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Neurological Development and the Potential for Conscious Perception after Birth Comparison between Species and Implications for Animal Welfare   A Thesis Presented in Partial  Fulfilment of the Requirements for the Degree of   Doctor of Philosophy  in  Physiology   Massey University Palmerston North New Zealand    Tamara Johanna Diesch MSc, BSc  2010

 I 
Widmung 
Ich widme diese Dissertation meiner Familie. Im Besonderen meinem Mann Cedric Priest, meiner Tochter Mackenzie, meinen Eltern Heidrun und Peter Diesch und meinen Grosseltern Liesbeth und Oskar Schienbein, Gertrud Kopp-Diesch und Josef Kesenheimer.   Ihr seid meine ganze Welt. Ohne Euch wäre dieses Unternehmen nicht möglich gewesen. Danke für Eure Liebe, Euer Verständis und Eure Unterstützung.     Dedication 
I would like to dedicate this thesis to my family.  In particular to my husband Cedric Priest, my daughter Mackenzie, my parents Heidrun and Peter Diesch and my grandparents Liesbeth and Oskar Schienbein, Gertrud Kopp-Diesch and Josef Kesenheimer.   You are my world. Without you this endeavour would not have been possible. Thank you so much for your love, understanding and support. 
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 Ethical Aff irmation   
As I embark on my career as a scientist I willingly pledge that I will conduct my research and my professional life in a manner that is always above reproach and I will seek to incorporate the body of ethics and moral principles that constitute scientific integrity into all that I do.   I will always strive to ensure that the results of my research and other scientific activities are ultimately beneficial – for animals and humans alike- and that they do not cause any harm.  With this affirmation I pledge to acknowledge and honour the contributions of ethical scientists who have preceded me, to seek the truth and the advancement of knowledge in all my work.     Adapted from Craig CR, Cather A & Culberson J. (2003). An ethical affirmation for scientists. Science 299, 1982-1983  
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Acknowledgments 
This thesis has been a fantastic, although at times trying, experience. I have learned so much over the last 5 years, on a scientific as well as on a personal basis, and this would not have been possible without the help of a great many special people and organisations.   First, I would like to express my deepest and sincere gratitude to my supervisor Prof David Mellor for his continual guidance and support. His enthusiasm has been infectious and has often saved me from the desire of wanting to give up.  I would like to thank him for teaching me that this journey was not only to learn about science and research, but also to learn about life in general. I owe him a lot of gratitude for his financial support, for believing in my abilities, for always being there for me when I had concerns, for being patient with me and for being such a wonderful and passionate person. It was an honour to have him as my mentor.   My special thanks also to my co-supervisor Assoc Prof Craig Johnson for teaching me how to undertake EEG recordings, how to analyse the data and for doing such a great job of explaining to me what all the ‘squiggly’ lines were all about. Thank you for your support, for your encouragement, financial help and for believing in my abilities.  I am also very grateful for Assoc Prof Roger Lentle (IFNHH) and his assistance with statistical matters, the support he gave unquestioningly and our stimulating conversations.   I would also like to express my gratitude to Dr Laura Bennet and Prof Alastair Gunn (Auckland University) for their collaboration on the literature review, which was the basis for this thesis, and for their encouragement.   Special thanks also to Assoc Prof David Walker (Monash University, Melbourne) for allowing me to join his laboratory on two occasions to undertake blood sample and brain tissue sample analyses. My thanks to Isabella Ciurey, Dr Tamara Yawno and Jan Loose (Monash University, Melbourne) for teaching me extraction and RIA procedures and for their help throughout my stay at Monash University.  
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  My thanks to Drs Paul Chambers and Joanna Murrell for assisting with anaesthesia matters, helpful discussions about planned projects, answering questions regarding anaesthesia and pharmacology and for their support.   Thank you to Jim Battye for his involvement with, his ideas for and helpful comments on the review presented in Chapter 8.   I am particularly grateful for the practical support by Corrin Hulls and Sheryl Mitchinson, without which none of my projects would have been possible.    I would also like to acknowledge the following people for their practical help and support with various parts of my projects: Nicola Bell, Leanne Betteridge, Helene Davesne, David DeAlmeida, Dephine Pernot and Marion Sandrin. Bruce Cann, Cathy Davidson, Dr Troy Gibson, Mike Hogan, Dr Ignancio Lizzaraga, Amanda McIlhone, Dr Vaughan Seed, Prof Kevin Stafford, Neil Ward (IVABS), Dr Sharon Hearne, Phil Pearce and Dr David Simcock (IFNHH), Matthew Levin (IT support; IFNHH), Lee-Anne Hannan, Yvonne Parkes, Christine Ramsay and Kathryn Tulitt (finance office and administration; IFNHH), Karen Pickering and Susan Sims (finance office and administration; Riddet Institute), Barry Evans (Engineering Services), Debbie Chesterfield (SAPU) and the following staff members of the Agricultural Services at Massey University: Byron Taylor, Geoff Warren, Phill Brooks (Tuapaka) and Peter Jessup (Haurongo).  Thank you to Mark Oliver from Auckland University for permission to use his photo of lambs at birth for the purposes of this thesis (Chapter 4).   Funding I gratefully acknowledge the Agricultural and Marketing Research and Development Trust (AGMARDT) for awarding me a Doctoral Scholarship and for being so patient with me.   I also acknowledge the following organisations for personal, project and travel funding 
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during the course of my PhD:  Ministry of Agriculture and Forestry, New Zealand Palmerston North Medical Research Foundation, New Zealand Institute of Food, Nutrition and Human Health, Massey University, New Zealand Riddet Institute, Massey University, New Zealand Animal Welfare Science and Bioethics Centre, Massey University, New Zealand Geoffrey Gardiner Foundation, Australia New Zealand Vice Chancellor Committee (Claude McCarthy Travel Grant) Education New Zealand (New Zealand Study Abroad Award 2005 and 2007)  Work Thank you to Dr David Bayvel, Dr Cheryl Conner and Dr Kate Littin for providing me with casual work with the Animal Welfare Group at the Ministry of Agriculture and Forestry, NZ.  Personal Without the support, love and understanding of my parents and family, my husband Cedric Priest and my best friend Nicole Tubach this endeavour would not have been possible. Thank you all from the bottom of my heart.  I would also like to thank my friends in New Zealand and Germany for believing in me, for encouraging me and for cheering me up when it was badly needed: NZ: Ngaio Beausoleil, Aurelie Castinel, Rene Corner, Amelie Deglaire, Daniel Gray and Abbie McKee, Carly Heatherwick, Sharon Henare, Janina Kuehn, Kate Littin, Megan McGregor, Amanda McIlhone, Anusiah Nicolls and Jeff Devey and Mischa and Paul Walton and Paul Wood. Germany: Tamara Diesch, Malta Fazzari, Elvira Fehringer, Carmen Haberland, Karsten Janz, Alexandra Schatz, Carmen Stoffel, Sabine Tutzauer and the girls from my former dancing group.  Thank you to Cathrin Klare and family, Kirsty Silcock and Joseph McGehan for helping me chase away the homesickness at my second stay in Melbourne. Special thanks also to Simon Verschaffelt and Amber Hays for allowing me to live with them during the 
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time of my projects and for being great friends.    Animals Last, but not least, I would like to acknowledge the animals that involuntarily partook in the experiments outlined in this thesis.   All the experiments presented here have been approved by the Massey University Animal Ethics Committee. 
