic input to the pFRG and the pre-B6tzinger complex that suppresses the pacemaker activity of those neurons in intact animals thus generating eupnea. If the pons is completely removed, the pacemaker activity of the pFRG and the pre-Botzinger complex then produces only the gasping pattern. In the context of the transformational hypothesis, it should be noted that there may be multiple regions in the brainstem that are able, in isolation, to depolarize rhythmically but when incorporated into a network or circuit, their firing pattern is modified and incorporated into the overall network.  In fact, Huang and St. John (1988)  demonstrated that after a transection at the pontomedullary junction, the mylohyoid branch of the trigeminal nerve acquired a rhythmic discharge pattern that was independent of the phrenic discharge. Respiratory Pattern Generation The shaping of the breaths, i.e. the tidal volume and timing of inspiration and expiration, which depend on the force and timing of the contraction of various muscle groups, is determined by the firing patterns of pre-motor neurons of the pattern generator. These, in turn, are determined by adjustments to the basic rhythm of respiration, which ensure that levels of oxygen and carbon dioxide are within the 'normal' range and that pH balance is maintained. The central neurons governing the pattern of respiration are found in the pons and medulla, with input from higher brain centres, the cerebellum, and peripheral afferent nerves (Bianchi et al., 1995). In the medulla, there are three regions that have been identified as having 'respiratory neurons' that may be involved in pattern generation. Respiratory neurons are  8  defined as "neurons exhibiting a rhythmic periodic activity in fixed relation with one phase of the respiratory cycle" (Bianchi and Pasaro, 1997). The regions are called the Ventral Respiratory Group (VRG), the Dorsal Respiratory Group (DRG) and, historically the Pneumotaxic Centre or Pontine Respiratory Group (PRG). The V R G is subdivided into five parts (Bianchi et al., 1995)(Fig. 1.1). The caudal V R G (cVRG) extends from the spinomedullary junction to the obex. This region overlaps with the nucleus retroambigualis, and contains a mixture of inspiratory (I) neurons and expiratory (E) neurons whose axons descend into the spinal cord. The intermediate V R G (iVRG) overlaps with the nucleus ambiguus and para-ambigualis. It contains both E and I neurons and motoneurons for laryngeal and pharyngeal muscles. In this region are also found some respiratory propriobulbar neurons (soma and projections are completely within the medulla) that are thought to be part of the rhythm generator. The rostral V R G (rVRG) overlaps with the retrofacial nucleus and contains pharyngeal motoneurons as well as a variety of E and I neurons and interneurons projecting to the caudal medulla and spinal cord. The Botzinger complex, found just caudal to the nucleus of C N VII (Fig. 1.1) contains a population of primarily expiratory neurons. The pre-Botzinger complex of the V R G (Smith et al., 1991) is just caudal to the Botzinger complex. (Fig. 1.1)  Fig 1.1. Pontine Respiratory Areas and VRG. A. Mapped neurons from FluoroGold injected in the BQtzinger/preBotzinger region. Pontine neurons from three adjacent sections were projected onto the single sagittal plane depicted. VRG neurons were mapped from a single section in the contralateral VRG. B. Schematic of lateral pontine respiratory compartments in the VRG. PBr parabrachial; mPBr medial PBr; lcPBr lateral crescent PBr; KF K6lliker-Fuse n.; ITR5 intertrigeminal n.; vlPons ventrolateral pons; rTZ retrotrapezoid n.; BC Botzinger complex; pBC preBotzinger complex; rVRG rostral VRG; cVRG caudal VRG; Mo5 motor n. trigeminal; 5n trigeminal nerve; 7 facial nucleus; 7n facial nerve; NAc compact part nucleus Ambiguus; LRt lateral reticular nucleus (from Alheid et al., 2004)  10  The Dorsal Respiratory Group (DRG), found in the ventrolateral portion of the Nucleus Tractus Solitarius (NTS) of the medulla, receives input from the vagus nerve (stretch receptor feedback from the lungs, diaphragm and intercostal muscles), the superior laryngeal nerve (part of the reflex arcs associated with coughing, swallowing, and irritation of the larynx), and chemoreceptor input from the carotid and aortic bodies via the glossopharyngeal and vagus nerves respectively (Heymans et al., 1935; Bianchi et al. 1995). The neurons of the D R G project to the premotor and motor neurons of the V R G , to the motor neurons of the phrenic and intercostal nerves in the spinal cord, and to the Kolliker Fuse nucleus of the pons (Bonham and McCrimmon, 1990, Ellenberger and Feldman, 1990; Herbert et al., 1990; Schwarzacher et al., 1991; St. -John, 1998). (Fig. 1.2)  11  ~1 mm  Fig. 1.2: Schematic of the organization of respiratory related regions in the brainstem of the rat. K F , Kolliker Fuse nucleus; PB, Parabrachial nucleus; R T N , retrotrapezoid nucleus; Bot C,B6tzinger complex; pre BotC, preBotzinger complex; rVRG, rostral ventral respiratory group; NTS, nucleus of the solitary tract; c V R G , caudal ventral respiratory group. (From Feldman and Smith, 1995).  12  In the pons there are a number of areas that have respiratory related neurons. Lumsden (1923) identified a pneumotaxic centre in the rostral pons and an apneustic centre in the caudal pons. The nuclei making up the pneumotaxic centre were later identified as the nucleus parabrachialis medialis (NPBM) and the Kolliker-Fuse (KF) nucleus (Cohen and Wang, 1959: Bertrand and Hugelin, 1971; Bianchi and St. John, 1982; and St. John, 1987). Subsequently, the lateral parabrachial nucleus was also shown to have neurons with respiratory related activity (Takayama and Miura, 1993), as was the A5 region located in the ventrolateral (vl) pons (Jodkowski et al., 1994 and 1997). Other pontine regions identified as having a role in the control of breathing are the locus coeruleus or the A6 region (Guyenet et al., 1993), the pontine reticular formation (PRF) (Fung and St. John, 1994b), the ventral reticular aspects of the caudal pons (Borday et al., 1997), and the intertrigeminal region (ITR), located ventral to the K F and between the principal sensory and motor trigeminal nuclei (Chamberlin and Saper, 1998) (Figure 1). Naming of the dorsolateral region of the pons has changed over time. Originally called the pneumotaxic centre (Lumsden, 1923) and identified with the N P B M and K F (Bertrand and Hugelin, 1971), Feldman (1976) suggested that it be called the Pontine Respiratory Group (PRG) because he did not believe that it was essential for breathing (as the name 'pneumotaxic' implies). As mentioned, however, there are other areas of the pons involved in the control of breathing that would be excluded by using this name. Harris and Milsom (2001) refer to the region as the parabrachial complex (PbC), but the PbC has many integrative functions beyond those involved in the control of breathing. These functions include integration of optic afferent information (Fite and Janusonis, 2002), of gustatory and visceral afferent input, and of cardiovascular, autonomic and  13  neuroendocrine information (Scalera et al. 1995; Bianchi et al. 1995; Hayward and Felder, 1995). Coordination of peripheral chemoreceptor input with sympathetic activity (Koshiya and Guyenet, 1994), control of hypoglossal motor output to tongue muscles (Kuna and Remmers, 1999), and production of expiratory apnea in the presence of noxious stimuli to the nasal mucosa (Dutschmann and Herbert, 1996) are also mediated via the K F nucleus. The necessity to coordinate breathing pattern to changes in cardiovascular system activity and other autonomically controlled activities makes the organization of neurons in close approximation seem reasonable. Thus, because there are a number of regions in the pons that are involved with respiratory control, and because the PbC itself has neurons with functions other than respiratory control, it would seem reasonable to refer to the region in the dorsolateral area of the pons, (comprised of the N P B M and K F nuclei) which is part of the parabrachial complex and involved in the control of breathing, as the parabrachial/Kolliker Fuse (PBrKF) respiratory complex (Alheid et al., 2004). Interconnections between Respiratory Related Neurons in the Brainstem The interconnections between medullary and pontine centres have been the subject of a large number of studies. As reviewed by Chamberlin (2004), retrograde tracing studies from injections into the vl medulla show densely labeled neurons in the K F and on the dorsolateral rim of the lateral parabrachial nucleus. Labeling was also seen in the medial parabrachial subnucleus, the adjacent reticular formation and the intertrigeminal region (ITR). The K F projects, via premotor neurons, to the phrenic, hypoglossal and facial motor nuclei (Yokata et al., 2001), and Herbert et al., (1990) demonstrated reciprocal projections between the K F and the NTS (DRG). These  14  interconnections between areas of the V R G , the NTS, the K F nucleus and the parabrachial nuclei of the pons would suggest that there is integration of action between the respiratory centres in the pons and the respiratory areas of the D R G and V R G . The Role of the Respiratory Related Neurons in the PBrKF What role then do the neurons of the PBrKF play in the control of respiration? This question has been approached in a number of different ways. The region Lumsden (1923) identified in the rostral pons as the pneumotaxic centre (now the PBrKF) seemed to be involved with the termination of inspiration. A transection just caudal to this area resulted in a breathing pattern characterized by prolonged inspiratory periods called apneuses. Apneusis was defined as "a holding of the breath" where the animal prolonged the inspiratory phase (with active inspiratory muscle recruitment) for varying periods of time (Lumsden, 1923). Stella (1938) subsequently found that cutting the vagus nerves was also required to produce apneusis, as has been confirmed by others (Wang et al.1957, Bertrand and Hugelin, 1971, St John and Wang, 1977 and St John, 1979). Many subsequent studies have employed electrolytic lesions of the PBrKF, both unilateral and bilateral, to explore the role that neurons in this area play in the control of breathing. Gautier and Bertrand (1975) studied breathing both before and after vagotomy. Prior to vagotomy the lesions increased inspiratory time (T|), expiratory time ( T E ) and tidal volume (Vj), and decreased the frequency of respiration (f ). R  Bilateral  vagotomy amplified the changes and led to apneusis. These findings were supported by those of others (Von Euler et a l , 1976: St. John, 1979: Caille et al., 1981; Oku and Dick, 1992; Morrison et al., 1994; and Fung and St. John, 1995), although the increase in T  E  15  was not consistent in all experiments (Oku and Dick, 1992; Fung and St. John, 1995; Mutolo et al., 1998; Chamberlin and Saper, 1994). Consistent with the lesion studies, electrical stimulation in the K F nucleus and the nucleus parabrachialis medialis (NPBM) caused phase switching. If the stimuli were given during inspiration, there was a decrease in phrenic discharge and a switch to expiration, however if the stimuli were given during the expiratory phase, there was early termination of that phase and a switch to inspiration (Cohen, 1971; Wang et a l , 1993). In addition, Cohen (1971) also found that electrical stimulation produced different effects depending upon where in the PBrKF the stimulus was delivered. Stimulation at dorsolateral points, caused increased phrenic discharge and shortening of the expiratory phase. Stimulation at ventrolateral points caused decreased phrenic discharge, shortening of the inspiratory phase and lengthening of the expiratory phase. The variation seen in lesion and stimulation experiments can potentially be explained in two ways. The PBrKF of 250 to 350 g rats extends 0.08 to 1.08mm longitudinally and 0.46 mm (KF) to 0.52 mm (lateral and medial parabrachial nuclei) vertically (see also Figure 1.1) (Paxinos and Watson, 1986). It is quite likely that attempts to stimulate, lesion or chemically block the PBrKF could affect only parts of the complex given its rostro-caudal extent. Furthermore, there is a heterogeneous population of respiratory neurons in this area including inspiratory (peak discharge frequency during inspiration), expiratory (peak discharge frequency during expiration), and phase spanning neurons (peak discharge frequency at the transition between phases) (Bertrand et al., 1973; Dick et al., 1994) which may also have been variously affected by attempts to stimulate, lesion or chemically block the PBrKF.  16  The balance of these studies, however, suggests a role for the PBrKF respiratory complex in respiratory phase switching, particularly in producing the switch from inspiration to expiration (the inspiratory off switch; IOS), particularly in the absence of vagal feedback. Its role in the presence of vagal feedback from pulmonary stretch receptors remains somewhat controversial. Feldman et al. (1976) found only tonic neuronal activity in the PBrKF when animals were vagally intact, suggesting that phasic PBrKF neuronal input to the IOS was redundant and present only in the absence of that from the vagus nerve. However, other groups have shown that neurons in the PBrKF have respiratory modulated activity even when the vagus is intact (Bertrand and Hugelin, 1971; Lydic and Orem, 1979: Sieck and Harper, 1980; St. John, 1987 and Dick et al., 1994) and that there is significant instability in the breathing pattern after even unilateral lesions of the PBrKF in vagally intact animals (Oku and Dick, 1992) suggesting that the respiratory neurons of the PBrKF stabilize the breathing pattern even when the vagi are intact. The controversies that surround the role of the PBrKF in function of the IOS are minor, particularly compared to those that surround the role of these neurons in respiratory rhythm generation per se. As discussed earlier (see 'Central Rhythm Generation'), St. John and colleagues (St. John, 1998; St. John and Paton, 2000; St. John and Paton, 2003) argue that the pons is essential for the generation of eupneic respiration. From the developmental viewpoint, Borday et al. (2003) have shown that inactivation of Krox-20 or Hox a l genes in mice leads to post-natal apnea. Since mutations in these genes lead to abnormal development of the neural tube associated with the pons, this  17  would support the belief that pontine respiratory neurons are vital for rhythm generation, at least in newborn rodents. The Role of Glutamate Glutamate is an important excitatory neurotransmitter in the CNS that causes a large majority of respiratory neurons to increase their discharge rate (McCrimmon et al, 1986, 1989; Greer et al, 1991; Kazemi and Hoop, 1991). There are both ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors are coupled to second messenger systems (Schoepp and Conn, 1993), while the ionotropic receptors, of which there are three types, are coupled to ion channels.  The three sub-types of the  ionotropic glutamate receptors are: 1) A M P A (a amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) [previously known as quisqualate (QA)] sensitive 2) Kainate (Ka) sensitive, and 3) N M D A (N-methyl-d -aspartate) sensitive The A M P A and Kainate sensitive receptors are commonly called non-NMDA receptors. (McCrimmon et al., 1995 for review). The N M D A type glutamate receptor ( N M D A receptor) is a receptor with a strong voltage dependence and high C a  ++  permeability. The A M P A and Ka sensitive glutamate receptor subtypes are permeable to N a and not voltage dependent. The functional N M D A receptor is composed of 5 +  heteromeric subunits of which one is always the N M D A R 1 ( N M D A receptor subunit 1) subtype, and the others (NMDAR2, a, b, c or d) vary according to the location of the receptor in the brain. These subunits form a pentamer with a central opening, the ionconducting pore.  18  N M D A receptors were found to have binding sites for two molecules, L-glutamate and L-glycine, on the external cell membrane adjacent to the opening of the ion channel. There would seem to be a negative allosteric interaction between the sites for glutamate and glycine. A binding site for M g  + +  was found in the ion channel. The activation of the  channel requires a displacement of the M g , which then allows the flow of C a + +  ++  ions  through the channel. The NMDA-type glutamate receptor is classified as a voltage-gated ion channel. It is thought that A M P A receptors are co-localized in the membrane and that when they are activated they generate the voltage needed to remove M g  + +  and open  the N M D A ion channel. (Pierrefiche et al. 1994) Location of the NMDA-type Glutamate Receptors The region with the highest density of N M D A receptors in the CNS is the CA1 region of the hippocampus, but receptors are also found in the cerebral cortex and basal ganglia, the thalamic regions, and in the midbrain and brainstem (Monaghan and Cotman, 1985). Within the midbrain and brainstem they are found in the parabrachial nucleus, especially the Kolliker-Fuse portion, and in the nucleus of the solitary tract, the inferior olivary complex, and the hypoglossal nucleus. N M D A receptors are also found in the rVRG, the Botzinger complex, the pre-B6tzinger complex and the phrenic motor nucleus (Robinson and Ellenberger, 1997). Since lesioning experiments have also identified neurons with respiratory-related activity localized to the parabrachial complex, especially the K F nucleus, as well as the NTS and the V R G , all of which are involved in the control of breathing, NMDA-type glutamate receptors are also implicated in respiratory control.  19  Blockade of NMDA Receptors Antagonists of glutamate receptor-mediated processes have been used extensively to differentiate the roles of NMDA-type glutamate receptors from other glutamate receptors. One of the important non-competitive antagonists to the NMDA-type glutamate receptor is MK-801, also known as dizocilpine maleate {(+)-5 methyl-10, 11dihydro-5H-dibenzo [a, d] cyclohepten-5, 10-imine} (Foster and Wong, 1987). The ability of MK-801 and related drugs (e.g. Phencyclidine, SKF 10,047 and Ketamine) to block N M D A action is increased when N M D A receptor agonists are added to the tissue (Fagg, 1987; Foster and Wong, 1987). This indicates that MK-801 binds noncompetitively to the open ion channel associated with the N M D A receptor, and not at the same site as N M D A or glutamate (Foster and Wong et al., 1987). Other antagonists of NMDA-type glutamate receptors are the competitive antagonists AP5 (2-amino-5phosphonopentanoic acid) and AP7 (2-amino-7-phosphonoheptanoic acid) (Evans et al., 1972). Application of these antagonists can be made either by systemic administration or by microinjection into an area of the pons or brainstem determined to have respiratoryrelated activity. Systemic Injections of Glutamate-receptor Antagonists Systemic injection of MK-801 increased the time of inspiration in nonvagotomized cats (Foutz et al., 1988b; Foutz et al., 1994; Foutz et al., 1989; Abrahams et al., 1993; and Borday et al., 1998) and rats (Monteau et al., 1990; Connelly et a l , 1992: Cassus-Soulanis et al., 1995; Ohtake et al., 1998). Subsequent vagotomy caused a further increase in Ti, and apneustic-breathing. The similarity of these effects to those produced by lesions in the PBrKF suggested that MK-801 was primarily acting at the  20  level of the PBrKF. The extent of the change tended to be dose dependent (Foutz et a l , 1988b; Foutz et al., 1989; Connelly et al., 1992). These effects, however, have also been shown to be dependent on the state of the animal (Cassus-Soulanis et al., 1995). When anaesthetized rats were both vagotomized and had NMDA-type glutamate receptors blocked with MK-801, dissipation of anaesthesia returned the apneustic-breathing pattern to normal. Just as with lesion studies in the PBrKF, the effects of MK-801 on expiratory time were variable with some authors reporting no change in T E (Foutz et al., 1988a,b; Monteau et al., 1990; Ohtake et al., 1998) others reporting a decrease (Foutz et al., 1989; Connelly et al., 1992; and Pierrefiche et al., 1992), and still others reporting an increase in T (Connelly et al., 1992 and Abrahams et al., 1993). The effects on overall E  ventilation varied as well, but with high doses of MK-801, there were small decreases in frequency of respiration and larger decreases in tidal volume (Connelly et al., 1992; Abrahams et al., 1993; Cassus-Soulanis et al., 1995 and Ohtake et al., 1998). Diaphragm E M G and phrenic nerve discharge amplitudes also decreased after systemic MK-801 injection in vagotomized adult cats and rats (Foutz et al., 1989; Karius et al, 1991; Connelly et al., 1992), indicating that N M D A receptor activation was required to relay inspiratory drive to bulbospinal neurons. When antagonists of both NMDA-type glutamate receptors (MK-801) and of nonN M D A receptors (NBQX) were used simultaneously, the combined blockade caused a profound depression of respiration and apneustic breathing (Foutz et al., 1994; and Borday et al., 1998). This would indicate that blockade of the N M D A receptors inactivates the PBrKF-mediated inspiratory termination mechanism, while blockade of  21  non-NMDA receptors inactivates the vagally mediated inspiratory termination mechanism. PBrKF Injections of Glutamate-receptor Antagonists Experiments in which MK-801 (Ling et al., 1994), MK-801 plus AP-5 ( N M D A receptor blockers), and C N Q X and D N Q X (non-NMDA receptor blockers) were injected into the PBrKF in rats (Fung et al., 1994) produced similar results. In all cases, there was a significant dose-related prolongation of Ti when there was no vagal feedback, suggesting that it is PBrKF NMDA-type glutamate receptor-mediated processes that are involved in the timing of inspiration. The non-NMDA antagonists alonehad no effect on the duration of inspiration (Fung et al., 1994), and input from the PBrKF to the cat medulla did not involve glutamatergic input to NMDA-sensitive neurons, indicating that the neurons with NMDA-type glutamate receptors involved with inspiratory termination and inhibited by systemic MK-801 were localized to the pons (Ling et al., 1993). Control of Inspiratory Timing: the Inspiratory Off-Switch (IOS) While the role of the PBrKF in the control of the inspiratory off-switch (IOS) has been well documented (Cohen, 1971; Wang et al., 1993), and there is strong evidence suggesting that the neurons involved have NMDA-type glutamate receptors (Ling et al., 1994; Fung et al., 1994). It has been noted that the increase in T was not nearly as great (  after PBrKF injection of MK-801 as it was with systemic injections of MK801 (Monteau et al., 1990; Connelly et al., 1992). This suggests there is another region involved in the control of the inspiratory off-switch. Neurons in the ventrolateral pons have also been shown to influence inspiratory timing in vagotomized rats through the activation of expiratory neurons (Jodkowski et al., 1994, 1997; Dick et al., 1995), and it has been  22  proposed that the 'pneumotaxic centre' may extend ventrally to include the vl pons (Dick et al., 1995). Neurons with NMDA-type glutamate receptors in the NTS are also involved in phase switching and in regulating the timing of inspiration (Berger, A. J., 1977; Berger and Dick, 1987; Gillis et al., 1997; Miyazaki et al., 1998, 1999; Wasserman et al., 2000). Injection of N M D A into the ventrolateral portion of the NTS increased the expiratory phase of the respiratory cycle and led to an apnea during expiration (Berger et al., 1995). Furthermore, in one study where transection of the rostral pons produced apneustic breathing in 1/3 of cats, subsequent systemic administration of MK-801 produced apneusis in all animals (Haji et al., 1998), suggesting there are neurons with NMDA-type glutamate receptors in both the PBrKF and the medulla that are involved in the timing of respiration and phase switching. It is not clear where the neurons are, but the data do suggest that neurons of the NTS also have an important role in phase switching. Effects of Cortical Activation State on Breathing Pattern There are differences in the breathing pattern of rats in the awake or cortically activated state and the slow wave sleep (SWS) state (Pappenheimer, 1977). As in most mammals, cortical activation results in an increase in frequency (f ), tidal volume ( V T ) , R  and total ventilation ( V TOT) (Pappenheimer, 1977; Megirian et al., 1980; Phillipson and Bowes, 1986; and Orem, 1994 for reviews). The increase in V TOT in the awake state is thought to result from the influence of behavioral factors that are superimposed on the metabolic factors (PaC02, pH, temperature) that control breathing pattern during SWS (Phillipson and Bowes, 1986; Orem, 1994; Hunter and Milsom, 1998; Hunter et al., 1998).  23  When a calculation of time-in-state in the light period was made, it was found that rats breathing air spent approximately 50% of their time sleeping, 35% in the awake state, and 15%) in R E M sleep (rapid eye movement sleep, also called paradoxical sleep (PS) (Pappenheimer, 1977; Megirian et al., 1980). The episodes of SWS were between 5 and 15 minutes in length, and a short period of R E M sleep often occurred toward the end of the SWS period. Ten percent oxygen in nitrogen was found to disrupt the time spent in the slow wave sleep state, reducing State III to between 27% and 34% of total time, and R E M sleep was not seen. The structure of the sleep was also disrupted so that instead of established intervals of sleep, SWS decreased to 2-3 minute "incompletely developed " episodes, and the mean rectified slow-wave voltage of the E E G did not reach the values seen when the rats were breathing air (Pappenheimer, 1977; Megirian et al., 1980; Ryan and Megirian, 1982; Laszy and Sarkadi, 1990). Sectioning of the carotid sinus nerve converted the sleep-wake pattern to one like that of rats in normoxia, indicating that input from peripheral chemoreceptors influenced the sleep-wake pattern. If CO2 was added to the hypoxic gas mixture, there was no increase in the time spent in SWS, indicating that the respiratory alkalosis that occurred during hypoxia was not causing the reduction of sleep during hypoxia (Pappenheimer, 1977; Megirian et a l , 1980; Ryan and Megirian, 1982). Sensitivity to hypoxia was greater during SWS. When rats were exposed to air and then 10%> oxygen in nitrogen, frequency increased in both states, but it increased more in SWS so that overall ventilation was not significantly different between the awake and SWS states (Pappenheimer, 1977).  24  Breathing 5% CO2, on the other hand, produced no change in the E E G pattern and slow-wave voltage from that seen when animals were breathing air, and the time spent in various sleep states did not change (Pappenheimer, 1977; Megirian et al., 1980). If the levels of hypercapnia increased above 5%, however, there was a reduction in the amount of slow wave sleep (Ioffe et al., 1984). Since the pontine reticular formation (PRF) has been linked to state changes, i.e. an active PRF resulted in wakefulness, and an inactive PRF resulted in sleep, it was hypothesized that there must be a connection between the PRF and respiratory areas of the brainstem to account for the changes in respiration with state changes (Lydic and Orem, 1979). Accordingly, correlations between the discharge of neurons in the PBrKF and SWS, R E M sleep and waking states have been found (Sieck and Harper, 1980; Gilbert and Lydic, 1994). The mean discharge rate of PBrKF neurons was significantly lower during SWS than during awake or R E M sleep, and it was suggested that these changes could account for the differences seen in respiratory patterns in different E E G states (Sieck and Harper, 1980; Gilbert and Lydic, 1994). In further support of this, reciprocal monosynaptic connections have been shown between the PRF and the PBrKF, as have monosynaptic connections between the PRF and the D R G and V R G (Herbert et a l , 1990; Lee et al, 1995). Anaesthesia, Breathing and the Urethane-Anaesthesia Model of Sleep-Wake The experiments presented in this thesis were conducted under anaesthesia, and anaesthesia has been shown to affect PBrKF function. The apneustic breathing pattern produced by pneumotaxic lesions has been shown to be more predominant in animals anaesthetized with pentobarbital, halothane, alphaxolone-alphadolone (Saffan), or chloral  25  hydrate (St. John, 1977; Foutz et al, 1988b, 1989: Monteau et al., 1990; Connelly et al., 1992; Foutz et al., 1994; Cassus-Soulanis et al., 1995: Borday et al., 1998). The effect was dose dependent, with low doses enhancing apneusis and larger doses reducing it (presumably due to an overall reduction of the activity of the respiratory network and its ability to control rhythm and pattern) (Cassus-Soulanis et al., 1995). Natural sleep has also been found to increase the effects of lesions of the PBrKF (Baker et al., 1981). This would indicate that the 'wakefulness stimulus', which is missing in anaesthetized and sleeping animals, also has a role to play in the inspiratory off-switch, adding a third level of control along with vagal feedback and PBrKF mechanisms. It is thought that the loss of the wakefulness stimulus in anaesthetized animals is due to reticular deactivation (Moruzzi, 1972) that then results in a reduction in activity of respiratory neurons (Lydic and Orem, 1979). Thus, there was a general assumption that, when anaesthetized, mammals would simply show a slow wave sleep-like E E G pattern, and that the depression of breathing observed would be due, in part, to loss of input from the PRF to the respiratory centres. The majority of anaesthetics, however, have been found to depress respiration more than occurs as a result of a transition into natural SWS (Gautier, 1976; Nunn, 1990). It has been shown that most anaesthetics, including halothane and barbiturates, potentiate the function of the G A B A  A  receptors (Krasowski  et al., 1999) and inhibit the function of nicotinic acetylcholine (nACh) receptors (Violet et al., 1997). Urethane anaesthesia is an exception in that at doses less than 1.4g/kg body weight, respiration is not depressed (Grahn and Heller, 1989; Maggi and Meli 1986 a and b; Hunter and Milsom, 1998). Urethane potentiates the effects of neuronal nicotinic acetylcholine, G A B A  A  and glycine, and produces a small inhibition of glutamate (at  26  N M D A and A M P A receptors) (Hara and Harris, 2002). The lack of a significant depression of breathing frequency by urethane is probably due to the lack of a single predominant target for its action; however, the fact that it targets many different channels would indicate that high doses might produce results that are not representative of unanaesthetized preparations. Of interest as well is the demonstration that urethane anaesthetized animals with body temperatures maintained at 37 °C continue to show cyclic changes in E E G pattern. These changes superficially resemble the E E G patterns in unanaesthetized animals as they cycle between awake, drowsy and slow wave sleep states (Grahn and Heller, 1989; Hunter and Milsom, 1998; Harris and Milsom, 2001). These patterns have been termed State I for the awake-like state, with a low amplitude high frequency desynchronized EEG; State II for the drowsy or light-sleep state, with a mixture of low and high amplitude waves and an intermediate frequency; and State III for the SWS-like state, with a high amplitude, low frequency synchronized E E G pattern (Grahn and Heller, 1989; Hunter and Milsom, 1998). If the changes in respiration between state I (awake-like E E G pattern) and state III (SWS-like E E G pattern) parallel those seen between the awake and sleeping state in an unanaesthetized animal, then this would be a good system in which to study respiratory pattern and the "state" effects on pattern under various conditions such as exposure to hypercapnia or hypoxia. This supposition appears to hold true for golden-mantled ground squirrels (Hunter and Milsom, 1998; Hunter et al., 1998).  27  Role of the Pons and NMDA-type Glutamate Receptor Mediated Processes in Hypoxic Ventilatory Response While both hypoxia and hypercapnia stimulate breathing, they do so in different ways. Usually hypoxia causes a greater increase in breathing frequency than in tidal volume, while hypercapnia causes a greater increase in tidal volume than in breathing frequency. The hypoxic ventilatory response (HVR) is more robust when isocapnia is maintained and decreases if CO2 levels are allowed to decrease with the increased respiratory frequency (Powell et a l , 1998). The range of values reported is wide, and the magnitude of the changes varies (Pappenheimer, 1977; Bartlett and Tenney 1970, Megirian et al., 1980; Walker et al., 1985; Mortola, 1991; Hayashi and Sinclair, 1991; Frappell et al., 1992; Strohl et al., 1997, 2001). The variation reported would seem to be due to differences in age, weight, species, gender and methodology (Strohl et al., 1997, 2001). It has become clear that the H V R is influenced by the pattern and intensity of the hypoxic stimulus, that the time course of the response has a number of phases, and that the tidal volume and frequency vary over the period of hypoxic exposure and in its aftermath. There are times when facilitation of respiration occurs, and others when depression occurs, and there is evidence for the involvement of a number of different neurotransmitters. The terminology to describe the H V R was clarified by Powell et al. (1998) (Fig. 1.3) Initially, the H V R (2-5 minutes) is characterized by an immediate increase in both f and V , termed the acute response. It is thought that the acute phase of the H V R is R  T  generated via chemoreceptor input to the NTS causing the release of glutamate (Ang et  28  al., 1992; Mizusawa et al., 1994; Soto-Arape et al., 1995; and Ohtake et al., 1998; Lin et al., 1996). The glutamate activates NMDA-type glutamate receptors and A M P A receptors in the NTS (Ohtake et al., 1998), and the excitatory stimulus is relayed to the V R G and the pons. NMDA-type glutamate receptors are also found in both these areas, especially the PBrKF (Richter et a l , 1999; Hoop et al., 1999). This is followed by a decrease in frequency, called short-term depression (STD), and an increase in tidal volume, called short-term potentiation (STP). If the hypoxia continues for more than a few minutes, there is a decrease in ventilation called hypoxic ventilatory decline (HVD) or roll-off. A n increase in the production of G A B A and other neuromodulators is thought to lead to the short-term depression of frequency and the short-term potentiation of tidal volume within the first few minutes of the response (Hoop et al., 1999; Richter et al., 1999). Then the neuroinhibitory influences start to predominate and hypoxic ventilatory decline occurs. When the hypoxic stimulus is removed, there is another acute response, called post-hypoxic frequency decline (PHFD), characterized by an immediate decrease in V TOT, V and f followed by a period in which frequency remains below base-line (due T  R  to increases in expiratory time, Coles and Dick, 1996). If the hypoxic exposure continues for days, there is ventilatory acclimatization to hypoxia (VAH) where the V TOT progressively increases to a level above that seen in the initial response. The mechanism for this has not been fully determined, but could involve augmentation of glutamate release and activation of NMDA-type glutamate receptormediated processes. However, if the hypoxia continues for months or years, hypoxic desensitization (HD), a decreased response, occurs.  29  Hypoxia or CSN Simulation  A.  