@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Science, Faculty of"@en, "Zoology, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Drummond, Peter Charles Patterson"@en ; dcterms:issued "2010-03-23T17:27:15Z"@en, "1980"@en ; vivo:relatedDegree "Doctor of Philosophy - PhD"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """Heart rate was found to be significantly lower in unrestrained diving muskrats than in those which were forced to dive. The response in the unrestrained animal represents a heart rate of about 9% of the resting rate and is similar to the cardiac responses recorded in freely diving pinnipeds. Apnea and bradycardia were initiated by water lapping the nares of the conscious animal. Anaesthesia abolished this narial reflex to submersion. In anaesthetized muskrats water was drawn into the nasal cavity causing transient apnea and prominent bradycardia by stimulating receptors located principally in the glottal and pharyngeal areas. Nerve blockade by reversible cooling and section demonstrated that these nasal receptors are innervated by the maxillary and inferior, laryngeal nerves. In the conscious animal trigeminal neurotomy failed to affect the course of the response confirming that the muskrat has a number of external sensory mechanisms capable of initiating the diving reflexes. Respiratory activity was shown to have a marked effect on heart rate when the muskrat was at rest and when water was passed through the nares. Cardioacceleration during nasal stimulation resulted from a central component and from neural input originating in fast adapting pulmonary receptors. Artificial ventilation not only increased heart rate but often tended to restore normal respiratory activity. Pulmonary deafferentation by steaming eliminated the Hering-Breuer reflex to maintained lung inflation as well as the cardioacceleration seen in response to artificial ventilation during nasal stimulation. The loss of the Hering-Breuer reflex occurred first suggesting that different receptors are involved. Lung deflation per se caused a reflex bradycardia but it appears that this does not potentiate the narial reflex since nasal bradycardia was not reduced when lung inflation was maintained. Central and peripheral components arising from respiratory activity have their greatest effect during the recovery period. Elimination of the carotid bodies delayed but did not abolish chemoreceptor driven bradycardia demonstrating that these are the most chemosensitive units but not the only ones responding to changes in blood gas tensions. No role however, has been found for the arterial baroreceptors. The barostatic reflex brought on by drug induced hypertension was triggered at a lower pressure than that found in the seal but it appears that this pressure would not be exceeded in the muskrat if heart rate remained low during a dive. It is concluded that the cardiac response to submersion in the muskrat results from at least three reflex arcs. These reflexes originate from the nares, the lungs and from peripheral chemoreceptors. Although the chemoreceptors act to maintain the prevailing diving responses, it is likely that the external narial reflex accounts for almost all of the cardiovascular adjustment brought about in normal foraging dives since these are usually of short duration. The chemoreflex could play a significant role in dives exceeding one minute by prompting the animal to resurface when oxygen stores are depleted."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/22344?expand=metadata"@en ; skos:note "r THE INITIATION OF AND RECOVERY FROM DIVING BRADYCARDIA IN THE MUSKRAT by PETER CHARLES PATTERSON DRUMMOND B.Sc., U n i v e r s i t y of B r i t i s h Columbia, 1967 M.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of ZOOLOGY We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1980 (c) Peter Charles Drummond, 1980 In presenting 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 the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the l i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representa-t i v e s . I t i s understood that copying or p u b l i c a t i o n of 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 w r i t t e n permission. Department of Zoology The U n i v e r s i t y of B r i t i s h Columbia Vancouver, B. C., Canada i i ABSTRACT Heart r a t e was found to be s i g n i f i c a n t l y lower i n u n r e s t r a i n e d d i v i n g muskrats than i n those which were forced to d i v e . The response i n the unrestrained animal represents a heart r a t e of about 9% of the r e s t i n g r a t e and i s s i m i l a r to the c a r d i a c responses recorded i n f r e e l y d i v i n g pinnipeds. Apnea and bradycardia were i n i t i a t e d by water l a p p i n g the nares of the conscious animal. Anaesthesia a b o l i s h e d t h i s n a r i a l r e f l e x to submersion. In anaesthetized muskrats water was drawn i n t o the n a s a l c a v i t y causing t r a n s i e n t apnea and prominent bradycardia by s t i m u l a t i n g receptors l o c a t e d p r i n c i p a l l y i n the g l o t t a l and pharyngeal areas. Nerve blockade by r e v e r s i b l e c o o l i n g and s e c t i o n demonstrated that these n a s a l receptors are innervated by the m a x i l l a r y and i n f e r i o r , l a r y n g e a l nerves. In the conscious animal t r i g e m i n a l neurotomy f a i l e d to a f f e c t the course of the response confirming that the muskrat has a number of e x t e r n a l sensory mechanisms capable of i n i t i a t i n g the d i v i n g r e f l e x e s . R e s p i r a t o r y a c t i v i t y was shown to have a marked e f f e c t on heart r a t e when the muskrat was at r e s t and when water was passed through the nares. C a r d i o a c c e l e r a t i o n during n a s a l s t i m u l a t i o n r e s u l t e d from a c e n t r a l component and from n e u r a l input o r i g i n a t i n g i n f a s t adapting pulmonary re c e p t o r s . A r t i f i c i a l v e n t i l a t i o n not only increased heart r a t e but o f t e n tended to r e s t o r e normal r e s p i r a t o r y a c t i v i t y . Pulmonary d e a f f e r e n t a t i o n by steaming el i m i n a t e d the Hering-Breuer r e f l e x to maintained lung i n f l a t i o n as w e l l as the c a r d i o a c c e l e r a t i o n seen i n response to a r t i f i c i a l v e n t i l a t i o n during n a s a l s t i m u l a t i o n . The l o s s of the Hering-Breuer r e f l e x occurred f i r s t suggesting that d i f f e r e n t receptors are i n v o l v e d . Lung d e f l a t i o n per se caused a r e f l e x bradycardia but i t appears that t h i s does not p o t e n t i a t e the i i i n a r i a l r e f l e x since nasal bradycardia was not reduced when lung i n f l a t i o n was maintained. Central and peripheral components a r i s i n g from res p i r a t o r y a c t i v i t y have t h e i r greatest e f f e c t during the recovery period. Elimination of the c a r o t i d bodies delayed but did not abolish chemorecep-tor driven bradycardia demonstrating that these are the most chemosensitive units but not the only ones responding to changes i n blood gas tensions. No r o l e however, has been found f o r the a r t e r i a l baroreceptors. The baro-s t a t i c r e f l e x brought on by drug induced hypertension was triggered at a lower pressure than that found i n the seal but i t appears that t h i s pressure would not be exceeded i n the muskrat i f heart rate remained low during a dive. It i s concluded that the cardiac response to submersion i n the muskrat r e s u l t s from at le a s t three r e f l e x arcs. These reflexes o r i g i n a t e from the nares, the lungs and from peripheral chemoreceptors. Although the chemorecep-tors act to maintain the p r e v a i l i n g diving responses, i t i s l i k e l y that the external n a r i a l r e f l e x accounts f o r almost a l l of the cardiovascular adjust-ment brought about i n normal foraging dives since these are usually of short duration. The chemoreflex could play a s i g n i f i c a n t r o l e i n dives exceeding one minute by prompting the animal to resurface when oxygen stores are depleted. i v TABLE OF CONTENTS General I n t r o d u c t i o n 1 Methods and M a t e r i a l s 14 Re s u l t s 26 1. Re s p i r a t o r y and Cardiac Responses to D i v i n g 26 1.1. Cardiac Response to Unrestrained D i v i n g 26 1.2. Cardiac Response to Restrained D i v i n g 34 1.3. Re s p i r a t o r y and Cardiac Responses to D i v i n g During Anaesthesia 34 2. Responses to Water S t i m u l a t i o n of the Nasal Area 43 2.1. E x t e r n a l N a r i a l S t i m u l a t i o n 43 2.2. I n t e r n a l Nasal S t i m u l a t i o n 47 3. A f f e r e n t and E f f e r e n t Pathways of the D i v i n g Reflexes 53 3.1. Morphology of the Nasal Receptor Areas 53 3.2. A f f e r e n t Pathway of the E x t e r n a l N a r i a l Reflexes 56 3.3. A f f e r e n t Pathway of the I n t e r n a l Nasal Reflexes 57 3.3.1. Nerve Blockade of the A f f e r e n t Pathway 57 3.3.2. E l e c t r i c a l S t i m u l a t i o n of the A f f e r e n t Pathway 63 3.3.3. Recordings from the I n f e r i o r Laryngeal Nerve 64 3.4. E f f e r e n t Pathway of the Cardiac R e f l e x 67 4. E f f e c t of Lung Input on R e s p i r a t i o n , Heart Rate and Blood Pressure 68 4.1. E f f e c t of A r t i f i c i a l V e n t i l a t i o n on Bradycardia Caused by Nasal S t i m u l a t i o n 68 4.2. E f f e c t of Maintained Lung I n f l a t i o n on the Cardiac Response to Nasal S t i m u l a t i o n 80 V 4.3. E f f e c t of Pulmonary Deafferentation on the Respiratory and Cardiac Responses to A r t i f i c i a l V e n t i l a t i o n 80 4.4. Discussion Summary E f f e c t of Lung D e f l a t i o n on Heart Rate and Blood Pressure Baroreceptor and Chemoreceptor Contributions to the Cardiovascular Responses 94 94 113 133 L i t e r a t u r e Cited 137 v i LIST OF FIGURES Figure 1. Diagram of the r e s p i r a t o r y v a l v e . 17 Figure 2. Mean heart r a t e during unrestrained d i v e s . 27 Figure 3. Comparison of ca r d i a c i n t e r v a l s i n t r a n s i t o r y and long d i v e s . 30 Figure 4. Electrocardiogram during u n r e s t r a i n e d d i v i n g . 32 Figure 5. Mean heart r a t e during r e s t r a i n e d d i v i n g . 35 Figure 6. Electrocardiogram during r e s t r a i n e d d i v i n g . 37 Figure 7. Mean heart r a t e during d i v i n g i n the anaesthetized muskrat. 39 Figure 8. R e s p i r a t o r y and car d i a c responses to submersion of the anaesthetized muskrat. 41 Figure 9. Cardiac response to n a r i a l water s t i m u l a t i o n i n the c u r a r i z e d a r t i f i c i a l l y v e n t i l a t e d muskrat. 45 Figure 10. Mean heart r a t e during apneic n a s a l water flow s t i m u l a t i o n . 48 Figure 11. Cardiac responses to non-apneic water flow s t i m u l a t i o n i n the anaesthetized and c u r a r i z e d anaesthetized muskrat. 51 Figure 12. In n e r v a t i o n of the nares and n a s a l and g l o t t a l mucosa. 54 Figure 13. E f f e c t of u n i l a t e r a l and b i l a t e r a l s e c t i o n of the m a x i l l a r y and i n f e r i o r l a r y n g e a l nerves on the r e s p i r a t o r y and car d i a c responses to n a s a l water flow s t i m u l a t i o n . 61 Figure 14. Nerve a c t i v i t y i n the i n f e r i o r l a r y n g e a l nerve i n response to pressure and water f l o w . s t i m u l a t i o n of the pharynx. 65 Figure 15. E f f e c t of a r t i f i c i a l v e n t i l a t i o n on the car d i a c response to non-apneic n a s a l s t i m u l a t i o n . 69 Figure 16. R e s p i r a t o r y and c a r d i a c responses to nasa l water and s a l i n e flow s t i m u l a t i o n . 72 v i i F igure 17. Cardiac response to n a s a l s t i m u l a t i o n during spon-taneous and a r t i f i c i a l v e n t i l a t i o n . 74 Figure 18. E f f e c t of a r t i f i c i a l v e n t i l a t i o n on the c a r d i a c response to apneic n a s a l s t i m u l a t i o n . 76 Figure 19. Cardiac response to v e n t i l a t i o n w i t h hyperoxic-normocapnic gas and a i r during apneic n a s a l s t i m u l a t i o n . 78 Figure 20. Cardiac response to a r t i f i c i a l v e n t i l a t i o n and maintained i n f l a t i o n during apneic n a s a l s t i m u l a t i o n . 81 Figure 21. Separation of c a r d i a c responses to v e n t i l a t i o n and maintained i n f l a t i o n . 83 Figure 22. Loss of c a r d i a c response to v e n t i l a t i o n during s e q u e n t i a l lung steaming. 85 Figure 23. E f f e c t of lung d e a f f e r e n t a t i o n on r e s p i r a t i o n , heart r a t e and the c a r d i a c response to n a s a l s t i m u l a t i o n . 87 Figure 24. A b o l i t i o n of the c a r d i a c response to a r t i f i c i a l v e n t i l a t i o n by lung steaming. 90 Figure 25. Loss of the c a r d i a c response to a r t i f i c i a l v e n t i l a t i o n i n 9 muskrats. 92 Figure 26. 'Cardiovascular e f f e c t s of lung inflation-'and deflation-. 95 Figure 27. E f f e c t of i n s p i r a t o r y pressure on the l a t e n t p e r i o d to bradycardia caused by lung i n f l a t i o n and d e f l a t i o n . 97 Figure 28. A b o l i t i o n of the b a r o s t a t i c r e f l e x by sinus nerve s e c t i o n . 100 Figure 29. E f f e c t of sinus nerve s e c t i o n on the c a r d i o v a s c u l a r responses to hypoxia. 102 Figure 30. Response of a r t e r i a l blood oxygen tension to asphyxia. 105 Figure 31. Mean heart r a t e during asphyxia and n a s a l s t i m u l a t i o n w i t h and without a r t i f i c i a l v e n t i l a t i o n . 107 Figure 32. Response of a r t e r i a l blood oxygen tension to n a s a l s t i m u l a t i o n . 109 Figure 33. Comparative p l o t of heart r a t e and a r t e r i a l blood oxygen during asphyxia and n a s a l s t i m u l a t i o n . I l l Figure 34. Neural mechanisms r e g u l a t i n g r e s p i r a t i o n and heart r a t e i n the muskrat. 130 v i i i LIST OF TABLES Table I . E f f e c t of nerve blockade and e l e c t r i c a l s t i m u l a t i o n on heart r a t e and r e s p i r a t i o n . Table I I . E f f e c t of n a s a l water flow on heart r a t e and r e s p i r a -t i o n before and a f t e r b i l a t e r a l s e c t i o n or c o l d blockade of the i n f e r i o r l a r y n g e a l and m a x i l l a r y nerves. Table I I I . Comparison of c a r d i a c responses. 129 i x ACKNOWLEDGEMENTS I wish to express my appreciation to Dr. David R. Jones f o r his advice and generous assistance throughout t h i s study. I also wish to thank Drs. William K. Milsom and Richard B r i l l f o r t h e i r h e l p f u l suggestions i n the preparation of the text and e s p e c i a l l y to my wife Judith who has borne much of the burden for far too long. F i n a n c i a l support was provided by Teaching Assistantships from the Department of Zoology and the University of B r i t i s h Columbia Graduate Fellowships. 1 General I n t r o d u c t i o n The r e s p i r a t o r y and c a r d i o v a s c u l a r events which occur during submersion of d i v i n g animals have a t t r a c t e d considerable a t t e n t i o n f o r over a century. These adjustments are w e l l known to play a major r o l e i n the a b i l i t y of the animal to t o l e r a t e the prolonged periods of asphyxia which can accompany submersion. This defense against asphyxia i s most prominent i n l a r g e aquatic mammals and i s manifested by apnea, a dramatic decrease i n car d i a c f u n c t i o n and an extensive r e d i s t r i b u t i o n of blood flow. Burne (1909) was the f i r s t to suggest that s e l e c t i v e r e d i s t r i b u t i o n of blood flow to the heart and b r a i n occurred during periods of asphyxia. He proposed that during asphyxia blood flow favoured t i s s u e s which are vuln e r a b l e to hypoxia and was reduced to those areas which can be supported by anaerobic metabolism. La t e r i t was found that many d i v e r s repay only a p o r t i o n of the oxygen debt which would be i n c u r r e d i f r e s t i n g metabolism was maintained during d i v i n g (Scholander et a l . , 1940; Andersen, 1961). In f a c t , metabolic r a t e d e c l i n e s during d i v i n g i n b i r d s ( P i c k w e l l and Douglas, 1968), r e p t i l e s (Andersen, 1961) and even i n f i s h exposed to a i r (Leivestad et a l . , 1957). Scholander et_ al. (1942a, b) were the f i r s t to e s t a b l i s h that anaerobic g l y c o l y s i s takes place i n t i s s u e s which are deprived of c i r c u l a t i o n during d i v i n g . More recent work has revealed that energy i n prolonged dives may be derived from carbohydrate and amino a c i d stores as w e l l as g l y c o l y t i c pathways (Hochachka et a l . , 1973). While there i s l i t t l e doubt that these biochemical processes p l a y a r o l e i n the defense against asphyxia, i t i s d i f f i c u l t to assess t h e i r c o n t r i b u t i o n s i n many animals. 2 However, i n at l e a s t one s p e c i e s , the sea t u r t l e which has been described: 1 as a \" f a c u l t a t i v e anaerobe 1', the biochemical adaptations seem to be the most important i n the defense against asphyxia (White and Ross, 1966; Hochachka and Storey, 1975). While the mechanisms of the d i v i n g r e f l e x e s have not been revealed u n t i l recent years, i t i s now c l e a r that t h e i r i n i t i a t i o n , at l e a s t i n mammals,\"is p r i m a r i l y of n a s a l o r i g i n (Andersen, 1966; A n g e l l James and Daly, 1972a; Daly and A n g e l l James, 1975). This recent work i n f a c t confirms i n p r i n c i p l e the work of Beau (1860) who f i r s t suggested that the responses were, i n p a r t , r e f l e x l y i n i t i a t e d by impulses c a r r i e d i n the t r i g e m i n a l nerve. His view was l a t e r disputed by Bert (1870) who b e l i e v e d that the c a r d i o v a s c u l a r adjustments r e s u l t e d from v o l u n t a r y breath h o l d i n g . At the same time Kratchmer (1870) found e l e c t r i c a l s t i m u l a t i o n of the t r i g e m i n a l root to be i n e f f e c t i v e i n evoking c a r d i o - r e s p i r a t o r y changes i n both cats and dogs. La t e r however, h i s observations were questioned by F r a n c o i s -Franck (1889) who was able to generate apneic and bradycardic responses from s t i m u l a t i o n of the same nerve; r e s u l t s which were u l t i m a t e l y confirmed by Richet (1899) and Brodie and R u s s e l l (1900). Richet was a l s o able to show that the e f f e r e n t pathway of :the cardio-depressant response i n the duck was c a r r i e d i n the vagus nerve. His work i n p a r t i c u l a r e s t a b l i s h e d the importance of n a s a l water contact i n the i n i t i a t i o n of the c a r d i o - r e s p i r a t o r y adjustments which are now known as the d i v i n g r e f l e x e s . A number of important elements of the d i v i n g r e f l e x e s were explored i n 1913. In that year Huxley (1913) demonstrated the involvement of a p o s t u r a l component thought to be a s s o c i a t e d w i t h the d i v i n g responses i n the duck. She was able to show i n decerebrated animals that both apneic and c a r d i a c 3 responses could be evoked by v e n t r i f l e x i n g the head; a r e f l e x that i s able to f u n c t i o n independently of higher centre c o n t r o l . In the same year t h i s p o s t u r a l r e f l e x was described as p a r t of l a b y r i n t h i n e f u n c t i o n and the a f f e r e n t pathway shown to be c a r r i e d by the c e r v i c a l nerves (Patton, 1913). This was followed by a c o n t r o v e r s i a l piece of work i n the duck concerning the e f f e c t of carbon d i o x i d e on r e s p i r a t i o n (Orr and Watson, 1913). The paper was p a r t i c u l a r l y s i g n i f i c a n t because i n s p i t e of t h e i r erroneous co n c l u s i o n that r e s p i r a t i o n i s i n h i b i t e d by carbon d i o x i d e , r e c o g n i t i o n was given to both the r o l e s of e x t e r n a l and humoral d r i v e s during periods of asphyxia. This was l a t e r to have a profound i n f l u e n c e on s t u d i e s r e l a t e d to the d i v i n g c o n d i t i o n . The r o l e of the t r i g e m i n a l r e f l e x i n d i v i n g was a l s o s t u d i e d i n the same year. Experimenting w i t h ducks, Lombroso (1913) demonstrated the presence of bradycardia when lung i n f l a t i o n was maintained and confirmed the e a r l i e r view h e l d by Beau and R i c h e t . Studies on the i n i t i a t i o n of the d i v i n g r e f l e x were not resumed u n t i l Vincent and Cameron (1920) and l a t e r Koppanyi and Dooley (1928) concluded that submersion apnea i n the duck was caused by wetting the beak, nares and mucous membranes of the n a s a l c a v i t y . In a subsequent paper, Koppanyi and Dooley (1929) suggested that a p o s t u r a l component was a l s o . i n v o l v e d i n the d i v i n g r e f l e x of the muskrat. Much l a t e r , Andersen (1963a,c) and F e i g l and Folkow (1963) confirmed that the r e f l e x i n the duck was i n i t i a t e d by s p e c i f i c receptors i n the nares and that the subsequent bradycardia was augmented by hypoxia and hypercapnia. Andersen found that bradycardia and apnea were unaffected by decerebration but were ab o l i s h e d by d i v i s i o n of the ophthalmic and mandibular branches of the t r i g e m i n a l nerve. He conclud-ed that the former branch was the major a f f e r e n t nervous pathway of the 4 card i a c response and was t r i g g e r e d by water immersion per se. In recent years, a t t e n t i o n has been focused on a v a r i e t y of f a c t o r s which c o n t r i b u t e to underwater endurance, p a r t i c u l a r l y i n aquatic mammals. Behavioural adaptations to d i v i n g have.a marked i n f l u e n c e on the responses i n many d i v i n g animals. B i r d s and mammals which are accustomed to aq u a t i c environments tend to r e l a x when they are f o r c i b l y submerged ( I r v i n g , 1939; Andersen, 1966) yet s t i l l e x h i b i t s t r i k i n g responses whether they s t r u g g l e or not. I r v i n g e_t a l . (1941b) f r e q u e n t l y observed that the d i v i n g a b i l i t y of the s e a l was not f i x e d but i n f a c t was l a b i l e and depended on a \"steady d i s p o s i t i o n \" . C o n d i t i o n i n g a l s o seems to play a r o l e i n the expression of c a r d i o v a s c u l a r responses to d i v i n g . I n experiments on human s u b j e c t s , t r a i n i n g has been shown to improve the d i v i n g r e f l e x e s and the d u r a t i o n of breath h o l d i n g during face immersion (Whayne and K i l l i p , 1967; C o r r i o l and Rohmer, 1968); a consequence probably r e l a t e d to the s t a t e of apprehension of the untrained group. In dolphins t r a i n e d to dive on command, d i v i n g bradycardia i s prominent, and s t a b l e but q u i t e u n l i k e the w i l d l y f l u c t u a t i n g heart r a t e which c h a r a c t e r i z e s spontaneous d i v i n g (Eisner e_t a l . , 1966b). I r v i n g et a l . (1941a) a l s o noted that the dolphin responds poorly to forced submersion and i s e a s i l y asphyxiated i n short dives of t h i s nature. While m o d i f i c a t i o n by c o n d i t i o n i n g tends to improve the tol e r a n c e to asphyxia, the d i v i n g r e f l e x seems to be f u l l y developed at b i r t h and i n some cases has been shown to decrease w i t h age. In the elephant s e a l , bradycardia r e s u l t i n g from forced submersions i s more pronounced i n the newborn c a l f than i n the a d u l t (Hammond et aL , 1969). In s p i t e of t h i s , the newborn c a l f appears incapable of dives exceeding two minutes. T