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Abstract 
In order for animals to experience pain and to suffer from it, they have to be capable of conscious perception. Recent evidence suggests that the fetus is maintained in a sleep-like unconscious state and that conscious perception therefore only occurs after birth. The timing of the onset of conscious perception depends on the maturation of underlying neurological processes and is anticipated to be species dependent. Pain-specific electroencephalographic (EEG) responses of lightly anaesthetised young of three species born at different levels of neurological development were investigated. The results of the present thesis are in agreement with published data on general neurological, EEG and behavioural development. This information, in addition to the present results, has been used to estimate the approximate time of the onset of conscious perception in tammar wallaby joeys, rat pups and newborn lambs.   In wallaby joeys (extremely immature at birth), the EEG remained isoelectric until about 100-120 days of in-pouch age and became continuous by about 150-160 days, with electroencephalographic and behavioural signs of conscious perception apparent by about 160-180 days. In rat pups (immature at birth), the absence of a differentiated EEG suggests that the ability for conscious perception in pups younger than 10-12 days is doubtful. The marginal EEG responses to noxious stimulation in 12-14 day-old pups and the pronounced EEG responses in pups 18-20 days suggest that rats may be capable of conscious perception from 12-14 days onwards. In lambs (mature at birth), full conscious perception is probably not apparent before 5 minutes after birth and may take up to several hours or days to become fully established. Its modulation by the residual neuroinhibitor allopregnanolone, if that occurs, would be highest over the first 12 hours after birth.  Overall, the onset of conscious perception does not seem to follow an “on-off phenomenon”, but seems to develop gradually, even in species born neurologically mature. Although conscious perception, and hence pain experience, may be qualitatively different in younger animals, on the basis of the precautionary principle, when significantly invasive procedures are planned, pain relief should be provided from those postnatal ages when pain may first be perceived – i.e. from about 120 days in the tammar wallaby joey, about 10 days in the rat pup and from soon after birth in the lamb. 
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Table of Contents 
CHAPTER 1 ......................................................................................................................................................1 
TABLE OF CONTENTS......................................................................................................................................2 1.1 INTRODUCTION .........................................................................................................................................4 1.2 DEFINITION OF CONSCIOUSNESS AND CONSCIOUS PERCEPTION .............................................................4 1.3 PHYSIOLOGICAL BASIS OF CONSCIOUSNESS ............................................................................................6 1.4 NEUROLOGICAL DEVELOPMENT AND THE ONTOGENY OF CONSCIOUS PERCEPTION ............................12 1.5 PURPOSE OF THESIS ................................................................................................................................25 1.6 USING PAIN PERCEPTION TO INVESTIGATE CONSCIOUS PERCEPTION....................................................25 1.7 EXPERIMENTAL OUTLINE .......................................................................................................................30 1.8 REFERENCES ...........................................................................................................................................34 
CHAPTER 2 ....................................................................................................................................................49 ABSTRACT .....................................................................................................................................................50 TABLE OF CONTENTS....................................................................................................................................51 2.1 INTRODUCTION .......................................................................................................................................53 2.2 MATERIALS AND METHODS ...................................................................................................................55 2.3 RESULTS..................................................................................................................................................67 2.4 DISCUSSION ............................................................................................................................................95 2.5 CONCLUSIONS.......................................................................................................................................111 2.6 REFERENCES .........................................................................................................................................113 CHAPTER 3 ..................................................................................................................................................121 
ABSTRACT ...................................................................................................................................................122 TABLE OF CONTENTS..................................................................................................................................123 3.1 INTRODUCTION .....................................................................................................................................125 3.2 MATERIALS AND METHODS .................................................................................................................127 3.3 RESULTS................................................................................................................................................134 3.4 DISCUSSION ..........................................................................................................................................149 3.5 CONCLUSIONS.......................................................................................................................................163 3.6 REFERENCES .........................................................................................................................................165 
CHAPTER 4 ..................................................................................................................................................175 
ABSTRACT ...................................................................................................................................................176 TABLE OF CONTENTS..................................................................................................................................177 4.1 INTRODUCTION .....................................................................................................................................179 4.2 MATERIALS AND METHODS .................................................................................................................183 4.3 RESULTS................................................................................................................................................194 4.4 DISCUSSION ..........................................................................................................................................227 4.5 CONCLUSIONS.......................................................................................................................................241 
