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Neural control of the cardiac response of the Pekin duck (Anas platyrhynchos) to forced submersion Gabbott, Geoffrey Roy Julian 1985

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s  NEURAL CONTROL OF THE CARDIAC RESPONSE OF THE PEKIN DUCK (ANAS PLATYRHYNCHOS) TO FORCED SUBMERSION by GEOFFREY ROY JULIAN GABBOTT B.Sc.(Hons) U n i v e r s i t y of East A n g l i a , Norwich, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA Zoology A p r i l 1985 $  ©  G e o f f r e y Roy J u l i a n Gabbott, 1985  _  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may department o r by h i s o r her  be granted by the head o f representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  Department of  "Z.OC>LOQ^f  The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main M a l l Vancouver, Canada V6T 1Y3  written  i i  ABSTRACT C a r d i o v a s c u l a r responses enable  evoked  during  forced  submersion  the Pekin duck (Anas platyrhynchos) to s u r v i v e p r o t r a c t e d  periods  of  asphyxia.  b r a d y c a r d i a and result  that  The  intense  blood  responses  peripheral  flow  is  via  the  caudal  v a s o c o n s t r i c t i o n with  favoured  s u s c e p t i b l e to lack of oxygen. mediated  include an e x t r a o r d i n a r y  to  those  organs  These adjustments  brainstem  p e r i p h e r a l and c e n t r a l a r t e r i a l  following  appear to  the most be  s t i m u l a t i o n of  chemoreceptors.  The minor r o l e that b a r o r e c e p t o r s play in the generation of these responses  was  cardiovascular baroreceptor circulation experiments  changes  from  the  located  A  appeared  in  of  carotid  arterial  especially  of  the  arterial  the  cephalic  bodies, within  P0  potent  the  in  2  and  the  in evoking 2  Similar  changes  in  the  c e p h a l i c c i r c u l a t i o n d i d not e l i c i t However,  both  receptor  groups  systemic bradycardia  blood  gas  significant to  by a c t i v a t i n g s u b s t a n t i a l r e d u c t i o n  vascular resistance.  Although  mitigate  arterial  in  resulted  responded  p e r i p h e r a l blood flow, as r e f l e c t e d by the r i s e  changes  from cranial  Increased a r t e r i a l PC0 , l i k e w i s e ,  a r t e r i a l hypoxic hypercapnia in  the  chemoreceptors  heart r a t e . the  bradycardia.  peripheral  Isolation  declining  during submersion.  of  persistance  systemic c i r c u l a t i o n enabled a s e r i e s of  central  circulation.  levels  the  to assess the r e l a t i v e c o n t r i b u t i o n s from p e r i p h e r a l  unidentified  in a reduced  by  following  denervation.  chemoreceptors,  circulation  demonstrated  baroreceptors blood  may  pressure  i n hind limb continue  to  and cause some  iii  change in heart rate appear  to  be  and  vascular  predominantly  resistance,  chemoreceptors  r e s p o n s i b l e for the changes during  submersion. The  c a r d i a c response to  submersion diving.  was  chemoreceptor  discovered  H a b i t u a t i o n was  several  training  forced  dives  to  the  following repetitive  bradycardia  abolished.  during  Habituation  response appeared dependent on the stimulation.  of  produced by  oxygen content,  or no c a r d i a c h a b i t u a t i o n was  conducted  to  chemoreceptors was breathing  tests  suggested before  observations i s w i t h i n the The  l e d to the suggestion  was  observed. of  the  sensitivity  change  in  oxygen  Furthermore,  established  of  and  the  these  that the l o c u s of h a b i t u a t i o n  CNS.  dependent on a r t e r i a l P0  that 2  Further  the demonstration  the  level  in both naive and  argues a g a i n s t the c o n t e n t i o n  intact  of  either  inspired  efficacy  after training.  intensity  demonstration  response.  pre-dive  Maintained  by the lack  and  p e r s i s t e n c e of stimulus  the  demonstrate  c a r d i o i n h i b i t o r y efferent discharge.  cardiac  chemoreceptor  or  were  reducing  40-second  the  p r o l o n g i n g d i v e times  Tests  by  of  intensity  With severe a r t e r i a l hypoxia,  little  during  so pronounced in some ducks that a f t e r  sessions  was  habituate  stimulation  evidence  of  bradycardia  habituated  was  animals  that the d i v i n g response i s a fear a g a i s t t h i s view was  that the d i v i n g response  remains  provided  by  essentially  f o l l o w i n g t r a n s e c t i o n in the r o s t r a l mesencephalon below  the l e v e l of the hypothalamus. It  i s concluded  that  chemoreceptor-driven  cardiovascular  iv  changes regions of  evoked  as  part of the d i v i n g response are mediated by  of the CNS below the r o s t r a l  these  responses  can  simple forms of l e a r n i n g . what l e v e l  be  brainstem.  produced  However,  t h i s influence a r i s e s .  it  Modification  i n the i n t a c t animal by remains  uncertain  at  V  TABLE OF CONTENTS  ABSTRACT  i i  LIST OF TABLES  x  LIST OF FIGURES  xi  ACKNOWLEDGEMENTS  xiii  GENERAL INTRODUCTION  1  The Responses  2  Response I n i t i a t i o n  7  Higher Centre Influence  11  Neural Substrate  14"  SECTION  1  20  The Role of C e n t r a l and P e r i p h e r a l Chemoreceptors i n the D i v i n g Response  20  Introduction  20  METHODS Effect  ' of U n i l a t e r a l Vagotomy  Surgical preparations Effect  23  of  Artificial  on D i v i n g Bradycardia  and experimental Perfusion  of  protocol  Carotid  . 24 .. 25  Body  Chemoreceptors  26  Surgical preparations  26  Measurements  • • • 30  Experimental P r o t o c o l Cross-Perfusion  Between Ducks  32 33  Surgical Preparations  34  Measurements  35  vi  Experimental p r o t o c o l  41  S t a t i s t i c s and  42  the A n a l y s i s of Data  RESULTS  44  Effect  of U n i l a t e r a l  Effect  of  Vagotomy on D i v i n g Bradycardia  Perfusion  of  the  Carotid  . 44  Body  Chemoreceptors Effect  44  of Changing Blood Gas  Tension  in the C e r e b r a l  Circulation  51  DISCUSSION  59  SECTION 2 The  66  Effect  of a Simple Form of Learning  on  the  Diving  Response  66  Introduction  66  METHODS  69  General P r o t o c o l  69  A) H a b i t u a t i o n of the Cardiac Response to D i v i n g B) E f f e c t C)  ....  of H a b i t u a t i o n on Blood V a r i a b l e s  i . Effect  of  Breathing  Pure  75  Oxygen on D i v i n g  Bradycardia ii.  Effect  D) E f f e c t  70  76  of Hypoxia on the Habituated  Response ..  of H a b i t u a t i o n on the Oxygen Breathing  79  Test  79 E) H a b i t u a t i o n of  the  Cardiac  Response  in  "True"  D i v i n g Ducks  82  RESULTS  84  A) H a b i t u a t i o n of the Cardiac Response to D i v i n g B) E f f e c t  ....  84  of H a b i t u a t i o n on some Blood V a r i a b l e s ....  91  vii  C)  i.) Effect  of  Breathing  Pure Oxygen on D i v i n g  Bradycardia i i . ) Effect D) E f f e c t  of  101 of Hypoxia on the Habituated Habituation  on  the Oxygen  Response. 101 Breathing  Test E)  1 08  Habituation  of  the Cardiac  Response i n D i v i n g  Ducks  1 08  DISCUSSION  114  SECTION 3  121  Demarcation  of  the Neural  Substrate  of  the D i v i n g  Response  121  Introduction  121  METHODS  125  P r e l i m i n a r y Surgery  125  Decerebration  126  R e v e r s i b l e S e c t i o n Of The Brainstem Mechanical  Brainstem  Experimental  Using Xylocaine .127  Transection  Protocol  .....129 129  RESULTS  131  X y l o c a i n e blockade  131  Plane  131  of t r a n s e c t i o n  D i v i n g performance Effect  136  of H a b i t u a t i o n T r a i n i n g of Decerebrate  METHODS  ,  Ducks ....146 146  RESULTS  147  DISCUSSION  150  GENERAL DISCUSSION  156  viii  LITERATURE APPENDIX  I  APPENDIX  II  CITED  161 184 .  ..187  ix  LIST OF TABLES  E f f e c t of C a r o t i d Body S t i m u l a t i o n  48  E f f e c t of P e r i p h e r a l and C e n t r a l Chemoreceptor S t i m u l a t i o n  53  E f f e c t of H a b i t u a t i o n on the C a r d i o v a s c u l a r System  99  Effect  of H a b i t u a t i o n on Breathing T e s t s  109  X  LIST OF FIGURES  C a r d i o v a s c u l a r responses to f o r c e d submergence  4  Diagram of arrangement f o r c r o s s - p e r f u s i o n  27  Diagram of the r e c i p i e n t  36  duck  Diagram of c r o s s - p e r f u s i o n  38  Effect  45  Effects  of u n i l a t e r a l vagotomy on heart rate of PaC0  2  at c e n t r a l  chemoreceptors  56  R e l a t i v e c o n t r i b u t i o n s of chemoreceptors  61  Diagram of duck p o s i t i o n  during t r a i n i n g  71  Diagram of remote d i v i n g  system  73  Diagram of a i r flow system in t r a i n i n g  77  Diagram of body plethysmograph  80  Effect  of t r a i n i n g  on d i v i n g  85  Effect  of t r a i n i n g  (composite f o r 13 ducks)  Effect  of i n t e r u p t i o n  Effect  of prolonged dive time  92  Effect  of shortened dive time  94  Effect  of h a b i t u a t i o n on blood pressure  97  Effect  of h a b i t u a t i o n on blood gas l e v e l s  102  Effect  of oxygen on d i v i n g  104  Effect  of oxygen on c a r d i a c h a b i t u a t i o n  106  Effect  of t r a i n i n g  in diving  112  Effect  of t r a i n i n g  on v a s c u l a r r e s i s t a n c e  116  Effect  of t r a n s e c t i o n on body temperature  132  bradycardia  of t r a i n i n g  bradycardia  ducks  S a g i t t a l view of t r a n s e c t i o n l e v e l s  87 89  134  xi  E f f e c t of b r a i n t r a n s e c t i o n on d i v i n g bradycardia  137  E f f e c t of b r a i n t r a n s e c t i o n on blood pressure  139  Effect  141  of b r a i n t r a n s e c t i o n on blood v a r i a b l e s  E f f e c t of b r a i n t r a n s e c t i o n on b r e a t h i n g  143  Effect  148  of t r a i n i n g on d e c e r e b r a t e ducks  Diagram of Xylocaine  i n j e c t i o n apparatus  185  ACKNOWLEDGEMENTS I  thank  Dr.  David R.  Jones  guidance throughout t h i s study. Milsom  I a l s o thank  f o r h i s h e l p with the f i r s t  h i s continued support l a t e r on. Bob  f o r h i s i n v a l u a b l e h e l p and  Furilla  Dr.  William  K.  part of t h i s study and f o r  I am g r a t e f u l to  Frank  Smith,  and the members of my research committee f o r t h e i r  help i n p r e p a r i n g and reviewing t h i s manuscript. I am g r a t e f u l B r i t i s h Columbia by  grants  to Dr. The  f o r the s c h o l a r s h i p s I have r e c e i v e d from the  Heart Foundation.  The research  was  supported  from the NSERC of Canada and the BC Heart Foundation  D.R.Jones. experiments  collaboration  with  in  D.R.Jones  n e c e s s a r i l y agrees with my these experiments.  Section and  1. were  performed  in  W.K.Milsom although n e i t h e r  interpretation  of  the  results  of  1  GENERAL INTRODUCTION It has long been  known  that  diving  animals  submerged f o r an i n o r d i n a t e p e r i o d of time. research  in t h i s  of e q u i v a l e n t as  field  water.  A few years  than the chicken  succumb  against  occlusion  than  by  triggering  asphyxia.  f a r sooner  pioneered  i f they forced  He  s u r v i v e as  much  (1894) e s t a b l i s h e d that extraordinary  discovered  were  head  will  when both are h e l d under  l a t e r Charles Richet  water played a key r o l e i n mechanisms  Paul Bert  remain  i n 1870 when he demonstrated that a duck  s i z e and weight to a chicken  seven times longer  can  that ducks would  asphyxiated  immersion.  defensive  by  tracheal  However, Laurence  Irving,  i n c o l l a b o r a t i o n with Per Scholander, was the  report  s t u d i e s that began to unravel  t h i s problem.  first  to  Scholander,  who was concerned with s p e c i a l p r o p e r t i e s i n the blood of d i v i n g seals, discovered  that l a c t a t e c o n c e n t r a t i o n  a remarkable i n c r e a s e f o l l o w i n g a d i v e increase  was  particularly  lactate concentration hardly other  hand,  was  are  that most  postulated  (Scholander  i n c r e a s e d at a l l .  of  concerned  with  the  imminent  on the  strategy  of the  asphyxia.  It  was  the c e n t r a l nervous system (CNS) and the myocardium susceptible a  to  differential  oxygen  deprivation;  distribution  blood.  of  of  thus  blood  Irving  flow  with a  during steady  Other t i s s u e s r e c e i v e d e i t h e r a c o n s i d e r a b l y  reduced flow or none at a l l ( I r v i n g appearance  This  Irving,  hypoxia so that the b r a i n and heart were favoured ration  1940).  unusual because throughout the dive  c a r d i o v a s c u l a r system t o cope with known  i n the blood showed  1937,1938).  The  post-dive  high l a c t a t e c o u l d thus be e x p l a i n e d by the f a c t  2  that d u r i n g a d i v e , l a c t a t e produced sequestered  as blood flow was  by  the  appear  ( I r v i n g e_t a_l.  The comprehensive and Scholander  remain  1942;  the  the  muscles  did  Scholander et a l .  ground-breaking  as  remained  stagnant, and only upon s u r f a c i n g  when normal blood flow was a b l e to f l u s h lactate  muscles  studies  greatest  of  the  1942).  Irving  contributions  to  and  diving  p h y s i o l o g y and subsequent work has continued to c h a r a c t e r i s e the d e t a i l s of the immersion  cardiovascular  addition  the  to  rate  apnoea,  falls  submergence l e v e l s . cardiac  output  Eisner  Holeton increase beds  elicited  the most e a s i l y observed feature  as  This f a l l  little  reflects  ( F r a n k l i n e_t a l .  et  1972). in  to  i n heart as a  rate.  In  most  10 to 20% of presimilar  change  a_l. In  the  1964;  1979;  Zapol et a l .  t h i s reduction function 1966).  in  Johansen  and  Aakhus  Shelton and Jones 1965; Jones and  conjunction resistance  1962;  with  the  bradycardia  is  1979; McKean 1982).  i n flow i s manifest i n the  during forced submersion Cerebral  (Kerem and E i s n e r  blood 1973;  flow,  an  to flow in many systemic v a s c u l a r  (Andersen 1959; B u t l e r and Jones 1971; Daly 1972; Jones  al.  by  because, i n most animals s t u d i e d , stroke volume  remains unchanged 1963;  are  Responses  during submergence i s the marked f a l l species  that  i n water.  The In  changes  by  i n t e n s i t y of  cessation  (Murdaugh contrast,  Zapol et a l .  The  et_ a l .  of  kidney  1961;  remains  1979) or may  et  even  Sykes  constant increase  3  during  the d i v e  1979).  Some u n c e r t a i n t y surrounds data of  brain  because  consequently on  (Irving  1938;  Dormer et a l .  blood  1978;  to  increase  circulation  Jones  though  it  (Jones and  1981).  appears  c a r d i a c output  Holeton  Coronary  ( B l i x et. a l .  and  flow  1976;  muscle  is  consequently  oxygen a v a i l a b i l i t y  beds  Jones et a l . predominant restriction  1979)  (Olsson  remaining 1979;  Zapol  result  of  unable  1981). may  patent et the  to  sustain  the  As  itself  In a d d i t i o n to the b r a i n be  the  i n a dive  al.  only  by to and  regions  with  ( E i s n e r e_t a_l.  1978;  1979;. McKean  vascular  with  remains subject  changes  is  1982).  The  a selective  in t i s s u e p e r f u s i o n which a c t s to conserve a v a i l a b l e  output  the r i s e  the h e a r t .  in s u f f i c i e n t harmony that mean a r t e r i a l blood unaltered  and  and  are  (Irving  Johansen 1972; comparison  to  i n systemic  et  Johansen and Aakhus 1963;  In  in  1979).  cardiac  Jones and  et' a l .  but  in  1963;  the  f o r c e d submersion  Zapol et a_l.  oxygen almost e x c l u s i v e l y for the CNS  usually  but  than  Sinnet  during  and  sparse,  less  1972;  cardiac effort  the heart, the adrenal glands vascular  the  to decrease in p r o p o r t i o n to the r e d u c t i o n in  tissue, cardiac  anaerobiosis  to  Information  is  considerably  remains unchanged in the duck (Jones e_t a_l. seal  flow  are not amenable to r i g o r o u s a n a l y s i s .  appears  systemic  Jones et. a l .  most measurements have been taken i n d i r e c t l y  flow r e s i s t a n c e in the pulmonary c i r c u l a t i o n  it  CNS  1977;  a_l.  1942;  Butler  more  fall  vascular resistance pressure  Hollenberg  and  Jones  Angell-James et a l . their  The  1978).  strictly  is  and Uvnas  1968,  1971;  (FIGURE 1) terrestrial  homologues, d i v i n g animals possess enhanced oxygen s t o r e s in the  gure 1. C a r d i o v a s c u l a r responses to f o r c e d submersion of Pekin duck (Anas p l a t y r h y n c h o s ) . 'Peripheral r e s i s t a n c e ' r e f e r s to the r e s i s t a n c e to blood flow i n one l e g . Blood oxygen tension was measured in the brachiocephalic artery. (from B u t l e r and Jones 1982)  5  air flow ( L/min )  +3 3  if  400r heart rate (beats/min)  200  0 20 peripheral resistance (pru)  0 a r t e r i a l blood pressure (mm Hg)  230T  •Iff  0 blood o x y g e n tension (mm Hg)  150 75  •  dive  surface  30 sec  6  form of large blood volumes and high haemoglobin (Irving also  1939;  M i l s o m et a l .  possess  large  Although,  for  important  source  stores  diving of  uncertain.  Gas  membranes;  therefore,  the  of  Lenfant et a l . oxygen  in  the  their  air  a  its  usefulness during diving i s  not  occur  across  potentially  the  air-sac  to be a v a i l a b l e to the animal, oxygen in  system  must  be  passed  through  the  lungs.  of gases between a i r sacs  a c t i v e l y swimming b i r d .  d i v i n g mammals, d i v i n g b i r d s may  Furthermore,  l i k e many  e x p i r e before submergence.  buoyancy i s a primary concern, many d i v i n g b i r d s possess air  sac  volumes  and F u r i l l a : important the  in preparation).  But by  circulation.  This  is  far  and  best demonstrated  for  8  and  after  will  and  well  alert,  submersion.  Indeed  would f i n a l l y if  the  responsive as Richet  moiety  duck  will  on  sufficient prepared average  of the d i v i n g response  i n h i b i t i o n of the sympathetic  1969;  noted,  by work i n which  easily  system  with  f o r another his  animals  Andersen  and  Blix  1974).  i s prevented  a-receptor  b a r e l y s u r v i v e 1 minute under water  any  recovery  succumb a f t e r p r o t r a c t e d d i v e s of 23 minutes.  vascular  ducks w i l l  most  to 10 minutes without  i n d i c a t i o n of damage to i t s CNS seem  the  substances.  Under normal circumstances, the domestic submersion  (Jones  i s the change that occurs in  t h i s change i s blocked by pharmacological  forced  away  As  reduced  compared to t h e i r non-diving r e l a t i v e s  s t r a t e g y a g a i n s t asphyxia  withstand  sacs.  oxygen, does  represents  Birds  this  However, i t i s not known i f " s t i r r i n g " occurs  in  1969).  birds,  exchange  respiratory  1973;  concentrations  by  blockade,  (Kobinger and  Such severe e f f e c t  Yet  on  Oda  diving  7  performance a l s o occurs i f the v a g a l l y mediated  bradycardia  abolished  and Jones 1968,  1971). in  by  atropine  (Richet  1894;  Butler  Bryan and Jones (1980) demonstrated a  surface  significant  oxygen  tension  of  greater  decrease  the c e r e b r a l hemispheres and a  increase i n c e r e b r a l NADH during submersion i n ducks  t r e a t e d with a t r o p i n e compared  Response  to c o n t r o l s .  Initiation  C o n s i d e r a b l e d i f f i c u l t y has been encountered i n e f f o r t s define  is  to  the p r e c i s e mechanisms whereby submersion i s able to s e t  in t r a i n  these c a r d i o v a s c u l a r defences a g a i n s t  asphyxia.  This  i s l a r g e l y because at the moment of submersion there are so many diverse  sensory  inputs impinging upon the animal, a l l of which  may p l a y a s i g n i f i c a n t The  effect  of  r o l e i n the i n i t i a t i o n  water  itself  r e c e p t o r s has been s t r o n g l y Andersen 1974;  Blix  et. a_l.  suspected (Koppanyi and Dooley 1928;  to  1975)  physical  regions  (Lombroso  and M c R i t c h i e 1973). voluntarily  development investigated  of  particularly  because  of  their  c o n t a c t and because of the apnoea and  c a r d i o v a s c u l a r r e f l e x e s so  by  as a stimulus t o f a c i a l or n a r i a l  I963a,b,c; H a r r i s o n and Kooyman 1968; Bamford and Jones  sensitivity  these  of the responses.  often  provoked  by  stimulation  1913; Tchobroutsky et. a l .  P o s t u r a l changes, such as  diving  animals,  diving  responses  may and  also this  1969; White  those  executed  contribute has  of  long  i n the l a b o r a t o r y by mimicking the changes  to the been i n head  and body p o s i t i o n that occur with submersion (Huxley 1913; Paton  8  1913;  Koppanyi and Kleitman  1927).  The  cessation  of  central  r e s p i r a t o r y a c t i v i t y and pulmonary feedback, both of which are a consequence  of  the apnoea, may serve  activity  ( I r v i n g et a l .  Taylor  1973;  importance of  1941; L i n et_ a l .  Bamford any  one  and of  (Butler  considerable  and  Jones  Jones  these  confounded by the p o s s i b i l i t y them  to depress  that  1982).  1972;  1976). sensory  cardiovascular Butler  Analysis inputs  and  of the  is  further  i n t e r a c t i o n may occur between It  i s also  apparent  i n t e r s p e c i e s v a r i a t i o n may e x i s t i n  the  that  preferred  a f f e r e n t pathway, and t h i s i s best demonstrated by the v a r i a t i o n in  response  patterns  elicited  by  submersion  of d i f f e r e n t  animals. Some of the e a r l i e s t responded  to  face  studies  showed  that  marine  immersion with almost immediate  mammals  bradycardia  and  t h i s s t r o n g l y suggested r e f l e x a c t i v i t y v i a p o s s i b l e  or  narial  1971;  receptors  (Angell-James and Daly  Dykes 1974; Drummond and  Jones  facial  l969;Folkow e_t a l .  1979).  Similar  studies  have shown that c e r t a i n d i v i n g b i r d s a l s o e x h i b i t r a p i d onset of bradycardia  i n d i c a t i v e of the r e f l e x a c t i o n of immersion per se  on the heart  r a t e response  and  1978;  Woakes  diving bradycardia  ( C a t l e t t and  Mangalam  and  i n the M a l l a r d  gradually  f a l l i n g heart  Jones or  shows  a  neural  l i n k between the water stimulus  Johnston 1984).  domestic  to  progressive  consequence of the  Butler  The p r o f i l e of duck,  however,  rate and t h i s argues against a and the c a r d i a c  An a l t e r n a t i v e h y p o t h e s i s was put forward that due  1974;  response.  bradycardia  was  hypoxia and hypercapnia that develops as a apnoea  which  is elicited  by  submergence  9  (Feigl  and  Jones and carotid  Folkow 1963;  Andersen  Purves (1970) who body  discovered  recently  nature  dives It was  duck. with  perpetuation  of  carotid  Woakes  body  response in the  attenuated  response was showed  immediate the  lowered  stimulation  that  have  that  though  bradycardia heart  (Huang  stages body  chemoreceptive  by no means e x c l u s i v e to  rate and  a c t i v a t i o n of v a s c u l a r  marine upon  was  Peng  the  mammals  immersion,  dependent 1976;  responses during  co-workers  (1968)  conducted  upon  Daly e_t a l .  bradycardia.  In ducks the  also implicated  dogs  that  of  independence of v a s o c o n s t r i c t i o n  was  Jones 1971).  chemoreceptor  hypercapnic blood  w i l l cause an  in blood  stimulation increase  in  r e s i s t a n c e when pulmonary v e n t i l a t i o n  (Daly and  Scott  On  Daly and  pressure any  by total  during  change  Moreover, i t has  vascular  1963;  indicated absence  by the s u b s t a n t i a l r i s e  ( B u t l e r and  been  Murdaugh  that  occur in the  f o r c e d submersion, in s p i t e of the a b o l i t i o n of rate  d i v i n g has  change.  