SP  SID  1 min B.  5 min  V  VAH  HVD mm  |  h r s - d ays  m o n t h s - years  Fig. 1.3. Time domains of the hypoxic ventilatory response. A. The response to a short episode of continuous hypoxia. B . The response to intermittent hypoxia. C. The response to long-term exposure to hypoxia. CSN, carotid sinus nerve; A R , acute response; STP, short-term potentiation; STD, short-term depression; PA, progressive augmentation; LTF, long-term facilitation; H V D , hypoxic ventilatory decline; V A H , ventilatory acclimatization to hypoxia; HD, hypoxic depression. (Adapted from Powell et al., 1998).  30  Are pontine respiratory neurons involved in the response to hypoxia? Guyenet et al. (1993) showed that neurons in the A5 and A6 (locus coeruleus) regions of the pons were activated by hypoxia and showed respiratory rhythmicity. Blockade of vl pontine activity, however, did not block the response to hypoxia (Koshiya and Guyenet, 1994a,b; Coles and Dick, 1996). Lesions or inhibition of neural activity in both the dl and the vl pons have been shown to abolish post-hypoxia frequency decline (Coles and Dick, 1995, 1996). Systemic MK-801 also blocks PHFD when given at high doses (Coles et al., 1998) by preventing the increase in T that is responsible for the decline in frequency. E  Interestingly, MK-801 has been shown to increase Ti during hypoxic exposure, resulting in a decrease in the peak frequency of respiration (Dick and Coles, 2000), but it is not clear whether the neurons affected are in the PBrKF or in other respiratory related areas. With short bouts of intermittent hypoxia (5 minutes, followed by a period of room air or hyperoxygenated air), it has been found that each subsequent exposure to hypoxia may produce a slightly greater response, a process called progressive augmentation (PA). After several episodes, if the hypoxic stimulus is not repeated, ventilation slowly increases to a level above baseline for at least an hour. This has been called long-term facilitation (LTF) (Powell et al., 1998) (Fig. 1.4). L T F has been seen as an enhancement of phrenic and hypoglossal nerve activities leading to an increase in V j for at least 60 minutes after episodic hypoxia or carotid sinus nerve (CSN) stimulation in awake and anaesthetized, vagotomized animals (Eldridge and Millhorn, 1986; Hayashi et al., 1993; Fregosi and Mitchell, 1994; Bach and Mitchell, 1996). The expression of L T F varies depending on the strain of rat, whether the rats are vagotomized, whether they are poikilocapnic or isocapnic during hypoxia, and whether they are anaesthetized or not  31  (Mitchell et al., 2001). Because LTF can be elicited by stimulating the C S N (carotid sinus nerve) without exposing the animal to hypoxia, and can also be elicited in chemodenervated rats exposed to hypoxia, it is possible that there are two mechanisms involved in its expression, "synaptic mechanisms involving chemoafferent neurons, and hypoxic effects on the C N S " (Mitchell et al., 2001). It has been shown that phrenic L T F requires the activation of 5HT receptors in the phrenic motor nucleus during, but not following, episodic hypoxia (Fuller et al., 2001). The role of other neurotransmitters in the production of L T F has not been ruled out at this point (Mitchell et al., 2001).  Long Term Facilitation long term facilitation baseline 50% 02  |0 min 11% 02  60 min  5min  Fig. 1.4. Long-term facilitation of phrenic motor activity after intermittent hypoxia. Hypoxia was administered for three five minute episodes followed by 5 minutes in 50% O2. In the 60 minutes following the episodic hypoxia, phrenic nerve activity increased until it was as high as during the hypoxic episodes. (From Mitchell et al., 2001)  32  Hypotheses The goal of this research was to explore the role of NMDA-type glutamate receptor-mediated processes in the Parabrachial/Kolliker Fuse region (PBrKF) of the pons in the control of "state-related" changes in breathing in rats. Given that rats have been shown to cycle through states of cortical activation during urethane anesthesia superficially similar to wake and slow wave sleep, and that golden-mantled ground squirrels have been shown to exhibit changes in breathing and respiratory sensitivity with these changes in state that were identical to those seen in animals cycling between sleep and wake, I hypothesized that: 1) Urethane anesthetized Sprague Dawley rats exhibit changes in breathing with changes in EEG activity. 2)  Urethane anaesthetized rats exhibit changes in respiratory sensitivity with changes in EEG activity.  3) The changes in respiration that accompany changes in EEG activity would be identical to those seen in unanaesthetized animals cycling between states with similar EEG profiles. 4) The changes in respiratory sensitivity that accompany changes in EEG activity would be identical to those seen in unanaesthetized animals cycling between states with similar EEG profiles. Then, since the Reticular Activating System (RAS) has been shown to be involved in sleep state regulation, and since this area has monosynaptic connections to the PBrKF respiratory complex, and since some nuclei of the R A S (the Pontine Reticular  33  Formation or PRF, and the Pedunculopontine tegmentum or PPT) and the PBrKF have a high density of NMDA-type glutamate receptors, I hypothesized that: 5) Changes in cortical activation state would involve NMDA-type glutamatergic processes and 6) NMDA-type glutamatergic processes in the PBrKF would be involved in producing a) The effects of changes in cortical activation state on breathing and b) The effects of changes in cortical activation state on respiratory sensitivity to hypoxia and hypercapnia. To test these hypotheses, I characterized the response of urethane-anaesthetized rats to the blockade of NMDA-type glutamate receptor-mediated processes with MK-801 (dizocilpine maleate - an N M D A receptor antagonist) administered systemically (Chapter 3) and then injected directly into the parabrachial/Kolliker Fuse region (PBrKF) of the pons (Chapter 4). In the course of these experiments I considered the role of NMDA-type glutamate receptor-mediated processes in the changes in breathing that occur on the transition between cortical activation states (Chapters 3, 4), and in the responses to hypoxia (delivered either continuously (Chapters 3, 4) or intermittently (Chapter 5) and hypercapnia (Chapters 3, 4), as a function of state. A further hypothesis derived from the results of the experiments of Chapter 4 was that: 7) NMDA receptor-mediated processes in the PBrKF play a role in returning tidal volume to normal following exposure to hypoxia. This hypothesis was tested in Chapter 5 and the results compared to those of Chapter 4.  34  References: Abrahams, T.P., Taveira DaSilva, A . M . , Hamosh, P., McManigle, J.E., Gillis, R.A., 1993. Cardiorespiratory effects produced by blockade of excitatory amino acid receptors in cats. Euro. J. Pharmacol. 238: 223-233. Alheid, G.F., Milsom, W.K., McCrimmon, D.R., 2004. Introduction: Lateral Pontine Influences On Respiratory Control. Respir Physiol Neurobiol. In press Ang, R.C., Hoop, B., Kazemi, H., 1992. Role of glutamate as the central neurotransmitter in the hypoxic ventilatory response. J. Appl. Physiol. 72, 1480-1487. Bach, K . B . , Mitchell, G.S., 1996. Hypoxia induced long term facilitation of respiratory nerve activity is serotonin dependent. Respir. Physiol. 104: 251-160. Baker, T.L. Netick, A., Dement, W.C. 1981. Sleep-related apneic and apneustic breathing following pneumotaxic lesion and vagotomy. Respir. Physiol. 46: 271-294. Ballanyi, K., Onimaru, H., Homma, I., 1999. Respiratory Network Function in the Isolated Brainstem-Spinal cord of Newborn Rats. Prog. Neurobiol. 59:583-634. Bartlett, D. Jr., Tenney, S.M., 1970. Control of breathing in experimental anemia. Respir. Physiol. 10, 384-395. Berger, A.J. (1977) Dorsal respiratory group neurons in the medulla of cat: spinal projections, responses to lung inflation and superior laryngeal nerve stimulation. Brain Res. 1135: 231-254. Berger, A.J. and Dick, T. E. (1987) Connectivity of slowly adapting pulmonary stretch receptors with dorsal medullary respiratory neurons. J. Neurophysiol. 58:11259-1274.) Bertrand, F., Hugelin, A., 1971. Respiratory synchronizing function of nucleus parabrachialis medialis: pneumotaxic mechanisms. J. Neurophysiol. 34: 189-207. Bertrand, R.A., Hugelin, Vibert, J.-F. 1973. Quantitative study of anatomical distribution of respiration-related neurons in the pons. Exp. Brain Res. 16: 383-399. Bianchi, A . L . , Denavit-Saubie, M . , Champagnat, J., 1995. Central control of breathing in mammal: neuronal circuitry, membrane properties, and neurotransmitters. Physiol. Rev. 75, 1-46. Bianchi, A . L . , St. John, W . M . , 1982. Medullary axonal projections of respiratory neurons of pontile pneumotaxic centre. Respir. Physiol. 48: 357-373.  35  Bianchi, A . L . , Pasaro, R., 1997. Organization of central respiratory neurons. In: Miller, A . D . Bianchi, A . L . (Eds) C R C Press Boca Raton Florida Neural Control of the Respiratory Muscles pp. 77-90. Bonham, A.C., McCrimmon, D.R., 1990. Neurones in a discrete region of the nucleus tractus solitarius are required for the Hering-Breuer reflex in rat. J. Physiol. 427: 261180. Borday, V., Kato, R., Champagnat, J., 1997. A ventral pontine pathway promotes rhythmic activity in the medulla of neonate mice. NeuroReport 8: 3679-3683. Borday, V., Foutz, A.S., Nordholm, L., Denavit-Saubie. 1998. Respiratory effects of glutamate receptor antagonists in neonate and adult mammals. Euro. J. Pharmacol. 348, 235-246. Borday C, Abadie V , Chatonnet F, Thoby-Brisson M , Champagnat J, Fortin G., 2003. Developmental molecular switches regulating breathing patterns in CNS. Respir Physiol Neurobiol.35 (2-3): 121-32. Caille, D., Vibert, J.F. and Hugelin, A., 1981, Apneusis and apnea after parabrachial or Kdlliker-Fuse n. lesion: influence of wakefulness, Respir. Physiol. 45, 79-95. Cassus-Soulanis, S., Foutz, A.S., Denavit-Saubie, M . , 1995. Involvement of N M D A receptors in inspiratory termination in rodents: effects of wakefulness. Brain Research 679,25-33. Chamberlin, N.L., Saper, C.B., 1994. Topographic organization of respiratory responses to glutamate microstimulation of the parabrachial nucleus in the rat. J. Neurosci. 14: 6500-6510. Chamberlin, N.L., and Saper, C.B., 1998. A brainstem network mediating apneic reflexes in the rat. J. Neurosci. 18: 6048-6056. Chamberlin, N . L., 2004. Functional organization of the parabrachial complex and intertrigeminal region in the control of breathing. Respir Physiol Neurobiol. In press. Cohen, M . I. and Wang, S. C , 1959. Respiratory neuronal activity in pons of cat. J Neurophysiol. 22, 33-50 Cohen, M.I., 1971. Switching of the respiratory phases and evoked phrenic responses produced by rostral pontine electrical stimulation. J. Physiol. (Lond). 217: 133-158. Coles, S.K. and Dick, T.E. 1995. Dorsolateral (dl) versus ventrolateral (vl) pontine influences on the response to hypoxia in adult rats (Abstract) Am. J. Respir. Crit. Care med. 151: A448.  36  Coles, S.K., Dick, T.E., 1996. Neurones in the ventrolateral pons are required for posthypoxic frequency decline in rats. J. Physiol. 497, 79-94. Coles, S.K., Ernsberger, P., Dick, T.E., 1998. A role for N M D A receptors in posthypoxic frequency decline in the rat. Am. J. Physiol. 274 (Regulatory Integrative Comp. Physiol. 43): R1546-R1555. Connelly, C.A., Otto-Smith, M.R., Feldman, J.L., 1992. Blockade of N M D A receptorchannels by MK-801 alters breathing in adult rats. Brain Research 596, 99-110. Dick, T.E., Bellingham, M.C., Richter, D.W., 1994. Pontine respiratory neurons in anesthetized cats. Brain Res. 636:259-269. Dick. T.E., Coles, S.K., Jodkowski, J.S., 1995. A 'pneumotaxic centre' in the ventrolateral pons of adult rats. In: Trouth, O., Millis, R . M . , Kiwull-Schone, H., Schlafke, M . E . (eds.), Ventral Brainstem Mechanisms and Control Functions. Marcel Dekker, New York, pp. 723-740. Dick, T.E., Coles, S.K., 2000. Ventrolateral pons mediates short-term depression of respiratory frequency after brief hypoxia. Respir. Physiol. 121, 87-100. Dutschmann, M , Herbert, H., 1996. The Kolliker-Fuse nucleus mediates the trigeminally induced apnoea in the rat. NeuroReport 7: 1432-1436. Eldridge and Millhorn, Eldridge, R.L and D.E. Millhorn (1986) Oscillation, gating and memory in the respiratory control system. In: handbook of Physiology. Section 3: The Respiratory System vol. 1: Circulation and nonrespiratory Functions. Edited by A.P. Fishmann and A . B . Fisher. Washington, D.C. American Physiological Society. Pp. 92114. Ellenberger, H.H., Feldman, J.L., 1990. Subnuclear organization of the lateral tegmental field of the rat. I: Nucleus ambiguus and ventral respiratory group. J. Comp. Neurol. 294: 202-211. Euler, C. von, Marttila, I., Remmers, J.E., Trippenbach, T., 1976. Effects of lesions in the parabrachial nucleus on the mechanisms for central and reflex termination of inspiration in cat. Acta Physiologica Scandinavica 96: 324-337. Fagg, G., 1987, Phencyclidine and related drugs bind to the activated N-methyl-Daspartate receptor-Channel complex in rat brain membranes. Neuroscience Letters, 76:221-227) Feldman, J.L., Cohen, M.I., Wolotsky, P., 1976. Powerful inhibition of pontine respiratory neurones by pulmonary afferent activity. Brain Res. 104: 341-346.  37  Feldman, J.L., 1976. A network model for control of inspiratory cutoff by the pneumotaxic centre with supportive experimental data in cats. Biol. Cybernetics, 21: 131— 138. Feldman, J.L., 1986.Neurophysiology of breathing in mammals. Handbook of Physiology. The Nervous System. Intrinsic Regulatory Systems of the Brain. Bethesda, American Physiological Society.Vol 4: 463-524. Feldman, J.L., Smith, J.C., Ellenberger, H.H., Connelly, C.A., Liu, G., Greer, J.J., Lindsay, A.D., Otto, M.R., 1990. Neurogenesis of respiratory rhythm and pattern: emerging concepts. Am. J. Physiol. 259: R879-R886. Feldman, J.L., Smith, J.C., 1995. Neural control of respiratory pattern in mammals:an overview. In: Dempsey, J.S., Pack, A.I., (Eds). Regulation of Breathing. Marcel Dekker, New York, pp. 39-69. Feldman, J.L., Mitchell, G.S., Nattie, E.E..2003. Breathing: Rhythmicity, Plasticity, Chemosensitivity. Annu.Rev. Neurosci. 26: 239-266. Fite, K . V . , Janusonis, S, 2002. Optic afferents to the parabrachial nucleus. Brain Res. 943: 9-14. Foster, A.C., and Wong, E.H.F., 1987. The novel anticonvulsant MK-801 binds t the activated state of the N-methyl-D-aspartate receptor in rat brain. Br. J. Pharmacol. 91:403-409) Foutz, A.S., Champagnat, J., Denavit-Saubie, M . , 1988a. N-methyl-D-aspartate (NMDA) receptors control respiratory off-switch in cat. Neuroscience Letters. 87: 221-226. Foutz, A.S., Champagnat, J., Denavit-Saubie, M . , 1988b. Respiratory effects of the N methyl-D-aspartate (NMDA) antagonist, MK-801, in intact and vagotomized chronic cats. Euro. J. Pharmacol. 154: 179-184. Foutz, A.S., Champagnat, J., Denavit-Saubie, M . , 1989. Involvement of N-methyl-Daspartate (NMDA) receptors in respiratory rhythmogenesis. Brain Res. 500: 199-208. Foutz, A.S., Pierrefiche, O., Denavit-Saubie, M . , 1994. Combined blockade of N M D A and non-NMDA receptors produces respiratory arrest in the adult cat. NeuroReport 5: 481-484. Frappell, P., Lanthier, C , Baudinetter, R.V., Mortola, J.P., 1992. Metabolism and ventilation in acute hypoxia: a comparative analysis in small mammalian species. Am. J. Physiol. 262: R1040-R1046.  38  Fregosi, R. and G.S. Mitchell (1994) Long-term facilitation of inspiratory intercostal nerve activity following repeated carotid sinus nerve stimulation in cats. J. Physiol. (London) 477/3: 469-479. Fuller, D.D., Zabka, A . G . , Baker, T.L., Mitchell, G.S., 2001. Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia. J. Appl. Physiol 90: 2001-2006. Fung, M . L . and St.-John, W. M . , 1994a. Electrical stimulation of pneumotaxic center: activation of fibers and neurons. Respir Physiol. 96: 71-82. Fung, M . L . and St.-John, W. M . , 1994b. Separation of multiple functions in ventilatory control of pneumotaxic mechanisms. Respir Physiol. 96: 83-98. Fung, M - L . , Wang, W., St. John, W., 1994. Involvement of pontile N M D A receptors in inspiratory termination in rat. Respir. Physiol. 96: 177-188. Fung, M . L . , St. John, W . M . 1995. The functional expression of a pontine pneumotaxic centre in newborn rats. J. Physiol. (Lond) 489: 579-591. Gautier, H., 1976. Pattern of breathing during hypoxia or hypercapnia of the awake or anesthetized cat. Respir. Physiol. 27: 193-206 Gilbert, K . A . and Lydic. R., 1994. Pontine cholinergic reticular mechanisms cause statedependent changes in the discharge of parabrachial neurons. Am. J. Physiol. 266: R136R150). Gillis, R.A., Hernandez, Y . M . , Bingaman, M . , Panico, W.H., Taveira da Silva, A . M . , 1997. N-Methyl-D-Aspartate Receptors (NMDA) at the ventrolateral nucleus tractus solitarius (NTS) play a role in the termination of inspiration. Neurosci. Abstracts 23:725. Gautier, H., Bertrand, F., 1975. Respiratory Effects of pneumotaxic center lesions and subsequent vagotomy in chronic cats. Respir. Physiol. 23: 71-85. Grahn, D.A., Redeke, C . M . , Heller, H.C., 1989. Arousal state vs. temperature effects on neuronal activity in subcoeruleus area. Am. J. Physiol. 256: R840-R849. Grahn, D.A., Heller, C., 1989. Activity of most rostral ventromedial medulla neurons reflect E E G / E M G pattern changes. Am. J. Physiol. 257: R1496-R1505. Greer, J.J., Smith, J.C., Feldman, J.L., 1991. Role of excitatory amino acids in the generation and transmission of respiratory drive in neonatal rat. J. Physiol. 437: 727-749. Guyenet, P.G. Koshiya, N . , Huangfu, D., Verberne, A.J., Riley, T.A. 1993. Central respiratory control of A5 and A6 pontine noradrenergic neurons. Am. J. Physiol. 264: R1035-44  39  Haji, A . , Okazaki, H., and Takeda, R., (1998) N D M A receptor-mediated inspiratory offswitching in pneumotaxic-disconnected cats. Neuroscience Research 32: 323-331. Hara, K., and Harris, R.A., 2002. The Anesthetic Mechanism of Urethane: The Effects on neurotransmitter-Gated Ion Channels. Anesth. Analg. 94: 313-318. Harris, M . B . , Milsom, W.K., 2001. The influence of N M D A receptor-mediated processes on breathing pattern in ground squirrels. Respir. Physiol. 125: 181-197 Hayashi, F., Coles, S.K., Back, K . B . , Mitchell, G.S., McCrimmon, D.R., 1993. Time dependent phrenic nerve responses to carotid afferent activation: Intact vs. decerebellate rats. Am. J. Physiol. 265: 811-819. Hayashi, F., Sinclair, J.D., 1991. Respiratory patterns in anesthetised rats before and after anemic decerebration. Respir. Physiol. 84: 61-76. Hayward, L.F., Felder, R.B., 1995. Peripheral chemoreceptor inputs to the parabrachial nucleus of the rat. Am. J. Physiol. 268: R707-R714. Herbert, H., Moga, M . M . , Saper, C.B., 1990. Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat. J. Compar. Neuro. 293: 540-580. Heymans, J.J., Bouckaert, J., Dautrebande, L., 1930. Sinus carotidien et reflexes respiratoires. Arch. Intern. Pharmacodyn. 39: 400-450. Hoop, B., Beagle, J.L. Maher, T.J., Kazemi, H., 1999. Brainstem amino acid neurotransmitters and hypoxic ventilatory response. Respir. Physiol. 118: 117-129. Huang, Q., St. John, W . M . 1988. Respiratory neural activities after caudal-to-rostral ablation of medullary regions. J. Appl. Physiol. 64: 1405-1411. Hunter, J.D., McLeod, J.Z., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia: respiratory reflexes. Respir. Physiol. 112: 83-94. Hunter, J.D., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia: cardio-respiratory effects. Respir. Physiol. 112: 71-81. Ioffe, S., Jansen, A . H . , Chernick, V., 1984. Hypercapnia alters sleep state pattern. Sleep 3: 219-222. Jodkowski, J.S., Coles, S.K., Dick, T.E., 1994. A 'pneumotaxic centre' in rats. Neurosci. Lett. 172: 67-72. Jodkowski, J.S., Coles, S.K., Dick, T.E., 1997. Prolongation in expiration evoked from  40  ventrolateral pons of adult rats. J. Appl. Physiol. 82: 377-381. Karius, D.R., Ling, L., Speck, D.F., 1991. Lesions of the rostral dorsolateral pons have no effect on afferent-evoked inhibition of inspiration. Brain Res. 559: 22-28. Karius, D.R. Ling, L . Speck, D.F. 1994. Nucleus tractus solitarius and excitatory amino acids in afferent-evoked inspiratory termination. J. Appl. Physiol. 76: 1293-1301 Kazemi, H., Hoop, B., 1991. Glutamic acid and y-aminobutyric acid neurotransmitters in central control of breathing. J. Appl. Physiol. 70: 1-7. Koshiya, N . , Guyenet, P.G., 1994. Role of the pons in the carotid sympathetic chemoreflex. A m . J. Physiol. 267: R508-R518. Koshiya, N . , Guyenet, P.G., 1994. A5 noradrenergic neurons and the carotid sympathetic chemoreflex. Am. J. Physiol. 267: R519-26 Krasowski, M . D . Harrison, N . L . 1999. General anesthetic actions on ligand-gated ion channels. Cell M o l Life S c i , 55: 1278-1303. Kuna, S.T. Remmers, J.E., 1999. Premotor input to hypoglossal motoneurons from Kolliker Fuse neurons in decerebrate cats. Respir. Physiol. 117: 85-95. LaszyJ., Sarkadi,A., 1990. Hypoxia-induced sleep disturbance in rats. Sleepl3:205-217. Lee, L . H , Friedman, D.B., Lydic, R., 1995. Respiratory nuclei share synaptic connectivity with pontine reticular regions regulating R E M sleep. Am. J. Physiol. 268: L251-L262. Lin, J., Suguihara, C , Huang, U . , Hehre, D., Devia, C , Bancalari, E., 1996. Effect of N methyl-D-aspartate-receptor blockade on hypoxic ventilatory response in unanaesthetized piglets. J. Appl. Physiol. 80 (5): 1759-1763. Ling, L., Karius, D.R., Speck, D.F., 1993. Pontine-evoked inspiratory inhibitions after antagonism of N M D A , G A B A A , or glycine receptor. J. Appl. Physiol. 74 (3): 12651273. Ling, L., Karius, D.R., Speck, D.E., 1994. Role of N-methyl-D-aspartate receptors in the pontine pneumotaxic mechanism in the cat. J. Appl. Physiol. 76: 1138-1143. Lumsden, T., 1923. Observations on the respiratory centres in the cat. J. Physiol. 57: 153160. Lydic, R., Orem, J., 1979. Respiratory neurons of the pneumotaxic center during sleep and wakefulness. Neurosci. Lett. 15:187-192.  41  Maggi, C.A., Meli, A., 1986a. Suitability of urethane anesthesia for physiopharmacological investigations in various systems. Part II: Cardiovascular systems. Experientia 42: 292-297. Maggi, C.A., Meli, A., 1986b. Suitability of urethane anesthesia for physiopharmacological investigations in various systems. Part III: Other systems and conclusions. Experientia 42: 531-537. Marder, E., Calabrese, R., 1996. Principles of rhythmic motor pattern generation. Physiol. Rev. 76: 687-717. McCrimmon, D. R., J.L. Feldman, and D.F. Speck (1986) Respiratory motoneuronal activity is altered by injections of picomoles of glutamate into cat brainstem. J. Neurosci. 6: 2384-2392. McCrimmon, D.R., J.C. Smith, and J.L. Feldman, (1989) Involvement of excitatory amino acids in neurotransmission of inspiratory drive to spinal respiratory motoneurons. J. Neurosci. 9:1920-1921. McCrimmon, D.R., Dekin, M.S., Mitchell, G.S., 1995. Glutamate, G A B A , and serotonin in ventilatory control. In: Dempsey, J.A. Pack, A.I., (Eds.) Lung Biology in Health and Disease. Regulation of Breathing vol. 79: Central Nervous System, New York: Marcel Dekker, pp. 151-218. Megirian, D., Ryan, A.T., Sherrey, J.H., 1980. A n eletrophysiological analysis of sleep and respiration of rats breathing different gas mixtures: diaphragmatic muscle function. Electroencephalography and Clinical Neurophysiol. 50: 303-313. Mitchell, F.S., Baker, T.L., Nanda, S.A., Fuller, D.D., Zabka, A . G . , Hodgeman, B.A., Bavis, R.W., Mack, K.J., Olson, E.B. Jr. 2001. Intermittent hypoxia and respiratory plasticity. J. Appl. Physiol. 90: 2466-2475. Miyazaki, M . , Arata, A., Tanaka, I., Ezure, K., 1998. Activity of rat pump neurons is modulated with central respiratory rhythm. Neurosci. Lett. 249: 61-64. Miyazaki, M . , Tanaka,!., Ezure, K. 1999. Excitatory and inhibitory synaptic inputs shape the discharge pattern of pump neurons of the nucleus tractus solitarii in the rat. Exp. Brain Res. 129: 191-200 Mizusawa, A., Ogawa, H., Kikuchi, Y., et al., 1994. In vivo release of glutamate in nucleus tractus solitarii of the rat during hypoxia. J. Physiol. Lond. 478: 55-66. Monaghan, D.T., Cotman, W., 1985. Distribution of N-methyl-D-aspartate sensitive L [3H] glutamate-binding sites in rat brain. J. Neurosci. 5: 2909-2919.  42  Monteau, R., Gauthier, P., Rega, R., Hilaire, G., 1990. Effects of N-methyl-D-aspartate (NMDA) antagonist MK-801 on breathing pattern in rats. Neurosci. Lett. 109:134-139. Morrison, S.F., Cravo, S.L., Wilfehrt, H . M . , 1994. Pontine lesions produce apneusis in the rat. Brain Res. 652: 83-86. Mortola, J.P., 1991. Hamsters versus rats: ventilatory responses in adults and newborns. Respir. Physiol. 85:305-317. Moruzzi, G., Magoun, H.W., 1949. Brain stem reticular formation and activation of the EEG. Electroencephalogr. Clin. Neurophysiol. 1: 455-473. Mutolo, D., Bongianni, F., Carfi, M . , Pantaleo, T., 1998. Respiratory changes induced by kainic acid lesions in rostral ventral respiratory group of rabbits. A m J. Physiol. 283: R227-R242. Nunn, J.F., 1990 Effects of anaesthesia on respiration. Brit. J. Anaesthes. 65: 54-62. Ohtake, P J . , Torres, J.E., Gozal, Y . M . , Graff, G.R., Gozal, D., 1998. N M D A receptors mediate peripheral chemoreceptof afferent input in the conscious rat. J. Appl. Physiol. 84: 853-861. Oku, Y . , Dick, T.E., 1992. Phase resetting of the respiratory cycle before and after unilateral pontine lesions in cat. J. Appl. Physiol. 72(2): 721-730. Onimaru, H., Arata, A., Homma, I., 1987. Localization of respiratory rhythm-generating neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats. Pflugers Arch. 420:399-406. Onimaru, H., Homma, I., 2003. A Novel Functional Neuron Group for Respiratory Rhythm Generation in the Ventral Medulla. J. Neurosci. 23(4): 1478-1486, Orem, J., 1994. The wakefulness stimulus for breathing. In: Saunders, N . A . , Sullivan, C.E. (Eds), Sleep and Breathing, 2 Edn. Marcel Dekker, New York, pp. 113-155. nd  Pappenheimer, J.R., 1977. Sleep and respiration of rats during hypoxia. J.Physiol. 266: 191-207. Paton, JFR. 1996. A working heart-brainstem preparation of the mouse. J. Neurosci. Meth. 65: 63-68. Paxinos and Watson, 1986. The Rat Brain in Stereotaxic Coordinates.2 edition. Academic Press. nd  Phillipson, E.A., Bowes, G., 1986. Control of breathing during sleep. Handbook of Physiology, Vol. 2. Cherniack, N.S. and J.G. Widdicomb. Washington D C : American Physiological Society, 649-689.  43  Pierrefiche, O., Foutz, A.S., Champagnat, J., Denavit-Saubie, M . , 1992. The bulbar network of respiratory neurons during apneusis induced by a blockade of N M D A receptors. Brain Res. 89: 623-639. Pierrefiche, O., Schwarzacher, S.W., Bischoff, A . M . , Richter, D.W., 1998. Blockade of synaptic inhibition within the pre-Botzinger complex in the cat suppresses respiratory rhythm generation in vivo. J. Physiol. 509.1: 245-254 Powell, F.L., Milsom, W.K., Mitchell, G.S., 1998. Time domains of the hypoxic ventilatory response. Respir. Physiol. 112: 123-134. Ramirez, J.M., Telgkamp, P, Elsen, F.P., Quelmallz, U.J.A., Richter, D.W., 1997. Respiratory rhythm generation in mammals: synaptic and membrane properties. Respir. Physiol. 110: 71-85. Ramirez, J.M., Zuperku, E.J., Alheid, G.F., Lieske, S.P., Krzysztof, P., McCrimmon, D.R., 2002. Respiratory Rhythm generation: converging concepts from in vitro and in vivo approaches? Respir. Physiol. Neurobiol. 131: 43-56. Richter, D.W., Lalley, P.M., Pierrefiche, O., Haji, A., Bischoff, A . M . , Wilken, B., Hanefeld, V., 1997. Intracellular signal pathways controlling respiratory neurons. Respir. Physiol. 110: 113-123. Richter, D.W., Schmidt-Garcon, P., Pierrefiche, 0., Bischoff, A . M . , Lalley, P.M., 1999. Neurotransmitters and neuromodulators controlling the hypoxic respiratory response in anaesthetized cats. J. Physiol. 514: 567-578. Richter, D.W., Spyer, K . M . 2001. Studying rhythmogenesis of breathing: comparison of in vivo and in vitro models. Trends in Neurosci. 24 (8): 464-472 Robinson, D., Ellenberger, H., 1997. Distribution of N-methyl-D-Aspartate and Non-NMethyl-D-Aspartate Glutamate Receptor Subunits on Respiratory motor and premotor Neurons in the Rat. J. Comp. Neurology, 389:94-116. Ryan, A.T., Megirian, D., 1982. Sleep-wake patterns of intact and carotid sinus nerve sectioned rats during hypoxia. Sleep 5:1-10. Rybak, I. A., Shevtsova, N . A., Paton, J. F. R., Dick, T. E., St.-John, W. M . , Morschel, M . and Dutschmann, M . , 2004. Modeling the ponto-medullary respiratory network. Respir Physiol Neurobiol. In Press. Scalera, G., Spector, A., Norgren, R., 1995. Excitotoxic lesions of the para-brachial nuclei prevent conditioned taste aversions and Na appetite in rats. Behav. Neurosci. 109: 997-1008.  44  Schwarzacher, S.W., Wilhelm, Z., Anders, K., Richter, D.W., 1991. The medullary respiratory network in the rat. J. Physiol. Lond. 435: 631-644. Schoepp, D.D., Conn, P.J., 1993. Metabotropic glutamate receptors in brain function and pathology. Trends Pharmacol. Sci. 14:13-20. Sieck, G.C., Harper, R . M . , 1980. Pneumotaxic area neuronal discharge during sleepwaking states in the cat. Exp. Neur. 67: 79-102, Smith, J.C., Ellenberger, H.H., Ballanyi, K.,.Richter, D.W., Feldman, J.L., 1991, PreBotzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254: 726-729. Smith, J.C., Butera, R J . Jr., Koshiya, N . , Del Negro, C , Wilson, C.G., Johnson, S.M., 2000. Respiratory rhythm generation in neonatal and adult mammals: the hybrid pacemaker-network model. Respir. Physiol. 122: 131-147. Soto-Arape, I., Burton, M.D., Kazemi, H., 1995. Central Amino acid neurotransmitters and the hypoxic ventilatory response. Am. J. Respir. Crit. Care Med. Apr. 151: 111 31120. St. John. W . M . and Wang. S.C., 1977. Alteration from apneusis to more regular rhythmic respiration in decerebrate cats. Respir. Physiol, 31: 91-106. St. John, W . M . , 1979. Differential alteration by hypercapnia and hypoxia of the apneustic respiratory pattern in decerebrate cats. J. Physiol. (Lond) 287: 467-491. St. John, W . M . 1987. Influence of pulmonary inflations on discharge of pontile respiratory neurons. J. Appl. Physiol. 63 (6): 2231-2239! St. John, W . M . , 1998. Neurogenesis of Patterns of Automatic Ventilatory Activity. Progress in Neurobiology. 56:97-117. St. John, W . M . , 1999. Rostral medullary respiratory neuronal activities of decerebrate cats in eupnea, apneusis and gasping. Respir. Physiol. 116: 47-65 St. John, W . M . , Paton, J.F.R., 2000. Characterizations of eupnea, apneusis and gasping in a perfused rat preparation. Respir. Physiol. 123: 201-213. St. John, W . M . , Paton, J.F.R., 2003. Respiratory-modulated neuronal activities of the rostral medulla which may generate gasping. Respir. Physiol. Neurobiol. 135: 97-101. Stella, G., 1938. On the mechanism of production, and the physiological significance of 'apneusis'. J, Physiol. 93: 10-23. Strohl, K.P., Thomas, A.J., St. Jean, P., Schlenker, E.H., Koletsky, R.J., Schork, N.J., 1997. Ventilation and metabolism among rat strains. J. Appl. Physiol. 82: 317-323.  45  Strohl, K.P., Thomas, A.J., 2001. Ventilatory behavior and metabolism in two strains of obese rats. Respir. Physiol. 124: 85-93. Suzue, T., 1984. Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat. J. Physiol. (Lond.) 354:173-183. Takayama, K., Miura, M . , 1993. Respiratory responses to microinjection of excitatory amino acid agonists in ventrolateral regions of the lateral parabrachial nucleus in the cat. Brain Res. 604: 217-223. Violet, J.M., Downie, D.L., Nakisa, R.C. Lieb, W. R., Franks, N . P., 1997. Differential sensitivities of mammalian neuronal and muscle nicotinic acetylcholine receptors to general anesthetics. Anesthes. 86:866-874. Walker, B.R., Adams, E. M . , Voelkel, N.F., 1985. Ventilatory responses of hamsters and rats to hypoxia and hypercapnia. J. Appl. Physiol. 59: 1955-1960. Wang, W., Fung, M . St. John, W . M . 1993. Pontile regulation of ventilatory activity in the adult rat. J. Appl. Physiol. 74 (6): 2801-2811. Wasserman, A . M . , Sahibzada, N . , Hernandez, Y . M . , Gillis, R.A., 2000. Specific subnuclei of the nucleus tractus solitarius play a role in determining the duration of inspiration in the rat. Brain Res. 880: 118-130 Wang, S.C., Ngai, S.H., Frumin, M.J., 1957. Organization of central respiratory mechanisms in the brain stem of the cat: genesis of normal respiratory rhythm. Am. J. Physiol. 190(2): 333-342. Yokota, S., Tsumori, T., Ono, K. and Yasui, Y . 2001. Phrenic motoneurons receive monosynaptic inputs from the Kdlliker-Fuse nucleus: a light- and electron-microscopic study in the rat. Brain. Res. 888: 330-335.  46  Chapter 2  Respiratory chemoreflexes and effects of cortical activation state in urethane anaesthetized rats  This chapter has been published. Boon, J.A., Garnett, N.B.L., Bentley, J.M., Milsom, W.K., 2004. Respiratory chemoreflexes and effects of cortical activation state in urethane anesthetized rats. Respir. Physiol. Neurobiol. 140 (3): 243-256.  47  2.1. Introduction There are significant differences in values reported in the literature for breathing frequency, tidal volume and total ventilation in anesthetized rats as a function of strain, gender, age, and the time of day that the measurements were made (Strohl et al., 1997; Mortola,  1991;  Peever and Stephenson,  1997;  Borday et al.,  1998).  Nevertheless,  hypercapnia and hypoxia always increase the frequency of respiration (fR) and the tidal volume  (V ) T  and hence, the total ventilation  (V OT) T  (Bartlett and Tenney,  1970;  Pappenheimer, 1977; Strohl et al., 1977; Walker, et al., 1985; Ohtake et al., 1998; Kondo et a l ,  2000).  In the case of hypoxia, the sudden (first  20  seconds) initial increase in both  frequency and tidal volume (the acute response (Powell et al.,  1998))  is followed by a  decrease in ventilation, the result of a short-term depression of frequency, which is modulated b y a small augmentation i n tidal volume. This is followed by a period of hypoxic ventilatory decline ( H V D ) that takes place over several hours to days (Powell et al., 1998).  In most mammals during slow wave sleep (SWS), respiratory frequency is slower and total ventilation is lower than during the awake state (See Orem, 1994 for reviews). Thus, in rats, waking (cortical activation) results in an increase in respiratory frequency (f ), tidal volume ( V R  T  ) and  total ventilation ( V TOT) (Pappenheimer,  1977).  The increase  in total ventilation in the awake state is thought to result from behavioral influences (e.g. increased vigilance, movement) being superimposed on the metabolic factors (Pa coi, pH, temperature) that control breathing during SWS (Orem,  1994).  Exposure to hypoxia in rats significantly decreases the amount of time spent in SWS and increases the amount of awake and disturbed sleep (Pappenheimer, 1977; Ryan  48  and Megirian, 1982; Laszy and Sarkadi, 1990). Although a decrease in time spent in SWS is also seen with exposure to low levels of hypercapnia, the effect is only significant at levels above 5% CO2 (Ioffe et al., 1984). While the influence of sleep on reflex ventilatory responses to hypoxia and hypercapnia is variable between species and studies, increased responses have been documented during sleep in rats (Pappenheimer, 1977). When animals are anaesthetized, behavioral factors are also removed, as in SWS and respiration declines. The majority of anaesthetics, however, have been found to depress respiration more than occurs as a result of the normal transition into natural SWS (Nurm, 1990). As reviewed by Neubauer et al. (1990), this depression can probably be attributed to a reduction in cortical and diencephalic influences. Urethane anaesthesia at a dosage of 1.3g/kg of body weight or less is an exception, having been shown to have very little effect on cardiorespiratory function (Maggi and Meli, 1986; Hunter and Milsom, 1998; Hunter et al., 1998). In addition, it has been shown that urethane anaesthetized animals continue to show cyclic changes in E E G pattern, similar to the E E G pattern changes recorded in unanaesthetized animals as they cycle between awake, drowsy and slow wave sleep (Grahn and Heller, 1989; Hunter and Milsom, 1998). The different patterns of E E G activity have been referred to as being State I for the awake like pattern, State II for the drowsy or light sleep pattern, and State III for the SWS-like pattern (Grahn and Heller, 1989; Hunter and Milsom, 1998). Similar changes in cardiovascular and respiratory pattern, as well as in the respiratory sensitivity to hypoxic and hypercapnic stimuli (Hunter et al., 1998), have been reported in unanaesthetized golden-mantled ground squirrels moving between natural arousal states, and states with similar E E G profiles under urethane anesthesia (Hunter and  49  Milsom, 1998). These studies suggested that the states observed under urethane anesthesia mimicked sleep/wake in terms of their effect on cardio-respiratory function. Our interest in the present study was to determine whether rats anaesthetized with urethane would show changes in breathing pattern and respiratory sensitivity to hypercapnia and hypoxia with changes in cortical activation state, similar to those that occur in unanaesthetized rats. Preparations employing neonatal and adult rats, in vivo (awake or anaesthetized), in situ (decerebrate, paralyzed or working heart brainstem preparation) and in vitro (isolated brainstem-spinal cord or brain slice preparations) have become standard models for studies of respiratory control. Our interest was to determine whether the urethane-anaesthetized rat would be a good model system for investigating the mechanistic basis of cortical activation states on breathing patterns and r espiratory sensitivity. 