h i s , however, may be due to the lower blood oxygen ca p a c i t y of the young animal and a f a r greater b r a i n to body weight r a t i o . In con t r a s t to the elephant s e a l , the sm a l l e r pinnipeds tend to increase the i n t e n s i t y of the r e f l e x to submersion as they mature (Harrison and Tomlinson, 1960) suggesting that experience improves d i v i n g performance i n some species. F e t a l and h i b e r n a t i n g animals respond i n much the same manner as the d i v i n g mammals to hypoxic s t r e s s (Scholander, 1960). Bradycardia o f t e n occurs during d e l i v e r y and i s i n v a r i a b l y accompanied by a post n a t a l r i s e i n blood l a c t a t e (James, 1962) s i m i l a r to-the changes which occur i n non-c i r c u l a t e d v a s c u l a r beds f o l l o w i n g submersion i n the d i v i n g animal (Scholander, 1940; Scholander et a l . , 1942a; Clausen and Er.sland, 1970/71). S e l e c t i v e r e d i s t r i b u t i o n of blood which favours the heart and b r a i n has a l s o been observed i n the f e t a l lamb during maternal hypoxia (Parker and Purves, 1967) although flow to the fetus i s maintained (Eisner jet juL. , 1969) . In other s t u d i e s on s e a l s , i t has been found that the onset of and recovery from asphyxia induced bradycardia i n the f e t u s l a g s w e l l behind that of the mother (Eisner e^ t a l . , 1969) . The maintenance of apnea i n utero has r a i s e d the question about whether the f e t a l and d i v i n g r e f l e x e s are s i m i l a r . Tchobroutsky at a l . (1969) noted that f e t a l apnea was r e l a t e d to an \" i n h i b i t o r y f a c t o r \" ( B a r c r o f t , 1946) and the absence of e x t e r n a l s t i m u l i (Reynolds, 1962) and suggested that i n i t i a t i o n of r e s p i r a t i o n at b i r t h may r e s u l t from t h e i r withdrawal. R e s p i r -a t o r y airway f l u i d , known to be present i n f e t a l development, i s thought to be the i n h i b i t o r y f a c t o r causing apnea. The onset of h i b e r n a t i o n i n c e r t a i n animals i s of p a r t i c u l a r i n t e r e s t because of the appearance of bradycardia before a decrease i n body temperature sets i n , i n d i c a t i n g that i t s cause might be a primary v a s o c o n s t r i c t i o n (Scholander, 1963). A r o u s a l from the 6 h i b e r n a t i n g s t a t e a l s o seems to be r e l a t e d to a primary vasomotor response r e s u l t i n g i n an increase i n blood f l o w to the limbs and an e l e v a t i o n i n metabolic r a t e ( E l i a s s e n , 1960b). From an e v o l u t i o n a r y sense, these r e f l e x e s which c h a r a c t e r i z e such a broad range of p h y s i o l o g i c a l behaviour have given r i s e to s p e c u l a t i o n that they form p a r t of a \"general adaptation syndrome\" common to a l l animals (Selye, 1949). Thus as i n the neonate and h i b e r n a t o r , the adjustments to d i v i n g i n the aquatic mammal do not imply a unique r e f l e x but rather suggest them to be a refinement of a s t e r e o t y p i c r e f l e x . P o i k i l o t h e r m s such as snakes (Johansen, 1959), t u r t l e s (White and Ross, 1966) and a l l i g a t o r s (Andersen, 1961) d i s p l a y marked responses to submersion even though they appear much more slowly than i n mammals. The r e f l e x e s have a l s o been observed i n anurans (Jones, 1966; West and Jones, 1976) i n s p i t e of t h e i r a b i l i t y to provide metabolic needs through cutaneous r e s p i r a t i o n , and i n f i s h exposed to hypoxia ( S a t c h e l l , 1961) or removed from water ( L e i v e s t a d et a l . , 1957). Homeotherms are v u l n e r a b l e to even short periods of asphyxia and not s u r p r i s i n g l y possess r e f l e x e s which are f a r more dramatic than those found i n the c o l d blooded species. In the harbour s e a l , f o r example, c a r d i a c responses occur before submersion and underwater endurance i s 4 to 5 times greater than that p r e d i c t e d from normal body oxygen st o r e s i f surface l e v e l s of metabolism are maintained ( I r v i n g et_ _ a l . , 1941b) and some of the l a r g e r pinnipeds are able to remain submerged f o r periods up to one hour (Kooyman and Andersen, 1969). The a b i l i t y of the animals to withstand lengthy periods of asphyxia yet remain a c t i v e during submersion has been shown to depend l a r g e l y on anatomical and p h y s i o l o g i c a l f e a t u r e s (Scholander, 1963; E i s n e r et a l . , 1966a; E i s n e r , 1969; Yonce and Folkow, 1970; Daly, 1972). 7 Anatomical adaptations f o r d i v i n g , e s p e c i a l l y those i n aquatic mammals, have been recognized f o r over a century (Hunter, 1787; Burow, 1838). M o d i f i -c a t i o n of the r e s p i r a t o r y organs i n these d i v e r s however, i s o f t e n misleading s i n c e some features are r e l a t e d to r e l i e f from the e f f e c t s of pressure r a t h e r than to conservation of oxygen (Harrison and Kooyman, 1971; Hempleman and Lockwood, 1978). As a r u l e l a r g e t i d a l volumes r e l a t i v e to body weight and a slow breathing frequency are a s s o c i a t e d w i t h the b e t t e r d i v e r s (Kooyman, 1973). While t h i s allows a higher degree of oxygen u t i l i z a t i o n during normal r e s p i r a t i o n , e v i d e n t l y none of the shallow d i v i n g animals takes advantage of t h i s p o t e n t i a l s t o r e . Shallow d i v e r s have been reported to exhale on submersion (Kooyman and Andersen, 1969) but t h i s may not always be the case. Ducks (Andersen, 1966), muskrats ( E r r i n g t o n , 1963) and s e a l s (Kooyman et a l . , 1971) have been observed to r e l e a s e a i r during shallow d i v e s . In c o n t r a s t to pinnipeds, the deep d i v i n g cetaceans have r e l a t i v e l y small lung volumes and d i v i n g o f t e n takes place w i t h the lungs f u l l y i n f l a t e d (Kooyman and Andersen, 1969). Because of the r e l i a n c e of aquatic b i r d s and mammals on a v a i l a b l e oxygen stores during d i v i n g , blood c h a r a c t e r i s t i c s of these d i v e r s have recei v e d s p e c i a l a t t e n t i o n . Richet (1899) was the f i r s t to note the s i g n i f i c a n c e of l a r g e blood volumes i n ducks accustomed to prolonged d i v e s . In some, such as the d i v i n g g u i l l e m o t , the blood volume i s n e a r l y double that i n s i m i l a r t e r r e s t r i a l species ( I r v i n g , 1939). Cetaceans and pinnipeds i n p a r t i c u l a r , possess l a r g e venous r e s e r v o i r s which are h i g h l y oxygenated and provide s i z e a b l e oxygen reserves which can be drawn upon i n asphyxic periods. Oxygen bi n d i n g c a p a c i t y and the Bohr f a c t o r have been considered 8 as l i k e l y adaptations to hypoxic environments s i n c e both ensure e f f i c i e n t u t i l i z a t i o n of hemoglobin bound oxygen. The oxygen b i n d i n g c a p a c i t y of the blood i n many of these animals i s unusually high and s i m i l a r to that found i n burrowing and high a l t i t u d e mammals . ( B a r t e l s , 1964; H a l l et a l . , 1936) but oddly there seems to be l i t t l e r e l a t i o n s h i p between t h i s adaptation and d i v i n g a b i l i t y . While the oxygen c a p a c i t y of s e a l blood i s approximately 45% higher than i n man, the bloods of the beaver, sea l i o n and some whales have been found to be no d i f f e r e n t from man ( I r v i n g , 1939). S i m i l a r l y , the blood of many aquatic mammals does not have a l a r g e Bohr f a c t o r (Clausen and Er s l a n d , 1968). Muscle t i s s u e may a l s o be a va l u a b l e source of oxygen during times of hypoxic s t r e s s . Robinson (1939) has estimated that oxygen i n the form of oxymyoglobin accounts f o r 47% of the st o r e a v a i l a b l e to the harbor ' s e a l during d i v i n g . I t has been claimed f o r many years that chemoreceptors i n d i v i n g animals are l e s s s e n s i t i v e to asphyxia than those' i n t e r r e s t r i a l s s p e c i e s . Apneic Weddell s e a l s f o r example, have been shown to t o l e r a t e a PaC^ of 8 mm Hg simultaneously w i t h a PaCC^ of 80 mm Hg without s t a r t i n g b r e a t h i n g (Eisner eX al., 1970). In the hooded s e a l , v o l u n t a r y breath h o l d i n g can be maintained u n t i l Pa02 i s 14-16 mm Hg before r e b r e a t h i n g begins. R e s p i r a t o r y i n s e n s i t i v i t y has a l s o been noted i n the duck (Orr and Watson, 1913), the beaver ( I r v i n g , . 1938) and a number of r e p t i l e s ( D i l l and Edwards, 1931; Johansen, 1959) suggesting that at l e a s t i n aquatic animals, the withdrawal of chemoreceptor d r i v e tends to promote d i v i n g responses. Andersen (1966) however, s t a t e d that on the b a s i s of two st u d i e s i n the s e a l (Robin et a l . , 1963) and duck (Andersen and Lv<&; 1964) that r e s p i r a t o r y d r i v e was l i k e l y to be increased over the range of carbon d i o x i d e tensions experienced i n 9 d i v i n g and i n d i r e c t l y r a i s e d questions concerning the mechanisms of chemo-receptor i n t e g r a t i o n w i t h the primary responses o c c u r r i n g e a r l y i n the d i v e . L i k e hypercapnia, hypoxia i n the absence of other f a c t o r s s t i m u l a t e s r e s p i r a t i o n (Andersen, 1959; Cherniak et a l . , 1970/71). Both Scholander (1940) and E i s n e r et_ al. (1970) have suggested that the t o l e r a n c e of s e a l s to hypoxia may be l e s s than to carbon d i o x i d e s i n c e the animals show sign s of d i s t r e s s a f t e r breathing n i t r o g e n f o r only a short time. E v i d e n t l y the t r a i n of oxygen sparing adjustments i s not i n i t i a t e d by v e n t i l a t i o n hypoxia. I t i s now g e n e r a l l y recognized that at l e a s t two inputs c o n t r i b u t e to the c a r d i o v a s c u l a r and r e s p i r a t o r y r e f l e x e s a s s o c i a t e d w i t h d i v i n g ( A n g e l l James and Daly, 1975) but only r e c e n t l y have t h e i r i n t e r a c t i o n s been examined. A t r i g e m i n a l r e f l e x , i d e n t i f i e d by Andersen (1963c) i s considered to i n i t i a t e the necessary adjustments to submersion before any marked changes occur i n blood gas tensions. A chemoreflex on the other hand, r e s u l t i n g l a r g e l y from hypoxic and hypercapriic s t i m u l a t i o n of the c a r o t i d bodies, i s thought to i n t e n s i f y the primary r e f l e x e s i n prolonged dives ( A n g e l l James and Daly, 1975). Thus i t i s c l e a r that a f i n e l y set c e n t r a l mechanism i s r e q u i r e d to i n t e g r a t e the a f f e r e n t inputs to evoke the appropriate c a r d i o v a s ^ c u l a r and r e s p i r a t o r y adjustments during d i v i n g . In view of the interdependence of the c a r d i o v a s c u l a r and r e s p i r a t o r y centres i t i s not s u r p r i s i n g t h a t a good deal of controversy s t i l l surrounds t h e i r r e s p e c t i v e r o l e s i n the c o n t r o l of the d i v i n g r e f l e x e s . In f a c t , i t i s the complexity of these c e n t r a l mechanisms which has o f t e n l e d to the confusion of cause and e f f e c t (Anrep e t a l . , 1936a). Thus through the work of Bainbridge (1920) and Hering (1933) there p e r s i s t e d a b e l i e f that r e s p i r a -tory movement accompanied c a r d i a c arrhythmia but was not i t s cause. 10 H i s t o r i c a l l y , many workers b e l i e v e d that the r e s p i r a t o r y i n f l u e n c e on heart r a t e was a p u r e l y c e n t r a l phenomenon independent of p e r i p h e r a l f a c t o r s (Traube, 1865; Snyder, 1915; Heymans, 1929). This u n c e r t a i n t y however, l e d Anrep et. a l . (1936a, b) to a s e r i e s of c l a s s i c experiments which c l e a r l y demonstrated that i n dogs, sinus arrhythmia has both c e n t r a l and p e r i p h e r a l o r i g i n s . The conclusions of Anrep jet a l . proved to be s i g n i f i c a n t i n the l i g h t of the work of Daly and Scott (1958, 1962) i n which they showed that primary c a r d i o v a s c u l a r r e f l e x e s evoked by c a r o t i d body s t i m u l a t i o n could be g r e a t l y a l t e r e d by lung v e n t i l a t i o n . Increases i n v e n t i l a t i o n of spontaneous-l y b r e a thing animals are known to o v e r r u l e bradycardia and v a s o c o n s t r i c t i o n caused by apneic asphyxia: both responses being regarded as primary r e f l e x e s i n the n o n - v e n t i l a t e d animal ( B e r n t h a l , 1938; B e r n t h a l et a l . , 1951; Daly and Daly, 1959; Daly and Hazeldine, 1963). Thus i t i s c l e a r that mechanical s t i m u l a t i o n of lungs may evoke concomitant changes i n c e n t r a l r e s p i r a t o r y a c t i v i t y which tend to reverse the primary consequences of chemoreceptor s t i m u l a t i o n . I t i s apparent from these f i n d i n g s that the s t a t e of r e s p i r a t o r y a c t i v i t y determines both the nature and the degree of c a r d i o v a s c u l a r responses to asphyxia. Recently considerable a t t e n t i o n has been devoted to the i n t e r a c t i o n of the r e f l e x e s during c o n d i t i o n s of acute s t r e s s such as hemorrhage, v e n t i l a -t i o n hypoxia and apneic asphyxia (Andersen, 1966; Daly, 1972; Daly and A n g e l l James, 1975): the l a t t e r c o n d i t i o n o c c u r r i n g most commonly during submersion. Much of the a t t e n t i o n however, has been focused on the i n f l u e n c e of chemoreceptor d r i v e ( F e i g l and Folkow, 1963; Kawakami et a l . , 1967; C o r r i o l and Rohmer, 1968; Daly and A n g e l l James, 1975) and l i t t l e c r e d i t has been given to the immediate e f f e c t s of r e f l e x apnea. I t i s now 11 c l e a r that apnea per se i n i t i a t e d by water contact not only serves as a p r o t e c t i v e r e f l e x by preventing the i n h a l a t i o n of water, but a l s o allows the expression of the e s s e n t i a l c a r d i o v a s c u l a r adjustments. In prolonged d i v i n g however, the primary chemoreceptor i n f l u e n c e on r e s p i r a t i o n has been questioned s i n c e i t f a i l s to produce r e s p i r a t o r y breakthrough as p r e d i c t e d from p r e l i m i n a r y s t u d i e s (Daly and Ungar, 1966). Attempting to r e s o l v e t h i s paradox, A n g e l l James and Daly (1972b) and Daly e_t a l . (1977) have shown that the chemoreceptors respond d i f f e r e n t l y during face submersion than during purely asphyxic c o n d i t i o n s . When t r i g e m i n a l and c a r o t i d body s t i m u l a t i o n were a p p l i e d together apnea was provoked i n f r e e l y breathing dogs which p e r s i s t e d much longer than when nasa l s t i m u l a t i o n was given alone. The bradycardia and vasomotor responses were a l s o found to increase i n i n t e n s i t y . These r e s u l t s i n d i c a t e that the t r i g e m i n a l r e f l e x not only f a c i l i t a t e s the ca r d i o v a s c u l a r responses o c c u r r i n g during asphyxia but a l s o tends to reverse the primary s t i m u l a t i n g e f f e c t of chemoreceptors on r e s p i r a t i o n and delays premature breakthrough. Thus i f t h i s c o n c l u s i o n i s extended to d i v i n g b i r d s and mammals, i t appears to r e s o l v e the paradox noted p r e v i o u s l y by Andersen (1966) that r e s p i r a t o r y d r i v e i s increased by asphyxia i n the duck and s e a l but not during asphyxia combined w i t h n a s a l s t i m u l a t i o n . Lung c o l l a p s e during submersion has l e d to s p e c u l a t i o n that i t might be a r e q u i s i t e f o r optimum c a r d i o v a s c u l a r adjustment during d i v i n g (Andersen, 1966; Yonce and Folkow, 1970; A n g e l l James and Daly, 1969b). Some avian d i v e r s when f a i l i n g to exhale before submersion, develop bradycardia and p o s s i b l y other responses only slowly (Andersen, 1963a; E l i a s s e r i et a l . , 1960a) which suggests that pulmonary receptors i n i t i a t e increases i n heart r a t e and tend to normalize blood flow f o l l o w i n g submersion. I t has been 12 p o s t u l a t e d f o r some time that the c a r d i o - i n h i b i t o r y centre i s under the i n f l u e n c e of two inputs from the lungs, one causing t a c h y c a r d i a during i n s p i r a t i o n and the other bradycardia on e x p i r a t i o n by way of the r e s p i r a t o r y centre (Cordier and Heymans, 1935). Thus aside from s t a t i c c o n d i t i o n s , lung receptors responding to expansion and c o l l a p s e of the lung are seen to have a dual c a p a c i t y i n s t i m u l a t i n g and i n h i b i t i n g the c a r d i o v a s c u l a r centre. Many animals d i s p l a y rhythmic bursts of c a r d i a c a c t i v i t y i n the f i r s t few breaths on emersion ( I r v i n g et a l . , 1941b; Johansen,. 1959; Andersen, 1963a, b; Tchobroutsky et a l . , 1969); a r e f l e x too r a p i d to be a s s o c i a t e d w i t h a change i n chemoreceptor a c t i v i t y . The a f f e r e n t arm of the c a r d i a c response i s considered to o r i g i n a t e i n the nervous plexus of the b r o n c h i o l e s s i n c e the r e f l e x i s a b o l i s h e d by steam i n h a l a t i o n (Hainsworth e_t a l . , 1972). I t would seem that lung i n f l a t i o n has i t s g r e a t e s t e f f e c t at the onset of breathing a f t e r a dive not only because of the pronounced r e s p i r a t o r y a c t i v i t y but a l s o because of the depressed c a r d i o v a s c u l a r a c t i v i t y . In summary, i t appears that w h i l e the t r i g e m i n a l and chemoreceptor r e f l e x e s have been w e l l documented, the involvement of s t a t i c and phasic lung i n f l a t i o n s i n the d i v i n g r e f l e x e s has been l a r g e l y s p e c u l a t i v e . I t has been claimed that the n a s a l and chemoreceptor r e f l e x e s i n t e r a c t to enhance the c a r d i o v a s c u l a r adjustments to d i v i n g ( A n g e l l James and Daly, 1973; Str^mme and B l i x , 1976; E i s n e r et a l . , 1977), the modulation of which r e s t s w i t h the occurrence of r e s p i r a t o r y a c t i v i t y ( A n g e l l James and Daly, 1978). The r o l e of a r t e r i a l baroreceptors i n the d i v i n g responses remains c o n t r o v e r s i a l . In b i r d s , s e l e c t i v e denervation of a o r t i c baroreceptors has no s i g n i f i c a n t e f f e c t on d i v i n g bradycardia (Jones, 1973; Jones and West, 1978), yet others have claimed that d i v i n g bradycardia r e s u l t s from the b a r o s t a t i c r e f l e x i n response to chemoreceptor induced p e r i p h e r a l v a s o c o n s t r i c t i o n (Andersen and 13 B l i x , 1974; B l i x et a l . , 1974; B l i x , 1975; B l i x et a l . , 1975). Baroreceptors appear to c o n t r i b u t e to the bradycardia caused by n a s a l s t i m u l a t i o n i n the r a b b i t (White and M c R i t c h i e , 1973); i n other mammals t h e i r r o l e has not been f u l l y e l u c i d a t e d ( A n g e l l James et a l . , 1978). The purpose of the present i n v e s t i g a t i o n was to examine the c a r d i o -depressant and cardiac s t i m u l a t i n g pathways i n an accomplished d i v e r , the muskrat (Ondatra z i b e t h i c a osoyoosensis), to e s t a b l i s h which are important i n the i n i t i a t i o n and maintenance of submersion bradycardia. Muskrats are e a s i l y obtained and managed i n the l a b o r a t o r y and d i v i n g bradycardia appears to be unaffected by anaesthesia. The study was a l s o undertaken i n the hope th a t i t would prove u s e f u l i n e l u c i d a t i n g c e n t r a l n e u r a l i n t e g r a t i o n of the r e f l e x e s bearing on c a r d i a c f u n c t i o n . 14 Methods and M a t e r i a l s Experiments i n t h i s study were c a r r i e d out on 162 a d u l t and subadult muskrats of both sexes v a r y i n g i n weight from 0.61 to 1.35 kg. The animals were trapped e i t h e r i n the sloughs and di t c h e s of the Fraser R i v e r d e l t a or i n the marshlands i n the v i c i n i t y of P i t t Meadows, B. C. Although experiment-a t i o n was c a r r i e d on throughout the year, v i r t u a l l y a l l the animals were trapped i n the winter months (November through February) as trapping was not u s u a l l y s u c c e s s f u l i n the s p r i n g and summer. The muskrats were he l d i n pens, u s u a l l y i n p a i r s , f o r up to 8 months and were sustained on a d i e t of high p r o t e i n r a b b i t \"chow\" (Purina) supplemented w i t h l e t t u c e and c a r r o t s . The r a b b i t chow was i n i t i a l l y medicated w i t h 0.03% s u l f a q u i n o x a l i n e to prevent suspected h e p a t i c c o c c i d i o s i s but the drug was found to be de t r i m e n t a l s i n c e a number of muskrats succumbed to Tyzzer's disease (Chalmers and M c N e i l l , 1977) , u s u a l l y between the f i r s t and second week of c a p t i v i t y . Each hol d i n g pen (3 x 1 x 1.5 om) was constructed of s t e e l and contained an enclosed p l a t f o r m and a wooden n e s t i n g box. Water under the p l a t f o r m was kept at a depth of 0.5 m w i t h a continuous flow. Operative Procedures and Recording Techniques A l l o p e r a t i v e procedures were performed under urethane (950-1350 mg/kg) or nembutal (60 mg/kg) anaesthesia administered by i n t r a p e r i t o n e a l i n j e c t i o n . P r i o r to i n j e c t i o n the muskrats were sedated w i t h e t h y l ether vaporized i n a closed j a r ; the procedure t a k i n g approximately 3 minutes. A number of experiments were c a r r i e d out on animals paralyzed by the i n j e c t i o n of curare (Tubocurarine, 2 mg/kg i.p.) and i n some cases the areas of i n c i s i o n were i n f i l t r a t e d w i t h l o c a l a n aesthetic (Xy l o c a i n e , 2%). During a l l s u r g i c a l procedures body temperature was maintained at 37.5±0.5°C w i t h an overhead lamp. 15 In p r e p a r a t i o n f o r r e c o r d i n g electrocardiograms i n u n r e s t r a i n e d d i v i n g , the muskrats were f i r s t anaesthetized w i t h nembutal and a s i l i c o n e d baseplate (1 x 1.5 cm) bearing female microconnector plugs was placed sub-cutaneously between the c l a v i c l e s . B i p o l a r e l e c t r o c a r d i o g r a p h i c e l e c t r o d e s (formex coated copper w i r e , 0.5 mm diameter) were l e d from the microconnector plugs, beneath the s k i n and sewn i n place at the chest and the base of the t a i l . The animals were allowed to recover overnight before experimentation. For r e s t r a i n e d d i v i n g , the muskrats were sedated w i t h ether and then f i t t e d w i t h surface chest and t h i g h copper wire e l e c t r o d e s . Submersion of the animal was done a f t e r recovery from anaesthesia and was made i n an open topped perspex box (8 x 10 x 28 cm) which was p e r f o r a t e d to a l l o w r a p i d entry of water. The h o l d i n g box was mounted above a l a r g e r perspex box c o n t a i n i n g water which could be r a i s e d q u i c k l y and locked at the d e s i r e d l e v e l . Anaesthetized muskrats were a l s o dived under the same c o n d i t i o n s was described f o r r e s t r a i n e d d i v i n g above. Breathing was monitored by one of three techniques: pneumotachograph recordings from an attachment to a t r a c h e a l cannula, impedance changes across the chest or by thermistor recordings of expired and i n s p i r e d a i r . A tygon cannula (2.5 mm o.d.) was inserted.through a t r a c h e a l i n c i s i o n 1.0-1.5 cm above the b r o n c h i a l b i f u r c a t i o n . The pneumotachograph output connections were made to a d i f f e r e n t i a l transducer (Hewlett Packard, type 268 B) and the s i g n a l a m p l i f i e d by a LVDT coupler (Beckman). In some cases the s i g n a l obtained from the coupler was passed through an i n t e g r a t i n g p r e a m p l i f i e r (Hewlett Packard 3700A) to give t i d a l volume. The pneumotacho-graph was of a minimum s i z e so that i t added l i t t l e to the volume of the dead space. Impedance changes were taken from a Harvard impedance pneumo-graph (Model 391). For thermistor r e c o r d i n g s , a bead thermistor was 16 mounted e i t h e r i n the t r a c h e a l cannula or i n the n a s a l c a v i t y through a small hole bored i n the nasa l bone. The leads from the thermistor were connected to a standard bridge c i r c u i t . A l l s i g n a l s were a m p l i f i e d by conventional means and d i s p l a y e d on a storage o s c i l l o s c o p e (Tektronix 564 B) and e i t h e r on a Harvard 4 channel (Model 486) or a Beckman 2 channel (RS type) pen recorder. A r t i f i c i a l v e n t i l a t i o n was regulated using a small p o s i t i v e pressure v a l v e ( F i g . 