experiments  systemic v a s o c o n s t r i c t i o n i n s e a l s can  in  (1982)  carotid  the  l e s s well s t u d i e d than a c t i v a t i o n of c a r d i a c  heart  heart  1979). The  and  the  the  later  following  also established  Studies  responded  1977,  is  of the c a r d i a c  domestic  of  by  shown that even with free-swimming d i v i n g ducks,  voluntary  denervation.  supported  that denervation  B u t l e r and  such as t u f t e d ducks, the c a r d i a c of  T h i s was  chemoreceptors almost t o t a l l y d i s r u p t e d  r a t e response to forced d i v i n g . more  1963).  in  been shown  hypoxic  and  peripheral  i s held  constant  Ungar 1966).  the s t r e n g t h of the observations  that  vasoconstriction  10  might be the d i r e c t consequence of chemoreceptor a c t i v a t i o n , causal  relation  chronotropic and  Blix  so  that  and  also c a l l e d 1974,  bradycardia  is  r e f l e x which responds caused  demonstrated  by  a  the  These  into  It was  the  of  prevented results, that  contention  are  surgical  denervation  ducks (Jones  not the cardiomotor  are e l i c i t e d  blood  vasoconstriction.  however,  at  b a r o r e c e p t o r s leaves the b r a d y c a r d i a  response  in  by a-adrenergic blockade  arterial  or  rise  of a  the bradycardiac response  demonstration  Whether  that  activated  this  earlier  in c h r o n i c a l l y denervated  proposed  expression  incipient  supporting  abolition  cardiac (Andersen  sequentially  merely to an  the  question  1975). are  and  chemoreceptor-driven  experiments  v a s o c o n s t r i c t i o n was 1974).  receptors  vasomotor responses  Pharmacological  al.  was  these  B l i x et a l .  diving  barostatic pressure  response  1974;  cardiomotor  between  independently  the  odds  when  ( B l i x et_ with  of  the  systemic  virtually  intact  1973). response  and  the vasomotor  or whether there i s a c a u s a l  r e l a t i o n between them i s f u r t h e r confounded by the  contribution  of  evidence  two  other  f a c t o r s to v a s o c o n s t r i c t i o n .  demonstrated the p o s s i b i l i t y chemoreceptor 1961;  activation  1982).  In  the  of vasomotor a c t i v i t y d e r i v e d  of the defence  H i l t o n and J o e l s 1964; decerebrate  First,  reaction  M a r s h a l l 1981; cat  chemoreceptor s t i m u l a t i o n w i l l evoke c h o l i n e r g i c in  the  hind  and  kidney.  submersion,  from  ( B i z z i e_t a l .  H i l t o n and  with an i n t a c t  has  Marshall  hypothalamus, vasodilatation  limb along with v a s o c o n s t r i c t i o n i n the mesentery It  could,  therefore,  chemoreceptor-  driven  be  reasoned  that  during  v a s o c o n s t r i c t i o n may  be  11  a t t r i b u t e d to 1981) .  activation  Second,  unidentified upon  certain  If  baroreceptor groups rise  the  which both  are  defense  reaction  (Marshall  evidence suggests the e x i s t e n c e of an  r e c e p t o r group  activation.  significant  of  stimulates peripheral  acutely  vasoconstriction  chemoreceptor  denervated  i n mean a r t e r i a l  in  the  and  duck,  a  blood p r e s s u r e p e r s i s t s when  the animal i s submerged (Jones and West 1978;  Lillo  and  Jones  1982) .  Higher Centre I n f l u e n c e R e f l e x o g e n i c e f f e c t s notwithstanding, i t i s w e l l documented how  pervasive  i s the extent of "higher CNS"  in any experiment  conducted  receptor  toward  groups  to  the  discern  control.  the  In  fact,  contributions  d i v i n g b r a d y c a r d i a , due  should be p a i d to the " p s y c h o l o g i c a l c o n d i t i o n "  of  of  attention  the  animal  both before and during d i v i n g . Once  again,  I r v i n g and Scholander were f i r s t  the d i s p o s i t i o n and nervous for  full  factors have  development  1941).  or  activity,  most, i f not immersion Steptoe  more  Folkow  and  that  important physical  co-workers  (1967)  concluded that "calm" ducks show more intense d i v i n g  responses than "alarmed" stress  was  of f o r c e d d i v i n g responses than  ( I r v i n g et a_l.  also  s t a t e of s e a l s  to note  a l l , of  (Paulev 1980).  animals.  Moreover,  in  man,  i n the form of mental a r i t h m e t i c , the  1969;  cardioinhibitory  Wolf et a l .  1975;  effects Wolf  1978;  mental prevents of  face  Ross and  12  In t h i s regard, have  had  the advances in r a d i o t e l e m e t r i c  considerable  physiological "naturally"  data  impact.  from  during  It  i s now  unrestrained  voluntary  t u f t e d ducks, the s t r i k i n g o b s e r v a t i o n was in  behaving  rate so c h a r a c t e r i s t i c  then,  the  heart  of  rate  in the d i v e , f e l l  the  during v o l u n t a r y  forcibly  rose  briefly  Butler  1980).  intense bradycardia Canada  geese  exhibited  recordings  forcibly  e_t  a_l.  more  rapid  however, in  the  voluntary  ( M i l l a r d et al.  the  ( B u t l e r and Woakes showing  a  lack of  1981 ).  The  fall  these  bradycardia  animals  in mean femoral  dives  compared  and  reported were  blood  with  to  diving flow  forced  was  dives  1973) .  Telemetrically  recorded  data  proven to be e q u a l l y as c o n f l i c t i n g Reports  to that when  submerged Papua penguins was  s l i g h t l y more intense than when  voluntarily;  animals.  were taken from f r e e l y d i v i n g cormorants  (Kanwisher  by  reductions  j u s t before d i v i n g and  back to l e v e l s s i m i l a r  Similar  from  diving,  submerged  animals were swimming a c t i v e l y on the surface 1979;  more  When recording  that the heart never d i s p l a y e d the e x t r a o r d i n a r y  Instead,  be  p o s s i b l e to o b t a i n  animals  submergence.  technology  indicate  that  degree of bradycardia  may  in  the  from f r e e - d i v i n g mammals has as  case  that  from  the  birds.  of the Weddell s e a l ,  the  be c o r r e l a t e d with the d u r a t i o n of  the  d i v e : i n short d i v e s the heart  rate may  fall  to  recorded  during short r e s p i r a t o r y  levels  similar  to  that  s l i g h t l y , but  pauses when breathing at the s u r f a c e ; in long d i v e s the f a l l heart dives  rate  is  more dramatic  (Kooyman and Campbell  and may  1972).  In  only  in  match that seen in f o r c e d unrestrained  muskrats,  13  large  and  very  rapid  falls  i n heart rate have been  recorded  which may exceed that evoked by f o r c e d dives  (Drummond and Jones  1979).  unrestrained,  However,  muskrats  were  in  this  actively  study,  provoked  other hand appear to develop trained  dives  than  though  when  to  more  dive.  intense  spontaneously  Porpoises,  the on the  bradycardia  during  d i v i n g (Eisner et_ a l .  1966). The  implications  characteristics higher  these  .discrepancies  in  the  neural c e n t r e s might exert responses,  significant  control  over  whether i t be to attenuate  from u n r e s t r a i n e d s e a l s which show decreases i n heart  the  or enhance  T h i s i s f u r t h e r e x e m p l i f i e d by some of the data  before  dive  between f r e e l y and forced d i v e d animals i s that  cardiovascular them.  of  collected rate  even  submergence and i n c r e a s e s before emergence (Jones e_t a l .  1973;  Casson and Ronald  heart  rate has a l s o been claimed  ducks, 1979). learning  such  as  These may  pochards  data be  1975).  This  and  suggest  involved  anticipatory  t o occur  change  in  in unrestrained d i v i n g  t u f t e d ducks ( B u t l e r and Woakes  that  some  form  of  associative  ( B u t l e r and Jones 1982), and by the  same token, the f a i l u r e of some animals to  display  bradycardia  during v o l u n t a r y d i v e s c o u l d mean that p e r i p h e r a l sensory  inputs  are "ignored" e i t h e r by c o n d i t i o n i n g or by h a b i t u a t i o n . Experiments  have  been  reported  i n which s e a l s have been  c o n d i t i o n e d to develop extreme b r a d y c a r d i a acoustic  command  signal  (Ridgway  development and even the degree of  et a_l.  in  response  1975).  bradycardia  h o l d i n g was enhanced by c o n d i t i o n i n g experiments.  to  an  The r a t e of  during  breath-  These r e s u l t s  14  suggest that the s e a l s may heart  rates.  However,  be capable  of some c o n t r o l over  r e s u l t s from t h i s kind of c o n d i t i o n i n g  experiment are e q u i v o c a l because animals may  perform an  in response to c o n d i t i o n i n g s t i m u l i which only the  desired  effect  a c t i v i t y which has  on heart  early  modelled  on  "depressor"  of  feedback  lowering  blood  on the heart, and  pressure, an  in such a system was  The  neural  confined  within  activity  (myelencephalon) vasomotor c e n t r e . century  Thus the  "pressor" and  delimited  the was  to  effects  The  reverse  C o n t r o l of in  fine the  Wang 1964).  the  envisaged  medulla  a p p r o p r i a t e l y designated  medulla  to  mediated  variations  Experiments in the l a t t e r p a r t the  pressure  believed  rose.  activation.  (Uvnas 1960;  of  the  r e f l e x e s , for example  consequently  restricted  domain  on  antagonism of the autonomic  s u b s t r a t e f o r such c o n t r o l was  and  operated  i n h i b i t i o n of vagal d e c e l e r a t o r y  blood pressure  were  that  caused a s y m p a t h e t i c a l l y  c o n d i t i o n occurred during depressor  magnitude of CNS  control  systems  A c t i v a t i o n of the pressor  v a s o c o n s t r i c t i o n and  tuning  not c a r d i a c c o n t r o l .  Furthermore, t h i s was  echo the sympathetic/parasympathetic  by  has  In other words, i t i s t h i s  seemed adequate to c o n t r o l blood  by t h e i r mutual antagonism.  nervous system.  secondarily  cardiovascular  e f f e c t o r processes.  reflexes  activity  Substrate  assumptions negative  e s s e n t i a l l y two  rate.  been c o n d i t i o n e d , and  Neural The  their  the of  to be  oblongata medullary the  last  as the region most c r u c i a l for  15  c o n t r o l of normal  blood pressure (Dittmar 1870; Owsjannikow 1871  : see review by Korner progressively  more  blood pressure f e l l  1979).  By s e c t i o n i n g  the  maintains  drastically.  Thus t h i s l e v e l ,  vasomotor tone.  nerve  arteries  stimulation  and  (Uvnas 1960; Wang  Research  vascular  to  of the major  1964). focussed on e l e c t r i c a l  centres  and  reports  responsible for  r a i s i n g blood pressure (Ranson and B i l l i n g s l e y  1916; Chen et a l .  The depressor c e n t r e  general  was  that  centre were  1936).  discrete  reflexes  occlusion  f o l l o w i n g these experiments  suggested  rostral  of a "centre"  vascular  s t i m u l a t i o n w i t h i n the medullary vasomotor soon  limit  just  Further s e c t i o n of the medulla  caused extreme hypotension and a b o l i s h e d both  at  caudal planes, a l e v e l was reached at which  to the obex i n the medulla, marked the upper that  brainstem  then  characterised  and the  d i s t r i b u t i o n of these areas was confirmed and mapped by  Alexander  (1946).  Progress c o n t i n u e d vascular  responses  and  to  characterise  i t became  the  apparent  details  that an animal i s  capable of much more r e f i n e d and s e l e c t i v e c o n t r o l of vascular  response  beds.  different,  f o r i n s t a n c e , i n e x e r c i s e when compared with that  Hilton  1965;  Moreover, when complexity  was  quite in  hypoxia, or i n the defence r e a c t i o n when compared with  v a s c u l a r changes i n response to heat 1960;  pattern  different  vascular  arterial  The  of the  Korner  examining  should  be  1971;  these  (Abrahams  Heistad  and  responses,  considered:  i n t e g r a t i o n of v a r i o u s somatomotor  stress  this  Abboud  another is  responses  e_t a l . 1980).  order  of  the c o o r d i n a t e d which  are  often  16  inextricably  l i n k e d to the autonomic responses (Hess 1957).  The  concept, h e l d for so long, of a d i a r c h i c a l c o n t r o l system seemed inadequate to account f o r both the complexity intact  systems.  and  flexibility  That the e a r l i e r model s u f f i c e d merely  the l i m i t e d scope of the t e s t s of v a s c u l a r v i a b i l i t y P e i s s 1964;  concepts  of  the  location  c a r d i o v a s c u l a r response p a t t e r n s and,  appropriate to  accordingly,  (Peiss 1964).  incorporate  occurring way,  higher  a  The  (Wang  shifted  regions  cardiovascular  brainstem,  the  the  rostrally  the  hierarchical  of  along  system seemed more was  considered  the b r a i n with  interaction  "downward" d i r e c t i o n s .  response p a t t e r n s evoked by,  defence r e a c t i o n are the product diencephalon  system ( H i l t o n and  of  and  Zbrozyna  interactions relevant 1963;  cardiac  Mesenteric muscles  output  vasodilatate for  so  Hilton  changes  in  those  flow  (Hilton  be regarded  as  to the  is  a  the The  flow.  1975).  functional  in These unit  of the defence  1964).  response  in one  the  skeletal  diverted  1965,  the  increase  blood  patterns  demanding  d i f f e r e n t . v a s c u l a r beds (that i s , the  vasoconstriction  say,  1975).  of  f o r the f u l l e x p r e s s i o n  (Abrahams et. a l .  Typically,  activity  can  must remain i n t a c t  blood  this  regions w i t h i n  redistribution  that  muscular  regions of the neuraxis  reaction  a  v a s c u l a r beds v a s o c o n s t r i c t and  preparation  which  and  In  between  response i n c l u d e s a m o b i l i s a t i o n of venous r e s e r v e s , an in  1964;  of the c o n t r o l of  neural substrate  in both "upward" and  the  limbic  reflects  H i l t o n 1975).  Current  neuraxis  of  bed  with  vasodilatation  bidirectional simultaneous in  another)  1 7  involve other  the  integrated  than  the  medulla  h y p o t h a l a m u s has and  its  effects (Hilton the of  be  demonstrated an  anything  and  It  everything  i s because  visceral  the  functions  suggest  that  r e s p o n s e s and  This  reasoning  of  diencephalon diving  type  Rubinstein elicited those  i n the  the  were not  diving  observed,  potentiated  area  dorsal  i n the  behavioural When t h i s  pecking studies  it  was  would  electrically immediately  b o t t o m of  have  also  not  the  pool  attempted  of  , on the  area  the  to  do  1964). influences  . in  was  many  area  in  locate  for  and  which  responses  hand,  forced Another elicited  responses.  free-swimming  i t s h e a d and  i f searching  elicit  resembled  during  the  the  (Folkow  responses.  other  the  which  closely  to  diving.  located  cardio-respiratory  submerge  to  system  behavioural  this  of  "stimulation  integrate  study  which  activated  as  or  may  cardiovascular  hypothalamus  r e s p o n s e s but  area  the  the  that  stimulation  Although  stimulation  region  f o r a r e a s w h i c h would  upon e l e c t r i c a l  response.  this  changes during  A v e n t r a l hypothalamic  submersion  animal,  probed  c a r d i o - r e s p i r a t o r y responses  in  parasympathetic  (Smith  area  cardiovascular  duck was  known,  i t seems r e a s o n a b l e  l e d to a remarkable  responses  1965).  this  the  in a discussion  regulates that  brainstem That  of  Smith  its limits"  within  the  i s well  cardiovascular  behaviours  behavioural line  the  hypothalamus  regions  and  control, stated  within  and  function  stimulation  example, O.A.  force  of  1965,1977).  sympathetic  by  cardiovascular  h y p o t h a l a m u s can  levels  regulatory  widespread  As  of  (Manning  autonomic of  1975).  l o c a t i o n of the  an  control  can  participation  swim a r o u n d  food.  suprabulbar  Other regions  18 integrating apnoea  diving  have  Putkonen  been  diving  compilation separate  of  complex  action  same  evoking while  on  leaving  Folkow  be  visualised  must  1963;  eliciting  Kotilainen  and  receives  barrage  a  host  glottal  from  networks from  (Korner need  hypothalamus,  supplying  of  et  for f o r the  of  the  experiments  an  1969;  The  afferent  or  capable  central  response  out  1971)  by  of  most nervous arising  receptors,  receptors  and  might  involve  This  concept  Korner  and  demonstrating  diencephalon,  expression  beds  centres.  integration. carried  of  vascular  information  higher  while  tissues  vestibular  Korner  intact  full  few  with  neurons.  slowing  appears  the  diving  for neural  a l .  heart  systemic  a  patterns  r e c e p t o r s , pulmonary  cognitive  control  support  crucial  diverse  baroreceptors, possibly  CNS  co-workers  and  maximal  deprivation.  interpret  Accordingly, distributed  those  being  vasomotor  activation  in  as  response  and  provokes  oxygen  narial  undefined  cardiomotor  open  chemoreceptors, an  sites  and  differential  shutdown  to  facial,  and  sympathetic  complete  controller  can  activation  time  susceptible  from  mesencephalic  (Feigl  response  Parasympathetic the  found  and  1972).  The  at  responses  especially  his the the  chemoreceptor-driven  bradycardia. In  the  responses with  of  present the  particular  initiation section,  and the  Pekin  study,  the  duck  submersion  regard  maintenance  to  to of  participation  the these of  the  control  of has  the been  mechanisms responses. barostatic  cardiovascular investigated  involved In reflex  with  the toward  the first the  19  development  of  investigation  the  cardiac  response  assessed.  submersion  responses.  circulation  i s separated, an attempt  infuences  Using  of  a  may  contribute  the  investigated submersion.  i s made  to  of  simple  form  the  In of  the  the  of  this  response  influence.  second  learning  the c o n s i d e r a t i o n of chemoreceptor  re-examines  particularly  a  i n the c o n t r o l of the c a r d i a c  a n a l y s i s i s made of the locus section  evaluate the  decreased a r t e r i a l oxygen and i n c r e a s e d  influence  With  to the  technique whereby the c e n t r a l  a r t e r i a l carbon d i o x i d e on each receptor group. section,  An  i s a l s o made i n t o the p o s s i b i l i t y of the presence  of a c e n t r a l chemosensitive s i t e which  relative  is  to  is  forced  input, an The  final  r o l e of the higher brainstem c e n t r e s ,  hypothalamic  region  developed whereby r e v e r s i b l e t r a n s e c t i o n  and  a  technique  i s accomplished.  is  20  SECTION  The  1.  R o l e o f C e n t r a l and P e r i p h e r a l C h e m o r e c e p t o r s  i n the D i v i n g  Response.  Introduction The  chemoreceptive  well established Daly  e_t  Purves and  al.  nature  1977;  Huang and Peng 1976)  1982).  of  1982).  and d u c k s  1972; J o n e s and West  much  that  the responses d u r i n g  submersion  seems  each  responses.  are  strongly  (Daly et a l .  little  doubt  et a l . rate  of  these  1977;  ducks  is  p e r i p h e r a l chemoreceptors arterial  PC0  2  influenced  B u t l e r and  (Feigl  of and  pulmonary  magnitude  arterial Folkow receptors  PC0  2  of  on  the  of the diving  oxygen  (Cohn  of  heart  p r e s e n c e of the  ( J o n e s and P u r v e s 1 9 7 0 ) .  investigators  role  of  to establish.  As  have  The  altered  the  by c h a n g i n g t h e c o n t e n t o f i n s p i r e d  1963;  Andersen  1963a,b);  are  unclear  the  1984) and e x p r e s s i o n  dependent  many  by  Woakes  i t is  of a r t e r i a l  has p r o v e n t o be more d i f f i c u l t  w i t h s t u d i e s on o x y g e n , level  the  1968; Mangalam and J o n e s in  later  that stimulus modalities  i s d e t e r m i n e d by t h e l e v e l  change  the  c o n t r i b u t e t o the development  I n d u c k s and g e e s e ,  bradycardia  ( J o n e s and  differ  t h e p r o g r e s s i v e h y p o x i a and h y p e r c a p n i a ; h o w e v e r , how  1973;  A l t h o u g h t h e i n i t i a t i n g m e c h a n i s m s may  stimulation  There  Daly  Lillo  prolonged  chemoreceptor  r e s p o n s e i s now  1978;  among a n i m a l s , i t i s c l e a r stages  the d i v i n g  f o r b o t h mammals ( A n g e l l - J a m e s and  1970; Holm and S o r e n s e n  Jones  of  however,  a r e i n h i b i t e d by h i g h l e v e l s o f C 0  2  C0  2  avian i n the  21  airways  (Fedde et a l .  linkage Taylor  1974a,b).  because  of  the  between v e n t i l a t o r y a c t i v i t y and heart r a t e ( B u t l e r 1973;  Bamford and Jones 1976), i t has been  distinguish changes due surrounds  between to  precise  of  HR  due  response  blood  baroreceptors  to  to a r t e r i a l PC0  and  ventilation.  nature of how  2  Controversy  also  the autonomic response  as  part  is  investigation  was  v a s o c o n s t r i c t i o n c o u l d cause an  incipient  that  would  and B l i x  designed  submersion before and a f t e r perfusing  to a more reflex.  (Andersen  the  to  barostatic  pressure  of  1974,  To e s t a b l i s h the r o l e of the b a r o s t a t i c  while  difficult  s t i m u l a t i o n alone, or whether i t i s due  Chemoreceptor-driven in  in  and  I t i s not c e r t a i n whether the b r a d y c a r d i a i s due  chemoreceptor indirect  changes  alteration  the  generated.  rise  Therefore,  the  stimulate  arterial  B l i x et al.1974, reflex,  part  1975).  of  this  to compare the c a r d i a c response denervation  peripheral  of  the  to  baroreceptors  chemoreceptors  with  blood  c o n t a i n i n g high or low oxygen t e n s i o n . Before attempts  can  be  made  to  establish  c o n t r i b u t i o n s of hypoxia and hypercapnia the  possibility  of. involvement  the  separate  to the "diving  response,  by receptor groups other  p e r i p h e r a l chemoreceptors must be examined. both  peripheral  denervated, occurred  a  chemoreceptor  marked  when  the  increase  baroreceptor  when  groups  were  in  mean  animal  was  submerged (Jones and West  As the  heart  arterial  example,  rise  L i l l o and Jones 1982). stimulation  and  For  rate  than  blood  remained  pressure 1978;  constant,  of other r e c e p t o r groups must have given r i s e to an  i n e i t h e r stroke volume or v a s c u l a r r e s i s t a n c e .  22  Since the d i s c o v e r y of influence al.  a  chemoreceptors  and  their  in both b i r d s and mammals ( M i t c h e l l et  1963; Loeshcke et a l .  1981) may  on r e s p i r a t i o n  central  1970;  Sebert  1979;  Milsom  et  al.  growing body of evidence suggests that these r e c e p t o r s  also  contribute  to  vascular  tone.  In  dogs,  after  denervation of p e r i p h e r a l chemoreceptors, modulation of a r t e r i a l carbon  dioxide  resistance the  is reflected  (Lioy e_t a_l.  assumption  that  1978;  in concomitant changes i n v a s c u l a r Hanna et a_l.  central  1979).  Based  on  chemoreceptors are r e s p o n s i v e to  e l e v a t e d a r t e r i a l PC0 , experiments were designed to assess  the  magnitude,  may  2  result  if  from t h e i r s t i m u l a t i o n d u r i n g  In order receptor each  any, of the c a r d i o v a s c u l a r adjustments which  to  sites  receptor  altering  blood  determine  i t was site gas  circulation perfusion  with  the  contributions  from  separate  necessary to i s o l a t e the v a s c u l a r and  examine  the  subsequent  t e n s i o n s on t h e i r  It was decided that while perfused  the  submersion.  