2.2. Methods 2.2.1. Animal Care Adult, male Sprague Dawley rats (250 ± 20 g) were obtained from the U B C animal care facility (University of British Columbia, Vancouver, B.C. Canada). The surgeries and protocols were carried out with the prior approval of the U B C Animal Care Committee and the Okanagan University College (OUC) Animal Care Committee. The rats were chronically instrumented in the U B C Zoology surgical facility, as described below, and were allowed to recover for at least one week, after which they were transported to OUC. There they were housed singly at 25°C and allowed access ad libitum to food and water, supplemented from time to time with sunflower seeds and fruit. The animals were maintained on a schedule of 12 hours of light and 12 hours of  50  dark (lights on at 8 a.m.). At the time of the experiments, the rats' average weight was 450g (range: 275g to 670g). A l l experiments were run between 9 a.m. and 6 p.m. 2.2.2. Surgical Preparation Animals were anaesthetized with 2% vaporous halothane.  Each animal was  placed in a stereotaxic head frame (Kopf), adjusted such that the skull surface landmarks lambda and bregma were on the same horizontal plane. After injections of xylocaine (2% lidocaine hydrochloride, Astra Pharmaceutical), a dorsal-longitudinal incision was made over the crown of the cranium, extending from the orbits to the mid-point of the neck. Four electroencephalographic (EEG) electrodes were implanted in the skull as described by Hunter and Milsom (1998). Each electrode was fashioned from a length of insulated, multi-stranded, stainless steel wire ( A M Systems) that was soldered to a self-tapping stainless steel screw (00 x 3/16, Fine Science tools). The other end was soldered to a gold Amphenol pin. When all 4 screws were in place, the Amphenol pins were inserted into an Amphenol pin strip, which was cemented to the skull with dental acrylic. The incision was then sutured shut, the wound dusted with antibiotic and the animal allowed to recover fully. 2.2.3. Experimental Protocol At the start of each experiment, each animal was anaesthetized with an intraperitoneal or subcutaneous injection of a 20% solution of urethane (Sigma, dose = l.Og/kg) in saline. Supplemental doses (O.lg/kg) were administered IP any time the rat responded to a noxious toe pinch. After one hour, a flow-through facemask was placed on the animal and air was administered at a rate of 3 L per minute. A pneumotach was attached to the outflow line and connected to a differential pressure transducer (Validyne,  51  DP 103-18). A cable was attached between the amphenol pin strip and an A C amplifier (P511, Grass Instruments) to obtain recording o f cortical activity (EEG). The pressure signal was amplified (Gould transducer amplifier) and • integrated (Gould integrating amplifier) and the integrated trace was recorded, along with the E E G signal, on a chart recorder and a data acquisition system (AT-CODAS, DataQ Instruments) sampling at a frequency of 120 samples per second. After an initial 30-minute adjustment period, the protocol began. With the animal breathing air, respiratory and E E G patterns were recorded while animals spontaneously cycled through different arousal states until recordings had been made during at least 5 episodes of established State I and State III. The air was then replaced with either 5% CO2 in air (hypercapnia) or 10% O2 in nitrogen (hypoxia), delivered from commercially mixed bottled gases. Again the respiratory and E E G patterns were monitored through at least 5 established episodes of each cortical activation state except in the case of the hypoxic gas where it was often difficult to obtain this many episodes of State III since hypoxia was a very strong stimulus for cortical activation. administered  air  until  the  breathing  pattern  The animal was then  approximated  initial  conditions  (approximately 20 minutes post hypercapnia and at least 60 minutes post hypoxia). Sixteen of the rats were then exposed to the second gas mixture (the hypoxic and hypercapnic gases were administered in random order), while 6 rats received air and either hypoxia (n=3) or hypercapnia (n=3) only.  There were no differences in the  responses to the various gas mixtures as a function of the order of presentation of the gases or whether the animal received only one or both of the hypoxic and hypercapnic gases and so all data has been combined.  52  After the experiment, the rats were euthanized with an overdose of halothane. 2.2.4. Data Analysis Cortical activation states were scored based  on E E G profiles: State I  (desynchronized cortical activity), State II (intermediate  activity), and State III  (synchronized activity), with E E G activity resembling that seen in wake, light sleep and slow wave sleep, respectively as scored according to conventional criteria (Rechtschaffen et al., 1968). A l l arousal state data were scored in 30 sec. epochs and classified according to the predominant state during that epoch. The percentage of total recording time spent in each state was then calculated from these data. Segments of 20 seconds in length of the recorded traces from each o f at least five separate episodes of established State I and State III, on each gas, were then analyzed and the values averaged. Calculations were made of the frequency of respiration (fR), as well as the time of inspiration (Ti) and the time of expiration ( T E ) .  The mask was calibrated using a hand-operated pump that  delivered known volumes at known frequencies, and the differential pressure signals generated by these volumes were used to make a calibration curve that allowed conversion of differential pressure into tidal volume (V ). The total ventilation was T  calculated by multiplying f x V . The values were then normalized to body weight R  T  (ml/1 OOg (V ), or ml/min/1 OOg ( V TOT) (STPD)). T  A spectral analysis of the E E G traces from areas identified subjectively as State I or State III was c arried o ut u sing a Fast Fourier T ransform function w ith a H amming window and 512 points (Windaq Analysis Program, D A T A Q Instruments).  The data  were then exported into a spreadsheet, averaged for five to eight 20-second recordings from each of seven animals in each State and graphed to give the spectral patterns for  53  both State I and State III. The results were expressed as the magnitude in D B against the frequency of the peaks in Hz. Statistical comparisons were made between groups using a Student T test for matched data, a Mann-Whitney rank sum test, or a one-way repeated measures A N O V A . Differences were considered to be significant when p<0.05. 2.3. Results 2.3.1. E E G and Respiratory Traces Adult male Sprague Dawley rats anaesthetized with urethane spontaneously cycled between a slow-wave sleep-like E E G pattern, termed State III, a "drowsy" pattern termed State II, and an awake-like E E G pattern, termed State I (Hunter and Milsom, 1998), whether breathing air or a hypoxic or a hypercapnic gas mixture. Fig. 2.1 shows both the respiratory and E E G patterns of one rat breathing each gas mixture in State I (characterized by a high frequency low amplitude E E G pattern), and State III (characterized by a low frequency high amplitude E E G pattern). The differences in the E E G patterns in these two states are shown quantitatively in Fig. 2.2, which shows the mean power spectrum of 5 to 8 20-second segments of E E G recording from each of 7 animals in each state. Note that the power of the waves in State III was 3 times larger than that of the waves in State I. In State I, the frequencies of highest power were above 4.5 Hz., corresponding to E E G frequencies seen in unanaesthetized rats that are awake or in R E M sleep. The major peak was in the 0 bandwidth (4-7.5 Hz); peaks in the a (7.5 to 13.5 Hz) and p (13.5 to 20 Hz) bandwidths were very small.  In State III, as in N R E M  sleep, the frequencies of highest power were in the bandwidth for 5 waves, 0.5 to 4Hz. (Hamrahi et al., 2001). There was suppression of activity in the 9, a and (3 bandwidths.  54  C:5^  CCX !I • I  * r;11 1  illllll  HIIII'  SO MS  Figure 2.1: Recordings of the E E G and integrated differential pressure signal (Respiration) during State 1 (desynchronized E E G activity) and State III (synchronized E E G activity) in a urethane anaesthetized Sprague Dawley rat breathing air, 10% O2 in nitrogen and 5% CO2 in air.  55  State I  0.004  0.003  CO. Q  —  0) TJ 3  E O) re  0.002  0.001  Frequency (Hz) 0.000  10  20  30  40  50  Frequency (Hz)  State III  0.004  0.003  a)  5"  TJ 3 0 002  Q  'E  d) "§  re 0.001  0.002  'E  O) re S  0  2  4  6  8 10  12  Frequency (Hz)  0.001  0.000 10  20  30  40  50  Frequency (Hz)  Figure 2.2: Spectral Analysis of the E E G patterns in State I and State III in urethane anaesthetized rats using a Fast Fourier Transform (Hamming window with 512 points). Inserts in the upper right of each panel show data from 0-12 Hz on an expanded time scale. Note the difference in the Y-axis scale for State I and State III. (n= 7).  56  Urethane-anaesthetized Sprague Dawley rats breathing air spent almost half their time in State III, and about equal amounts of time in State I and State II (Fig. 2.3). In hypercapnia, the amount of time spent in State III was reduced to 38.4 + 5.8%, but this was not significant. Rats breathing air or 5% CO2 alternated between extended periods of State I and State III, which varied from approximately 5 to 20 minutes. Poikilocapnic hypoxia (10% oxygen in nitrogen), on the other hand, significantly disrupted the sleepwake pattern and reduced periods of time in State III to short periods ranging from 20 to 80 seconds, primarily by extending the amount of time spent in State I (Fig. 2.3). In hypoxia, the total amount of State III was significantly reduced to only 16.0 ± 3.9%. In some rats, the disruption was so severe there was no established synchronized E E G activity. 2.3.2. Effects of cortical activation In rats breathing air, on cortical activation (switch from State III to State I) there was a significant increase in frequency of respiration of 18.0 ± 2.6% (p<0.001) due to a significant decrease in both Ti and T E (Table 1) (n=18). Tidal volume and total ventilation also increased significantly on cortical activation by 11.7 ± 3.5% (p=0.032) and 31.5 ± 5.2% (p=1.4 x 10" ), respectively (Fig. 2.4, Table 2.2). The change in breathing pattern on 5  cortical activation in rats breathing 10% oxygen in nitrogen (n=10) was not as pronounced. The increase in frequency was not significant, but there were significant increases i n t idal v olume and t otal v entilation o f 7.9 ± 2 % (p=0.04) and 11.6 ± 4.1 % (p=0.01) respectively (Fig. 4, Table 2). When rats breathing 5% CO2 in air (n=14) made the transition from State III to State I, there were small but significant increases in breathing frequency (6.7 ± 2.1% (p=0.01)) due to a significant decease in Ti but not T E .  57  (Table 2.1). Total ventilation also increased (11.8 ± 2.9% (p=lxlO")) while the increase in tidal volume was not significant (7.12% ± 3.4% (p=0.1)) (Fig. 2.4, Table 2.2).  Figure 2.3: Distribution of time spent in arousal states I ggg] II ^ g ] and III I  I as a  function of respiratory gas in urethane anaesthetized Sprague-Dawley rats. (n= 8 in each group)  * Indicates significant difference from times for rats breathing air.  58  Table 2.1: Summary of times of inspiration (Ti) and expiration (TE) in States I (SI) and III (SHI) in rats breathing air, 10% 0 or 5% C 0 . 2  2  T, SI Air 10% o 5% C 0  2  2  T  SIII  E  SI  SIII  0.27±0.01*  0.32±0.01  0.32±0.01*  0.37±0.01  0.22±0.01#  0.23±0.01#  0.30±0.02#  0.32±0.02#  0.26±0.02*  0.27±0.01#  0.30±0.01  0.32±0.025#  Values are means given in seconds ±SEM. *Indicates a significant change from State III, # indicates a significant change from air.  59  40 30 -  Air  10%  5%  o  co  2  2  Figure 2.4: The effect of cortical activation (the change from state III to state I) on frequency, tidal volume and total ventilation in urethane anaesthetized rats breathing air  ^  (n=18), 10% 0 in nitrogen ™ 2  (n=10), and 5% C 0  2  ™  in air (n=14).  The values are given as % change from State III. * Indicates a significant difference from State III.  60  Table 2.2: A comparison of the absolute values of f , V T and V TOT from urethane R  anaesthetized rats with those from unanaesthetized rats in a plethysmograph  Gas  State  fR  (bpm) Air  10% 0  5% C 0  2  2  V (mls/lOOg) T  V TOT (mls/min/lOOg)  Authors  State I  101 ± 2 . 4  0.46 ± 0.02  46 ± 2.5  1  Awake  83 ± 1.6  0.57  47 ± - 1 . 4  2  Awake  83-113  0.29-0.81  27-78  3 to 8  State III  86 ± 2 . 3  0.42 ± 0.03  36 ± 2 . 7  1  SWS  76 ± 1.1  0.51  39 ± 1.1  2  State I  117 ± 3.6  0.61 ± - 0.05  71 ± 6 . 4  1  Awake  103 ± 1.9  0.67  69 ± 2.4  2  Awake  103-132  0.37-0.75  48-98  3 to 8  State III  110.1 ± 0 . 2  0.55 ± 0.04  62 ± 6 . 3  1  SWS  117 ± 1.7  0.58  68 ± 2 . 7  2  State I  109 ± 4 . 0  0.69 ± 0.06  75 ± 6.5  1  Awake  113 ± 2 . 4  0.81  91 ± 4 . 8  2  Awake  113-138  0.45-1.0  58-142  3 to 8  State III  102 ± 2 . 9  0.66 ± 0.06  68 ± 6 . 5  1  SW  115± 1.9  0.73  84 ± 3 . 8  2  Authors: 1 = present study; 2 = Pappenheimer (1977); 3 =Bartlett & Tenney (1970); 4 = Lai et al., (1978); 5 = Lai et al., (1981); 6 =Peever & Stephenson (1997); 7 - Strohl et al. (1997); 8 = Walker et al., (1997)  61  2.3.3. Effects of hypoxia and hypercapnia Hypoxia in State I significantly increased breathing frequency, due to decreases in both Ti and T E (Table 2.1), and also increased tidal volume and total ventilation (Fig. 2.5, Table 2.2). Frequency increased 16.6 ± 2.1% (p=1.3 x 10" ), tidal volume increased 28.8 6  ± 2.6% (p=1.3 x 10" ), and total ventilation increased 49.9 ± 5.8% (p=2.7 x 10" ). There 4  5  was also a significant increase in frequency of 37.8 ± 5.6% (p=5.8 x 10" ), in tidal volume 5  of 31.5 ± 8.3% (p=4xl0" ), and in total ventilation of 81.5 ± 15.8% (p=4.5xl0~ ) in State 3  4  III (Fig. 2.5, Table 2.2). The increases seen in frequency and total ventilation in State III were larger than those seen in State I. Hypercapnia in State I also significantly increased breathing frequency, (6.9 ± 3.1%) (p=0.04), tidal volume (47.8 ± 6.7%) (p=1.89 x 10" ), and total ventilation (57.9 ± 5  8.7%>) (p=4.56 x 10" ) (Figure 2.5), and again, the increases were somewhat larger in 6  State III.  However, only the increase in frequency in State III (16.2 ± 3.2%) was  significantly larger than that in State I (p=lxl0~ ). The increases in tidal volume (49.1 ± 4  5.2%) (p= 3.8 x 10" ) and total ventilation (77.3 ± 10.2%) (p=l.l x 10" ) were not 7  6  significantly larger than those in State I. Note that the increase, in total ventilation for both states was primarily due to an increase in tidal volume (Fig. 2.5, Table 2.2). The hypoxic response has time domains (Powell et al., 1998), but our measures of the various respiratory variables were taken based on cortical activation state, not time, and there was great variation in the amount and location of State III during the hypoxic exposure. As a consequence, the values for f , V T and V TOT reported here are averaged R  over the whole period of the hypoxic exposure (on average 1 h.). In either state, the average frequency v alues m easured d uring h ypoxia w ere m uch higher in the first few  62  minutes of exposure and decreased over time, most likely reflecting hypoxic ventilatory decline (Powell et al., 1998), although they always remained above the baseline frequencies measured in animals breathing air. Sighing was common in animals in hypoxia, and Fig. 2.6A shows a compressed sample of the breathing and E E G traces recorded in one rat illustrating the regular pattern of sighing that was usually seen under this condition (see also Fig. 2.1). The sighs became less frequent when slow waves appeared in the E E G trace (see also Fig. 2.1). Fig. 2.6B is a plot of the duty cycle  (TI/TTOT)  against breath number for the trace shown in  Fig 2.6A while Fig 2.6C is a plot of tidal volume versus breath number for the same data set.  Fig. 2.7 plots the tidal volume of each breath versus the tidal volume of the  following breath. The cluster of breaths in the lower left hand corner of the plot illustrates the low variability in V r between breaths. During a sigh, V r increases and the difference between these breaths and the preceding breaths gives rise to the cluster in the lower right hand corner. After the sigh, the following breath tends to be smaller than the breaths that preceded the sigh giving rise to the cluster in the upper left hand quadrant of the graph. Figures 2.6 and 2.7 s how t hat t here i s a trend for i nspiration t o b ecome p rogressively longer relative to expiration during the pause between sighs, and for the tidal volume of each breath following a sigh to be small.  63  ATotal Ventilation  Figure 2.5: Respiratory responses in urethane anaesthetized rats breathing 10 % 0  2  (n=14 State I, n=10 State III), and 5 % C 0 (n=14 for both) expressed as the % increase 2  in frequency, tidal volume and total ventilation from air, in both State I and State III l l s l * Indicates a significant difference from the value for rats breathing air, # indicates a significant difference from State I.  M  state i  0  i  11:  i i l i i l l  M  '  •»  fefaml i  1*5  Inl 1  m \i  *M  BfMlh Numfrtr  Figure 2.6: A n analysis of the breathing pattern in hypoxia in States I, II and III. A. A representative trace showing the respiratory pattern and the E E G trace for a 250 breath interval in one rat breathing a hypoxic gas mixture (10% O2 in nitrogen). The cortical activation state is shown below the E E G trace. B. The 'duty cycle' for the 250 breath interval shown in A. The duty cycle represents the time during which the muscles of inspiration are active. C. The changes in tidal volume during hypoxia are shown for the same 250-breath interval shown in A .  65  oo  cP*  CD  O  i  o 3 -  oo 2 -  1 1  4  *  5  7  8  Breath N  Figure 2.7: A Pointe Carre plot in which the tidal volume of breath N is plotted against that of breath N - l to show the clustering of tidal volumes during the inter-sigh interval, as well as during the sighs and the after sigh breaths. Open circles represent breaths taken in States I and II, while closed circles represent breaths taken in State III.  66  2.4. Discussion 2.4.1. State Distribution Grahn et al. (1989) defined two stable E E G states in lightly urethaneanaesthetized Wistar rats.  The first was called State I and was characterized by a  desynchronized, high frequency, low amplitude E E G trace that was similar to that seen in unanaesthetized animals that were awake or in R E M sleep. The second, called State III, was characterized by a synchronized, low frequency, high amplitude E E G that was similar to that seen in unanaesthetized animals in SWS. There was a third 'transitional' state (State II) that was a mixture of States I and III and resembled the E E G seen during drowsiness or light sleep (Grahn et al., 1989).  The E E G pattern changed from State III  to State I whenever the animal was exposed to a temperature differential between the skin and the core, as well as to noxious stimuli such as a tail pinch, or a loud noise. It also cycled between states on a regular basis without any external stimulation. These E E G patterns have also been seen in urethane-anaesthetized  golden-  mantled ground squirrels (Hunter and Milsom, 1998; Hunter et al., 1998) and, now, in Sprague Dawley rats. As in the ground squirrels (Hunter and Milsom, 1998; Hunter et al., 1998), State I and State III were seen in rats breathing air (normocapnic normoxia), 10% oxygen in nitrogen (poikilocapnic hypoxia) or 5% carbon dioxide in air (hypercapnic normoxia) in the present study. Furthermore, the animals cycled between arousal states in the same manner as has been documented in unanaesthetized rats (Pappenheimer, 1977); episodes of State I would lead to a transitional period of State II during which slow waves would begin to predominate and the animals would enter State  67  III. The transition from State III to State I was quite abrupt. These patterns closely resemble those seen in rats falling asleep and waking up (Pappenheimer, 1977). Spectral analysis of the E E G patterns for States I and III indicate that State I is characterized by major peaks with frequencies above 4.0 Hz (in the 9 band), just as is seen during wakefulness and R E M sleep in rats. State III is characterized by waves of higher power and a major peak in the frequency band below 4 Hz (the 5 band), as is seen in N R E M or slow wave sleep in freely behaving rats (Campbell and Feinberg, 1996, Stephenson et al., 2001). The major peak in the 9 band (4-7.5 Hz) in State I may represent hippocampal brainwave activity as reported by Keita et al. (2000) and the very small magnitude peaks in the a (7.5-13.5 Hz) and P (13.5-20 Hz) ranges would suggest that the cortical activity that generates these brain waves is suppressed by urethane anaesthesia. Anaesthetized rats spontaneously cycled between states in a regular fashion while they were breathing air or a hypercapnic gas mixture, but this pattern was disrupted when they were breathing 10% oxygen. During hypoxia, the incidence of time spent in State III was significantly reduced and individual bouts of State III were dramatically shortened. These results were similar to those documented for unanaesthetized rats where the time spent in SWS was also significantly reduced during hypoxia.  As well, the length of  episodes of SWS was reduced from 5 to 15 minutes to brief 2-3 minute bouts of incompletely developed SWS (Pappenheimer, 1977; Ryan and Megirian, 1982; Laszy and Sarkadi, 1990). There was no significant difference between the times spent in each state on 5% CO2 compared to air, which is also similar to reports for time spent in sleep/wake in unanaesthetized rats (Pappenheimer, 1977; Megirian et al., 1980) and  68  ground squirrels (Hunter and Milsom, 1998; Hunter et al., 1998). Ioffe et al. (1984), on the other hand, reported that 6%-8% CO2 increased the amount of time spent in N R E M sleep in unanaesthetized, rats suggesting that 5% CO2 is just below the threshold for the level of CO2 that will promote sleep. Given the lack of effect of hypercapnia on sleep state distribution described above, it is not too surprising that the respiratory alkalosis that accompanies the hypoxic ventilatory response does not have any effect on the time spent in awake and SWS states (Pappenheimer, 1977). The disruption in SWS/State III seen in hypoxia is thought to be due to the direct action o f hypoxia on neurons i n the brain (Pappenheimer, 1984). Of interest is the finding by Ryan and Megirian (1982) that peripheral chemoreceptor denervation in rats had no effect on sleep-wake pattern in animals breathing air, but did alter the frequency of state changes during hypoxia, suggesting that peripheral 0  2  chemoreceptors are somehow involved in this process. 2.4.2. Effects of changes in state on breathing The values for Ti, T E , f , V j and R  V OT T  recorded in the present study for rats  breathing air fall within the range reported for unanaesthetized rats breathing air (Bartlett and Tenney, 1970; Pappenheimer, 1977; Lai et al., 1978; Peever and Stephenson, 1979; Strohl et al., 1997; Walker et al., 1997). Unanaesthetized rodents (both rats and goldenmantled ground squirrels (Spermophilus lateralis)) show an increase in ventilation with arousal from sleep (see Hunter and Milsom, 1998; Hunter et al., 1998 for review). Similar changes were also seen with arousal from State III to State I i n urethane anesthetized ground squirrels (Hunter and Milsom, 1998; Hunter et al., 1998), and we have now found this to be true in urethane-anaesthetized rats. In rats breathing air or 5% C 0 , this was 2  69  due t o i ncreases i n b oth f and V r , while in rats breathing 10% oxygen this was due R  solely to increases in V j , possibly because the respiratory drive was already so high with this level of hypoxia that breathing frequency could not increase much further. 2.4.3. Effects of hypoxia/hypercapnia on breathing The respiratory sensitivities of urethane-anaesthetized rats to hypercapnia and hypoxia were also within the range found by other researchers for unanaesthetized rats (Bartlett and Tenney, 1970; Pappenheimer, 1977; Lai et al., 1978; Peever and Stephenson, 1997 and Strohl et al. 1997). The greater response to hypoxia in State III than State I, due to a greater increase in f in State III, is identical to the changes reported R  by Pappenheimer (1977) in rats, and by Hunter and Milsom (1998) and by Hunter et al. (1998) in golden-mantled ground squirrels, in SWS versus wakefulness. Also, in hypercapnia, the increase in frequency was more robust in State III than in State I, while the majority of the response to hypercapnia in both states was due primarily to increases in V T rather than f . This pattern of response fits within the highly variable results found R  by other researchers in unanaesthetized rats and ground squirrels (Waldrop, 1982, Hayashi and Sinclair, 1991 and Hunter and Milsom, 1998; Hunter et al., 1998). In unanaesthetized Wistar rats, Walker et al. (1985) found a greater increase in V  T  than in  f . Pappenheimer (1977) found almost equal increases in f and V T , and Ohtake et al. R  R  (1998) found that the increase in ventilation in hypercapnia was almost entirely due to increases in f . R  Finally, a regular pattern of sighing occurred in hypoxia in urethane-anesthetized rats i n b oth State I and State III, and the rate of sighing decreased when slow waves appeared in the E E G . Sighs or augmented breaths occur spontaneously in man and other  70  mammals, and their incidence increases in hypoxia and hypercapnia (Bartlett, 1971; Cheraiack et al., 1981) and decreases during non-REM sleep (Issa and Porostocky, 1993). They can be easily provoked by rapid lung inflation (Thach and Tausch, 1976) and routinely occur following periods of airway occlusion (Sant'Ambrogio et al., 1971). They are usually followed by breaths of smaller amplitude and shorter duration. They normally appear to be initiated by excitation of either 'defence' type receptors in the lungs or airways, and/or peripheral chemoreceptors, and lead to an increase in lung c ompliance and an increase in end expiratory volume (Glogowska et al., 1972). It has been suggested that they are normally triggered by atelectasis and/or counteract tendencies to develop atelectasis (Glogowska et al., 1972). In light of this, our data (Fig. 2.6) suggest that hypoxia leads to a fall in lung volume (perhaps due to a mismatch between the amount of air inspired versus that expired) and progressive increases in inspiratory time and tidal volume until a threshold is reached and a sigh is triggered, and that this occurs less frequently in states with a synchronized EEG.  71  2.5. Conclusions Rats under urethane anesthesia have levels of resting ventilation that are similar to those seen in unanaesthetized rats. They spontaneously cycle through periods with E E G profiles (States I and III) that closely resemble periods of wake and SWS. The effects of hypoxia and hypercapnia on the time spent in each state, and the respiratory responses to hypoxia and hypercapnia in each state under urethane anesthesia mimic the changes that are seen in these same variables in unanaesthetized rats cycling between sleep and wake. The sum of these data indicate that the brain activity states observed under urethane anesthesia mimic sleep/wake in terms of their e ffect on respiratory function and, thus, that urethane anaesthetized rats would be a good model for invasive, mechanistic studies of the processes that produce such state effects on respiration.  72  2.6. References: Bartlett, D. Jr., Tenney, S.M., 1970. Control of breathing in experimental anemia. Respir. Physiol. 10: 384-395. Bartlett, D. Jr., 1971. Origin and regulation of spontaneous deep breaths. Respir. Physiol. 12: 230-238. Borday, V., Foutz, A.S., Nordholm, L., Denavit-Saubie, M . 1998. Respiratory effects of glutamate receptor antagonists in neonate and adult mammals. Eur. J. Pharmacol. 348: 235-246. Campbell, I.G., Feinberg, I., 1993. A cortical E E G frequency with a REM-specific increase in amplitude. J. Neurophysiol. 69: 1368-1371. Cherniack, N.S., von Euler, C , Glogowska, M . , Homma, I., 1981 . Characteristics and rate of occurrence of spontaneous and provoked augmented breaths. Acta. Physiol. Scand. I l l : 349-360. Glogowska, M . , Richardson, P.S., Widdicombe, J.G. and Winning, A.J., 1972. The role of the vagus nerves, peripheral chemoreceptors and other afferent pathways in the genesis of augmented breaths in cats and rabbits. Respir. Physiol. 16: 179-196. Grahn, D.A., Heller, C , 1989. Activity of most rostral ventromedial medulla neurons reflect E E G / E M G pattern changes. Am. J. Physiol. 257: R1496-R1505. Hamrahi H., Chan, B., Horner, R.L., 2001. On-line detection of sleep-wake states and application to produce intermittent hypoxia only in sleep in rats. J. Appl. Physiol. 90:2130-2140. Hayashi, F., Sinclair, J.D., 1991. Respiratory patterns in anesthetized rats before and after anemic decerebration. Respir. Physiol. 84: 61-76. Hunter, J.D., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia: cardio-respiratory effects. Respir. Physiol. 112: 71-81. Hunter, J.D., McLeod, J.Z., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia: respiratory reflexes. Respir. Physiol. 112: 83-94. Ioffe, S., Jansen, A . H . , Chernick, V., 1984. Hypercapnia alters sleep state pattern. Sleep 3:219-222. Issa, F.G., Porostocky, S., 1993. Effect of sleep on changes in breathing pattern accompanying sigh breaths. Respir. Physiol. 93: 175-187.  73  Keita, M.S., Frankel-Kohn, L., Bertrand, N . , Lecanu, L., Monmaur, P., 2000. Acetylcholine release in the hippocampus of the urethane anaesthetized rat positively correlates with both peak theta frequency and relative power in the theta band. Brain Res. 887: 323-334. Kondo, T., Kumagai, M . , Ohta, Y . , Bishop, B., 2000. Ventilatory responses to hypercapnia and hypoxia following chronic hypercapnia in the rat. Respir. Physiol. 122: 35-43. Lai, Y . L . , Tsuya, Y . , Hildebrandt, J. 1978. Ventilatory responses to acute CO2 exposure in the rat. J. Appl. Physiol. 45(4): 611-618. Laszy, J., Sarkadi, A., 1990. Hypoxia-induced sleep disturbance in rats. Sleep. 13: 205217. Maggi, C.A., Meli, A., 1986. Suitability of urethane anesthesia for physiopharmacological investigations in various systems. Part III: Other systems and conclusions. Experientia 42: 531-537. Megirian, D., Ryan, A.T., Sherrey, J.H., 1980. A n electrophysiological analysis of sleep and respiration of rats breathing different gas mixtures: diaphragmatic muscle function. Electroencephalogr. Clin. Neurophysiol. 50: 303-313. Mortola, J.P., 1991. Hamsters versus rats: ventilatory responses in adults and newborns. Respir. Physiol. 85: 305-317. Neubauer, J. A., Melton, J.E., Edelman, N.H., 1990. Modulation of respiration during brain hypoxia. J. Appl. Physiol. 68: 441-451. Nunn, J.F., 1990 Effects of anaesthesia on respiration. Br. J. Anaesth. 65: 54-62. Ohtake, P J . , Torres, J.E., Gozal, Y . M . , Graff, G.R., Gozal, D., 1998. N M D A receptors mediate peripheral chemoreceptor afferent input in the conscious rat. J. Appl. Physiol. 84: 853-861. Orem, J., 1994. The wakefulness stimulus for breathing. In: Saunders, N . A . , Sullivan, C.E. (Eds), Sleep and Breathing, 2 Edn. Marcel Dekker, New York, pp. 113-155. nd  Pappenheimer, J.R., 1977. Sleep and respiration of rats during hypoxia. J.Physiol., London, 266: 191-207. Pappenheimer, J.R., 1984. Hypoxic Insomnia: effects of carbon monoxide and acclimatization. J. Appl. Physiol. 57:1696-1703. Peever, J.H., Stephenson, R., 1997. Day-night differences in the respiratory response to hypercapnia in awake adult rats. Respir. Physiol. 109: 241-248.  74  Powell, FL, Milsom, W.K., Mitchell, G.S., 1998. Time domains of the hypoxic ventilatory response. Respir. Physiol. 112: 123-134. Rechtschaffen, A., Kales, A., Berger, R.J., Dement, W.C., Jacobson, A., Johnson, L.C., Jouvet, M . , Monroe, L.J., Oswald, I., Roffward, H.P., Roth, B., Walter, R.D., 1968 A Manual of Standardized Terminology, Techniques and Scoring System for Sleep Stages of Human Subjects. US Government Printing Office, Washington, DC. Ryan, A.T., Megirian, D., 1982. Sleep-wake patterns of intact and carotid sinus nerve sectioned rats during hypoxia. Sleep 5: 1-10. Sant'Ambrogio, G., Milic-Emili, J., Camporesi, E., 1971. Occurrence of a deep breath after a period of airway occlusion. Pflugers Arch 327: 95-104. Stephenson, R., Liao, K.S., Hamrahi, H., and Horner, R.L. 2001. Circadian rhythms and sleep have additive effects on respiration in the rat. J. Physiol., London, 536.1. 225-235. Strohl, K.P., Thomas, A.J., St. Jean, P., Schlenker, E.H., Koletsky, R.J., Schork, N.J., 1997. Ventilation and metabolism among rat strains. J. Appl. Physiol. 82: 317-323. Thach, B.T., Taeusch, H.W. Jr., 1976. Sighing in newborn human infants: role of inflation augmenting reflex. J. Appl. Physiol. 41: 502-507. Waldrop, T.G., 1982. Posterior hypothalamic modulation of the respiratory response to C 0 in cats. Pflugers Arch. 418: 7-13. 2  Walker, B.R., Adams, E. M . , Voelkel, N.F., 1985. Ventilatory responses of hamsters and rats to hypoxia and hypercapnia. J. Appl. Physiol. 59: 1955-1960. Walker, J.K.L., Lawson, B.L., Jennings, D.B. 1997. Breath timing, volume and drive to breathe in conscious rats: comparative aspects. Respir. Physiol. 107: 241-250.  