1). The v a l v e c o n s i s t e d of a two chambered t e f l o n b a r r e l (2.5 x 5.0 cm) and a brass casing (4.4 x 6.0 cm) which allowed the passage of a i r to and from a Y piece f i x e d to the t r a c h e a l cannula. F i v e e q u a l l y spaced holes (30° apart) and two o f f s e t holes were d r i l l e d i n the casing and . occupied 120° of the perimeter so that the i n s p i r a t o r y and e x p i r a t o r y d u r a t i o n times could be v a r i e d between 33 and 67% of the r e s p i r a t o r y c y c l e . The b a r r e l was grooved to correspond w i t h the two sets of holes i n the casing and each groove was m i l l e d through 240° of the b a r r e l perimeter so that the two were o f f s e t by 120° i n the c r o s s - s e c t i o n a l a x i s . E x t e r n a l connections were made w i t h p o l y v i n y l tubing (4 mm i.d.) to the two va l v e chambers ( e x p i r a t i o n and i n s p i r a t i o n ) and the remaining holes were stoppered. E x p i r a t i o n was e i t h e r passive (normal) or induced ( a s p i r a t i o n ) . The va l v e and the s h a f t of the b a r r e l were h e l d . i n place by a brass cover f i x e d to the casing and were r o t a t e d by a v a r i a b l e speed motor attached by a f l e x i b l e hose. Gas flow s u p p l i e d to the i n s p i r a t o r y chamber was regulated by having the gas overflow from a s i d e arm of the i n l e t tube. The s i d e arm was placed so that the opening was at various depths i n the water f i l l e d c y l i n d e r and a b a l l o o n was attached on the i n l e t s i d e of the va l v e to l e s s e n pressure f l u c t u a t i o n s caused by va l v e c l o s u r e . 17 Figure\"1. Diagram of the r e s p i r a t o r y valve. Refer to text f o r d e s c r i p t i o n . 18 RESPIRATORY PRESSURE VALVE top view ' O 1 o o O 2 O inspO 3 Oexp O 4 O o O 5 O cross section of valve chamber aperture percent pairing resp. cycle insp exp 15 11 25 21 35 3 ' 45 41 55 51 insp exp 33 67 42 58 50 50 58 42 67 33 19 The v e n t i l a t i o n v a l v e possessed a number of advantages i n that i t could be placed some di s t a n c e from the animal as w e l l as a l l o w i n g quick and easy adjustment of the v e n t i l a t i o n r a t e , t i d a l volume and i n s p i r a t o r y - e x p i r a t o r y r a t i o . In many experiments i n f l a t i o n and d e f l a t i o n of the lungs was achieved by stopping the v a l v e i n the i n s p i r a t o r y or e x p i r a t o r y p o s i t i o n r e s p e c t i v e l y . A more s u i t a b l e technique however, was to simply clamp e i t h e r the i n s p i r a t o r y or e x p i r a t o r y tubes l e a d i n g to the v a l v e . In experiments i n v o l v i n g the determination of the Hering-Breuer r e f l e x to constant I n f l a t i o n , the l a t t e r was the only method used. P r i o r to experiments i n v o l v i n g c r a n i a l nerve s e c t i o n , the animals were anaesthetized w i t h urethane and a de n t a l burr was used to remove a p o r t i o n (1 x 1.5 cm) of the p a r i e t a l bone. Normally the s a g g i t a l sinus was l i g a t e d , the dura r e f l e c t e d and the c e r e b r a l hemispheres a s p i r a t e d w i t h a curved p i p e t . O c c a s i o n a l l y the f r o n t a l bone was pared to the f l o o r of the c r a n i a l c a v i t y to expose the m a x i l l a r y nerve trunk and i t s branches. Bleeding was c o n t r o l l e d by c a u t e r i z a t i o n . The o l f a c t o r y nerve, f a c i a l and p e t r o s a l nerves, mandibular and m a x i l l a r y d i v i s i o n of the t r i g e m i n a l nerve were exposed i n t r a c r a n i a l l y a f t e r decerebration. The i n f e r i o r and s u p e r i o r l a r y n g e a l , glossopharyngeal, s i n u s , vagus and phrenic nerves were approached v e n t r a l l y through an i n c i s i o n running from the lower jaw to the thorax. Nerve blockade was accomplished e i t h e r by s e c t i o n or by c o o l i n g i n a s p e c i a l l y designed thermode s i m i l a r to that described by Douglas and Malcolm (1955). The thermode c o n s i s t e d of an i n s u l a t e d s i l v e r probe (7 x 4 x 1-3 cm) s l o t t e d to accommodate a nerve, and a supporting handle through which c o o l i n g f l u i d could be c i r c u l a t e d . The device was cooled by drawing e i t h e r i c e water or dry i c e cooled acetone through to an evacuated f l a s k , and the temperature 20 c o n t r o l l e d by v a r y i n g the flow r a t e . Thermode temperature was normally h e l d at 5-6°C u n t i l nerve blockade was complete as judged by the response to water flow s t i m u l a t i o n of the nas a l passages. Nerve recordings were made w i t h conventional s i l v e r w i r e e l e c t r o d e s . The s i g n a l s were a m p l i f i e d (Tektronix a m p l i f i e r , type FM 122) and d i s p l a y e d on a storage o s c i l l o s c o p e ( T e k t r o n i x , 564 B). The output was, i n t u r n , connected i n p a r a l l e l w i t h a two t r a c k tape recorder (Tandberg, model 64) and a window d i s c r i m i n a t o r (F. Haer, No. 40-75-1). The s i g n a l s from the l a t t e r were r e l a y e d to an audio a m p l i f i e r and speaker. Taped nerve discharge was passed through a ratemeter (EKEG, model RT 682) and recorded on a Brush 220 pen w r i t e r (Gould). Ele c t r o d e s s i m i l a r to that described f o r r e c o r d i n g were a l s o used f o r nerve s t i m u l a t i o n . The e l e c t r i c a l stimulus was s u p p l i e d by a Grass S 6 s t i m u l a t o r coupled w i t h an i s o l a t i o n u n i t (Grass model S1U5). Dry g l o t t a l s t i m u l a t i o n was achieved by i n f l a t i o n of a small b a l l o o n attached to a polyethylene tube i n s e r t e d through a t r a c h e a l i n c i s i o n . D i v i n g c o n d i t i o n s were simulated by s i t u a t i n g a second t r a c h e a l cannula ( o r a l f a c i n g cannula) j u s t a n t e r i o r to the f i r s t and a l l o w i n g water or s a l i n e to flow from a r e s e r v o i r u s u a l l y set one meter ;above the animal. The mouth was not occluded and permitted some f l u i d to escape during n a s a l s t i m u l a t i o n . Pulmonary d e a f f e r e n t a t i o n was c a r r i e d out by f o r c i n g steam from a b o i l i n g f l a s k through an i n s u l a t e d tube (1 cm o.d., 3 mm i.d.) and i n t o a small nylon tube (1.5 mm i.d.) i n s e r t e d through a hole midway along the t r a c h e a l cannula. A r i g i d p l a s t i c sleeve w i t h a s i d e opening was placed over the j u n c t i o n to prevent leakage during steaming. The sleeve was r o t a t e d to s e a l the opening when the steaming cannula was withdrawn. A three-way tap was i n s e r t e d between the l a r g e and small steam l i n e s to ensure that maximum 21 temperature of steam at the e x i t was reached before i t s a p p l i c a t i o n to the lungs. Steam temperature normally exceeded 98°C a few m i l l i m e t e r s from the t i p i f t h i s procedure was followed. D e a f f e r e n t a t i o n was most s u c c e s s f u l when the steam was d e l i v e r e d by a r t i f i c i a l v e n t i l a t i o n at a pressure at 12 cm s i n c e i t precluded the p o s s i b i l i t y of animals becoming apneic. A r t e r i a l blood pressure and oxygen tension i n the muskrats were measured by means of a cannula forming an a r t e r i a l - a r t e r i a l or a r t e r i a l - v e n o u s loop, u s u a l l y on the l e f t s i d e . The c a r o t i d a r t e r y was exposed by a m i d - l i n e i n c i s i o n i n the neck and was cannulated w i t h polyethylene tubing (PE 90) to make a connection w i t h a pressure transducer (Harvard Apparatus, type 377) and a flow-through cuvette c o n t a i n i n g an oxygen e l e c t r o d e (Beckman, No. 315752). When the cuvette was i n use the pressure transducer was connected to a s i d e arm of the c a r o t i d loop. In some experiments, a c a r o t i d a r t e r y -j u g u l a r v e i n loop was used to improve the loop c i r c u i t time and when t h i s was used the response time of the system depended on the nature of the membrane covering the e l e c t r o d e . Using a t e f l o n membrane reduced the 90% response time of the e l e c t r o d e to 1.7 seconds. The e l e c t r o d e was c a l i b r a t e d w i t h a i r or n i t r o g e n e q u i l i b r a t e d s a l i n e a p p l i e d to the si d e arm of a T-piece on the upstream end of the cannula and removed through a second T-piece on the downstream loop. C a l i b r a t i o n s a l i n e s were flowed past the electrode from a pressure head which was adjusted to be c l o s e to that of the animal's blood pressure. The cuvette h o l d i n g the e l e c t r o d e was enclosed i n a water j a c k e t which was maintained at 37°C by the flow of water from a r e s e r v o i r c o n t a i n i n g a h e a t e r - s t i r r e r u n i t . 22 Experimental P r o t o c o l Muskrats instrumented to give EKGs i n unr e s t r a i n e d dives were t r a n s -f e r r e d the day a f t e r the operation to a 3 meter diameter wooden stave tank f i l l e d w i t h water to a depth of one meter. Approximately 2/3 of the tank area was covered w i t h wire mesh a few centimeters below the water l i n e to encourage long d i v i n g periods. A n e s t i n g . p l a t f o r m (0.5 x 0.5 m) was f l o a t e d on the uncovered water and the animals were connected to a p r e a m p l i f i e r w i t h a t h i n i n s u l a t e d w i r e which was maneuvered by a l i g h t rod during d i v i n g . As a r u l e , two muskrats were he l d i n the tank at the same time f o r the d i v i n g t r i a l s . D i v i n g was prompted by a scare stimulus from the operator and the po i n t of submersion was recorded by an operator a c t i v a t e d event marker on the chart recorder. S u c c e s s f u l recordings of the EKG were obtained only up to the s i x t h p ost-operative day. For muskrats dived i n the r e s t r a i n i n g box, submersion was c a r r i e d out by q u i c k l y r a i s i n g the lower water f i l l e d box u n t i l the animal was f u l l y submerged. In these experiments the d i v e was g e n e r a l l y terminated at 40 seconds although some animals were held under water f o r periods of up to 5 minutes without drowning. To. ensure complete recovery,\" a p e r i o d of at l e a s t 15 minutes was allowed between successive d i v e s . A group of anaesthetized animals were a l s o dived as described f o r r e s t r a i n e d d i v i n g . This group however, was . not able to t o l e r a t e submersions as w e l l as when they were conscious and as a r e s u l t only a few of these dives exceeded one minute. Nasal s t i m u l a t i o n was e f f e c t e d by passing water or s a l i n e through the o r a l f a c i n g cannula w i t h the animal i n the supine p o s i t i o n . Normally a flow of 32 ml/min was generated when the r e s e r v o i r was kept at a height of 1 m. 23 When r e q u i r e d , the flow r a t e was reduced by lowering the pressure head. P r i o r to n a s a l s t i m u l a t i o n , water was run to the end of the cannula and the tube was clamped so that maximum flow r a t e s were achieved i n l e s s than a second when the clamp was removed. Fol l o w i n g a p e r i o d of s t i m u l a t i o n ( u s u a l l y 20 seconds or l e s s ) a i r was forced through the cannula to remove r e s i d u a l f l u i d from the n a s a l passages. F a i l u r e to remove the water r e s u l t e d i n decreased responsiveness to subsequent s t i m u l a t i o n . Water s t i m u l a t i o n of the e x t e r n a l n a r i a l r e g i o n alone was c a r r i e d out i n c u r a r i z e d a r t i f i c i a l l y v e n t i l a t e d muskrats. In each case the animal was h e l d i n the prone p o s i t i o n and the nose drawn through a hole i n a sheet of d e n t a l dam which formed one side of a small box (10 x 10 x 8 cm). A i r was continuously blown through the o r a l f a c i n g cannula and e x i t e d through the nares to prevent the entry of water. The water l e v e l i n the box was r a i s e d by pouring water from a beaker i n t o the open top or by r a i s i n g the water l e v e l from a r e s e r v o i r and drained by removing a plug from the bottom. The e f f e c t of decerebration and of s e c t i o n i n g or c o o l i n g the c r a n i a l nerves on the r e s p i r a t o r y and c a r d i o v a s c u l a r responses to n a s a l s t i m u l a t i o n was st u d i e d . The d e l i m i t a t i o n of the neural pathways f o r the responses was confirmed by recording from nerves i n n e r v a t i n g the areas which gave apneic and bradycardic responses to punctate s t i m u l a t i o n and by e l e c t r i c a l l y s t i m u l a t i n g the c e n t r a l ends of these nerves. Nerve recordings of discharge i n the phrenic, n a s o c i l i a r y and i n f e r i o r l a r y n g e a l nerves were taken from the a c t i v e cut ends of the nerves. The nerve ends were prepared f o r recording by removing the sheath and d i v i d i n g the nerve body i n t o s m a l l bundles. In a l l preparations the i n d i f f e r e n t e l e c t r o d e was grounded to an und e r l y i n g baseplate w i t h a p o r t i o n of extraneous nerve of the same diameter as that of 24 the a c t i v e bundle. The baseplate was earthed, the recording area immersed in a pool of mineral o i l and a r i n g of s i l i c o n e sealant was applied to the area of i n c i s i o n to prevent the seepage of o i l into the f u r . The e f f e c t of lung receptor input on the responses to nasal stimulation was studied i n anaesthetized muskrats which were a r t i f i c i a l l y v e n t i l a t e d over a range of pressures from 4 to 22 cm t^O. V e n t i l a t i o n was maintained throughout periods of nasal stimulation with water and s a l i n e , before and a f t e r lung deafferentation. The e f f e c t of maintained i n f l a t i o n and d e f l a t i o n of the lungs on heart rate, blood pressure and PaC^ were investigated i n animals paralyzed with curare. Steam deafferentation was c a r r i e d out i n steps. Normally, two inhalations of steam were given i n succession and the Hering-Breuer r e f l e x and the cardiac response to v e n t i l a t i o n during nasal stimulation were checked before further steaming. Generally 3-6 breaths of steam was s u f f i c i e n t f o r pulmonary deafferentation but i n a few cases further steaming was necessary. Baroreceptor stimulation was produced by the r i s e i n blood pressure following i n t r a - a r t e r i a l i n j e c t i o n of 5 yg/kg of adrenaline, while c a r o t i d body stimulation was achieved by i n j e c t i o n of 80-200 yg/kg of potassium cyanide. The c a r o t i d sinus baroreceptors were denervated by section of the sinus nerve and was judged to be successful when the blood pressure r i s e following adrenaline f a i l e d to cause bradycardia. Denervation of the c a r o t i d body chemoreceptors was also c a r r i e d out by sinus nerve section and was considered complete i f i n j e c t i o n of cyanide produced no increase i n r e s p i r a -t i o n . The e f f e c t s of baroreceptor and chemoreceptor denervation on the responses to nasal stimulation and maintained lung i n f l a t i o n and d e f l a t i o n 25 were i n v e s t i g a t e d . In some experiments chemoreceptors were st i m u l a t e d by a r t i f i c i a l v e n t i l a t i o n at a c o n t r o l l e d frequency and t i d a l volume, w i t h a gas mixture of n i t r o g e n and 5% carbon d i o x i d e . Blood oxygen was continuously recorded during t h i s procedure. The c a r d i o v a s c u l a r responses to normocapnic anoxia were recorded before and a f t e r sinus nerve s e c t i o n . Heart r a t e and a r t e r i a l oxygen t e n s i o n were a l s o monitored during n a s a l s t i m u l a t i o n w i t h and without maintained v e n t i l a t i o n during asphyxia. In the t e x t and f i g u r e s , the numerical values used i n r e f e r r i n g to determinations of v a r i a b l e s i n a group of animals, are given as means ± the standard e r r o r of the means (±S.E.M.). The standard student t - t e s t was a p p l i e d i n a few cases to determine the s t a t i s t i c a l s i g n i f i c a n c e of the d i f f e r e n c e between groups. In such cases, 95% was considered the f i d u c i a l l i m i t of confidence. In t r i a l s demonstrating a decrease i n heart r a t e , bradycardia was recognized when the c a r d i a c i n t e r v a l lengthened by more than 10% from the value i n the c o n t r o l p e r i o d . 26 Results 1. R e s p i r a t o r y and Cardiac Responses to D i v i n g . 1.1. Cardiac Responses to Unrestrained D i v i n g . Under the c o n d i t i o n s imposed by the dimensions of the d i v i n g ' tank and i t s covering w i r e set beneath the surface of the water, the muskrats d i s p l a y e d two d i s t i n c t types of d i v i n g behaviour to a scare stimulus. In approximately one h a l f the d i v e s , the animals resurfaced immediately a f t e r c r o s s i n g the \"open\" water which was approximately 1/3 the tank area. In such cases (n = 50) the dives never exceeded 5 seconds i n length. The second type of dive was c h a r a c t e r i z e d by the muskrats swimming d i r e c t l y under the wire n e t t i n g and r e s u r f a c i n g a f t e r a r e s t p e r i o d which v a r i e d from 6 to 63 seconds. While i t was d i f f i c u l t to determine i f the animals dived a f t e r i n s p i r a t i o n , i t was obvious that they continued to expel a i r during the course of the d i v e . This was a l s o noted i n muskrats which dived v o l u n t a r i l y f o r periods of up to 10.5 minutes without r e c o r d i n g attachments. In a l l cases the animals were allowed to resume normal behaviour (grooming, feeding, etc.) before a second scare stimulus was given. The EKG of the muskrats during r e s t i n g periods and moderate a c t i v i t y was reg u l a r and d i s p l a y e d no sinus arrhythmia. On average, heart r a t e f e l l from 310±3 to 54±3 beats/min (n = 102) i n the f i r s t measurement f o l l o w i n g submersion (1-2 seconds) and d e c l i n e d to 27±3 beats/min (n = 3) at 40 seconds ( F i g . 2). Due to the u n r e s t r i c t e d nature of the t r i a l s the mean d u r a t i o n of dives exceeding 5 seconds (long dives) was 17.5±4.1 seconds and thus the time course of heart r a t e represents a decreasing number of di v e s . Recovery of 27 Figure 2. Mean heart r a t e during u n r e s t r a i n e d dives exceeding 5 seconds. Each po i n t represents the mean r a t e f o r a l l dives (n = 102 at time 0, n = 3 at 40 seconds) i n f i v e muskrats. Standard e r r o r of the mean i s given f o r S.E.M. greater than 4. 28 29 heart r a t e on r e s u r f a c i n g was marked by a short p e r i o d sinus arrhythmia and normal r a t e s were reached a f t e r 5 to 10 seconds. P o s t - d i v i n g t a c h y c a r d i a was not observed i n any of the t r i a l s . Evidence was obtained from one muskrat that not only d i d the c a r d i a c response precede submersion but al s o that the degree of response was r e l a t e d to d i ve d u r a t i o n but because of e l e c t r i c a l i n t e r f e r e n c e caused by swimming these observations could not be confirmed i n the other muskrats. In t r a n s i t o r y dives of 4 seconds or l e s s the f i r s t c a r d i a c i n t e r v a l a f t e r submersion was the lon g e s t , averaging 0.82±0.03 seconds (n = 50) wh i l e i n those dives 5 seconds or longer the f i r s t c a r d i a c i n t e r v a l was n e a r l y doubled to 1.56±0.08 seconds (n = 37, F i g . 3). The same r e l a t i o n s h i p was evident f o r the remaining c a r d i a c i n t e r v a l s up to 4 seconds f o r the s h o r t e r dives and i n each case the groups were s i g n i f i c a n t l y d i f f e r e n t at the 99% confidence l i m i t . In dives which were 5 seconds or longer there was no c o r r e l a t i o n between the i n i t i a l c a r d i a c response and the time underwater. The mean time periods by which bradycardia preceded submersion were 0.30±0.02 and 0.43±0.02 seconds f o r the short and long dives r e s p e c t i v e l y . These i n t e r v a l s were measured from the time of the f i r s t appearance of bradycardia to the event marker a c t i v a t e d at the p o i n t when the head touched the water. The animals however, d i d not show a r e l i a b l e a n t i c i p a t o r y response to r e s u r f a c i n g and when t h i s response was present, i t was not pronounced ( F i g . 4 ). When the muskrats were sti m u l a t e d to r a p i d successive d i v e s , the i n t e r v e n i n g recovery of heart r a t e was s l i g h t and the i n i t i a l c a r d i a c i n t e r v a l of the second dive was increased. Although heart r a t e i n the r e s t -ing animal was u s u a l l y s t a b l e , i n many instances the muskrats d i s p l a y e d 30 Figure 3. Duration of the f i r s t four c a r d i a c i n t e r v a l s of t r a n s i t o r y ( s t i p p l e d ) and long dives (unstippled) ±S.E.M. U n l i k e the long dives (6-63 seconds), the muskrat d i d not swim under the covering wire i n the t r a n s i t o r y d i v e s (2-4 seconds). T r i a l s were on one muskrat. 31 • • < rm • . i • • . • • i i • • • • i I • • • • • • • i • • • • • i I • • • • i I • • • • » v.1 • • • • • • < • • _ * * 4 > • i • • ^ • • i 1 2 cardiac 3 interval 4 32 Figure 4. Electrocardiogram during unrestrained d i v i n g . Top t r a c e , during a t r a n s i t o r y dive (duration 2.5 seconds); bottom t r a c e s , continuous r e c o r d i n g during an 11 second d i v e . In the l a t t e r dive the muskrat swam without pause beneath the covering wire (see t e x t ) . Note that the e l e c t r o m y o c a r d i a l a r t i f a c t on both tr a c e s s i g n i f i e s the movement preceding the p o i n t of submersion at downward pointed arrows. Resurfacing i s i n d i c a t e d by the upward p o i n t i n g arrows. Time marker, 1 second. 33 I I I I 1 second 34 t r a n s i e n t bradycardia when d i s t u r b e d , most notably i n response to t a c t i l e , noise and v i s u a l s t i m u l i ( F i g . 9). 