the  individual  systemic  animal's  own  to the head could be  best  effects  could  isolation  accomplished  of blood from a donor animal.  In t h i s way,  of  by  varying  the  oxygen  and  carbon  i n s p i r e d gas i n the i n d i v i d u a l animals.  dioxide  be the  cross-  the blood  gas t e n s i o n s i n each c i r c u l a t i o n c o u l d be independently by  of  circulations.  circulation  blood,  flow to  contents  altered of the  23  METHODS Experiments were done at room temperature pairs  of  White  Pekin  ducks  weight from 2.5 to 3.8 kg.  (Anas  platyrhynchos)  The ducks were  cardiovascular  anaesthesia, animals.  responses  a l l experiments  Only minor surgery  to  were  done  on  unanaesthetised  f o r implantation of cannulae,  on  the  of  experiment,  minimise s t r e s s to the animals limitation  of  experiment  day,  anaesthesia Artificial  major  or  was  of  areas of these  was done  to  handling 2  days  performed  30mg/kg  or  and  general  1g/kg  i.v.).  regions  of the  e x t e n s i v e use of heparin to prevent making i t important day  of  by  before the  under  urethan  restricted  to heal before the  under  great care was taken to  gentle  One  i n the p e r f u s i o n cannulae,  i n c i s i o n s had time The  through  surgery  cross-perfusion  requires  and  noise.  (pentobarbital  vasculature clotting  excessive  this  to d i v i n g are a b o l i s h e d by  (2% X y l o c a i n e , A s t r a Pharmaceuticals)  day  22  experiments.  l o c a l anaesthesia the  on  varying in  acclimated  temperature f o r at l e a s t one week before any Since  (20-22°C)  blood  that a l l  the  experiment.  i n c i s i o n s were a l s o p e r i o d i c a l l y  infiltrated  with a l o c a l a n a e s t h e t i c d u r i n g the course of the experiment. In a l l experiments hyperoxic  blood r e f e r s to a r t e r i a l  with p a r t i a l pressures of oxygen (Pa0 ) at l e a s t 2  normal l e v e l s ; mean values were 373 ±49 mm values  were  ±1.7 mm Hg. partial  at l e a s t  of  200 mm Hg above Hypoxic  blood  50 mm Hg below normal with a mean of 43.6  Hypercapnic  pressures  Hg.  blood  blood r e f e r s carbon  to  arterial  blood  d i o x i d e (PaC0 ) at l e a s t  above normal values and hypocapnic  2  with  10 mm Hg  blood values were at l e a s t 10  24  mm Hg below normal; mean values of PaC0 ±2.5  and  21.4  ±1.9  mm  Hg.  blood from a donor animal, used,  and  were r e s p e c t i v e l y  62.9  When r e f e r r i n g to p e r f u s i o n with  the term  "cross-perfusion"  will  and when a l l regions of the c i r c u l a t i o n are perfused  the animal's The  2  own blood, the term " a u t o - p e r f u s i o n " w i l l  adjective "intact"  be  be with  used.  "normal" r e f e r s to ducks which were autoperfused  r e f e r s to ducks before baroreceptor  denervation.  Ef f e c t of U n i l a t e r a l Vagotomy on D i v i n g Bradycardia C a r o t i d body chemoreceptors i n b i r d s are l o c a t e d low i n the neck and are innervated by branches ganglion arterial are nerve  of  the  vagus  (Jones  arising  and  from  Purves  each  1970).  nodose Systemic  b a r o r e c e p t o r s are l o c a t e d at the root of the a o r t a  similarly (Jones  innervated  by  a  d i f f e r e n t branch  of the vagus  1973).  In order to assess the r o l e of experiments  were  designed  peripheral  chemoreceptors,  to i s o l a t e and a r t i f i c i a l l y  perfuse  one c a r o t i d body with blood of v a r i o u s blood gas t e n s i o n s . contralateral vagus nerve. entailed artery.  carotid  body  was  denervated  I s o l a t i o n of the c i r c u l a t i o n  disruption  of  blood  The  by s e c t i o n of the  to one  carotid  flow i n the i p s i l a t e r a l  body  carotid  Richards and Sykes (1967) showed t h a t , i n b i r d s , normal  flow was p r o v i d e d to the head occluded.  when  a l l but  one  artery  were  T h i s maintenance of flow was a t t r i b u t e d to extensive  i n t e r c a r o t i d and e x t r a c r a n i a l anastamoses 1967;  and  Baumel  and  (Richards  Gerchman 1968; West et a l .  1981).  and  Sykes  Only the  25  afferent left was  f i b r e s from the a r t i f i c i a l l y  intact.  For t h i s reason, an  performed  cardiac  i n i t i a l s e r i e s of  to assess the e f f e c t  response  perfused c a r o t i d body were experiments  of u n i l a t e r a l vagotomy on  to d i v i n g by comparing the response  up to 1 or 2 days a f t e r nerve  the  before  and  rate  was  section.  S u r g i c a l preparat ions and exper imenta1 p r o t o c o l Six  ducks were used  determined  from  the  in t h i s  experiment.  electrocardiogram  Heart  (ECG)  obtained  from  subcutaneous e l e c t r o d e s p l a c e d on each side of the c h e s t . animal was  lightly  positioned  in  restrained  the  wide  d i v i n g , the funnel was 14°C) was  to completely  Each  in a supine p o s i t i o n with i t s head  mouth of a l a r g e p l a s t i c  f i l l e d with s u f f i c i e n t  submerge the head.  tap  funnel. water  For  (10  Termination of the d i v e  achieved a f t e r 2 minutes by d r a i n i n g the funnel through  the  spout. Following  dives  with  i n t a c t animals,  exposed under l o c a l a n a e s t h e s i a high i n the segment of the nerve c o o l e d with a thermode. cut  just  promptly  distal  to  the  cooled  c l o s e d with sutures and  Two-minute the next day  the r i g h t vagus neck The  and  a  short  nerve was  s e c t i o n and the i n c i s i o n  the animal allowed to  was  then was  recover.  t e s t d i v e s were c a r r i e d out l a t e r that day or w i t h i n or  two.  26  Effect  of A r t i f i c i a l  Perfusion  of C a r o t i d Body Chemoreceptors  S u r g i c a l preparat ions The  cardiovascular  through the  a  ventral  carotid  body  a i r sac.  To  prepare  a l l a r t e r i e s i n the region  The  thyroid  artery  was l i g a t e d d i s t a l  suface of the c e r v i c a l a i r sac.  punctured  to  arteries, junction artery  access  region  usually  two  to  dorsal in  freed  from  artery.  This  a i r sac was  the v e r t e b r a l a r t e r y and other to  the c a r o t i d  body.  These  number, were l i g a t e d c l o s e to t h e i r  with the common c a r o t i d was  carotid  to the t h y r o i d gland on  the d o r s a l  allow  body  of the c a r o t i d body on the  s i d e were l i g a t e d except f o r the common  i n the  exposed  for carotid  left  arteries  was  i n c i s i o n i n the skin and through the w a l l of  interclavicular  perfusion  area of the  artery.  The  common  carotid  c o n n e c t i v e t i s s u e on both sides of the  c a r o t i d body region.  A pneumatic or "snare-type"  occluder  around t h i s a r t e r y proximal to the c a r o t i d  was  body ( F i g u r e in  placed  2 ) , while the a r t e r y on the d i s t a l  rubber d e n t a l  i d e n t i f i c a t i o n for cannulation  In  some  left  the  a s t a i n l e s s s t e e l snare was placed  on the day of the experiment.  overlying The  with d e n t a l  or  t i s s u e and the s k i n was sutured  freed  dam.  around the of  nerve  The c e r v i c a l a i r sac and closed.  r i g h t vagus was exposed, high i n the neck,  sectioned  and to  on the day of the experiment.  b a r o r e c e p t o r nerve to p r o v i d e a convenient means  section  vessel  side was wrapped  dam to i s o l a t e i t from t i s s u e regrowth  aid  animals  blood  and  either  from surrounding t i s s u e s and wrapped around  In the l a t t e r case the vagus was re-exposed on  27  F i g u r e 2. Schematic diagram i l l u s t r a t i n g the experimental arrangement f o r a r t i f i c i a l p e r f u s i o n of one c a r o t i d body. L e f t vagus nerve i n t a c t ; l e f t c a r o t i d body perfused from i n f u s i o n s y r i n g e . Right vagus nerve sectioned; right c a r o t i d artery i n t a c t .  28  29  the day of the experiment, under cooled,  as  described  this region.  above,  local  anaesthesia,  T h i s type of u n i l a t e r a l vagal  artery  was  section  exposed  in  the  with d e n t a l dam.  denervates  i n ducks.  One  l e g , f o r f u t u r e blood  pressure r e c o r d i n g s on the day of the experiment, around  then  before s e c t i o n i n g j u s t d i s t a l to  the c a r o t i d body and b a r o r e c e p t o r s , on that s i d e , ischiatic  and  and  wrapped  The s k i n over a l l i n c i s i o n s was c l o s e d  using s t a i n l e s s s t e e l wound c l i p s . On  the day of the  c a r e f u l l y re-exposed, both  upstream  (Intramedic pulled  of  downstream  over  a  Tygon  flame  to  tubing  blood  i n f u s i o n pump was  (i.d.  The opened P.E. was  reduce  solution  the  160 t u b i n g . positioned  This cannula  with tubing  i n t o the ends  3mm; Norton,  was  to  a c t , on  To i n h i b i t adhesion a l l cannulae  P.E. 2 4 0 which  to f i t the  of  2 5 cm  a  the T-pieces A  were  variable  the Tygon  flow  tubing,  of blood c e l l s t o the  and tubing were p r e - t r e a t e d  ( P r o s i l , PGR Research Chemicals a i r sac  Inc.). was r e -  l e f t common c a r o t i d a r t e r y was cannulated The t i p of the cannula close  to  was connected  was  Ohio) which had T-  transducers.  a n t e r i o r t i p of the i n t e r c l a v i c u l a r and  artery  i t s diameter  The s i d e arms of  positioned  of the cannulae,  with a s i l i c o n  to  pressure  between the T - p i e c e s . walls  directions  were i n s e r t e d  pieces c l o s e t o each end. connected  ischiatic  under l o c a l a n a e s t h e s i a , and cannulated i n  and  The cannulae  length  the  P o l y e t h y l e n e t u b i n g , Clay Adams)  out  vessel.  experiment,  was advanced u n t i l  the d i s t a l end of the t h y r o i d  with it  gland.  t o a s y r i n g e i n f u s i o n / w i t h d r a w a l pump  (Harvard Apparatus Inc., M i l l i s , Massachusetts.  Figure 2 ) .  The  30  pump's syringe was surrounded maintained  blood  by a  temperature  copper  heating  coil  which  i n the syringe at 41°C over a 30  minute t e s t p e r i o d (Figure 2 ) . One b r a c h i a l v e i n was cannulated for  i n j e c t i n g a d r e n a l i n e to t e s t  f o r baroreceptor  denervation.  Measurements A r t e r i a l blood pressure a r t e r y cannula while hind  the  perfusion flow  monitored  was  adjusted  in  the  perfused at a flow to  ischiatic  When so  was used to monitor the the  carotid  body  mean  flow was reduced  was  that the p e r f u s i o n p r e s s u r e ,  i n the c a r o t i d a r t e r y cannula, was equal to  the  mean  B i o t e c BT-70 pressure t r a n s d u c e r s were  f o r a l l measurements.  similar  cannula  pressure.  a r t e r i a l blood p r e s s u r e . used  monitored  upstream of the pump used f o r hind limb p e r f u s i o n  downstream i s c h i a t i c  limb  perfused,  was  rate  which  Between yielded  a r t e r i a l pressure  throughout  a  (MAP).  and p e r f u s i o n p r e s s u r e  T h i s flow was maintained  d i v e s the hind limb was  fell  perfusion  pressure  Just before a d i v e , to about 50 mm  the dive and recovery.  Hg. In a  s e r i e s of p r e l i m i n a r y experiments i t was e s t a b l i s h e d that at a l l flows  above  9 ml/min, hind limb v a s c u l a r r e s i s t a n c e (HLVR) was  independent of flow r a t e . the  r e d u c t i o n i n flow.  maintained HLVR  g r e a t e r than  Below 9 ml/min HLVR rose Consequently,  9 ml/min to a v o i d p a s s i v e  increases  being added to the values f o r HLVR obtained p r e - d i v e .  an  instantaneous  with  hind limb flow was always  was obtained from subcutaneous e l e c t r o d e s and from  along  heart  rate  in ECG (HR)  rate meter t r i g g e r e d by the QRS wave of  31  the  ECG. The  trachea was  bifurcation.  The  connection,  the  cannula  of which was  supply  be  i n s p i r a t o r y gas c o u l d be Breathing was the 2).  tracheal The  tracheal Packard flow  pressure air  of  gas  the  composition  the  from b r e a t h i n g was  was  fed  through  preamplifier  composition  of  the  monitored  with  the  pneumotach A547.  Figure  pneumotachograph  during  recorded with a  Hewlett-  to  a  give  Hewlett-Packard tidal  volume.  the  Centronic  200  MGA  air  350-3700A The  r e s p i r a t o r y gases s u p p l i e d v i a the a  flow  of  Model 270 d i f f e r e n t i a l pressure transducer and signal  the  with a pneumotachograph attached to  across  integrating  was  thus,  system  T  flowing past the end of the  (Hewlett-Packard  'drop  flow  attached to a  controlled.  monitored  cannula  With a  of the gas  altered;  r o s t r a l to i t s  open to the atmosphere and  ( F i g u r e 2).  composition  could  i n the neck, j u s t  d i s t a l end of the cannula was  one arm  other to a gas meters  cannulated  gas  T-piece  clinical  mass  from the animals  before  spectrometer. Arterial  blood  d i v i n g and a f t e r  samples were taken  55 or 90 seconds of d i v i n g .  measured using an Instrumentation gas  analyser  which  was  The  samples  were  L a b o r a t o r i e s IL micro  13 blood  c a l i b r a t e d with p r e c i s i o n gas  mixtures  before each a n a l y s i s . All  s i g n a l s were a m p l i f i e d using c o n v e n t i o n a l means and  blood p r e s s u r e , t r a c h e a l a i r flow, ECG,  and  C0  a  2  compositions  thermal  pen  were  recorder  displayed  writing  on  on  respiratory Technirite  rectilinear  0  2  the and  8-channel  coordinates  and  32  stored  on  computer.  an  8-channel  The s t o r e d  FM  data  tape system f o r l a t e r a n a l y s i s by  were  analysed  using  a  specially  prepared computer programme f o r a D i g i t a l PDP Lab 8e computer.  Experimental P r o t o c o l On lightly The  the day  of the experiment,  r e s t r a i n e d on an o p e r a t i n g t a b l e i n the supine  head was p o s i t i o n e d f i r m l y  diving  unanaesthetised ducks were  was  accomplished  temperature  i n the mouth  as  described  of  a  position.  funnel  previously.  The body  of a l l b i r d s used was c o n t i n u o u s l y monitored  experiments  and maintained  and  during  at 41±1°C by i n f r a - r e d lamps mounted  above the b i r d s . In normal d i v e s , the c a r o t i d body was autoperfused and MAP  and  HLVR  submersion, infusion  were  recorded.  After  a blood sample was taken  pump,  2).  the d i v e s were extended  analysis. artery  A  sub-sample  Some time l a t e r  was  occluded  of  to  1.5  for a n a l y s i s .  blood was withdrawn from the c a r o t i d (Figure  1  into  blood  ( 5 - 1 0 minutes),  When  the  breathing  minutes.  the  the common  syringe f o r gas carotid  on the s i d e with the i n t a c t c a r o t i d body,  stimulation  hypoxic, hypercapnic  the  was taken  proximal t o the c a r o t i d body, and p e r f u s i o n from started.  To f i l l  of  beyond 1 minute before  artery  this  minutes  HR,  end-dive  trace,  of  the pump  was  c a r o t i d chemoreceptors by t h i s  blood was s t a b l e , as  the animal  was  from  submerged f o r 1 t o 1.5  C a r o t i d body p e r f u s i o n was maintained  i n t o the recovery p e r i o d a f t e r the d i v e .  judged  for 2  minutes  The p e r f u s i o n rate was  33  set  to  yield  a p e r f u s i o n pressure which was equal to a r t e r i a l  blood p r e s s u r e . giving  Hyperoxic  the duck  withdrawn to f i l l breathing  a  few  blood f o r p e r f u s i o n was breaths  of  a i r for 5 - 1 0  diving  2  minutes.  was used  by  Blood was then  the pump r e s e r v o i r and the animal  p e r f u s i o n of the c a r o t i d bodies with during  100% 0 .  obtained  returned  to  The same procedure f o r  hypoxic-hypercapnic  blood  for p e r f u s i o n with hyperoxic blood  from  the pump. Baroreceptors were denervated the  left  by p u l l i n g the  cuts  through  depressor  nerve  right  s i d e were p r e v i o u s l y denervated  snare,  ( b a r o r e c e p t o r s on the  by vagotomy) and a f t e r  - 60 minutes recovery the above p r o t o c o l was repeated. baroreceptor  denervation  cardiac chronotropic  was  response  which  confirmed during  by  the  Complete  lack  hypertension  30  of any  evoked  by  i n j e c t i o n of 5 ug/kg a d r e n a l i n e i n t o the b r a c h i a l v e i n .  C r o s s - P e r f u s i o n Between Ducks To  enable  investigation  p e r i p h e r a l and separate  the  central blood  of  the r e l a t i v e c o n t r i b u t i o n of  chemoreceptors,  i t was  flow to these s i t e s .  cannulated  were cannulated connections  to  carotid  arteries.  f o r flow  through  The c a r o t i d  arteries  i n both upstream and downstream d i r e c t i o n s the v a s c u l a r system of a donor animal.  arrangement p e r i p h e r a l  chemoreceptors  to  T h i s was achieved by  p r e v e n t i n g a l l blood flow to the head except previously  necessary  could  be  either  with  By t h i s auto-  perfused or c r o s s - p e r f u s e d with blood from the donor animal.  34  Surgical  Preparations  In was  one  animal,  necessary  and  the  to completely  body  jugular v e i n s . vagi  designated h e r e a f t e r as the r e c i p i e n t , i t  except  d i s r u p t blood flow between the  f o r the common c a r o t i d a r t e r i e s and the  To accomplish  t h i s the  jugular  veins  were exposed high i n the neck and c a r e f u l l y  surrounding region  tissue.  were  The s k i n and  superficial  were  then  sewn  separated  muscle  jugular  e n c i r c l e the neck  back t o g e t h e r .  these t i s s u e s was d i s r u p t e d . exposed  and the  in  from this  i n f i l t r a t e d with l o c a l a n a e s t h e t i c and clamped.  cut was c a r e f u l l y made to completely edges  head  veins,  A vagi  cord and  A  and the  In t h i s way a l l flow i n was  passed  trachea,  beneath  the  encircling  the  oesophagus, v e r t e b r a l column and a s s o c i a t e d muscles and the ends were l e d to the s u r f a c e at the back of the neck.  On the day of  the experiment t h i s cord c o u l d be t i g h t e n e d to occlude any blood flow  to  the  arteries  were  head  through  exposed  low  these t i s s u e s .  The common c a r o t i d  in  by  the  neck  opening  the  i n t e r c l a v i c u l a r a i r sac and again, high i n the neck, by d i v i d i n g skin  and  muscle j u s t behind  the a r t i c u l a t i o n of the lower jaw.  The common c a r o t i d a r t e r i e s were wrapped i n rubber The  dental  dam.  v e r t e b r a l , c e r v i c a l and thyroidean a r t e r i e s were exposed and  ligated  bilaterally.  The i s c h i a t i c a r t e r y i n the l e g was a l s o  exposed and wrapped with d e n t a l dam. were c a r e f u l l y and a l l other In  The w a l l s of the  a i r sac  sewn back together and the s k i n sutured over  this  incisions.  a second animal, h e r e a f t e r designated as the donor, one  i s c h i a t i c a r t e r y was exposed i n the l e g and wrapped with  dental  35  dam.  A s e a l e d l a r g e bore cannula  (1.0 cm i.d.) was i n s e r t e d and  sewn i n t o the i n t e r c l a v i c u l a r a i r s a c . On the  the day of the experiment, the donor duck was i n t u b a t e d ,  interclavicular  cannula  unidirectional ventilation carefully  opened  (UDV).  and The  and  under  l o c a l anaesthesia,  with P.E. was  240 tubing  flow  from  i s c h i a t i c cannula recipient  to  the donor  limb  and cannulated in  re-opened  Tygon tubing  (i.d.  3  the c a r o t i d a r t e r y of the r e c i p i e n t to  of the donor  hind  was  low and high i n the neck  the i s c h i a t i c a r t e r y of the donor duck. carrying  artery  In the r e c i p i e n t duck,  ( F i g u r e s 3 and 4).  used to connect  p l a c e d on  the neck was c a r e f u l l y  the r i g h t c a r o t i d a r t e r y cannulated  mm)  ischiatic  re-exposed, under l o c a l a n a e s t h e s i a ,  both upstream and downstream d i r e c t i o n s . also  the animal  of  was (flow  donor.  The tube  indicated  as  connected t o the upstream from  carotid  Figures  3  artery and 4).  of The  i s c h i a t i c a r t e r y of the r e c i p i e n t was prepared  f o r pump  p e r f u s i o n as d e s c r i b e d above to measure HLVR.  A l l ducks used i n  cross  perfusion  experiments  driven  had  i n t a c t vagi and baroreceptor  was  monitored  innervat i o n .  Measurements A r t e r i a l blood cannulae  pressure  in  the  upstream  of the c a r o t i d and i s c h i a t i c a r t e r i e s i n the r e c i p i e n t  and donor ducks r e s p e c t i v e l y and p e r f u s i o n pressure was measured in the c r o s s - p e r f u s i o n cannulae s u p p l y i n g the Biotec  BT-70  pressure  transducers).  recipient  When  (using  perfusion  was  36  Figure  3. Schematic diagram of the r e c i p i e n t duck in c r o s s p e r f u s i o n experiments.  37  38  F i g u r e 4. Schematic diagram i l l u s t r a t i n g the experimental set-up f o r c r o s s - p e r f u s i o n between r e c i p i e n t and donor ducks.  monitored g a s mixtures (UDV)  RECIPIENT hind limb perfusion pressure  blood pressure heart rate  occluder on right carotid artery  spontaneously breathing j monitored g a s ' H mixtures  DONOR  *  DIVE  blood p r e s s u r e a n d blood g a s composition monitored CO  40  e s t a b l i s h e d , flow pressure arterial perfusion  to  was  adjusted  so  that  the c r o s s - p e r f u s i o n  the head of the r e c i p i e n t duck matched the c a r o t i d  blood pressure of the r e c i p i e n t . pressure  was  measured  ECG  and  hind  limb  as i n c a r o t i d body p e r f u s i o n  exper iments. In the r e c i p i e n t one common c a r o t i d a r t e r y was carried  flow  to  the head during a u t o - p e r f u s i o n .  before, normal flow was provided to the head of  a l l but  one  artery  (Richards  Gerchman 1968; West et a l . was  established  forceps.  this  anaesthetic. Harvard  the  the  area  Model  1210  cross-perfusion  c l o s e d o f f with  the  haemostatic  with the mass blood  first  ligature  peristaltic  with  pump.  carefully a  to  the  driven  monitored  Composition  donor  local  Breathing  i n the r e c i p i e n t were  supplied  duck  and as  of the  v i a UDV  was  spectrometer.  samples  were  taken  from the animals f o r  55 or 90 seconds of  c a r o t i d body p e r f u s i o n experiments.  that blood c r o s s - p e r f u s e d drained  after  the c r o s s - p e r f u s i o n tubes was  a n a l y s i s before d i v i n g and a f t e r the  Soon  c a r o t i d body p e r f u s i o n experiments.  Arterial  in  occlusion  and Sykes 1967; Baumel and  was  under  Flow through  r e s p i r a t o r y gases monitored  As mentioned  despite  was t i g h t e n e d , a f t e r  i n s p i r a t o r y gas composition in  and  The cord p r e v i o u s l y placed around the v e r t e b r a l column  infiltrating  a  1981).  vessel  and a s s o c i a t e d musculature  by  intact  to  the  diving  as  I t should be noted  recipient  would  have  been  back i n t o the systemic c i r c u l a t i o n by the j u g u l a r veins  and thus would a f f e c t  PV0  2  and  extent of t h i s contamination  PVC0  2  of  the  recipient.  