 IX 
4.6 REFERENCES......................................................................................................................................... 243 
CHAPTER 5.................................................................................................................................................. 253 
ABSTRACT .................................................................................................................................................. 254 TABLE OF CONTENTS ................................................................................................................................. 255 5.1 INTRODUCTION..................................................................................................................................... 257 5.2 MATERIALS AND METHODS................................................................................................................. 264 5.3 RESULTS ............................................................................................................................................... 279 5.4 DISCUSSION.......................................................................................................................................... 302 5.5. CONCLUSIONS ..................................................................................................................................... 319 5.6 REFERENCES......................................................................................................................................... 321 
CHAPTER 6.................................................................................................................................................. 331 
ABSTRACT .................................................................................................................................................. 332 TABLE OF CONTENTS ................................................................................................................................. 333 6.1 INTRODUCTION..................................................................................................................................... 335 6.2 MATERIALS AND METHODS................................................................................................................. 337 6.3 RESULTS ............................................................................................................................................... 349 6.4 DISCUSSION.......................................................................................................................................... 372 6.5 CONCLUSIONS ...................................................................................................................................... 384 6.6 REFERENCES......................................................................................................................................... 385 
CHAPTER 7.................................................................................................................................................. 391 
TABLE OF CONTENTS ................................................................................................................................. 392 7.1 THE ONSET OF CONSCIOUS PERCEPTION AFTER BIRTH: MAJOR FINDINGS AND CONCLUSIONS OF THE PRESENT THESIS.......................................................................................................................................... 393 7.2 IMPLICATIONS FOR ANIMAL WELFARE ................................................................................................ 396 7.3 EXPERIMENTAL DESIGN AND LIMITATIONS......................................................................................... 398 7.4 WHERE DO WE GO FROM HERE: FUTURE STUDIES .............................................................................. 399 7.5 REFERENCES......................................................................................................................................... 402  APPENDIX 1: LABORATORY PROTOCOLS .................................................................................................. 407 APPENDIX 2: FULL PUBLICATIONS RELATED TO THE PRESENT THESIS .................................................... 408  
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Table of Figures 
FIGURE 2.1: EEG TRACES OF JOEYS APPROXIMATELY 94 DAYS, 124 DAYS, 145 DAYS, 173 DAYS, 198 DAYS AND 261 DAYS OF IN-POUCH AGE (FROM TOP TO BOTTOM), SHOWING AN ISOELECTRIC EEG AT 94 DAYS AND ISOELECTRIC EPOCHS FOR JOEYS AT 124 AND 145 DAYS OF IN-POUCH AGE. NOTE THE DIFFERENT SCALE FOR THE TWO OLDEST JOEYS – THE FIRST SCALE SHOWN RELATES TO THE FOUR TRACES ABOVE IT AND THE SECOND TO THE TWO TRACES ABOVE IT. ...................................................62 FIGURE 2.2: CHANGES IN THE PROPORTION OF TIME OCCUPIED BY NON-ISOELECTRIC EEG PATTERNS AT DIFFERENT IN-POUCH AGES FOR BASELINE (1.0% ENDTIDAL HALOTHANE) OBSERVATIONS IN ANAESTHETISED JOEYS. EACH DATA POINT REPRESENTS ONE ANIMAL. ...............................................69 FIGURE 2.3: MEANS OF LOG-TRANSFORMED POWER IN FREQUENCIES (1-30HZ) OF THE EEG POWER SPECTRUM AT BASELINE (1.0% ENDTIDAL HALOTHANE CONCENTRATIONS) FOR THE TWO AGE GROUPS OF ANAESTHETISED JOEYS. STANDARD ERRORS OF THE MEANS ARE SHOWN AS VERTICAL BARS. ........................................................................................................................................................72 FIGURE 2.4: MEANS OF LOG-TRANSFORMED POWER IN FREQUENCIES (1-30HZ) OF THE EEG POWER SPECTRUM OF ANAESTHETISED JOEYS AGED 140-181 DAYS BEFORE, DURING AND AFTER CLAMPING. STANDARD ERRORS OF THE MEANS ARE SHOWN AS VERTICAL BARS. ...................................................75 FIGURE 2.5: MEANS OF LOG-TRANSFORMED POWER IN FREQUENCIES (1-30HZ) OF THE EEG POWER SPECTRUM OF ANAESTHETISED JOEYS AGED 187-260 DAYS BEFORE, DURING AND AFTER CLAMPING. STANDARD ERRORS OF THE MEANS ARE SHOWN AS VERTICAL BARS. ...................................................76 FIGURE 2.6: FACTOR SCORES CALCULATED FOR FREQUENCIES (1-30HZ) BY PRINCIPAL COMPONENT ANALYSIS PLOTTED AGAINST THE 1ST AND 2ND PRINCIPAL COMPONENT AXES, SHOWING SEPARATION ACCORDING TO AGE ALONG THE FIRST AXIS (1ST PRINCIPAL COMPONENT) AND SEPARATION ACCORDING TO FREQUENCIES OF 1-12HZ ALONG THE SECOND AXIS (2ND PRINCIPAL COMPONENT). FACTOR SCORES FOR BOTH AGE GROUPS AND TREATMENTS (BEFORE, DURING AND AFTER CLAMPING) ARE SHOWN AND INCLUDE 20 DATA POINTS PER JOEY FOR EACH TREATMENT.....................................79 FIGURE 2 7: MEANS OF LOG-TRANSFORMED POWER IN FREQUENCIES (1-30HZ) OF THE EEG POWER SPECTRUM OF JOEYS AGED 140-181 DAYS FOR 1.0%, 1.2% AND 1.4% ENDTIDAL HALOTHANE EEGS. STANDARD ERRORS OF THE MEANS ARE SHOWN AS VERTICAL BARS. ...................................................83 FIGURE 2.8: MEANS OF LOG-TRANSFORMED POWER IN FREQUENCIES (1-30HZ) OF THE EEG POWER SPECTRUM OF JOEYS AGED 187-260 DAYS FOR 1.0%, 1.2% AND 1.4% ENDTIDAL HALOTHANE EEGS. STANDARD ERRORS OF THE MEANS ARE SHOWN AS VERTICAL BARS. ...................................................84 FIGURE 2.9: FACTOR SCORES CALCULATED FOR FREQUENCIES (1-30HZ) BY PRINCIPAL COMPONENT ANALYSIS PLOTTED AGAINST THE 1ST AND 2ND PRINCIPAL COMPONENT AXES, SHOWING SEPARATION ACCORDING TO AGE ALONG PC1 AND SEPARATION ACCORDING TO FREQUENCIES OF 1-11HZ ALONG PC2. FACTOR SCORES FOR BOTH AGE GROUP AND TREATMENTS (1.0%, 1.2% AND 1.4% ENDTIDAL HALOTHANE) ARE SHOWN AND INCLUDE 20 DATA POINTS PER JOEY PER EACH TREATMENT. .............87 FIGURE 2.10: MEANS OF LOG-TRANSFORMED POWER IN FREQUENCIES (1-30HZ) OF THE EEG POWER SPECTRUM FOR THE THREE NON-ANAESTHETISED JOEYS. STANDARD ERRORS OF THE MEANS ARE SHOWN AS VERTICAL BARS......................................................................................................................90 FIGURE 2.11: FACTOR SCORES CALCULATED FOR FREQUENCIES (1-30HZ) BY PRINCIPAL COMPONENT 