1.2 Cardiac Response to Restrained D i v i n g The mean r e s t i n g heart r a t e of 11 r e s t r a i n e d muskrats was s i g n i f i c a n t l y lower (266±3 beat/min, n = 66) than those i n the \" n a t u r a l \" environment (310±3 beat/min, n = 102). During r e s t r a i n e d d i v e s , n o s t r i l c l o s u r e and apnea occurred immediately on submersion and brad y c a r d i a f o l l o w e d a f t e r a l a t e n t p e r i o d of 300+10 msec (n = 60); w i t h heart r a t e f a l l i n g to 78±4 beat/ min w i t h i n 1 to 2 seconds ( F i g . 5). A r a t e of 51±2 beat/min was reached a f t e r 5 seconds and t h e r e a f t e r heart r a t e remained r e l a t i v e l y s t a b l e u n t i l the animals were resurfa c e d . Heart r a t e increased r a p i d l y on emersion, reaching the pre-dive r a t e a f t e r 10 seconds. The increases were always a s s o c i a t e d w i t h the recovery of normal r e s p i r a t i o n ; heart r a t e rose markedly during the i n s p i r a t o r y phase. As i n unr e s t r a i n e d d i v i n g , heart r a t e seemed unaffected by movement or s t r u g g l i n g ( F i g . 6, top) and no tachycardia was evident i n the recovery p e r i o d . N e i t h e r the l a t e n t p e r i o d to the onset of bradycardia nor the time course of the response were changed when the unanaesthetized animals were forced dived i n t o s a l i n e (0.9%) or when the water temperature was v a r i e d between 4 and 38°C. 1.3 Re s p i r a t o r y and Cardiac Responses to D i v i n g During Anaesthesia Both the r e s t i n g heart r a t e and the car d i a c response to d i v i n g w h i l e the muskrat was anaesthetized (urethane, 1250 mg/kg, i.p.) were s i m i l a r to those t r i a l s on the r e s t r a i n e d animals ( F i g . 7). In 9 experimental animals, mean 35 Figure 5. Mean heart r a t e during r e s t r a i n e d d i v i n g . Each p o i n t represents the mean response i n 11 muskrats (n = 60) f o r dives terminated at 40 seconds. The arrows i n d i c a t e the po i n t s of submersion and r e s u r f a c i n g . Standard e r r o r of the mean i s given f o r S.E.M. greater than 4. 37 Figure 6. Electrocardiogram during d i v i n g i n the r e s t r a i n e d (above) and anaesthetized muskrat (below). The p o i n t s of submersion and r e s u r f a c i n g are i n d i c a t e d by the arrows. Sustained apnea d i d not occur i n the anaesthetized animals. Time marker i s 1 second. t M i ' i i ' i il i I i j» {—1 •« | O D 3 1 ' 1 ' 1 ' ' M I M M M M ! M M M I I M M I i I I I H M ' ' i i i | i M H H ! M ! M I M J9|66rujs 1 1 1 1 1 1 1 1 1 ^ 1 ^ 1 1 1 1 1 m i i M 1 1 1 1 1 i 1 1 M 111 11 i 11 i n n 11 | 1 1 11 | 1 1 11 111 39 Figure 7. Mean heart r a t e during d i v i n g i n the anaesthetized muskrat. Each p o i n t represents the mean value of a minimum of 39 t r i a l s i n 9 muskrats i n which a l l dives were terminated at 40 seconds. The arrows i n d i c a t e the p o i n t s of submersion and r e s u r f a c i n g . Standard e r r o r of the mean i s given f o r S.E.M. greater than 4. 41 Figure 8. Respiratory and cardiac responses to submersion i n the anaesthetized muskrat. Traces top to bottom, time (1 second marker), tracheal a i r flow ( i n s p i r a t i o n downwards), EKG and event trace. Respiration recorded from a thermistor set i n the nasal passage indicated the d i r e c t i o n a l flow of a i r i n the pre-dive state, and i n h a l a t i o n of water during submersion. Note that a s i g n i f i c a n t bradycardia occurs despite continued r e s p i r a t o r y e f f o r t s . The arrow marks the point of submersion. 42 43 heart r a t e f e l l from 281±5 to 84±4 beats/min (n = 56) a f t e r an i n i t i a l l a t e n t p e r i o d of 591±40 msec and continued to d e c l i n e to a r a t e of 39±2 beats/ min (n = 39) a f t e r 40 seconds of submersion. As a r u l e however, r e s p i r a t o r y movements continued during submersion and these tended to increase w i t h dive d u r a t i o n . That water was taken i n t o the n a s a l c a v i t y i n dives i n which the animals were anaesthetized was confirmed by recordings taken from a ther m i s t o r set i n the n a s a l c a v i t y ( F i g . 8). A second thermistor placed i n the trachea and la r y n x e s t a b l i s h e d that i n h a l e d water was not drawn past the g l o t t i s but was ex p e l l e d during the e x p i r a t o r y phase of the c y c l e . Heart r a t e on r e s u r f a c i n g was slow to recover and g e n e r a l l y r e q u i r e d 60 seconds to reach the pre-dive r a t e . I n t e r e s t i n g l y , the anaesthetized muskrats could not t o l e r a t e sub-mersion i n i s o t o n i c s a l i n e (0.9%) f o r more than a short time. On three occasions muskrats were drowned a f t e r s a l i n e submersions of l e s s than 40 seconds and were found to have taken the f l u i d i n t o the lungs. 2. Responses to Water S t i m u l a t i o n of the Nasal Area. 2.1. E x t e r n a l N a r i a l S t i m u l a t i o n . V i s u a l observations on the normal muskrat at r e s t on the surface of the water i n d i c a t e d that apnea was t r i g g e r e d by water contact w i t h the nares. The n a r i a l f l a p s adjacent to the nares seemed to f u n c t i o n i n both a sensory and motor c a p a c i t y . Not only could the d i v i n g responses be evoked from t h i s area but i t was a l s o apparent that the f l a p s prevented water from en t e r i n g the n a s a l passages. In the unanaesthetized animal, water was never observed to enter the nares. F o l l o w i n g anaesthesia however, the n a r i a l r e f l e x was l o s t s i n c e water was taken i n t o the n a s a l passages. Anaesthesia 44 also abolished the r e s p i r a t o r y and car d i a c responses to n a r i a l water stimu-l a t i o n when the animals were allowed to breathe through an exposed cannula. In unanaesthetized c u r a r i z e d muskrats which were a r t i f i c i a l l y v e n t i l a t e d (8-12 cm R^O), pouring water over the e x t e r n a l nares or r a i s i n g the water l e v e l over the nose r e s u l t e d i n a moderate bradycardia which was not sustained ( F i g . 9). In these experiments water was excluded from the n a s a l passages by a continuous but gentle flow of a i r through the o r a l f a c i n g cannula. Heart r a t e f e l l from a mean of 292±6 to 76±12 beats/min (n = 6) on submersion. As a r u l e , the response was b e t t e r sustained when the nose was kept underwater than when water was poured on the nares. Stopping v e n t i l a t i o n i n e x p i r a t i o n j u s t before water s t i m u l a t i o n of the nares caused heart r a t e to f a l l f u r t h e r than when v e n t i l a t i o n was maintained. V i r t u a l e l i m i n a t i o n of the response was obtained i f the nares was covered w i t h a f i n e l a y e r of v a s e l i n e ( F i g . 9) p r o v i d i n g t h a t water was excluded from the n a s a l passages. I t i s a l s o noteworthy that c u r a r i z a t i o n preserved the c a r d i a c responses to various s t i m u l i ; sudden s t i m u l i such as n o i s e , l i g h t f l a s h e s and hide s t r o k i n g could a l s o evoke t r a n s i e n t responses even when a r t i f i c i a l v e n t i l a -t i o n was continued. P o s t u r a l changes had l i t t l e e f f e c t on the heart r a t e when the muskrats were subjected to sham (dry) d i v e s . P a s s i v e head ducking i n e i t h e r the normal or anaesthetized animal d i d not evoke bradycardia when t h i s was c a r r i e d out i n a gentle manner s i m i l a r to that observed i n un r e s t r a i n e d d i v e s . Furthermore, i n animals which were f o r c e dived i n a v e r t i c a l head-down p o s i t i o n , c a r d i a c responses were no d i f f e r e n t from those which were dived i n the prone p o s i t i o n and suggest that the p o s t u r a l responses noted by Koppanyi 45 Figure 9. Response of heart rate to n a r i a l water stimulation i n a curarized a r t i f i c i a l l y v e n t i l a t e d muskrat. Top to bottom, time marker (1 second s i g n a l ) , v e n t i l a t i o n ( i n s p i r a t i o n downwards) and e l e c t r o -cardiogram. V e n t i l a t i o n was given at an i n s p i r a t o r y pressure of - 8 cm f^O and a frequency of 1 hz. A gentle flow of a i r passing from the o r a l facing cannula out the nares was maintained throughout to prevent water from entering the nasal passages. In the top and bottom ( l e f t ) recordings, water l e v e l was slowly ra i s e d above the head and then drained r e s u l t i n g i n a moderate but maintained bradycardia. Vaseline applied to the nares abolished the response u n t i l water was r a i s e d near eye l e v e l . The traces at the bottom r i g h t i n d i c a t e that the response to t a c t i l e s t i m u l i remains i n s p i t e of c u r a r i z a t i o n and forced v e n t i l a t i o n . LU-I-I 1 ) 1 1 1 1 1 1 II 1 II 1 I 1 1 I M I 1 I I I 1 I I I I I I I 1 M M I 1 1 I M I I M 1 1-LJ-l J_i-t-l-l--L4-l-J J-i-ULI J-fill ^ m m ^ drain ^ resp N O R M A L R E S P O N S E e.c.g. base top of of nose nares nose base of nares nose fmrnmrmftt-T -4-1 I l-I I I I I I I I I I I I 1 I 1 I 1 1 I i-l resp. V A S E L I N E O N N A R E S e.c.g. WTTTWtTTTTTTm^^ i i i i i i > >—i-1 i i i i i i i i i i i i i i i i i i i i i i i i i i i i i hide stroking T i i i i i i i i i 47 and Dooley (1929) have l i t t l e s i g n i f i c a n c e i n the d i v i n g a b i l i t y i n the muskrat. 2.2 I n t e r n a l Nasal S t i m u l a t i o n . Passing water or s a l i n e through the n a s a l passages to e x i t at the nares i n c u r a r i z e d or anaesthetized muskrats caused v a r y i n g degrees of apnea and bradycardia depending on the flow r a t e and the i o n i c composition of the f l u i d . Small j e t s of a i r (50 u l ) d i r e c t e d i n t o the nares could a l s o produce a bradycardic response but t h i s tended to be s l i g h t and short l i v e d . Nasal water flow d r i v e n by a 100 cm head provided a flow r a t e of 32 ml/min and gave a pronounced bradycardia and an apnea which l a s t e d from 15 to 40 seconds w i t h maintained s t i m u l a t i o n i n the anaesthetized animal. In 12 anaesthetized animals which remained apneic over the experimental t r i a l p e r i o d (20 seconds), heart r a t e f e l l from a mean p r e - s t i m u l a t i o n r a t e of 211+5 to 20±2 beats/min (n = 34) when water flow began ( F i g . 10). Brady-c a r d i a occurred at a mean of 320±27 msec a f t e r the onset of water flow but heart r a t e g r a d u a l l y rose during s t i m u l a t i o n and reached a r a t e of 33±2 beats/ min a f t e r 20 seconds. When water flow was stopped, the recovery to pre-s t i m u l a t i o n heart r a t e although much slower than i n the conscious animals, was always simultaneous w i t h the re-establishment of bre a t h i n g ; u s u a l l y beginning 5 to 10 seconds a f t e r water flow. The water d r i v e n responses were preserved by c l e a r i n g the n a s a l passages of water between t r i a l s and these could normally be maintained over a f i v e or s i x hour experimental p e r i o d . I f during extended periods (6 to 10 hours) the responses to water flow s t i m u l a t i o n decreased, they were soon l o s t a l t o g e t h e r . In such cases, r e s p i r a t o r y and c a r d i a c responses were l o s t 48 Figure 10. Mean heart r a t e i n the muskrat during apneic n a s a l water flow s t i m u l a t i o n . Each po i n t represents the mean response i n 34 t r i a l s i n 12 muskrats tS.E.M. The onset and termina t i o n of water flow (32 ml/min) are i n d i c a t e d by the arrows. Water remained i n the n a s a l passages approximately 30 seconds a f t e r water flow was stopped. 49 50 simultaneously i n s p i t e of surface r e s p i r a t i o n and heart r a t e remaining normal. The l o s s of the r e f l e x e s appeared to be r e l a t e d to c e n t r a l impairment of the bradycardia r e f l e x arc s i n c e a s p h y x i a t i o n a l s o f a i l e d to produce r e f l e x slowing of the heart. This phenomenon has a l s o been noted i n anaesthetized dogs i n which the n a s a l responses are prone to disappear spontaneously ( A n g e l l James and Daly, 1972a). The s u b s t i t u t i o n of 0.9% s a l i n e f o r water or lowering the water flow r a t e always caused a l e s s intense and more unpredictable bradycardia than that caused by 32 ml/min water flow. Apnea at the onset of s t i m u l a t i o n was n e i t h e r immediate nor l a s t i n g ( F i g . 11) and r e s p i r a t o r y breakthrough occurred throughout, which i n t u r n increased heart r a t e w i t h each breath. During non-apneic s t i m u l a t i o n , a w e l l defined r e l a t i o n s h i p e x i s t e d between the i n s p i r a t o r y phase and the appearance of EKG s p i k e s . In the i n i t i a l p e r i o d of s t i m u l a t i o n an EKG spike always occurred simultaneously w i t h i n s p i r a t i o n ( F i g . 11, t o p ) , but as r e s p i r a t o r y d r i v e increased a second (and o c c a s i o n a l l y a t h i r d ) spike appeared near the peak of i n s p i r a t i o n . This r e l a t i o n s h i p continued u n t i l sinus arrhythmia was no longer present. The p a t t e r n of a c t i v i t y revealed that the s i n g l e spikes preceded the r e s p i r a t o r y d e f l e c t i o n by 200-300 msec but when p a i r e d spikes were present, the f i r s t advanced c l o s e r to the beginning of lung i n f l a t i o n w h i l e a second appeared near to but not at maximal i n f l a t i o n . As the stimulus continued, a s l i g h t phase s h i f t occurred i n which the second of the two advanced on the f i r s t to give a double spike that was a s s o c i a t e d w i t h e a r l y i n s p i r a t i o n . The p a t t e r n of sinus arrhythmia i n the a r t i f i c i a l l y v e n t i l a t e d animal suggested that the i n f l u e n c e of r e s p i r a t i o n on heart r a t e combined both a c e n t r a l and a p e r i p h e r a l component as noted by Anrep et a l . (1936b). 51 Figure 11. Cardiac responses to non-apneic water flow stimulation i n the anaesthetized and curarized anaesthetized muskrat. Top to bottom i n each recording from time trace (1 second marker); pneumotachogram ( i n s p i r a t i o n downwards), electrocardiogram and ( event trace. The upper recording shows the response to low water flow stimulation (10 ml/min) i n which the integrated tracheal a i r flow trace i s punctuated by breakthrough i n s p i r a t i o n s (A). The bottom recording shows the cardiac response of a curarized muskrat i n which v e n t i l a t i o n was stopped during and a f t e r water flow. Forced v e n t i l a t i o n was delivered at 10 cm H„0 and 1 hz. Water flow i s indicated by arrows. 52 t llfliMlllWiffillilillllillllll 1 1 1 — i i i i i 11 I I I I I I n i n I I 1 1 1 i n n u n 11 m HI HI I t WWyWWVWVYvVvY' -I 1 1 I I I I I—UI I I I I I I I I I i 1 f 53 Observations i n the c u r a r i z e d animal during asphyxia however, tend to show that the t y p i c a l p a i r e d sequence may a r i s e e x c l u s i v e of lung i n f l a t i o n ( F i g . 11, bottom t r a c e ) . 3. A f f e r e n t and E f f e r e n t Pathways of the D i v i n g R e f l e x e s . 3.1. Morphology of the Nasal Receptor Areas. While the i n f e r i o r l a r y n g e a l d i v i s i o n of the vagus was r e a d i l y a c c e s s i b l e , blockade of the m a x i l l a r y branch of the t r i g e m i n a l was hampered by i t s l o c a t i o n deep w i t h i n the basisphenoid bone and the v a r i a b i l i t y of the a n c i l -l a r y branches near the a n t e r i o r l a c e r a t e d foramen and w i t h i n the o r b i t . Cardiac and r e s p i r a t o r y responses to water flow were not a f f e c t e d when the m a x i l l a r y d i v i s i o n was sectioned or cooled r o s t r a l to the e x i t of the naso-p a l a t i n e and sphenopalatine branches. A b o l i t i o n of the m a x i l l a r y c o n t r i b u t i o n to the n a s a l responses however, occurred when blockade was attempted at a l e v e l j u s t caudal to the foramen where the m a x i l l a r y and ophthalmic d i v i s i o n s of the t r i g e m i n a l u n i t e to form the main t r i g e m i n a l trunk ( F i g . 12). Microscopic examination of the nasopalatine branches revealed that these nerves are given o f f from one or two sub-maxillary bundles which pass v e n t r a l l y and m e d i a l l y through the basisphenoid, presphenoid and m a x i l l a bones. The branches emerge w i t h i n the n a s a l c a v i t y to innervate the n a s a l mucosa and the base of the molar teeth and reappear beneath the e p i t h e l i a l t i s s u e s of the hard p a l a t e . From t h e i r e x i t on the medial s i d e of the t h i r d molar, two branches were d i s t i n c t , one of which passed forward to innervate the a n t e r i o r p o r t i o n of the hard p a l a t e w h i l e the second ran p o s t e r i o r l y along the medial s i d e of the m a x i l l a r y r i d g e to give o f f f i n e r branches to the s o f t p a l a t e . In two operations i n which the pharynx and p a l a t e were 54 Figure 12. Major nerves supplying the nares and the nasal and g l o t t a l mucosa. Top, cutaway diagram of the f r o n t a l portion of the s k u l l i n which the p a r i e t a l bone, f r o n t a l lobe, o l f a c t o r y lobe, r i g h t eye and l a c r i m a l gland have been removed to show some branches of the maxillary and ophthalmic d i v i s i o n s of the trigeminal nerve. Bottom, v e n t r a l exposure of the g l o t t a l area. frontal nasociliary nasopalatine ophthalmic maxillary -epiglottal cart, thryoid cart. - inferior laryngeal - trachea - recurrent laryngeal 56 exposed by mid-ventral jaw s e c t i o n , the d o r s a l a n t e r i o r surface of the s o f t p a l a t e appeared to the the most s e n s i t i v e to probe, s u c t i o n or e l e c t r i c a l s t i m u l a t i o n of the e p i t h e l i a l t i s s u e s as judged by r e s p i r a t o r y and c a r d i a c responses. As a r u l e s t i m u l a t i o n of these areas evoked t r a n s i e n t apnea and s l i g h t bradycardia. S i m i l a r but l e s s intense responses could be e l i c i t e d from the pharyngeal and r o s t r a l n a s a l c a v i t y areas. The i n n e r v a t i o n of the l a r y n x and p o s t e r i o r pharyngeal mucosa by the i n f e r i o r and r e c u r r e n t l a r y n g e a l nerves i s shown i n the lower h a l f of F i g . 12. The muscles of the e p i g l o t t a l c a r t i l a g e appeared to c o n t a i n f i b e r s from both d i v i s i o n s but only the a n t e r i o r l y d i r e c t e d branches of the i n f e r i o r l a r y n g e a l nerve were found to extend to the pharyngeal musculature where they terminate on the v e n t r a l and l a t e r a l s i des of the e p i t h e l i u m . The area most s e n s i t i v e to probe and e l e c t r i c a l s t i m u l a t i o n was found to be near the a n t e r i o r end of the g l o t t i s but the more a n t e r i o r e p i t h e l i u m , innervated by the s u p e r i o r l a r y n g e a l d i v i s i o n f a i l e d to cause any c a r d i a c or r e s p i r a t o r y responses on s t i m u l a t i o n . 3.2 A f f e r e n t Pathway of the E x t e r n a l N a r i a l Reflexes. Blockade of the a f f e r e n t pathway of the e x t e r n a l n a r i a l r e f l e x e s was attempted i n three unanaesthetized muskrats. In each case the animals were denervated under general anaesthesia (nembutal) and were allowed to recover f u l l y before experimentation. M a x i l l a r y nerve s e c t i o n at the l e v e l of the zygomatic arch and i n f i l t r a t i o n of the n a r i a l r e g i o n w i t h 2% x y l o c a i n e f a i l e d to a f f e c t the r e s p i r a t o r y and c a r d i a c responses to forced d i v e s . As i n the normal animals, apnea occurred when the water lapped the nares and none appeared to enter the n a s a l passages. 57 In two animals v i s u a l input was e l i m i n a t e d by sewing the eyes close d i n a d d i t i o n to f r o n t a l m a x i l l a r y s e c t i o n but both responded i n a manner s i m i l a r to that i n normal forced submersions. I t was concluded that w h i l e these r e s u l t s do not r u l e out f r o n t a l m a x i l l a r y nerve involvement i n the i n i t i a t i o n of the primary n a s a l r e f l e x e s , the a f f e r e n t limb l i k e l y i n c l u d e s many of the small branches which r e s i d e i n the nas a l passages and u n i t e w i t h the ophthalmic d i v i s i o n and p o s s i b l y the m a x i l l a r y d i v i s i o n w i t h i n the o r b i t . Because of the i n a c c e s s i b l e l o c a t i o n of these nerves w i t h i n the n a s a l c a v i t y , no r e l i a b l e r e s u l t s were obtained to confirm t h e i r involvement i n the r e f l e x e s . 3.3 A f f e r e n t Pathway of the I n t e r n a l Nasal Reflexes. 3.3.1 Nerve Blockade of the A f f e r e n t Pathway. The a f f e r e n t pathway of the nas a l water flow r e f l e x e s was i n v e s t i g a t e d by nerve s e c t i o n , r e v e r s i b l e nerve c o o l i n g , i r r e v e r s i b l e freeze blockade and by e l e c t r i c a l s t i m u l a t i o n of the c e n t r a l ends of the cut nerves. Of the 17 muskrats examined, 10 were decerebrated. In animals which were decerebrated by a s p i r a t i o n to the l e v e l of the thalamus, normal r e s p i r a t i o n r a t e , t i d a l volume and heart r a t e were unaffected although the c a r d i a c response to n a s a l water flow was s i g n i f i c a n t l y reduced. When water flow of 32 ml/min was allowed to pass through the nares heart r a t e rose to 31±5.8% of the normal r a t e from the pre-decerebrate response of 8.3±1.0%. Section or c o o l i n g blockade of the f o l l o w i n g nerves had no e f f e c t on e i t h e r the r e s p i r a t o r y or c a r d i a c responses to the water flow s t i m u l u s : o l f a c t o r y (n-= 10), ophthalmic (n = 10), mandibular (n = 10), f a c i a l (main branch, n = 2), s u p e r i o r p e t r o s a l (n = 3 ) , glossopharyngeal (main branch 58 d i s t a l to sinus nerve, n = 3), sinus (n = 5), superior laryngeal (n = 6), recurrent laryngeal (n = 3). In 4 of 6 muskrats i n which both the maxillary d i v i s i o n of the trigeminal nerve and the i n f e r i o r laryngeal branches of the vagus were sectioned or cooled, the resp i r a t o r y and cardiac responses were f u l l y abolished. In the remaining two animals section of the two d i v i s i o n s resulted i n only s l i g h t decreases (less than 5%) i n normal heart and r e s p i r a t i o n rates during the water flow stimulus. Table I summarizes the mean r e s u l t s of maxillary and i n f e r i o r laryngeal nerve blockade. I t i s evident from these data that while the maxillary nerve dominates cardiac i n h i b i t o r y input, i t has less e f f e c t than the i n f e r i o r laryngeal nerve on the res p i r a t o r y response (Table I ) . It was cl e a r from the r e s u l t s that response l o s s was dependent on the order of blockade of the two nerves. Table II compares the mean data of the cardiac and re s p i r a t o r y responses when the sequence of nerve blockade was reversed i n one h a l f the t r i a l s . T y p i c a l l y , when nerve blockade was c a r r i e d out a f t e r the loss of the f i r s t nerve, the loss of the response was greater than i f nerve blockade had been done alone. The table includes data from denervations by section and cold blockade since there was no differe n c e i n response loss between the two techniques. In two of eight animals i n which i n f e r i o r laryngeal blockade was c a r r i e d out f i r s t , there was no noticeable decline i n the cardiac response to water flow even though there was an obvious decrease i n the resp i r a t o r y response. In the remaining animals the loss of the cardiac response due to blockading the i n f e r i o r laryngeal nerve f i r s t never exceeded that when the maxillary nerve was blockaded f i r s t . Figure 13 shows the r e s u l t s from one muskrat which demonstrate that the sequential elimination of the water driven responses was achieved by b i l a t e r a l Table I . E f f e c t of nerve blockade and e l e c t r i c a l s t i m u l a t i o n on heart r a t e and r e s p i r a t i o n . Blockade was t e s t e d by determining response l o s s to nasal water flow. Average l o s s of response N u m b e r to blockade (%) of C r a n i a l animals nerve Branch Resp. H.R. 10 I o l f a c t o r y 0 0 0 10 V ophthalmic 0 0 t r a n s i e n t decrease 10 V m a x i l l a r y 44.5 68.5 sustained decrease 10 V mandibular 0 0 0 2 VII main 0 0 0 3 VII s u p e r i o r p e t r o s a l 0 0 0 3 IX main ( d i s t a l ) 0 0 0 5 IX sinus 0 . 