as i t a f f e c t e d systemic Pa0  2  The of the  41  recipient  i n d i v e s , was assessed by using an i n d w e l l i n g oxygen  e l e c t r o d e i n the hind limb p e r f u s i o n c i r c u i t .  The decrement  Pa0  compared  in a  2  dive  with  cross-perfusion  was  o c c u r r i n g when the r e c i p i e n t was auto-perfused. s i g n i f i c a n t d i f f e r e n c e between Pa0 it  was judged  to that  As there was no  i n both types of  2  in  that the extent of any contamination  experiment  was s m a l l .  Experimental p r o t o c o l  Gas  The  donor  duck  was  administered  interclavicular  was p l a c e d on u n i d i r e c t i o n a l v i a the  trachea  and  ventilation.  vented  a i r s a c . Changing the gas composition  r a p i d a l t e r a t i o n of the oxygen and carbon  r e c i p i e n t was allowed to breathe Auto-perfusion r e c i p i e n t animal  allowed  d i o x i d e content  a r t e r i a l blood f o r c r o s s - p e r f u s i o n to the r e c i p i e n t  Under  v i a the  of the  duck.  The  spontaneously.  represents  the  i s in t o t a l control  normal c o n d i t i o n where the of  i t s own  blood  flow.  t h i s c o n d i t i o n , a s e r i e s of 2 minute d i v e s was done a f t e r  the animal least  5  had been breathing e i t h e r a i r or pure oxygen minutes  pre-dive.  These  dives  f o r at  were done randomly,  i n t e r s p e r s e d with d i v e s i n which the head was c r o s s - p e r f u s e d . C r o s s - p e r f u s i o n was e s t a b l i s h e d by o c c l u d i n g the intact  common  carotid  p e r f u s i o n pump. Heparin remainder minutes,  was  artery  first  switching  on the  Once c r o s s - p e r f u s i o n was e s t a b l i s h e d , 1000 i . u .  administered  every  of the experiments. either  after  remaining  first  two  hours  throughout  the  R e c i p i e n t ducks were d i v e d f o r 2  having breathed a i r or pure oxygen,  while  42  their  heads were perfused with blood  from donors which had  v e n t i l a t e d with a i r , pure oxygen, high oxygen and high high was  C0  and  2  taken  low oxygen gas mixtures.  from the donor before, d u r i n g and  to ensure that blood gases remained blood  samples were taken  minutes  HR,  HLVR, MAP At  was  and a r t e r i a l  the  deeply  end  the p e r f u s i o n pump was died  within  1  cross-perfusion recipient's  minute  turned o f f . (as  considered  arterial  cerebral  blood  be  flow  1.5  r e c o r d i n g Pa0  were  allowed  2  at  recovery as i n d i c a t e d by  tensions.  cross-perfusion,  animal  and  Only i f the r e c i p i e n t  then animal  judged by r e s p i r a t o r y f a i l u r e ) to  or  sample  of the day's experiments the r e c i p i e n t  a n a e s t h e t i s e d , p l a c e d on  ,  before and a f t e r  Recipients  blood gas  blood  Carotid  of the d i v e , in a d d i t i o n to c o n t i n u o u s l y  30 minutes between d i v e s f o r f u l l  2  at the end of a d i v e  stable.  from the r e c i p i e n t  in the hind limb p e r f u s i o n l i n e . least  An a r t e r i a l  C0  been  supplying and  all  of  the experimental  was the  results  acceptable.  S t a t i s t i c s and  the A n a l y s i s of Data  In the t e x t and to  determinations  f i g u r e s , numerical  in  the  when  of v a r i a b l e s in a group of animals  N, are given as means ±S.E. Only  values,  of the  mean  of  n  referring of number  determinations.  c a r o t i d body p e r f u s i o n s e r i e s of experiments were  r e p l i c a t e s c a r r i e d out.  Data from the v a r i o u s groups,  s e r i e s of experiments, were compared at each sampling a one-way a n a l y s i s of v a r i a n c e  in  each  time using  (ONEWAY, SPSS; Nie et a l .  1975).  43  In the case of s i g n i f i c a n t  F values  (P<0.05), p a i r e d comparisons  of means were done with S c h e f f e ' s method (Scheffe 1959).  44  RESULTS  E f f e c t of U n i l a t e r a l Vagotomy on D i v i n g Pre-dive section  heart  of  one  rates  (right)  significant  (P>0.05).  did  a  cause  completely  of  Pekin  vagus  but  Bradycardia ducks the  were  increase  substantial  elevation  of  the  eliminated diving bradycardia.  has  been observed  p r e v i o u s l y i n ducks (Johansen and R e i t e  1964;  study.  In the remaining  Jones  1968).  These ducks were not i n c l u d e d i n the  ducks  unilateral  d i v i n g b r a d y c a r d i a , heart rate being after  only  203  beats/min;  compared n=22;  to  N=4)  the  control  (38  ±4 beats/min, p r e - d i v e  (Figure 5 ) .  to only 26% of  in  vagotomised ducks compared with a d e c l i n e to 17.6% in  diminished  s i g n i f i c a n t l y above that i n  r a t e had f a l l e n  (60 ±4 beats/min ±12  vagotomy  15 seconds of submersion  A f t e r 2 minutes submergence heart  of  rate and  fibres  and  value  resting  inhibitory  Butler  the pre-dive value  not  T h i s phenomenom where  vagus c a r r i e s a l l the c a r d i a c c h r o n o t r o p i c  ducks  was  However, i n two ducks u n i l a t e r a l vagotomy  one  intact  e l e v a t e d by  the  pre-dive  unilaterally of  pre-dive  187 ±13 beats/min;  n=l0; N=4).  Effect  of P e r f u s i o n of the C a r o t i d Body Chemoreceptors  The  mean pre-dive HR  in  bodies  ±28  beats/min.  seconds of submergence, HR f e l l  r a p i d l y at  carotid  was  328  intact  ducks  with  autoperfused  During  the f i r s t 3  a  mean  rate  of  45  Figure  5. E f f e c t of u n i l a t e r a l vagotomy on d i v i n g bradycardia. Time= 0 seconds r e p r e s e n t s the s t a r t of a 2 minute d i v e . Values for the i n t a c t animal are represented by c l o s e d c i r c l e s ( n = 2 2 ) , those f o r vagotomised animals by open c i r c l e s (n=lO).(bars ±S.E.)  in CM  47  24.7±6  beats/s.  beats/s  i n the p e r i o d from 3 to 6s, to  period  from  beats/min decline  The r a t e of f a l l  0.5  to  1.0  (Table 1). HR to  65±7  a  dive  was  not  the dive  the p r e - d i v e value a f t e r but  at  above the p r e - d i v e v a l u e  (Pa0  =  2  significantly increase  272  mm  reduced  yet  beats/s  stable,  f o r the  continuing  1.5 minutes underwater.  increasing  to HLVR  significantly  1 minute of submergence.  from  MAP a l s o rose  no time was the i n c r e a s e s i g n i f i c a n t l y (Table 1).  P e r f u s i o n of the one i n t a c t blood  -1.53  minute, reaching a l e v e l of 91.3 ±10  beats/min a f t e r  rose slowly throughout  during  then d e c l i n e d from -10.9 ±2.5  Hg;  carotid  PaC0  =  2  body  with  hyperoxic  27.2 mm Hg; pHa = 7.67)  both d i v i n g b r a d y c a r d i a  (P< 0.05) and the  i n HLVR (P< 0.05) a f t e r a 1 minute d i v e , compared with  an autoperfused  dive  (Table  1).  although HR d e c l i n e d s i g n i f i c a n t l y was no s i g n i f i c a n t carotid  In  fact  in  these  from the p r e - d i v e value  i n c r e a s e i n HLVR.  There was l i t t l e  dives, there  e f f e c t of  body p e r f u s i o n on c a r d i o v a s c u l a r v a r i a b l e s i n b r e a t h i n g  ducks (Table 1). P e r f u s i n g c a r o t i d bodies with hypoxic Hg;  PaC0  gave  = 44.4 mm Hg; pHa = 7.36) throughout  2  mean  obtained  blood  values  f o r HR  from a u t o - p e r f u s e d  with hyperoxic  blood  (Pa0 1  2  = 35.4 mm  minute  and HLVR i n t e r m e d i a t e between and from p e r f u s i o n of c a r o t i d  (Table 1). A f t e r  1  minute  dives those bodies  underwater  HR  was  s i g n i f i c a n t l y below that i n ducks in which the c a r o t i d body  was  perfused  different (Table  with  hyperoxic  blood  but  not  from v a l u e s i n ducks with auto-perfused  1).  HR f e l l  rapidly  i n the f i r s t  significantly carotid  bodies  3 seconds of the dive  48  Table  I. E f f e c t of C a r o t i d Body S t i m u l a t i o n and Withdrawal on C a r d i o v a s c u l a r Responses to Submergence i n i n t a c t and Barodenervated Ducks. Values are means ±S.E.. HR denotes heart r a t e (beats/min); MAP denotes mean a r t e r i a l pressure (mm Hg); HLVR denotes hind limb v a s c u l a r r e s i s t a n c e (PRU's); N=animals, n=total d i v e s .  Table I. E f f e c t of Carotid Body Stimulation and Withdrawal on Cardiovascular Responses to Submergence i n i n t a c t and Barodenervated Ducks Surface Intact Duck, Normal Dive N=8 n=15  Dive 30 s  60 s  HR  328.0+27.7  132.9+11.6  MAP  142.9+7.6  165.1+5.3  168.3+9.8  8.93+1.81  10.01+1.66  16.71+2.37  HLVR  91.3+10.2  Baroreceptors Denervated Normal Dive N=5 n=10  HR  383.0+22.1  247.0+25.1  137.8+15.8  MAP  167.8+19.2  206.5+13.8  205.4+16.7  7.39+1.03  13.19+2.53  18.44+4.41  Intact Duck, Hypoxic-Hypercapnic Perfusion N=7 n=10  HR  313.0+23.2  150.0+15.6  126.0+13.7  MAP  144.8+6.7  153.1+3.9  146.8+6.7  8.30+1.18  10.27+1.18  12.72+3.00  Baroreceptors Denervated Hypox i c-Hypercapni c Perfusion N=5 n=8  HR  408.8+21.3  227.5+25.6  197.5+22.5  MAP  194.0+19.2  211.7+16.8  207.6+9.4  8.99+1.33  13.01+1.43  16.26+3.55  Intact Duck, Hyperoxic Perfusion N=7 n=12  HR  330.8+25.0  220.8+2.1  179.2+14.3  MAP  139.1+9.4  144.9+7.2  158.6+8.4  Baroreceptors Denervated Hyperoxic Perfusion N=4 n=5  HLVR  HLVR  HLVR  HLVR  6.88+1.05  8.14+1.11  10.75+2.17  HR  400.0+10.9  297.6+23.7  280.0+33.5  MAP  153.3+8.5  157.5+23.7  160.8+14.3  10.23+2.30  12.60+4.99  HLVR  7.30+1.99  50  at  a rate of -19.8±2.5 beats/s which p e r s i s t e d  seconds  of  the  dive  u n l i k e the s i t u a t i o n  f o r the  next  i n auto-perfused or  hyperoxic blood perfused d i v e s i n which the r a t e of f a l l was u s u a l l y halved from the i n i t i a l  3 second  dive,  perfusion  body  little  e f f e c t on c a r d i o v a s c u l a r v a r i a b l e s i n s p i t e of  that  minute  of  the  ventilation  d u r i n g the d i v e d i d not values  although  carotid  increased increase  significantly different  period.  by at l e a s t  in  blood had the  2 times.  significantly  HR  Before the  with hypoxic  i t increased s u f f i c i e n t l y  3  from  fact HLVR  pre-dive  so that HLVR was not  from that i n auto-perfused ducks  (Table  1) . Denervation caused when  of  systemic  i n c r e a s e s i n HR, MAP and HLVR before the  carotid  body  was  c a r o t i d body was  perfused  However,  cases  diving  a r t e r i a l baroreceptors generally  in  no  auto-perfused  with  were  hypoxic  the  auto-perfused  during  or when one i n t a c t  or  hyperoxic  underwater  MAP i n denervates  was a l s o  r e s p e c t i v e c o n t r o l s except  perfusion  c a r o t i d body with hyperoxic blood  the  Nevertheless, a f t e r  hyperoxic  significant group  blood  perfusion,  difference existed  (Table  in  of  in intact  the  case  of  (Table 1).  1 minute, the only s i g n i f i c a n t d i f f e r e n c e i n  HR e x i s t i n g between a baro-denervate for  before  significantly  above that i n t h e i r of  blood.  a l l groups  ducks had HR s i g n i f i c a n t l y above those ducks.  diving  increases s i g n i f i c a n t  (Table 1). A f t e r 0.5 minute  baro-denervated  and  in  and i t s c o n t r o l while  the  was above that i n t h e i r  f o r MAP  hypoxic  1). HLVR i n baro-denervates,  group  blood  the  was only  perfused  before submergence,  r e s p e c t i v e c o n t r o l group except  i n auto-  51  perfused animals. all  denervated  cases  were  animals  the  significant effect during d i v i n g , dives  However, a f t e r  1 minute underwater,  was above that i n c o n t r o l s although  differences on blood  significant.  gas  i n auto-perfused  was reduced  tensions  ducks.  or  The  dive  the trends  HR  was  in  excess  first  12  seconds  was p a r t i c u l a r l y n o t i c e a b l e of 250 beats/min.  of  dive. after  either  dive  fall  when p r e -  Pre-dive HR (y) was which  (r=0.82).  occurred  in  The r e g r e s s i o n  In other words, i f the  pre-dive  HR  around 188 beats/min there was no change i n the HR e a r l y i n  the d i v e . any  the  HR  breathed  or c r o s s - p e r f u s e d were c h a r a c t e r i s e d by a very r a p i d  equation was y = mx - 188. was  in  i n the C e r e b r a l C i r c u l a t i o n  e a r l y p e r i o d i n a l l d i v e s i n which both ducks  The f a l l  or  i n HLVR i n c r e a s e d .  s t r o n g l y c o r r e l a t e d to the change i n HR (x) the  had no  Expressing HR or HLVR i n  while the p r o p o r t i o n a t e r i s e  in heart r a t e .  in  i n no  before  or 100% oxygen p r e - d i v e and the r e c i p i e n t ' s head was  auto-  yet,  pHa,  In a l l three groups, the p r o p o r t i o n a t e f a l l  E f f e c t of Changing Blood Gas Tension  air  Denervation  as a p r o p o r t i o n of the p r e - d i v e r a t e confirmed  noted above.  HLVR  There was no c o r r e l a t i o n between  pre-dive  change i n HLVR that occurred i n the f i r s t  was obtained  12 seconds of submergence as e x i s t e d before the  The  aim  the e f f e c t s of  of  and  12 seconds of the  In f a c t , the same mean value of HLVR (n =112)  MAP rose s i g n i f i c a n t l y by, on average,  HLVR  dive  and  8% of p r e - d i v e .  these experiments was to i s o l a t e and i d e n t i f y  hypoxia  and  hypercapnia  at  the  central  and  52  peripheral  chemoreceptors,  alone  achieved s i n c e a l l combinations subjected  or  of  perfusing  breathing  Eleven  to  central  on measurements of blood gas  the r e c e p t o r s , i t was decided to merge some  these combinations,  ducks  and c r o s s - p e r f u s i o n  of gas t e n s i o n s were a p p l i e d  and p e r i p h e r a l r e c e p t o r s but, based  of  auto-  This was not  the p e r i p h e r a l chemoreceptors to hypercapnia.  d i f f e r e n t combinations  tensions  together.  along with  the  data  from  autoperfused  a i r or oxygen p r e - d i v e , to give 5 experimental  groups (Table 2 ) . During d i v e s i n  which  the  head  was  auto-perfused  both  p e r i p h e r a l and c e n t r a l r e c e p t o r s i t e s were exposed to high PaC0 in  combination  with  low  b r a d y c a r d i a and l a r g e s t HR  fell  rapidly  stabilise after significantly times.  Pa0 .  developed  (Table  2).  from the p r e - d i v e r a t e of 342±22 beats/min to  1 minute underwater at 43±4 beats/min.  MAP  fell  by 22 mm Hg and HLVR i n c r e a s e d s i g n i f i c a n t l y by  Continuously  initial  these d i v e s the g r e a t e s t  i n c r e a s e i n HLVR  measured  p r e v i o u s l y by Jones and Purves an  In  2  2  Pa0  2  3  showed the f e a t u r e s noted  (1970); Pa0  2  fell  rapidly  after  l a t e n t p e r i o d of 5 - 10 seconds but once bradycardia  was f u l l y developed,  only f e l l  rest  In one group of c r o s s - p e r f u s i o n experiments  of  the d i v e .  both receptor s i t e s tensions, recipient  as  in  were diving  slowly ( <5 mm Hg/min )  stimulated  auto-perfused  to breath a i r before the  the head with low Pa0  2  with  dive  and h i g h PaC0 . 2  similar  f o r the  blood  ducks, by a l l o w i n g the while  cross-perfusing  There was no s i g n i f i c a n t  d i f f e r e n c e between the c a r d i o v a s c u l a r v a r i a b l e s monitored cross-perfused  and  auto-perfused  gas  i n the  animals d u r i n g d i v e s , so the  53  Table  II. E f f e c t of Change i n Blood Gas Tensions at C a r o t i d Body and C e n t r a l Chemoreceptors on C a r d i o v a s c u l a r Responses to Forced D i v i n g . Values are means ±S.E.. Animals were d i v i d e d i n t o 5 groups a c c o r d i n g to which r e c e p t o r s were s t i m u l a t e d (*) d u r i n g the d i v e . In a l l groups the c a r o t i d bodies were autoperfused. In group 1 the head was e i t h e r auto- or c r o s s - p e r f u s e d ; i n a l l other groups the head was c r o s s perfused. A b b r e v i a t i o n s are the same as i n Table 1.  54  Table II. Effect of Change in Blood Gas Tensions at Carotid Body and Central Chemoreceptors on Cardiovascular Responses to Forced Diving  Group 1  Pre-Dive  Dive,30s  Dive, 60s  Dive,90s  Dive,120s  2  3  4  5  HR  304.7+25.7  371.1+38.0  338.1+20.5  356.0+64.9  255.6+25.1  MAP  131.1+4.1  130.7+7.7  122.3+5.0  133.8+11.B  119.3+11.2  HLVR  4.49+0.45  3.76+0.53  4.65+0.40  3.94+0.76  5.03+0.68  HR  94.0+16.1  92.2+10.4  207.4+16.8  256.0+59.5  178.9+59.5  135.6+6.3  136.2+13.4  126.4+12.1  6.96+0.70  4.64+0.77  6.76+0.94  170.6+14.9  220.0+49.7  151.2+20.6  131.4+6.4  133.4+13.0  127.3+10.6  5.22+0.69  6.55+0.69  MAP  129.9+5.9  127.3+7.7  HLVR  8.47+0.82  7.54+0.B7  HR  41.3+B.6  65.6+6.0  MAP  114.5+5.5  110.0+8.1  HLVR  13.09+1.31  9.82+0.76  7.79+0.73  HR  43.3+7.9  53.3+6.5  157.1+15.0  214.0+53.8 . 150.0+17.4  MAP  109.6+5.8  96.5+5.2  124.9+6.1  124.4+10.6  122.9+11.8  HLVR  13.29+1.13  11.63+0.86  8.62+0.74  6.38+1.68  6.32+0.65  HR  52,0+9.2  70.0+6.8  161.3+15.6  180.0+42.4  157.7+13.6  MAP  111.6+5.9  93.4+6.7  126.0+5.6  119.8+10.1  121.6+11.7  HLVR  13.40+1.17  9.76+1.07  8.32+0.80  5.75+1.47  6.03+0.53  15 * * * *  8-9 * *  28-31  4-5  9  *  * *  *  n Low 02 Peripherally High C02 Peripherally Low 02 Centrally High C02 Centrally  *  55  groups were combined these no  to form Group  1 i n Table 2.  Consequently,  experiments confirmed that the c r o s s - p e r f u s i o n per se had  effect  on  Furthermore,  the  cardiovascular  responses  to  diving.  since d i v i n g heart r a t e i n auto-perfused ducks was  not s i g n i f i c a n t l y d i f f e r e n t  from that  in the p r e v i o u s experiment), the  i n i n t a c t ducks  extensive  (described  surgical  procedure  a s s o c i a t e d with c r o s s - p e r f u s i o n experiments had no e f f e c t on the d i v i n g response. Although different minutes  the  pre-dive  from each other of  values  i n Groups  submergence,  1  HR of Group  below the HR's of Groups 3 to 5. Group  other groups except Group 2.  in  MAP  hypercapnic  difference (Table  blood  yet  5,  after  On  the  other  s i g n i f i c a n t l y above that submergence.  only  0.5  1 minute, HR and HLVR of  above,  respectively, a l l  At no time d i d s i g n i f i c a n t  among the groups.  there  was  In group 2 ducks  excited never  hand,  i n Group 5  HLVR ducks  in  by  hypoxic  any s i g n i f i c a n t  i n HR and HLVR d u r i n g d i v e s by Groups  2).  significantly  1 ducks was s i g n i f i c a n t l y  only the p e r i p h e r a l chemoreceptor zone was and  not  These s i g n i f i c a n t d i f f e r e n c e s were  maintained f o r the r e s t of the d i v e . exist  to  After  1 were s i g n i f i c a n t l y below and  differences  were  1  or  2  ducks  Group 2 ducks was  after  1  minute  of  In Group 5 animals, p e r i p h e r a l chemoreceptors were  e x c i t e d by hypercapnic blood alone  (Table 2; F i g u r e 6 ) .  Even though no s i g n i f i c a n t d i f f e r e n c e s e x i s t e d among groups 3,  4  and  5  in  terms of HR and HLVR before and d u r i n g  diving  (Table 2 ) , n o r m a l i s a t i o n of the data by comparing the changes i n HR and HLVR i n dives r e v e a l e d one i n t e r e s t i n g d i f f e r e n c e  between  56  F i g u r e 6. E f f e c t s of v a r y i n g PaC0 in the r e c i p i e n t duck during 2 P e r i p h e r a l chemoreceptors were hypercapnia i n both A and B. 0 and low C0 gas. B: donor high C0 gas. 2  2  2  2  at c e n t r a l chemoreceptors minutes submersion. subjected to p r o g r e s s i v e A: donor b r e a t h i n g high breathing high 0 and 2  57  A.  air flow (L/min )  • 3 0 - 3  DONOR breathing hyperoxic-hypocapnic gas RECIPIENT breathing oxygen  taw  V;  225r mean arterial blood 112 pressure (mmHg) 20 peipheral resistance (pru)  10  t  0  dive  B.  surface  DONOR breathing hyperoxic - hypercapnic gas RECIPIENT breathing oxygen  air flow (L/min )  mean arterial blood pressure (mmHg)  225r ^ ^ ^ ^ ^ ^ l'  2  0 20  peripheral resistance (pru)  r  io dive  f surface  30 seconds  58  Groups 3 and 5. peripheral  The i n c r e a s e  chemoreceptors  (Figure 7a and b) peripheral  was  receptors  difference  between  blood  that  stimulated  effect  mean  HR  when  both  stimulated  significantly  beats/min) and HR at other confirmed  HLVR  were  were  attempt to look at the  in  of  with  greater  than  (Table 2 ) .  chemoreceptors  high  and PaC0  when  2  only  In a f u r t h e r on  HR,  the  at 12 seconds of submergence (188  times i n the dive was  excitation  central  of  peripheral  low i n oxygen gave the only s i g n i f i c a n t  in the l a t e r p e r i o d of the d i v e .  tested.'  This  chemoreceptors with reductions  in  HR  59  DISCUSSION The  chemoreceptive  nature  of  the d i v i n g response i n the  Pekin duck has been confirmed i n t h i s i n v e s t i g a t i o n . in  addition  to  the  p e r i p h e r a l chemoreceptors,  c a r o t i d bodies, a c e n t r a l chemoreceptor role  in  establishing  the  full  submersion has been r e v e a l e d .  From  group with an  the  major  of b r a d y c a r d i a and the  occur  in  spite  diving  1983).  Though they may  of  increase  their  (Kobinger and Oda  of  the  significant  role  cardiac  adjustments  in  not  diving  contribute  response  limited  role  1973; Jones et. a l . the  cardiovascular  itself,  they  of  during  MAP  these  peripheral i s in  their  to  these  and t h i s  may  retain a submersion.  output i s g r e a t l y reduced i n the d i v e , even output  in  the  face  of  the  increased  (TPR) w i l l have marked a e f f e c t on  small total MAP.  to the c a r d i a c c h r o n o t r o p i c response, there are  important c o n t r i b u t i o n s from r e c e p t o r groups not yet in  the  submersion.  in  absence  1969; Jones  in the r e g u l a t i o n  peripheral resistance regard  to  of  that  serve to m i t i g a t e the responses evoked by  during  In  part  inferred  agreement with p r e v i o u s r e p o r t s i n d i c a t i n g  When  response  performances of animals during submersion before  The development  changes  important  The c o l l e c t i v e response from both  and a f t e r baroreceptor d e n e r v a t i o n i t was  resistance  i n the  changes.  the  receptors  located  cardiovascular  these groups e s s e n t i a l l y accounts f o r cardiovascular  Moreover,  experiments.  If the p r e - d i v e HR was  unusual f o r HR to d e c l i n e as much i n the  first  identified  h i g h i t was 12  seconds  submergence as i t d i d i n the r e s t of the 2 minute d i v e .  not of  A rapid  60  early  fall  others  ( F e i g l and Folkow  reflex,  i n HR, when p r e - d i v e HR was high, has been noted by  although  the  1963) and a t t r i b u t e d t o a " n a r i a l - t y p e " sudden  withdrawal  of  i n t e r a c t i o n between feedback from pulmonary respiratory  neuron  controlling  neurons,  involved  (Bamford  and  medullary  consequent  upon  apnoea,  and Jones 1976).  HLVR,  as  a  in  beds,  indication  of  chronotropic  the  of  the  change  response  similar  with  1979). dive in  from  during  For t h i s  i s the cardiac  12  both low Pa0  proportionate  significantly  of  increased.  i n the d i v e , has been  other  Although diving  in  2  increase  different  from  r e p o r t e d by Jones reason,  change  only  in  i n HR a good  output.  The  cardiac  to 120 seconds submergence, i n  ducks i n which both p e r i p h e r a l and c e n t r a l stimulated  (TPR), lags  i n a s u b s t a n t i a l number  (Jones e_t a l .  