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ANALYSIS PLOTTED AGAINST THE 1ST AND 2ND PRINCIPAL COMPONENT AXES FOR BOTH AGE GROUP (137-145 DAYS = YOUNG; 189-196 = OLD) AND TREATMENTS (ANAESTHESIA OR NO ANAESTHESIA) ARE SHOWN AND INCLUDE 20 DATA POINTS PER JOEY PER TREATMENT. ............................................. 94 FIGURE 3.1: EEG TRACES OF 5-7DAY-OLD RAT PUPS (A, B AND C; TRACE C SHOWING ECG ARTEFACT), OF 12-14 DAY RATS (D AND E) AND OF 21-22DAY-OLD RATS (G AND H). TRACE F SHOWS THE EEG (BACKGROUND ELECTRICAL NOISE) AFTER INJECTION OF PENTABARBITONE. NOTE THE DIFFERENCES IN SCALE FOR TRACES A-F (50MV) AND G-H (100MV). .................................................................... 132 FIGURE 3.2: MEAN POWER (LOG) IN FREQUENCIES (INCLUDING STANDARD ERROR OF THE MEAN BARS) OF THE EEG SPECTRUM OF 12-14 DAY PUPS BEFORE AND AFTER CLAMPING FOR ALL EPOCHS OF EEG ACTIVITY................................................................................................................................................ 141 FIGURE 3.3: MEAN POWER (LOG) IN FREQUENCIES (INCLUDING STANDARD ERROR OF THE MEAN BARS) OF THE EEG SPECTRUM OF 21-22 DAY PUPS BEFORE AND AFTER CLAMPING FOR ALL EPOCHS OF EEG ACTIVITY................................................................................................................................................ 142 FIGURE 3.4: MEAN POWER (LOG) OF THE EEG SPECTRUM FREQUENCIES (INCLUDING STANDARD ERROR OF THE MEAN BARS) FOR ALL EPOCHS OF EEG ACTIVITY DURING BASELINE OBSERVATIONS FOR 12-14 AND 21-22 DAY PUPS. ........................................................................................................................... 143 FIGURE 3.5: FACTOR SCORES PLOTTED AGAINST THE 1ST AND 4TH PRINCIPAL COMPONENT AXES SHOWING SEPARATION ACCORDING TO AGE (12-14 DAY PUPS VERSUS 21-22 DAY PUPS) AND TAIL CLAMPING (12-14 AND 21-22 DAY PUPS BEFORE VERSUS AFTER CLAMPING). ..................................................... 148 FIGURE 4.1: EEG TRACES OF NEWBORN (3 TO 30 MINUTES) AND YOUNG (1 TO 4 HOURS) LAMBS SHOWING THE DIFFERENCES BETWEEN LVHF, HVLF AND INT EEGS.............................................................. 190 FIGURE 4.2: PRINCIPAL COMPONENT FACTOR SCORES PLOTTED AGAINST THE 1ST AND 2ND PRINCIPAL COMPONENT AXES ACCORDING TO AGE GROUP FOR INT EEGS OF LAMBS OVER THE FIRST 30 MINUTES AFTER BIRTH. ......................................................................................................................... 209 FIGURE 4.3: TOP TRACE: ISOELECTRIC EEG TRACE OF A NON-BREATHING LAMB 3 MINUTES AFTER COMMENCING EEG RECORDINGS (I.E. ~ 5:00 MIN AFTER BIRTH). MIDDLE TRACE: LVHF EEG OF LAMB BREATHING SUCCESSFULLY DURING THE SAME PERIOD. BOTTOM TRACE: INT EEG OF THE SAME SUCCESSFULLY BREATHING LAMB. ............................................................................................ 221 FIGURE 4.4: MEANS AND SEMS FOR OCCURRENCE OF BEHAVIOURAL STATES (EYES OPEN OR EYES CLOSED AND HEAD UP OR HEAD RESTING) PER MINUTE OF BEHAVIOURAL OBSERVATIONS IN LAMBS AT 3 TO 15 MINUTES, 15 TO 30 MINUTES AND 1 TO 4 HOURS. THERE WERE NO SIGNIFICANT CHANGES WITH TIME................................................................................................................................................................ 225 FIGURE 4.5: MEANS AND SEMS FOR BEHAVIOURS PER MINUTE OF BEHAVIOURAL OBSERVATIONS IN LAMBS AGED 3 TO 15 MINUTES, 15 TO 30 MINUTES AND 1 TO 4 HOURS. DIFFERENT LETTERS INDICATE SIGNIFICANT DIFFERENCES P<0.05. ...................................................................................................... 226 FIGURE 5.1: NEUROSTEROID BIOSYNTHESIS AND METABOLISM SHOWING THE PRECURSORS FOR ALLOPREGNANOLONE AND PREGNANOLONE AND THE ASSOCIATED ENZYMES (ADAPTED FROM BIRZNIECE ET AL. (2006))..................................................................................................................... 263 FIGURE 5.2: MEAN AND SEM OF PLASMA ALLOPREGNANOLONE CONCENTRATIONS OF FETAL LAMBS ~130 DAYS GESTATIONAL AGE (YAWNO ET AL., 2007) AND THE PRESENT NEWBORN LAMBS AGED 
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BETWEEN 12HRS OR LESS AND 9 DAYS AFTER BIRTH (N=5 PER AGE GROUP). THE FETAL DATA HAVE BEEN INCLUDED WITH PERMISSION BY TAMARA YAWNO. VALUES THAT DO NOT SHARE THE SAME LETTER ARE SIGNIFICANTLY DIFFERENT FROM EACH OTHER (P<0.05)................................................282 FIGURE 5.3:  MEAN AND SEM OF ALLOPREGNANOLONE CONCENTRATIONS (PMOL/G WET WEIGHT) OF THE SPINAL CORD IN LAMBS AGED UP TO 12HRS (N=5), 3 (N=3) AND 7 (N=3) DAYS AFTER BIRTH. NUMBER OF LAMBS WITH VALUES BELOW THE DETECTION LIMIT WERE 0, 5, 3, 2, 2 AND 4 FOR LAMBS AGED 12HRS OR LESS, 1, 1.5, 3, 7 AND 9 DAYS, RESPECTIVELY....................................................................284 FIGURE 5.4: MEAN AND SEM OF ALLOPREGNANOLONE CONCENTRATIONS (PMOL/G WET WEIGHT) IN THE MEDULLA OF LAMBS AGED BETWEEN 12HRS OR LESS AND 9 DAYS AFTER BIRTH (N=5 FOR ALL BUT 9-DAY LAMBS WHERE N=4). VALUES THAT DO SHARE THE SAME LETTER ARE SIGNIFICANTLY DIFFERENT FROM EACH OTHER, P<0.05). THERE WERE NO VALUES BELOW THE DETECTION LIMIT. .285 FIGURE 5.5: MEAN AND SEM OF ALLOPREGNANOLONE CONCENTRATIONS (PMOL/G WET WEIGHT) OF THE PONS IN LAMBS AGED UP TO 12HRS (N=5) AND 3 DAYS (N=3) AFTER BIRTH. NUMBER OF LAMBS WITH VALUES BELOW THE DETECTION LIMIT WERE 0, 5, 5, 2, 3 AND 4 FOR LAMBS AGED 12HRS OR LESS, 1, 1.5, 3, 7 AND 9 DAYS, RESPECTIVELY...................................................................................................287 FIGURE 5.6: MEAN AND SEM OF ALLOPREGNANOLONE CONCENTRATIONS (PMOL/G WET WEIGHT) OF THE CEREBELLUM OF LAMBS AGED UP TO 12HRS TO 9 DAYS AFTER BIRTH (N=5 FOR ALL BUT 9-DAY-OLD LAMBS WHERE N=4). THERE WERE NO VALUES BELOW THE DETECTION LIMIT. .................................288 FIGURE 5.7: MEAN AND SEM OF ALLOPREGNANOLONE CONCENTRATIONS (PMOL/G WET WEIGHT) OF THE BASAL GANGLIA IN LAMBS AGED UP TO 12HRS (N=5) AND 7 DAYS (N=3) AFTER BIRTH. NUMBER OF LAMBS WITH VALUES BELOW THE DETECTION LIMIT WERE 0, 3, 3, 5, 2 AND 4 FOR LAMBS AGED 12HRS OR LESS, 1, 1.5, 3, 7 AND 9 DAYS, RESPECTIVELY....................................................................289 FIGURE 5.8: MEAN AND SEM OF ALLOPREGNANOLONE CONCENTRATIONS (PMOL/G WET WEIGHT) OF THE CEREBRAL CORTEX IN LAMBS AGED UP TO 12HRS (N=4) AND 1.5 DAYS (N=3) AFTER BIRTH. NUMBER OF LAMBS WITH VALUES BELOW THE DETECTION LIMIT WERE 1, 4, 2, 3, 5 AND 4 FOR LAMBS AGED 12HRS OR LESS, 1, 1.5, 3, 7 AND 9 DAYS, RESPECTIVELY....................................................................290 FIGURE 5.9: MEAN AND SEM OF PROGESTERONE CONCENTRATIONS (PMOL/G WET WEIGHT) OF THE MEDULLA OF LAMBS AGED UP TO 12HRS TO 9 DAYS AFTER BIRTH (N=5 FOR ALL BUT LAMBS UP TO 12HRS AND 9 DAYS OF AGE WHERE N=3 AND 4, RESPECTIVELY). NUMBER OF LAMBS WITH VALUES BELOW THE DETECTION LIMIT WERE 2, 0, 0, 0, 0 AND 1 FOR LAMBS AGED 12HRS OR LESS, 1, 1.5, 3, 7 AND 9 DAYS, RESPECTIVELY. ................................................................................................................293 FIGURE 5.10: MEAN AND SEM OF PROGESTERONE CONCENTRATIONS (PMOL/G WET WEIGHT) OF THE CEREBELLUM OF LAMBS AGED UP TO 12HRS TO 9 DAYS AFTER BIRTH (N=5 FOR ALL BUT 9-DAY-OLD LAMBS WHERE N=4). THERE WERE NO VALUES BELOW THE DETECTION LIMIT. .................................294 FIGURE 5.11: MEAN AND SEM OF PROGESTERONE CONCENTRATIONS (PMOL/G WET WEIGHT) OF THE BASAL GANGLIA IN LAMBS AGED UP TO 12HRS (N=5) AND 7 DAYS (N=4) AFTER BIRTH. NUMBER OF LAMBS WITH VALUES BELOW THE DETECTION LIMIT WERE 0, 3, 5, 5, 1 AND 3 FOR LAMBS AGED 12HRS OR LESS, 1, 1.5, 3, 7 AND 9 DAYS, RESPECTIVELY.....................................................................................295 FIGURE 5.12: MEAN AND SEM OF PROGESTERONE CONCENTRATIONS (PMOL/G WET WEIGHT) OF THE HIPPOCAMPUS IN LAMBS AGED UP TO 12HRS (N=4), 1 DAY (N=4) AND 3 DAYS (N=3) AFTER BIRTH. 