0 t r a n s i e n t decrease 3 X c e r v i c a l (main) 0 100 sustained decrease 4 X , super i o r l a r y n g e a l 0 0 0 11 X i n f e r i o r l a r y n g e a l 55.5 31.5 sustained decrease 3 X re c u r r e n t l a r y n g e a l 0 0 0 E f f e c t of low voltage s t i m u l a t i o n on r e s p i r a t i o n and heart r a t e Table I I . E f f e c t of nasa l water flow on heart r a t e and r e s p i r a t o r y minute volume i n 14 muskrats before and a f t e r b i l a t e r a l s e c t i o n or c o l d blockade of the i n f e r i o r l a r y n g e a l and m a x i l l a r y nerves ±S.E.M. Each water flow p e r i o d was 20 sec and heart rates and minute volumes are the averages f o r t h i s p e r i o d . On average each t e s t was done 3 times on each animal. Normal animals C o n t r o l Water flow Decerebrate Water Co n t r o l flow B i l a t e r a l IL blockade B i l a t e r a l IL and max blockade B i l a t e r a l max blockade B i l a t e r a l max and IL blockade Heart r a t e beats/min 277±13 25±4 286±13 53±10 93±18 270±18 177±14 275±8 Minute volume 214±8 ml/min 189±12 22±10 101±15 171±9 89±18 181±17 Number of 14 14 11 11 animals 61 Figure 13. The e f f e c t of u n i l a t e r a l and b i l a t e r a l s e c t i o n of the m a x i l l a r y (max) and i n f e r i o r l a r y n g e a l (IL) nerves on the r e s p i r a t o r y and car d i a c responses to n a s a l water flow. Traces from top to bottom; time (1 second marker), pneumotachogram ( i n s p i r a t i o n downward) and electrocardiogram. A, decerebrate response to n a s a l water fl o w (32 ml/min); B, a f t e r l e f t max s e c t i o n ; C, a f t e r b i l a t e r a l max s e c t i o n ; D, a f t e r b i l a t e r a l max and l e f t IL s e c t i o n ; E, a f t e r b i l a t e r a l max and IL s e c t i o n . The s l i g h t downward d e f l e c t i o n s i n the t r a c h e a l a i r flow t r a c e during the c o n t r o l s t i m u l a t i o n r e s u l t e d from c a r d i a c c o n t r a c t i o n s and not from r e s p i r a t o r y breakthrough. 62 i 1- ^ H C 1 U X J . . . I . U . . U . J I I . . . . . . . . . 1 t M i l t *—t—*— 1 i n n i m i i i i i i I i i i i L U _ L i U U U u 11111 l l l l l 1 1 1 1 1 1 1 1 1 1 1 1 1 ( 111 t . L . i I I I ! 1 l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l L j _ J - i j K i l l rnltttTr 11 l l l l l l l l l l l l l l 63 s e c t i o n of the m a x i l l a r y and i n f e r i o r l a r y n g e a l d i v i s i o n s . In the pre-s e c t i o n c o n t r o l t r a c e (A) apnea due to the water flow was maintained f o r 15 seconds f o l l o w i n g decerebration but heart r a t e had increased from 13 to 34 beats/min. When the r i g h t (B) and l e f t (C) m a x i l l a r y nerves were d i v i d e d , apnea and a pronounced bradycardia d i d not occur as the r e c o r d i n g was marked by breakthrough i n s p i r a t i o n s and sinus arrhythmia. Following the s e c t i o n of the r i g h t (D) and l e f t (E) i n f e r i o r l a r y n g e a l nerves, n a s a l s t i m u l a t i o n caused only a s l i g h t i r r e g u l a r i t y of the r e s p i r a t o r y p a t t e r n and no a p p r e c i -able change i n heart r a t e or minute volume. For t r i a l s of t h i s nature a f f e r e n t blockade was considered complete i f minute volumes decreased by l e s s than 5% during the 15 second p e r i o d of water s t i m u l a t i o n . In a l l cases, a short p e r i o d of asphyxia by t r a c h e a l tube clamping was c a r r i e d out to confirm that the chemoreceptor r e f l e x e s remained i n t a c t a f t e r the a b o l i t i o n of the water d r i v e n responses. 3.3.2. E l e c t r i c a l S t i m u l a t i o n of the A f f e r e n t Pathway. Table I i n c l u d e s the e f f e c t s of low v o l t a g e (1 V) s t i m u l a t i o n of the c e n t r a l ends of 12 nerves considered as p o s s i b l e c o n t r i b u t o r s to the a f f e r e n t pathway. Although the responses to nerve s t i m u l a t i o n were always l e s s intense than those i n i t i a t e d by n a s a l water flow, apneic periods of 5 to 10 seconds could be evoked by s t i m u l a t i o n of both the i n f e r i o r l a r y n g e a l nerve and the main m a x i l l a r y trunk of the t r i g e m i n a l nerve. E l e c t r i c a l s t i m u l a t i o n of the m a x i l l a r y d i v i s i o n a n t e r i o r to the mid-point of the o r b i t had no e f f e c t on e i t h e r r e s p i r a t i o n or heart r a t e but s t i m u l a t i o n of branches l o c a t e d i n the rear of the o r b i t u s u a l l y gave t r a n s i e n t responses. These were found to be the nasopalatine and sphenopalatine nerves which, enter the 64 o r b i t from the nasa l passages and j o i n the m a x i l l a r y bundle on the f l o o r of the c r a n i a l c a v i t y . On the b a s i s of e l e c t r i c a l s t i m u l a t i o n , the involvement of other small branch nerves could not be r u l e d out. Higher voltage s t i m u l a t i o n (1 to 5 v o l t s ) of the n a s o c i l i a r y branch of the ophthalmic d i v i s i o n of the t r i g e m i n a l nerve evoked t r a n s i e n t r e s p i r a t o r y and c a r d i a c responses i n two of the f i v e animals i n v e s t i g a t e d . A response to 1 v o l t s t i m u l a t i o n of the glossopharyn-geal nerve r e s u l t e d only i f the e l e c t r o d e was placed c e n t r a l to the entry o f , or i n contact_with the sinus nerve. As w i t h water flow s t i m u l a t i o n , when the stimulus s t r e n g t h was s u f f i c i e n t to evoke a response, some i n h i b i t i o n of breathing always accompanied the c a r d i a c response. R e s p i r a t o r y responses however, could o f t e n be evoked without an obvious decrease i n heart r a t e . 3.3.3. Recordings from the I n f e r i o r Laryngeal Nerve. Nerve a c t i v i t y from one of the two a f f e r e n t limbs was monitored to support i t s involvement i n the i n t e r n a l n a s a l r e f l e x . The response to s t i m u l a t i o n by water flow, punctate s t i m u l a t i o n and constant pressure s t i m u l a t i o n of the g l o t t a l area was recorded from s i l v e r wire e l e c t r o d e s placed on the d i s t a l end of the d i v i d e d i n f e r i o r l a r y n g e a l nerve. G l o t t a l s t i m u l a t i o n by water flow was achieved by the technique p r e v i o u s l y described f o r n a s a l s t i m u l a t i o n ( v i a the o r a l f a c i n g cannula) w h i l e constant pressure was a p p l i e d by a b a l l o o n i n the n a s a l c a v i t y i n f l a t e d to 10 cm ^ 0 . The patterns of nervous discharge from a m u l t i - f i b e r p r e p a r a t i o n to both s t i m u l i were s i m i l a r i n onset and peak discharge but water flow always maintained a c t i v i t y much longer than d i d c u f f i n f l a t i o n ( F i g . 14). In f a c t , i n water flow t r i a l s , nerve a c t i v i t y remained above the p r e - s t i m u l a t i o n l e v e l 65 Figure 14. Integrated discharge f rom the l e f t i n f e r i o r l a r y n g e a l nerve i n response to pressure and water flow s t i m u l a t i o n of the pharynx. Top, response to c u f f i n f l a t i o n (10 cm H^O) of the g l o t t i s ; middle, response to a water flow of 32 ml/min; bottom, response to water flow a f t e r two minute t o p i c a l anaesthesia ( x y l o c a i n e , 1%) and during recovery from the anaesthetic (X-W). The v e r t i c a l a x i s measures discharge r a t e of the m u l t i p l e - f i b e r p r e p a r a t i o n i n spikes per second (SPS). The periods of water flow are i n d i c a t e d between the arrows. Water was not f l u s h e d from the nas a l passages i n the middle and bottom t r a c e s . Note the break i n the middle t r a c e . Time, 1 second marker. \" I \" ' \" f \" • i . i m i i i i i i i i i i i i i i i i i i i n n i i m i i n m m i i i m 67 u n t i l the second minute a f t e r water flow was stopped. Cuff i n f l a t i o n caused a d e c l i n e i n spike a c t i v i t y over a one to two minute period and at t h i s time discharge became l e s s than i n the unstimulated c o n d i t i o n . In each p r e p a r a t i o n (n = 3), the a b o l i t i o n of both the nervous a c t i v i t y and the r e s p i r a t o r y and car d i a c responses to water flow s t i m u l a t i o n was complete f o l l o w i n g the two minute i n s t i l l a t i o n of x y l o c a i n e (1%) i n t o the g l o t t a l area ( F i g . 14, bottom t r a c e ) . The recovery of discharge a c t i v i t y from the anaesthetic was r a p i d , beginning about one minute a f t e r i t was flushed out and reached p r e - s t i m u l a t i o n l e v e l a f t e r approximately 5 minutes. 3.4. E f f e r e n t Pathway of the Cardiac R e f l e x Vagal blockade by s e c t i o n and cooling.was attempted i n 5 muskrats but only the former procedure r e s u l t e d i n complete a b o l i t i o n of r e f l e x bradycard-i a . In urethane anaesthetized animals, d i v i s i o n of the v a g i at the l e v e l of the l a r y n x increased heart r a t e by 15±4 beats/min from a normal r a t e of 286±6 (n = 3) and abolished the car d i a c response to nasa l s t i m u l a t i o n and asphyxia. In both cases a t e s t p e r i o d of 30 seconds was used. Blockade of the i n t a c t vagal nerve trunks by thermode c o o l i n g w h i l e r e v e r s i b l e , was l e s s s u c c e s s f u l i n a b o l i s h i n g r e f l e x bradycardia. On c o o l i n g to 5-6°C the i n i t i a l response to nasa l water flow was absent but p e r i o d i c bradycardia occurred i f water flow was continued. The normal response was r e - e s t a b l i s h e d when the thermodes were returned to ambient temperatures. I t was concluded that because of the r e l a t i v e l y l a r g e diameter of the vagal trunk, core tempera-tures of l e s s than 8°C were not achieved over the nerve channel length i n the thermode. The f a i l u r e of complete blockade may have been caused by nerve \" h u r d l i n g \" , noted by Douglas and Malcolm (1955) and would l i k e l y be prevented by decreasing thermode temperatures or i n c r e a s i n g the length of the nerve channel. 68 4. E f f e c t of Lung Input on R e s p i r a t i o n , Heart Rate and Blood Pressure. 4.1. E f f e c t of A r t i f i c i a l V e n t i l a t i o n on Bradycardia Caused by Nasal S t i m u l a t i o n . A r t i f i c i a l v e n t i l a t i o n of seventeen anaesthetized muskrats caused s l i g h t c a r d i o a c c e l e r a t i o n when minute volume exceeded that during u n a s s i s t e d v e n t i l a -t i o n . At four times the minute volume of 150±4 ml/min (n = 10) heart r a t e increased about 10 beats/min. This e f f e c t was independent of CO2 washout as i t occurred i n animals v e n t i l a t e d w i t h 95% 0^ and 5% C0^. l f c w a s evident that a r t i f i c i a l v e n t i l a t i o n drove r e s p i r a t o r y motor neurone output, monitored by phrenic nerve discharge, when the r a t e was c l o s e to the normal breathing r a t e (70±6 per min, n = 10). This response was evoked w i t h t i d a l volumes (1.6 ml) l e s s than that measured i n the f r e e l y b r e athing animal (2.3±0.2 ml, n = 10) arid motor discharge increased w i t h i n c r e a s i n g i n s p i r a t o r y pressure. Slowing a r t i f i c i a l v e n t i l a t i o n below the normal r a t e of f r e e breathing caused phrenic discharge to occur synchronously w i t h both spontaneous and forced i n h a l a t i o n s . Since the e f f e c t of a r t i f i c i a l lung i n f l a t i o n on the car d i a c response may a l s o depend on the s t a t e of c e n t r a l r e s p i r a t o r y neurone a c t i v i t y , a comparison was made between the responses during apneic and non-apneic c o n d i t i o n s . Non-apneic responses were e l i c i t e d by s a l i n e flow (32 ml/min) or by low water flow (15 to 25 ml/min). These s t i m u l i r e s u l t e d i n h i g h l y v a r i a b l e c a r d i a c responses. M a i n t a i n i n g a normal or elevated l e v e l of v e n t i l a t i o n throughout the pe r i o d of nasa l water or s a l i n e flow always a f f e c t e d the l e v e l of bradycardia, although no l e v e l of v e n t i l a t i o n was found which e l i m i n a t e d the bradycardia at the beginning of s t i m u l a t i o n ( P i g . 15). I n d i v i d u a l v a r i a t i o n of the c a r d i a c response to weak nasa l s t i m u l a t i o n was 69 Figure 15. E f f e c t of a r t i f i c i a l v e n t i l a t i o n on the ca r d i a c response to non-apneic n a s a l s t i m u l a t i o n . Top to bottom from time tr a c e (1 second marker) pneumotachogram ( i n s p i r a t i o n downwards), electrocardiogram and event tr a c e i n d i c a t i n g water flow. A, na s a l s a l i n e flow (32 ml/min) i n the f r e e l v breathing animal: B. s a l i n e flow during 4 cm H^ O v e n t i l a t i o n ; C, s a l i n e flow during 8 cm R^O v e n t i l a t i o n ; D, s a l i n e flow during 12 cm R^O v e n t i l a t i o n ; E, s a l i n e flow during 16 cm R^O v e n t i l a t i o n ; F, n a s a l water flow (32 ml/min) i n the f r e e l y breathing animal. Arrows accentuate the period of n a s a l s t i m u l a t i o n . R e s p i r a t o r y traces i n A and F are given i n the i n t e g r a t e d form. 70 M I I I I I M I I , i i i i i i i M i i I , i , M I I I I I 1 I I I I I I L U - U - t - U - U I I I I I I I I I I I B j wmmmmm n i H i i i i m i i i i i i i i i i i n - u i i i i i m n m m \\ \\ m i ! ! : c I 1 71 p a r t i c u l a r l y l a r g e during a r t i f i c i a l v e n t i l a t i o n . S i x of eigh t muskrats became apneic during a 20 second p e r i o d of low water flow but only one was c o n s i s t e n t l y apneic during s a l i n e flow ( F i g . 16). In the l a t t e r animal, when apnea occurred at the onset of s t i m u l a t i o n , the cardiac response was always l e s s w i t h s a l i n e flow than w i t h water flow. When spontaneous breathing occurred during n a s a l s t i m u l a t i o n , the response of heart r a t e was much greater than when a r t i f i c i a l v e n t i l a t i o n was given during apneic s t i m u l a t i o n i n the same animals. Heart r a t e responses to low water flow r a t e or to s a l i n e flow at any given l e v e l of spontaneous (breakthrough) v e n t i l a t i o n were achieved only when a r t i f i c i a l minute volume was increased 4 to 5 times i n the apneic t r i a l s ( F i g . 17). With strong n a s a l s t i m u l a t i o n (water f l o w 32 ml/min) a r t i f i c i a l v e n t i l a t i o n had no e f f e c t i f t i d a l volume was kept below 4 ml. When t i d a l volume exceeded t h i s , heart r a t e became \"locked\" to the r e s p i r a t o r y frequency i n the range of the normal r a t e of breathing ( F i g . 18). S u b s t i t u t i n g 5% carbon d i o x i d e i n oxygen f o r a i r during a r t i f i c i a l v e n t i l a t i o n had no e f f e c t on the r e l a t i o n s h i p between the ca r d i a c and v e n t i l a -t i o n frequencies during strong n a s a l s t i m u l a t i o n ( F i g . 19). Slowing the v e n t i l a t i o n r a t e during s t i m u l a t i o n caused the heart to beat i r r e g u l a r l y , but at a f a s t e r r a t e than i n n o n - v e n t i l a t e d p r e p a r a t i o n s , w h i l e a t higher r a t e s and volumes of v e n t i l a t i o n i t was not unusual f o r heart r a t e to remain high i n the i n i t i a l p e r i o d of s t i m u l a t i o n , f a l l i n g to the v e n t i l a t i o n frequency a f t e r a period which v a r i e d from 3 to 10 seconds. 72 Figure 16. Resp i r a t o r y and c a r d i a c responses to 32 ml/min water and s a l i n e n a s a l flow i n 8 muskrats. Average minute volumes (M.V.) and heart r a t e s (H.R.) are expressed as percent i n h i b i t i o n of the c o n t r o l r a t e s . The t r i a l p e r i o d was 20 seconds i n le n g t h . An apneic response to water flow i s shown i n 6 of the 8 animals. 74 Figure 17. The e f f e c t of spontaneous v e n t i l a t i o n and a r t i f i c i a l v e n t i l a t i o n on the cardiac response to weak nasal stimulation. Open (water) and closed (saline) c i r c l e s describe the r e l a t i o n s h i p between heart rate and minute volume during periods when nasal stimula-t i o n f a i l e d to cause apnea. The t r i a n g l e s describe the r e l a t i o n s h i p between heart rate and a r t i f i c i a l v e n t i l a t i o n at the minute volume shown when the nasal stimulation caused apnea. Open squares ind i c a t e the control values i n the non-stimulated spontaneously breathing animal. The l i n e s were f i t t e d to the points by eye. 75 • a a I I a • ; • 300 r 200 c E tn O -Q X 100 h (t!A 0 1 1 1 ' 0 200 400 600 M.V. ml./min. 76 Figure 18. E f f e c t of a r t i f i c i a l v e n t i l a t i o n on the c a r d i a c response to strong n a s a l s t i m u l a t i o n . Top to bottom from time trace (1 second marker), pneumotachogram ( i n s p i r a t i o n downwards), EKG and event trace i n d i c a t i n g water flow (32 ml/min). A, a r t i f i c i a l v e n t i l a -t i o n at 4 cm H^O; B, a r t i f i c i a l v e n t i l a t i o n at 8 cm H^O; C, a r t i f i c i a l v e n t i l a t i o n at 16 cm R^O; D, maintained lung i n f l a t i o n at 16 cm H^O. V e n t i l a t i o n was given at 1 hz. M W W W V V V V W V W V V W W W W 1 i H i l U l l H i l l i l i m i U l i l n i l i l n i i n l i -• |. , , l m l I 11111 i| il II i| 11 II 1 I M l I 1 I i l M I 1 i I I i 1 i I II I I I I—I i I I V I I I II I I I I I I I I 1 I I I M I i I 1,1.1.1 M I II I I I I I I I I M II II I II M I I I I I I I II H II I II I M M II M M II I I I I H I I I II LL 78 Figure 19. Cardiac response during v e n t i l a t i o n w i t h 95% oxygen-5% carbon d i o x i d e gas mixture and a i r . V e r t i c a l bars represent means of the c o n t r o l bradycardic response, 5% CC^ v e n t i l a t e d and a i r v e n t i l a t e d t r i a l s ±S.E.M. I n s p i r a t o r y pressures of 16 cm R^O at 1 hz were given i n a l l t r i a l s i n which the response to v e n t i l a t i o n was t e s t e d . Water flow of 32 ml/min caused apnea i n each of the 15 second t r i a l p e riods. 79 1 0 0 i c _S50 | i r n w a t e r o n l y • 9 5 0 2 - 5 C 0 2 • a i r A 12 m u s k r a t 13 14 80 4.2 E f f e c t of Maintained Lung I n f l a t i o n .on the.Cardiac7 Response to Nasal S t i m u l a t i o n . The e f f e c t of maintained lung i n f l a t i o n on bradycardia caused by n a s a l water flow was i n v e s t i g a t e d i n t r i a l s on three muskrats by comparing heart r a t e during constant i n f l a t i o n w i t h that i n animals which were allowed to breathe normally before water flow began. Apneic responses to constant i n f l a t i o n (Hering-Breuer r e f l e x ) were normally achieved at 10-12 cm ^ 0 and at t h i s pressure were maintained f o r approximately 15 seconds. When pressure was a p p l i e d simultaneously w i t h n a s a l s t i m u l a t i o n the heart r a t e was no d i f f e r e n t from that when n a s a l s t i m u l a t i o n was given alone ( F i g . 20). A l l data were c o l l e c t e d from animals which were apneic over the 15 second t r i a l p e r i o d . 4.3 E f f e c t of Pulmonary Deafferentation-: on the R e s p i r a t o r y and Cardiac Responses to A r t i f i c i a l V e n t i l a t i o n . D e a f f e r e n t a t i o n of the lungs was achieved by a l l o w i n g the animals to breathe steam. As a r u l e , two breaths of steam abolished the Hering-Breuer r e f l e x to constant i n f l a t i o n yet heart r a t e was s t i l l a f f e c t e d by a r t i f i c i a l v e n t i l a t i o n frequency during strong n a s a l s t i m u l a t i o n ( F i g s . 21 and 22). However, a f t e r 4 breaths •; of steam, the \" r a t e l o c k i n g \" e f f e c t of v e n t i l a t i o n frequency was abolished and lung i n f l a t i o n had no apparent e f f e c t on heart r a t e . D e a f f e r e n t a t i o n , as judged by the l o s s of the c a r d i a c response to v e n t i l a t i o n , was followed by spontaneous breathing during a r t i f i c i a l v e n t i l a -t i o n and the pacing of the normal breathing by lung i n f l a t i o n was no longer evident ( F i g . 21E). Lung d e a f f e r e n t a t i o n by steaming was attempted i n 10 muskrats ( F i g . 23). In f r e e l y breathing animals complete d e a f f e r e n t a t i o n had l i t t l e e f f e c t on 81 Figure 20. Cardiac response to a r t i f i c i a l v e n t i l a t i o n and maintained i n f l a t i o n during strong n a s a l s t i m u l a t i o n i n three muskrats. The v e r t i c a l bars represent the means of the c o n t r o l response +S.E.M. (un a s s i s t e d , l e f t ) and the response to lung i n f l a t i o n and v e n t i l a t i o n at 16 cm R^O. Animals were apneic i n a l l t r i a l s . 82 60 r 40 o X 20 w a t e r o n l y • c o n s t , i n f l . • a r t . v e n t . A 13 m u s k r a t 14 83 Figure 21. Separation of the responses to a r t i f i c i a l v e n t i l a t i o n and maintained i n f l a t i o n . Top to bottom from time t r a c e (1 second marker), pneumotachogram ( i n s p i r a t i o n downwards), EKG and event trac e (dots i n B and C). A, c o n t r o l response to 12 cm H^O, 1 hz v e n t i l a t i o n during apneic water flow s t i m u l a t i o n ; B, Hering-Breuer r e f l e x to 15 cm I^O maintained i n f l a t i o n i n the f r e e l y breathing animal; C, l o s s of the Hering-Breuer r e f l e x a f t e r one breath of steam (15 cm ^ 0 ) ; D, 12 cm ^ 0 a r t i f i c i a l v e n t i l a t i o n a f t e r one breath of steam; E, 12 cm ^ 0 a r t i f i c i a l v e n t i l a t i o n a f t e r three breaths of steam. Nasal water flow (32 ml/min) i s i n d i c a t e d by the arrows. Note the i r r e g u l a r i t y of t r a c h e a l a i r flow before water flow and the l o s s of the pacing of heart r a t e during water flow i n the l a t t e r r e c o r d i n g . 84 I I I I I I I I ! i I I I ! I I M I i I I I : i I I I I I I I I I I I I I I I I I I I I i I I I I I I I I I i I I I I I I I I I I I i I I mmmmmiwmmt-I.I 1111 I II i i 1 1 1 1 1 1 1 1 1 liiiiiiiiiiiiiiliiliiliiii'r.iliiiii iMAAAAAAA^AAAAAAAAVWvAJ|^_. 15 cm. inflation i!!!.,. • • I liiiLlJlittliUlilldiJ,!! j ! i I j 1 1 1 1 1 1 | ! | | f l>ii'|\"ifili|li| !iii|^ ! l | l l | l l l f i l « l l « i f | l l | 15 cm. inflation -nnttmttr-rr tmm |+ttmtttttrtrhni^^ MUUt • n — i r r i i - i r i i i i i i ' 11 i i i i i 11 11 | T < - i | i i II n | i i II ! i II n m r 85 Figure 22. Loss of cardiac response to v e n t i l a t i o n a f t e r s e q u e n t i a l steaming i n one muskrat. Histograms show c a r d i a c response to 12 cm H2O v e n t i l a t i o n at 1 hz during water flow s t i m u l a t i o n (32 ml/min) ±S.E.M. Top, c o n t r o l response w i t h p e r i o d of asphyxia i n d i c a t e d above i n diagrammed a r t i f i c i a l v e n t i l a t i o n trace (A.V.); middle, a f t e r one breath of steam; bottom, a f t e r three breaths of steam. Water flow marker corresponds to a period of 30 seconds. The increase i n heart r a t e a f t e r secondary steaming i s the normal recovery p a t t e r n described e a r l i e r . 