stages  either  1966; Jones and Holeton 1972), an increase  stroke volume, e a r l y  later  be  12 seconds  Consequently,  or stroke volume must have  (Folkow e_t a_l.  also  interesting,  measure of t o t a l p e r i p h e r a l r e s i s t a n c e  and co-workers the  could  i n the f i r s t  most authors f i n d stroke volume i s unchanged ducks  cardiovascular  What was most  yet HLVR d i d not change.  behind the r e s i s t a n c e changes vascular  stimulatory  a f f e r e n t s or c e n t r a l  activity  however, was that MAP rose s i g n i f i c a n t l y of submergence,  a  receptor  zones  and high PaC0 , was matched 2  in  HLVR,  and  MAP  was  were by a not  pre-dive MAP throughout the dive  (Figure 7,A and B). From comparisons between the baroreceptor denervated groups and t h e i r that  r e s p e c t i v e c o n t r o l groups (Table  baroreceptors  are  capable  of  1)  i t was  enhancing  deduced  submersion  61  Figure  7. Assessment of the c o n t r i b u t i o n of c e n t r a l (c) and p e r i p h e r a l (p) chemoreceptors to bradycardia and changes in hind limb vascular r e s i s t a n c e (HLVR) in ducks during the p e r i o d from 12 to 120 seconds of submersion, (baros.) denotes the presumed c o n t r i b u t i o n of the barostatic reflex. (PRU's) = p e r i p h e r a l r e s i s t a n c e units.  H-  60  SECONDS  1  63  bradycardia increased  by about  12%.  30  intact  HR,  MAP  and  HLVR  were a l l  f o l l o w i n g barodenervation, but these changes were not  significantly different After  Resting  from  the  intact  seconds of submersion, HR was  controls  significant  (P>0.05)  but  difference  hyperoxia d u r i n g The present  after  remained  condition  (P>0.05).  s i g n i f i c a n t l y above the 60  in  seconds  the  the  group  only  exposed  to  submersion. study shows that a c e n t r a l  chemoreceptor  zone  in  the c e p h a l i c c i r c u l a t i o n makes a s i g n i f i c a n t c o n t r i b u t i o n to  the  increase  prove  i n HLVR during  stimulatory,  the  diving.  rising  e f f e c t on r e s i s t a n c e changes. the change i n HLVR i n d i v e s al.  1983);  central  it  seems  Nevertheless,  PaC0  a greater  elicits  2  blood  gases  the more potent  I t has p r e v i o u s l y been shown that  r e f l e c t s the change i n TPR  reasonable  chemoreceptors  Although both  to  assume,  also contribute  (Jones e_t  therefore,  to the increase  p o r t i o n of the i n c r e a s e  is  that  in TPR.  attributable  to the p e r i p h e r a l chemoreceptors. By  30  seconds  cardiovascular to  develop  receptor  immersion  changes have occurred  and  Consequently,  after  the g r e a t e s t  but the responses  assessments  of  made p r i m a r i l y a f t e r t h i s point  the  relative  chronotropic  Table  peripheral 2).  responses  were  blood  identical  of  gases were  peripheral  when  only  and when both c e n t r a l  chemoreceptors were s t i m u l a t e d  Clearly,  contributions  i n the d i v e .  p e r i p h e r a l chemoreceptors were s t i m u l a t e d , and  continue  s t a b i l i s e only a f t e r 60 seconds of submersion.  groups in response to the s t i m u l a t o r y  Cardiac  part of the  (Groups 1 and 2,  chemoreceptors  are  mainly  64  responsible  f o r submersion  bradycardia  and  this  i s further  emphasized by the lack of d i f f e r e n c e between auto-perfused ducks and Group 2 ducks (Table 2). stimulated  only  by  By comparing responses  hypercapnia  s t i m u l a t e d by hypoxic hypercapnic possible PaC0 . four  animals  and  animals  sites  it is  peripherally  blood  at  both  to d i s c e r n the changes due to the p e r i p h e r a l a c t i o n of S i m i l a r comparisons between  2  of  permutations  individual  other  groups  representing  of receptor s t i m u l a t i o n evince the a c t i o n of  stimulus-receptor  pairing.  These  estimates  assessed with due c o n s i d e r a t i o n of the baroreceptor  were  contribution  and are summarised i n F i g u r e 7A. In  ducks  in  which  both  p e r i p h e r a l and c e n t r a l receptor  s i t e s were s t i m u l a t e d simultaneously with Pa0 ,  there  2  high  PaC0  low  was a change i n HLVR, from p r e - d i v e to end-dive of  some nine p e r i p h e r a l r e s i s t a n c e u n i t s  (PRU)(Table  in Table 2 the changes i n HLVR apportioned among and  and  2  stimulatory  2 ) . From data the  receptors  blood gases are represented i n F i g u r e 7B.  The  g r e a t e s t c o n t r i b u t i o n to the t o t a l change was a t t r i b u t e d to the stimulation Together  of  with  peripheral  peripheral  an  receptors  estimated  11%  a c t i o n of hypercapnia,  -is r e s p o n s i b l e f o r two-thirds little  less  than  by  of  the  9%  (67%) of  much  or so i s caused  more  due  to the  the  total  change.  A  a t h i r d of the change c o u l d be a t t r i b u t e d to  than  in  hypercapnia  while  the  by the c e n t r a l a c t i o n of hypoxia.  C a r o t i d body p e r f u s i o n i n barodenervated increase  change  the p e r i p h e r a l receptor group  the s t i m u l a t i o n of c e n t r a l r e c e p t o r s by remaining  blood low i n oxygen.  ducks  ducks  caused  with an i n t a c t  HLVR  to  barostatic  65  reflex  (Table  1). This  barodenervates  is  (Jones  unlike  1973 ;  the  Jones et a_l.  does not r i s e anywhere near as much as portion  of  situation  1982) i n which HLVR  in  intact  Figure  7B  increase i n HLVR which would occur i n t h e i r brought  into  which  i s below  the  88%  of  the  HR  and  s t i m u l a t i o n of c e n t r a l advancing  hypoxic  an  incipient  HLVR  and  chemoreceptors forced  200  non-chemoreceptor  peripheral  dive,  chemoreceptors  Hence,  85  by the  diving bradycardia in r e f l e x set  serve  1974;  to  Blix  modulate  el  in  train  a_l.  1974).  the c a r d i o v a s c u l a r  responses provoked by submersion and may h e l p  These  to  i n MAP caused by a chemoreceptor-driven  baroreceptors  v i a the b a r o s t a t i c  overall  responses w i l l be caused by the  increase i n TPR (Andersen and B l i x Instead,  The  absence.  When these animals  hypercapnia.  rise  the  i n the range of 150  ducks i s not an expression of a b a r o s t a t i c by  as  l e v e l where  inputs c o n t r i b u t e to b r a d y c a r d i a . to  ducks.  the l a b o r a t o r y without any p r e p a r a t i v e  o p e r a t i o n s , ducks have HR's u s u a l l y beats/min,  chronic  change due to b a r o r e c e p t o r s i n each group was taken  i n t o account and i s represented i n  When  in  to  regulate  MAP  importance  of  reflex.  results  have  in activating  submersion.  Scholander and G r i n n e l  confirmed the  However,  the  cardiovascular as  was  (1941) f o r s e a l s  reported and  later  responses  to  by  Irving,  by  Folkow,  N i l s s o n and Yonce (1967) f o r ducks, animals may be a b l e t o exert a  c o n s i d e r a b l e degree of i n f l u e n c e over the f u l l development of  these responses.  In the f o l l o w i n g s e c t i o n , an attempt  to assess the scope of t h i s  control.  i s made  66  SECTION 2.  The  E f f e c t of a Simple Form of Learning  on the D i v i n g Response  Introduct ion The  purely  reflexogenic  aspects  of  the  response  submersion can be r e a d i l y examined i n the l a b o r a t o r y . interpretation  the  The most evanescent  literature,  when r e s t r a i n e d . arousal  is  arises  et  I t appears that  a_l.  1941;  Blix  successive  animal's  abolishing  some 1967).  wild,  voluntary  as  well  Simple  vertebrate  learning  influences  on  forms as  of  crucial  aspects The  cardiovascular  as  CNS  of i t  discrepancy  the  1973; B u t l e r and 1984).  Randall  of  variability  d i v e s may be accounted f o r by t h i s  f a c t o r (Jones et_ a_l.  controlling  level  submerged i n the l a b o r a t o r y and animals  1984; Kanwisher and G a b r i e l s e n  considered  and  the  Folkow et a l .  v o l u n t a r i l y d i v i n g in the  CNS a r o u s a l  according  capable of e i t h e r maximising the development of the  between animals f o r c i b l y  between  interference,  from the animal's s t a t e of a r o u s a l  d i v i n g response or completely (Irving  However,  of the data must i n c l u d e the i n f l u e n c e of higher  l e v e l s of the CNS. to  to  function  1982;  have r e c e n t l y been  higher  (Galosy  Jones  nervous  et a l .  1984; Engel and Schneiderman 1984).  centres  1981; Cohen  With regard  to  diving,  i t has been demonstrated i n s e a l s that both the rate and  degree  of b r a d y c a r d i a  conditioning  can be increased  (Ridgway et a l .  1975) and  by c l a s s i c a l and operant this  may  account  a n t i c i p a t i o n or enhancement of some of the responses.  for  67  The never  CNS  been  permanent  attenuation demonstrated.  forms  proposed  that  in of  Habituation,  of  learning  may  habituation  have  ranging  invertebrate,  from  Aplysia  However,  1956)  play  and  relatively  it  the  primarily gill  has  an important r o l e  s i t u a t i o n s (Glaser  dealt  with  withdrawal  been i n the  especially  1966).  Studies  defence-related reflex  of  the  (Kandel 1976), to complex behaviour such  as the mobbing responses  flexion  the  of an organism to i t s environment,  innocuous or unimportant  responses  (Thorpe  habituation  adaptation  1970).  or  waning of responses, has been d e s c r i b e d as one of the  simplest  general  of the d i v i n g response has, however,  of  several  chaffinches simple  to  predators  reflexes,  (Hinde  such as hind  i n the c a t have a l s o been examined (Thompson and  limb  Spencer  1966). Various  cardiovascular  responses have been i n v e s t i g a t e d and  found amenable to h a b i t u a t i o n . r a t e response to noise temperature  (Glaser  responses i n man,  (Raskin and  1982;  to  ubiquitous  resistant  a  et a_l.  Griffin  included  1969)  1968);  Zbrozyna  1982).  and  heart  blood  pressure  response"  (Krebbel  Although h a b i t u a t i o n  phenomenon,  to h a b i t u a t i o n .  the  or sudden change i n  such as the " c o l d pressor  and Zbrozyna be  These have  certain  responses  For i n s t a n c e , Furedy  unable to observe h a b i t u a t i o n of the d i g i t a l  appears appear  (1969,1971)  vasomotor  was  component  of the human o r i e n t i n g r e f l e x . Long  term  habituation  has been s t u d i e d and a l s o demonstrated.  of the defence r e a c t i o n i n the cat  independence It  was  of the response components  discovered  that  the  was  vasodilatory  68  response  habituated  components, such Martin  et_ a_l.  noise and  posture  1976).  readily  than  (Sutherland  1976;  variability both these  the  and  behavioural  Zbrozyna  Renal v a s o c o n s t r i c t i o n evoked  in baboons by fear has a l s o been  (Zbrozyna  To  as  more  Seal  and Zbrozyna  shown  1978).  i n dogs by  to  habituate  In a d d i t i o n , marked  in the h a b i t u a t i o n of these responses was  noted  date, h a b i t u a t i o n of the responses to forced adequately  carried  by  out  Rey  investigated,  (1971)  in  although  which  submersion  a  ducklings  study were  repeatedly f o r many t r i a l s without observing any change bradycardia.  T h i s study was  not undertaken  examine h a b i t u a t i o n , but the p r o t o c o l response  decrement.  could  The present study was  whether the c a r d i a c c h r o n o t r o p i c response habituate  and,  features.  Although d a b b l i n g ducks were  this  to  have  was dived  in  the  to d i r e c t l y resulted  in  designed to examine submersion  would  i f so, to c h a r a c t e r i s e some of the p h y s i o l o g i c a l  investigation, a parallel  to e s t a b l i s h diving  in  studies.  has not been  diving  1974;  the  main  set of experiments  subjects was  i f h a b i t u a t i o n would occur, i n a species  ducks (Aythya americana)  of  conducted of  "true"  known to possess r a p i d onset of  b r a d y c a r d i a : i n other words, ducks that respond to immersion se with an immediate change i n heart r a t e .  per  69  METHODS F i f t e e n a d u l t domestic Pekin ducks (Anas female, 7 male) were used in the f i r s t and  three  adult  Redheads (Aythya  were used i n the l a s t  platyrhynchos;  8  four s e t s of experiments,  americana; 2 female, 1 male)  set of experiments.  F i v e s e t s of experiments were c a r r i e d  out:  a) H a b i t u a t i o n of the Cardiac Response to D i v i n g ; b) E f f e c t of H a b i t u a t i o n on Blood V a r i a b l e s ; c) i . E f f e c t ii.  of Breathing  Effect  Pure Oxygen on D i v i n g  of Hypoxia on the Habituated  Bradycardia;  Response;  d) E f f e c t of H a b i t u a t i o n on the Oxygen Breathing  Test  e) H a b i t u a t i o n of the Cardiac Response in "True" D i v i n g Ducks  General The of  Protocol animal's  breathing  positioned prevent the The  body was  movements,  secured to  a  padded p l a t f o r m .  c r a n i a l oedema, the p l a t f o r m was  beak was  head kept  The  head  was  In order  to  i n c l i n e d at 25° to keep  p a r t i a l l y open by means of a 2 cm  out of the mouth.  trainer.(Figure  section  of  i n p o s i t i o n to allow water to d r a i n The  w i t h i n a l i g h t - t i g h t chamber which the  restriction  l e v e l with the r e s t of the body (Figure 8 ) .  s o f t l a r g e bore tubing taped completely  without  i n a padded brace a l l o w i n g no movement.  animal's  seeing  firmly,  9)  e n t i r e apparatus was prevented The  the  situated  animal  d i v i n g bucket was  from  operated  70  remotely by a l e v e r . head the  R a i s i n g the  bucket  immersed  the  duck's  in water to a l e v e l above i t s eyes; at the end of the dive bucket was  drain and  lowered.  A l a r g e diameter hose was a t t a c h e d to a  in the bottom of the bucket so that filled  without  disturbing  the  it  could  animal.  be  emptied  Sham d i v e s were  performed by r a i s i n g and lowering an empty bucket. Heart rate was determined from the e l e c t r o c a r d i o g r a m which  was  obtained  from  three  subcutaneously in the abdominal  wire e l e c t r o d e s : one  a  right  third  "grounding"  side of  the  e l e c t r o d e a t t a c h e d to the web  foot by means of an a l l i g a t o r c l i p .  amalgamated  inserted  w a l l adjacent to the l e f t  second i n s e r t e d subcutaneously i n the r i g h t and  (ECG)  These ECG  leg; a chest; of the  leads  were  i n t o a c a b l e which l e d out of the chamber through a  small opening i n the w a l l .  A) H a b i t u a t i o n of the C a r d i a c Response to D i v i n g The d a i l y t r a i n i n g second  head  immersions.  immersions  schedule c o n s i s t e d of 15 t r i a l s with  Each s e s s i o n was  5  to 6 minute  preceded by  a  of  40-  i n t e r v a l s between 20  to  30  minute  q u i e t p e r i o d to allow the animal to s e t t l e down. In for  the  case of two animals (D-1, D-2), the immersion  the f i r s t 9 days of t r a i n i n g was  usual 40 second t r i a l s commenced.  60 seconds, a f t e r which the  In the case of one animal ( J -  22) an i n i t i a l 7-day t r a i n i n g schedule of 40 second followed by a f u r t h e r 3 days of 60-second ECG was  time  trials  was  trials.  recorded on a Physiograph Six c h a r t  recorder  (E & M  71  Figure  8. Diagram i l l u s t r a t i n g during training.  the  body  position  of  the  duck  73  F i g u r e 9. for  Diagram i l l u s t r a t i n g training.  the remote d i v i n g  system used  74  75  Instrument  Co.,  Houston,  (GX-280D-SS, Akai) through Rebersburg, the  Pa.).  Texas) and a 4-channel tape an,FM c o n v e r t e r  (A.R.  recorder  Vetter  Co.,  Pre-dive heart rate (HR) was determined  over  10 second p e r i o d j u s t before immersion and dive HR from  final  10 seconds j u s t before the d i v i n g bucket  was  the  lowered.  B) E f f e c t of H a b i t u a t i o n on Blood V a r i a b l e s To  o b t a i n blood samples and monitor  the r i g h t or l e f t local  blood pressure,  b r a c h i o c e p h a l i c a r t e r y was cannulated.  anaesthesia  (2%  and  the  Under  X y l o c a i n e , A s t r a Pharmaceuticals)  s e c t i o n of the a r t e r y was exposed i n the region adjacent femur  either  a 2cm  to  the  f l a r e d end of a 50cm l e n g t h of PE 90 tubing was  i n s e r t e d 7.5 to 8.0cm i n t o the a r t e r y so that the t i p l a y w i t h i n the ascending skin  aorta.  A f t e r the cannula  was sutured c l o s e d .  was  well  secured,  the  Animals were allowed at l e a s t one day  to recover from the e f f e c t s of surgery. During through  experiments the t r a i l i n g  l e n g t h of cannula  the chamber w a l l and was connected  way stopcock. Pasadena, remaining  A BT-70 pressure transducer  California)  was  connected  extended  to a port on a t h r e e (Bio-Tec  Instruments,  to a second port and the  port was used f o r withdrawing blood samples.  Pre-dive  blood samples were taken a few seconds before  immersion and end-  dive blood samples were taken  7 seconds  dive. blood  Each gas  sample analyser  Massachusetts).  was then  w i t h i n the l a s t immediately  (Instrumention  Blood  gas  of  the  analysed on an IL-813  Laboratories,  Lexington,  analyses were done on a maximum of  76  s i x 0.5ml samples from each animal closely  monitored  series  of  frequent  flushed  slightly with  animals throughout  reduced.  i n t o a small loop that  In an  subjected  clear  (100  more  could  cannula  i.u./ml), sealed off  be  tucked  bag was  under  the  cannula.  t i g h t l y attached to the top of  The duck's head was  animal's  neck.  inserted  into  In  this  by mixing  way,  at  10).  flow  around  r a t e s of 10 l i t r e s / m i n ,  to breathe anything but the  A i r of v a r i o u s oxygen content was  flows of pure oxygen and n i t r o g e n gas using  and  the percentage  MGA  gas a n a l y s e r (T.C.  possibility served  v a l v e system  bag  Gases of v a r i o u s oxygen contents entered at  impossible f o r the animal (Figure  the  then l o o s e l y secured around  water l e v e l and e x i t e d v i a the loose f i t t i n g c o l l a r  have  to  Between s e s s i o n s , the  through a hole i n the base which was  gases  earlier  the t r a i n i n g s e s s i o n and  then taped down to p r o t e c t the  polythene  the d i v i n g bucket.  neck.  was  E f f e c t of Breathing Pure Oxygen on D i v i n g Bradycardia A  the  hematocrit  period. were  heparinised saline  b i r d ' s wing which was  i^.  training  two  blood gas sampling  and c o i l e d  C)  the  experiments  hematocrit was was  over  each day and  composition  was  checked  i t was  inflowing obtained flowmeters  with a Centronic  Centronic L t d . , England).  the  To reduce  200 the  that the noise a s s o c i a t e d with s w i t c h i n g gases might as was  a  conditioned devised  v a r i a t i o n s when gas mixtures  which  stimulus (CS) to the animal, a reduced  noise  due  to  flow  were a l t e r e d .  A p r e l i m i n a r y s e r i e s of experiments  was  done to examine the  77  Figure  10. Diagram i l l u s t r a t i n g the system f o r c o n t r o l l i n g the a i r breathed by the duck before d i v e s .  78  79  effect HR  of  and  Ducks  end-dive  were  oxygen the  breathing  once  after  day.  water  before  which  began  began  with  i i.  trials  oxygen  low  To  test  and  three placed  the  a  before  high  room  recorded  To  each  dive.  breathing  pure  the  following  day  assess  the  effect  the  after  the  test  emptying 6  on  for  Pre-dive  On  done  dive.  dives  after  air.  quietly the  bradycardia.  once  were  on  ducks  the  the were  first  of  sequence  bucket tested:  day,  effect  the  Habi tuated  Response  of  breathing  hypoxic  response,  five  procedure  substantial  level  the  a i r was  a i r of  10,  15,  20  ducks,  for  of  the  of 3  other  of 3  a  of  prior  to  sufficient  habituation  then or  gas  altered  100%  so  oxygen  the  testing, number  was  of  achieved.  that  for  on  at  the  ducks  least  3  response  to  habituation  in  immersion.  for oxygen  within  was  reversed.  i t for  Habituation  animals.  day:  entailed  training  breathing  of  was  diving  dive)  breathing  Hypoxia  content  Effect  each  oxygen  diving the  second  on  dives.  of  until  minutes  D)  with  examine  underwent  oxygen  "sham" d i v e s  raising  air  habituated  were  dives  This  Ef f e c t  To  The  of  procedure,  each  (40  twice  sequence  for  HR  dived  and  diving  pure  on  the  Oxygen  chemosensitivity, was  measured  Breathing  the  before  these  Test  ventilatory and  To  accomplish  a  temperature-controlled  after  tests,  the  body  animals  were  plethysmograph.  80  Figure  11. Schematic diagram i l l u s t r a t i n g the body Detachable plethysmograph f o r measur ing v e n t i l a t i o n . face mask i n p o s i t i o n f o r the oxygen breathing t e s t .  82  Pure oxygen, a i r or administered  at  attached  the  to  Breathing  was  hypoxic  drop  recorded and  front  tidal  times,  during or  and  a  port  in  transducer  volume  (Vt).  the  each trials  fed  to dive  animal's  a  Gould  animal,  the  of the  before  and  to a  level  by  firmly  cold  water.  the  Pekin  ducks.  face  (f)  100% 0  mask  2  was  response  were  platform  in  a  (experimental  unrestrained.  The  but g e n t l y f o r c i n g  their  The  training  protocol  w i t h 5 m i n u t e s b e t w e e n e a c h t r i a l and  an a v e r a g e o f 30 t r i a l s a d a y . for  frequency  f o r the Pekin ducks  accomplished  20-second t r i a l s  calculated,  R e s p o n s e i n " T r u e " D i v i n g Dueks  t h a t used  of  to  response.  trials  beaker  Northridge)  a f t e r a s u f f i c i e n t p e r i o d of  However, t h e i r h e a d s were l e f t  heads i n t o a  was  Integrator  breathing  protocol A). were  The  h e a d immersed i n t o a b e a k e r o f w a t e r  to establish a habituated  to  (Fleisch,  30 s e c o n d s o f b r e a t h i n g  the  Two t e s t s  animal  similar  11).  plethysmograph.  ( V a l i d y n e DP103,  into  r e d h e a d d u c k s were s e c u r e d  involved  (Figure  pneumotachograph the  was  2  a f a c e mask  M i n u t e v e n t i l a t i o n was  E) H a b i t u a t i o n o f t h e C a r d i a c  manner  N )  the pneumotachograph d u r i n g b r e a t h i n g  was  from t h i s p o s i t i o n .  The  plethysmograph  with  signal  In order  2  on  balance  2  f r o m t h e change i n V t a n d r e s p i r a t o r y  10% 0 .  diving  the  a i r b r e a t h i n g and a f t e r  removed  run  to a  with a pressure  provide two  of  monitored  across  the a i r f l o w  (10% 0 ;  flow r a t e s of 6 l i t r e s / m i n through  Switzerland) attached pressure  air  ECG was  Pre-dive  recorded  as  described  HR and e n d - d i v e HR were a l s o  83  determined.  84  RESULTS  A) Habituat ion of the Cardiac  Response to D i v i n g  Naive or non-habituated ducks e x h i b i t e d rate  to  dive.  about 70% of pre-dive  fall  in  with  an  increase  The rate and amount of h a b i t u a t i o n  Recovery  over s u c c e s s i v e  blocks  (Figure  12  of t r i a l s  and 14).  p o t e n t i a t i o n of h a b i t u a t i o n of  the  cardiac  recovery  was never complete and the e f f e c t of t h i s  tooth" curve of the d i m i n i s h i n g trials  was to produce a cardiac  rising  response with  increasing  With repeated t r a i n i n g s e s s i o n s a  r e s u l t e d i n the v i r t u a l  response.  "saw-  elimination  In f i v e animals h a b i t u a t i o n  was so  pronounced that the HR response to immersion was transformed a sustained One sufficient good  submersion t a c h y c a r d i a  animal  (Figure  (D-4)  t r a i n i n g to  retention 14).  and  allowed abolish  rapid  to  rest  for  submersion  habituation  Recovery to naive  48  hours a f t e r  bradycardia  i n subsequent  showed sessions  l e v e l s of d i v i n g bradycardia of  was rest  t r a i n i n g s i n c e t e s t d i v e s were i n d i s t i n g u i s h a b l e from p r e -  habituation  dives.  Although training  the  pre-dive  progressed,  animals before mean  to  (Figure 13).  complete, however, i n two ducks t e s t e d a f t e r one month from  in  varied  among animals and a l l showed some degree of spontaneous overnight.  heart  l e v e l s by the end of a 40 second  T h i s response g r a d u a l l y diminished  the number of t r i a l s .  a  pre-dive  the  HR  in  difference  and a f t e r h a b i t u a t i o n rate  for  most  the  first  in  ducks  mean values  was not trial  decreased  as  from a l l  significant.  The  of a l l animals was  85  Figure  12. E f f e c t of h a b i t u a t i o n t r a i n i n g on d i v i n g bradycardia. End-dive heart rate i s expressed as a percentage of the Pre-dive heart r a t e . ( O ) r e p r e s e n t s end-dive v a l u e s ; (®) r e p r e s e n t s end-dive values obtained for the f i r s t t r i a l of each t r a i n i n g s e s s i o n ; (•) represents end-dive values obtained when the animal breathed 15% oxygen before the d i v e .  a o  o CO  o  o  o CM  20  60  40 Trials  80  100  gure 13. E f f e c t of t r a i n i n g on d i v i n g b r a d y c a r d i a f o r 13 ducks. The s o l i d l i n e s represent i n t e r p o l a t i o n between the means of the l a s t 3 t r i a l s of each t r a i n i n g s e s s i o n . (The l i n e s begin with the mean of the f i r s t 3 t r i a l s of the f i r s t t r a i n i n g s e s s i o n . )  Percentage of Pre-Dive Heart  o  Rate  89  Figure  14. E f f e c t of i n t e r r u p t i o n of t r a i n i n g on h a b i t u a t i o n of b r a d y c a r d i a . C i r c l e d points the f i r s t t r i a l of each t r a i n i n g s e s s i o n .  the represent  06  91  176.9±40 beats/min and was  142.7±36  beats/min  from the p r e - d i v e HR significant in  HR  the  showed an  commenced  The  Of  As  trial  seconds. training  soon as the  The  slowly two  immersion  training  periods  schedule,  cardiac  demonstrated  d e f i n e d as a  with  fall  training,  2  immersion p e r i o d (D-1,  D-2)  immersion p e r i o d was  schedule  once again  that  for s e v e r a l days, with  was  altered  the  to  40  Conversely, to  response was  with  60-  cardiac  shortened  (Figure 15).  (J-22) the h a b i t u a t e d  response  i f the  l i t t l e a t t e n u a t i o n of the  However, as the t r a i n i n g progressed the  HR  animals  seconds, h a b i t u a t i o n proceeded r a p i d l y when the normal  which  arbitrarily  pre-dive  very  second immersions showed very response.  training  1 remained unchanged.  proceeded  their  after  the 9 ducks used i n t h i s p a r t of  6 showed a decreased  than 40  value  l a t t e r value was c a l c u l a t e d  first  ( t h i s was  10%).  i n c r e a s e and  longer  the  habituation  Habituation was  (N=9).  of  of no more than  study,  the mean p r e - d i v e  80-second abolished.  extended  showed some r e d u c t i o n  trials, (Figure  16).  B) E f f e c t  of H a b i t u a t i o n on  To assess  some Blood  the e f f e c t s of h a b i t u a t i o n , comparisons were made  between measurements obtained training than all  or  s e s s i o n and equal  the  to 20%  first  from the  first  dive of  d i v e to e x h i b i t a f a l l  of the p r e - d i v e HR.  the animals achieved  days.  Variables  From t h i s  the i n HR  first less  criterion,  t h i s l e v e l of h a b i t u a t i o n w i t h i n 4 to 5  92  Figure  15. E f f e c t of p r o l o n g a t i o n of dive time on habituation. The s o l i d l i n e represents i n t e r p o l a t i o n between the means of the l a s t 3 t r i a l s of each t r a i n i n g session. The v e r t i c a l broken l i n e i n d i c a t e s the change from 60 to 40 second d i v e s .  ure  16. E f f e c t of r e d u c t i o n of d i v e time on h a b i t u a t i o n The v e r t i c a l broken l i n e i n d i c a t e s the change from 40 60 second d i v e s .  S6  96  Despite the c o n s i d e r a b l e b r a d y c a r d i a which developed 40  seconds  submersion  in  the  unchanged from p r e - d i v e l e v e l s . bradycardia  diminished,  naive  animal,  As t r a i n i n g  MAP  remained  progressed  and the  MAP continued t o match pre-dive  (Figure  17; Table 3 ) . The  before  habituation  mean  was  end-dive  158±7mm  Hg  MAP  of  and a f t e r  after  levels  five  ducks  h a b i t u a t i o n was  I57±12mm Hg (Figure 17). The d i r e c t i o n before was  and a f t e r the  mean end-dive habituation  of  the  different Pa0  was  of  2  change  (Table  Hg  Hg.  and  The  and end-dive  2  between  submersion  before  training  Pa0  2  habituation  some  0.04  after  significantly  PaC0  rose  2  from  A f t e r h a b i t u a t i o n , however, the  27.8±5.0mm Hg to 35.3±5.8mm Hg. only  value  (P<0.05).  amount of r i s e i n the d i v e was s l i g h t l y reduced:  to  2  s e s s i o n had no  was not  habituation,  29.2±2.4mm Hg to 38.5±2.2mm Hg.  amounted  same  conditions  this  between naive and h a b i t u a t e d animals  During  the  3 ) . Naive ducks e x h i b i t e d a  55.7±3.0mm  56.5±2.7mm  on p r e - d i v e Pa0  different  was  h a b i t u a t i o n ; however, only i n the case of Pa0  magnitude  insignificantly  effect  of change i n blood gas l e v e l s  The i n c r e a s e pH  units  PaC0  in  2  rose from  end-dive  pHa  both before and a f t e r  (Table 3 ) . The d i f f e r e n c e s i n the  change  of  both  these v a r i a b l e s were not s i g n i f i c a n t at P=0.05 l e v e l s . The  hematocrits  not change over the However,  in  two  of  the.four ducks used i n t h i s study d i d  training  ducks  sampled  frequently during t r a i n i n g , days.  The  blood  gas  sessions  f o r blood  hematocrit  values  and  fell  remained  at 41%.  gas a n a l y s i s more by  1-3%  f o r these two animals  over  six  were not  gure  17. E f f e c t of h a b i t u a t i o n t r a i n i n g on a r t e r i a l blood pressure. A: Recordings from the f i r s t dive of the f i r s t day of t r a i n i n g . B: Recordings from a d i v e on the f i f t h day of t r a i n i n g . Top t r a c e of each r e c o r d i n g i s blood pressure, bottom t r a c e i s ECG.  I  A.  FIRST  DIVE  ,  I  I  I  I  I  I  i i i  ^ f i m m t r n t t f t f i r t-r  H  ft  n  t t  ttttt i i  DIVE  SURFACE  • I l  • B.  HABITUATED  DIVE  I I  I  i  i  I I  i  i 10 seconds  99  Table I I I . E f f e c t of H a b i t u a t i o n on the C a r d i o v a s c u l a r System. Values are means ±S.E.. Naive values were obtained from the f i r s t dive of the f i r s t . t r a i n i n g s e s s i o n . Habituated values were obtained from the f i r s t dive to e x h i b i t a f a l l i n HR l e s s than or equal to 2 0 % of p r e d i v e HR. Pa0 , PaC0 and MAP i n mm Hg. (n=N) 2  2  Table  III.  E f f e c t of System  H a b i t u a t i o n T r a i n i n g on  the  Cardiovascular  n  Pre-Dive  Dive  Pre-Dive  Dive  PaO*  4  95.13+1.42  55.70+2.99  93.83+7.30  56.48+2.74  PaCOa  4  29.15+2.39  38.50+2.19  27.80+4.96  35.33+5.78  pHa  4  7.44+0.02  7.40+0.01  7.45+0.04  7.41+0.03  MAP  5  157.5+7.4  157.2+12.4  101  included f o r s t a t i s t i c a l the  other  analysis  trends  as  ducks  animals  i s g r a p h i c a l l y represented  but  they  showed  In the p r e l i m i n a r y study before  substantially  submersion,  i n Figure 18.  on the  of  fall  day.  In  the  breathing  i n heart  l e s s than a f t e r the same animals  the  Bradycardia  s i x animals  This was true whether the oxygen dive was the dive  difference  had breathed a i r . first  m a j o r i t y of cases,  the end-dive HR's of a i r - d i v e s  was 70%  fall  f o r the  training,  the  animals  fall  of  studied the  fall  low  (P<0.05)  from  (Figure  20).  After  response was 22%  were then given a i r  2  I f the oxygen content  (10% 0 ) , pre-dive b r e a t h i n g 2  fall  no  oxygen (15% 0 ) p r i o r to f o r c e d submergence, the mean  i n HR was 69%.  submersion.  There was  Response.  habituated  (Figure 20). When these h a b i t u a t e d animals with  the heart rate  i n HR during submersion before h a b i t u a t i o n  five  mean  second  (Figure 19).  i i • ) Ef f e c t of Hypox i a on the Habituated mean  or  i n the end-dive HR's between oxygen and  sham-dives, but a l l were s i g n i f i c a n t l y d i f f e r e n t  The  pure  rate was always  during oxygen dives exceeded the pre-dive v a l u e s . significant  same  and the r e s u l t s from one of these  C) j^. ) Ef f ect of Breathing Pure Oxygen on D i v i n g  oxygen  the  After  was  lowered  increased and HR f e l l  breathing  i n the h a b i t u a t e d animals  pure  further  by 70% during  oxygen (100% 0 ) , the mean 2  only amounted to 24%, s i m i l a r  to  1 02  Figure  18. E f f e c t of h a b i t u a t i o n on end-dive blood gas levels. Graph (A) represents the changes of end-dive PaC0 and graph (B) represent the changes of end-dive Pa0 . The s o l i d l i n e in each graph represents heart rate. The changes in blood gas l e v e l s are p l o t t e d as the d i f f e r e n c e s ( i n mm Hg) from the value obtained f o r the f i r s t d i v e on the f i r s t day. 2  2  P e r c e n t a g e of P r e - D i v e H e a r t 0  A Pa  20  0 (mm Hg) 2  40  60  BO  Rate 100  1 04  Figure  19. E f f e c t of breathing pure oxygen before 40 second dives. '1st','2nd' and '3rd' r e f e r to the sequence of t e s t s as d e s c r i b e d in the t e x t . ( E r r o r bars ±S.E.; n=6)  1st  2nd  AIR DIVES  1st  2nd  OXYGEN DIVES  3rd  SHAM DIVES  1 06  F i g u r e 20. E f f e c t of v a r i o u s i n s p i r e d oxygen contents on the h a b i t u a t e d response to submersion. The hatched bar r e p r e s e n t s values f o r u n t r a i n e d animals; open bars represent values for h a b i t u a t e d animals. Values beneath each bar represent i n s p i r e d oxygen content. (n = 6)  100  CD  o  80  £  60  o  LO  20  21%  21% Pre-dive  15% inspired  10% O 2 content  100%  1 08  the The next  values  for  habituated  e f f e c t of the hypoxic trial  with  animals  t e s t was  normal  not  oxygen  evoked the usual h a b i t u a t e d  on normal a i r (Figure permanent  content  response (Figure  10%  a decrease  comparison achieved elicited and the  Test.  the v e n t i l a t o r y response to  to  was air  breathing.  and  very  12).  In naive animals, oxygen  the  (breathing room a i r )  D) Ef f e c t of Habi tuat ion on the Oxygen Breathing  and  as  20).  pure  oxygen  i n c r e a s e , r e s p e c t i v e l y , in  After  training  each  animal  a l e v e l of h a b i t u a t i o n where only a 10% drop in HR by submersion.  When these animals  were given  was  the  low  high oxygen t e s t s , the changes in minute v e n t i l a t i o n were of same  magnitude and  d i r e c t i o n as before.  t e s t s were compared using a two-way a n a l y s i s P<0.05.  The  significantly  ventilatory different to  responses  from  one  The of  to  data from the variance  the  another,  tests  but  obtained  prior  h a b i t u a t i o n t r a i n i n g were not  different  from those a f t e r h a b i t u a t i o n (Table 4).  the  with were values  significantly  E) H a b i t u a t i o n of the Cardiac Response in D i v i n g Ducks. The  redhead duck responds to submersion with  and  r a p i d drop in HR.  Although  is  considerably  130  beats/min) they achieved a 70%  lower than  the pre-dive HR  an  immediate  in these  animals  in Pekin ducks (ranging from 100 fall  in HR  within  the  to  first  109  Table IV. E f f e c t of H a b i t u a t i o n on the Oxygen Breathing Test. ' A f t e r H a b i t u a t i o n ' values were obtained a f t e r 5 t r a i n i n g s e s s i o n s f o r each animal  Table IV. Effect of Habituation on the Oxygen Breathing  Test.  AIR  100'/. Oa  10% 0  Before Habituation  0.32+0.02  0.22+0.05  0.57+0.03  After Habituation  0.33+0.04  0.23+0.04  0.59+0.06  N=3sn=6. Values are Ve (1 <BTPS>/min/kg) + S . E .  2  111  10 seconds of immersion. repetitive  As with the Pekin ducks, the e f f e c t of  submersion was the gradual r e d u c t i o n  bradycardia.  A l l animals  showed  some  i n the degree of  spontaneous  recovery  overnight but i t was i n s u f f i c i e n t to prevent them from a c h i e v i n g almost complete h a b i t u a t i o n of the c a r d i a c response about  100 t r i a l s ,  over 3 days of t r a i n i n g  after  (Figure 21).  only  1  12  Figure 21. E f f e c t of t r a i n i n g on d i v i n g Redhead ducks (Aythya americana).  b r a d y c a r d i a in 3  0  0  I  I  I  20  40  BO  3  4i  S  f  4  1  10  1  20  •  90 Trials  40  1  BO  BO  1  80  Trials  1  100  1  120  140  100  1 1 4  DISCUSSION Repeated results  in  exposure  to  habituation  chronotropic  response  the  of  stimuli  the  normal  (bradycardia).  bradycardia  The  submersion,  r e f l e c t i o n of activation  that  the of  naive  r a t e response appears  that  vagal  having  abolished  deceleratory  system,  the  form  cardiac  disinhibition)  effects.  reverse  chemoreceptor  i s the r e s u l t of i n h i b i t i o n of the vagal  acceleratory  abolished. to  bradycardia of  sessions,  i s , to develop t a c h y c a r d i a , may be a  fact  the  cardiac  between t r a i n i n g  i n many animals could be completely  i n s t a n c e s where the heart  during  or  by submersion  T h i s i s so pronounced that  in s p i t e of some spontaneous recovery the  evoked  Before  or  reduced  system  (a  direct  sympathetic  the  acceleratory  habituation  e f f e c t s on the heart due to a g i t a t i o n or s t r u g g l i n g were l a r g e l y overridden  by  vagal  deceleratory  control,  but  h a b i t u a t i o n , the vagal d r i v e i s e i t h e r diminished In a previous not  day  regime of t h i r t y  f o r up to 5 months (Rey  at the end of such an e x t e n s i v e  it  and  their untrained  training period,  was shown that stimulus  habituation.  If  controls.  12  abolished  and or  there  20),  habituation  considerably  of  reduced.  dives  a  crucial  no  study,  factor  was only s l i g h t l y diving  was  between t r a i n e d  dive times exceeded 40 seconds (Figure  16), or i f the i n s p i r e d oxygen content (Figure  60-second  From the present  intensity is  was  I t i s s u r p r i s i n g that  1 9 7 1 ) .  s i g n i f i c a n t d i f f e r e n c e i n the d i v i n g b r a d y c a r d i a animals  or a b o l i s h e d .  study, h a b i t u a t i o n of d i v i n g bradycardia  observed d e s p i t e a t r a i n i n g  each  following  for  15 and reduced  bradycardia  was  I t seems l i k e l y that the  11 5  d i v e time i n the study done by Rey (1971) was too long and arterial  P0  The to  was consequently  2  When  too low f o r h a b i t u a t i o n to occur.  argument c o u l d be r a i s e d that t r a i n i n g s e s s i o n s  f a m i l i a r i s e the animals viewed  that  to an otherwise  i n t h i s l i g h t , the gradual  "fearful"  served  situation.  r e d u c t i o n i n the d i v i n g  b r a d y c a r d i a c o u l d be because the animals  are  As  sounds, i t ignores the  accomodating  following  as  this interpretation  less  frightened.  observations:  a) Naive ducks show l i t t l e  or no b r a d y c a r d i a  during  submersion  a f t e r b r e a t h i n g pure oxygen; b) The  same  animals  not h a b i t u a t e  that h a b i t u a t e with 40 second t r i a l s  will  i f the time i s extended to 60 seconds, even i f the  number of t r a i n i n g s e s s i o n s i s i n c r e a s e d ; c) A f u l l y h a b i t u a t e d animal rate  as  a  reduction  naive  animal  develops after  i n oxygen content  as deep a  breathing  of  either  the  equal  constrictor  reduction and  in  parallel  habituation  to maintain  MAP.  component  of  submersion  response  training.  T h i s was demonstrated f o r one of  both  the  unaltered  T h i s suggests of  the  also  habituated  that  vascular  the vasomotor  attenuates  animal  which  with showed  b r a d y c a r d i a and the i n c r e a s e i n HLVR observations).  e f f i c a c y of e f f e r e n t c o n t r o l was confirmed  where the  in  the b a r o s t a t i c  In any event  (Figure 22; Gabbott and Smith unpublished The  remains  bradycardia.  r e f l e x can continue  habituation  heart  a i r with only a 5%  response must occur, or a l t e r n a t i v e l y  the  in  before the d i v e .  Over the p e r i o d of h a b i t u a t i o n , MAP spite  fall  animals  breathed  slightly  i n the t e s t s hypoxic  gas  116  Figure 22. E f f e c t of t r a i n i n g on hind limb v a s c u l a r flow. Open c i r c l e s r e p r e s e n t values for end-dive heart r a t e . Closed c i r c l e s represent values f o r blood flow measured with a Doppler flow probe on the i s c h i a t i c a r t e r y .  0  P e r c e n t a g e of P r e - D i v e H e a r t R a t e and P r e - D i v e H i n d Limb V a s c u l a r R e s i s t a n c e 20 40 60 80  100  1 18  mixtures  before  prior  submergence  to  submersion. was  Though  the  negligible,  effect  levels  on  of  breathing  bradycardia  during  dives  were  approximately  the same as i n naive c o n t r o l  dives.  Only  when  the  oxygen  exceeded  21%  did  inspired  habituation  effect  of hypoxia t r i a l s was  trial  with  normal  air  r e t u r n of the habituated output  of  content  the b r a d y c a r d i a  not permanent s i n c e the  (21%  oxygen  response.  remains j u s t as potent  matched occur.  or The  very  next  content) r e s u l t e d in the  This  suggests  in the h a b i t u a t e d  that  vagal  c o n d i t i o n as in  the naive c o n t r o l . Tests  of  accomplish.  receptor  sensitivity  are  However, an attempt was  Test"  before  minute v e n t i l a t i o n oxygen  appear  and  after  be  the  on  breathing  habituation.  in response to  to  either  same  to  accept  the  notion  after  that  The  pure five  s e s s i o n s which r e s u l t e d i n s u c c e s s f u l c a r d i a c order  difficult  of  the  sensitivity effector carotid  carotid  involves  system. body  ventilatory 1975).  and  Nevertheless,  chemoreceptors change  in  afferent  in  an  to  are  during  the to  is  to the  effects  i n d i r e c t measure of entirely  prime the  In  examine  different  i t i s w e l l e s t a b l i s h e d that  response  As with b r a d y c a r d i a body  changes  i s an  10%  training  sensitivity  for chemosensitivity test  or  habituation.  receptor  the  changes in  days of  the p r o p r i e t y of such a t e s t Obviously  of  oxygen  v i r t u a l l y u n a l t e r e d by h a b i t u a t i o n , i t i s necessary  submersion.  to  made to d e t e c t a change in  c h e m o s e n s i t i v i t y by comparing the e f f e c t "Oxygen  more  mediators  Oxygen Test  submersion,  the  the of  (Dejours  removal  of  a c t i v i t y a l s o a b o l i s h e s the v e n t i l a t o r y  1 19  response to hypoxia 1970;  (Bouverot et a_l.  Bouverot and L e i t n e r During  a  dive  conserve oxygen;  1965;  1972; L i l l o and Jones  cardiovascular  the  elimination  of  these  ( i . e . habituation)  depletion.  However, the f a l l  habituated  c o n d i t i o n as i n the c o n t r o l .  within  adjustments loss.  40 are  This  not  is  cardiovascular atropine  seconds  of  Pa0 ,  changes  to  40  cardiovascular time  is  2  responses  the  during oxygen  same  i n the  the  blood  flow  experiments  eliminated  where  the  pharmacologically  ( B u t l e r and  Jones  Thus the r a t e of oxygen d e p l e t i o n  seconds  to  developed to prevent oxygen  in  are  function  T h i s suggests t h a t , at  submersion,  corroborated  Purves  to provoke greater  and a-adrenoreceptor b l o c k e r s  30  this  in  sufficiently  Bryan and Jones 1980). first  ought  and  1982).  adjustments  submersion  least  Jones  by  1971; i n the  of submersion i s s i m i l a r whether the  responses are present  or not.  I t i s only  p e r i o d when the d i v i n g responses are f u l l y  after  developed  that the l o s s of oxygen i s r e t a r d e d . Since, eliciting be  i n these experiments, the c a r d i o v a s c u l a r  maintained,  chemosensitivty habituated within diverse  because  nor  efferent  stimulus  intensity  for  responses to submersion appears to decrement  in  neither  receptor  potency could be demonstrated i n  animals, i t suggests that the locus of h a b i t u a t i o n i s  the CNS. studies  demonstrated Spencer  and  the  a  This of CNS  1966; Kandel  Habituation  i s i n agreement with evidence habituation mechanism  that of  support  habituation  from  and  other  have even  (Thompson  and  1976).  has been c o n s i d e r e d  one of the main mechanisms  1 20  by which an animal copes with the onslaught of stimuli  to  1966).  which  i t i s exposed  trivial  in i t s natural habitat  (Glaser  Each stimulus may evoke complex p a t t e r n s of somatic  visceral  responses,  the magnitude  p h y s i o l o g i c a l parameters behavioural elicit  diverse  context  of  which  of the sensory input  of  the s t i m u l u s .  depend as  and  on the  well  as the  Often these s i g n a l s may  responses of increased magnitude d i s p r o p o r t i o n a t e t o the  situation.  Habituation,  sensitivity  to  however,  biologically  suppresion  of  more  (including  reflexes).  mechanisms  are  serve  significant  meaningless When  readily  may  and  animals  evoked  to maintain CNS stimuli  inappropriate dive,  to r e s t r i c t  can  be  suppressed,  whether  responses  asphyxic  defense  the l o s s of oxygen.  However, t h i s study has revealed that the c a r d i a c submersion  through  they  responses  to  are provoked i n  d i v i n g ducks by " n a r i a l type" r e c e p t o r s or i n d a b b l i n g ducks  by  chemoreceptors. Data c o l l e c t e d by telemetry from animals d i v i n g has  shown  provoked. brief  that This  the  cardiovascular  variability  excursions  under  water  reasonable to suggest that unnecessary  particularly  (Jones et a_l.  oxygen  are not always  conserving  common f o r  1973).  adjustments  C l e a r l y the means by which  are determined  are many  forced  demonstrated.  submersion  that  has  are  within  behavioural  and v a r i e d , however, t h i s  study has suggested an option i n the c o n t r o l of c a r d i a c during  I t seems  i n animals d i v i n g f o r p e r i o d s which are w e l l  their aerobic capacity. responses  appears  responses  voluntarily  hitherto  function  never  been  121  SECTION 3.  Demarcation  of the Neural S u b s t r a t e of the D i v i n g Response.  Introduct ion The  complex  temporal  and  spatial  organisation  c a r d i o v a s c u l a r response p a t t e r n to submersion and demonstrates Evidence  the involvement of higher l e v e l s  indicates  that  induced by submersion, (Kobinger  and  i s well  Oda  during  Jones  et  al.  mediated 1970;  suggests  that  also  Suprabulbar been  regions  implicated  et a l .  