 XIII 
NUMBER OF LAMBS WITH VALUES BELOW THE DETECTION LIMIT WERE 1, 1, 5, 2, 3 AND 4 FOR LAMBS AGED 12HRS OR LESS, 1, 1.5, 3, 7 AND 9 DAYS, RESPECTIVELY. ........................................................ 295 FIGURE 5.13: MEAN AND SEM OF PROGESTERONE CONCENTRATIONS (PMOL/G WET WEIGHT) OF THE CEREBRAL CORTEX IN LAMBS AGED UP TO 12HRS, 1, 1.5, 3 AND 9 DAYS AFTER BIRTH (N=5, 4, 4, 3 RESPECTIVELY). NUMBER OF LAMBS WITH VALUES BELOW THE DETECTION LIMIT WERE 0, 1, 1, 2, 3 AND 0 FOR LAMBS AGED 12HRS OR LESS, 1, 1.5, 3, 7 AND 9 DAYS, RESPECTIVELY. ......................... 296 FIGURE 5.14: MEAN AND SEM OF PLASMA PREGNENOLONE AND PREGNENOLONE SULFATE CONCENTRATIONS OF NEWBORN LAMBS AGED BETWEEN 12HRS OR LESS AND 9 DAYS AFTER BIRTH (N=5 FOR ALL AGES BUT AT 12HRS OR LESS N=4). VALUES THAT DO NOT SHARE THE SAME LETTER ARE SIGNIFICANTLY DIFFERENT FROM EACH OTHER, P<0.05)............................................................. 301 FIGURE 6.1: EXPERIMENTAL DESIGN FOR CONTROL, PREGNANOLONE AND PICROTOXIN GROUPS. ............ 346 FIGURE 6.2: MEAN AND STANDARD ERROR OF THE MEAN (SEM) OF F50 FOR CONTROL, PREGNANOLONE AND PICROTOXIN LAMBS COMPARING PRE- AND POST-STIMULATION PERIODS. ................................ 355 FIGURE 6.3: MEAN AND STANDARD ERROR OF THE MEAN (SEM) OF F95 FOR CONTROL, PREGNANOLONE AND PICROTOXIN LAMBS COMPARING PRE- AND POST-STIMULATION PERIODS. ................................ 356 FIGURE 6.4: MEAN AND STANDARD ERROR OF THE MEAN (SEM) OF PTOT FOR CONTROL, PREGNANOLONE AND PICROTOXIN LAMBS COMPARING PRE- AND POST-STIMULATION PERIODS.TABLE 6.5: SUMMARY OF THE RESULTS OF THE PAIRED-SAMPLE WILCOXON SIGNED RANK TESTS SHOWING SIGNIFICANT CHANGES IN EEG PARAMETERS FOR THE DIFFERENT GROUPS IN RESPONSE TO THE THREE STIMULI  (+ DENOTES AN INCREASE IN A PARAMETER, WHILE  - DENOTES A DECREASE IN A PARAMETER). ........ 357 FIGURE 6.5: MEANS AND STANDARD ERROR OF THE MEAN (SEM) OF HEART RATE FOR PRE- AND POST-STIMULUS ECGS FOR THE THREE ELECTRICAL STIMULI FOR CONTROL, PREGNANOLONE AND PICROTOXIN LAMBS 4-24 HOURS AFTER BIRTH. THE MEAN PERCENTAGE CHANGE IN HEART RATE BETWEEN PRE- AND POST STIMULUS IS ALSO PRESENTED. .................................................................. 362 FIGURE 6.6: MEANS AND SEM FOR HEART RATE FROM PRE- AND POST-STIMULUS ECGS FOR THE THREE ELECTRICAL STIMULI FOR CONTROL, PREGNANOLONE AND PICROTOXIN LAMBS 7-11 DAYS AFTER BIRTH.  THE MEAN PERCENTAGE CHANGE IN HEART RATE BETWEEN PRE- AND POST STIMULUS IS ALSO PRESENTED. ........................................................................................................................................... 363 FIGURE 6.7: RECTAL TEMPERATURE (°C) OF INDIVIDUAL 4-24HR (TOP) AND 7-11DAY (BOTTOM) LAMBS AT THE FIVE SAMPLING PERIODS (1 = START OF ANAESTHESIA; 2 = AT INITIAL BASELINE, 3 = PRE STIMULUS 1, 4 = PRE STIMULUS 2; 5  = PRE STIMULUS 3). .................................................................. 365 FIGURE 6.8: FACTOR SCORES PLOTTED AGAINST THE 1ST AND 2ND PRINCIPAL COMPONENT AXES SHOWING SEPARATION ACCORDING TO AGE (4-24HR LAMBS VERSUS 7-11DAY LAMBS..................................... 371  
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Table of Tables 
TABLE 1.1: BRAIN REGIONS, PARTICULAR NUCLEI AND ASSOCIATED TRANSMITTERS INVOLVED IN THE SLEEP-WAKE CYCLE, WHICH ALL DIRECTLY OR INDIRECTLY PROJECT TO VARIOUS AREAS OF THE CEREBRAL CORTEX. .................................................................................................................................10 TABLE 2.1: GENERAL INFORMATION ON THE ANAESTHETISED WALLABY JOEYS USED IN THE PRESENT STUDY.......................................................................................................................................................57 TABLE 2.2: PERCENTAGE OF TIME OCCUPIED BY ISOELECTRIC EEG PERIODS (%), AVERAGE DURATION OF ISOELECTRIC PERIODS (MSEC) AND NUMBER OF ISOELECTRIC PERIODS PRESENT (N) DURING 3 MINUTES OF EEG RECORDINGS DURING BASELINE, POST CLAMPING AND AT ENDTIDAL HALOTHANE CONCENTRATIONS OF 1.2% AND 1.4%. JOEYS THAT COULD NOT BE INTUBATED (N=3) AND HENCE ONLY HAD A 5-MINUTE EEG RECORD TAKEN ARE NOT INCLUDED IN THIS TABLE, BUT HAD AN ISOELECTRIC EEG THROUGHOUT THE 5 MINUTES (100%). ...................................................................63 TABLE 2.3: RESULTS OF THE NON-PARAMETRIC FRIEDMAN TEST, INCLUDING P-VALUES, CHI-SQUARE STATISTIC, DEGREES OF FREEDOM (DF), MEANS AND STANDARD ERROR OF THE MEAN (SEM) FOR EEG PARAMETERS FROM ANAESTHETISED JOEYS BEFORE, DURING AND AFTER CLAMPING. MEANS DENOTED WITH THE DIFFERENT LETTERS INDICATE SIGNIFICANT DIFFERENCES BETWEEN MEANS WITHIN AN AGE GROUP WITH SIGNIFICANT LEVELS AS FOLLOWS: * P<0.001; + P=0.001-0.01 AND ‘ P=0.01-0.05). ...........................................................................................................................................71 TABLE 2.4: RESULTS (SIGNIFICANT P-VALUES) OF THE TWO-TAILED KOLMOGOROV-SMIRNOFF TEST COMPARING FREQUENCY SPECTRA BETWEEN TREATMENTS (BEFORE, DURING AND AFTER CLAMPING) IN THE TWO AGE GROUPS OF ANAESTHETISED JOEYS. ............................................................................74 TABLE 2.5: EIGENVALUES AND COMPONENT SCORES OF THE COMPONENT MATRIX OF ALL FREQUENCIES FOR PRINCIPAL COMPONENTS (PCS) 1 TO 3 CALCULATED BY PRINCIPAL COMPONENT ANALYSIS FOR EEG TRACES BEFORE, DURING AND AFTER CLAMPING..........................................................................78 TABLE 2.6: RESULTS OF THE NON-PARAMETRIC