86 A . V . H 2 0 con t ro l i mm 1° s team 1 1 t i l l 2 ° steam i il m 87 Figure 23. E f f e c t of lung steaming on r e s p i r a t i o n and heart r a t e and t h e i r responses to strong nasal flow s t i m u l a t i o n (32 ml/min). V e r t i c a l bars i n d i c a t e the means of the heart r a t e (H.R.), r e s p i r a t o r y r a t e (R.R.), t i d a l volume (T.V.) and minute volume (M.V.) before (b) and a f t e r (a) the l o s s of the ; Hering-Breuer r e f l e x to constant i n f l a t i o n (12 cm ^ 0 ) i n 10 muskrats. S t i p p l e d bars show responses to water flow s t i m u l a t i o n i n a 20 second time p e r i o d . Note that on average, the animals were non-apneic during n a s a l s t i m u l a t i o n both before and a f t e r steaming. Values are the means ±S.E.M. normal water b 20 c E X Q. 89 minute volume (before 150±4 ml/min, n = 10; a f t e r 161±24 ml/min, n = 10) sinc e although t i d a l volume increased by 3 times (before 2.3±0.2 ml/min, n = 10; a f t e r 6.7±0.3 ml/min, n = 10) r e s p i r a t o r y frequency f e l l to one t h i r d the normal r a t e (before 70+6 per min, n = 10; a f t e r 24±2 per min, n = 10). Heart r a t e i n the f r e e l y breathing c o n t r o l s was 286±6 (n = 10) and t h i s f e l l to 197±22 (n = 10) i n the lung denervates. Strong n a s a l s t i m u l a t i o n caused heart r a t e to f a l l to 21.3±3 beats/min (n = 10) i n i n t a c t f r e e l y breathing animals yet i n the denervates heart r a t e only f e l l to 48.5±2 beats/min (n = 9). There was no apparent e f f e c t of d e a f f e r e n t a t i o n on the r e s p i r a t o r y response to n a s a l water flow. The l o s s of the car d i a c response to a r t i f i c i a l v e n t i l a t i o n was demon-s t r a t e d by p l o t t i n g the heart r a t e response r e s u l t i n g from n a s a l s t i m u l a t i o n combined w i t h a r t i f i c i a l v e n t i l a t i o n ranging to a maximum of 324 ( F i g . 24) to 875 ml/min. In each case v e n t i l a t i o n frequency was one hz and d e a f f e r e n t a t i o n was considered complete when the heart f a i l e d to respond to i n c r e a s i n g v e n t i l a t i o n ( i . e the heart rate-minute volume slope approached zero, F i g . 25). The f a i l u r e to a b o l i s h the response i n two animals apparently r e s u l t e d from i n h a l i n g steam too r a p i d l y (1 hz) sin c e those steamings c a r r i e d out at a slower r a t e (0.3 to 0.5 hz) were more e f f e c t i v e . Figure 25 al s o i n d i c a t e s the v a r i a b i l i t y of the pre-treatment response to a r t i f i c i a l v e n t i l a t i o n . The higher slopes given f o r muskrats 5, 9, 13 and 14 were due to the f a c t that heart r a t e was not paced by v e n t i l a t i o n frequency even at l a r g e v e n t i l a t i o n pressures w h i l e i n the other animals pacing of heart r a t e by v e n t i l a t i o n was pronounced during strong n a s a l s t i m u l a t i o n . 90 Figure 24. A b o l i t i o n of the c a r d i a c response to a r t i f i c i a l v e n t i l a t i o n by steaming. The response to 1 hz a r t i f i c i a l v e n t i l a t i o n i s shown before ( t r i a n g l e s ) and a f t e r ( c i r c l e s ) 4-6 breaths of steam. V e n t i l a t i o n pressures ranged up to 16 cm H^O. The s t i p p l e d blocks i n d i c a t e the range of c o n t r o l heart rate-minute volume p l o t s before (above) and a f t e r (below) the a p p l i c a t i o n of steam i n one animal. Nasal s t i m u l a t i o n was achieved w i t h a water flow which caused apnea f o r 15 seconds. The l i n e s were f i t t e d by l i n e a r r e g r e s s i o n techniques and were based on a minimum of 6 p o i n t s i n each animal (r = 0.99, m = 0.255, and r - 0.99, m = -0.007 r e s p e c t i v e l y f o r the pre- and post-treatment l i n e s ) . 91 o L 0 200 400 600 M . V . m l . / m i n . 92 Figure 25. Loss of the c a r d i a c response to a r t i f i c i a l v e n t i l a t i o n by steaming i n 9 muskrats. Each v e r t i c a l l i n e represents the extent of the response l o s s taken from the heart rate-minute volume slopes during n a s a l s t i m u l a t i o n ( F i g . 24). The normal response i s given at the top and the post-steam values near the dotted l i n e ; i n d i c a t i n g complete a b o l i t i o n . Open c i r c l e s represent response to water flow s t i m u l a t i o n and the clos e d c i r c l e s to s a l i n e flow. Q N H.R.-lung inf slope o o O 94 4.4 E f f e c t of Lung D e f l a t i o n on Heart Rate and Blood Pressure. The e f f e c t of lung d e f l a t i o n per se on normal heart r a t e was i n v e s t i g a t e d i n 9 a r t i f i c i a l l y v e n t i l a t e d c u r a r i z e d muskrats. The animals were u s u a l l y v e n t i l a t e d u w i t h oxygen and PaC^ remained constant throughout the t e s t periods. Stopping v e n t i l a t i o n w i t h the lungs i n f l a t e d had l i t t l e e f f e c t on heart r a t e but lung c o l l a p s e from any i n s p i r a t o r y pressure caused an immediate brady-c a r d i a which u s u a l l y p e r s i s t e d from 5 to 20 seconds or u n t i l v e n t i l a t i o n was resumed ( F i g . 26). Lung d e f l a t i o n caused heart r a t e to f a l l from 268±7 to 59±4 beats/min w i t h i n 0.97±0.17 seconds (n = 28) and n e i t h e r the l a t e n t p e r i o d nor the l e v e l of bradycardia was a f f e c t e d by the i n s p i r a t o r y pressure over the range of 5 to 15 cm ^ 0 . Blood pressure changes i n response to i n f l a t i o n (decrease) or d e f l a t i o n (increase) were dependent on the i n s p i r a t o r y pressure but s t a b i l i z e d w i t h i n 5 seconds and were unaffected by b i l a t e r a l sinus nerve s e c t i o n . Maintained lung i n f l a t i o n by clamping the t r a c h e a l cannula at f u l l i n s p i r a t i o n caused a bradycardia a f t e r a p e r i o d which was p r o p o r t i o n a l to the i n f l a t i o n pressure ( F i g . 27). When i n f l a t i o n pressures were v a r i e d between 5 and 15 cm ^ 0 , the time to the f i r s t appearance of bradycardia increased from 6.8±1.8 (n = 5) to 35.0±7.0 (n = 8) seconds r e s p e c t i v e l y . The onset of bradycardia caused by d e f l a t i o n of the lungs however, was not changed when the i n s p i r a t o r y pressures were t e s t e d over the same range (0.76±0.15 and 1.28±0.20 (n = 8) seconds r e s p e c t i v e l y ) . 5. Baroreceptor and Chemoreceptor C o n t r i b u t i o n s to the Cardiovascular Responses. An i n d i c a t i o n of baroreceptor a c t i v i t y was obtained from the degree of bradycardia which r e s u l t e d from an increase i n blood pressure caused by i n t e r a r t e r i a l i n j e c t i o n of a d r e n a l i n . I n j e c t i o n of 5 ug/kg of a d r e n a l i n 95 Figure 26. Cardiovascular e f f e c t s of lung i n f l a t i o n and d e f l a t i o n . Top to bottom; a r t e r i a l blood pressure ( c a r o t i d ) , a r t e r i a l blood oxygen, pneumotachogram ( i n s p i r a t i o n upwards), electrocardiogram and time trace (1 second marker). I n f l a t i o n and d e f l a t i o n of the lungs were brought about by clamping the e x p i r a t o r y (upward arrow) and i n s p i r a t o r y (downward arrow) tubes of the r e s p i r a t o r y v a l v e . I n s p i r a t o r y pressure was set at 12 cm H„0 throughout. 96 i 100r BLOOD PRESSURE mm Hg 100 \"o2 mm Hg V e n , i l a , i° S n '/lMMMM/^^ EXP. E. K. G. TIME sec * INFLATED • DEFLATED * • T T T T Y V J r> » l >T r rryr l l M i i r l y» M l l rnrryrm i i W I | f i ' r r i i . . l f iiivrttrtytf ryryyrryrryirnrn 97 Figure 27. E f f e c t of i n s p i r a t o r y pressure on the l a t e n t p e r i o d before onset of bradycardia caused by lung i n f l a t i o n and d e f l a t i o n . I n f l a t i o n i s i n d i c a t e d by^ t h e . u n s t i p p l e d bars; d e f l a t i o n by the s t i p p l e d bars ±S.E.M. In'jboth s e r i e s v e n t i l a t i o n at 1 hz preceded the event. *^»; •• '. . 1 •» • 4.14 98 40 p 30 to Q Z o u LU CO Q 2 0 O z LU I— < 10 8 VENT. PRESS. 10 CM. H 2 0 15 99 caused the mean blood pressure to r i s e from 63±5 to 118±5 mm Hg (n = 6). A mean pressure of 115±4 mm Hg (n = 6) was re q u i r e d to e l i c i t the ca r d i a c b a r o s t a t i c response and at t h i s pressure heart r a t e f e l l to 90±12 from a c o n t r o l r a t e of 253±6 beats/min. Bradycardia was maintained as long as the blood pressure was elevated i n s p i t e of continued a r t i f i c i a l v e n t i l a t i o n ( F i g . 28). Denervation of the c a r o t i d sinus baroreceptors by sinus nerve s e c t i o n e l i m i n a t e d the b a r o s t a t i c r e f l e x but had no e f f e c t on bradycardia caused by n a s a l s t i m u l a t i o n or lung d e f l a t i o n ( F i g . 28, r i g h t ) . C a r o t i d body chemoreceptor s t i m u l a t i o n due to i n j e c t i o n of 80-200 ug/kg potassium cyanide i n t o the c a r o t i d a r t e r y caused hyperpnea and tach y c a r d i a i n spontaneously breathing muskrats'..and\" t r a n s i e n t bradycardia i n c u r a r i z e d a r t i f i c i a l l y v e n t i l a t e d animals. B i l a t e r a l sinus nerve s e c t i o n abolished both r e s p i r a t o r y and car d i a c responses to the i n j e c t i o n of cyanide. A r t i f i c i a l v e n t i l a t i o n of c u r a r i z e d muskrats w i t h anoxic-normocapnic gas caused bradycardia when Pa02 reached an average of 63+6 mm Hg (n = 6). Heart r a t e f e l l from an i n i t i a l r a t e of 277±11 to 76±7 beats/min at a Pa02 of 29 mm Hg ( F i g . 29). Mean a r t e r i a l pressure was l i t t l e a f f e c t e d i n the i n i t i a l p e riod of anoxic-normocapnic v e n t i l a t i o n but rose when bradycardia occurred. B i l a t e r a l s e c t i o n of the sinus nerve delayed chemoreceptor d r i v e n bradycardia u n t i l Pa02 had f a l l e n to a' p a r t i a l pressure of 34+4 mm Hg (n = 9). To f u r t h e r study the c o n t r i b u t i o n of the chemoreceptors during simulated d i v i n g c o n d i t i o n s , Pa02 was continuously monitored i n paralyzed muskrats which were asphyxiated w i t h and without n a s a l water f l o w , and a l s o when a r t i f i c i a l v e n t i l a t i o n was maintained during water flow. F i v e muskrats were prepared f o r t h i s study by s i t u a t i n g a temperature regulated oxygen el e c t r o d e i n a loop formed by cannulation of the r i g h t c a r o t i d a r t e r y . A s p h y x i a t i o n 100 Figure 28. A b o l i t i o n of baroreceptor r e f l e x by sinus nerve s e c t i o n . Top to bottom, c a r o t i d a r t e r y blood pressure (mm Hg), a r t e r i a l blood oxygen (mm Hg), pneumotachogram ( i n s p i r a t i o n upwards), electrocardiogram and time tr a c e (1 second marker). L e f t , normal response to i n j e c t i o n of 5 yg a d r e n a l i n i n t o a r t e r i a l loop; r i g h t , response to 5 yg a d r e n a l i n f o l l o w i n g b i l a t e r a l s e c t i o n of sinus nerves. Recording a l s o i n c l u d e s responses to 10 second p e r i o d of asphyxia i n the e x p i r a t o r y p o s i t i o n , ( r i g h t ) . V e n t i l a t i o n a t 8 cm H o0. vrrvrim tnrryt r 102 Figure 29. E f f e c t of sinus nerve s e c t i o n on the responses to hypoxia. Top to bottom, c a r o t i d a r t e r i a l blood pressure, c a r o t i d a r t e r i a l blood oxygen, pneumotachogram ( i n s p i r a t i o n upwards) and EKG. A, normal response; B, f o l l o w i n g b i l a t e r a l sinus nerve s e c t i o n . A r t i f i c i a l v e n t i l a t i o n was set at 10 cm 1^0 and 1 hz. V e n t i l a -t i o n w i t h 5% CO2 i n n i t r o g e n and 100% oxygen are i n d i c a t e d by the arrows. The onset of.,hypoxic bradycardia i s i n d i c a t e d by the h o r i z o n t a l l i n e . Time,'1 second marker. 103 100i BLOOD PRESSURE mm Hg I N S P . Ventilation 9 5 N 2 - 5 C 0 2 -N-E. K. G. 100p BLOOD PRESSURE mm Hg 100i P0°2 mmHg I N S P . Ventilation 95 N 2 - 5 C 0 2 -M-E.K.G. 1-4! TIME sec pTTrrrrrryTi f tnrnyii 111 n tr^YrrTTrrrryrrTrrirrY^ 104 was brought on by clamping the t r a c h e a l cannula a f t e r e x p i r a t i o n . Figures 30 and 31 compare the ca r d i a c response to asphyxia w i t h that when a r t i f i c i a l v e n t i l a t i o n was maintained during water flow s t i m u l a t i o n and when water flow and asphyxia were combined. Bradycardia caused by a s p h y x i a t i o n alone d i d not begin u n t i l 8.3±0.6 seconds (n = 18) a f t e r t r a c h e a l clamping and heart r a t e continued to f a l l to a r a t e of 78±8 a f t e r one minute. The response to water flow s t i m u l a t i o n was immediate ( l a t e n t p e r i o d 0.55±0.07 seconds, n = 10) w i t h or without v e n t i l a t i o n and heart r a t e f e l l to 53±7 (apneic) and 110±5 beats/ min ( v e n t i l a t e d ) a f t e r 10 seconds of s t i m u l a t i o n . In view of the pronounced c a r d i a c response to water s t i m u l i i n the anaesthetized muskrat, the r a t e of d e c l i n e of a r t e r i a l blood oxygen was great e r than expected when compared to the asphyxic c o n t r o l s and the r e l a t i v e gradual f a l l found i n PaO^ i n other animals of comparable d i v i n g a b i l i t y ( I r v i n g et a l . , 1941b; Clausen and E r s l a n d , 1968; Eerrante, 1970). Nevertheless, when the trachea was clamped PaO^ began to f a l l sooner and more r a p i d l y than i f water was begun simultaneously w i t h a s p h y x i a t i o n and the PaO^ throughout the one minute t r i a l s were s i g n i f i c a n t l y d i f f e r e n t (95% confidence l i m i t ) i f the c o n t r o l was normalized to 100%-(Fig. 32). Normaliz-a t i o n was re q u i r e d because of the v a r i a b i l i t y of c o n t r o l Pa02 to moderate l e v e l s of a r t i f i c i a l v e n t i l a t i o n . The l a t e n t p e r i o d before a measured f a l l i n Pa02 was 7.3±1.2 (n = 15) and 11.4±1.5 (n = 14) seconds r e s p e c t i v e l y f o r the asphyxic and water s t i m u l a t e d asphyxic responses. Figure 33 i l l u s t r a t e s the r e l a t i o n s h i p of heart r a t e and Pa02 based on the data given i n Figure 32. The graphs underline the dependence of heart r a t e on blood oxygen tension to about 40 mm Hg when the animal i s asphyxiated. However, during water stimu-l a t i o n of the nares, cardiac f u n c t i o n i s apparently t o t a l l y independent of Pa0 o during the e n t i r e experimental p e r i o d . 105 Figure 30. Response of a r t e r i a l blood oxygen tension to asphyxia i n a c u r a r i z e d muskrat. A, n a s a l water flow (32 ml/min) coupled w i t h a r t i f i c i a l v e n t i l a t i o n at 8 cm H^O and 1 hz; B, asphyxia; C, n a s a l water flow w i t h asphyxia. I n i t i a t i o n and t e r m i n a t i o n of events are i n d i c a t e d by the arrows. Time marker i s 60 seconds. The dip i n Pa02 at the onset of bradycardia (C) was a r t i f a c t u a l and due to the flow s e n s i t i v e nature of the oxygen e l e c t r o d e . 106 107 Figure 31. Mean heart r a t e during asphyxia and water flow s t i m u l a t i o n w i t h and without a r t i f i c i a l v e n t i l a t i o n i n f i v e c u r a r i z e d muskrats. a, asphyxia brought on by t r a c h e a l clamping (n = 18); w, n a s a l s t i m u l a t i o n w i t h water flow of 32 ml/min coupled w i t h a r t i f i c i a l v e n t i l a t i o n at 12 cm H^ O and 1 hz (n = 16); w & a, n a s a l water s t i m u l a t i o n during a s p h y x i a t i o n • ( n = 22). A l l t r i a l s were 60 seconds i n d u r a t i o n , beginning and ending at the markers. 108 r 109 Figure 32. Response of a r t e r i a l blood oxygen tension to water flow induced asphyxia i n f i v e muskrats. w, n a s a l water flow (32 ml/min) coupled w i t h a r t i f i c i a l v e n t i l a t i o n at 8 cm 1^0 and 1 hz (n = 10); w & a, n a s a l water flow during a s p h y x i a t i o n (n = 14); a, asphyxia (n = 15). Events were i n i t i a t e d and terminated at the arrows.- A l l c o n t r o l values are normalized to 100%. 110 0 I 1 1 1 1 1 1 1 1 L_ 0 20 40 60 80 seconds I l l F igure 33. P l o t of heart r a t e and a r t e r i a l blood oxygen during asphyxia and n a s a l water flow. P o i n t s are as i n F i g . 38 (5 muskrats). w, n a s a l water flow (32 ml/min) coupled w i t h a r t i f i c i a l v e n t i l a -t i o n at 8 cm and 1 hz; w & a, n a s a l water flow during a s p h y x i a t i o n ; a, asphyxia. P o i n t s are p l o t t e d at 5 second i n t e r v a l s during 60 second events s t a r t i n g from c o n t r o l ( c l o s e d markers) and ending at arrows. 112 w&a 0 20 40 60 80 100 P 0 i 0 2 mm Hg. 113 D i s c u s s i o n The r e s u l t s from the present study show that even though the muskrat i s not noted f o r i t s underwater endurance, i t d i s p l a y s c a r d i o v a s c u l a r r e f l e x e s that are as profound as any found among marine mammals. The animal normally i n h a b i t s shallow waters and i n the w i l d i s r a r e l y observed to d i v e f o r periods longer than 30 seconds although a tolerance of 12 to 17 minutes underwater has been reported ( I r v i n g , 1939; E r r i n g t o n , 1963). In t h i s study, provoked submersions l a s t i n g up to 10 minutes were observed i n Ondatra z i b e t h i c a osoyoosensis which s t i l l f a r exceed that p r e d i c t e d from c a l c u l a t e d oxygen stores i f r e s t i n g metabolism was maintained during the div e . Based on a blood oxygen c a p a c i t y of 25% ( I r v i n g , 1939), a lung volume of 20 ml and a r e s t i n g metabolic r a t e of 0.73 ml/gm hr (McEwan e_t a l . , 1974) the upper l i m i t of d i v i n g time i s about 2.7 minutes. L i k e the shallow d i v i n g s e a l however, the muskrat g e n e r a l l y exhales a i r p r i o r to d i v i n g and thus apneic endurance time without c a r d i o v a s c u l a r adjustment would reduce t h i s estimate to approximately 2.3 minutes and near :the d i v i n g endurance times found i n t e r r e s t r i a l mammals of equal s i z e ( I r v i n g , 1939). As a r e s u l t i t i s not s u r p r i s i n g that changes i n heart r a t e and p e r i p h e r a l blood fl o w i n the muskrat are dramatic (Lord, unpublished) and r e f l e c t an unusual a b i l i t y to conserve oxygen. In c o n t r a s t to the w e l l documented l a b i l i t y of heart r a t e i n the marine mammal (Scholander, 1940; I r v i n g et a l . , 1942; Van C i t t e r s et^ al., 1965; Eisn e r et a l . , 1966b), the r e s t i n g muskrat d i s p l a y e d a r e g u l a r EKG which was r a p i d and without any s i g n of the sinus arrhythmia which i s so prominent i n the s e a l . Sinus arrhythmia was found to occur only i n the f i r s t few seconds of recovery ifrom d i v i n g . The maximum c a r d i a c response to submersion i n the 114 f r e e l y d i v i n g animal averaged from 8 to 17% of the r e s t i n g heart r a t e i n a s e r i e s of dives ranging up to one minute. Restrained dives on the other hand, y i e l d e d responses of 19 to 27% of the c o n t r o l r a t e but the d i f f e r e n c e between the two types of dives was l a r g e l y a r e s u l t of a s i g n i f i c a n t l y lower r e s t i n g r a t e i n the r e s t r a i n e d animals. Nevertheless, w h i l e the s t a b i l i z e d underwater r a t e during forced d i v i n g was s t a t i s t i c a l l y higher than i n the unrestrained animals, t h i s i n d i c a t e s that the muskrat responds q u i t e d i f f e r e n t -l y to r e s t r a i n t than e i t h e r the s e a l of the porpoise. In f a c t , a greater response occurs during forced d i v i n g i n the s e a l ( E i s n e r , 1965; H a r r i s o n et a l . , 1972) while the porpoise d i s p l a y s s u r p r i s i n g l y weak c a r d i a c responses under these c o n d i t i o n s ( E i s n e r e_t a l . , 1966b) . In the f r e e l y d i v i n g s e a l , bradycardia has been v a r i o u s l y measured at 5% (Harrison, 1960), 12% (Harrison and Tomlinson, 1960), 10% (Murdaugh et a l . , 1961), 14-28% (Harrison et a l . , 1972), 18% (Kooyman and Campbell, 1972) and 5-8% (Dykes, 1974a) of the pre-dive r a t e . I n most cases however, the d u r a t i o n of these dives was much longer than those reported here and heart r a t e l i k e l y r e f l e c t s to a greater degree, d i r e c t asphyxic responses. This i s p a r t i c u l a r l y evident i n the elephant s e a l whose heart r a t e f a l l s from 80 to 4 beats/min a f t e r a 40 minute dive w h i l e i t reaches only one h a l f the r e s t i n g r a t e a f t e r one minute underwater (Van C i t t e r s _et aL,, 1965). T e r r e s t r i a l species such as man (Kawakami et a l . , 1967; Heistad and Wheeler, 1970; Whayne and K i l l i p , 1967; C o r r i o l and Rohner, 1968), cats (Lisander, unpublished observations) and the white r a t show responses which are f a r slower i n onset and much l e s s intense than i n aquatic or marine species. In the r a t , submersion bradycard-i a reaches j u s t 29-44% of the c o n t r o l r a t e a f t e r 30 seconds of dive time ( L i n and Baker, 1975; Huang and Peng, 1976). 