The  of  (areas  in  the  et  bradycardia following  a_l. in  interaction i s  1966; Gebber and Snyder Manning  1973;  integration  cardiovascular  involvement  of  responses  of  the  chemoreceptor-driven  1970).  They  response  transection  to  of  hypothalamus (Korner et a l . 1971).  some  Coote  r o s t r a l to the pons) have  r e p o r t e d by Korner and h i s co-workers Uther  Though the  unclear,  similar  (Hilton  changes  arterial  in  the cat  1964,1965; Thomas and C a l a r e s u 1973).  specific  modulation  a  1971; Takeuchi and  chemoreceptor-driven (Bacelli  mammals  w i t h i n the hypothalamus  H i l t o n and Spyer  1978).  in  is  CNS.  attenuated  1982).  n e u r o p h y s i o l o g i c a l b a s i s of t h i s a t t e n u a t i o n evidence  the  chemoreceptor-driven be  the  integrated  of  b a r o r e c e p t o r r e f l e x e s may  1969;  of  Certainly,  hypothalamus bradycardia (Korner  demonstrated arterial  the  brain  anatomical  studies  first  e_t a l .  1969;  hypoxia  1969; Uther et  in rabbits,  was  the al.  have  the  was  that,  below  in  abolished  l e v e l of the 1970;  revealed  Korner direct  1 22  hypothalamic  projections  to  and  from  areas  brainstem that mediate c a r d i o v a s c u l a r f u n c t i o n 1976;  Evans  1976;  and Caverson  1984).  Swanson 1977;  of  1977;  Ricardo and Koh  Furthermore, hypothalamic  1980).  Ciriello  These  and  observations  hypothalamic  control  another  cardiovascular similarity reaction et a l .  1978;  neurones  appear  (Brickman et  the  cardiovascular  Ciriello  concept  function  of  (Hilton  1974).  point  of  responses  (Smith et_ a l .  to  Pfaff  Ciriello  C a l a r e s u and  support  view,  higher  responses to submersion  to  1981;  lend  of  1965,1975; Smith et a l . From  C a l a r e s u 1980,  caudal  (Conrad and  to be s e n s i t i v e to c a r d i o v a s c u l a r - r e l a t e d a f f e r e n t s al.  the  for  1974;  CNS  of  has been suggested by the  a particular  form of the defence  Smith and Woodruff  Smith and Tobey 1983).  control  1980;  Kanwisher  Often d e s i g n a t e d the  Type  II fear response or " f r e e z i n g " response, t h i s behaviour has only recently  become  Evans 1976; Instead  of  classically  the  focus of a t t e n t i o n  G a b r i e l s e n et a l . the  changes  1977;  evoked  named the " f l i g h t  (Folkow and N e i l  Smith  in  et  al.  1981a,b).  the Type I fear  response,  or f i g h t " response, the changes i n  Type II are almost d i a m e t r i c a l l y o p p o s i t e .  Whereas the  response  hypertension  features  1971;  tachycardia,  Type  and  h y p e r v e n t i l a t i o n , Type II i n v o l v e s b r a d y c a r d i a , hypotension often 1977;  greatly  reduced  Azevedo et a l .  ventilation  1980;  (Zanchetti  Smith et a l .  1981a  and  are  and  Bartorelli  and b ) .  for the c o n t e n t i o n that l a b o r a t o r y - i n d u c e d submersion  I  Support responses  o s t e n s i b l y the r e s u l t of a Type II fear response comes from  a study i n which  r a t s were s t a r t l e d when  either  restrained  or  1 23  unrestrained the  fear  (allowed  response  unrestrained In  to escape).  included  condition,  the  cat,  the  In the  restrained  bradycardia  startle  regions  have  whereas  r e s u l t e d in been  condition, in  the  tachycardia.  mapped  in  the  rostral  grey  matter  brainstem  ( i n c l u d i n g the hypothalamus, the c e n t r a l  and  mesencephalic tegmentum) from which a c t i v e c h o l i n e r g i c  the  vasodilatation stimulation regions  in  the  (Abrahams  are not merely  characteristic elicited,  muscles  can  et  1960,  al.  of  the  evoked  1964;  "vasodilator"  features  including  be  defence in  electrical  Uvnas 1960).  areas,  vasoconstriction  by  as  many  reaction skin  and  These of  are  the also  intestine,  p u p i l d i l a t a t i o n , p i l o - e r e c t i o n , r e s p i r a t o r y changes, a r c h i n g the  back  and  swishing movements of the t a i l .  does not  normally d i s p l a y the Type II response,  certain  areas  sympathetic resulting  of the a n t e r i o r c i n g u l a t e  inhibition in  together  hypotension.  s k e l e t a l muscle tone and In  another  study,  hypothalamus  of  hypertension  and  stimulation  of  vagal  i s reduction  1961).  regions  the  caudal  of  in  'defence areas' of  vasoconstriction resulting  other  responses  exopthalmus, p u p i l d i l a t a t i o n and  e r e c t i o n of ears  From  likely  these  studies,  defence r e a c t i o n s particular In  a  it  i s mediated  the hypothalamic study  seems via  of  (Lofving  r a b b i t c o r r e s p o n d i n g to  various  bradycardia,  of breathing  the c a t , provoke apnoea, b r a d y c a r d i a , in  cat  In a d d i t i o n , there  stimulation  the  Although the  gyrus cause widespread  with  depression  of  the  including  (Evans 1976).  that expression rostral  of  the  brainstem,  in  area.  attempting  to  resolve  the  question  of  the  1 24  v o l u n t a r y or  r e f l e x nature of the apnoeic response of  submersion,  Huxley  demonstrated  bradycardiac response p e r s i s t e d both  cerebral  hemispheres  (1963) demonstrated that even  after  section  diencephalon. Andersen's the  be  r a i s e d that  were s t i l l This  diving  Xylocaine of the  brain  below  the  at the  of b r a i n also  and  destruction  of  the  was  level  unfortunate  transection, removed,  persisted  was  diving  designed  nervous technique  was  was  level  and  argument  the  re-examine  in the  the  tissue  whereby  Schiller  to  the  examine  1979).  effect  of  t r a i n i n g on d i v i n g --bradycardia in decerebrate ducks.  the  local  transection  mesencephalon  inactivation  ( M a l p e l i and  the  c o n t r o l of  X y l o c a i n e blocks both  to cause r e v e r s i b l e  centres  response.  developed  of  of  clear i f  to  centres  the  i t i s not  used to impose a r e v e r s i b l e  rostral  conducted  of  inadequacy  conduction along f i b r e s of passage and  used s u c c e s s f u l l y  were  the  hypothalamic region.  t r a n s m i s s i o n and  areas  A  infusion  below  m a i n t a i n i n g the  higher  responses.  to  region  investigation of  apnoeic  some remaining caudal d i e n c e p h a l i c  v i a b l e and  involvement  the  to  Some time l a t e r , Andersen  brain  of b r a i n  entire diencephalic  could  following  (1913).  the  owing  description  the  both responses to submersion  of  But  that  ducks  of  just  synaptic has  been  discrete  Experiments habituation  125  METHODS Experiments  were performed  platyrhynchos) weighing male  ducks  were  between 2.2 and 4.0kg.  used.  Experiments  temperature  (21-22°C) and the  temperature  f o r at l e a s t  Preliminary  Surgery  Decerebrations  ducks  the  animals. cm  air  were  stream  of  and 5  acclimated  to  halothane  this  experimentation.  (Fluothane, Ayerst  unidirectionally  induced  Laboratories)  ventilated  For t h i s purpose a r e s e a l a b l e l a r g e bore cannula  would serve as the  (Anas  out at room  were done under general a n a e s t h e s i a  i . d . ) was f i r s t  artery  Two female  were c a r r i e d  1 week before  by a d m i n i s t r a t i o n of Halothane into  on seven White Pekin ducks  (UDV) (1.0  sewn i n t o the i n t e r c l a v i c u l a r a i r sac which exit  c o u l d be s a f e l y  port  from  which  eliminated.  expired  gases  and  The r i g h t b r a c h i o c e p h a l i c  and v e i n were cannulated with PE 90 t u b i n g .  Cannulation  techniques were d e s c r i b e d i n the Methods f o r S e c t i o n I I , and a l l surgery up t o t h i s stage was done under Xylocaine,  Astra  minimum of  2  experiments.  Pharmaceuticals).  hours  to  recuperate  local The  from  anaesthesia  (2%  animal was allowed a surgery  before  any  1 26  Decerebration The  animal  stereotaxic  was  table  intubation  positioned  and  arranged  of the trachea  and  administered  Air)  by  means  W.Germany).  i n t o the of  To  a  5%  dextrose)  was  1.8  Halothan the  Ml/ml  rate of  Co.,  (Figure  the  were 24a)  the  amount  rapidly  action  Millis,  of  the  by  force of  with  suction of  pressure  With  could  be  from b r a i n t i s s u e .  l i g a t e d and to  a  a  being  of a small  In t h i s way  was  brainstem  The  above  blunt  the  tipped  controlled side-hole  brain tissue  to  opened,  removed.  level  through  suction  occlusion  minimal  Mass.).  Once the dura  extirpated  handle c a r r y i n g the needle. removed  oxygen:balance  performed, t a k i n g care not  segment of the s i n u s was  hypodermic needle, varying  Halothane  (Dragerwerk, Lubeck,  i n f u s i o n , blood  craniotomy was  c e r e b r a l hemispheres thalamus  (50%  %  sodium n i t r o f e r r i c y a n i d e s o l u t i o n ( i n  enough to reduce b l e e d i n g  extensive  central  entailed  interclavicular  volumes  hypotensive  damage the u n d e r l y i n g d o r s a l s i n u s . the  This  of the  Vaporiser  Narishige  i n f u s e d i n t r a v e n o u s l y by means of an i n f u s i o n  c a r e f u l c o n t r o l of the  An  UDV.  to 2.2  pump (Model"901 Harvard Apparatus  maintained low  modified  inspired airstream  augment  a n a e s t h e t i c , a 0.142  for  a  connection  cannula to an exhaust system. was  within  by  in the  could  d i s t o r t i o n and  be  blood  loss. After bleeding raised bleeding pressure  by  had  reducing  occurred again  the until  stopped, blood  the  rate  of  ferricyanide  i n f u s i o n r a t e was bleeding  pressure  was  gradually  infusion.  i n c r e a s e d to drop  ceased.  The  operation  If  blood usually  1 27  r e q u i r e d an hour and a h a l f ferricyanide  solution.  The  edges  cut  v a s e l i n e and prevent  of  the  scalp  dehydration.  Several  layers  of  secured with  c o n t i n u o u s l y monitored  maintained  at  41±1.0°C  via  plastic  the  decerebration Training these  scalp and  these  experiments  animals  transection  (DC5  gauze  tape.  a  animal  was  Body  temperature  thermistor  and  incision  was  animals  were  DC8)  sutured used  in  continued  (DC5,  DC8  closed  Subsequently,  was  infra  after  Habituation use of two of  for  the  brain  the  use  of  experiments.  Most  experiments  temperature-  controlled  Section  and  II)  were  conducted  body  plethysmograph  consequently  (Tb),  blood  with  (described  in the l a t t e r  the animals were kept i n the plethysmograph temperature  were  The  rectal  to  padding  In three animals  (see l a t e r ) . and  ml  film  with an e l e c t r i c h e a t i n g pad and  red lamps mounted over the animal. H),  20  were l i b e r a l l y coated with  to recuperate f o r a minimum of 24 hours.  (Tb) was  and  and no more than  the c r a n i a l opening covered with  placed over the s k u l l and left  to complete  a in  stages of recovery so  that  the  body  p r e s s u r e , HR and r e s p i r a t i o n c o u l d be  monitored.  R e v e r s i b l e Sect ion Of The Areas 1971)  Bra instem Using X y l o c a i n e  of the hypothalamus in the  and the duck (Simon et a l .  thermoregulatory  control.  For  chicken  (Richards  1970,  1978,1981) are r e s p o n s i b l e f o r this  reason,  c o n f i r m a t i o n of  128  complete t r a n s e c t i o n across the brainstem  below the hypothalamus  would be i n d i c a t e d by l o s s of thermoregulatory Decerebrate apparatus.  animals  Since  the  were  positioned  brainstem  micropipette  (See  from  I  for  vertical  below  advanced  in  the  8  and  lifted advanced  deposited  and  Xylocaine-fi1led  glass  the  as  clear into before  The  with  from the p o s t e r i o r  by  the  brain  of one  intended  The  to  a  it  mm  the  with  Tb was  head was  point was  raised.  moved 1.5  laterally  Xylocaine  u l q u a n t i t i e s f o r every  1mm  raised.  in a planar array took  being  across  up  to  during  the  injection period.  f r e e d from the apparatus and  Xylocaine  immerse the head to j u s t above eye water s p i l l e d  8  c o n t i n u o u s l y monitored and u s u a l l y  immersed  i n t o a beaker of water for d i v i n g , but only i f a drop in Tb occurred  of  slowly  the m i c r o p i p e t t e was  deposited  54°  was 1 mm  in 0.5  at  plane  for every  again,  lobe.  micropipette As  a  injecting  deposited  tissue  of  optic  tilted  commissure  tissue.  began to drop toward the end of the animal's  the  i n j e c t i o n procedure u s u a l l y  to complete.  means  micropipette  foramen.  nl Xylocaine was  the brainstem.  The  a  the The  positioned  of  alignment  into  stereotaxic  monitored.  the caudal end  details  In t h i s manner, X y l o c a i n e was  minutes  Tb only were  30jum)  hypophyseal  mm  withdrawn, 0.5 Once  the  l o n g i t u d i n a l a x i s of the p i p e t t e was  transection passing just  exposed  d i r e c t l y over  Appendix  system.) The  was  ( t i p diameter:  micromanipulator  in  plethysmograph could not be used at  same time, blood pressure, HR and dorsal  function.  blockade. l e v e l and  i n t o the c r a n i a l c a v i t y .  Care  was  to ensure  had  taken  to  that  no  C o n t r o l experiments were  1 29  carried  out u s i n g s a l i n e  injections  d i d not drop, d i v e s were done saline  at  about  the  same  time  i n j e c t i o n that Tb would have f a l l e n were X y l o c a i n e  Mechanical  Brainstem  To accomplish diencephalon,  the animals were r e - p o s i t i o n e d i n the s t e r e o t a x i c  micropipette  i n the micromanipulator  narrow  scalpel  blade  was  exchanged  f o r the  and the blade drawn  i n e x a c t l y the same plane as the X y l o c a i n e  S e c t i o n of t i s s u e down to the v e n t r a l completed  used.  t r a n s e c t i o n between the mesencephalon and the  A  brain  after  Transect ion  apparatus.  the  (0.9% NaCl) and although Tb  with a hand-held  side  of  the  through  blockade. brain  was  scalpel.  Exper imental P r o t o c o l Prior  to  decerebration,  temperature-regulated 2  minute  body-plethysmograph and were subjected  to  submersions during which HR, BP and Tb were recorded.  Pre-dive and p o s t - d i v e during  the animals were placed w i t h i n a  dives  ventilation  following  was  also  X y l o c a i n e blockade.  recorded  except  Samples f o r blood  gas a n a l y s i s were taken before submersion and i n the 10  seconds  j u s t before the end of the d i v e . The  animals  were allowed a minimum of 1 day to recuperate  from d e c e r e b r a t i o n d u r i n g which they water.  were  regularly  force-fed  F o l l o w i n g b r a i n t r a n s e c t i o n , the animals were allowed at  1 30  least  6  hours  to  recover  monitored and maintained heating pads and The animal.  dives  at  during 41±1.0°  infra  red lamps.  were  replicated  in  C  means  by  of  closely electric  c o n d i t i o n f o r every  In a d d i t i o n to the t e s t d i v e s , two  of the mesencephalic  At the end  removed and  in  placed in 5%  by  s t a i n e d with  Appendix  inspection luxol  were k i l l e d  (Sodium P e n t o b a r b i t a l i . v . ) formal-saline.  the b r a i n s were removed and processed described  4 minute submersions.  of the experiments, the animals  an overdose of a n a e s t h e t i c  confirmed  time Tb was  each  p r e p a r a t i o n s were subjected to a few  heads  which  II. of  f a s t blue  The  for  Two  paraffin  plane midline  of  and  /urn  'G'  and n e u t r a l red.  the  weeks l a t e r , section  transection  12  with  sagittal  as was  sections  131  RESULTS  Xylocaine  blockade  The  temporary  Xylocaine  of  blockade was marked  function.  The  20 minutes in  removal  fall  hypothalamic c o n t r o l  by  a  loss  of  following  thermoregulatory  i n Tb reached a maximum approximately 18 -  from the end of the i n j e c t i o n p e r i o d .  The mean  fall  Tb f o r a l l animals was 1.2±0.2°C and t h i s value i s s i m i l a r to  the  fall  in  transection gradually  showed  a  produced approximately 20 minutes a f t e r b r a i n  (Figure to  injection.  Tb  23).  normal  Control drop  by  Body about  experiments  in  Tb.  temperature  usually  45  to  minutes  with  saline  an hour a f t e r  injection  never  In one animal Tb was monitored f o r 2.5  hours a f t e r death and i t was found that for  returned  i t took 1hour 20 minutes  Tb to f a l l 1°C.  Plane of t r a n s e c t ion Figure  24a i s a composite diagram of a s a g i t t a l view of the  b r a i n of a Pekin duck showing plane of s e c t i o n regions. from  a  between  Figure brain  mesencephalon.  the l e v e l of d e c e r e b r a t i o n  the  mesencephalic  and  diencephalic  24b i s a photograph of a m i d - s a g i t t a l  transected  at  the  rostral  and the  level  section of  the  1 32  F i g u r e 23. E f f e c t of X y l o c a i n e blockade and t r a n s e c t i o n on body temperature. Closed c i r c l e s represent values obtained a f t e r i n f u s i o n of X y l o c a i n e across the r o s t r a l mesencephalon. Open c i r c l e s represent values obtained a f t e r mechanical t r a n s e c t i o n . V e r t i c a l dashed l i n e i n d i c a t e s the time of Xylocaine i n j e c t i o n and mechanical transect ion.  133  000000 0 C\J  •  a  0  I  CD  c_  O  •  Q  0 00 00  "3  o  00 •  trj  c_  •  CD  •  a  O  o  S  o  •  o o  E  0)  o o  n o  CD  00  O  00  o  o o  01  cn  GO  cn  10  20  30  Time. (minutes)  40  50  60  1 34  Figure  24. A) Schematic view of a m i d - s a g i t t a l s e c t i o n of i n t a c t b r a i n from the Pekin duck (Anas platyrhynchos) i n d i c a t i n g l e v e l of d e c e r e b r a t i o n and mesencephalic transection. B) Photograph of m i d - s a g i t t a l s e c t i o n from a mesencephalic p r e p a r a t i o n . (Stained with l u x o l blue and n e u t r a l red)  136  D i v i n g performance Resting  HR  and  MAP u s u a l l y decreased a f t e r d e c e r e b r a t i o n  and  the ducks, though s t i l l  24  hours a f t e r surgery, they would o f t e n walk spontaneously and  would o c c a s i o n a l l y below  the  activity  their  diencephalon,  although  Differences animals  flex  wings.  Following  the ducks never  they  between  r e s p o n s i v e , were much more  would  resting  transection  e x h i b i t e d spontaneous  sometimes v a l u e s from  passive.  stand  i f provoked.  i n t a c t and t r a n s e c t e d  f o r HR, MAP and blood gases were not s i g n i f i c a n t at the  0.05 p r o b a b i l i t y The  level.  profile  of  the  fall  i n HR during submersion was the  same f o r a l l i n t a c t and experimental c o n d i t i o n s . significant  difference  There  between the r a t e s of f a l l  was  no  f o r the f i r s t  30 seconds nor the maximum l e v e l s of b r a d y c a r d i a achieved at the end  of  the  dives  (Figure  25).  b r a d y c a r d i a , MAP f o r a l l animals dive  levels  Despite  the  considerable  was maintained and matched p r e -  (Figure 26). The mean f a l l  i n Pa0  2  by the end of 2  minutes submersion f o r the i n t a c t , decerebrate and mesencephalic ( i n c l u d i n g both X y l o c a i n e and mechanically was  43.5,  40.6  and  43.1mm  significantly different during  submersion  Hg  (P>0.05).  and  the  transected)  respectively; PaC0  rise  2  was  animals  these were not  rose some 13 - 16mm identical  Hg  under a l l  experimental c o n d i t i o n s ( F i g u r e 27). There was a profound during  recovery  the  breathing  pattern  from submersion between i n t a c t and decerebrate  animals when compared with 28).  difference in  mesencephalic  T y p i c a l l y , minute v e n t i l a t i o n  preparations  (Figure  i n c r e a s e d immediately  after  1 37  Figure  25. E f f e c t of v a r i o u s l e v e l s of b r a i n t r a n s e c t i o n on the c a r d i a c response to 120 seconds submersion. A. I n t a c t animals (N=7, n=14), B. Decerebrate animals (N=7, n=14), C. 'Xylocaine' animals (N=6, n=6), D. Mesencephalic animals (N=7, n=14). (Error bars ±S.E.)  o o  o o  o  a in  CM  cn  ID  cn 4-»  ro  ro  n  CD  O O  JQ  4-)  ro cr  ro  c_ ro  o o  CD  CD  4->  4->  B.  CM  4-1  CD  138  O If)  4-1  ro  CD X  o  un  CD X  o  0 20 Time  60  100  0 20 Time  (Seconds)  a o  a o  O If)  o in  CM  cn  cn 4J  CD  n  O O  4J  c ro  D.  o o  CD  cu  4->  4->  ro cr  (Seconds)  ro  ro  n  100  CM  4->  CD  60  ro  cr  O  4-1  in  c_ ro  CD X  o in  CD  x  0 20 Time  60  100  (Seconds)  a  0 20 Time  60  100  (Seconds)  1 39  F i g u r e 26. E f f e c t of v a r i o u s l e v e l s of b r a i n t r a n s e c t i o n on a r t e r i a l blood pressure during 120 seconds submersion. A. I n t a c t (N=3, n=6) B. Decerebrate (N=3, n=6) C. 'Xylocaine' (N=3, n=6) D. Mesencephalic (N = 3, n=6) ( E r r o r bars ±S.E.)  140  O O CVJ  O  O ID  a  O O  O O  B.  a  OJ  ID  cn X E E  Q. <  0_ <  O ID  O ID  0 20  60  0 20  100  100  Time (Seconds)  Time (Seconds)  a a ru  60  D.  C. O O  O ID  a ID  X  x  o o  E E  a. <  O O  CL <  o  O ID  ID  0 20  60 Time (Seconds)  100  0 20  60 Time (Seconds)  100  141  Figure 27. E f f e c t of b r a i n t r a n s e c t i o n on end-dive blood gas l e v e l s and pH. Hatched bars represent values obtained at the end of 120 seconds submersion. ( E r r o r bars ±S.E.)  1 43  Figure  28. E f f e c t of brain t r a n s e c t i o n on breathing in the recovery p e r i o d f o l l o w i n g 120 seconds submersion. A, I n t a c t animals B. Decerebrate animals C. Mesencephalic animals V e r t i c a l dashed l i n e i n d i c a t e s the end of submersion; the point l e f t of t h i s l i n e on each graph represents pre-dive values f o r v e n t i l a t i o n . (N=3, n=6. E r r o r bars ±S.E.)  144  A.  0  50 Time After End of Olve  100 (Seconds)  ISO  200  1 45  s u r f a c i n g by about 4 to 5 times,  falling  after  not  about 3 minutes.  preparations  which  never  p a t t e r n of b r e a t h i n g was The all  mesencephalic  T h i s was  the while maintaining  i r r e g u l a r and  values.  to  normal  f o r mesencephalic  hyperventilation. sometimes m i l d l y  The  gasping.  s u r v i v e d 4 minute submersion p e r i o d s  b r a d y c a r d i a , but again  extended d i v e s , minute v e n t i l a t i o n resting  the case  exhibited  animals  gradually  was  little  f o l l o w i n g these different  from  1 46  E f f e c t of H a b i t u a t i o n T r a i n i n g of Decerebrate Ducks  METHODS Three  ducks (DC5,DC8 and H) were decerebrated as d e s c r i b e d  in the preceeding experiments minimum  of  6  weeks.  65  Laboratories) dissolved intramuscularly were r e g u l a r l y temperature all  for  and  noises.  mg 1.5  the f i r s t  controlled  would  room.  allowed  antibiotic mis  saline  to  recover  for a  (Penbritin,  Ayerst  were  6 days of r e c o v e r y .  f o r c e - f e d food and  these animals walked  cages  in  and  water  and  were  administered The animals kept  W i t h i n a few days a f t e r surgery  spontaneously when r e l e a s e d from  respond,  though  a  animal  (H) was•eventually s e l f  their  somewhat s l u g g i s h l y to loud  One animal was allowed to recover f o r much  after  in a  longer  few months was feeding and d r i n k i n g v o l u n t a r i l y . sufficient  enough to be  and This  returned  to the Animal Care U n i t with the stock animals where i t remained for 4 y e a r s . Each described trials  of  the  ducks  underwent  habituation  i n S e c t i o n II Methods.