FRIEDMAN TEST, INCLUDING P-VALUES, CHI-SQUARE STATISTIC, DEGREES OF FREEDOM (DF), MEANS (M) AND STANDARD ERROR OF THE MEAN (SEM) FOR EEG TRACES AT 1.0%, 1.2% AND 1.4% ENDTIDAL HALOTHANE CONCENTRATIONS. MEANS DENOTED WITH DIFFERENT LETTERS INDICATE SIGNIFICANT DIFFERENCES BETWEEN MEANS WITHIN AN AGE GROUP WITH SIGNIFICANT LEVELS AS FOLLOWS: * P<0.001; + P=0.001-0.01 AND ‘ P=0.01-0.05).....81 TABLE 2.7: RESULTS (P-VALUES) OF THE TWO-TAILED KOLMOGOROV-SMIRNOFF TEST COMPARING FREQUENCY SPECTRA BETWEEN TREATMENTS (1.0%, 1.2% AND 1.4% ENDTIDAL HALOTHANE) IN THE TWO AGE GROUPS OF ANAESTHETISED JOEYS..................................................................................82 TABLE 2.8 EIGENVALUES AND COMPONENT SCORES OF THE COMPONENT MATRIX OF ALL FREQUENCIES FOR PRINCIPAL COMPONENTS (PCS) 1 TO 4 CALCULATED BY PRINCIPAL COMPONENT ANALYSIS FOR EEG TRACES AT 1.0%, 1.2% AND 1.4% ENDTIDAL HALOTHANE CONCENTRATION. ...........................86 TABLE 2.9: MEAN AND STANDARD ERROR OF THE MEAN (SEM) FOR F50, F95 AND PTOT (FIVE EEG PERIODS EACH OF 30 SEC DURATION) FOR NON-ANAESTHETISED JOEYS (N = 1 FOR EACH AGE). ........88 TABLE 2.10: MEAN, STANDARD ERROR OF THE MEAN (SEM) AND NUMBER OF DATA POINTS USED (N) FOR F50, F95 AND PTOT CALCULATIONS FOR YOUNGER (137-145 DAYS) AND OLDER (189-196 DAYS) JOEYS THAT WERE ANAESTHETISED (BASELINE – 1.0% ENDTIDAL HALOTHANE) OR NOT 
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ANAESTHETISED. ..................................................................................................................................... 91 TABLE 2.11: EIGENVALUES AND COMPONENT SCORES OF THE COMPONENT MATRIX OF ALL FREQUENCIES FOR PRINCIPAL COMPONENTS (PCS) 1 TO 5 CALCULATED BY PRINCIPAL COMPONENT ANALYSIS FOR COMPARISON OF ANAESTHETISED AND NON-ANAESTHETISED JOEYS. .................................................. 92 TABLE 3.1: DETAILS OF WEIGHT AND RECTAL/SKIN TEMPERATURE OF RAT PUPS OF THE THREE AGES...... 135 TABLE 3.2: HEART RATE DATA (BPM) OF RAT PUPS OF THE THREE AGES DURING THE PRE-BASELINE, BASELINE AND POST-CLAMP PHASES OF OBSERVATION. DATA INCLUDE DETAILS FOR THE ENTIRE 5-MINUTE PRE-BASELINE, 5-MINUTE BASELINE AND 5-MINUTE POST-CLAMP PERIODS, AS WELL AS DATA FOR THE PERIOD IMMEDIATELY PRIOR TO (BASELINE) AND IMMEDIATELY AFTER (POST CLAMP) THE CLAMPING, WHICH WERE USED TO DETERMINE THE IMMEDIATE SHORT-TERM EFFECT OF CLAMPING ON HEART RATE. .................................................................................................................................... 137 TABLE 3.3: P-VALUES AND KOLMOGOROV-SMIRNOFF Z-STATISTICS (K-STATISTIC) CALCULATED BY THE TWO-TAILED KOLMOGOROV-SMIRNOFF TEST COMPARING POWER OF FREQUENCY SPECTRA BETWEEN 12-14 DAY PUPS AND 21-22 DAY PUPS (BASELINE OBSERVATIONS) BEFORE AND AFTER CLAMPING................................................................................................................................................................ 146 TABLE 3.4: EIGENVALUES AND COMPONENT SCORES OF ALL FREQUENCIES FOR PRINCIPAL COMPONENTS (PCS) 1 TO 5 CALCULATED BY PRINCIPAL COMPONENT ANALYSIS................................................... 147 TABLE 4.1: GENERAL INFORMATION FOR LAMBS USED IN DATA ANALYSIS. PCV=PACKED CELL VOLUME; B=BEHAVIOUR; 1ST= EEG 3 TO 30MIN AFTER BIRTH, 2ND=EEG 1 TO 4HRS AFTER BIRTH. ............... 195 TABLE 4.2: MEANS AND SEMS OF LAMB BIRTH PARAMETERS COMPARING LAMBS THAT WERE ASSISTED WITH THOSE THAT WERE NOT, FEMALE WITH MALE LAMBS, AND SINGLE LAMBS WITH MULTIPLES. PCV = PACKED CELL VOLUME ............................................................................................................. 197 TABLE 4.3: PERCENTAGE CONTRIBUTION OF THE THREE EEG STATES (LVHF = LOW VOLTAGE HIGH FREQUENCY, INT = INTERMEDIATE AND HVLF = HIGH VOLTAGE LOW FREQUENCY) TO THE TOTAL NUMBER OF SECONDS AVAILABLE FOR EEG ANALYSIS FROM 16 LAMBS FOR EACH TIME PERIOD INVESTIGATED OVER THE FIRST 30 MINUTES AFTER BIRTH AND PERCENTAGE CONTRIBUTION OF THE THREE EEG STATES AS WELL AS ARTEFACT TO THE TOTAL NUMBER OF SECONDS RECORDED FOR THE 16 LAMBS............................................................................................................................................... 199 TABLE 4.4: NUMBER OF LAMBS WHOSE EEGS SHOWED THE CHARACTERISTICS OF LVHF, INT AND HVLF STATES OR WHOSE EEG COULD NOT BE USED AT THE SELECTED TIME POINTS AFTER BIRTH DUE TO MOVEMENT ARTEFACT, ASSESSED BY SCAN SAMPLING AT THE STIPULATED TIME. ........................... 200 TABLE 4.5: MEANS AND SEMS FOR F50, F95 AND PTOT AND THE TEN FREQUENCY BANDS FOR THE LVHF AND INT EEGS OF THE FIRST 30 MINUTES AFTER BIRTH. N=NUMBER OF ANIMALS ......................... 201 TABLE 4.6: RESULTS OF THE INDEPENDENT SAMPLE T-TESTS AND MANN-WHITNEY TESTS COMPARING SPECTRAL PARAMETERS BETWEEN LVHF AND INT EEGS FOR LAMBS 3 TO 30 MINUTES AFTER BIRTH. * Z-STATISTIC............................................................................................................................ 202 TABLE 4.7: RESULTS OF THE INDEPENDENT SAMPLE T-TESTS, ONE-WAY ANOVAS, MANN-WHITNEY TESTS AND RELATED SAMPLES WILCOXON SIGNED RANK TESTS COMPARING SPECTRAL PARAMETERS IN INT EEGS BETWEEN THE DIFFERENT AGE GROUPS BETWEEN 3 AND 30 MINUTES AFTER BIRTH.............. 203 TABLE 4.8: MEAN AND STANDARD ERROR OF THE MEAN (SEM) FOR F50, F95 AND PTOT OF EEGS 