115 The r e l a t i o n s h i p between the c a r d i a c and vasomotor responses to d i v i n g i s s t i l l a matter of s p e c u l a t i o n but i t i s c l e a r that both are important p a r t i c u l a r l y i n prolonged d i v e s . Species i n which intense c a r d i a c responses are recorded a l s o show the most prominent increases i n p e r i p h e r a l r e s i s t a n c e ( I r v i n g et a l . , 1941b; Ferrante and F r a n k e l , 1971; Folkow et a l . , 1971) yet the vasomotor response does not depend on the development of bradycardia (Murdaugh at a l . , 1968). Even though there may be an innate d i s s o c i a t i o n of the two r e f l e x e s i n d i v i n g , heart r a t e i s presumed to be an accurate r e f l e c -t i o n of the t o t a l d i v i n g response ( I r v i n g et_ a l . , 1941b; L e i v e s t a d , 1960; B l i x et a l . , 1975; B l i x et a l . , 1976a). I t has been suggested that the major r o l e of the bradycardic response to submersion i s not r e l a t e d d i r e c t l y to the conservation of oxygen si n c e d i v i n g without a c a r d i a c response i n the s e a l does not a f f e c t short term d i v i n g a b i l i t y (Murdaugh et a l . , 1968). Rather, these workers p o i n t out, that the r e f l e x may be necessary to equate c a r d i a c output w i t h a reduced area of t i s s u e p e r f u s i o n . I r v i n g jst a l . (1941b) however, c l a i m that bradycardia per se i n the s e a l accounts f o r a saving of approximately h a l f the oxygen s t o r e i n longer d i v e s . This conclusion i s supported by White and McRitchie (1973) who demonstrated that oxygen conservation i n unanaesthetized r a b b i t i s only e f f e c t i v e when both c a r d i a c and v a s o c o n s t r i c t o r responses to nasopharyngeal s t i m u l a t i o n are i n t a c t . They suggested that because vagal blockade r e s u l t s i n a f a s t e r d e c l i n e of blood oxygen tensions during s t i m u l a t i o n , bradycardia aids i n oxygen conservation by reducing pulmonary blood flow. P e r i p h e r a l r e s i s t a n c e changes i n the unanaesthetized n u t r i a are abrupt and have been shown to increase 10 f o l d i n 10 seconds of submersion (Folkow ej: a l . , 1971) w h i l e i n the s e a l the response i s maximal a f t e r l e s s than 20 seconds ( I r v i n g _et a l . , 1942; E i s n e r et a l . , 1966a) suggesting that the r a p i d onset of 116 bradycardia i s c l o s e l y followed by a r a p i d increase i n p e r i p h e r a l r e s i s t a n c e . This view i s al s o supported by work i n the r a b b i t i n which the c a r d i o v a s c u l a r e f f e c t s to smoke i n h a l a t i o n were found to be f u l l y e s t a b l i s h e d a f t e r 7-10 seconds of s t i m u l a t i o n (White and McRi t c h i e , 1973). While i t proved d i f f i c u l t i n t h i s study to i s o l a t e the e x t e r n a l n a r i a l c o n t r i b u t i o n from the i n t e r n a l n a s a l r e f l e x e s by denervation alone, one can s a f e l y conclude that n a r i a l receptors p r e c i p i t a t e the t r a i n of d i v i n g r e f l e x e s i n the muskrat. Apnea and bradycardia were caused by water l a p p i n g the nares of the conscious animal; a r e f l e x which does not depend on the a n t e r i o r p o r t i o n of the m a x i l l a r y d i v i s i o n s i n c e b i l a t e r a l s e c t i o n of t h i s nerve at the l e v e l of the zygomatic arch had no e f f e c t on the responses. Studies on the i n n e r v a t i o n and s t i m u l a t i o n of nerves l e a d i n g to the nares i n d i c a t e that the n a s o c i l i a r y nerve may be a major c o n t r i b u t o r to the r e f l e x e s o r i g i n a t i n g from t h i s area. On the other hand, a b o l i t i o n of these r e f l e x e s by anaesthesia e x p l a i n s the low s e n s i t i v i t y of the n a s o c i l i a r y nerve to e l e c t r i c a l s t i m u l a -t i o n . Sensory i n n e r v a t i o n and motor c o n t r o l of the nares r e s i d e s i n the n a s o c i l i a r y but l i k e the sphenopalatine and nasopalatine branches df the m a x i l l a r y d i v i s i o n which supply the nasa l mucosa, i t i s not e a s i l y a c c e s s i b l e and consequently i t s involvement i n the r e f l e x e s was not t e s t e d d i r e c t l y . I t i s i n t e r e s t i n g to note that v i s u a l input seems to play no r o l e i n the generation of the responses during forced dives i n the muskrat, but t h i s should not be assumed to be the case i n the w i l d . In marine mammals v i s u a l cues are known to have a profound e f f e c t on heart r a t e ( E i s n e r , 1969). While the a f f e r e n t limb of the n a r i a l r e f l e x e s probably l i e s • i n branches of the m a x i l l a r y or ophthalmic d i v i s i o n s of the t r i g e m i n a l nerve and incl u d e s c e n t r a l suprabulbar connections found to be i n v o l v e d i n r e f l e x e s at the conscious l e v e l (White e_t a l . , 1974), the f i n d i n g s do not n e c e s s a r i l y c o n f l i c t 117 with those of Dykes (1974b) who demonstrated that f a c i a l neurotomy i n the s e a l a l t e r s b ehavioural responses but not the time course of bradycardia to submersion. Even i n the absence of the n a r i a l r e f l e x e s , secondary sensory pathways w i t h i n the nares and i n the n a s a l passages would most l i k e l y produce responses no d i f f e r e n t from those i n i t i a t e d by n a r i a l r e c e ptors. V a r i a t i o n s i n water temperature had no n o t i c e a b l e e f f e c t on the d i v i n g responses i n the muskrat. This i s c o n s i s t e n t w i t h r e s u l t s from ducks (Ander-sen, 1963a; B u t l e r and Jones, 1968) and s e a l s (Dykes, 1974b) but not i n man i n which bradycardia prompted by face immersion i s temperature dependent (Kawakami et a l . , 1967; Song et a l . , 1969; Whayne and K i l l i p , 1967; Moore et a l . , 1972). The r e s u l t s however, do not appear to agree w i t h Thornton et a l . (1978) who found a greater c a r d i a c response i n f o r c e dived muskrats when water temperature was lowered from 32 to 2°C. Nevertheless, since these animals were immersed i n water p r i o r to the head being submerged, responsive-ness to the c o l d e r temperature may have been increased. As i n the anaesthetized dog (Ang e l l James and Daly, 1972a), no temperature dependency of the r e f l e x caused by n a s a l i r r i g a t i o n was found i n the muskrat. The f i n d i n g that the a f f e r e n t limb of the i n t e r n a l \" n a s a l r e f l e x e s i n muskrats r e s i d e s i n the m a x i l l a r y and i n t e r n a l l a r y n g e a l nerves i s somewhat d i f f e r e n t from that found i n sheep, r a b b i t s and dogs. In sheep, c a r d i o r e s p i r a -tory responses induced by p u l s a t i l e water flow introduced i n t o a t r a c h e a l cannula were abolished by s e c t i o n of the e x t e r n a l ( g l o t t a l ) and s u p e r i o r l a r y n g e a l nerves (Tchobroutsky _et a l . , 1969) but i n r a b b i t s n a s a l r e f l e x e s to i r r i t a n t vapours seem to be c a r r i e d i n the o l f a c t o r y and t r i g e m i n a l nerves ( A l l e n , 1928). On the other hand, r e s p i r a t o r y and c a r d i o v a s c u l a r responses to n a s a l water flow i n the dog are abolished by c u t t i n g the m a x i l l a r y and ethmoidal nerves ( A n g e l l James and Daly, 1972a); the l a t t e r i n n e r v a t i n g the 118 mucosa of the n a s a l sinuses and s i m i l a r i n sensory f u n c t i o n to the spheno-p a l a t i n e branches r e f e r r e d to e a r l i e r . The l o c a t i o n of the a f f e r e n t limb on the medial aspect of the eye e x p l a i n s the o c u l o - c a r d i a c r e f l e x prompted by pressure on the e y e b a l l (Aserinsky and Debias, 1961; Ganderia et a l . , 1978b). In the duck the most l i k e l y c o n t r i b u t o r s to the n a s a l r e f l e x are the ophthalmic d i v i s i o n of the t r i g e m i n a l (Andersen, 1963c) or the glossopharyn-geal nerve (Bamford and Jones, 1974), the l a t t e r of which i s considered to be s t i m u l a t e d by water drawn to the g l o t t i s i n forced d i v e s . B l i x et a l . (1976b) on the other hand, b e l i e v e that the t r i g e m i n a l as w e l l as the g l o s s o -pharyngeal nerves are i n v o l v e d i n the e l i c i t a t i o n of the d i v i n g responses. Reflexes o r i g i n a t i n g from the nasopharyngeal area are known to evoke r e s p i r a t o r y and c a r d i o v a s c u l a r responses s i m i l a r i n magnitude to those found during d i v i n g . Experimentally these r e f l e x e s may be e x c i t e d by f l u i d flow through the n a s a l passages (Tchobroutsky et a l . , 1969; A n g e l l James and Daly, 1972a) and a v a r i e t y of noxious vapours ( A l l e n , 1928; 1929; Ebbecke, 1944; A n g e l l James'and Daly£969a>; White, arid McRitehiej '1973). Even though water i s not taken i n t o the nares of the normally d i v i n g mammal, the r e f l e x e s are presumed to prevent f u r t h e r i n h a l a t i o n of water and set i n motion the same oxygen sparing mechanisms and thus provide a l a t e n t a f f e r e n t limb d i s t i n c t from the n a r i a l r e f l e x limb, which p o t e n t i a l l y o f f e r s a defense against drowning. Bamford and Jones (1974) and L e i t n e r et a l . (1974) have considered the p o s s i b i l i t y that the g l o t t i s of the duck may be regarded as a true r e f l e x o g e n i c s i t e f o r d i v i n g apnea but conclude that the g l o t t a l receptors probably only c o n t r i b u t e to the r e s p i r a t o r y r e f l e x i n i t i a t e d by t r i g e m i n a l l y innervated r e c e p t o r s . In a d d i t i o n to the d i f f e r e n c e s i n the a f f e r e n t arms of the n a r i a l and n a s a l r e f l e x e s , evidence given here a l s o p o i n t s to d i s s i m i l a r i t i e s among t h e i r 119 c e n t r a l connections. Anaesthesia or decerebration has a profound e f f e c t on the responses to submersion but w i t h the exception of the r a b b i t , probably does not have a conspicuous e f f e c t on the c a r d i a c and r e s p i r a t o r y r e f l e x e s o r i g i n a t i n g from w i t h i n the nasal passages. Oddly apnea d i d not occur i n the anaesthetized muskrat when dived but nevertheless the animal d i s p l a y e d a pronounced bradycardia caused by water s t i m u l a t i o n of the n a s a l airways. Water s t i m u l a t i o n of these areas i n c l u d i n g the g l o t t i s caused an apneic r e f l e x which excluded water from the lungs. On the other hand, c u r a r i z e d and a r t i f i c i a l l y v e n t i l a t e d animals were s t i l l able to give s t r i k i n g c a r d i a c responses to submersion but l o s t the r e f l e x i f the nares was covered and water was not allowed to enter the i n t e r n a l n a s a l passages. The anaesthetized muskrat however, showed no c a r d i a c responses during submersion i f i t was permitted to breathe f r e e l y through an exposed cannula, confirming that the n a r i a l r e f l e x i s abolished by anaesthesia. The independence of the n a s a l r e f l e x e s from the higher b r a i n centres i n the muskrat i s not s u r p r i s i n g i n view of the work by Huxley (1913) and Andersen (1963) on the d i v i n g duck. In both cases the authors reported only a s l i g h t decrease i n r e f l e x a c t i v i t y a f t e r decerebration or anaesthesia and there was no evidence that e i t h e r procedure a l t e r e d the a f f e r e n t connections. P o s t u r a l e f f e c t s have long been regarded as a p o s s i b l e c o n t r i b u t o r to the d i v i n g r e f l e x e s ; a c l a i m f i r s t advanced by Huxley (1913) and l a t e r supported by Koppanyi and Dooley (1929) i n muskrats. Both accounts reported that p o s t u r a l changes induce apnea and bradycardia. Other i n v e s t i g a t i o n s however, f a i l to confirm these conclusions and f i n d no marked responses on v e n t i f l e x i n g the head of the duck (Reite et. a l . , 1963) or s e a l ( I r v i n g et a l . , 1935). The r e s u l t s reported here suggest that w h i l e bradycardia may be i n i t i a t e d by q u i c k l y v e n t r i f l e x i n g the head of the muskrat, the response i s shallow and 120 transient. Moreover, whether the animal begins the normal dive from i n the water or on a perch, i t appears not to v e n t r i f l e x the head s u f f i c i e n t l y to evoke a response that would contribute to an oxygen sparing adjustment. The exclusion of water from the nasal passages of the muskrat i s of course, a feature common to a l l diving mammals. But while the anaesthetized animals took water i n to the l e v e l of the g l o t t i s , t h e i r intolerance to s a l i n e suggests that the affer e n t limbs of the n a r i a l and nasal r e f l e x e s are triggered by d i f f e r e n t types of receptors. Unlike the n a r i a l receptors those i n the nasal passages are osmosensitive, slow adapting and can be found i n t e r r e s t r i a l species (Harding et. a l . , 1976; Harding et a l . , 1978). The maintained responsiveness of the pharyngeal receptors i n sp i t e of an increasing heart rate throughout water stimulation implies that some cent r a l adaptation process must e x i s t which i s s i m i l a r to that i n the cat. Berger (1977) has demonstrated that the same c e n t r a l units which he believes are involved i n the slowly adapting Hering-Breuer r e f l e x , are also activated by the stimulation of the i n f e r i o r laryngeal nerve and suggests that integra-t i o n of these reflexes takes place at the medullary l e v e l . Receptors i n the lower pharyngeal area are known to be related to apnea i n the fetus (Barcroft, 1946) as well as i n the adult (Boushey et a l . , 1972; Boushey e_t a l . , 1974; Storey and Johnson, 1975; Berger, 1977) and to be e f f e c t i v e must be slowly adapting. Nasal receptors responding to water flow are considered to be free nerve endings situated i n the e p i t h e l i a l mucosa or lamina propria (Cauna et_ a l . , 1969) but i t i s l i k e l y that these are mixed with other types (Boushey et a l . , 1974). The d i s s i m i l a r i t y of the contributions of the maxillary and i n f e r i o r laryngeal nerve to the re s p i r a t o r y and cardiac responses indicates that the two afferent limbs have d i f f e r e n t c e n t r a l connections and that g l o t t a l -121 pharyngeal input i s more i n v o l v e d w i t h r e s p i r a t o r y c o n t r o l than w i t h heart r a t e . Based on nerve blockade, the m a x i l l a r y d i v i s i o n accounted f o r 68.5% of the c a r d i a c response to n a s a l water flow w h i l e the i n f e r i o r l a r y n g e a l branch c o n t r i b u t e d the m a j o r i t y of the r e s p i r a t o r y response (55.5%). These d i f f e r -ences are not e a s i l y explained i n terms of the f r e e l y d i v i n g animal because the means of s t i m u l a t i o n was severe and u n l i k e l y to occur i n the n a t u r a l s t a t e of d i v i n g . At best, under the l a t t e r c o n d i t i o n s , the e f f e c t on heart r a t e of i n t e r n a l n a s a l s t i m u l a t i o n can be seen only as a marginal c o n t r i b u t i o n to the muskrat and perhaps only underlines the c l o s e r e l a t i o n s h i p between the r e s p i r a t o r y and c a r d i o v a s c u l a r centres. I t i s obvious from the r e s u l t s given here that apneic tendencies, although, t r a n s i e n t , which can be i n i t i a t e d w i t h as l i t t l e as 50 ull.of water may be an e s s e n t i a l mechanism against water i n h a l a t i o n i n aquatic species. A s u r p r i s i n g observation i n the u n r e s t r a i n e d dives was the apparent dependency of the c a r d i a c response on the dive d u r a t i o n . In one animal the i n i t i a l c a r d i a c i n t e r v a l s on submergence seemed to i n d i c a t e the l e n g t h of dive to f o l l o w thus g i v i n g r i s e to s p e c u l a t i o n that the muskrat matches the degree of response w i t h dive time. A s i m i l a r r e l a t i o n s h i p has been noted by Kooyman and Campbell (1972) who concluded that the Weddell s e a l prepares i t s e l f f o r d i v i n g a c t i v i t i e s . Jones et a l . (1973) however, b e l i e v e that the s e a l a djusts dive d u r a t i o n to correspond to the degree of response set e a r l y i n the d i v e . The r e s u l t s given here show that r a p i d f l i g h t dives were i n v a r i a b l y a s s o c i a t e d w i t h a weaker c a r d i a c response r a t h e r than the \"maximum\" i n i t i a l bradycardia recorded from those dives i n which the muskrat swam and remained under the covering wire before r e s u r f a c i n g . These data, w h i l e confined to one animal, were h i g h l y p r e d i c t a b l e and p o i n t to a c a r d i o v a s c u l a r adjustment which i s not a p u r e l y conditioned 122 response i n s o f a r as the animal f a i l s to respond c o n s i s t e n t l y to a given stimulus. R e l a t i v e to the s e a l t h i s suggests some form of poorly conditioned c o n t r o l of heart r a t e ; e i t h e r a f u l l response i s evoked when an oxygen sparing adjustment i s r e q u i r e d or a p a r t i a l response occurs and heart r a t e i s lowered to approximately 60-100 per minute; Since oxygen conservation i s not req u i r e d i n sho r t e r d i v e s , i t may be that the reduced response represents a conditioned o v e r r u l i n g of the primary r e f l e x . In e i t h e r of the two types of d i v i n g found here, i t i s obvious that r e s u r f a c i n g was not prompted by the d e p l e t i o n of oxygen s t o r e s even i f e s t a b l i s h e d heart r a t e was high. I t i s noteworthy however, that the d i s t i n c t i o n drawn between the two types was dependent on the \"open\" water a v a i l a b l e to the muskrat and thus the response may not have r e s u l t e d from a n t i c i p a t i o n of d i v i n g time per se but rat h e r from the nature of the div e . A n t i c i p a t o r y r e f l e x e s to d i v i n g have been observed i n some pinnipeds (Murdaugh et a l . , 1961; Kooyman and Campbell, 1972; Jones et a l . , 1973) and the t r a i n e d porpoise ( E i s n e r , 1965) and when present are much more prominent than those noted i n the muskrat. In the f r e e l y d i v i n g duck a n t i c i p a t o r y r e f l e x e s i n c l u d e a t a c h y c a r d i a , which e v i d e n t l y r e s u l t s from h y p e r v e n t i l a t i o n p r i o r to immersion ( B u t l e r and Woakes, 1976; 1979). The r e s u l t s shown here i n d i c a t e only an o c c a s i o n a l bradycardic response before immersion of the nares but even then do not n e c e s s a r i l y imply v o l i t i o n a l c o n t r o l of heart r a t e since many s t i m u l i evoke s i m i l a r responses i n the conscious animal. By the same token, a n t i c i p a t o r y increases i n heart r a t e before r e s u r f a c i n g are a common occurrence i n most aquatic mammals ( I r v i n g e t a l . , 1941a; Murdaugh et a l . , 1961; Kooyman and Campbell, 1972). Although t h i s was not found i n the muskrat, i t s presence i n higher Vertebrates suggests some o v e r r u l i n g of the primary and 123 secondary r e f l e x e s by c o n d i t i o n i n g through higher centres and i s reminscent of the modified c a r d i a c response found i n t r a n s i e n t d i v i n g i n the muskrat. The c a r d i a c response of the muskrat during short v o l u n t a r y dives a l s o c o n t r a s t s to the l a b i l e nature of heart r a t e i n the s e a l (Harrison and Tomlinson, 1960) and porpoise (Eisner et a l . , 1966b) i n which i t i s sometimes not g r e a t l y a f f e c t e d by b r i e f submersions or i n feeding dives (Jones at al., 1973). On r e s u r f a c i n g , recovery of heart r a t e was always c o i n c i d e n t w i t h the re-establishment of the breathing p a t t e r n but when the animal was prompted to r a p i d successive dives heart r a t e d i d not increase a p p r e c i a b l y between dives. The abnormal course of recovery was considered to be due to continu i n g apnea and thus underscores the importance of r e s p i r a t o r y a c t i v i t y on the ca r d i o v a s c u l a r r e f l e x e s , p a r t i c u l a r l y i n the recovery p e r i o d . Since the muskrat t y p i c a l l y dives f o r only short p e r i o d s , the primary r e f l e x e s are l i k e l y to account f o r almost a l l of the c a r d i o v a s c u l a r responses brought on i n a normal escape or f o r a g i n g d i v e . The i n t e n s i t y of these r e f l e x e s leaves one to speculate that the chemoreceptors may f u n c t i o n l a r g e l y to maintain the p r e - e s t a b l i s h e d responses r a t h e r than to induce f u r t h e r changes i n dives of t h i s l e n g t h . This reasoning i s supported by the observa-t i o n here that a s u b s t a n t i a l bradycardia was maintained f o r at l e a s t one minute when v e n t i l a t i o n i n the c u r a r i z e d but unanaesthetized animal was continued during submersion. While t h i s may be true i n the muskrat, s e a l and other d i v e r s i t c l e a r l y does not apply to a l l mammals. In the rat,, f o r example, c a r o t i d body denervation v i r t u a l l y a b o l i s h e s the e n t i r e c a r d i a c response to submersion (Huang and Peng, 1976). In dives exceeding one minute, however, chemoreceptor reinforcement of the c a r d i o v a s c u l a r adjustments i s probably much greater. Because heart r a t e increases during d i v i n g when 124 chemoreceptor Input i s reduced or withdrawn, while i n the f r e e l y d i v i n g animal i t continues to f a l l , suggests i t may be that muskrats cannot maintain the i n i t i a l responses but must r e l y on a secondary mechanism i n long periods of asphyxia. I f blood pressure remains r e l a t i v e l y constant i n the f r e e l y d i v i n g muskrat as i t does i n the s e a l (Murdaugh at a i . , 1961; A n g e l l James e_t a l . , 1976) then baroreceptor d r i v e n responses are u n l i k e l y to be i n v o l v e d . Thus i n d i r e c t l y , i t may be concluded that the greater c a r d i a c response seen i n u n r e s t r a i n e d d i v i n g i n the muskrat probably r e f l e c t s f a c i l i t a t i o n of the t r i g e m i n a l r e f l e x by chemoreceptors such as the case i n the dog ( A n g e l l James and Daly, 1973) and s e a l ( A n g e l l James et a l . , 1978). I t i s w e l l e s t a b l i s h e d that a c t i v a t i o n of lung receptors by h y p e r v e n t i l a -t i o n can i n i t i a t e t a c h y c a r d i a and systemic v a s o d i l a t a t i o n (Anrep et a l . , 1936a, b; Daly and S c o t t , 1958; Daly et a l . , 1967; Daly and Robinson, 1968). While t h i s a l s o appears to be the case i n the muskrat, the e f f e c t of v e n t i l a t i o n on heart r a t e i s f a r more evident under c o n d i t i o n s which favour bradycardia as forced v e n t i l a t i o n even at high minute volumes i n the anaesthetized animal r e s u l t e d i n only marginal increases i n heart r a t e . Consequently high vagal tone seems to be necessary f o r the complete expression of r e s p i r a t o r y e f f e c t s on heart r a t e . This co n c l u s i o n i s supported by Anrep ej; a l . (1936b) and Levy et. a l . (1966) who observed that sinus arrhythmia occurs i n the dog only when vagal tone was present and subsequently found that the phenomenon was based l a r g e l y on the diminution of vagal tone during the i n s p i r a t o r y phase. Their c l a i m that the lung i n f l a t i o n r e f l e x disappears i n the presence of strong vagal a c t i v i t y however, does not apply to the muskrat. As i n dogs (Ange l l James and Daly, 1978a; Gandevia e t . , 1978a; Aserinsky and DeBias, 1961), r a b b i t s (White et a l . , 1974) and s e a l s (Daly et 125 a l . , 1977) a r t i f i c i a l v e n t i l a t i o n i n the muskrat tends to reverse the brady-c a r d i c response to n a s a l s t i m u l a t i o n ; the degree of which i s approximately p r o p o r t i o n a l to the minute volume. U n l i k e ducks (Bamford and Jones, 1976), no l e v e l of v e n t i l a t i o n was found to f u l l y o v e r r u l e the c a r d i a c response to apneic s t i m u l a t i o n . Apneic s t i m u l a t i o n given simultaneously w i t h normal or above normal t i d a l volume v e n t i l a t i o n most o f t e n y i e l d e d a 1:1 r e l a t i o n s h i p between heart r a t e and the frequency of v e n t i l a t i o n and i s seen to r e f l e c t the p e r i p h e r a l component of sinus arrhythmia f i r s t i n v e s t i g a t e d by Anrep at a l . (1936b). The purpose of such a r e f l e x i s s p e c u l a t i v e but i n the d i v e r there seems l i t t l e doubt that the re-establishment of normal heart r a t e and blood flow d i s t r i b u t i o n i s g r e a t l y a c c e l e r a t e d upon r e s u r f a c i n g by pulmonary in p u t . Thus i t may be that the r e f l e x permits a more r a p i d repayment of an i n c u r r e d oxygen debt and a s h o r t e r p e r i o d at the surface between d i v e s . At l e a s t i n the muskrat, pulmonary input a l s o seems to be necessary to s u s t a i n normal heart r a t e as lung d e a f f e r e n t a t i o n r e s u l t e d i n s i g n i f i c a n t decreases i n both a r t i f i c i a l l y v e n t i l a t e d and spontaneously breathing animals. Based on s e q u e n t i a l e l i m i n a t i o n of the r e s p i r a t o r y and c a r d i a c responses to a r t i f i c i a l v e n t i l a t i o n by the i n h a l a t i o n of steam, the receptors i n i t i a t i n g the apneic response to maintained i n f l a t i o n are d i f f e r e n t from those causing