75  Chapter 3  Respiratory Chemoreflexes and Effects of Cortical Activation State: Role of NMDA-type Glutamate Receptor Mediated Processes  76  3.1. I n t r o d u c t i o n  Cyclic changes in cortical activation state and breathing pattern continue to occur in rats during low dose urethane anaesthesia (Grahn et al., 1989, Hunter et al., 1998;Boon et al., 2004, Chapter 2). When body temperature is maintained at 37°C, animals typically cycle between two stable E E G states; State I, an awake-like state and State III, a slow wave sleep-like state with a transitional state (State II, - a drowsy state) between (Hunter and Milsom, 1998; Boon et al., 2004). Several studies have now shown that the changes in respiration and respiratory chemosensitivity that accompany changes from State III to State I closely parallel those seen as unanaesthetized animals awaken from slow wave sleep, validating this as a useful model for studies of the control of breathing during sleep (Hunter and Milsom, 1998; Hunter et al., 1998; Boon et al., 2004). Glutamate is an important excitatory neurotransmitter in the CNS, and both N M D A and non-NMDA-type ionotropic glutamate receptors play key roles in respiratory control in mammals (Bonham, 1995 and Bianchi et al., 1995 for review). While the highest density of NMDA-type glutamate receptors (NMDAr) are localized to the CA1 region of the hippocampus, the cerebral cortex and the basal ganglia, there are still relatively high numbers of N M D A r located within the midbrain and brainstem (Monaghan and Cotman, 1985; Petralia et al., 1994; Connelly et al., 1992). They are localized to the ventrolateral (vl) and dorsolateral (dl) pons, especially in the Kdlliker-Fuse portion of the parabrachial nucleus, the nucleus of the solitary tract (NTS), and the rostral ventral respiratory group (rVRG), all regions involved with the control of breathing. They are also found in the pontine reticular formation, which has a role in the determination of cortical activation  77  state (Monaghan and Cotman, 1985; Petralia et al., 1994; Connelly et al., 1992 and Coles et al., 1998; Lydic and Baghdoyan, 2002). Specific antagonists have been used to separate out the roles of the N M D A sensitive receptors from those of other glutamate receptors in respiratory control, particularly the role of these receptors in the switch between inspiratory and expiratory phases. Systemic administration of the non-competitive antagonist of N M D A r , MK-801 (dizocilpine maleate ((+)-5 methyl-10, ll-dihydro-5H-dibenzo [a,d] cyclohepten-5,10imine)) increases the breathing frequency in mice (Cassus-Soulanis et al., 1995; Borday et al., 1998) 15 day-old rats (Ohtake et al., 2000) and ground squirrels (Harris and Milsom, 2001). However, in adult rats the most significant effect is a decrease in tidal volume with no change in breathing frequency (Cassus-Soulanis et al., 1995; Ohtake et al., 1998 and 2000). While Ti (inspiratory time) is consistently lengthened after systemic MK-801 administration in adult rats (Cassus-Soulanis et al., 1995; Ohtake et al., 1998 and 2000), the effects on T E (expiratory time) are varied. Some authors report no change (Connelly et al., 1992; Ohtake et al., 1998) and others report a decrease in T (Monteau et E  al., 1990; Connelly et al., 1992; Cassus-Soulanis et al., 1995) depending on the strain of rat and whether or not the animals were anaesthetized. Blocking N M D A r in conscious animals not only affects their breathing pattern, but also affects their cortical activation state (Lydic and Baghdoyan, 2002). Systemic MK-801 administration produced a predominance of high amplitude slow waves in the E E G record, as seen in slow-wave sleep, in rats and cats (Foutz et al., 1994; Campbell and Feinberg, 1999), although in rats this slow wave sleep followed a period of motor disturbance called motor intoxication (Campbell and Feinberg, 1996 a, b).  78  In addition, N M D A r play a role in determining the response to hypoxia and possibly hypercapnia. A systematic evaluation of the various time domains of the hypoxic ventilatory response (HVR) (Powell et al., 1998) after systemic blockade of N M D A r has not been carried out. However, it is known that systemic MK-801 injections (>0.3 mg/kg) significantly reduce the ventilatory response to hypoxia (8-10% O2 in nitrogen) (Mizusawa et al., 1994; Ohtake et al., 1998; Coles et a l , 1998; Gozal et al., 2000; Ohtake et al., 2000). At least a portion of this reduction results from an effect on N M D A r processing of peripheral chemoreceptor afferent input to the NTS (Mizusawa et al., 1994). Local application of glutamate into the NTS increased ventilation during hypoxia, but pretreatment with MK-801 reduced the H V R primarily through a reduction in tidal volume. While it is not clear where the remainder of the effect arises from, lesions and N M D A r blockade in the vl pons do not alter the H V R although they do eliminate the post-hypoxic frequency decline in rats (Coles et al., 1998; Dick and Coles, 2000). MK-801 doesn't completely eliminate the H V R , however, indicating that nonN M D A receptors are also involved (Vardhan et al., 1993; Coles et al., 1998). While N M D A r antagonism has been reported to affect the hypercapnic ventilatory response in some studies (Nattie et al., 1993; and Harris and Milsom, 2001), it has not in others (Pierrefiche et al., 1996; Ohtake et al., 1998). This study was designed to further elucidate the role of NMDA-type glutamate receptor mediated processes on the pattern of breathing in vagally-intact urethaneanaesthetized rats during different 'states' of E E G activity, breathing different gases. Reports of the effects of N M D A r blockade on breathing have not taken into account cortical activation state, and it would seem that this is particularly important if N M D A r  79  blockade, while having a direct affect on breathing, could also have an indirect effect through state changes. First we compared the breathing patterns in urethaneanaesthetized rats breathing air, hypoxic and hypercapnic gas mixtures in States I and III. Then we examined the effect of systemic MK-801 on state, on breathing patterns and on chemoreflexes in these states. These results will serve as a basis for the investigation of the role of neurons with N M D A r in the parabrachial/Kdlliker Fuse respiratory complex in the pons in the control of breathing pattern, cortical activation state and the response to hypoxia and hypercapnia. 3.2. Methods 3.2.1. Animal Care Male Sprague Dawley rats were obtained from the U B C animal care facility (University of British Columbia, Vancouver, B.C. Canada). The surgeries and protocols were carried out with the prior approval of the U B C Animal Care Committee and the Okanagan University College (OUC) Animal Care Committee. Animals were housed singly in the OUC animal care facility and allowed access ad libitum to food and water, supplemented from time to time with sunflower seeds and fruit. The rats were kept at 25°C, with a light/dark cycle of 12:12; lights on at 8 a.m. Experiments were run from approximately 9 a.m. to 6 p.m. At the time of the experiments, the average weight of the rats was 390 g, and the range was 325-452 g. After experiments, the rats were euthanized with an IP injection of 1 ml of Somnotol (Sodium pentobarbital, 65 mg/ml, M T C pharmaceuticals).  80  3.2.2. Experimental Protocol 3.2.2.1. Surgical Preparation The animals were anaesthetized with 2% vaporous halothane administered through a mask. The head of the rat was sealed into the mask using absorbent cotton impregnated with petroleum jelly. The mask was attached to an airline via a T tube, and the flow was vented to a fume hood. The rate of gas flow was 2 L per minute flowing from a cylinder through a bubble valve to reduce the chance of flow fluctuations. A pneumotach connected to a Validyne differential pressure transducer (Validyne, DP 10318) was installed in the outflow line from the T-tube. The rat was then given an intraperitoneal (IP) injection of the appropriate volume of a 20% solution of Urethane (Sigma) dissolved in sterile saline to give a final concentration of 1.3 g/kg. As the surgery progressed and the urethane started to take effect, the dose of the halothane was reduced. When the surgery was completed, and at least one hour after the injection of urethane, the halothane was discontinued After injections of xylocaine (2% lidocaine hydrochloride, Astra Pharmaceutical), a dorsal-longitudinal incision was made over the crown of the cranium, extending from the orbits to the lambdoid suture. Four electroencephalographic (EEG) electrodes were implanted in the skull as described in Hunter and Milsom (1998). Each electrode was fashioned from a length of insulated, multi-stranded, stainless steel wire ( A M Systems), which was soldered to a self-tapping stainless steel screw (00 x 3/16, Fine Science tools). E K G needle electrodes were inserted into the skin on the sides of the rat. The positive and negative electrodes were inserted into the area of the left shoulder'and the lower right side of the abdomen, while the ground was in the area of the right shoulder.  81  The pneumotach signal was amplified with a Gould amplifier and then transmitted to a two-channel data acquisition system (AT-CODAS, DataQ Instruments) sampling at a frequency of 120 Hz on each channel. The breathing signal was also recorded on a chart recorder. The E E G leads were soldered to gold amphenol pins that were inserted into an amphenol pin strip connected to a lead to the E E G amplifier. The E E G signal was amplified and recorded on a chart recorder and on the data acquisition system on the second channel. The signal was filtered with both low and high pass filters and a 60 Hz filter to reduce noise. The E K G signal was amplified by a Grass amplifier, and recorded on a Harvard Apparatus chart recorder at a speed of 5 cm per second to allow for manual counting of heart rate. 3.2.2.2. Recordings in Animals Breathing Air, 10%O in N and 5% C 0 2  2  2  After an initial half-hour stabilization period, the protocol began. Additional doses of urethane (0.2 ml of 20% solution by weight) were administered IP whenever the rat responded to a noxious toe pinch. Body temperature was maintained between 36 and 37 °C with a servo-controlled heat lamp. With the animal breathing air, the respiratory, E K G and E E G patterns were monitored until periods of both established State III and established State I, were recorded. The air was then replaced with a gas mixture of either 10% (n=5), or 12% (n=8) 0 in nitrogen (hypoxia), or 5% C 0 in air (hypercapnia) (n=8). The respiratory, 2  2  E K G and E E G patterns were monitored for at least 45 minutes on each gas while attempting to ensure that recordings were made in both States I and III. The animal was then switched back to air, and breathing was monitored until the breathing pattern approximated that seen before the animal was exposed to hypoxia or hypercapnia; at least  82  20 minutes post-hypercapnia, and at least 60 minutes post-hypoxia. Each animal was exposed to only one experimental gas. 3.2.2.3. Injection of Saline and MK-801 After the animals had been returned to air, post hypoxia or post hypercapnia, saline was injected intraperitoneally in a volume equivalent to the volume of MK-801 to be subsequently administered. The animals were monitored for at least 45 minutes and until both States I arid III were observed. Then, MK-801 was injected intraperitoneally at a dose of 2 mg/kg. Breathing and E E G were monitored for at least one hour. In 5 rats, an attempt was made to administer 10% 0 in nitrogen, but within 3-5 minutes after 2  exposure to hypoxia the rats died unless switched back to air. As a result, the protocol was changed to use 12% O2 in nitrogen (n=8) as well as the 5% C 0 gas (n=8). 2  Following gas administration, the animal was then switched back to air, and breathing and E E G were monitored for at least one hour. E K G recordings were taken at intervals during the procedure to monitor heart rate during all states and all gases before and after MK-801. 3.2.3. Data Analysis Cortical activation states were scored, based on E E G profiles, as State I (desynchronized cortical activity), State II (intermediate activity), and State III (synchronized activity), with E E G activity resembling that seen in wake, light sleep and slow wave sleep, respectively according to conventional criteria (Rechtschaffen et al., 1968). States IV and V , which were only seen after injection of MK-801, were scored according to the criteria of Grahn et al. (1989). A l l arousal state data were scored in 30  83  sec. epochs and classified according to the predominant state during that epoch. The percentage of total recording time spent in each state was then calculated from these data. To assess the breathing pattern during different E E G states, five to six segments of the recorded trace, 20 seconds in length each, were chosen from areas of the established E E G states, and the data were analyzed and averaged. To assess the breathing pattern during the response to hypoxia and after discontinuation of hypoxia, files were taken at timed intervals: 20 seconds immediately prior to hypoxia, at 20 second intervals for 200 seconds after the start of hypoxia, then every five minutes until the end of the hypoxic exposure; 20 seconds immediately prior to the return to air, at 20 second intervals following the return to air for 200 seconds and then at 5 minute intervals until the end of the experiment. Calculations were made of the frequency of respiration (fR), the voltage corresponding to the tidal volume, the time of inspiration (T) and the time of expiration (T ). Tidal volume calibrations were carried out using an electrically operated E  pump that delivered known volumes into the mask at a constant frequency. The voltage generated by these volumes was used to make a calibration curve that allowed the conversion of the experimental voltages into tidal volumes (V ). The total ventilation T  ( V TOT) was calculated by multiplying the f x V . The values were converted to R  T  volumes in ml/1 OOg (V ), or ml/min/lOOg ( V TOT) STPD. T  A spectral analysis of the E E G traces from defined states was carried out using a Fast Fourier Transform function with a Hamming window and 512 points. The data were exported to an Excel spreadsheet where they were averaged, pooled and graphed to give the spectral patterns for the various states before and after MK-801. The results were  84  expressed as the magnitude in D B plotted against the frequency of the peaks in Hz. The state data from at least 8 rats were averaged for each of States I, and III. Comparisons were made between treatments in each animal using a Student's T test for matched data with two tails. Means presented are overall estimated marginal mean values ± S E M .  A l l of the.data were then analyzed using the General Linear  Model multivariate analysis of variance (SPSS version 11.5, SPSS Inc. Chicago, Illinois). Multiple comparison tests (Bonferonni) were used to separate significant mean values. The timed sequences were analyzed using paired Student T Tests for matched data with two tails and with a One Way Repeated Measures A N O V A with a Bonferonni correction (Sigma Stat, Jandel Scientific). Differences were considered to be significant when p<0.05. 3.3. Results 3.3.1. Respiratory and E E G Traces for Rats Breathing Air Fig. 3.1 shows representative traces demonstrating the respiratory and E E G patterns of rats breathing air (normocapnic normoxia) in States I, II and III before M K 801, and States III and IV after MK-801. State I was characterized by low amplitude, high frequency E E G waves, similar to those seen in unanaesthetized awake animals. State III, was characterized by high amplitude, low frequency E E G waves similar to those seen in animals in slow wave sleep. State II is a transition state between the two. After MK-801 injection, there was very little established State I. Within 5-8 minutes post-injection, all animals had cycled into State III, and in all but 3 rats this was followed by periodic episodes of State IV. In State TV the E E G waves were clustered into groups of high amplitude, low frequency waves separated by short periods of almost  85  no (isoelectric) activity.  N o t e that b o t h the tidal v o l u m e a n d f r e q u e n c y o f respiration  decreased f r o m State I to State III i n air.  86  State I Respiration  t, ,  '/ •! L J  ,',/ /U* A''<!1" J  JI (  1 .  1  > ' '  '  V  EEG  5 s.  State II  State IV Post M K 801  Fig. 3.1: Recordings of the E E G and the differential pressure signal (respiration) during States I, II and III in urethane anaesthetized rats breathing air before injection of MK-801 and in States III and IV after injection of MK-801.  87  3.3.2. Time in State Fig. 3.2 shows the time spent in each state in animals breathing air and hypoxic and hypercapnic gases before and after MK-801 injection. Rats breathing air before M K 801 administration spent 51.5 ± 5.4 % of the time in State I, 21.0 ± 5.8 % of the time in State II, and 27.5 ± 4.8% of the time in State III. Poikilocapnic hypoxia significantly disrupted the sleep-wake pattern. There was a less fully developed SWS-like state, and when State III occurred it was seen in short 23 minute periods followed by cortical activation to State I or II. Cumulatively, the rats spent 42.6 ± 7.9% in State I, 40.1 ± 7.2 % of the time in State II, and 17.3 ± 1.9 % of the time in State III. After hypoxia was discontinued, the rats immediately entered State III for a prolonged period, after which cycling between States I, II and III recommenced. Post-hypoxia, the rats spent 56.2 ± 6.5 % of the time in State III, 9.5 ± 4.1% of the time in State II, and 34.3 ±10.1% of the time in State I. Exposure to 5% CO2 increased the time spent in State I to 66.1 ± 7.8 %, and reduced the time spent in State II to 4.2 ± 1.5%. After the hypercapnic stimulus was removed, the rats immediately entered State III, but cycled into States I and II, as exposure to air continued. In the post-hypercapnic period, they spent 44.7 ± 7.5 % of the time in State III, and 48.2 ± 7.4 % of the time in State I. MK-801 injected into rats breathing air caused the rats to enter State III and in 14 out of 16 rats, to cycle into State IV. The rats spent significantly more time in State III after MK-801 whether they were breathing air, a hypoxic gas mixture or a hypercapnic gas mixture (p O.05). While breathing air, only 5.2 + 1.7% of the time was spent in  88  State I, while the rats were in State III for 63.9 ± 3.2% of the time. State IV was seen for 24.6 ±3.0%) of the time. When the MK-801 treated rats were then exposed to either hypoxia or hypercapnia, very little State I was seen (<1%). The hypoxia exposed rats (n=8) spent 64.6 ±10.9 % of the time in State III and 23.6 ± 7.7 % of the time in State IV. The hypercapnia exposed rats spent 68.5 ± 4.5 % of the time in State III and 19.4 ± 6 . 1 % of the time in State IV. On the return to air after gases, the hypoxia and hypercapnia exposed rats cycled primarily between States II, III and IV, with the amount of State IV decreasing and the amounts of States III and II increasing. State I was seen in 4 rats during this period.  Fig. 3.2: Distribution of time spent in each arousal state in urethane-anaesthetized rats. The top graph shows the data for rats breathing air followed by 12% oxygen in nitrogen , followed by air (n=8). Saline was injected while the rats were breathing air after hypoxia (not shown), and then MK-801 was injected IP (Air +MK-801). After 1 hour, these rats were exposed to hypoxia again, and breathing and E E G were monitored for 1 hour (Hypoxia + MK-801). Air was then reintroduced. In the lower graph are the results for rats exposed to 5% CO2 in air (n=8). The same procedure was followed as for rats breathing a hypoxic gas mixture. State I State IV  I , State III  VSSSA  * Significant difference from values before MK-801. # Significant  difference from values in air pre-MK801. post-MK801.  , State II 1  A  Significant difference from values in air  MK-801  90  3.3.3. Effects of MK-801 on the E E G It appeared that systemic MK-801 blockade of N M D A r blocked cortical activation, and that the rats remained primarily in State III, even in the presence of hypoxia and hypercapnia. To determine i f there was a difference in the spectral pattern of the E E G before and after MK-801, we compared the spectral analyses of E E G traces from States I and III of animals breathing air before MK-801 injection and from State III after MK-801 injection (Fig. 3.3). In State I, pre-MK801, the frequencies of highest power were in the 9 bandwidth, around 5 Hz, and the magnitude of the peaks was low (Fig. 3.3A). Waves with these frequencies are found in unanaesthetized animals in both the awake and R E M sleep states, although the awake state is also characterized by activity in the a (7.5-13.5 Hz) and (3 (13.5-20 Hz) bandwidths, which were suppressed in the urethane-anaesthetized rats. In State III, before MK-801, the frequencies of highest power were in the bandwidth for 5 waves, below 4Hz as in N R E M sleep (Fig. 3.3B). There was suppression of activity in the 9 (4-7.5 Hz) bandwidth. MK-801 administration caused an increase in the magnitude of the slow waves initially, but did not change the frequency of the waves (Fig. 3.3C).  The magnitude decreased over the time of the  exposure to MK-801, but did not decrease below the magnitude seen in State III prior to MK-801. Fig. 3.3C (State III with MK-801) was taken in the first half hour after M K 801 administration. The spectral analyses of E E G records from rats breathing hypoxic and hypercapnic gas mixtures after MK-801 administration did not differ from those seen in rats breathing air after injection of MK-801.  91  State I  0.004  _  0.0015  n Q  "ST 0.0010  0.003  TJ 3 c 0.0005  <> i 0.002 TJ =  O)  to  0.0000  5  0.001  0  TO 0.000  3  6  9  12 15  Frequency (Hz) 10  20  30  40  State III  0.008  50 _  .a a  0.006  0.004  TJ 3 0.002 +J 'E  <u 0.004  TO CO  §, 0.002  0.000 0  0.000  6  9  12 15  Frequency (Hz) 10  0.008  3  20  30  40  50  State III with MK-801 _  0.006  n  0.006  Q  <u  <u 0.004 TJ =  Ui  3 'E  0.002  0.003  G)  ro 0.000  S  0.000  0  3  6  9  12 15  Frequency (Hz) 10  20  30  40  50  Frequency (Hz)  Fig. 3.3: Spectral analysis of the E E G patterns in urethane anaesthetized rats using a Fast Fourier Transform (Hamming window with 512 points) for rats breathing air in States I and III before MK-801 injection, and in State III after MK-801 injection (n=9). The inserts in the upper right of each panel show data from 0 to 15 Hz on an expanded time scale.  92  3.3.4. Effects of MK-801 on Breathing Pattern In addition to blocking cortical activation, the injection of MK-801 caused a reduction in tidal volume, and within 8-10 minutes the breathing pattern included augmented breaths with a lengthened inspiratory time and increased tidal volume followed by a series of breaths with very small tidal volumes (Fig. 3.4B). The tidal volume then gradually returned to the pre-sigh value and remained constant until the next augmented breath. The overall pattern was reminiscent of the pattern of sighing seen in rats breathing a hypoxic gas mixture, although the sighs were not as frequent (Fig. 3.4A). In hypoxia after MK-801, tidal volume and frequency increased, and the frequency of sighs increased (Fig. 3.4C), but not to the level seen before MK-801 (Fig. 3.4A). In rats breathing air in State I, (n=6) MK-801 produced a small, non-significant decrease in frequency of 4.3 ± 2.7% (p= 0.19), due to a significant increase in Ti of 10.8 ± 2.8%> (p=0.01), but T did not change (Fig. 3.5; see also Table 3.1). Tidal volume E  decreased significantly by 23.5 ± 7.1% (p=0.03) and V TOT decreased by 27.3 ± 6.0% (p=0.01).  In State III (n=17) there was no change in frequency of respiration from that  seen prior to MK-801 injection. The increase in Ti of 8.4 ± 2.6% (p=0.003) was offset by .a decrease in T of 6.5 ± 2.9 % (p= 0.03). Tidal volume decreased by 15.7 ± 4.2% E  (p=0.0007) and total ventilation decreased by 16.4 ± 4.3 % (p=0.0008) (Fig. 3.5).  Fig. 3.4 The pattern of breathing and the E E G trace for one rat: A) breathing 12% oxygen in nitrogen, B) breathing air after injection of MK-801, and C) breathing 12% oxygen in nitrogen after injection of MK-801.  94  Fig. 3 . 5 . The effects of MK-801 on breathing pattern of urethane anaesthetized rats breathing air in States I (n=6), and III (n=17). The changes are shown as % change from air before MK-801. * Significantly different from values prior to injection of MK-801  95  Table 3.1. Inspiratory (Ti) and expiratory (T ) times before and after MK-801 injection E  Before MK-801 injection  After MK-801 injection  Air  Ti (sec)  T (sec)  Ti (sec)  T (sec)  State I  0.219 ±0.003  0.255 ± 0.006  0.234 ± 0.008#  0.247 ± 0.02  State III  0.245 ± 0.005 *  0.289 ± 0.007 *  0.263 ± 0.007#*  0.271 ± 0.008 #  State I  0.203 ± 0.001  A  0.228 ± 0.01  0.287 ± 0.01 #  0.274 ± 0.02 #  State III  0.205 ± 0.012  A  0.237 ± 0 . 0 1 *  0.258 ± 0.01 #  0.263 ±0.015  N/A  12%0  E  E  2  A  A  5% CO 2 State I  0.226 ±0.006  0.261 ±0.004  N/A  State III  0.238 ±0.015  0.288 ± 0.006 *  0.257 ± 0.02  0.308 ± 0.02  Times are in seconds ± S.E.M. * Significantly different from State I. # Significantly different from before MK-801 injection. state.  A  Significantly different from air in the same  A  96  3.3.5. Effects of MK-801 on Cortical Activation Although blockade of N M D A r seems to suppress cortical activation, there were small periods of State I seen. Does MK-801 have an effect on breathing pattern on the transition from State III to State I? Fig. 3.6 shows the effects of cortical activation on animals breathing air and hypoxic and hypercapnic gases before and after MK-801. When rats breathing air before the MK-801 injection (n=18) made the transition from State III to State I, there were significant increases in frequency, tidal volume and total ventilation (13.1±1.8% ( p « 0 . 0 0 1 ) , 6.2 ± 1.8% (p= 0.004), and 20.0 ± 2.6% ( p « 0 . 0 0 1 ) respectively). The inspiratory and expiratory times decreased by 10.2 ± 1.7% and 11.5 + 1.9% (see Table 1). Cortical activation during hypoxia before MK-801 injection caused small increases in f (2.4 ± 1.6%), due to a decrease in expiratory time, and in V T (2.7 ± R  1.7%), but these were not significant. Additively they caused V O T to increase slightly T  but significantly by 5.0 ± 2.0 % (p=0.009). Rats breathing 5% C 0 showed significant 2  increases i n f (8.2 ± 1.7'%, p=0.0026), V R  T  (11.4 ± 2.8%, p=0.005) and V TOT (20.5 ±  4.9%, p=0.0026) on cortical activation. Once again, Ti did not change on the state transition, but T decreased from 0.288 ± 0.006 to 0.261 ± 0.004 sec, a change of 9.4 ± E  l.l%(p=0.0005). Systemic MK-801 in rats breathing air (n=6) significantly reduced the increase in ventilation normally seen on cortical activation by limiting the increases in both frequency and tidal volume. The increase in total ventilation was only 7.4 ± 1 . 9 %, (p=0.03) compared to a 20% increase prior to MK-801.  After MK-801 in rats breathing  a hypoxic gas mixture, the pattern of breathing on cortical activation changed. Although there wasn't a significant increase in total ventilation, frequency decreased (7.0 ± 2.7%  97  (p=0.058)) and tidal volume increased (14.0 ± 2.7% (p= 0.01)) due to a significant increase in inspiratory time. (After MK-801, T i increased 34.8 ± 13.4 % in State I, and 20.1 ± 4.9 % in State III, see Table 3.1). The fact that we did not observe any State I in rats breathing 5% CO2 after injection of MK-801 leaves us unable to assess the effects of cortical activation on breathing pattern in hypercapnia.  98  Pre-MK801  Air  12% 5% 02 CO2  Air  12% 5% 0 2 CO2  Fig. 3.6: The effects of cortical activation (the change from State III to State I), on breathing pattern in rats breathing air, 12% 0 in nitrogen and 5% C 0 in air. The 2  2  changes are shown as % change from State III to State I. * Significantly different from State III.  99  3.3.6. Effects of MK-801 on the Hypoxic Ventilatory Response Since systemic MK-801 had caused a significant decrease in ventilation in rats breathing air, would it also decrease the response to hypoxia? To answer this question, I compared the response to hypoxia before and after MK-801. Prior to injection of M K 801 the response to hypoxia in State I was characterized by increases in frequency, tidal volume and total ventilation (11.9 ± 3.4% (p=0.006), 19.2 ± 5.6% (p=0.007) and 32.9 ± 6.9% (p=0.002) respectively) (Fig. 3.7). In State III the increase in total ventilation was greater at 43.4 ± 8.2% (p=0.0008), caused by increases in frequency of 22.8 ± 6.5% (p=0.006) and tidal volume of 18.0 ± 6.1% (p=0.017). Inspiratory and expiratory times decreased by 16.6 ± 5 . 6 % and 20.2 ± 3 . 1 % respectively, which accounts for the greater frequency increase in State III. (See Table 3.1) The hypoxic ventilatory response after administration of MK-801 was measured as the difference between the breathing pattern in rats breathing air after MK-801 and breathing a hypoxic gas mixture after MK-801. In State I, there was a trend to an increase in tidal volume and total ventilation on the introduction of the hypoxic gas, but no increase in frequency of breathing (n=3)(Fig. 3.7). In State III (n=9), there was an increase in f of 10.6 ± 2.3% (p=0.001) and V of 12.9 ± 4.6 % (p=0.03) to give an R  T  overall increase in V TOT of 25.1 ± 4.6% (p=0.0002). Ti and T both decreased E  significantly (10.2 ± 3.7 % (p=0.001) and 6.4 ± 4.8% (p=0.01) respectively). (Table 3.1) The overall effect of the hypoxic exposure in State III was to bring the total ventilation up to the level seen in rats breathing air before the injection of MK-801; ventilation in State III in air was not significantly different from ventilation in State III hypoxia after M K 801.  100  Fig. 3.7. The effect of 12% oxygen in nitrogen on breathing pattern in urethane anaesthetized rats in both States I and III before and after the injection of MK-801. The values are given as % change from air to a hypoxic gas mixture and from air after M K 801 injection to hypoxia after MK-801. * Significantly different from air or air plus M K 801. (n=ll)  101  3.3.7. The Effect of MK-801 on the Time Domains of the HVR It has been shown that blockade of N M D A r reduces the response to hypoxia, but an analysis of the effects of MK-801 on the time domains of the H V R has not, to our knowledge, been carried out. In Fig. 3.8 the changes in ventilation occurring during exposure to hypoxia and during the post-hypoxic period (n=6) before and after MK-801 injection are compared. Before MK-801, breathing frequency (Fig 3.8A) increased immediately and maximum frequency was reached within 40 seconds, accompanied by a change into State I. This increase in frequency was maintained for up to 120 seconds and then declined slowly, although it always remained above the frequency of respiration of rats breathing air. After the hypoxic gas was discontinued, the frequency of respiration declined immediately and fell below the frequency of breathing prior to hypoxia (post hypoxic frequency decline; PHFD), accompanied by a change into State III. After 45 minutes post-hypoxia the frequency had still not returned to the pre-hypoxia level, although short intervals of cortical activation were seen after -20 minutes. The patterns of change seen in tidal volume and total ventilation before MK-801 mirrored those seen in breathing frequency, an abrupt increase followed by roll-off. The changes in tidal volume were not significant, but V TOT increased significantly reaching a maximum at 40 seconds. The decline over the course of the hypoxic exposure was small and not significant. Post-hypoxia there were significant and immediate decreases in tidal volume and total ventilation, with no significant changes over the post-hypoxic period. MK-801 caused the rats to go into State III. When hypoxia was administered there was not cortical activation and the acute response was slower, with the maximum increase in frequency now occurring at 80 sec. Frequency then declined until it was not  102  significantly different from the pre-hypoxic frequency. The breathing frequencies during the post-drug hypoxic response were significantly lower than those seen pre MK-801. On return to breathing air, the frequency of respiration of the MK-801 treated rats did not change significantly, and they remained in State III. As with the changes in breathing frequency following MK-801, tidal volume and total ventilation increased transiently on exposure to hypoxia, but by the end of the hypoxic exposure both variables had decreased to pre-hypoxic levels. Again, on return to breathing air, tidal volume and total ventilation remained at these initial levels.  103  A  Hypoxia  Post-hypoxia  160 -,  Time (sec)  T i m e (sec)  Fig. 3.8: Changes in ventilation during exposure to hypoxia and during the post-hypoxic period before and after injection of MK-801 (n=7). The first value was the value 20 seconds before the start of the hypoxic exposure. The last value during hypoxia was the value 20 seconds before hypoxia was discontinued. * Values post MK-801 that are significantly different from values pre MK-801. Q pre-MK-801  ^  post-MK-801  104  3.3.8. The Effects of MK-801 on the Hypercapnic Ventilatory Response The finding that systemic MK-801 had an effect on the response to hypoxia led to an evaluation of the effect of MK-801 on the response to CO2. Prior to injection of M K 801 the response to hypercapnia in State I was characterized by increases in tidal volume and total ventilation (54.4 ± 5.8% (p=0.00008) and 57.0± 6.6% ( p « 0 . 0 0 1 ) respectively) (Fig. 3.9). Frequency did not change significantly, nor did Ti or T E .  In State III the  increase in total ventilation was 61.5 ± 12.4% (p=0.006), caused by an insignificant increase in frequency of 5.3 ± 2.3% (p=0.07) and a significant increase in tidal volume of 52.7 ± 10.4% (p=0.005). Inspiratory and expiratory times did not change significantly (See Table 3.1). The hypercapnic response after administration of MK-801 was measured as the difference between the breathing pattern in rats breathing air after MK-801 and hypercapnia after MK-801. We did not see State I after MK-801 so cannot comment on the effects of drug in that state. In State III, there was an increase in V of 33.0 ± 12.5 % T  (p=0.003) to give an overall increase in V TOT of 31.25 ± 6.1% (p=0.01). Frequency did not change significantly, nor did T and T (Table 3.1). Although MK-801 caused a r  E  decrease in total ventilation in rats breathing air, the relative increase in ventilation on exposure to C 0 was of the same magnitude and pattern as before MK-801 injection. 2  105  State I Pre-MK801 80 -,  State III Pre-MK801  P o s t MK801  Fig. 3.9: The effect on breathing of exposure to 5% CO2 in air before and after MK-801 injection. Note that no State I is seen during hypercapnia after the injection of MK-801. The values are given as a % change from values in air. * Significant difference from values in air or values in air plus MK-801 (n= 6).  106  3.3.9. The response of Heart Rate to State Changes and MK-801 In rats breathing air, heart rate was not significantly different between States I and III, before or after MK-801 (Table 3.2). Heart rate was significantly greater in State III in hypoxia than in State I, although this difference was lost after MK-801. Rats breathing a hypercapnic gas mixture showed a significant decrease in heart rate on going from State I to State III but MK-801 injection did not change heart rate in State III.  Table 3.2: Heart Rate measured before and after the administration of MK-801  Before MK-801  After MK-801  State I  State III  State I  State III  Air  346 ± 5  341 ± 7  364 ± 19  339 ± 1 1  Hypoxia  360 ±10  414±25*  368 ± 13  357 ±13.5  Hypercapnia  3  5  8 ±  4  333 ± 8*  A  N/A  330 ± 7  Heart rates are given as beats/minute ± S.E.M. * significantly different from State I. A  Significantly different from air in the same state.  