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RECORDED IN 16 LAMBS AGED UP TO 30 MINUTES AFTER BIRTH USING MEAN DATA FOR INDIVIDUAL LAMBS. VALUES FOR THE INT EEG STATE ARE PRESENTED. N=NUMBER OF ANIMALS....................204 TABLE 4.9: RESULTS OF THE INDEPENDENT SAMPLE T-TESTS, ONE-WAY ANOVAS, MANN-WHITNEY TESTS AND RELATED SAMPLES WILCOXON SIGNED RANK TESTS COMPARING RELATIVE POWER IN THE TEN FREQUENCY RANGES IN INT EEGS BETWEEN THE DIFFERENT AGE GROUPS BETWEEN 3 AND 30 MINUTES AFTER BIRTH. *NON-PARAMETRIC TEST STATISTIC ..............................................................205 TABLE 4.10: MEANS AND SEMS OF RELATIVE EEG POWER (%) OF THE TEN FREQUENCY BANDS FOR INT EEGS FOR LAMBS 3 TO 30 MINUTES AFTER BIRTH. N=NUMBER OF ANIMALS ....................................206 TABLE 4.11: EIGENVALUES AND PRINCIPAL COMPONENT SCORE CALCULATED BY PCA FOR INT EEGS OF THE FIRST 30 MINUTES AFTER BIRTH. ...................................................................................................208 TABLE 4.12: MEANS AND SEMS FOR LVHF, INT AND HVLF FOR LAMBS 1 TO 4 HOURS AFTER BIRTH. N=NUMBER OF ANIMALS .......................................................................................................................211 TABLE 4.13: RESULTS OF THE ONE-WAY ANOVA (F STATISTIC) AND NON-PARAMETRIC KRUSKAL WALLIS TEST (Z-STATISTIC) COMPARING LVHF, INT AND HVLF EEGS IN LAMBS 1 TO 4 HOURS AFTER BIRTH. *Z-STATISTIC .............................................................................................................................212 TABLE 4.14: MEANS AND SEMS FOR F50, F95 AND PTOT AND THE TEN FREQUENCY BANDS FOR THE INT AND HVLF. N=NUMBER OF ANIMALS ..................................................................................................214 TABLE 4.15: RESULTS OF THE INDEPENDENT SAMPLE T-TEST (T-STATISTIC) AND NON-PARAMETRIC MANN-WHITNEY TEST (Z-STATISTIC) COMPARING INT AND HVLF EEGS IN LAMBS 1 TO 2 DAYS AFTER BIRTH. *Z-STATISTIC .............................................................................................................................215 TABLE 4.16: PERCENTAGE CONTRIBUTION OF THE THREE EEG STATES TO EEG DATA USED FOR STATISTICAL ANALYSIS COMPARING THE THREE AGE GROUPS AND TO OVERALL EEG DATA AVAILABLE (NOTE THAT HVLF EEG WAS NOT USED FOR STATISTICAL ANALYSIS IN LAMBS 3 TO 30 MINUTES AFTER BIRTH AND NO STATISTICAL ANALYSIS WAS UNDERTAKEN TO DETERMINE THE CHANGES IN EEG MOVEMENT ARTEFACT WITH AGE). .........................................................................216 TABLE 4.17: RESULTS OF THE ONE-WAY ANOVAS AND INDEPENDENT T-TESTS AND THE NON-PARAMETRIC MANN-WHITNEY AND KRUSKAL-WALLIS TESTS COMPARING F50, F95 AND PTOT BETWEEN EEGS RECORDED FROM NEWBORN LAMBS UP TO 30 MINUTES, 1 TO 4 HOURS AND 1 TO 2 DAYS AFTER BIRTH. *Z-STATISTIC FOR INDIVIDUAL MEANS, ^CHI-SQUARE STATISTIC FOR INDIVIDUAL MEANS, DF DEGREES OF FREEDOM ...........................................................................................................................217 TABLE 4.18: MEANS AND SEMS FOR F50, F95 AND PTOT OF EEGS RECORDED IN LAMBS UP TO 30 MINUTES AFTER BIRTH, 1 TO 4 HOURS AFTER BIRTH AND 1 TO 2 DAYS AFTER BIRTH FOR INT AND HVLF EEGS. N=NUMBER OF ANIMALS ...........................................................................................................218 TABLE 4.19: RESULTS OF THE ONE-WAY ANOVAS AND INDEPENDENT T-TESTS AND THE NON-PARAMETRIC MANN-WHITNEY AND KRUSKAL-WALLIS TESTS COMPARING THE RELATIVE POWER OF THE TEN FREQUENCY BANDS BETWEEN EEGS RECORDED FROM NEWBORN LAMBS UP TO 30 MINUTES, 1 TO 4 HOURS AND 1 TO 2 DAYS AFTER BIRTH. *Z-STATISTIC FOR INDIVIDUAL MEANS, ^CHI-SQUARE STATISTIC FOR INDIVIDUAL MEANS, DF DEGREES OF FREEDOM...........................................................219 TABLE 4.20: MEANS AND SEMS FOR THE TEN EEG FREQUENCY BANDS RECORDED IN LAMBS UP TO 30 MINUTES AFTER BIRTH, 1 TO 4 HOURS AFTER BIRTH AND 1 TO 2 DAYS AFTER BIRTH FOR THE INT AND 
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HVLF EEGS. N=NUMBER OF ANIMALS............................................................................................... 220 TABLE 4.21: RESULTS OF THE SPEARMAN RANK CORRELATION TEST FOR ASSESSING INTRA-OBSERVER RELIABILITY........................................................................................................................................... 222 TABLE 4.22: SPEARMAN RANK CORRELATIONS (R) AND P-VALUES OF BEHAVIOURS AND STATES LINKED TO AROUSAL AND NUMBER OF DATA POINTS (N). ONLY THOSE CORRELATIONS WITH COEFFICIENTS >0.500 ARE PRESENTED (MODERATE AND STRONG CORRELATIONS).................................................. 223 TABLE 4.23: RESULTS OF THE NON-PARAMETRIC FRIEDMAN TEST SHOWING P-VALUES, CHI-SQUARE STATISTIC AND DEGREES OF FREEDOM (NUMBER OF GROUPS AND LAMBS PER GROUP) FOR BEHAVIOURS AND STATES COMPARING LAMBS 3 TO 15 MINUTES, 15 TO 30 MINUTES AND 1 TO 4 HOURS AFTER BIRTH.............................................................................................................................. 224 TABLE 4.24: PERCENTAGE CONTRIBUTION OF THE THREE EEG STATES TO EEG DATA IN THE THREE AGE GROUPS (WITH ^ AND WITHOUT + CONSIDERATION OF MOVEMENT ARTEFACT). THE DATA PRESENTED HERE ARE IDENTICAL TO THOSE IN TABLE 4.16 IN THE RESULTS SECTIONS OF THIS CHAPTER. THE DATA HAVE BEEN RELOCATED HERE FOR CONVENIENCE. ................................................................... 232 TABLE 5.1: ALLOPREGNANOLONE ANTISERUM CROSS REACTIVITY WITH CLOSELY RELATED STEROIDS AS CHARACTERISED BY BERNARDI ET AL. (1998). ................................................................................... 