(DC5 and  per day; H f o r 2 days,  DC8  for  training  as  3  15  days,  15 t r i a l s per day) Heart r a t e was  obtained from the ECG i n the usual manner.  147  RESULTS The two operative diving  ducks  recovery  bradycardia  substantial  (DC5  and  showed (Figure  reduction  of  DC8)  very  allowed  little,  29).  f u r t h e r h a b i t u a t i o n on the second day. evoked  a  shorter  post-  i f any, h a b i t u a t i o n of  Duck  bradycardia  a  H, on  however, the  first  Whereas the  70% drop i n HR, by the s i x t h t r i a l  showed day and  first  dive  of the second day,  HR dropped by only 4% of the pre-dive HR (Figure 29).  1 48  Figure 29. E f f e c t of h a b i t u a t i o n t r a i n i n g on three decerebrate animals. Duck H: 4 years a f t e r d e c e r e b r a t i o n , ducks DC5 and DC8: 6 weeks a f t e r decerebrat ion.  duck:  duck: H  O  duck:  DCS  0C5  0 0 0  O O O  0  0  o o  O  1  1  10  IS Trials  20  j  i  SB  30  J  °  L 20  30 Trials  40  90  0  0  0 0  o  o  0  00  0  0  0  t0  10  20  30 Trials  40  50  1 50  DISCUSSION The r e s u l t s of t h i s study c o n f i r m those and  Andersen(1963).  r e g i o n s , i t was during  submersion, MAP  that  in  the  diving  Similar  to  mesencephalic to  i s maintained at p r e - d i v e l e v e l s d e s p i t e  response, blood  minute  values  confirm  that  the a b i l i t y of these  periods  the  and  of submersion  control  of  were  the  fact  demonstrates  to  border  r e s u l t s argue driven  of  the  survive  and  Jones  adjustments  against  mesencephalon.  a  (mediated  through  component  of the d i v i n g response  Woodruff  1980;  the  Kanwisher  et a l .  is  not  (Smith e_t a_l.  an 1974;  diving  response  must  that suprabulbar involvement and  differential  changes  and apnoea below  the these  that  fear  essential Smith  and  1981).  The h y p o t h e s i s that suprabulbar r e g i o n s are the  1980).  chemoreceptor-  and they demonstrate  diencephalon)  four-  Consequently,  diencephalon-mediated  b r a d y c a r d i a (Korner 1971)  lost.  the e f f e c t i v e n e s s of  Bryan  cardiovascular  from  response i s not  preparations  1969;  that  adjustments  appears to be l o c a l i s e d w i t h i n the caudal brainstem, rostral  part  indistinguishable  that the d i v i n g  the responses (Kobinger and Oda Thus,  serves, as  l i m i t the l o s s of stored oxygen.  gas  submersions  animals,  Moreover,  to  increase i n  preparations exhibited cardiovascular  two-minute  intact  bradycardia  This suggests that a compensatory  end-dive  (1913)  of d i e n c e p h a l i c  addition  p e r i p h e r a l v a s c u l a r r e s i s t a n c e must occur which of  Huxley  Regardless of the absence  demonstrated  a 70% drop in HR.  of  be r e a s s e s s e d . i s necessary  essential  for  I t has been assumed for  multidirectional  in vascular resistance  (Uther et a l .  151  1970;  Korner  present  1971; J a n i g  data  1975; Manning  be r e j e c t e d .  beds i n the d i v i n g  i s the  widespread  increase  throughout  the  vascular  system.  sympathetic a c t i v a t i o n would shutdown  of v a s c u l a r  and  vasodilatatory  vessels,  sympathetic  be  fibres  and  are is  densely  other . hand are s p a r s e l y i n n e r v a t e d reported  remaining the same or decreasing autoregulatory  the  heart  and  brain and  vessels  cet a l .  hypercapnia  1975).  during  as  the  greatest  1979; Zapol  flow  either  et  to these  increasing,  I t a l s o seems l i k e l y  counter blood  reported  response of r e n a l and limb Heistad  in  with  t o hypoxia and hypercapnia i n  will  increase  cerebral  and  mesenteric and  and blood  slightly.  adjustments  extreme hypoxia has been and  adrenergic  C e r e b r a l , coronary and lung v e s s e l s on  variously  that  of  Complete  innervated  reflected  the  vasoconstriction  of  i n these t i s s u e s (Jones et. a_l.  been  that,  level  maximum.  Muscular,  1979; McKean 1982).  has  made  i s , the  presence  fibres.  this  be  i s increased to v e s s e l s  to  al.  coronary  should  because of the uneven d e n s i t y  the  for instance,  decrease to flow  tissues  response  could  That  close  does not occur  innervation  cholinergic renal  differential  in peripheral vascular  Consequently, the suggestion  submersion, sympathetic discharge  vascular  the  The predominant f e a t u r e of vasomotor change during  resistance. during  however,  suggest that the concept of a h i g h l y  c o n t r o l of v a r i o u s v a s c u l a r  submersion  1977);  to in  vessels  neurally  mediated  flow.  Dilatation  occur  predominantly  contrast  to  (Daugherty  et  the al.  during in  minimal 1967;  Thus, one of the consequences of hypoxic submersion  i s almost  complete  vascular  152  sympathetic  activation  excluding  a  few  i n c r e a s e d blood  causing  specific  widespread  vasoconstriction,  t i s s u e s r e q u i r i n g maintained  or even  flow.  In d i v i n g some degree of d i f f e r e n t i a l c o n t r o l , mediated higher  nervous  c e n t r e s , may e x i s t  i n i n t a c t animals  the c a r d i o v a s c u l a r adjustments but i t s occurrence  is  be  how  limited.  For  instance,  v a s c u l a r beds respond  it  is  to submersion.  uncertain  by  as part of likely  to  cutaneous  D j o j o s u g i t o and co-workers  (1969) have demonstrated that blood flow i n the web of a duck i s preserved d u r i n g shunts.  submersion  Maintained  diving seal  primarily  al.  (Zapol et al_.  1975)  1979).  Studies of s k i n blood flow i n  have  revealed  1966) and dog  vasodilatation  chemoreceptor s t i m u l a t i o n by a r t e r i a l hypoxia. has  been  obtained  sympathetic 1976).  to suggest  activity  (Iriki  in  (Heistad  response to  Strong  evidence  et_ a_l.  1972; I r i k i  and Kozawa 1975,  A r e d i s t r i b u t i o n of blood flow away from s k e l e t a l muscle  adjustments  control  suprabulbar  oxygen  of  this  consumption differential  regions as i t i s  hypothalamus ( I r i k i proportion  to  lost  of  blood flow  oxygen  of  this  conserving  tissue.  activity  following  and Kozawa 1976).  cutaneous v a s c u l a r r e s i s t a n c e contributes  oxygen  i n view of the abundance of A-V anastomoses i n s k i n  the low rate of  rabbits,  what  paw  that t h i s i s due to d i f f e r e n t i a l  toward the s k i n can be v i s u a l i s e d as part of  and  arterio-venous  cutaneous flow was a l s o demonstrated i n the  the r a b b i t ear (Chalmers and Korner et  through  In  resides  removal  in  of the  I t i s u n c e r t a i n , however,  i s a f f e c t e d by these changes i n  and  therefore  conservation.  Much  how  much  less  is  of i t known  1 53  regarding  parasympathetic a c t i v i t y ,  output appears maximal as  picture  that  submersion, both the are  maximally  differential brain,  owing  to  autoregulatory the  begins  activated,  to  with  is  that  parasympathetic little  perfusion  sparse v a s c u l a r  control  to decrease heart  evolve  very  Adequate  its  cardioinhibitory rate  escape.  sympathetic and  control.  vagal  i t is d i f f i c u l t  f u r t h e r without provoking vagal The  but  (Berne e_t §_1.  in  divisions  the  way  of  continues  to  the  innervation  1981;  during  and  Kontos  powerful  1981).  If  d i v i n g response demands some b i d i r e c t i o n a l c o n t r o l , such as  a p o s s i b l e v a s o d i l a t a t i o n of the adrenal and  cutaneous  beds,  then  the  glands  effect  on  (McKean overall  1982) MAP  is  much  of  immeasurable.  the  Stimulation  induced by submersion does not  CNS:  maximal  just  system, q u i t e w i t h i n Small  regional  the  differences  large scale  reveal  the  the  case  highly  An  brainstem  Transection of  control.  be p e c u l i a r to the  insignificant  responses  by comparison  experiments  higher  diving  CNS  will  control,  requiring  greater  to not  unless In  vascular  such as the defence r e a c t i o n , v a s o d i l a t a t i o n i s  abolished  interesting  hyperventilation.  that may  features  more e a s i l y measured in the noticeably  lower  autonomic  l o c a l i s e d changes in flow are measured.  of  differentiation,  of  relatively  shutdown.  detailed  s p e c i f i c and  widespread a c t i v a t i o n of the  the c a p a c i t y  response would thus be  demand  large  following  muscle  beds  and  would  be  transection.  observation  was  Whereas the p a t t e r n  the  loss  of b r e a t h i n g  of  post-dive  a f t e r a dive  1 54  appeared s i m i l a r for each l e v e l of s e c t i o n , only  the  mesencephalic p r e p a r a t i o n s .  known how return  long  to  recovery the may  period.  rostral  as  these  the  with  (Richards  midbrain  respect  1971)  and  to  pH the  stimulation  1974). in  mediating  The  control  polypnoea  thermoregulation  Putkonen  of  in  these  the  in  to dive to  sites  duck  mesencephalic  animals  centres. have  the  been pigeon  r e s u l t s in  (Kotilainen  l o s s of the normal p o s t - d i v e confirms  r e s p i r a t o r y c o n t r o l r e s i d e s in regions  that  and  respiratory important  at or above t h i s l e v e l  of  brainstem. With regard  to the e f f e c t of h a b i t u a t i o n  bradycardia  in decerebrate animals, the  tested  f a r from c o n c l u s i v e .  are  allowed to recover habituation  when  for  suggests  d r i v e n bradycardia allowed  a  6  training. first  From two  The  weeks  that  far  these  t r a i n i n g on d i v i n g  r e s u l t s from the  post-decerebration  modification  greater  of  recovery  habituation  the  duck H.  The  suggestion  However, one  duck  second day  of  to say whether  suffering post-surgical  or that perhaps some degree of " p l a s t i c i t y " may  no  chemoreceptor  period,  by only the  ducks  showed  regions.  results, i t is d i f f i c u l t  animals were s t i l l  animals  f i n d i n g that the two  o r i g i n a t e s in r o s t r a l CNS  demonstrated c o n s i d e r a b l e  the  l e v e l s and  to known r e s p i r a t o r y  r e s p i r a t o r y frequency  the  i t i s not  were not measured during  increased  pattern  gas  in  border of the mesencephalic r e g i o n , c e r t a i n damage  within  identified  absent  Although the plane of s e c t i o n corresponded  have spread c a u d a l l y  Areas  was  Unfortunately,  i t took f o r a r t e r i a l blood  normal  it  trauma  have occurred  in  c o u l d even be made t h a t , on the b a s i s of  1 55 the  speed  bradycardia the  CNS.  of  habituation  duck  H,  habituation  i n i n t a c t ducks i s i n h i b i t e d by r o s t r a l C l e a r l y , these f i n d i n g s  i n v e s t i g a t i o n of CNS brain  in  transection.  of  regions  the of  i n d i c a t e the need f o r f u r t h e r  control following  long  term  recovery  from  1 56  GENERAL DISCUSSION From  the  cardiovascular submersion  preceeding adjustments  are  experiments to  instigated  prevent  primarily  the  l o c a t e d within  rising  PaC0  bradycardia vascular  for  the  duck,  the  during  stimulation  The p e r i p h e r a l  declining  and a s u b s t a n t i a l part resistance.  potent stimulus  Pekin  receptor  The  fall  Pa0 ,  and  2  of the increase of Pa0  to the p e r i p h e r a l  causes  appears to be the more  2  chemoreceptors  and  The r i s e of PaC0 , on the other hand, c o n t r i b u t e s  by  2  rate  a f u r t h e r 20%.  Other inputs,  r e f l e x e s , account f o r the remaining 12%).  Peripheral  increase  vascular  such as  bradycardia  chemoreceptor  both  in peripheral  accounts  approximately two-thirds of the t o t a l change i n heart  heart  of  the c a r o t i d bodies, i s responsive to both  and  2  the  asphyxiation by  c e n t r a l and p e r i p h e r a l chemoreceptors. group,  in  activation  rate.  decreasing  baroreceptive (approximately  also  r e s i s t a n c e , with an apparently  serves  similar  to  ratio  of c o n t r i b u t i o n  from both Pa0  and PaC0 .  The cumulative change  in  resistance  caused by these receptors  amounts to some 60% of  the  total.  to  be  due  increase  10%  attributed appears  resistance  to c e n t r a l chemoreceptors.  c e n t r a l region and  2  The r e s t of the v a s c u l a r  s e n s i t i v e to an increase total  2  or  in  of  to low Pa0 far  cardiovascular  i n PaC0 , and causes some  less  the 2  30%  2  vascular  resistance.  change  acting  responses to  in  submersion  a  falling  the Pa0 , 2  r e s i s t a n c e can be  Baroreceptor  the and  of the  Surprisingly,  in vascular  centrally.  significant  appears  This group i s extremely  a l s o appears to be a f f e c t e d by so  change  input  development serves  mainly  of to  1 57  mitigate the  barostatic  are to  the  changes  and maintain a r t e r i a l blood pressure v i a  reflex.  Peripheral  and  central  chemoreceptors  thus of paramount importance in the g e n e r a t i o n of responses forced  submersion.  Although c e n t r a l and p e r i p h e r a l chemoreceptors for  the  cardiovascular  difficult  responses  seen in forced d i v e s ,  to q u a n t i f y and assess the r o l e of  animals i n n a t u r a l s e t t i n g s because they may many  more  diverse  inputs.  sensory  receptors, On the  The  basis  submersion,  of it  other recent  than  b r a d y c a r d i a evoked following  that (such as  on  the  from  in  ducks  Pekins),  when  diving  as  nasal  hypoxia  submersion.  The  demonstrated  in  rate  diving.  to  receptors  T h i s i s supported by where  the  of  a  local  passages  anaesthetic  sectioned.  (Furilla  and  role  Jones  of  experiments  The  1985).  r e f l e x e s remain  and hypercapnia develop during prolonged peripheral studying  initial  u n a l t e r e d while b r a d y c a r d i a was longer d i v e s  initial  to the mucous  chemoreceptors the c a r d i a c  drop  slightly  in  carotid  heart rate appeared  reduced at the  (Butler and Woakes 1982 a,b).  was  response of  spontaneously d i v i n g ducks that had the nerves to t h e i r bodies  with  (such as redheads),  respond  ducks,  receptors.  heart  However, there i s l i t t l e doubt that chemosensory important  pulmonary  in f o r c e d submersion i s s u b s t a n t i a l l y reduced  application  membranes of  in  be responding to so  that  change  diving  chemoreceptors  experiments  including  immediate  seems  u n l i k e d a b b l i n g ducks  chemoreceptors  r e c e p t o r s and f a c i a l or n a r i a l  the  it is  moment of submergence i s crowded with  information  vestibular  predominate  end  of  1 58  It  i s apparent that d i v e r s e  r e f l e x pathways c o n t r i b u t e to  the development of the d i v i n g responses; certain  that  modification Folkow  higher  centres  of the c a r d i o v a s c u l a r  and N e i l 1971; Korner  reduction  i n heart  surfacing  is  mammals  to  voluntary under  1973).  exhibit  "ignored" The  1965;  For i n s t a n c e , a n t i c i p a t o r y increase  before  some form of a s s o c i a t i v e l e a r n i n g  great  a  fall  forced  conditions,  in  dives  peripheral  birds  and  heart  rate  during  could  mean  that,  sensory  inputs  are  e i t h e r by c o n d i t i o n i n g or by h a b i t u a t i o n .  chronotropic  response  following  contention It seems  as  also  system (Folkow e_t a l .  Moreover, the f a i l u r e of some  demonstration,  habituate  i t is  powerful c o n t r o l or  submergence and  of  d i v e s compared with  natural  impose  1971).  r a t e before  indicative  (Jones e_t a l .  that  Habituation  this  to  diving to  evoked  f o r periods  in  study,  forced  repetitive  reasonable  adjustments diving  CNS  however,  lends  cardiac  will  readily  support  to the  responses can be modified suggest  that  the  by l e a r n i n g .  oxygen  conserving  by submersion may be unnecessary i n animals which are w e l l w i t h i n  t h e i r aerobic  therefore  means by which these animals adapt t o t h e i r though  perhaps  In t h i s regard,  be  an  capacity. important  environment.  l e s s t a n g i b l e , f e a t u r e of higher  c e n t r a l nervous c o n t r o l i s the a r o u s a l the animal.  the  submersion  diving  of these responses may  Another,  that  s t a t e or  disposition  of  suggestions have been made that the  responses i n c u r r e d by f o r c e d submersion may have l e s s to do with diving Woodruff  than  with  the  defence  1980; Kanwisher et a l .  or  fear  1981; Smith  reaction and  (Smith and  Tobey  1983).  1  However, the demonstration the  rostral  border  of  that decerebrate animals s e c t i o n e d at the  mesencephalon continue to e x h i b i t  profound d i v i n g b r a d y c a r d i a and can even  withstand  periods  of  to  response  i s a defence mechanism.  T h i s i s a l s o supported by  finding  that  not  submersion  clear  that  seems  bradycardia  submerged a f t e r  59  contrary  does  the  develop  four-minute view  in  that the  naive  they have been b r e a t h i n g pure oxygen.  the observed responses are engendered  per se and t h a t , on o c c a s i o n , these responses may  ducks  It  by  the  seems  submersion  be  profoundly  a l t e r e d by higher c e n t r e s and not v i c e v e r s a . In view substrate  of the f i n d i n g that only a minimal  is  required  to  responses to submersion, degree  of  Descriptions "selective  generate  i t is  control  in  literature  the  redistribution"  effective  tempting  neural  amount of neural  to  involved  cardiovascular  speculate in  the  frequently  lack  of oxygen.  The  response  1965).  the  susceptible  i s c h a r a c t e r i s e d as being the  d i s c r i m i n a t i v e v a s o c o n s t r i c t i o n produces or  to  of blood away from h y p o x i a - t o l e r a n t  r e s u l t of d i f f e r e n t i a l output of sympathetic  to  the  responses.  refer  t i s s u e s to the b r a i n and heart which are e s p e c i a l l y to  on  activity  so  that  r e g i o n a l i s e d blood flow  from s e l e c t c a p i l l a r y beds (Johansen  1964;  Folkow et a l .  B i d i r e c t i o n a l c o n t r o l of t h i s type i s f u r t h e r  suggested  to be d e r i v e d from suprabulbar c e n t r e s , such as the hypothalamus (Korner  1971;  Hilton  1975;  Manning 1977).  of the i n t a c t d i v i n g response  in the absence  brainstem  an  centres  suggests  Thus, the occurrence of  these  rostral  a l t e r n a t i v e hypothesis f o r the  g e n e r a t i o n of blood r e d i s t r i b u t i o n .  For example, v a s o d i l a t a t i o n  1 60  evoked l o c a l l y by hypoxia and  is  instead  instance, cerebral and  i s not uniform  selective  dilatation  (Heistad  occurs  and  1977,1978). innervation  a_l.  1967;  Heistad  In a d d i t i o n to t h i s , of  the  in  a  Pa0  et. aJL.  the  vasculature  both  submersion components  Parasympathetic sympathetic the  more  may  provoke  of discharge  density varies  and  20  mm  Hg  of  sympathetic  on  tissues. the  CNS  a c t i v a t i o n of  nervous  system.  cause extreme b r a d y c a r d i a and  d i s c h a r g e would produce intense v a s o c o n s t r i c t i o n of densely  innervated v e s s e l s .  of the blood flow i s thus achieved powerful  coronary  between  autonomic  would  For  1975; Grubb e_t a l .  the simultaneous  the  1980).  of  2  Therefore, the chemoreceptor i n f o r m a t i o n impinging during  beds,  i n limb v e s s e l s i s weak,  r e n a l v e s s e l s do not even d i l a t e at et  vascular  Abboud  predominantly  v e s s e l s , whereas the response  (Daugherty  in a l l  autoregulatory  vasoconstriction.  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(1982) H a b i t u a t i o n of C a r d i o v a s c u l a r R e s p o n s e s t o A v e r s i v e S t i m u l a t i o n and I t s S i g n i f i c a n c e f o r t h e D e v e l o p m e n t of E s s e n t i a l H y p e r t e n s i o n . C o n t r i b u t i o n s t o N e p h r o l o g y 30: 78-81.  184  APPENDIX I A method i s d e s c r i b e d whereby l o c a l i s e d b r a i n t i s s u e were lidocaine The  reversibly  hydrochloride  injection  (P.E.  port of syringe with  the  90, stopcock  i n f i g u r e 24.  was  connected  was  connected  mineral  oil.  It consisted  by  polyethylene  to  a  the  this  Xylocaine  way,  micrometer-driven  A f t e r the m i c r o p i p e t t e  s y r i n g e , the stopcock  with the mineral as  oil  Advancing  0.1mm  micrometer  a l i q u o t s of x y l o c a i n e mounted  in  by  the  along  filled  backfilled  second  to connect syringe.  the o i l / x y l o c a i n e the  increments  from the p i p e t t e t i p .  i n a standard  was  was turned  the micrometer was turned, moving  the  One  The other port was connected to a syringe  i n t e r f a c e c o u l d be e a s i l y seen  was  Pharmaceuticals).  (AGLA, Burroughs Wellcome Co., England) which was  the m i c r o p i p e t t e In  which  i n j e c t i o n s of  Clay Adams) to a three-way stopcock.  f i l l e d with X y l o c a i n e . from  with  (Xylocaine 2%, A s t r a  system i s i l l u s t r a t e d  of a g l a s s m i c r o p i p e t t e tubing  inactivated  regions deep w i t h i n  The  P.E. t u b i n g . e j e c t e d 0.5M1 micropipette  micromanipulator clamped on one r a i l  of the s t e r e o t a x i c apparatus ( N a r i s h i g e , Japan).  185  F i g u r e 30. Appendix I. Schematic diagram arrangement f o r Xylocaine i n j e c t i o n .  illustrating  Xylocaine reservoir-  glass syringe (mineral oil )  micrometer J|  nn<m l^ V  oil/Xylocaine meniscus  glass micropipette  ear bar (stereotaxic  apparatus)  187  APPENDIX II Immediately a f t e r death f e a t h e r s , and s o f t much  the animal  was d e c a p i t a t e d .  t i s s u e around the s k u l l  were  removed.  b r a i n t i s s u e as p o s s i b l e was exposed by c a r e f u l l y  away s k u l l bone with ronguers.  The lower  jaw  was  the  beak,  clipping  The r e s t  the neck muscles, trachea and e y e b a l l s were a l s o  removed and the head was then 7.5%  As  completely  removed and the v e n t r a l s u r f a c e of the b r a i n exposed. of  Skin,  immersed i n 4-5%  formaldehyde  in  saline. After  3  weeks,  the  hardened b r a i n was c a r e f u l l y  from the s k u l l and r e p l a c e d i n f r e s h f i x a t i v e  until  removed  ready  for  processing. C l e a r i ng:  The  brain  was  washed  i n running water f o r 24  hours. Dehydrat i n g : Soaked i n the f o l l o w i n g sequence of s o l u t i o n s : 50% ethanol o v e r n i g h t ; 70% ethanol f o r 24 hours; 80%  ethanol f o r 24 hours;  95% ethanol f o r 24 hours; repeat; repeat; 85% amyl a c e t a t e f o r 24 hours; repeat; toluene f o r 24 hours; repeat; 50%  t o l u e n e : 50% melted  p a r a f f i n overnight at 60°C;  188  1) melted p a r a f f i n (paraplast  "+")  for 24 hours at 60°C under  15  l b s vacuum; m)  repeat with f r e s h p a r a f f i n ;  n)  repeat;  o) embed in f r e s h de-gassed p a r a f f i n . Sectioning: rotary  Saggital  microtome  (Spencer  water bath at 54°C and subbed s l i d e s . Staining: and  Neutral  Red  sections "820).  mounted  on  were cut,  12 urn t h i c k ,  Sections double  Sections  were  counterstain.  a  were f l o a t e d in a  coated  S l i d e s a i r d r i e d overnight at  on  chrome/alum  35°C.  s t a i n e d with Luxol Fast  Blue  'G'  

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