107  3.4. Discussion Our goal was to determine the effects of blockade of N M D A r through systemic administration of MK-801 on breathing pattern and cortical activation state using the urethane anaesthesia model of sleep and wake. As has been previously reported, the urethane anaesthetized rats cycled between E E G states that looked similar to arousal states seen in unanaesthetized rats. States I and III resembled the E E G traces of awake (high frequency, low amplitude waves) and slow wave sleep  (low frequency, high amplitude waves), respectively (Grahn and Heller,  1989; Grahn et al., 1989; Hunter and Milsom, 1998, Boon et al., 2004). The breathing pattern also varied with these states such that rats in State I breathing air had a higher frequency and larger tidal volume than did rats in State III. 3.4.1. Role of NMDA-type glutamate receptors in determining state It has been reported that neurons with N M D A r are found in the pontine reticular formation, which is involved with the determination of cortical activation state (Steriade 1988). It has also been shown that cortical activation state influences breathing pattern in both freely behaving and urethane-anaesthetized rats and ground squirrels (Pappenheimer, 1977; Hunter and Milsom, 1998; Hunter et al., 1998; Boon et al., 2004, Chapter 2). We examined these two findings by assessing breathing pattern in relation to cortical activation state before and after blockade of N M D A r with MK-801. After systemic injection of MK-801, rats cycled into States III and IV. The finding of an almost immediate transition into State III differs from previous reports in unanaesthetized and pentobarbital anesthetized rats (Campbell and Feinberg, 1996). While previous studies report a dominance of N R E M delta sleep following MK-801  108  injection, this occurred after a period of wakefulness with motor intoxication (increased locomotion, stereotypic head swinging and ataxia) that lasted about 3 hours. If the M K 801 was injected during pentobarbital anaesthesia (40 mg/kg), enhanced N R E M delta sleep was not observed until the animals came out of anaesthesia, and progressed through the same motor intoxication, in the same time frame as MK-801 injected rats that had not been anaesthetized (Feinberg and Campbell, 1997). With MK-801 administration during urethane anaesthesia in the present study, motor intoxication was not seen and the stimulation of N R E M delta-like activity occurred immediately. The action of urethane differs from that of the barbiturates in that pentobarbital enhances the activity of  GABAA  receptors by more than 100% with little effect on glycine or N M D A r , while urethane affects both excitatory and inhibitory neurotransmitter receptors (Hara and Harris, 2002). At deep anaesthetic doses (higher than the doses used in the present study) urethane potentiates the function of nicotinic acetylcholine receptors, G A B A A receptors and a i glycine receptors, but inhibits the N M D A and A M P A receptors (Hara and Harris, 2002). Thus MK-801 and urethane would have synergistic actions on the N M D A r ' s , and the potentiation of the G A B A  A  receptors could prolong and magnify the effects of the M K -  801. Campbell and Feinberg (1999) found that the N R E M delta sleep pattern of MK-801 treated rats closely resembled the N R E M sleep pattern seen in sleep-deprived rats. Our results are similar. The power spectra of the State III E E G before and after MK-801 were similar, with highest power in the bandwidth for 5 waves, below 4Hz and a suppression of activity in the 8, a and (3 bandwidths (as is seen in N R E M sleep). These data suggest that cortical activation is, in part, mediated via NMDA-type glutamatergic processes, presumably in the pontine reticular formation.  109  The appearance of State IV was most likely due to a decrease in brain temperature as has been reported by others (Grahn et al., 1989; Buchan and Pulsinelli, 1990; Busto et al., 1987). 3.4.2. Role of NMDA-type glutamate receptors in determining the effects of changes in state on breathing Cortical activation (i.e. the transition from State III to State I) in animals breathing air was accompanied by significant increases in all respiratory variables. M K 801 almost eliminated this effect due to a greater depression of breathing in State I than State III, suggesting that the effects of cortical activation on breathing are mediated by N M D A r stimulation. Since MK-801 blockade of N M D A r depressed breathing within minutes, as reported by Cassus-Soulanis et al. (1995), and also caused a transition into State III, which is also accompanied by a reduction in breathing (Lydic and Baghdoyan, 2002; Boon et al., 2004, Chapter 2), it is likely that part of the decrease in ventilation due to blockade of N M D A r is due to the effect of MK-801 on arousal state. Most reports on the effects of MK-801 on breathing have not considered cortical activation state as a factor. However, Lydic and Baghdoyan (2002) reported that systemic Ketamine (a dissociative anaesthetic known to block N M D A r ) and MK-801 reduced breathing frequency and decreased acetylcholine release in the medial PRF. In addition, Gilbert and Lydic (1994) reported that pontine cholinergic transmission altered the excitability of pontine respiratory neurons in the parabrachial region. These findings lead to the conclusion that blockade of N M D A r will affect breathing not only through a direct effect on respiratory  110  neurons, but also indirectly through the reduction in acetylcholine in the mPRF and its effects on respiratory-related neurons in the adjacent parabrachial respiratory region. 3.4.3. The effects of changes in state on the HVR While systemic MK-801 largely eliminated the effects of cortical activation on ventilation in animals breathing air, cortical activation in hypoxic animals caused a change in breathing pattern. Before MK-801, the effect of cortical activation on ventilation in hypoxic animals was small. The small increases in f and V T seen when R  the animals went from State III to State I under control hypoxic conditions were replaced, in rats injected with MK-801, by a decrease in frequency due to a longer Ti in State I than in State III and a large increase in tidal volume. Thus, while overall ventilation did not change between states, the change in breathing pattern was significant. The normal activation of N M D A r requires three events to occur within a short time period. Glutamate must occupy the glutamate-binding site on the N M D A r . Glycine must bind to the glycine-binding site on the N M D A r and a non-NMDA receptor must be activated to depolarize the neuronal membrane and release the M g  + +  from the ion  channel. MK-801 works by binding within the ion channel and preventing the flow of ions through the channel (Foster and Wong, 1987), but MK-801 can only enter the ion channel when it is open. The longer inspiratory time seen in State I of the hypoxic response after MK-801 injection would support the idea that blockade of N M D A r might be more effective during situations where there is strong stimulation of neurons by glutamate, known to be released during the H V R , and by acetylcholine known to be released during wakefulness. The wakefulness stimulus coupled with the hypoxic stimulus together could result in the opening of the ion channel, allowing the MK-801 to  Ill  enter resulting in a more effective blockade of N M D A r in State I than in State III. As the MK-801 reduces the production of acetylcholine in the mPRF, the wakefulness stimulus would be lost and the effectiveness of the blockade could decrease. 3.4.4. Role of NMDA-type glutamate receptors in determining the hypoxic ventilatory response As reported by Powell et al. (1998) the hypoxic response has time domains, with an acute response occurring within the first 20 seconds of exposure, followed by a small decrease in frequency (short term depression), and an increase in tidal volume (short-term potentiation). Thereafter, there is a decrease in both tidal volume and frequency resulting in hypoxic ventilatory decline (HVD), although the ventilation still remains above that of an animal breathing room air. When the hypoxic stimulus is removed, there is a decrease in frequency to levels below those seen prior to hypoxia. This post-hypoxic frequency decline is alleviated over time. On the basis of our data, it appears that NMDA-type glutamate receptor-mediated processes play a role in the acute response to hypoxia, in the continuing response to hypoxia and in the post-hypoxic frequency decline. The acute response (immediate increases in frequency and tidal volume) was delayed after MK-801 injection, and the increases in frequency and tidal volume were not maintained but declined steadily to pre-hypoxia levels. As a consequence, in the presence of MK-801, the mean H V R was eliminated over time in both states. It has been reported that G A B A and taurine levels increase over the course of the H V R (Hoop et al., 1999), and that MK-801 blockade of N M D A r inhibits the production of N O in the NTS (Haxhiu et al., 1995). Gozal et al. (1996) have shown that N O is important in sustaining ventilatory output during hypoxia. It would seem reasonable to  112  suggest that the increase in levels of inhibitory neurotransmitters caused by the hypoxia, and the decrease in N O release due to MK-801 blockade of N M D A r on neurons in the NTS together result in the decrease in frequency and tidal volume. Therefore N M D A type glutamate receptor-mediated processes play a role in maintaining the frequency and tidal volume responses to hypoxia. The fact that there is still an acute H V R would indicate that other neurotransmitters and receptors are also involved, but respond more slowly than the N M D A r . In addition, after MK-801 injection there are changes in cortical activation state distribution. During hypoxia before MK-801, the animals were constantly being aroused by the hypoxia and showed only short periods of State III. After MK-801, the arousal potential of hypoxia was diminished, and the animals were in States III or IV all of the time. Since hypoxic sensitivity is normally greater in State III, the decline in ventilation seen over time cannot be attributed to state changes. In the presence of MK-801, post-hypoxia frequency decline was not seen, as has also been reported by Coles et al. (1998). They reported that the frequency of respiration after hypoxia when MK-801 was present was higher than that seen after hypoxia when no MK-801 had been injected. Our results were different, but there were also a number of differences in our experimental preparation. In their experiments the rats were anaesthetized with chloral hydrate and pentobarbital, they were vagotomized and their hypoxia level was severe (8% oxygen in nitrogen). They used a very short exposure time, 30-90 sec, so their measurements were taken primarily during the acute response to hypoxia when build up of inhibitory neurotransmitters would not have taken place. In our experiments, the rats were not vagotomized so the frequency of respiration was not  113  initially reduced, and we used 12% oxygen in nitrogen because we found that if we used a lower oxygen concentration, the rats would die after 5 to 10 minutes because of the large suppression of breathing. The time course of our hypoxic exposure was quite long, ~ 40 minutes, and we saw a decrease in frequency of breathing over time until the frequency at the end of the hypoxic exposure was at the same level as that prior to hypoxia. In effect, the hypoxic response was lost. Since the rats were in State III for the whole time, this decline is not due to state changes. When air was re-introduced, frequency remained at the same low level that it had reached during hypoxia and did not decline further. In untreated rats the frequency and tidal volume fell to the same levels as those of the MK-801 treated rats, accompanied by a change into State III. This suggests that a change of state could contribute to post-hypoxia frequency decline. It should be noted that prior to MK-801, removal of the hypoxic gas mixture caused the rats to cycle into State III immediately. Some of the decline in frequency after hypoxia could be due to the change in cortical activation state. With time, the untreated rats cycled into States I and II, which would account for some of the increase in frequency and tidal volume during the post-hypoxia air exposure. MK-801treated rats were in State III or IV during hypoxia and remained in those states even after air was re-introduced. This could account for some of the depression of breathing and the lack of recovery. 3.4.5. Role of NMDA-type glutamate receptors in determining the hypercapnic ventilatory response and the effects of changes in state on the hypercapnic ventilatory response Since CO2 (5% in air) has not been found to produce cortical arousal, (Pappenheimer, 1977), and since we found very little State I even with hypoxia after M K -  114  801, we were not surprised at the lack of State I in CO2 after MK-801. There were significant increases in tidal volume and total ventilation before and after MK-801 in State III, and no change in the response to hypercapnia after MK-801 as also reported by Pierrefiche et al. (1996) and Ohtake et al. (1998). In studies where a decrease was seen in ventilation with hypercapnia plus MK-801 (Nattie et al., 1993; Harris and Milsom, 2001) it is possible that this arose as a result of the reduced ventilation that occurs in State III since cortical activation states were not being monitored. It is also likely that there are species differences in the response to MK-801. For example, Harris and Milsom (2001) found that MK-801 caused cortical activation in squirrels as opposed to the sedative effects that are found in rats. In addition, they reported no change in total ventilation during hypercapnia after MK-801, but a difference in the pattern of breathing. That being said, while MK-801 has significant effects on the response to hypoxia, it does not seem to have had direct effects on the response to hypercapnia in the rats in the present study. 3.5. Conclusions In summary, our results indicate that systemic blockade of N M D A r results in a reduction in ventilation, primarily through a decrease in tidal volume in both States I and III. It would seem that while NMDA-type glutamate receptor mediated processes are involved in the inspiratory off-switch in both States I and III, changes in T E are seen primarily when the animals cycle into State III. Blockade of N M D A r also causes a loss of the wakefulness stimulus, which contributes to the depression of breathing seen in rats breathing air or a hypoxic gas mixture. Neurons with NMDA-type glutamate receptors are also involved in the H V R , in the acute phase, in the continuing response and in the  115  post-hypoxia period, some of which is due to the sedative effects of the MK-801. The response to carbon dioxide does not seem to require the activity of N M D A r except insofar as this response is modified by the effects of wakefulness on the inspiratory offswitch. Having examined the effects of systemic MK-801, we can now proceed to an evaluation of which of these effects are due to NMDA-type glutamate receptor-mediated processes in the Parabrachial/Kolliker Fuse respiratory complex in the pons.  116  3.6. References Bianchi, A . L . , Denavit-Saubie, M . , Champagnat, J., 1995. Central control of breathing in mammal: neuronal circuitry, membrane properties, and neurotransmitters. Physiol. Rev. 75: 1-46. Bonham, A . C . 1995. Neurotransmitters in the CNS control of breathing. Respir. Physiol. 101:219-230. Boon, J.A., Garnett, N.B.L., Bentley, J.M., Milsom, W.K., 2004. Respiratory chemoreflexes and effects of cortical activation state in urethane anesthetized rats. Respir. Physiol. Neurobiol. 140 (3): 243-256. Borday, V., Foutz, A.S., Nordholm, L., Denavit-Saubie. 1998. Respiratory effects of glutamate receptor antagonists in neonate and adult mammals. European Journal of Pharmacology 348: 235-246. Buchan, A., Pulsinelli, W.A., 1990. Hypothermia but not the N-Methyl-D-Aspartate antagonist, MK-801, attenuates neuronal damage in Gerbils subjected to transient global ischemia. J. Neurosci. 10(1): 311-316. Busto, R. Dietrich, W.D., Globus, M , Y . -T., Vlades, I., Scheinberg, P., Ginsberg, M.D., 1987. Small Differences in Intra-ischemic Brain Temperature Critically Determine the Extent of Ischemic Neuronal Injury. J. Cerebral Blood Flow and Metab. 7:729-738. Campbell, I.G.,and Feinberg, I., 1996. Noncompetitive N M D A channel blockade during waking intensely stimulates N R E M 8 '. J..Pharmacol. & Exp. Ther. 276:737-742. Campbell, I.G. and Feinberg, I. 1996 N R E M Delta stimulation following MK-801 is a response of sleep systems. J. Neurophysiol. 76(6): 3714-3720. Campbell, I.G, Feinberg, I. 1999. Comparison of MK-801 and sleep deprivation effects on N R E M , R E M , and waking spectra in the rat. Sleep, 22 (4): 423-432. Cassus-Soulanis, S., Foutz, A.S., Denavit-Saubie, M . , 1995. Involvement of N M D A r ' s in inspiratory termination in rodents: effects of wakefulness. Brain Res. 679: 25-33. Coles, S.K., Ernsberger, P., Dick, T.E., 1998. A role for N M D A r ' s in posthypoxic frequency decline in the rat. Am. J. Physiol. 274: R1546-R1555. Connelly, C.A., Otto-Smith, M.R., Feldman, J.L., 1992. Blockade of NMDAr-channels by MK-801 alters breathing in adult rats. Brain Res. 596: 99-110. Dick, T.E., Coles, S.K., 2000. Ventrolateral pons mediates short-term depression of respiratory frequency after brief hypoxia. Respir. Physiol. 121: 87-100.  117  Feinberg, I., Campbell, I. G., 1997. Co-administered pentobarbital anesthesia postpones but does not block the motor and sleep E E G responses to MK-801. Life Sciences: 60 (12) P L 217-222. Foutz, A.S., Pierrefiche, O., Denavit-Saubie, M . , 1994. Combined blockade of N M D A and non-NMDAr's produces respiratory arrest in the adult cat. NeuroReport: 481-484. Foster, A.C., and Wong, E.H.F., 1987. The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain. Br. J. Pharmacol. 91: 403-409. Gilbert, K.S., Lydic, R., 1994. Pontine cholinergic reticular mechanisms cause statedependent changes in the discharge of parabrachial neurons. Am. J. Physiol. 266: R13650. Gozal, D., Torres, J.E., Gozal, Y . M . , Littiwin, S.M., 1996. Effect of nitric oxide synthase inhibition on cardiorespiratory responses in the conscious rat. J. Appl. Physiol. 81:20682077. Gozal, D., Gozal, E., Simakajornboon, N . , 2000. Signaling of the acute ventilatory response in the nucleus tractus solitarius. Respir. Physiol. 121: 209-221. Grahn, D.A., Redeke, C M . , Heller, and H.C., 1989. Arousal state vs. temperature effects on neuronal activity in subcoeruleus area. A m J. Physiol. 256: R840-R849. Grahn, D.A., Heller, C , 1989. Activity of most rostral ventromedial medulla neurons reflect E E G / E M G pattern changes. Am. J. Physiol.257: R1496-R1505. Hara, K., and Harris, R.A. 2002. The anesthetic mechanisms of urethane; the effects on neurotransmitter-gated ion channels. Anesth Analg. 94: 313 -8 Harris, M.B., Milsom, W.K., 2001. The influence of NMDAr-mediated processes on breathing pattern in ground squirrels. Respir. Physiol. 125: 181-197 Haxhiu, M . A . , Chang, C.H., Dreshau, L A . , Erokwu, B., Prabhakar, N.R. and Cherniack, N.S. 1995. Nitric oxide ventilatory response to hypoxia. Respir. Physiol. 101:257-266. Hoop, B., Beagle, J.L. Maher, T.J., Kazemi, H., 1999. Brainstem amino acid neurotransmitters and hypoxic ventilatory response. Respir. Physiol. 118: 117-129. Hunter, J.D., McLeod, J.Z., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia: respiratory reflexes. Respir. Physiol. 112: 83-94. Hunter, J.D., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia: cardio-respiratory effects. Respir. Physiol. 112: 71-81.  118  Lydic, R. and Baghdoyan, H.A. 2002. Ketamine and MK-801 decrease acetylcholine release in the pontine reticular formation, slow breathing, and disrupt sleep. Sleep, 25:617-622. McCrimmon, D.R., Dekin, M.S., Mitchell, G.S., 1995. Glutamate, G A B A , and serotonin in ventilatory control. In: Dempsey, J. A. Pack, A.I., (Eds.) Lung Biology in Health and Disease. Regulation of Breathing vol. 79: Central Nervous System, New York: Marcel Dekker, pp. 151-218. Mizusawa, A., Ogawa, H., Kikuchi, Y . , et al., 1994. In vivo release of glutamate in nucleus tractus solitarii of the rat during hypoxia. J. Physiol. Lond. 478: 55-66. Monaghan, D.T., Cotman, W., 1985. Distribution of N-methyl-D-aspartate sensitive L [3H] glutamate-binding sites in rat brain. J. Neurosci. 5: 2909-2919. Monteau, R., Gauthier, P., Rega, R., Hilaire, G., 1990. Effects of N-methyl-D-aspartate (NMDA) antagonist MK-801 on breathing pattern in rats. Neuroscience Letters 109: 134139. Nattie, E.E., Godvin, M . , L i , A., 1993. Retrotrapezoid nucleus glutamate receptors: control of C 0 2 sensitive phrenic and sympathetic output. J. Appl. Physiol. 74: 29582968. Ohtake, P.J., Torres, J.E., Gozal, Y . M . , Graff, G.R., Gozal, D., 1998. N M D A r s mediate peripheral chemoreceptor afferent input in the conscious rat. J. Appl. Physiol. 84: 853861. Ohtake, P.J., Simakajornboon, N . , Fehniger, M.D., Xue, Y . D . , Gozal, D., 2000. N Methyl-D-aspartate receptor expression in the nucleus tractus solitarii and maturation of hypoxic ventilatory response in the rat. A m J. Respir. Crit. Care Med. 162: 1140-1147. Pappenheimer, J.R., 1977. Sleep and respiration of rats during hypoxia. J.Physiol. 266: 191-207. Petralia, R.S., Yokotani, N . , Wenthold, R.J., 1994. Light and electron microscope distribution of the N M D A r subunit N M D A R 1 in the rat nervous system using a selective antibody. J. Neurosci. 14: 667-696. Pierrefiche, O., Foutz, A.S., Champagnat, J., Denavit-Saubie, M . , 1996. N M D A and nonN M D A receptors may play distinct role in time mechanisms and transmission in the feline respiratory network. J. Physiol. (Lond.) 474: 509-523. Powell, F L , Milsom, W.K., Mitchell, G.S., 1998. Time domains of the hypoxic ventilatory response. Respir. Physiol. 112: 123-134.  119  Rechtshaffen, A., Kales, A., Berger, R.J., et al., 1968. A Manual of Standardized Terminology, Techniques and Scoring system for Sleep Stages in Human Subjects. U.S. Government Printing Office, Washington, D.C. Schoepp, D.D. and Conn, P J . , 1993. Metabotropic glutamate receptors in brain function and pathology. Trends Pharmacol. Sci. Jan: 14(1): 13-20 Steriade, M . , 1988. New vistas on the morphology, chemical transmitters and physiological actions of the ascending brainstem reticular system. Archs. Ital. Biol. 126: 225-238. Vardhan, A., Kachroo, A., Sapru.N., 1993. Excitatory amino acid receptors in commissural nucleus of the NTS mediate carotid chemoreceptor responses. Am. J. Physiol 264: R41-R50.  120  Chapter 4  Effects of Cortical Activation State on Respiration and Respiratory Chemoreflexes: Role of the Parabrachial/Kolliker Fuse Complex  121  4.1. Introduction: The production of an eupneic breathing pattern in intact animals requires the normal functioning of a ponto-medullary respiratory network (St. John and Paton, 2004). While there are neurons in the medulla that have a pacemaker function in reduced in vitro preparations (the pre-Bdtzinger complex, Smith et al., 1991, and the para-facial respiratory group (pFRG) Onimaru and Homma, 2003), in vivo the rhythm generated by these neurons is modified by, amongst other things, pulmonary stretch receptor feedback from the lungs via the Hering-Breuer reflex (vagal input to the NTS pump neurons), and by neural input from respiratory-related neurons in the pons, the rostral portion of the ventral respiratory group (rVRG) and the Botzinger complex (Alheid et al., 2004, for review). In vagotomized animals, without input from the rostral pons, the respiratory rhythm is characterized by apneustic breaths with prolonged discharges of inspiratory neurons in the pre-Bdtzinger complex and the r V R G . In some studies, if the input from both the rostral and caudal pons and the vagus are removed, the respiratory pattern becomes one of gasp-like breaths (Lumsden, 1923; St. John and Paton, 2000) while in other studies, breathing returns towards eupnea (Lieske et al., 2000). Neurons with respiratory-related activity have been localized to the dorsolateral pons (dl pons) in the region of the medial and lateral parabrachial complexes and in the Kolliker Fuse nucleus (KF) (Alheid et a l , 2004 for review). These neurons have been identified by single unit recordings and found to have a phasic discharge in relation to respiratory cycles superimposed on a tonic firing level (Cohen & Wang, 1959; Alheid et al., 1994, for review). Historically this region was called the pneumotaxic centre (Lumsden, 1923), then the pontine respiratory group (PRG) (Feldman et al., 1976) and,  122  more recently, the parabrachial/Kdlliker-Fuse complex (PBrKF) (Alheid et al., 2004). Respiratory related neurons have also been identified in other parts of the pons including the ventrolateral pons (vl pons) (the A5 region) (Jodkowski et al., 1994), in the intertrigeminal region (ITR) (Chamberlin, 2004), and in the pontine reticular formation (PRF) (Fung and St. John, 1994b). Lesioning, electrical stimulation, chemical stimulation, and neurotransmitter blockade of the dorsolateral pons (dl pons) (Alheid et al., 2004) have identified a heterogeneous population of neurons, some of which are involved in modulating respiratory frequency through connections with neurons in the D R G and the V R G (Herbert et al., 1990; Chamberlin, 2004 for review). Localization of NMDA-type glutamate receptors (NMDAr) to the PBr and K F areas (Monaghan and Cotman 1985; Petralia et al., 1994) has led to numerous experiments that have explored the role of the NMDA-type glutamate receptor-mediated processes in the control of respiratory pattern (Foutz et al., 1989; Connelly et al., 1992; Ling et al., 1994; Cassus-Soulanis et al., 1995; Ohtake et a l , 1998). The consensus is that neurons with NMDA-type glutamate receptors are part of the inspiratory off-switch mechanism, which along with vagally mediated stretch-receptor input to the D R G , control inspiratory time. Blockade of N M D A r in the PBrKF leads to an increase in Ti, and to apneusis in vagotomized animals, although other site-specific effects have been documented (Chamberlin, 2004, for review). The pontine reticular formation also has a high concentration of neurons with N M D A r (Monaghan and Cottman, 1985), and it has been shown that there are neurons in the pedunculopontine tegmentum (PPT) in the peribrachial region of the pons that have  123  N M D A r . When these neurons in the PPT are blocked with MK-801, the secretion of Acetyl choline in the PRF decreases, and both wakefulness and R E M sleep are reduced (Datta et a l , 2001). State also has an effect on breathing pattern and total ventilation in both anaesthetized and unanaesthetized animals (Hunter and Milsom, 1998; Hunter et al., 1998; Boon et al., 2004, Chapter 2). Blocking N M D A r in conscious animals affects both their cortical activation state and their breathing pattern (Lydic and Baghdoyan, 2002; Chapter 3). Few experiments in which N M D A r antagonists (e.g. MK-801) are used consider the effect of state on the documented reduction in ventilation that accompanies blockade of N M D A r . In a previous study (Chapter 3) we examined the effects of systemic MK-801 on state in the urethane model of state changes, and on breathing pattern in rats breathing air, 10% oxygen in nitrogen and 5% carbon dioxide in air. Systemic MK-801 was found to reduce overall ventilation primarily by reductions in tidal volume accompanying the descent into State III. It was also found to slow the acute response to hypoxia, reduce the magnitude of the hypoxic response generally, and eliminate post-hypoxia frequency decline (PHFD). There was no effect on the response to hypercapnia other than that caused by the loss of State I. In this chapter, we report on experiments in which we injected MK-801 directly into the PBrKF to look at the specific effects of N M D A r blockade in this region on changes in state, and on state effects on ventilation and chemoresponses to hypoxia and hypercapnia.  124  4.2. Methods 4.2.1. Animal Care Sprague Dawley rats were obtained from the U B C animal care facility (University of British Columbia, Vancouver, B.C. Canada). The surgeries and protocols were carried out with the prior approval of the U B C Animal Care Committee and the Okanagan University College Animal Care Committee. Measurements were made using adult male Sprague Dawley rats in a time period from approximately 9 a.m. to 6 p.m. They were housed singly in the OUC animal care facility and allowed access ad libitum to food and water, supplemented from time to time with sunflower seeds and fruit. The rats were kept at 25° C, with a light/dark cycle of 12 hours. The lights came on at 8 a.m. At the time of the experiments, the average weight was 390 g, and the range was 325-452 g. After the experiment, the rats were euthanized with an IP injection of 1 ml. of Somnotol (65 mg/ml; Sodium pentobarbital, M T C pharmaceuticals). 4.2.2. Experimental Protocol: 4.2.2.1. Surgical preparation The animals were anaesthetized with 2% vaporous halo thane administered through a mask. The mask was attached to the airline with a T tube and the flow was vented to a fume hood. Then the rat was given an intraperitoneal (IP) injection of a 20% solution of Urethane (Sigma) in saline to a final dose of 1.3g/kg). The trachea was canulated below the larynx for the measurement of airflow. The canula was attached to the airline from which the mask had been removed and the animal was placed in a stereotaxic head frame (Kopf), adjusted such that the skull surface landmarks lambda and bregma were on the same horizontal plane. The halothane anaesthesia was slowly decreased, but was  125  maintained until all surgery was completed and the urethane had become effective; at least 45 minutes. Additional doses or urethane were administered IP if the rat responded .to a noxious toe pinch. Body temperature was maintained between 36 and 37° C by placing the rat on a servo-controlled heating pad. After injections of xylocaine (2% Lidocaine hydrochloride, Astra pharmaceuticals), a dorsal-longitudinal incision was made over the crown of the cranium, extending from the orbits to the lambdoid suture. Four electroencephalographic (EEG) electrodes were implanted in the skull as described in Hunter and Milsom (1998). Each electrode was fashioned from a length of insulated, multi-stranded, stainless steel wire ( A M Systems) soldered to a self-tapping stainless steel screw (00 x 3/16, Fine Science tools). The other end was soldered to a gold Amphenol pin. The Amphenol pins were inserted into an Amphenol pin strip. A pneumotach was installed in the outflow of the gas line attached to a Validyne pressure transducer (Validyne, DP 103-18). The signal was amplified with a Gould amplifier and then transmitted to a two-channel data acquisition system (AT-CODAS, DataQ Instruments) sampling at a frequency of 120 samples per second on each channel. The breathing signal was also recorded on a chart recorder. The E E G signal was amplified and recorded on a chart recorder and on the data acquisition system on a second channel. The signal was filtered with both low and high pass filters and a 60 Hz. filter to reduce noise. 4.2.2.2. Monitoring breathing pattern in air, hypoxia and hypercapnia. With the animal breathing air, the respiratory and E E G patterns were monitored until there were periods of both established State III and established State I (for  126  description of States, see Data Analysis below), and representative E K G traces were taken during both states. The air was then replaced with a gas mixture of 10% 02 in nitrogen (hypoxia), or 5% CO2 in air (hypercapnia)(each animal was exposed to only one of these gas mixtures). The respiratory and E E G patterns were monitored for at least 45 minutes on the gas. The animal was then switched back to air, and breathing was monitored until the breathing pattern approximated that seen before the animal was exposed to hypoxia or hypercapnia, at least 20 minutes post-hypercapnia, but at least 60 minutes post-hypoxia. Injections of saline and/or MK-801 were made as described in the following section while the animals were breathing air, and breathing was monitored for a further 45 minutes. Then the air was replaced with the same gas mixture of either 10%> oxygen in nitrogen or 5% CO2 in air used previously. Breathing was again monitored for approximately 1 hour and then the animal was returned to air. At the end of the experiment, Pontamine Sky Blue was injected bilaterally to mark the position of the canula. 4.2.2.3. Injection of Saline and MK-801 For injections of saline, MK-801 (dizocilpine maleate ((+)-5 methyl-10, 11dihydro-5H-dibenzo [a,d]cyclohepten-5,10 imine, Sigma) and Pontamine sky blue, the rat's head was rotated 24° around ear-bar zero (EBZ). Using a drill, windows in the bones of the skull were opened from just behind the lambdoid suture extending ~ 4 mm caudally and from ~ l m m to 3 mm on either side of the midline. The meninges were carefully dissected away to expose the top of the brain. Using a calibrated micromanipulator, the needle of a 10 pi Hamilton syringe (#701 with a 26s needle) was dropped into the opening in the skull 1.37 mm behind E B Z and 2 mm on either side of  127  midline to a depth of ~ 7.9 mm from the top of the brain. B y our calculations, this meant that we were injecting saline or drug into the region of the PBrKF (Paxinos and Watson, 1986). Both saline and MK-801 were injected in 0.5 ul increments over a period of about 3-4 minutes to give'a total volume of 1-2 ul of each on one side and then the needle was slowly withdrawn and repositioned on the other side where the injection protocol was repeated. After the animals were euthanized, the brains were removed and fixed in a solution of 10% formalin. The brains were then frozen and either serially sectioned at 100u intervals (n=13) or serially sectioned between the 7 cranial nerve and the 4 th  th  cranial nerve with sections of-30 um taken every 0.1mm (n=7). The slides were then stained with Cresyl Violet for Nissl substance. In 5 rats, the injections were found to have been placed more rostrally or more caudally than the desired area and they served as controls. There was no effect on breathing with these injections. 4.2.3. Data Analysis Cortical activation states were scored based on E E G profiles: State I (desynchronized cortical activity), State II (intermediate activity), and State III (synchronized cortical activity), with E E G activity resembling that seen in wake, light sleep and slow wave sleep, respectively as scored according to conventional criteria (Rechtschaffen et al., 1968). A l l arousal state data were scored in 30sec. epochs and classified according to the predominant state during that epoch. The percentage of total recording time spent in each state was then calculated from these data. To assess the breathing pattern during different E E G states, five to six segments of the recorded trace, 20 seconds in length, were chosen from areas of the establishedE E G states, and the data were analyzed and averaged. To assess the breathing pattern  128  during the response to hypoxia and after discontinuation of hypoxia, files were taken at timed intervals: 20 seconds immediately prior to hypoxia, at 20 second intervals for 200 seconds, then every five minutes until the end of the hypoxic exposure; 20 seconds immediately prior to the return to air, at 20 second intervals for 200 seconds following the return to air, and then at 5 minute intervals until the end of the experiment. Calculations were made for the frequency of respiration (fR), the voltage corresponding to the tidal volume, the time of inspiration (Ti) and the time of expiration ( T E ) . Calibrations were carried out using an electrically operated pump that delivered known volumes into the mask at a constant frequency. The voltage generated by these volumes was used to make a calibration curve which allowed the conversion of the experimental voltages into tidal volumes (Vj). The total ventilation ( V TOT) was calculated by multiplying the f x V . The values were converted to volumes in ml/1 OOg ( V T ) , or R  T  ml/min/lOOg ( V TOT) (ATPS). 4.2.4. Statistical Analysis: Comparisons were made between treatments in each animal using a Student T test for matched data with two tails. Means presented are overall estimated marginal mean values + S E M .  A l l of the data were then analyzed using the General Linear Model  multivariate analysis of variance (SPSS version 11.5, SPSS Inc. Chicago, Illinois). Multiple comparison tests (Bonferonni) were used to separate significant mean values. The timed sequences of the hypoxic and hypercapnic responses were analyzed using paired Student T Tests for matched data with two tails and with a One Way Repeated Measures A N O V A with a Bonferonni correction (Sigma Stat, Jandel Scientific). Differences were considered to be significant when p< 0.05.  