268 TABLE 5.2: PROGESTERONE ANTISERUM CROSS REACTIVITY WITH CLOSELY RELATED STEROIDS (RICE ET AL., 1986; BILLIARDS, 2003). .............................................................................................................. 270 TABLE 5.3: PREGNENOLONE ANTISERUM CROSS-REACTIVITY WITH CLOSELY RELATED STEROIDS (BILLIARDS, 2003). ............................................................................................................................... 274 TABLE 5.4: GENERAL INFORMATION FOR LAMBS AGED BETWEEN 12HRS OR LESS AND 9 DAYS AFTER BIRTH (N = 5 PER GROUP)................................................................................................................................. 280 TABLE 5.5: NUMBER OF LAMBS WITH ALLOPREGNANOLONE CONCENTRATIONS BELOW THE DETECTION LIMIT FOR EACH AGE GROUP AND EACH BRAIN REGION. ..................................................................... 283 TABLE 5.6: MEANS AND SEM ALLOPREGNANOLONE CONCENTRATIONS (PMOL/G WET WEIGHT) AND NUMBER OF LAMBS PER AGE GROUP IN THE BRAIN REGIONS INVESTIGATED (N=5 FOR EACH AGE GROUP BUT 9-DAY-OLD LAMBS WHERE N=4). ONLY VALUES OF LAMBS WITH ALLOPREGNANOLONE CONCENTRATIONS ABOVE THE DETECTION LIMIT ARE PRESENTED..................................................... 286 TABLE 5.7: NUMBER OF LAMBS WITH PROGESTERONE CONCENTRATIONS BELOW THE DETECTION LIMIT FOR EACH AGE GROUP AND EACH BRAIN REGION........................................................................................ 291 TABLE 5.8: MEANS AND SEM PROGESTERONE CONCENTRATIONS (PMOL/G WET WEIGHT) AND NUMBER OF LAMBS PER AGE GROUP IN THE BRAIN REGIONS INVESTIGATED (N=5 FOR EACH AGE GROUP APART FROM 9-DAY-OLD LAMBS WHERE N=4). ONLY VALUES OF LAMBS WITH PROGESTERONE CONCENTRATIONS ABOVE THE DETECTION LIMIT ARE PRESENTED..................................................... 292 TABLE 5.9: GENERAL INFORMATION FOR PROTEIN CONCENTRATIONS (MG/G WET WEIGHT) PER AGE GROUP. N= NUMBER OF TISSUE SAMPLES PER AGE ........................................................................................... 297 TABLE 5.10: MEANS AND SEMS FOR PROTEIN CONCENTRATIONS (G WET WEIGHT) AND ALLOPREGNANOLONE (AP) CONCENTRATIONS (PMOL) PER MG OF PROTEIN FOR EACH BRAIN REGION IN EACH AGE GROUP FOR THOSE SAMPLES WHERE THE AP CONCENTRATIONS WERE ABOVE THE DETECTION LIMIT. N=NUMBER OF ANIMALS........................................................................................ 298 
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TABLE 5.11: MEANS AND SEMS OF PROTEIN CONCENTRATIONS (G WET WEIGHT) AND PROGESTERONE (PROG) CONCENTRATIONS (PMOL) PER MG OF PROTEIN FOR EACH BRAIN REGION IN EACH AGE GROUP FOR THOSE SAMPLES WHERE THE PROG CONCENTRATIONS WERE ABOVE THE DETECTION LIMIT. N=NUMBER OF LAMBS................................................................................................................299 TABLE 6.1:GENDER DISTRIBUTION ACCORDING TO AGE AND TREATMENT GROUP. N=NUMBER OF ANIMALS................................................................................................................................................................350 TABLE 6.2: RESULTS OF THE NON-PARAMETRIC TWO-RELATED SAMPLES WILCOXON TESTS AND PAIRED-SAMPLE T-TESTS FOR ALL TREATMENT GROUPS COMPARING PRE-INFUSION AND INFUSION (LAST 30 SECONDS) EEGS FOR 4-24HR LAMBS AND 7-11DAY LAMBS................................................................351 TABLE 6.3: RESULTS OF THE REPEATED MEASURES ANOVAS FOR ALL TREATMENT GROUPS COMPARING EEG PARAMETERS OF THE THREE BASELINE PERIODS (DF = 2 FOR EACH COMPARISON). * INDICATES CHI-SQUARE STATISTIC CALCULATED BY NON-PARAMETRIC FRIEDMAN TEST ..................................352 TABLE 6.4: RESULTS OF THE NON-PARAMETRIC TWO-RELATED SAMPLES WILCOXON TEST FOR F50, F95 AND PTOT FOR ALL TREATMENT GROUPS COMPARING PRE-STIMULUS AND POST-STIMULUS EEGS FOR ALL STIMULI. ..........................................................................................................................................354 TABLE 6.6: RESULTS OF THE TWO-TAILED KOLMOGOROV-SMIRNOFF TEST COMPARING FREQUENCY SPECTRA (1-30HZ) PRE- AND POST-STIMULATION FOR THE THREE STIMULI FOR CONTROL, PREGNANOLONE AND PICROTOXIN LAMBS OF BOTH AGES. ..................................................................359 TABLE 6.7: RESULTS OF THE PAIRED SAMPLE T-TEST FOR HEART RATE FOR 4-24HR AND 7-11DAY LAMBS OF ALL TREATMENT GROUPS [CONTROL, PREGNANOLONE (PREGNAN) AND PICROTOXIN (PICROTOX)], COMPARING PRE-STIMULUS AND POST-STIMULUS ECGS FOR THE THREE STIMULI. ...........................361 TABLE 6.8: MEAN, SEM AND NUMBER OF ANIMALS (N) FOR ENDTIDAL CO2 PARTIAL PRESSURE, RECTAL TEMPERATURE AND BLOOD GLUCOSE CONCENTRATIONS FOR 4-24HR LAMBS AND 7-11DAY LAMBS DURING THE COURSE OF THE STUDY. APPROXIMATE TIMING OF MEASUREMENTS IS GIVEN IN BRACKETS IN MINUTES FROM TIME 0 (START OF STABILISATION PERIOD, SEE FIGURE 6.1)...............366 TABLE 6.9: P-VALUES AND KOLMOGOROV-SMIRNOFF TEST STATISTIC (K-STATISTIC) CALCULATED BY TWO-TAILED KOLMOGOROV-SMIRNOFF TEST COMPARING FREQUENCY SPECTRA BETWEEN 4-24HR AND 7-11DAY LAMBS AT THE INITIAL BASELINE. .................................................................................369 TABLE 6.10: EIGENVALUES AND COMPONENT SCORES FOR ALL FREQUENCIES FOR PRINCIPAL COMPONENTS (PCS) 1 TO 4 CALCULATED BY PRINCIPAL COMPONENT ANALYSIS FOR AGE COMPARISON OF BASELINE EEG SPECTRA. ......................................................................................................................370