129  4.3. Results 4.3.1. Respiratory and E E G traces As shown previously for ground squirrels (Hunter and Milsom, 1998; Hunter et al., 1998) and rats (Boon et al., 2004, Chapter 2) the urethane anaesthetized animals breathing air cycled between two stable E E G states, State I (Fig. 4.1 A) was characterized by low amplitude, high frequency E E G waves, with low 8 power, superficially similar to those seen in unanaesthetized awake animals. State III (Fig. 4.IB) was characterized by high amplitude, low frequency E E G waves, with high 8 power, similar to those seen in animals in slow wave sleep. The exposure to 10% oxygen in nitrogen also produced a characteristic pattern of sighs as has been shown previously (Boon et al., 2004) (Fig 4.1C). Injection of saline caused an initial disruption of the E E G trace and a small depression in ventilation, but this resolved in ~ 10-15 minutes and-ventilation was not significantly different from before the saline injection. Subsequent injections of MK-801 did not disrupt the E E G trace, however, after MK-801 injection, there was very little established State I. Within 5-8 minutes post-injection, all animals had cycled into State III whether they were breathing air or the hypoxic or hypercapnic gas mixtures. Sighing also became more frequent after MK-801, although not as frequent as during hypoxia (Fig 4. ID). Fig. 4.IE illustrates the respiratory and E E G traces for the same rat breathing a hypoxic gas mixture after injection of MK-801. Frequency of respiration and tidal volume both increased, as did the number of sighs (see also Table 4.1).  130  Fig. 4.1. Recordings of the E E G and the differential pressure signal (respiration) from one rat before and after the injection of MK-801 into the PBrKF. The top two traces were made during States I and III in a urethane-anaesthetized rat breathing air. The third trace was taken when the rat was breathing a hypoxic gas mixture (10% oxygen in nitrogen), before MK-801 injection into the PBrKF, and transitions between States I and III can be seen. The 4 and 5 traces are from the same rat after PBrKF injection of MK-801 in th  th  State III breathing air, and in State III breathing the hypoxic gas mixture.  131  A  State I Air Pre MK-801 Respiration  0.6 ml  EEG  <WllNfo^^ 10sec  B  State III Air Pre MK-801  C  State I & III Hypoxia Pre MK-801  D  State III Air Post MK-801  E  State III Hypoxia Post MK-801  Table 4.1: Mean values ± S E M for Frequency of respiration (fR.), Tidal volume ( V T ) , Total ventilation ( V TOT), Inspiratory time (Ti) and Expiratory time ( T E ) for Sprague Dawley rats breathing air, 10% O2 in N2 or 5% CO2 in air.  Gas Air  Hypoxia  State  Drug  fR  V  State I  Pre M K 801 Post MK-801  bpm 118.7 ±2.0 87.3* ±3.5  mls/lOOg 0.349 ±0.014 0.330 * ±0.014  State III  Pre M K 801  102.9# ± 1.8  Post MK-801 State I  State III  Hypercapnia  State I  State III  Ti sec  TE  mis/1 OOg 40.3 ± 1.8 28.5 * ± 1.3  0.249 ± 0.006 0.371 * ± 0.032  0.267 ± 0.008 0.331* ±0.017  0.342 ±0.012  34.2 # ± 1.3  0.303 # ± 0.008  0.289 # ±0.010  79.9*# ±3.0  0.360 *# ±0.016  27.5* ±2.0.  0.415*# ±0.03  0.35*# ±0.019  Pre M K 801  151.6 ±4.1  0.370 ±0.013  51.6 ±2.8  0.177 ± 0.005  0.218 ± 0.007  Post MK-801  122.3* ±5.3  0.452 * ±0.018  50.6 ±4.0  0.227* ±0.010  0.254 * ±0.012  Pre M K 801  125.7# ±2.2  0.397 ±0.016  45.7 ±3.3  0.214 # ± 0.009  0.259# ± 0.007  Post MK-801  114.9* ±5.1  0.456 * ± 0.026  52.3 * ±4.0  0.25* ±0.015  0.270 ±0.010  Pre M K 801  124.0 ±4.7  0.566 ± 0.059  71.0 ±8.1  0.218 ± 0.007  0.268 ±0.015  Post MK-801  104.6* ±6.6  0.48 ±0.03  48.4* ±3.2  0.267* ±0.02  0.327* ±0.021  Pre M K 801  115.3# ±3.9  0.51# ± 0.047  59.4# ±5.4  0.237 # ± 0.009  0.286# ±0.015  Post MK-801  93.5 ±5.8  0.464 ±0.05  43.6 ±5.3  0.279 ±0.03  0.367 ±0.03  # Significantly different from State I  T  V TOT  * Significantly different from pre-MK-801  sec  133  4.3.2. Time in State Because the injection of MK-801 suppressed state changes, we calculated the time spent in each of the states. Prior to the injection of MK-801, animals breathing air spent 44.5 ± 5.5% of the time in State I, 20.8 ± 4.3 % of the time in State II, and 34.7 ± 5.5 % of the time in State III (Fig. 2). After MK-801 injection the animals cycled into State III, where they spent 88.7 ± 4.5% of the time, significantly more than pre MK-801. Very small amounts of States I and II were seen, 1.7 ± 1.1% (significantly less than before drug) and 9.6 ± 4.5% respectively. During the exposure to 10% oxygen in nitrogen, although the pattern of the cycling between states was very different (hypoxia significantly disrupted the sleep-wake pattern and reduced the time in State III to short periods ranging from 20 to 80 seconds), the time in each state was not significantly different than was seen in air: State I, 47.8 ± 7.7%; State II, 22 ± 4.5 %; and State III, 30.2 ± 6.1%. Injection of MK-801 again caused the animals to cycle into State III where they spent 73.2 ± 11.8% of the time. Hypoxia did have some arousal potential, however, since after MK-801 the rats spent more (23.3 ±11.4 %) time in State II than when breathing air. We found that 5% C 0 in air caused significant arousal prior to injection 2  of MK-801. The animals spent 68.5 ± 5.7% of the time in State I, and only 16.45 ± 2.0% of the time in State III. Injection of MK-801 increased the time spent in State III to 61.9 ± 3.3%, while time spent in State I was decreased to 15.4 ± 3 . 1 % .  134  AIR  120 100 80 60 40 20 0 • State III 3 State II • State I  HYPOXIA  120 100 -*ro —•  w 80 c 60 <L> E F 40  T Or  20 0  co  120 100 • 80 •  5 *  2  5  mm  60 40 •  •  *  20 •  8  0 Pre MK-801  Post MK-801  Fig. 4.2. The percent time spent in States I, II and III in rats breathing air, 10% oxygen in nitrogen and 5% CO2 in air is shown before and after injection of MK-801 into the PBrKF region. * Indicates a significant difference from the time in this state before M K 801. S Indicates significant differences from air.  135  4.3.3. Placement of Injections Histological examination of the injected brains indicated that the injections (as indicated by the canula tract and the blue dye) were in the desired region (-8.72 to -9.16 mm caudal to bregma at a depth below the surface of the brain of between 6 and 9 mm) bilaterally in 13 animals and unilaterally in 7 animals (Fig. 4.3). The dye diffused out from the injection site in a circle with an average radius of ~ 0.5 mm. In all of these rats, there was a noticeable decrease in breathing frequency within 5 minutes of injection of MK-801. Since we did not find a significant difference between the reduction in frequency of respiration between the unilateral and bilateral injections orbetween the land 2 ul injections, the data from all of these animals has been combined. Larger injection volumes of saline disrupted the E E G trace for longer. Injections that were either more rostral or more caudal to sections shown in Fig. 4.3 did not cause a significant change in breathing.  Fig. 4.3: The position of injections is shown superimposed on schematic diagrams of serial sections of the brain (Paxinos and Watson, 1986). The first section is 8.72 mm caudal from Bregma, 0.28 mm rostral to the interaural line. The second section is 8.80 mm caudal from Bregma and 0.20 mm rostral to the interaural line. The third section is 9.16 mm caudal from Bregma and 0.16 mm caudal to the interaural line. The fourth section is 9.30 caudal to Bregma and 0.30 mm caudal to the interaural line. Successful bilateral injections are shown with a circle while unilateral injections are shown with a triangle. Abbreviations: A5 = A5 noradrenaline cells; K F = Kolliker Fuse nucleus; L P B = Lateral parabrachial nucleus; mcp = middle cerebellar peduncle; Mo5 ^ motor trigeminal nucleus; PnC = pontine reticular nucleus, caudal; PnO = pontine reticular nucleus, oral; py  =  pyramidal tract; s5 = sensory root trigeminal nerve.  Interaural 0.28 mm Bregma -8.72 mm  Interaural 0.20 mm Bregma -8.80 mm  Interaural -0.16 mm Bregma -9.16 mm  Interaural -0.30 mm Bregma -9.30 mm  137  4.3.4. Effect of MK-801 in rats breathing air Since systemic MK-801 injections (Chapter 3) resulted in a large decrease in tidal volume accompanied by an increase in inspiratory time, we analyzed the effects of PBrKF injection of MK-801 in an attempt to define the role of neurons with N M D A r in this region.  MK-801 injected into the pons in rats breathing air caused a 26.1 ± 2.5%  decrease in frequency due to increases in both Ti (47.5 ± 8.0%) and T  E  (25.8 ± 5.6%) in  State I (all p< 0.01) (Fig. 4.4). Tidal volume normalized for a lOOg rat increased by 10.8 ± 4.2 % (p= 0. 04) which resulted in a 17.0 ± 2.9% decrease in total ventilation (p< 0.01)(Fig. 4.4; see also Fig. 4.1 and Table 4.1). In State III, the pattern of decrease was similar in that f decreased by 21.9 ± 1.9% due to increases in Ti and T of 38.3 ± 6.1% R  E  and 22.9 ± 4.9% respectively (all p<0.01). Tidal volume increased by 5.8 ±3.6 % (which was not significant) to give an overall decrease in V TOT of 18.6 ± 3.6 % (p<0.01). These results were based on n=12 rats. 4.3.5. Effect of MK-801 on changes in breathing with changes in state Systemic MK-801 suppresses state transitions (Chapter 3). This is most likely due, at least in part, to its effects on N M D A r in the pontine reticular formation and/or the PPT. To determine if neurons in the PBrKF either directly affect state transitions or act as a relay centre for PRF and/or PPT stimulation of the transition to State I, we compared the changes in breathing pattern on the transition from State III to State I before and after MK-801 injection. Before MK-801 blockade of NMDA-type glutamate receptors in the PBrKF, the change from State III to State I in rats breathing air was accompanied by an increase in f of 15.7 ± 1.5% due to decreases in Ti and T of 17.2 ± 1.6% and 6.8 ± 1.6% respectively E  R  138  (all p<0.01) (n-29). Tidal volume did not change significantly on the transition from State III to State I, but there was an overall increase in total ventilation of 17.6 ± 1.9% (p < 0.01) (Fig. 4.5). After injection of MK-801, the change fromStatelll to State I still caused an increase in frequency of respiration but this was only half of the increase prior to M K 801 (8.1 ± 1.4%). Inspiratory time decreased by 14.9 ± 1.8% (p O.01), but expiratory time did not change and the small decrease in tidal volume of 6.3 ± 3.5%> was due to significant decreases in 5 rats while in the other 5 rats there was no change. Total ventilation did not change. In rats breathing a hypoxic gas mixture before MK-801, the change from State III to Statel caused a significant increase in frequency (15.4 ± 2.5%) and a decrease in tidal volume of 8.6 ± 2.6 % with the result that there was no significant change in total ventilation. (These measurements were made in the latter part of the exposure to hypoxia, not during the acute response). Inspiratory time decreased by 14.3 ± 2.4% and expiratory time decreased by 11.6 ± 2.0% (all p < 0.01) (fig. 5). After the injection of MK-801 into the PBrKF, there were no significant differences in f , V T or V TOT between States I and R  III (Fig. 4.5). When rats were breathing 5%> CO2 in air, there were significant increases in frequency of respiration, tidal volume and total ventilation with cortical activation (8.3 ± 1.5%, 9.0 ± 3.1% and 18.0 ± 3.1% respectively) due to decreases in Ti of 7.9 ± 2.0 and in T of 6.0 ± 1.8% (p< 0.05). Once again, injection of MK-801 removed the difference in E  ventilation that characterized the cortical activation before administration of the drug.  139  60 i  60 i  State I  State III  fR  V  T  TOT  V  "*"|  T  E  Fig. 4.4: The effects of MK-801 injected into the PBrKF are shown for rats breathing air in State I and State III. The changes are shown as % change from air before MK-801. * Indicates values that are significantly different from those prior to injection of MK-801. Abbreviations: f = frequency of respiration; V = tidal volume/1 OOg; V TOT = total R  T  ventilation/ lOOg; Ti = inspiratory time; T = expiratory time. E  A Pre MK-801  o — < <u  « CO  £ o o *o = 2 « at +s  3  0  B P o s t MK-801  1  coco c J=  o  20  10  -10 f  R  V  T  V  T 0 T  f  R  V  T  V  TOT  fR  V  T  VTOT  Fig. 4.5: The effects of the change from State III to State I are shown for rats breathing air, 10% oxygen in nitrogen and 5% CO2 in air both before and after MK801 injection into the PBrKF. * Indicates a significant difference from State III. Abbreviations as in Fig. 4.4  141  4.3.6. Effect of MK-801 in hypoxic rats The switch from air to hypoxia caused a hypoxic ventilatory response that did not differ from that seen in unanaesthetized rats (Pappenheimer, 1977). When rats breathing air were made hypoxic in State I, there was a 27.4 ± 2.3% increase in frequency due to a 27.1 ±2.2% decrease in Ti and a 16.7 ± 1.9% decrease in T E . Tidal volume increased by 14.8 ± 2.2%o to give a total increase in ventilation of 41.2 + 5.3% (p < 0.01 for all) (n=15). When the H V R was measured in rats in State III, f increased by 30.7 ± 5.7% R  due to a decrease in Ti of 28.4 ± 4.1% and a decrease in T E of 17.2 ± 2.8%>. Tidal volume increased by 20.5 ± 2.6% to give an overall increase in ventilation of 53.6 ± 6.0%> (p< 0.01 for all) (n=8). In State I, following injection of MK-801, exposure to hypoxia still caused f to R  increase by 34.7 ±7.0%. due to decreases in Ti and T o f 33.8 ± 5.2%. and 15.8 ± 4.0% E  respectively. Tidal volume increased by 21.2 ± 5.0% to give an overall increase in V TOTof 65.5 ± 7.9% (all p < 0.01). In State III after MK-801, hypoxia caused the frequency of respiration to increase by 52.4± 5.9% due to a large decrease in Ti of 44.8 ± 3.5% (p < 0.01 for both) while T  E  decreased by only 17.0 ± 5.8% (p = 0.07). Tidal volume increased by 27.8 ± 8.3% (p= 0.05) and total ventilation increased by 102.1 ± 15.1% (p < 0.01). Overall, injection of MK-801 into the PBrKF increased the sensitivity to hypoxia. The total ventilation was significantly higher in State I after MK-801 (p=0.01), and in State III both the increase in frequency and the total ventilation were significantly greater (p-0.01 and 0.006 respectively) when the animals were exposed to the hypoxic gas mixture.  142  A State I Post MK-801  Pre MK-801  CO  E 2 **— CD CD  C  80  80  60  o 60 oo  O  _r_  40 -I  40 CO  £ 20 o  20  CO  SI  *s  0  0  CD  c  CO  -20  O -20  -40  -40  5s  B  State  120 •]  120  100 •  o 100 oo  80 -  I  CO  E 60 • o **— 40 o > CD c 20 • CO  sz  o  •-•9  0• -20 • -40 • -60 -  l~!  80  + 60  * 8  y  co 40 £ o 20 |  0  1-20 o  # -40  *8  -60  Fig. 4.6: The effect of breathing 10% oxygen in nitrogen on breathing pattern in urethane anaesthetized rats in both States I and III before and after the injection of MK-801 into the PBrKF. The values are given as % change from air to a hypoxic gas mixture and from air after MK-801 injection to hypoxia after MK-801. * Indicates values that are significantly different from air or air plus MK-801. 8 Indicates values that are significantly different from before MK-801 injection. Abbreviations as in Fig.4. 4.  143  4.3.7. Effect of MK-801 on the timing of the hypoxic ventilatory response Since systemic MK-801 had caused changes in the time domains of the H V R (Chapter 3), we examined the hypoxic response in this group of rats before and after M K 801 injection into the PBrKF. There was no difference in the timing of the acute change in fR in response to hypoxia post-MK801, and the frequency remained above the prehypoxia frequency during the hypoxic exposure. After hypoxia, a post-hypoxic frequency decline was seen both before and after MK-801 (Fig. 4.7). Injection of MK-801 also did not alter the increase in tidal volume during hypoxic exposure. In the post-hypoxic period, however, tidal volume did not decrease from the values seen during hypoxia and so was significantly higher than the tidal volume posthypoxia before MK-801. Over time, the tidal volume of the MK-801 injected rats decreased and that of the control rats increased so that after about 10 minutes there was no significant difference in the volumes pre and post-MK-801. Total ventilation during the acute response to hypoxia was less after MK-801 injection, due to a non-significant decrease in the frequency response, but otherwise the acute response was not different than before injection of the drug. The post-hypoxic decline in ventilation was the same both before and after MK-801 injection (Fig. 4.7).  Fig. 4.7: The time course of the hypoxic ventilatory response is shown with changes in breathing in the post-hypoxic period before and after injection of MK-801 (n=7) into the PBrKF. The first value was the value 20 seconds before the start of the hypoxic exposure. The last value during hypoxia was the value 20 seconds before hypoxia was discontinued. Fig 4.7A: changes in breathing frequency, Fig 4.7B: changes in tidal volume and Fig 4.7C: changes in total ventilation. * Indicates values that are significantly different from the values with MK-801.  144  Hypoxia  180 160  ** * TT*  -I  *. *  Post-hypoxia *  * * *  T  T  T  ?  4  *  £= 140  Q. £1  > 120  o c  Q> 100 3 C7  2!  80  u.  60 40 -50  0  50  1 00  0  SO  100  ISO  300 ~Z>  'Sao  1Q00  1SD0  2000  2500 -50  0  50  100  150  200  36 0 'SOO 1000 1 500  2000 2500 3000 3500  0.35 0.30  -SO  1S0  300  2SD  500  1000  150O  2000  3500-50  0  SO  100  ISO  100 -i  ^  300  z 3S0  -*—  80  SOD 1000 1500 3000 2SM) X00 3S0O  pre MK-801 post MK-801  o o  £  60  o r-  >  40  20  Fig. 4.7  •50  —i1 r 0  50  100  15D  200  250  500  1000  Time (sec)  1500  2000  2500 -5D  0  50  100  1 50  200  Z  250  500  1000  Time (sec)  1 500  2000 2500 3000 3500  145  4.3.8. Effect of MK-801 in hypercapnic rats Systemic MK-801 did not cause changes in the response to hypercapnia (Chapter 3). Nevertheless, given the differences in breathing pattern during air between rats given systemic MK-801 and PBrKF MK-801, we looked at the effects of MK-801 on breathing pattern on the change from air to 5% CO2 before and after MK-801. When rats breathing air in State I were exposed to 5% CO2 in air, there was a 56.8 ± 4.5% (p< 0.01) increase in tidal volume. The increase in frequency was not significant but the overall increase in total ventilation was (63.6 + 6.4%; p<0.01).  Injection of MK-801 into the PBrKF had  no effect on the response to CO2 in State I. In State III, before MK-801, there were increases in both frequency of respiration and tidal volume (10.5 ± 2.3 % and 56.3 ± 5.7% respectively) to give an overall increase in total ventilation of 73.1 + 8.0% (p<0.01 for all). . After MK-801 frequency of breathing increased significantly by 21.9 ± 7.7% while tidal volume increased by 38.3 ± 8.9% to give an overall change in ventilation of 68.0 ± 15.2% (all p<0.01-for the comparison to air)(Fig. 4.8; Table 4.1). These increases were not significantly different from those seen prior to MK-801injection into the PBrKF.  146  A State I Pre MK-801  80  o  60  00  CO  *8  om  'co  20  CO  £  60 -  + 40 •  E 40 o |  Post MK-801  80  20 •  o>  o> c 0• CO  0  O -20 -  -20 -40  -40  J  B State III 80  80  ^ so  60 CO  E 40 o  -I  + 40  'co  I  CO  u> 20 c CO  O  *  20  CO  0  o  _  £  0  co x: O  -20 •  SS -20  -40 •  -40  J  f  Fig. 4.8: T h e effect o f breathing 5% C 0  2  R  V V T  T O T  T,  T  E  i n air o n breathing pattern i n States I a n d III,  before a n d after injection o f M K - 8 0 1 into the P B r K F r e g i o n . T h e values are g i v e n as % change f r o m values i n air. * Indicates a significant difference f r o m values i n air or values i n air plus M K - 8 0 1 .  5 Indicates significant differences between values p r e - M K - 8 0 1 .  A b b r e v i a t i o n s as i n F i g . 4.4  147  4.4. Discussion: The purpose of this study was to determine the effects of MK-801 when injected into the PBrKF region on state, and on the changes in breathing pattern and chemoresponses that occur as a function of changes in state in urethane-anaesthetized Sprague Dawley rats. 4.4.1. Role of NMDAr in Establishing State Systemic application of MK-801 has been shown to promote slow wave sleep in unanaesthetized animals, with its accompanying increase in N R E M delta in the E E G (Campbell and Feinberg, 1996). We have also found that MK-801 administered systemically to urethane-anaesthetized rats promotes State III, also characterized by an increase in N R E M delta-like activity in the E E G (Chapter 3). In the present study, injections of MK-801 into the PBrKF also caused the rats to enter into State III. This suggests that N M D A r in the PBrKF promote State I and it is also possible that blockade of neurons with N M D A r in the PBrKF interrupts input from the pontine reticular formation and/or the PPT that excites NMDA-type glutamate receptor-mediated process in the PBrKF to promote wakefulness in unanesthetized rats. We can't rule out the possibility that our injections spread from the PBrKF region into the pedunculopontine tegmentum (PPT) where there are neurons with N M D A r (Datta et • al., 2001). Whatever the case, it is clear that NMDA-type glutamate receptor-mediated processes promote State I, and when these receptors are blocked, the animals spend more time in the sleep-like state of urethane anaesthesia (State III).  148  4.4.2. Role of NMDAr in the PBrKF in producing changes in breathing with changes in state State changes affect breathing such that the transition from SWS/State III to awake/State I is accompanied by an increase in ventilation (Hunter and Milsom, 1998; Hunter et a l , 1998; Boon et al., 2004, Chapter 2). It has been shown that MK-801 and Ketamine injected into the medial PRF depress the frequency of breathing (Lydic and Baghdoyan, 2002) and that sleep-waking state modulates the activity of neurons in the PBrKF (Gilbert and Lydic, 1994). It has been shown that there are monosynaptic connection's between the pontine reticular formation and the P B r K F (Herbert et al., 1990), and that neurons of the PBrKF are closely associated with neurons of the pedunculopontine tegmentum (PPT) (Datta et al., 1995). Together this suggests that there are interactions between the mPRF and/or the PPT and the P B r K F regions that account for state effects on breathing. In the present study, MK-801 injected into the PBrKF region significantly reduced the change in ventilation that was seen with the transition between State III and State I in rats breathing any of the gas mixtures. This means that MK-801 was more effective in depressing ventilation in State I than in State III, which may be explained by the method of action of MK-801. In order for MK-801 to enter the ion channel of the N M D A r and block it, the neuron must first be activated by the binding of glutamate to a non-NMDA receptor, glycine must bind to the glycine binding site of the N M D A r , and glutamate must bind to the glutamate site of the N M D A r . These three events result in depolarization of the neuronal membrane and the release of M g  + +  from the ion channel,  which then allows the MK-801 to enter (Foster and Wong, 1987). The stimulation  149  provided in the waking state could mean that in State I, more neurons would have open ion channels allowing the MK-801 to enter, thus blocking the action of a larger number of neurons. Phrased differently, this suggests that neurons with N M D A r in the PBrKF are more active in State I than State III, accounting in large part for the increase in ventilation associated with wakefulness. 4.4.3. Role of NMDAr in the PBrKF under resting conditions Systemic MK-801 caused a decrease in total ventilation due to a significant decrease in tidal volume in both unanaesthetized and urethane anaesthetized animals (Cassus-Soulanis et al., 1995; Ohtake et al., 1998; Chapter 3). Targeted injection of M K 801 into the PBrKF, on the other hand, reduced the frequency of breathing (f^, due to significant increases in both T and T E , in both unanaesthetized (cats, Ling et al., 1994) L  and urethane anesthetized animals (present study). The net result was a reduction in total ventilation with both systemic and PBrKF microinjections of MK-801, in the urethane anesthetized and the unanaesthetized animals (Ling et al., 1994; Cassus-Soulanis et al., 1995; Ohtake et al., 1998; Chapter 3), but due to different effects. The difference in response to systemic versus PBrKF injection of MK-801 reinforces the idea that the PBrKF is involved in the inspiratory off-switch mechanism to control respiratory frequency and that N M D A type glutamate receptor mediated processes represent the neural mechanism for accomplishing this. When the N M D A r in the PBrKF are blocked with MK-801, frequency decreases, and if the vagus is sectioned (as in the study on cats by Ling et al., 1994) apneusis ensues. Since the vagus nerves were intact in our rats we didn't see apneusis because volume-related feedback from the lungs was still able to terminate inspiration when the pontine neurons were non-functional. The changes that  150  occurred, however, indicate that while these pontine neurons have a similar role to vagal feedback in terminating inspiration. The effects are additive in part, rather than redundant, at least in urethane-anaesthetized rats. There was an increased variability between rats after MK-801 injection, particularly in hypoxia and hypercapnia, perhaps reflecting the role of the PBrKF in stabilizing the respiratory pattern (Oku and Dick, 1992). It is also of interest that there was an increase in tidal volume after MK-801 blockade of PBrKF neurons in the present study. While this undoubtedly reflects the prolongation of inspiratory time, it also indicates that the effects of systemic MK-801 on tidal volume (large decrease in both States I and III, Boon et al., 2004) were mediated by neurons outside the PBrKF, as has been shown to be the case in unanaesthetized animals (Ling et al., 1994; Cassus-Soulanis et al., 1995; Ohtake et al., 1998). Finally, given that MK-801 produced both an increase in time spent in State III and a reduction in the effects of state change on ventilation, it is clear that MK-801 administration (systemically or microinjected into the PBrKF region) will reduce ventilation in part by its effects on state. Since there was a significant depression of respiration in both States I and III after MK-801 injection however, the data indicate that reductions in ventilation following N M D A r blockade are also due in part to direct effects on the PBrKF region. 4.4.4. Role of NMDAr in the PBrKF in the hypoxic ventilatory response and changes in hypoxic sensitivity with changes in state N M D A type glutamate receptor mediated processes in the PBrKF do not seem to be involved in the hypoxic ventilatory response. MK-801 did not reduce the overall  151  hypoxic ventilatory response when injected into the PBrKF regardless of state. This is in contrast to the results obtained with systemic MK-801, where there were significant reductions in the magnitude of the hypoxic ventilatory response (Chapter 3). Furthermore, with systemic MK-801 there was a delay in the acute response to hypoxia, and after the initial increase in both frequency and tidal volume there was a reduction in the H V R such that frequency declined to pre-hypoxia levels, as did tidal volume and total ventilation. With MK-801 injected into the PBrKF the acute response was not delayed, and the increases in frequency, tidal volume and total ventilation were all maintained over the course of the response. In the post-hypoxic period, a frequency decline (PHFD) was evident as has been reported by Powell et al., (1998), and this was not affected by administration of MK-801. Systemic MK-801 has been shown to block PHFD (Coles et al., 1998), and the fact that we did not find this in the present study would indicate that our injections of MK-801 did not diffuse to the vl pons and the A5 nucleus, sites where MK-801 has been shown to block PHFD. After the discontinuation of hypoxia in the control rats, tidal volume decreased, but the rate of decrease was not as great as that seen with frequency and did not fall below the pre-hypoxia value. This is an example of short-term potentiation of tidal volume (Powell et al., 1998).  After MK-801, when air was re-administered, tidal  volume remained at the same level as during hypoxia for about 10 minutes before declining to the pre-hypoxia value. Thus it would appear that N M D A r in the PBrKF region are involved in suppressing the short-term potentiation of tidal volume following hypoxia.  152.  Unanaesthetized rats and urethane-anaesthetized rats and ground squirrels have been shown to be more sensitive to hypoxia during SWS/State III (Pappenheimer, 1977; Hunter et al., 1998; Boon et al., 2004, Chapter 2). The present study shows that blockade of N M D A r in the PBrKF increased the overall sensitivity to hypoxia, and it increased more in State III than in State I. This would indicate that not only were NMDA-type glutamate receptor- mediated processes in the PBrKF not involved in the increase in sensitivity with transition from State I to State III, they may actually have acted to reduce sensitivity. 4.4.5. Role of NMDAr in the PBrKF in the hypercapnic ventilatory response and changes in hypercapnic sensitivity with changes in state As with systemic MK-801, we did not find that injection of MK-801 into the PBrKF region had any effect on the response to 5% CO2 in air, as has also been reported by Ohtake et al. (1998). Kainic acid mediated destruction of neurons in the PBrKF region reduced the hypercapnic response in cats (Fung and St. John, 1994b), indicating that neurons with kainate receptors might be responsive to CO2. While there was a trend to greater sensitivity to hypercapnia in State III compared to State I, the differences were not significant in this study, and MK-801 did not alter this. The lack of change in sensitivity to C 0 seen in the present study is similar to that 2  shown in a previous study in rats (Boon et al., 2004, Chapter 2), although work in ground squirrels has shown an increased sensitivity to CO2 in State III (Hunter et al., 1998).  153  4.5. Conclusion Microinjection of MK-801 into the PBrKF region of the pons, in intact Sprague Dawley rats, reduced ventilation by reducing the frequency of respiration while allowing a small increase in tidal volume to occur. In our experiments, we also found that the blockade of N M D A r caused the urethane-anaesthetized animals to cycle into State III, the slow-wave sleep-like state, which could indicate that state effects, primarily modulated by neurons in the pontine reticular formation and or the pedunculopontine tegementum, may be routed through the PBrKF and thus affect breathing pattern. The reduction in ventilation due to MK-801 injection occurred in both States I and III, indicating that the MK-801 had its effects on breathing both indirectly via changes in State, but also directly by reducing breathing frequency through an elongation of both inspiratory and expiratory times. The fact that the rats were not vagotomized is important because it indicates that neurons in the pontine respiratory centres of the dl pons, the K F and parabrachial nuclei have a role to play in normal eupneic breathing. While this role may be interdependent with that of vagal feedback, it is not inconsequential or inactive when the vagus is intact. It would seem more likely that, as suggested by Oku and Dick (1992), respiratory neurons in the PBrKF region play a role in stabilization of the respiratory pattern generator and are an important part of the ponto-medullary circuit that controls respiration. Our results indicate that NMDA-type glutamate receptor-mediated processes in the dl pons do not play a role in the response to hypercapnia. However, they do reduce the increase in sensitivity to hypoxia that occurs with the transition from State I to State  154  III and they suppress the short-term potentiation of tidal volume that is seen after hypoxia t in some strains of rats.  155  4.6. References Alheid, G.F., Milsom, W.K., McCrimmon, D.R., 2004. Introduction: Lateral Pontine Influences On Respiratory Control. Respir Physiol Neurobiol. 143: 105-114. Boon, J.A., Garnett, N.B.L., Bentley, J.M., Milsom, W.K. 2004. Respiratory chemoreflexes and effects of cortical activation state in urethane anesthetized rats. Respir. Physiol. Neurobiol. 140: 243-256. Campbell, I.G., Feinberg, I. 1996. N R E M Delta stimulation following MK-801 is a response of sleep systems. J. Neurophysiol. 76(6): 3714-3720. Cassus-Soulanis, S., Foutz, A.S., Denavit-Saubie, M . , 1995. Involvement of N M D A r s in inspiratory termination in rodents: effects of wakefulness. Brain Res. 679: 25-33. Chamberlin, N . L., 2004. Functional organization of the parabrachial complex and intertrigeminal region in the control of breathing. Respir Physiol Neurobiol. 143: 115-125 Cohen, M . I. and Wang, S. C , 1959. Respiratory neuronal activity in pons of cat. J Neurophysiol. 22: 33-50. Coles, S.K., Ernsberger, P., Dick, T.E., 1998. A role for N M D A r s in posthypoxic frequency decline in the rat. Am. J. Physiol. 274: R1546-R1555. Connelly, C.A., Otto-Smith, M.R., Feldman, J.L., 1992. Blockade of NMDAr-channels by MK-801 alters breathing in adult rats. Brain Res. 596: 99-110. Datta, S., 1995. Neuronal activity in the peribrachial area relationship to behavioral state control. Neruosci. Biobehavior.Rev. 19: 67-84. Datta, S., Patterson, E.H., Spoley, E.E., 2001. Excitation of the pedunculopontine tegmental N D M A Receptors Induces Wakefulness and Cortical Activation in the Rat. J. Neurosci. Res. 66: 109-116. Feldman, J.L., Cohen, M.I., Wolotsky, P. 1976, Powerful inhibition of pontine respiratory neurons by pulmonary afferent activity. Brain Res. 104: 341-346. Foster, A.C., Wong, E.H.F., 1987. The novel anticonvulsant MK-801 binds to the activated state of the N-methyl-D-aspartate receptor in rat brain. Br. J. Pharmacol. 91: 403-409. Foutz, A.S., Champagnat, J. Denavit-Saubie, M . , 1989. Involvement of N-methyl-Daspartate (NMDA) receptors in respiratory rhythmogenesis. Brain Res. 500: 199-208 Fung, M . L . and St.-John, W. M . , 1994. Separation of multiple functions in ventilatory control of pneumotaxic mechanisms. Respir Physiol. 96: 83-98.  156  Gilbert, K.S., Lydic, R., 1994. Pontine cholinergic reticular mechanisms cause statedependent changes in the discharge of parabrachial neurons. Am. J. Physiol. 266:R13650. Herbert, H., Moga, M . M . and Saper, C. B., 1990. Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat. J Comp Neurol. 293: 540-580. Hunter, J.D., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia. I: Cardio-respiratory effects. Respir. Physiol. 112: 71-81. Hunter, J.D., McLeod, J.Z., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia. II: Respiratory reflexes. Respir. Physiol. 112: 83-94. Lieske, S.P., Thoby-Brisson, M . , Telgkamp, P., Ramirez, J.M., 2000. Reconfiguration of the neural network controlling multiple breathing patterns: eupnea, sighs and gasps. Nature Neurosci. 3(6): 600-607. Ling, L., Karius, D.L., Speck, D.F., 1994. Role of N-methyl-D-aspartate receptors in the pontine pneumotaxic mechanism in the cat. J. Appl. Physiol. 76 (3): 1138-1143. Lumsden, T., 1923. Observations on the respiratory centres in the cat. J Physiol (Lond). 57, 153-160. Lydic, R., Baghdoyan, H.A. 2002. Ketamine and MK-801 decrease acetylcholine release in the pontine reticular formation, slow breathing, and disrupt sleep. Sleep 25:617-622. Monaghan, D.T., Cotman, W., 1985. Distribution of N-methyl-D-aspartate sensitive L [3H] glutamate-binding sites in rat brain. J. Neurosci. 5:2909-2919. Oku, Y . , Dick, T.E., 1992. Phase resetting of the respiratory cycle before and after unilateral pontine lesion in cat. J. Appl. Physiol. 72: 721-730. Onimaru, H., Homma, I., 2003. A Novel Functional Neuron Group for Respiratory Rhythm Generation in the Ventral Medulla. J Neurosci. 23:1478-1486. Ohtake, P.J., Torres, J.E., Gozal, Y . M . , Graff, G.R., Gozal, D., 1998. N M D A r s mediate peripheral chemoreceptor afferent input in the conscious rat. J. Appl. Physiol. 84: 853861. Paxinos, G., Watson, C. 1986. The rat brain in stereotaxic coordinates. 2 Academic Press.  nd  edition.  Petralia, R.S., Yokotani, N . , Wenthold, R.J., 1994. Light and electron microscope  157  distribution of the N M D A r subunit N M D A R 1 in the rat nervous system using a selective antibody. J. Neurosci. 14:667-696. Powell, F L , Milsom, W.K., Mitchell, G.S., 1998. Time domains of the hypoxic ventilatory response. Respir. Physiol. 112:123-134. Rechtshaffen, A., Kales, A., Berger, R.J., et al., 1968. A Manual of Standardized Terminology, Techniques and Scoring system for Sleep Stages in Human Subjects. Us Government Printing Office, Washington, D.C. Smith, J. C , Ellenberger, H . H., Ballanyi, K., Richter, D. W. and Feldman, J. L., 1991. Pre-Botzinger Complex: A brainstem region that may generate respiratory rhythm in mammals. Science. 254:726-729. St. John, W . M . , Paton, J.F.R., 2000. Characterizations of eupnea, apneusis and gasping in a perfused rat preparation. Respir. Physiol. 123: 201-213. St.-John, W. M . and Paton, J. F. R., 2004. Role of pontile mechanisms in the neurogenesis of eupnea. Respir Physiol Neurobiol. 143: 321-332.  158  Chapter 5  The Effects of CorticalActivation State on the Response to Intermittent Hypoxia and the Role of the PBrKF  159  5.1. Introduction:  The hypoxic ventilatory response (HVR) is characterized by a complex interplay of physiological mechanisms that preferentially alter the components of ventilation (frequency and tidal volume) in different ways at different times depending on whether the hypoxia is delivered continuously or intermittently (Powell et al., 1998 for review). Thus, in rats during brief exposure to hypoxia (2-5 minutes) there is an initial acute response (AR), where frequency of breathing (f ) and tidal volume ( V ) increase R  T  immediately, (within seconds), often followed in the next few seconds to minutes by a further increase in tidal volume (short-term potentiation; STP) and/or a decline in frequency (short-term depression; STD). If the hypoxic stimulus is prolonged, there is a secondary decrease in tidal volume (hypoxic ventilatory decline; HVD). When the hypoxic stimulus is removed, there is an acute "off response as both f and V T decline. R  Frequency often declines to levels below the frequency of breathing prior to the hypoxic exposure (post-hypoxia frequency decline; PHFD) (Coles and Dick, 1996), which may reflect the continuing STD. The initial decline in tidal volume and total ventilation ( V TOT), however, are to levels above those seen prior to the hypoxic exposure, and they only then slowly decrease to initial levels, reflecting removal of the STP that developed during the H V R (Powell et al., 1998 for review). Episodic hypoxia (repeated brief bouts of hypoxia) in some animals results in a progressive augmentation (PA) of the H V R , and a slowly developing increase in V T after the hypoxic stimulus has been removed and after V T initially returns to pre-hypoxia levels. This can progress for many minutes to several hours (long term facilitation; LTF). LTF is not seen in some strains of rat (Bach and Mitchell, 1996), is smaller in awake animals than in anaesthetized animals (Powell et al.,  160  1998) and is enhanced by vagotomy that removes the inhibition from pulmonary stretch receptors (Mateika and Fregosi, 1997; Powell et al., 1998). There has been much interest in determining the mechanistic basis for these processes as well as the conditions that promote/extinguish them in different species. In the present study we were interested in one such possible condition, the state of cortical activation. In unanaesthetized animals it has been shown that the changes in state that occur as animals go back and forth between sleep and awake states lead to changes in total ventilation, breathing pattern and hypoxic sensitivity (Pappenheimer, 1970). It has also been shown that hypoxia leads to changes in sleep state distribution (increased wakefulness) and thus, indirectly to the changes in ventilation that are associated with the changes in state (Pappenheimer, 1977; Laszy and Sarkadi, 1990). Given this, in the present study we wished to examine the effects of intermittent hypoxic exposure on cortical state distribution (especially during the period when L T F would be developing) as well as the effect of changes in cortical state distribution during intermittent hypoxia on the various components of the H V R . For these studies we chose to use urethane-anesthetized animals. It has now been shown that urethane anesthetized rats (Boon et al., 2004; Chapter 2), and other species (Hunter and Milsom, 1998) cycle between states that, based on E E G criteria, closely resemble wake (State I) and slow wave sleep (State III). It has also been shown that these species undergo changes in total ventilation, breathing pattern and hypoxic sensitivity with these changes in state that are identical to those shown when unanaesthetized animals awaken or fall asleep (Hunter and Milsom, 1998;.Hunter et a l , 1998; Boon et al., 2004; Chapter 2). Thus, while these states may not be the same as slow wave sleep and  161  wake in other aspects, they do produce similar changes in ventilation, and offer a model for study of the control of breathing as a function of cortical activation state in which state can be carefully monitored and controlled. We also wanted to repeat these experiments in animals in which MK-801 was microinjected into the parabrachial and Kolliker Fuse nuclei (PBrKF). We have recently shown that, while such injections do not alter the magnitude or onset of the hypoxic ventilatory response, they do amplify the effects of changes in cortical activation state on hypoxic sensitivity, and reduce the rate at which tidal volume returns to normal following hypoxic exposure (Chapter 4). The latter would suggest that N M D A sensitive neurons in the PBrKF may influence STP and or LTF, phenomena involving changes in tidal volume during and following the return to breathing air after exposure to hypoxia. We have also recently shown that MK-801 microinjections into the region of the parabrachial and Kolliker Fuse nuclei (PBrKF) (presumably acting by blockade of N M D A type glutamate receptors) promotes State III (the slow-wave sleep-like state) even during hypoxia, and largely eliminates the effects of cortical activation (transition into State I, the wake-like state) on total ventilation and breathing pattern (Chapter 4). As a consequence, these animals are largely in a constant state, but show very small changes in breathing even i f state does change. This provides a means of controlling for state effects without disrupting the H V R itself and may provide us with a comparison group for determining the extent to which any changes in state that occur during episodes of intermittent hypoxia effect the expression of the different components of the hypoxic ventilatory response.  162  5.2. Methods 5.2.1. Animal Care: Sprague Dawley rats were obtained from the U B C animal care facility (University of British Columbia, Vancouver, B.C. Canada). The surgeries and protocols were carried out with the prior approval of the U B C Animal Care Committee and the Okanagan University College Animal Care Committee. Measurements were made using adult male Sprague Dawley rats in a time period from approximately 9 a.m. to 6 p.m. The rats were housed singly in the OUC animal care facility and allowed access ad libitum to food and water, supplemented from time to time with sunflower seeds and fruit. The rats were kept at 25° C, with a light/dark cycle of 12 hours. The lights came on at 8 a.m. At the time of the experiments, their average weight was 390 g, and the range was 325-452 g. After the experiment, the rats were euthanized with an IP injection of 1 ml. of Somnotol (Sodium pentobarbital, 65 mg/ml, M T C pharmaceuticals). 5.2.2. Experimental Protocol: 5.2.2.1. Surgical preparation The animals were anaesthetized with 2% vaporous halothane administered through a mask. The mask was attached to the airline with a T tube and the flow was vented to a fume hood. Then the rat was given an intraperitoneal (IP) injection of a 20% solution of Urethane (Sigma) in saline to a final dose of 1.3g/kg. The trachea was canulated below the larynx for the measurement of airflow. The canula was attached to the airline from which the mask had been removed and the animal was placed in a stereotaxic head frame (Kopf), adjusted such that the skull surface landmarks lambda and bregma were on the same horizontal plane. The halothane anaesthesia was slowly  163  decreased, but was maintained until all surgery was completed and the urethane had become effective; at least 45 minutes. Additional doses of urethane were administered IP if the rat responded to a noxious toe pinch. Body temperature was maintained between 36 and 37° C by placing the rat on a servo-controlled heating pad. After injections of xylocaine (2% Lidocaine hydrochloride, Astra pharmaceuticals), a dorsal-longitudinal incision was made over the crown of the cranium, extending from the orbits to the lambdoid suture. Four electroencephalographic (EEG) electrodes were implanted in the skull as described in Hunter and Milsom (1998). Each electrode was fashioned from a length of insulated, multi-stranded, stainless steel wire ( A M Systems) soldered to a self-tapping stainless steel screw (00 x 3/16, Fine Science tools). The other end was soldered to a gold Amphenol pin. The Amphenol pins were inserted into an Amphenol pin strip. A pneumotach was installed in the outflow of the gas line attached to a Validyne pressure transducer (Validyne, DP 103-18). The signal was amplified with a Gould amplifier and then transmitted to a two-channel data acquisition system (AT-CODAS, DataQ Instruments) sampling at a frequency of 120 samples per second on each channel. The breathing signal was also recorded on a chart recorder. The E E G signal was amplified and recorded on a chart recorder and on the data acquisition system on a second channel. The signal was filtered with both low and high pass filters and a 60 Hz. filter to reduce noise. 5.2.2.2. Injection of Saline and MK-801 For injections of saline, MK-801 (dizocilpine maleate ((+)-5 methyl-10, 11dihydro-5H-dibenzo [a,d]cyclohepten-5,10 imine, Sigma) and Pontamine sky blue, the  164  rat's head was rotated 24° around ear-bar zero (EBZ). Using a drill, windows in the bones of the skull were opened from just behind the lambdoid suture extending ~ 4 mm caudally and from ~ l m m to 3 mm on either side of the midline. The meninges were carefully dissected away to expose the top of the brain. Using a calibrated micromanipulator, the needle of a 10 ul Hamilton syringe (#701 with a 26s needle) was dropped into the opening in the skull 1.37 mm behind E B Z and 2 mm on either side of midline to a depth of - 7.9 mm from the top of the brain. By our calculations, this meant that we were injecting saline or drug into the region of the PBrKF (Paxinos and Watson, 1986). Both saline and MK-801 were injected in 0.5 ul increments over a period of about 3-4 minutes to give a total volume of 1-2 ul of each on one side and then the needle was slowly withdrawn and repositioned on the other side where the injection protocol was repeated. After the animals were euthanized, the brains were removed and fixed in a solution of 10% formalin. The brains were then frozen and serially sectioned between the 7 cranial nerve and the 4 cranial nerve with sections o f - 3 0 um taken every 0.1mm th  th  (n=5). The slides were then stained with Cresyl Violet for Nissl substance. In 5 rats, the injections were placed more rostrally or more caudally than the desired area to serve as controls. There was no effect on breathing with these injections. 5.2.2.3. Monitoring breathing pattern in air and hypoxia. With the animal breathing air, the respiratory and E E G patterns were monitored until they were stable and the halothane had been discontinued for at least 1 hour. The air was then replaced with a gas mixture of 10% O2 in nitrogen (hypoxia), which was administered for 5 minutes followed by 5 minutes of normoxia. This was repeated 5 times. After the 5 exposure to hypoxia, the breathing pattern in air was monitored for 1 th  165  hour.  In the treated animals, the injections of saline or MK-801 were made while the  animals were breathing air 10-15 minutes prior to exposure to hypoxia, a time determined from previous experiments to allow for blockade of NMDA-type glutamate receptormediated processes, as evidenced by a significant decrease in the frequency of respiration, an increase in inspiratory time, and transition into State III. 5.2.3. Data Analysis Cortical activation states were scored based on E E G profiles: State I (desynchronized cortical activity), State II (intermediate activity), and State III (synchronized cortical activity), with E E G activity resembling that seen in wake, light sleep and slow wave sleep, respectively as scored according to conventional criteria (Rechtschaffen et al., 1968). A l l arousal state data were scored in 30sec. epochs and classified according to the predominant state during that epoch. The percentage of total recording time spent in each state was then calculated from these data. To assess the breathing pattern during the response to hypoxia and after discontinuation of hypoxia, files were taken at timed intervals: 20 seconds immediately prior to hypoxia, at 20 second intervals for 200 seconds, then for the last 20 seconds of the exposure to the 10% oxygen in nitrogen i.e. 20 seconds immediately prior to the return to air. During the exposure to air, one file was taken in the first 20 seconds of air and then files were taken at 0.5 minutes, 1 minute, 2.5 minutes, and 3.5 minutes and in the last 20 seconds of air, just prior to the next episode of the hypoxia. After 5 episodes of hypoxia, when the animals were again breathing air, files of 20 seconds in length were taken during the first 20 seconds of exposure to air and then at 0.5 minutes, 1 minute, 2 minutes, 5 minutes, 30 minutes and 60 minutes. Calculations were made from the  166  computer-generated files (Dataq) for the f r e q u e n c y o f respiration (f ) a n d the voltage R  c o r r e s p o n d i n g to the tidal v o l u m e . C a l i b r a t i o n s were carried out u s i n g a n electrically operated p u m p that d e l i v e r e d k n o w n v o l u m e s into the tracheal tube at a constant frequency. T h e voltage generated b y these v o l u m e s was u s e d to m a k e a c a l i b r a t i o n curve w h i c h a l l o w e d the c o n v e r s i o n o f the experimental voltages into tidal v o l u m e s ( V T ) . total ventilation ( V TOT) was calculated b y m u l t i p l y i n g the f  R  x VT.  The  T h e values were  converted to v o l u m e s i n m l / l O O g ( V ) , or m l / m i n / l O O g ( V TOT) ( A T P S ) . T  5.2.4. Statistical Analysis: M e a n s presented are o v e r a l l estimated m a r g i n a l m e a n values ± S E M .  A l l o f the  data were a n a l y z e d u s i n g the G e n e r a l L i n e a r M o d e l multivariate analysis o f variance ( S P S S v e r s i o n 11.5, S P S S Inc. C h i c a g o , Illinois). M u l t i p l e c o m p a r i s o n tests ( B o n f e r o n n i ) were u s e d to separate significant m e a n values. V a l u e s at each point d u r i n g the time course o f h y p o x i a a n d air after h y p o x i a were a n a l y z e d w i t h a Student T test w i t h two tails.  D i f f e r e n c e s were c o n s i d e r e d to be significant w h e n p < 0.05.  5.3. Results In this study, w e e x a m i n e d the effects o f intermittent h y p o x i a exposure o n cortical state distribution a n d the effects o f c o r t i c a l activation state o n b r e a t h i n g pattern d u r i n g and  after h y p o x i c exposure.  5.3.1. The effect of intermittent hypoxia on cortical activation state and breathing pattern in control rats The  first episode o f intermittent h y p o x i a caused animals to i m m e d i a t e l y enter into  State I i f they w e r e not i n State I already ( F i g 1 A ) . State I was then the p r e d o m i n a n t state seen d u r i n g the first 5-minute exposure. N e a r the e n d o f the 5-minutes o f h y p o x i a , s o m e  167  slow waves appeared in the E E G trace, but there was no established State III (Fig. 5.IB, Fig 5.4). In the following interval on air, the rats went immediately into State III (Fig. 5.IB) and remained in that state for the full 5 minutes (Fig. 5.1C, Fig. 5.4), and this was the case for all subsequent intervals during which animals were returned to breathing air. In the second and subsequent episodes of hypoxia, the State III that occurred during the air episodes between the bouts of hypoxia persisted into the next hypoxic period for between 20 seconds and 40 seconds before the animals entered into State I (Fig 5.ID). After the final episode of intermittent hypoxia the rats went into State III and this state predominated for between 20 and 25 minutes. Note the frequency of augmented breaths (sighs) during hypoxia (Fig. 5.1 A), and the immediate decrease in both tidal volume and frequency of respiration on the transition to air (Fig. 5. IB). Both tidal volume and frequency of respiration remained significantly different in the air intervals (Fig. 5.1C). On the transition to the second hypoxic episode, tidal volume and frequency both increased within the first 10 seconds and sighing started about 30 seconds after the hypoxia was initiated.  168  A First Minute of First Hypoxic Episode  B Transition Minute:Hypoxia to Air  C Final Minute of Air 2 mis  Respiration  EEG  D Transition to Second Hypoxic Episode  lNll!i'''1'li|\i|l'll'-'lll'v,'||  10 sec.  Fig. 5.1. Respiratory and E E G traces from a control urethane-anesthetized Sprague Dawley rats. Fig. 5.1 A shows the first minute of the first exposure to 10% oxygen in nitrogen. The top trace is the respiration; the bottom trace is the E E G recording. Fig. 5. IB shows the transition from hypoxia to air after the first episode of hypoxia. Fig. 5.1C shows the last minute of the exposure to air. Fig. 5. ID shows the transition from air to hypoxia after the third episode of air.  169  5.3.2. The effect of intermittent hypoxia on cortical activation state and breathing in MK-801 treated rats Since MK-801 has been found to suppress cortical activation (Chapter 4), I investigated the result of injecting MK-801 into the PBrKF and its effects on both cortical activation state and breathing over the course of an exposure to intermittent hypoxia. After injection of MK-801 into the PBrKF region of the pons, animals entered State III and remained in that state regardless of which gas they were breathing (Fig. 5.2). The frequency of sighing was reduced during hypoxia, and in the air intervals, and while frequency decreased immediately, tidal volume was maintained for much longer than in the control rats (compare Fig. 5.IB and 5.2B). 5.3.3. The placement of MK-801 injections Histological examination of the injected brains indicated that the injections (as indicated by the canula tract and the blue dye) were in the desired region (-8.72 to -9.16 mm caudal to bregma at a depth below the surface of the brain of between 6 and 9 mm) (Fig. 5.3). The Pontamine sky blue was clearly visible in the fixed brain tissue and did not appear to extend beyond a radius of ~2 mm around the injection site. In all of these rats, there was a noticeable decrease in breathing frequency within 5 minutes of injection of MK-801. Injections of saline in the same area were without effect.  170  A. First Minute of Hypoxia  Respiration  EEG B. First Minute of Air  1.6 mis  C. Last Minute of Air  1  Fig. 5.2. Respiratory and E E G traces from a urethane-anesthetized Sprague Dawley rat after an injection of MK-801 into the PBrKF region of the pons. Fig. 5.2A shows the first minute of the first exposure to 10% oxygen in nitrogen. The top trace is the respiration; the bottom trace is the E E G recording. Fig. 5.2B shows first minute of air after the first episode of hypoxia. Fig. 5.3C shows the last minute of the exposure to air.  171  Fig. 5.3. The position of injections shown superimposed on serial sections from "The Rat Brain in Stereotaxic Coordinates" Paxinos and Watson, 1986. Fig. 5.3A shows the position of the injection superimposed on a section taken 8.80 mm caudal from Bregma and 0.20 mm rostral to the interaural line. Fig. 5.3B shows the position of the injection superimposed on a section taken 9.16 mm caudal from Bregma and 0.16 mm caudal to the interaural line. Abbreviations: A5 = A5 noradrenaline cells; K F = Kolliker Fuse nucleus; L P B = Lateral parabrachial nucleus; mcp = middle cerebellar peduncle; Mo5 = motor trigeminal nucleus; PnC = pontine reticular nucleus, caudal; PnO = pontine reticular nucleus, oral; py = pyramidal tract; s5 = sensory root trigeminal nerve.  A. Interaural 0.2, Bregma -8.8  B. Interaural -0.16 Bregma - 9.16  173  5.3.4. The time domains of the exposure to intermittent hypoxia with air intervals. We looked at the structure of the response to intermittent hypoxia to determine i f repeated exposure to hypoxia in short intervals was the same as in unanaesthetized rats. The changes in frequency of breathing, tidal volume and total ventilation for the control rats are shown for the 5 episodes of 10% hypoxia followed by air (n=8) in Fig. 5.4. While 3 of the 8 rats were in State III prior to hypoxia, the initial bout of hypoxia caused all rats to go into State I. As shown for the one rat in Figure 5.1, there was very little State III during hypoxia in the entire group, but as soon as the hypoxia was discontinued all of the rats cycled into State III and remained in State III for the full 5 minutes of air. Note in the figure that filled circles indicate times when the majority of rats were in State III, and the open circles indicate times when the majority of rats were in State I. A n acute response to hypoxia can clearly be seen with significant increases in frequency of breathing, tidal volume and total ventilation. There was no significant roll-off in the response for all three components. After the hypoxia was discontinued, an acute " o f f response occurred in which there was an immediate decrease in both frequency and tidal volume to give an overall decrease in total ventilation. There was a post-hypoxic frequency decline (PHFD) in which frequency decreased to levels below the frequency of breathing prior to hypoxia. While tidal volume fell during the exposure to air, it did not decrease significantly below the tidal volume value in State III prior to hypoxia. Total ventilation was significantly lower during the air intervals due to the depression of frequency. We did not see long-term facilitation of tidal volume in the averaged data for these rats.  174  160  0  4  8  12 .16 20 24 28 32  36 40 44 48  30  60  0  4  8  12  36 40 44 48  30  ., 60  16 20 24 28 32 Time (min)  Fig. 5.4. The effects of intermittent hypoxia and the post-hypoxia periods of air on breathing in the control rats (n=8) shown using absolute values. The black bars under the graphs indicate the time of the hypoxic exposure. The top figure shows the effects on frequency of respiration. The middle figure shows the effects on the tidal volume, and the bottom figure shows the effects on total ventilation. Open circles indicate times when the rats were in State I and filled circles indicate times when the rats were primarily in State III.  175  5.3.5. An examination of the possibility of long-term facilitation of breathing It has been reported that in some strains of rats and in some preparations, a significant long term facilitation of tidal volume or frequency occurs after the discontinuation of the hypoxic episodes. Fig. 5.5 shows the breathing pattern in the post hypoxic period for one of the control rats, which went into State I after about 25 minutes breathing air. While this rat was primarily in State I after 30 minutes of air, there was some State III. The graph shows the absolute values for fR, V and V TOT from the last t  twenty seconds of hypoxia through to 1 hour after hypoxia ended. For reference, the percent change was calculated based on the pre-hypoxia value in the corresponding state. For State I, at 60 minutes post hypoxia, frequency had increased by 6.3 % and by 9.6 % in State III. Tidal volume had increased by 2.9 % in both States I and III, while total ventilation had increased by 8.5 % in State I and 1.4 % in State III.  176  £ O  140  |  120  $  100 ^  f in State I pre-hypoxia R  -f in State III pre-hypoxia  c  R  0) 3  S"  80 -\ -4 -2 0  2  4  20  6  30  40  50  60  0.50 i 0.45  >  0.40  V in State I ' pre-hypoxia  0.35  _V in State III pre-hypoxia  T  T  0.30  -2  0  2  4  20  6  30  40  50  60  60  V in State I pre-hypoxia TOT  50  > "55 O  40 J  'V inStatel pre-hypoxia T0T  30 1  /y^-2  0  2  4  6  8  20  • 30  40  50  60  Time (minutes)  Fig. 5.5. The values for the frequency of respiration, tidal volume, and total ventilation for one representative rat in the post-hypoxic period is shown. Filled symbols represent values taken when the rat was in State III; open symbols represent values taken when the rat was in State I. O Represents the value prior to hypoxia in State I, H . Represents the value prior to hypoxia in State III.  177  5.3.6. The effect of MK-801 injection on the response to intermittent hypoxia. A comparison of the time course of the H V R for both control and MK-801 treated rats (n=5) is shown in Fig. 5.6. The values are given as percent change in frequency, tidal volume and total ventilation from pre-hypoxia values. The starting values are listed in Table 5.1. Note that while MK-801 caused a significant reduction in all three components of breathing pattern in rats breathing air (Table 5.1), the proportionate change in ventilation due to hypoxia was not significantly different, nor was the pattern of change with both the control and MK-801 treated rats showing an acute response followed by a short-term potentiation of frequency, tidal volume and total ventilation, followed by a trend to roll off in all three variables (Fig. 5.6). Note however, that in the post hypoxic period, tidal volume in the MK-801 treated animals declined significantly more slowly than in the control rats and remained above the pre-hypoxia tidal volume in the MK-801 treated rats. After the last of the 5 exposures to hypoxia, there was another PHFD followed by an increase in frequency over the next hour until the frequency was approximately equivalent to that before the first hypoxic exposure for both the control and the MK-801 treated rats. It should be noted that the control rats cycled into State III immediately after the hypoxia was discontinued and remained in State III for at least 20-25 minutes. After this time, 3 of the rats started cycling between States I and III, although they still tended to spend more time in State III than in State I. The MK-801 treated rats were in State III for the entire hour after hypoxia.  Table 5.1: A comparison of values for f , V and V TOT pre and post MK-801 R  T  Pre-MK-801  Post-MK-801  Frequency of Respiration  116.1 ± 1.1  106.1 ± 1.4 *  Tidal Volume  0.455 + 0.004  0.399 ± 0.005 *  Total Ventilation  53.2 ± .6  42.3 ± .8 *  * Indicates values that are significantly different from before, MK-801.  179  160  0  4  8  12  16 20  24  28 32  36 40  44 48  30  60  Time (min)  Fig. 5.6. A comparison of the pattern of the hypoxic and post-hypoxic response in control and MK-801 treated rats is shown as a % change (normalized to 100%) from values in State III prior to hypoxic exposure. Black bars indicate the time of hypoxic exposure. * Indicates significant differences between control and MK-801 treated rats. 5 Indicates significant differences from values prior to hypoxia.  180  5.3.7. An analysis of the breathing pattern at critical time points during the intermittent hypoxia exposure The finding that tidal volume did not decrease as much in the MK-801 treated rats in the aftermath of hypoxia led us to examine certain critical time points in more detail to compare the breathing pattern between the control and MK-801 treated rats. Fig. 5.7 shows the per cent change in frequency at the peak of the acute response, at the end of the hypoxic episode (5 minutes), in the first 20 seconds of the post-hypoxic period ("off, Fig. 5.7), one minute into the post-hypoxic period and at the end of the post-hypoxic period (5 min Post) for each of the five episodes.  Once again, there were no differences in the  response to hypoxia between the control and MK-801 treated rats. There were also no significant differences in either frequency or total ventilation during the post-hypoxic air exposure. However, in the post-hypoxia period, the MK-801 treated rats maintained a tidal volume that was significantly greater than the pre-hypoxia tidal volume, while the tidal volume of the control rats returned to the pre-hypoxia level (Fig. 5.8). At 5 minutes post-hypoxia, the tidal volume of the MK-801 treated rats was still greater than prehypoxia. For the control rats, in the 5-minute post-hypoxia period, tidal volume actually dropped significantly below the pre-hypoxia value. In episodes 2, 3 and 4, there were significant differences between control and MK-801 treated rats. Following the final episode of intermittent hypoxia, tidal volume in the MK-801 treated rats declined slowly over the first five minutes but increased again and by 60 minutes, was just significantly greater (p = 0.045) than pre-hypoxic values, suggestive of LTF (Fig. 5.8).  181  40  Peak  5 min  Off  1 min Post  , 5 min Post  20  -20 5 5 5  -40  5 5  S 8 5  •ti i i  .....  5  5 5  5  5  5  5  Juki  • H Control tSSSm MK-801  a. S  mi Episode: 1 2 3 4 5  1 2  3 4 5  I •I 1 2 3 4 5  1 2 3 4 5  1 2 3 4 5  Fig. 5. 7. Values shown are taken at the peak of the acute response to hypoxia, just prior to the end of the hypoxic episode, in the first 20 seconds of air after hypoxia, at 1 minute post hypoxia and in the last 20 seconds of air after hypoxia. *Indicates values that are significantly different between the control and MK-801 treated rats. 8 Indicates significant differences from values prior to hypoxia.  182  Fig. 5.8. A comparison of the post-hypoxic tidal volume between control rats and rats treated with MK-801. 8 Indicates significant differences from values prior to hypoxia.  183  5.4. Discussion: 5.4.1. The Effects of Intermittent Hypoxia on Cortical Activation State Continuous hypoxia disrupts sleep and leads to increased periods of wakefulness in unanaesthetized rats (Pappenheimer, 1977; Laszy and Sarkadi, 1990). In urethaneanaesthetized rats, hypoxia causes a similar disruption in the E E G pattern, such that when hypoxia starts the rats go into State I. During the initial 5 to 10 minutes, State II appears for only very short periods, followed by longer periods of State I and II (Boon et al., 2004, Chapter 2). As the exposure to hypoxia continues, there are more episodes of State III, and although they tend to be slightly longer they don't usually exceed -40-60 seconds in length (Boon et al., 2004, Chapter 2). Intermittent hypoxia caused a similar disruption of the normal cycling between states such that during hypoxia the animals were primarily in State I. After the hypoxia was discontinued, the rats went into State III, and this persisted for the entire 5 minutes of air exposure. This is consistent with the observation that hypoxia administered during sleep caused increased wakefulness in rats and that non-REM periods were shorter and less frequent (Hamrahi et al., 2001). When the hypoxic stimulus was discontinued, the rats entered a recovery period where there was decreased wakefulness and an increase in the length of the non-REM sleep periods. In our experiments, after the final episode of hypoxia, the control rats cycled into State III and most remained there for the following hour, although some started to exhibit periods of State I during the last half hour. MK-801 injected into the region of the PBrKF caused the rats to cycle into State III. Introduction of the hypoxic stimulus in an intermittent manner, while showing some potential for arousal (some State II appeared near the end of the episode), did not cause  184  the rats to cycle into State I. After the final episode of hypoxia, the MK-801 treated rats entered and remained in State III for the balance of the experiment. Similar results were found after continuous hypoxia in rats in which MK-801 was injected into the PBrKF (Chapter 4). 5.4.2. Effects of MK-801 on resting breathing pattern prior to hypoxic exposure Ventilation has been found to decrease after systemic MK-801 and Ketamine (Lydic and Baghdoyan, 2002). In part, the decrease in ventilation is due to the fact that the rats cycle into N R E M 8 slow- wave sleep (Campbell and Feinberg, 1996; Lydic and Baghdoyan, 2002; Chapter 3). Blockade of N M D A r in the region of the PBrKF also reduced total ventilation through a reduction in the frequency of breathing although there was a small increase in tidal volume (Chapter 4). With systemic blockade of N M D A r or injection of MK-801 into the PRF, the reduction in breathing is thought to be due in part to a decreased release of acetylcholine in the medial pontine reticular formation (mPRF) (Lydic and Baghdoyan, 2002). It is possible that our injections, being of relatively large volume, were diffusing into the mPRF where N M D A r have been found thus causing the change to State III (Monaghan and Cotman, 1985; Chapter 4). It has been reported that changes in pontine reticular activity cause a reduction in the excitability of pontine respiratory neurons in the parabrachial region (Gilbert and Lydic, 1994). However, the Pontamine sky blue was clearly visible in the fixed brain tissue and did not appear to extend into the region of the PRF. It has been reported that activation of neurons in the pedunculopontine tegmentum (PPT) results in release of acetyl choline in the PRF (Lydic and Baghdoyan, 1993), and given the proximity of the PPT to the PBrKF we could not  185  rule out diffusion of the MK-801 into that region thus having an indirect effect on the PRF. There are also N M D A r in the PBr and K F regions (Monaghan and Cotman 1985; Petralia et al., 1994), and the consensus is that neurons with these receptors are part of the inspiratory off-switch mechanism, which along with vagally mediated stretch-receptor input to the D R G , control inspiratory time. Blockade of N M D A r in the PBrKF leads to an increase in Ti and a reduction in breathing frequency, leading to apneusis in vagotomized animals (Ling et al., 1994). Thus the injection of MK-801 into the region of the PBrKF would decrease frequency of breathing due to its effects on the inspiratory offswitch, but may also change breathing pattern because of changes in cortical activation state. 5.4.3. Time domains of the HVR before and after blockade of NMDAr in the PBrKF 5.4.3.1. The Acute Response (AR) Both control and MK-801 injected rats showed a robust acute response to hypoxia, with increases in both frequency and tidal volume within the first 10 seconds after application of the hypoxic gas, as has been described previously (Powell et al., 1998; Chapters 3, 4). This acute response to the onset of hypoxia was maintained and did not change in the subsequent episodes of hypoxia. This indicates that the magnitude of this response was not affected by changes in state since there was no significant difference between the response to hypoxia in episode 1 and that in episodes 2 through 5. There was also an acute " o f f response at the end of hypoxia, on the switch to air, with the frequency of breathing decreasing within the first 20 seconds. While there was a decrease' in tidal  186  volume during the first 20 seconds on return to air in both groups of rats, this decrease was not as pronounced in the MK-801 treated rats as in the control rats. 5.4.3.2. Short Term Potentiation (STP) There was a progressive increase in both breathing frequency and tidal volume during the next 1-2 minutes after the onset of hypoxia in both control and MK-801 treated rats in all episodes of hypoxia, indicative of STP. The STP of tidal volume was prolonged in both the control and MK-801 treated rats in the post-hypoxic period, and there was a noticeable difference in the magnitude of STP in the MK-801 treated rats. Tidal volume remained higher than the pre-hypoxia tidal volume in the treated rats for 1 minute after hypoxia was discontinued, while it had decreased to, and even dropped below, the pre-hypoxia levels in the control rats by this time. The maintenance of an elevated tidal volume following hypoxia was also found after continuous hypoxia (Chapter 4) and would suggest that N M D A r in the PBrKF have a role in suppressing STP after hypoxia. Given that both groups of animals were in State III at this time, this suggests that the role of PBrKF neurons in suppressing STP is not state dependent. 5.4.3.3. Short Term Depression (STD) While there was a trend for both frequency and tidal volume to decrease over the 5 minutes of each episode of hypoxia, these changes were not significant. Following hypoxia, however, there was a depression of breathing frequency to values significantly below pre-hypoxia levels for at least 5 minutes for both control and MK-801 treated rats. Control animals cycled from State I to State III at this time while the MK-801 injected animals were continuously in State III, and yet STD (or PHVD) occurred in both groups. While it would seem reasonable to assume that a change from State I to State III  187  following hypoxia would contribute to PHFD, in the MK-801 treated rats where this state change did not occur, there was no difference in the magnitude of post-hypoxia frequency decline. 5.4.3.4. Progressive Augmentation (PA) While progressive augmentation of the hypoxic response is sometimes seen when hypoxia repeats in an intermittent manner (Powell et al., 1998), this has only been shown to occur when the animals are maintained at isocapnic levels. Since we did not control for the decrease in CO2 during hypoxia, it isnot surprising that P A did not occur. 5.4.3.5. Long Term Facilitation (LTF) LTF is "a persistent change in the neural control system based on prior experience" (Mitchell and Johnson, 2003, for review) and has been demonstrated in some experimental preparations after intermittent hypoxia or following episodic stimulation of the carotid sinus nerve (Milhorn et al., 1980; Hayashi et al., 1993). It can be seen as an enhanced respiratory motor output in the phrenic and hypoglossal nerves, (Bach and Mitchell, 1996) and the inspiratory intercostal nerves (Fregosi and Mitchell, 1994), or it can be seen as an increase in ventilation that continues after the hypoxic stimulus has been removed (Olson et al., 2001).  What is often not clearly understood is that  immediately after episodic hypoxia, tidal volume falls to the same level as was seen prehypoxia. Then, in some situations, tidal volume starts to increase again until it reaches a level that can be as great as that seen during hypoxia. Ventilatory LTF of frequency rather than tidal volume has also been demonstrated (Olson et al., 2001; McGuire et al., 2002).  188  There are a number of factors that influence the appearance and magnitude of LTF. These include the number of episodes of hypoxia (3-10), the level of hypoxia (10% but not 12%, Jannsen and Fregosi, 2000), the experimental preparation used i.e. whether rats are anaesthetized or not, whether they are vagotomized or not, whether they are poikilocapnic or isocapnic, and whether they are ventilated or spontaneously breathing can have an effect on LTF (Mitchell et al., 2001). LTF is not seen after continuous hypoxia (Dwinell et al., 1997). In addition, the substrain of rat used, the gender, and for female rats, the time during the estrus cycle, can all have an effect on whether L T F is observed and how large an effect is produced. It has been reported that humans show LTF during N R E M sleep, but not while awake (Babcock and Badr, 1998). In our preparation, we found that when state effects were taken into account there was no LTF of either frequency or tidal volume in the control rats. Note however, that it is important that state effects are considered. Given the depression in breathing that occurs when animals go from the awake state to the slow wave sleep state, if measurements were made prior to hypoxia in sleeping rats and compared to those in awake rats after hypoxia, it would appear that there was significant LTF. The same holds true for urethane anaesthetized rats and the breathing changes that occur when the rats go from State I to State III. It is interesting to note that we did see LTF of tidal volume in the MK-801 treated rats. While tidal volume dropped to levels that were not significantly different from the pre-hypoxia values during the first 5 minutes, by 60 minutes post-hypoxia, tidal volume had increased to levels that were significantly greater than the pre-hypoxia levels.  189  This would indicate that neurons with N M D A r in the PBrKF must suppress long-term facilitation in urethane-anaesthetized rats. 5.5. Conclusions:  Intermittent hypoxia, administered to poikilocapnic urethane-anaesthetized Sprague Dawley rats, produced cortical activation (State I) during the episodes of hypoxia, and cortical synchronization (State III) during recovery on air. This closely parallels the hypoxic wakefulness and the "recovery sleep" seen after exposure to hypoxia in unanaesthetized animals. There was a robust acute response to hypoxia followed by short-term potentiation of both fR and V T and there were no differences in the acute response between State I and State III. There was a trend to short term depression but the values were not significantly different over the course of the hypoxic exposure. After hypoxia short-term depression was evident as fR fell to levels below prehypoxia values and the animals went into State III. Short-term potentiation of tidal volume was also present, however, when the effects of state were factored in, we did not see LTF in the control rats. Finally, blockade of N M D A type-glutamate receptormediated processes in the PBrKF region of the pons, while causing the animals to cycle into State III, did not affect the overall response to intermittent hypoxia. It did, however, sustain the short-term potentiation of tidal volume in the intervals between the hypoxic episodes and produced a significant LTF following intermittent hypoxia. This supports the idea that the neurons of the PBrKF are involved in stabilizing the breathing pattern via control of the inspiratory off-switch (Chapter 4) and act to return and maintain tidal volume at resting levels following hypoxic exposure.  190  5.6 References: Babcock, M . A . , Badr, M.S., 1998. Long-term facilitation of ventilation in humans during N R E M sleep. Sleep 21:709-716. Bach, K . B . Mitchell, G.S., 1996. Hypoxia-induced long-term facilitation of respiratory activity is serotonin dependent. Respir. Physiol.104: 251-160. Boon, J.A., Garnett, N.B.L., Bentley, J.M., Milsom, W.K. 2004a. Respiratory chemoreflexes and effects of cortical activation state in urethane anesthetized rats. Respir. Physiol. Neurobiol. 140:243-256. Campbell, I.G. and Feinberg, I., 1996. Noncompetitive N M D A channel blockade during waking intensely stimulates N R E M 8 J. Pharmacol. & Exp. Ther. 276:737-742. Coles, S.K., Dick, T.E., 1996. Neurones in the ventrolateral pons are required for posthypoxic frequency decline in rats. J. Physiol. 497:79-94. Dwinell, M.R., Janssen, P.L., Bisgard, G.E., 1997. Lack of long-term facilitation of ventilation after exposure to hypoxia in goats. Respir. Physiol. 108: 1-9. Fregosi, R., Mitchell, G.S., 1994. Long term facilitation of inspiratory intercostals nerve activity following repeated carotid sinus nerve stimulation in cats. J. Physiol. (London) 477.3: 469-479. Gilbert, K.S., Lydic, R., 1994. Pontine cholinergic reticular mechanisms cause statedependent changes in the discharge of parabrachial neurons. Am. J. Physiol. 266:R13650. Hamrahi, Ff., Stephenson, R., Mahamed, S., Liao, K.S., Horner, R.L., 2001. Regulation of sleep-wake states in response to intermittent hypoxic stimuli applied only in sleep. J. Appl. Physiol. 90: 2490-2501. Hayashi, F., Coles, S.K., Bach, K . B . , Mitchell, G.S., McCrimmon, D.R., 1993.Timedependent phrenic nerve responses to carotid efferent activation: intact vs. decerebellate rate. A m J. Physiol. 265: R811-R819. Hunter, J.D., McLeod, J.Z., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia: respiratory reflexes. Respir. Physiol. 112: 83-94. Hunter, J.D., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia: cardio-respiratory effects. Respir. Physiol. 112: 71-81. Janssen, P.L., and Fregosi, R.F., 2000. No evidence for long-term facilitation after episodic hypoxia in spontaneously breathing, anesthetized rats. J. Appl. Physiol. 89: 1345-1351.  191  Laszy, J., Sarkadi, A., 1990.Hypoxia-Induced Sleep Disturbance in Rats. Sleep. 13(3): 205-217. Ling, L., Karius, D.R., Speck, D.E., 1994. Role of N-methyl-D-aspartate receptors in the pontine pneumotaxic mechanism in the cat. J. Appl. Physiol. 76:1138-1143. Lydic, R., Baghdoyan, H.A., 1993. Pedunculopontine stimulation alters respiration and increases Ach release in the pontine reticular formation. A m J. Physiol. 264: R544-R554. Lydic, R. and Baghdoyan, H.A. 2002. Ketamine and MK-801 decrease acetylcholine release in the pontine reticular formation, slow breathing, and disrupt sleep. Sleep. 25:617-622. Mateika, J.H., Fregosi, R.F., 1997. Long-term facilitation of upper airway muscle activities in vagotomized and vagally intact cats. J. Appl. Physiol. 82:419-425. McGuire, M . , Zhang, Y . , White, D.P., Ling, L., 2002. Effect of hypoxic episode number and severity on ventilatory long-term facilitation in awake rats. J. Appl. Physiol. 93: 2155-2161 Millhorn, D.E. Eldridge, F.L., Waldrop, T.G. 1980. Prolonged stimulation of respiration by a new central neural mechanism. Respir. Physiol.41: 171-198. Mitchell, G.S., Baker, T.L. Nanda, S.A., Fuller, D.D., Zabka, A . G . , Hodgeman, B.A., Bavis, R.W., Mack, K J . and Olson E.B. Jr., 2001. Invited Review: Intermittent hypoxia and respiratory plasticity. J. Appl. Physiol. 90:2566-2475. Mitchell, G.S., Johnson, S.M., 2003, Invited Review: Neuroplasticity in respiratory motor control. J. Appl. Physiol. 94: 358-374. Monaghan, D.T., Cotman, W., 1985. Distribution of N-methyl-D-aspartate sensitive L [3H] glutamate-binding sites in rat brain. J. Neurosci. 5: 2909-2919. Olson, E.B. Jr., Bohne, C.J., Dwinell, M.R., Podolsky, A., Vidruk, E.H., Fuller, D.D., Powell, F.L., Mitchell, G.S., 2001. Ventilatory long-term facilitation in unanaesthetized rats. J. Appl. Physiol. 91:709-716. Pappenheimer, J.R., 1977. Sleep and respiration of rats during hypoxia. J.Physiol. 266: 191-207. Petralia, R.S., Yokotani, N . , Wenthold, R.J., 1994. Light and electron microscope distribution of the N M D A r subunit N M D A R 1 in the rat nervous system using a selective antibody. J. Neurosci. 14: 667-696. Powell, FL, Milsom, W.K., Mitchell, G.S., 1998. Time domains of the hypoxic ventilatory response. Respir. Physiol. 112: 123-134.  192  Rechtshaffen, A., Kales, A., Berger, R J . , et al., 1968. A Manual of Standardized Terminology, Techniques and Scoring system for Sleep Stages in Human Subjects. U.S. Government Printing Office, Washington, D.C.  Chapter 6  General Conclusions  194  6.0 Introduction The purpose of this research was to study the role of the PBrKF respiratory complex of the pons in the control of respiration. In this regard, there were several hypotheses that I addressed. 1) The PBrKF region plays an important role in relaying information about cortical activation state to the medullary respiratory centres to cause the depression of ventilation that is commonly seen when animals go from waking to sleeping states. 2) The PBrKF region is involved in mediating the changes in chemoresponses to hypoxia and hypercapnia that occur on changes in cortical activation state. 6.1 Preliminary considerations: Urethane anaesthesia The experiments were conducted using urethane-anaesthetized Sprague Dawley rats, which introduces several issues that must be addressed. Urethane was used as the anaesthetic for a number of reasons. Urethane does not depress respiration or cardiac function as do other anaesthetics (Maggi and Meli, 1986a,b). In addition, under low doses of urethane anaesthesia animals continue to show cyclic changes in E E G pattern that superficially resemble those seen when animals cycle between natural sleep and awake states (Grahn and Heller, 1989; Grahn et al., 1989; Hunter and Milsom, 1998; and Hunter et al., 1998). In Golden-mantled ground squirrels, ventilation changed with changes in the E E G pattern in the same way that it did with natural state transitions (Hunter and Milsom, 1998). Thus it seemed that urethane anaesthesia could offer a good model system for studying changes in breathing with changes in cortical activation state in rats, without having to deal with the problems associated with freely behaving animals.  195  This model could also potentially allow me an opportunity to investigate the role of the PBrKF using more invasive techniques than could be used in unanaesthetized animals. I found that urethane anaesthetized rats had E E G patterns that superficially resembled the patterns seen in waking and sleeping animals and that there was a regular cycling between these patterns. In addition, ventilation and chemosensitivity changed with changes in E E G pattern in the same way that it does in unanaesthetized rats, as has been documented by other authors (Bartlett and Tenney, 1970; Pappenheimer, 1977; Lai et al., 1978; Peever and Stephenson, 1979; Strohl et al., 1997; Walker et al., 1997). In discussing the E E G patterns that occur during urethane anaesthesia, the terms 'sleep-like ' and 'awake-like' have been used. The 'sleep-like' E E G pattern, with low frequency, high-amplitude waves, has been called State m. The 'awake-like' E E G pattern, with high frequency, low-amplitudes waves, has been called State I (Grahn and Heller, 1989). The transition between these States has been referred to as 'cortical activation' (Hunter and Milsom, 1998). Although I have used this terminology in my thesis, it is appropriate to revisit it in light of the results of the spectral analyses of the E E G patterns. The State I pattern consisted of high frequency, low amplitude waves which superficially appeared to be similar to those seen during either the waking or R E M sleep states. However, the spectral analysis of this pattern shows that there is suppression of power in both the a and the P bands that would normally be seen during wakefulness and R E M sleep (Hamrahi et al., 2001), and in addition, there are no pontogeniculoccipital (PGO) waves that are one of the characteristics of R E M sleep in rats (Datta and Hobson, 2000) and other mammals (Steriade, 1993 for review). R E M sleep is also normally  196  characterized by muscle atonia in rats, as in other mammals (Datta and Hobson, 2000, England and Strobel, 1996). Grahn and Heller (1989) showed that there were two subclasses of E E G State I, defined by E M G activity. One showed continuous low E M G activity and the other showed an increased level of E M G activity that was also continuous. Whether these different levels of E M G activity could be taken as indications that within State I there was an 'awake-like' component and a ' R E M sleeplike' component is open to speculation. However, they indicated that the State I pattern with low E M G component preceded the transition to State III, which would not correspond to the usual appearance of R E M sleep in an unanaesthetized animal which normally appears after a period of SWS. It has been shown that urethane potentiates the function of nicotinic acetyl choline receptors (Hara and Harris, 2002), and that Ach release in the hippocampus is correlated with power in the 9 band (Keita et al., 2000). M y finding that the waves with the highest power in State I were in the 0 range indicates that there is hippocampal activity in urethane anaesthetized rats that is not suppressed by the anaesthetic, but since 9 waves are characteristic of both waking and R E M sleep (Hamrahi et al., 2001) this does not allow us to differentiate. The State III pattern is characterized by very high amplitude waves in the 8 range (0.5 - 4 Hz), which is typical of what is seen in sleeping rats. However, true SWS is also characterized by waves in the a band (Hamrahi et al., 2001), and these are suppressed in urethane-anaesthetized rats. This brings into question whether referring to these states as 'awake-like' and 'sleep-like' is appropriate and I think that the conclusion is that it is not appropriate.  197  This in turn leads to the question of whether the term 'cortical activation' is really indicative of what is happening in the transition from State III to State I. The desynchronized E E G of a true awake state appears when excitatory impulses from the Reticular Activating System (RAS) disrupt the thalamocortical oscillations that produce the synchronized 5 waves of SWS (Steriade, 1993). This desynchronized E E G is, as mentioned previously, characterized by an increase in the power of the waves with frequencies in the a and the |3 bands. In urethane-anaesthetized rats, the difference between States I and III is marked by the presence or absence of waves in the 8 band. While it can probably be stated that the transition from State III to State I is marked by the disruption of the thalamocortical oscillatory generator, which accounts for the loss of the synchronized wave pattern, there is no indication that there has been an activation of the cortex per se. Therefore it would be preferable to refer to State I as having a desynchronized E E G pattern with a low 5 power and State III as having a synchronized E E G pattern with a high 8 power. The transition between states should not be called cortical activation but could be referred to as brainstem reticular activation. The sensory inputs (temperature changes, loud noises, or touch) that disrupt the synchronous E E G pattern must do so by increasing the activity of neurons in the RAS that project to the thalamus. This critique of the terms used to describe the E E G States seen in urethaneanaesthetized animals does not invalidate the observations of this thesis. Whether the cortex is activated during the changes in E E G pattern is irrelevant to the findings that ventilation and chemosensitivity change in response to the changes in 8 power in a manner that is identical to the changes observed in unanaesthetized animals that go from  198  waking to sleep. In fact, this finding suggests that changes in the activity of neurons in the brainstem and thalamus are responsible for the changes in ventilation and chemosensitivity that occur due to peripheral sensory input, and that activation of the cortex is not required. 6.2 The Role of the PBrKF as a relay for State-related information This then allows us to focus attention on the role of neurons in the P B r K F in the control of ventilation and chemosensitivity in response to changes in the activity of neurons in the RAS. This led to the formulation of the additional hypotheses: 4) Changes in E E G state will involve NMDA-type glutamatergic processes and 5) NMDA-type glutamatergic processes in the PBrKF will be involved in the effects of changes in E E G state on breathing How is the information from the neurons of the R A S channeled to the rhythm generator in the medulla to change ventilation in response to changes in RAS activity? M y hypothesis was that the PBrKF in the pons is the integrating and relay centre that is responsible for conveying this information. M y results give support to one aspect of this hypothesis in that the blockade of neurons in the PBrKF that had NMDA-type glutamate receptors eliminated the effects of cortical activation on ventilation. It has been reported that fetal sheep show episodic, irregular breathing movements only during periods of low voltage E E G activity, with apnea during high voltage E E G periods (Dawes et al., 1972). Lesions in the dl pons of fetal sheep produced periods of continuous breathing during the high voltage E E G activity (Gluckman and Johnston, 1987). This would indicate that there must be an area in the dl pons that inhibits breathing during what would be the equivalent of SWS in the fetal lambs. After birth this inhibition is relieved, which could be due to  199  the development of an excitatory neural network, or changes in the balance of neurotransmitters that results in the disinhibition of the dl pons. The inhibition during sleep is not entirely removed in that there is a depression of ventilation during sleep, but this does not normally lead to apnea. I cannot draw any specific conclusions as to the origin of the impulses that are relayed through the PBrKF. Determining the source of the input, whether it might be from the Raphe nuclei, the Locus Coeruleus the pontine reticular formation (PRF), or the Pedunculopontine Tegmentum (PPT) is a topic worthy of research. There is evidence however to indicate that glutamate acting via N M D A receptors on neurons in the PPT, which is located just rostral to the PBrKF, increases the secretion of Acetyl Choline (ACh) in the PRF, and this activation leads to wakefulness and R E M sleep (Datta et al., 2001a). When glutamate is given in high doses, it increases wakefulness and at low doses it increases R E M sleep (Datta et al., 2001b). There are also reports that neurons of the PPT are intermingled with neurons of the PBrKF (Datta, 1995). This information leads to the possibility that the neurons with N M D A r that overlap the PBrKF and the PPT may have a dual function. They may stimulate release of A C h in the PRF and stimulate the respiratory neurons in the PBrKF that are responsible for increasing the stimulation of pacemaker neurons in the pre-Bdtzinger complex in the medulla, and motor neurons in the phrenic motor nucleus to increase frequency and tidal volume (respectively) during wakefulness. Due to equipment limitations, my microinjections were of relatively large volume, and further experiments with lower volumes of MK-801, along with recordings from individual neurons in the area would be needed to test this hypothesis.  200  6.3 The Role of the PBrKF in Chemosensitivitv Since I had found that chemosensitivity decreased in urethane anaesthetized rats when the E E G state changed from synchronized to desynchronized, I tested an additional hypothesis. 6) NMDA-type glutamatergic processes in the PBrKF will be involved in the changes in respiratory sensitivity to hypoxia and hypercapnia.that occur on changes in E E G state. I did not find any evidence that neurons with N M D A r in the PBrKF are involved in the response to hypoxia or hypercapnia when there are changes in state, but since I only blocked NMDA-type glutamate receptor mediated processes, I can't rule out the possibility that neurons responsive to other neurotransmitters, or with A M P A or kainate glutamate receptors in this region might alter chemosensitivity with state changes. Lesions in the dl pons of fetal sheep have been found to abolish the hypoxic respiratory depression that is seen in unanaesthetized lambs in utero (Gluckman and Johnston, 1987). Therefore it seems that during development of the fetus there is a structure in the dl pons that inhibits breathing movements when the mother is exposed to hypoxia and once again this inhibition is relieved after birth. Adult mammals show strong and immediate responses to hypoxia (Powell et al., 1998). It may be that the carotid and aortic bodies take over the function of O2 sensing and these and other centres in the brain develop sensitivity to hypercapnia. There seem to be multiple chemoreceptor sites for CO2 in the brainstem in neonates and adults (Nattie, 2000). If this is the case, then it is not surprising that changes in chemosensitivity with changes in E E G state are not mediated by the PBrKF.  201  6.4 The Role of the PBrKF in stabilizing the breathing pattern after hypoxia Although neurons with N M D A r in the PBrKF did not have a role in the overall response to hypercapnia or hypoxia, there were effects in the post-hypoxic period that would indicate a role for the PBrKF in the recovery from hypoxia. This led to my last hypothesis: 6) N M D A receptor-mediated processes in the PBrKF have a role in returning tidal volume to normal following exposure to hypoxia. Blockade of N M D A r delayed the return of tidal volume to normal after hypoxia and caused a significant long-term facilitation of tidal volume in the post-hypoxic period. This information contributes to the knowledge about the role of the PBrKF in the pons as a mediator of respiratory stability. It suggests that there is an interaction between hypoxic stimuli and pulmonary stretch receptor feedback that is permissive of increases in tidal volume but that once the hypoxic stimulus is removed the PBrKF acts to quickly restore V j to normal. The use of urethane anaesthetized rats also allowed me to determine i f there were state effects in the response to hypoxia. Since blockade of N M D A r in the PBrKF caused animals to go into State III, I was able to evaluate what role E E G state played in the response to hypoxia. The control rats showed the typical transition into State I on hypoxic exposure and a robust acute response. The MK-801 treated rats showed the same acute response to hypoxia even though they were in State III, which would indicate that E E G state does alter the hypoxic response, at least in urethane-anaesthetized rats. I was also able to show that it is important to monitor E E G state when assessing the possible appearance of LTF after intermittent hypoxia. If ventilation prior to hypoxia is  202  measured in an animal in State Ul and then compared to ventilation after hypoxia in an animal in State I, it would be very easy to mistake the difference in ventilation that occurs naturally between states for the appearance of LTF. Whether this is true in unanaesthetized animals should be explored. 6.5 The Role of the PBrKF in the Generation of Respiratory Rhythm An addition question that was discussed in the introduction to this thesis was: Is the PBrKF part of the respiratory rhythm generator? There is evidence to show that the neurons of the PBrKF have both tonic and phasic activity (Cohen and Wang, 1959; Cohen and Shaw, 2004). It has been suggested that there are pacemaker neurons in the pre-Botzinger complex that operate at a subthreshold level, and that tonic input from both the PBrKF and the D R G is required to raise the membrane potential of these neurons to threshold to generate the action potentials that drive respiratory rhythm (von Euler, 1986). However, it would seem to be a joint effort in that lesion or blockade of N M D A r in the PBrKF alone, although it increases the length of inspiration and expiration, does not stop respiratory activity (Gautier and Bertrand, 1975; Ling et al., 1994)). The same is true of removal of stretch receptor input from the lungs via vagotomy. However, when pulmonary stretch receptor (PSR) input to the D R G is removed via vagotomy, and PBrKF input via either lesion of the PBrKF or blockade of N M D A r is also removed, then apneusis occurs (Oku and Dick, 1992; Fung et al., 1994). This means that the PBrKF and PSR feedback to the D R G function together to control the inspiratory off-switch (IOS) (Cohen and Wang, 1959; Cohen and Shaw, 2004). When both are removed, respiration is far from eupneic, which would indicate the importance of both the PBrKF and PSR feedback in the control of respiratory rhythm.  203  Can it then be concluded that the roles of PSR feedback to the D R G , and the role of the PBrKF in the inspiratory off-switch mechanism are redundant to each? I don't think this is the case, because it is clear that the PBrKF has numerous functions in addition to its role in the inspiratory off-switch mechanism. The D R G , as the recipient of stretch receptor input from the lungs via the vagus nerve, has the dominant role in terminating inspiration when the lungs start to reach their maximal inflation level. During quiet breathing, however, when pulmonary stretch receptor feedback to the D R G via the vagus nerve is minimized, the PBrKF dominates in controlling the inspiratory offswitch (Cohen and Shaw, 2004). This control of the IOS involves the relay of information about cortical activation state (as shown by my data) and I would speculate that it also relays information about body temperature (which should be the topic of a further study). The PBrKF has also been shown to have a role in mediating the effects of cutaneous nociceptor input on breathing (Jiang et al., 2004), airway protective responses (Dutschmann and Herbert, 1996) and to play an integral part in the integration of sensory and autonomic activity (Dutschmann et al., 2004). I think that my results are important in that they provide more evidence that respiratory rhythm is controlled by a pontomedullary circuit of which the PBrKF is an integral part. 6.6. Summary Urethane anaesthesia is useful in the study of E E G state differences in breathing, but it cannot be directly equated to awake and sleeping states. Nevertheless, changes in ventilation with changes in the E E G states seen during urethane anaesthesia are the same as the changes in breathing that are seen when unanaesthetized rats go from sleep to  204  wakefulness, making this a good model system for studying changes in breathing with changes in E E G state. The PBrKF respiratory complex plays an important role as an integrative and relay centre for the changes in breathing that occur on changes in state. While the PBrKF is not directly involved in the response to hypoxia or hypercapnia or on the changes in ventilation that occur on changes in state during hypoxia or hypercapnia, it does function to stabilize the breathing pattern in the aftermath of hypoxia. The PBrKF functions with the PSR feedback to the D R G in the inspiratory offswitch and as such is an important part of a pontomedullary circuit that sets the rhythm and pattern of respiration in response to peripheral sensory input, as well as input from the Reticular Activating System and other higher brain centres.  205  6.6 References Bartlett, D. Jr., Tenney, S.M., 1970. Control of breathing in experimental anemia. Respir. Physiol. 10: 384-395. Cohen, M . I., Wang, S. C , 1959. Respiratory neuronal activity in pons of cat. J Neurophysiol. 22, 33-50 w  Cohen, M.I., Shaw, C , 2004. Role of the inspiratory off-switch of vagal inputs to rostral pontine inspiratory-modulated neurons. Respir. Physiol. Neurobiol. 143: 127-140. Datta, S., 1995. Neuronal activity in the peribrachial area: relationship to behavioral state control. Neurosci. Biobehav. Rev. 19: 67-84. Datta, S., Hobson, J.A., 2000. The Rat as an Experimental Model for Sleep Neurophysiology. Behav. Neurosci. 114: 1239-1244. Datta, S., Patterson, E.H., Spoley, E.E., 2001a. Excitation of the pedunculopontine tegmental N D M A Receptors Induces Wakefulness and Cortical Activation in the Rat. J. Neurosci. Res. 66: 109-116. Datta, S., Spoley, E.E., Patterson, E.H., 2001b. Microinjection of glutamate into the pedunculopontine tegmentum induces R E M sleep and wakefulness in the rat. A m J. Physiol. 280: R752-R759. Dawes, G.S., Fox, H.E., Leduc, B . M . , Liggins, G.C., Richards, R.T., 1972. Respiratory movements and rapid eye movement sleep in the foetal lamb. J. Physiol. 220: 119-143. Dutschmann, M , Herbert, H., 1996. The Kdlliker-Fuse nucleus mediates the trigeminally induced apnoea in the rat. NeuroReport 7: 1432-1436. Dutschmann, M . , Morschel, M . , Kron, M . , Herbert, H., 2004. Development of adaptive behaviour of the respiratory network: implications for the pontine Kdlliker-Fuse nucleus. Respir. Physiol. Neurobiol. 143: England, S.J., Strobel, R.J., 1996. Respiratory Changes During Sleep. In: Neural Control of the Respiratory Muscles. D.D. Miller (Ed.). CRC Press. von Euler, C , 1986. Brain stem mechanisms for generation and control of breathing pattern. Handbook of Physiology, Section3, The Respiratory system, Vol. II: Control of Breathing, American Physiological Society, Bethesda (1986). Fung, M - L . , Wang, W., St. John, W., 1994. Involvement of pontile N M D A receptors in inspiratory termination in rat. Respir. Physiol. 96: 177-188.  206  Gautier, H., Bertrand, F., 1975. Respiratory Effects of pneumotaxic center lesions and subsequent vagotomy in chronic cats. Respir. Physiol. 23: 71-85. Gluckman, P.D., Johnson, B . M . , 1987. Lesions in the upper lateral pons abolish the hypoxic depression of breathing in unanaesthetized fetal lambs in utero. J. Physiol. 382: 373-383. Grahn, D.A., Redeke, C M . , Heller, H.C., 1989. Arousal state vs. temperature effects on neuronal activity in subcoeruleus area. Am. J. Physiol. 256: R840-R849. Grahn, D.A., Heller, C , 1989. Activity of most rostral ventromedial medulla neurons reflect E E G / E M G pattern changes. Am. J. Physiol. 257: R1496-R1505. Hamrahi, H., Chan, B., Horner, R.L., 2001. On-line detection of sleep-wake states and application to produce intermittent hypoxia only in sleep in rats. J. Appl. Physiol. 90: 2130-2140. Hara, K., and Harris, R.A., 2002. The Anesthetic Mechanism of Urethane: The Effects on neurotransmitter-Gated Ion Channels. Anesth. Analg. 94: 313-318. Hunter, J.D., McLeod, J.Z., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia: respiratory reflexes. Respir. Physiol. 112: 83-94. Hunter, J.D., Milsom, W.K., 1998. Cortical activation states in sleep and anesthesia: cardio-respiratory effects. Respir. Physiol. 112:71-81. Jiang, M . , Alheid, G.F., Calandriello, T., McCrimmon, D.R., 2004. Parabrachial-lateral pontine neurons link nociception and breathing. Respir. Physiol. Neurobiol. 143: 215233. Keita, M.S., Frankel-Kohn, L., Bertrand, N . , Lecanu, L., Monmaur, P., 2000. Acetylcholine release in the hippocampus of the urethane-anaesthetized rat positively correlates with both peak theta frequency and relative power in the theta band. Brain Res. 887: 323-334. Lai, Y . L . , Tsuya, Y . , Hildebrandt, J. 1978. Ventilatory responses to acute CO2 exposure in the rat. J. Appl. Physiol. 45(4): 611-618. Ling, L., Karius, D.R., Speck, D.E., 1994. Role of N-methyl-D-aspartate receptors in the pontine pneumotaxic mechanism in the cat. J. Appl. Physiol. 76: 1138-1143. Maggi, C.A., Meli, A., 1986a. Suitability of urethane anesthesia for physiopharmacological investigations in various systems. Part II: Cardiovascular systems. Experientia 42: 292-297.  207  Maggi, C.A., Meli, A., 1986b. Suitability of urethane anesthesia for physiopharmacological investigations in various systems. Part III: Other systems and conclusions. Experientia 42: 531-537. Nattie,E., 2000. Multiple sites for central chemoreception: their roles in response sensitivity and in sleep and wakefulness. Respir. Physiol. 122: 223-235 Oku, Y . , Dick, T.E., 1992. Phase resetting of the respiratory cycle before and after unilateral pontine lesions in cat. J. Appl. Physiol. 72(2): 721-730. Pappenheimer, J.R., 1977. Sleep and respiration of rats during hypoxia. J.Physiol., London, 266: 191-207. Peever, J.H., Stephenson, R.S., 1997. Day-night differences in the respiratory response to hypercapnia in awake adult rats. Respir. Physiol. 109: 241-248. Steriade, M . , McCormick, D.A., Sejnowski, T.J., 1993. Thalamocortical Oscillations in the Sleeping and Aroused Brain. Science, 262: 679-685. Strohl, K.P., Thomas, A.J., St. Jean, P., Schlenker, E.H., Koletsky, R.J., Schork, N.J., 1997. Ventilation and metabolism among rat strains. J. Appl. Physiol. 82: 317-323. Walker, B.R., Adams, E. M . , Voelkel, N.F., 1985. Ventilatory responses of hamsters and rats to hypoxia and hypercapnia. J. Appl. Physiol. 59: 1955-1960.  

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