c a r d i o a c c e l e r a t i o n s i n c e the a b o l i t i o n of the Hering-Breuer r e f l e x occurred f i r s t . There a l s o seems to be l i t t l e doubt that the same receptors which cause tac h y c a r d i a a l s o have a s t i m u l a t i n g e f f e c t on r e s p i r a t i o n s i n c e even moderate t i d a l volumes tended to pace spontaneous breathing. E p i t h e l i a l \" i r r i t a n t \" receptors are known to promote r e f l e x s t i m u l a t i o n of breathing but they may a l s o respond to maintained lung i n f l a t i o n and d e f l a t i o n ( M i l l s j2t a l . , 1970). The i n s p i r a t o r y - e x c i t i n g r e f l e x resembles that found i n cats (Larrabee and Knowleton, 1946; Glogowska et a l . , 1972) and r a b b i t s (Davies 126 and Roumy, 1977) which i s claimed to r e f l e x l y improve lung f i l l i n g by a p o s i t i v e feedback mechanism and supports the conclusions of Knowleton and Larrabee (1946) that the p r i n c i p a l a c t i o n of these receptors on r e s p i r a t i o n i s to r e i n f o r c e breathing rhythm e s t a b l i s h e d through c e n t r a l connections. The phrenic nerve response to a r t i f i c i a l v e n t i l a t i o n however, i n d i c a t e s that eupneic breathing i n the muskrat evokes the i n s p i r a t o r y - e x c i t i n g r e f l e x at a lower receptor threshold than that found i n other mammals. Lung d e a f f e r e n t a t i o n a l s o produced some s t r i k i n g changes i n normal heart r a t e as w e l l as the expected slowing of spontaneous r e s p i r a t i o n . During lung steaming heart r a t e d e c l i n e d before a l o s s i n the. c a r d i a c response to v e n t i l a -t i o n was obvious but the cause of t h i s was not c l e a r . Repeated high minute volume v e n t i l a t i o n was found to temporarily r e s t o r e normal r a t e i n d i c a t i n g that there may be an incomplete anatomical separation of the two groups of receptors which l e a d to r e f l e x apnea and c a r d i o a c c e l e r a t i o n . U n l i k e the muskrat, the slowing of heart r a t e caused by pulmonary d e a f f e r e n t a t i o n by e i t h e r steam i n h a l a t i o n (Hainsworth et a l . , 1973) or by nerve s e c t i o n (Gandevia et a l . , 1978a) does not occur i n the dog. The c a r d i a c response to n a s a l water flow i n the muskrat i s unaffected by constant lung i n f l a t i o n to pressures w e l l beyond that r e q u i r e d to i n i t i a t e the Hering-Breuer r e f l e x . This i s a s i m i l a r observation to that i n dogs i n which rhythmic i n f l a t i o n was a f a r more e f f e c t i v e means of r e v e r s i n g vasomotor responses to t r i g e m i n a l s t i m u l a t i o n (Davidson.et.al. , 1976; A n g e l l James.and Daly, 1978a). Constant i n f l a t i o n of the lungs however, d i d not always produce tachycardia i n muskrats as shown'.in the dog (Hainsworth et al_. , 1973) and when present was never maintained f o r more than a few seconds. In f a c t , bradycardia developed w i t h ongoing i n f l a t i o n a f t e r a p e r i o d which was propor-t i o n a l to the i n f l a t i o n pressure. Baroreceptor denervation does not a l t e r the 127 course of heart r a t e change i n the dog during i n f l a t i o n of the lungs (Hains-worth et a l . , 1973; Hainsworth, 1974) even though increases i n i n t r a t h o r a c i c pressure are known to impede venous r e t u r n and thereby tend to lower blood pressure and i n i t i a t e the b a r o s t a t i c r e f l e x . The \"secondary\" slowing of heart r a t e during lung i n f l a t i o n i s considered to a r i s e from pulmonary receptor adaptation s i n c e i t i s abolished by s e c t i o n of the pulmonary v a g i (Anrep e_t a l . , 1936a). In the absence of n a s a l s t i m u l a t i o n the s t a t e of lung i n f l a t i o n has a marked e f f e c t on a r t e r i a l blood pressure. I n f l a t i o n causes an immediate hypotension whereas d e f l a t i o n r e s u l t s i n an approximately equal change i n pressure; both responses being independent of i n h a l e d carbon d i o x i d e and c a r o t i d sinus i n n e r v a t i o n . The hypotensive response to constant lung i n f l a -t i o n i s not unique to the muskrat s i n c e i t i s a l s o found to occur i n the beaver ( I r v i n g , 1937), dog (Hainsworth, 1974) and r a b b i t (Ott and Shepard, 1971). The slowing of heart r a t e i n response to d e f l a t i o n on the other hand, has been the subject of a number of studies (Nahas, 1956; A n g e l l James and Daly, 1969b; White and M c R i t c h i e , 1973) but i t s r e l a t i o n s h i p to the vasomotor response has not been determined. In the muskrat, the cause of these responses was not t e s t e d f u r t h e r but i t i s presumed that^both the b r a d y c a r d i a and vasomotor responses to s t a t i c lung volume changes a r i s e from pulmonary receptors s i n c e they are e l i m i n a t e d by a few breaths of steam (Hainsworth et a l . , 1973) and are unaffected when baroreceptor a c t i v i t y i s held constant (Hainsworth, 1974). The bradycardic response to d e f l a t i o n i n the r a b b i t , however, r e q u i r e s an i n t a c t b a r o s t a t i c r e f l e x (White et a l . , 1974). The b a r o s t a t i c r e f l e x causing bradycardia i n the muskrat occurred at about 115 mm Hg when hypertension was induced by a d r e n a l i n ; a pressure which i s considerably l e s s than i n the non-diving s t a t e i n the s e a l . Baroreceptor 128 d r i v e n bradycardia i n the d i v i n g mammal however, i s probably t r i g g e r e d at a lower pressure than that i n d i c a t e d here f o r water a p p l i e d to the face of the anaesthetized s e a l r e s e t s the b a r o s t a t i c r e f l e x towards bradycardia and thus p o t e n t i a t e s a r e f l e x which may already c o n t r i b u t e to the d i v i n g adjustments (Angell James et a l . , 1978b). There i s l i t t l e doubt that chemoreceptors are inv o l v e d i n the cardiovas-c u l a r responses i n prolonged d i v i n g but whether t h i s a c t i v i t y o r i g i n a t e s from s i t e s other than the c a r o t i d body i s not known. B i l a t e r a l sinus nerve s e c t i o n i n the anaesthetized muskrat delayed chemoreceptor d r i v e n bradycardia from a PaO^ of 63;. mm Hg to 34' mm Hg i n d i c a t i n g that the c a r o t i d bodies are by f a r the most chemosensitive u n i t s but not the only ones responding to hypoxia and hypercapnia. In prolonged s t i m u l a t i o n s of the nasal mucosa PaO^ de c l i n e d to 44 mm Hg w i t h i n 20 seconds, so i n s p i t e of anaesthesia i t i s reasonable to assume that the chemoreceptors w i l l c o n t r i b u t e to the c a r d i o v a s c u l a r responses at t h i s time. I t should be pointed out however, that chemoreceptor bradycard-i a was i n v e s t i g a t e d during a r t i f i c i a l v e n t i l a t i o n and there f o r e i t i s l i k e l y that t h i s c o n t r i b u t i o n w i l l be made at an even higher Pa02 during apnea. The present r e s u l t s suggest that i n the normal d i v i n g mammal the car d i a c response to submersion could be an expression of at l e a s t three groups of receptors. I n d i v i d u a l l y each group seems to have a d i r e c t i n f l u e n c e on both the r e s p i r a t o r y and c a r d i o i n h i b i t o r y centres ( P i g . 34) and i n the absence of other s t i m u l i , each r e f l e x i s p o t e n t i a l l y able to evoke c a r d i a c responses i n the same order of magnitude as that observed i n the f r e e l y d i v i n g muskrat (Table I I I ) . Lung d e f l a t i o n per se provoked bradycardia but f a i l e d to increase the car d i a c r e f l e x when coupled w i t h n a s a l s t i m u l a t i o n . No conclu-s i o n may be reached as to whether the d e f l a t i o n r e f l e x f a c i l i t a t e s the response to n a r i a l s t i m u l a t i o n at the poin t of submersion. While the chemo-Table I I I . Experimental t e s t s causing bradycardia i n the muskrat. Mean values of heart r a t e are shown before ( c o n t r o l ) and at a time of maximum response. Mean heart r a t e during d i v i n g of r e -s t r a i n e d animals i s compared to response i n other t e s t s and the s i g n i f i c a n t d i f f e r e n c e s noted. X, d i f f e r e n c e between means i s s i g n i f i c a n t (P<0.05%). 0, d i f f e r e n c e i s not s i g n i f i -cant (P>0.05%). n = number of observations. S i g n i f i c a n t C o n t r o l Test d i f f e r e n c e ... Notes Restrained dive 266±3 (n = 66) 51±2 (n = 60) -Unrestrained dive 310±3 (n = 102) 27±3 73+3 (n = (n = 3) 50) X X Dives >40 sec Dives <4 sec Lung d e f l a t i o n 268±7 (n = 23) 59±4 (n = 23) 0 Paralyzed Nasal water flow (32 ml/min) 277±5 (n =34) 20+2 (n = 34) X Anaesthetized and apneic Nasal s a l i n e flow 273±9 (n = 12) 38±3 (n = 12) X Anaesthetized and apneic Nasal water flow w i t h lungs deafferentated 195±21 (n = 10) 48±2 (n = 9) 0 Anaesthetized and apneic Forced dive under anaesthesia 281±5 (n = 56) 39±2 84±5 (n = (n = 39) 56) X X Non-apneic (40 sec) Non-apneic (1-2 sec) Water on nose 292+6 (n = 6) 76±2 (n = 6) X Paralyzed and v e n t i l a t e d Hypoxia 277±11 (n = 6) 76±7 (n = 6) X Paralyzed and v e n t i l -ated;. Pa0 2 = 44 mm Hg B a r o s t a t i c r e f l e x 253±6 (n = 6) 90112 (n = = 6) X Paralyzed and v e n t i l a t e d 130 Figure 34. Neural mechanisms r e g u l a t i n g r e s p i r a t i o n and heart r a t e i n the muskrat. RC, r e s p i r a t o r y centre; DNV, d o r s a l nucleus of the vagus; NR, n a s a l r e c e p t o r s ; CB, c a r o t i d bodies. The inputs from the i n f l a t e d lungs ( r i g h t ) to the r e s p i r a t o r y centre r e f e r to the Hering-Breuer r e f l e x (top arrow) and to the e f f e c t of lung v e n t i l a t i o n . The e f f e c t of i n f l a t i o n on heart r a t e appears to be d i r e c t s i n c e c a r d i a c pacing by lung i n f l a t i o n occurred i r r e s p e c t i v e of apnea induced by n a s a l s t i m u l a t i o n . Sudden lung d e f l a t i o n a l s o seems to have d i r e c t l i n k s to both centres. Increases i n r e s p i r a t o r y a c t i v i t y were observed f o l l o w i n g d e f l a t i o n ( d e f l a t i o n r e f l e x ) but t h i s a l s o caused a bradycardia. The diagram a l s o i l l u s t r a t e s the p a r a d o x i c a l e f f e c t s of n a s a l and c a r o t i d body s t i m u l a t i o n on r e s p i r a t i o n and heart r a t e . Although bradycardia always accompanied' apnea during n a s a l stimu-l a t i o n , a decrease i n heart r a t e a l s o occurred under c o n d i t i o n s i n which r e s p i r a t o r y rhythm was continued w i t h forced v e n t i l a t i o n . C a r o t i d body s t i m u l a t i o n alone increased r e s p i r a t o r y a c t i v i t y but a l s o y i e l d e d bradycardia during v e n t i l a t i o n hypoxia when there was no conspicuous i n c r e a s e i n r e s p i r a t i o n . 131 132 receptor r e f l e x no doubt f a c i l i t a t e s the trigeminally induced responses l a t e r i n the dive, the baroreceptor function i n diving i s speculative. Baroreceptors i n the muskrat have been shown to cause bradycardia i n response to drug induced hypertension but beyond a possible need to make s l i g h t r e -adjustments to the more dramatic changes caused by the n a r i a l and chemorecep-tor r e f l e x e s , no r o l e f or them i s evident i n diving. On the other hand, the cardiac response to lung v e n t i l a t i o n seems to be necessary for the maintenance of normal heart function while i t s primary contribution to diving i s confined to the recovery period. 133 SUMMARY Heart r a t e i n the muskrat was recorded during r e s t r a i n e d and unrestrained d i v i n g . In dives which were 40 sec i n d u r a t i o n , the unr e s t r a i n e d animals developed a r e l a t i v e l y s t a b l e heart r a t e which was s i g n i f i c a n t l y lower (27±3) than i n those t r i a l s i n which they were r e s t r a i n e d (56±4). There was no d i f f e r e n c e i n the onset of bradycardia i n the two types of d i v e s . The u n r e s t r a i n e d response represents a heart r a t e of about 9% of the r e s t i n g r a t e and i s s i m i l a r to that found i n f r e e range d i v i n g i n pinnipeds. Anaesthesia f a i l e d to a b o l i s h the car d i a c response to submersion and i n f a c t , heart r a t e followed approximately the same course as i n the forced dives a f t e r a s i g n i f i c a n t l y longer l a t e n t p e r i o d (591±40 vs 300±10 msec). Breathing movements continued during these dives p a r t i c u l a r l y when the muskrats were dived i n t o s a l i n e . Evidence was presented that the muskrat e x h i b i t s some form of poor l y con-d i t i o n e d response i n the i n i t i a l phase of submersion. The animal does not show s i g n i f i c a n t a n t i c i p a t o r y responses to submersion or emersion such as those which c h a r a c t e r i z e d i v i n g i n the s e a l . I t was concluded that the muskrat probably responds to the nature of the dive rather than to \" a n t i c i p a t e d \" d i v i n g time. I t was concluded that w h i l e higher centre input plays a r o l e i n d i v i n g a b i l i t y , the primary r e f l e x to submersion o r i g i n a t e s i n receptors l o c a t e d at the nares and i s probably c a r r i e d i n i t s a f f e r e n t limb by the naso-c i l i a r y nerve. D i v i s i o n of the main m a x i l l a r y nerve trunk which inner^r 134 vates the n a r i a l f l a p s , f a i l e d to a f f e c t the cardiac response to submersion. Anaesthesia blocks the external n a r i a l r e f l e x and allows nasally inhaled water to i n i t i a t e a nasal r e f l e x . I t i s thought that the p r i n c i p a l function of t h i s r e f l e x i s to exclude water from the trachea but nasal stimulation i n t h i s manner also evokes s t r i k i n g cardiorespiratory responses. Bradycardia i s the most prominent of these but transient apnea and vasomotor responses are also evident. The nasal r e f l e x originates i n the g l o t t i s and pharynx and i s c a r r i e d by the i n f e r i o r laryngeal and maxillary nerves. The former appears to provide the greater r e s p i r a t o r y response (55%) while the l a t t e r i n i t i a t e s most (68%) of the cardiac response to nasal water flow. The laryngeal receptors which contribute to the afferent limb are slow adapting and osmosensitive. The e f f e c t of r e s p i r a t o r y a c t i v i t y on heart rate was shown and the s i g n i f i c a n c e of the i n t e r a c t i o n i n regards to the d i v i n g mammal was d i s -cussed. The muskrat l i k e the dog, demonstrates the f u l l e s t expression of the cardiac response during apnea. However, i t may be at the point of resurfacing that c e n t r a l and peripheral input leading to cardiovascu-l a r changes have t h e i r greatest e f f e c t . Lung deafferentation by steaming r e s u l t s i n a s i g n i f i c a n t decrease i n normal heart rate suggesting that pulmonary input i n the muskrat i s required f o r the maintenance of normal cardiovascular function. 135 The pulmonary receptors i n i t i a t i n g the Hering-Breuer r e f l e x to constant i n f l a t i o n are d i s t i n c t from those which increase heart r a t e caused by v e n t i l a t i o n . The receptors which have t h i s c a r d i a c i n f l u e n c e however, are probably the same as those which augment the r e s p i r a t o r y c y c l e s i n c e both responses are l o s t simultaneously during p r o g r e s s i v e d e a f f e r e n t a t i o n by steam. Although the Hering-Breuer receptors are slow adapting, they have no d i r e c t e f f e c t on the c a r d i a c response during submersion. Bradycardia may be evoked by lung d e f l a t i o n per se. The slowing of heart r a t e began 0.97±0.17 seconds a f t e r d e f l a t i o n and t h i s time p e r i o d was independent of the i n f l a t i o n pressure. Hypertension induced by a d r e n a l i n caused a b a r o s t a t i c r e f l e x at 115±4 mm Hg i n d i c a t i n g that blood pressure increases due to p e r i p h e r a l vaso-c o n s t r i c t i o n during a dive would be l i m i t e d at t h i s pressure and tend to lower heart r a t e even f u r t h e r . B i l a t e r a l sinus nerve s e c t i o n i n the anaesthetized muskrat delayed chemoreceptor d r i v e n bradycardia from a PaG^ of 63 mm Hg to approximate-l y 34 mm Hg and i n d i c a t e s that the c a r o t i d bodies are the most chemo-s e n s i t i v e u n i t s but not the only ones responding to changes i n blood gas tensions. Although chemoreceptors undoubtedly act to maintain the d i v i n g responses, i t i s l i k e l y that the e x t e r n a l n a r i a l r e f l e x accounts for. almost a l l of the c a r d i o v a s c u l a r adjustments brought about i n a normal fora g i n g or escape dive s i n c e these are u s u a l l y of short d u r a t i o n . In dives approach-i n g one minute or longer, the chemoreflex probably plays a s i g n i f i c a n t 136 r o l e i n these responses and to prompt the animal to resurface before the ex p i r a t i o n of oxygen stores. The present study shows that i n the muskrat, the cardiac response to submersion r e s u l t s from at l e a s t three groups of receptors. These cause the primary n a r i a l r e f l e x i n i t i a t e d by water contact at the nares, a lung d e f l a t i o n r e f l e x and a chemoreflex both r e s u l t i n g from the ensuing apnea. The i n t e r a c t i o n of these reflexes was discussed. 137 L i t e r a t u r e C i t e d A l l e n , W. F. (1928). E f f e c t on r e s p i r a t i o n , blood pressure, and c a r o t i d pulse of various i n h a l e d and i n s u f f l a t e d vapors when s t i m u l a t i n g one c r a n i a l , nerve and v a r i o u s combinations of c r a n i a l nerves. I . Branches of the trigeminus a f f e c t e d by these s t i m u l a n t s . Am. J . P h y s i o l . 87, 319-325. A l l e n , W. F. (1929). E f f e c t of various i n h a l e d vapors on r e s p i r a t i o n and blood pressure i n anaesthetized, unanaesthetized, s l e e p i n g and anosmic s u b j e c t s . Am. J . P h y s i o l . 88, 620-632. Andersen, H. T. (1959). Depression of metabolism i n the duck during experimental d i v i n g . Acta p h y s i o l . scand. 46, 234-239. Andersen, H. T. (1961). P h y s i o l o g i c a l adjustments to prolonged d i v i n g i n the American a l l i g a t o r , A l l i g a t o r m i s s i s s i p p i e n s i s . Acta p h y s i o l . scand. 53, 23-45. Andersen, H. T. (1963a). Factors determining the c i r c u l a t o r y adjustments to d i v i n g . I . Water immersion. Acta p h y s i o l . scand. 58, 173-185. Andersen, H. T. (1963b). Factors determining the c i r c u l a t o r y adjustments to d i v i n g . I I . Asphyxia. Acta p h y s i o l . scand. 58, 186-200. Andersen, H. T. (1963c). The r e f l e x nature of the p h y s i o l o g i c a l adjustments to d i v i n g and t h e i r a f f e r e n t pathway. Acta p h y s i o l . scand. 58, 263-273. Andersen, H. T. (1966). P h y s i o l o g i c a l adaptations i n d i v i n g v e r t e b r a t e s . P h y s i o l . Rev. 46, 212-243. Andersen, H. T. and A. L^v^. (1964). The e f f e c t of carbon d i o x i d e on the r e s p i r a t i o n of avian d i v e r s (ducks). Comp. Biochem. P h y s i o l . 12, 451-456. Andersen, H. T. and A. S. B l i x . (1974). Pharmacological exposure of components i n the autonomic c o n t r o l of the d i v i n g r e f l e x . Acta p h y s i o l . scand. 90, 381-386. A n g e l l James, J . E. and M. deB. Daly. (1969a). Nasal r e f l e x e s . Proc. Roy. Soc. Med. (London) 62, 1287-1293. A n g e l l James, J . E. and M. deB. Daly. (1969b). Cardiovascular responses i n apnoeic asphyxia: Role of a r t e r i a l chemoreceptors and the m o d i f i -c a t i o n of t h e i r e f f e c t s by a pulmonary vagal i n f l a t i o n r e f l e x . J . P h y s i o l . (London) 201, 87-104. 138 A n g e l l James, J . E. and M. deB. Daly. (1972a). R e f l e x r e s p i r a t o r y and ca r d i o v a s c u l a r e f f e c t s of s t i m u l a t i o n of receptors i n the nose of the dog. J . P h y s i o l . (London) 220, 673-696. A n g e l l James, J . E. and M. deB. Daly. (1972b). The i n t e r a c t i o n of r e f l e x e s e l i c i t e d from the c a r o t i d bodies and nose a f f e c t i n g r e s p i r a t i o n and pulse i n t e r v a l . J . P h y s i o l . (London) 226, 78-79P. A n g e l l James, J . E. and M. deB. Daly. (1973). The i n t e r a c t i o n of r e f l e x e s e l i c i t e d by s t i m u l a t i o n of the c a r o t i d body chemoreceptors and receptors i n the n a s a l mucosa a f f e c t i n g r e s p i r a t i o n and pulse i n t e r v a l i n the dog. J . P h y s i o l . (London) 229, 133-149. A n g e l l James, J . E. and M. deB. Daly. (1975). I n t e r a c t i o n s between car d i a c r e f l e x e s from the lungs and those e l i c i t e d from the c a r o t i d bodies v and the l a r y n x i n the dog. J . P h y s i o l . (London) 254, 55-57P. A n g e l l James, J . E., M. deB. Daly and R. E i s n e r . (1976). C a r o t i d sinus baroreceptor r e f l e x i n the s e a l and i t s m o d i f i c a t i o n during d i v i n g . Proc. Aust. P h y s i o l . Pharm. Soc. 7, 145-146P. A n g e l l James, J . E. and M. deB. Daly. (1978). The e f f e c t s of a r t i f i c i a l lung i n f l a t i o n on r e f l e x l y induced bradycardia a s s o c i a t e d w i t h apnoea i n the dog. J . P h y s i o l . (London) 274, 349-366. A n g e l l James, J . E., M. deB. Daly and R. E i s n e r . (1978). A r t e r i a l baro-receptor r e f l e x e s i n the s e a l and t h e i r m o d i f i c a t i o n during experimental d i v i n g . Am. J . P h y s i o l . 234(6), H730-H739. Anrep, G. V., W. Pascual and R. R b s s l e r . (1936a). R e s p i r a t o r y v a r i a t i o n s of the heart r a t e . I . The r e f l e x mechanism of the r e s p i r a t o r y arrhythmia. Proc. Roy. Soc. B (London) 119, 191-217. Anrep, G. V., VJ. Pascual and R. Ro s s l e r . (1936b). R e s p i r a t o r y v a r i a t i o n s of the heart r a t e . I I . The c e n t r a l mechanism of the r e s p i r a t o r y arrhythmia and the i n t e r - r e l a t i o n s between the c e n t r a l and the r e f l e x mechanisms. Proc. Roy. Soc. B (London) 119, 218-230. As e r i n s k y , E. and D. DeBias. (1961). Suppression of the oc u l o - c a r d i a c r e f l e x by means of r e s p i r a t o r y movements. P h y s i o l o g i s t 4, 5. Bainbridge, F. A. (1920). The r e l a t i o n between r e s p i r a t i o n and p u l s e - r a t e . J . P h y s i o l . (London) 54, 192-202. Bamford, 0. S. and D. R. Jones. (1974). On the i n i t i a t i o n of apnoea and some c a r d i o v a s c u l a r responses to submergence i n ducks. Resp. P h y s i o l . 22, 199-216. Bamford, 0. S. and D. R. Jones. (1976). R e s p i r a t o r y and c a r d i o v a s c u l a r i n t e r a c t i o n s i n ducks: The e f f e c t of lung denervation on the i n i t i a t i o n of and recovery from some ca r d i o v a s c u l a r responses to submergence. J . P h y s i o l . (London) 259, 575-596. 139 B a r c r o f t , J . (1946). Researches on p r e - n a t a l l i f e . Oxford. B l a c k w e l l . B a r t e l s , H. (1964). Comparative physiology of oxygen t r a n s p o r t i n mammals. Lancet 2, 599-604. Beau, J . H. S. (1860). Recherches experimentales sur l a mort par submersion. Arch. Gen. Med. 2, 64-76. Berger, A. J . (1977). Dorsal r e s p i r a t o r y group neurons i n the medulla of c a t : s p i n a l p r o j e c t i o n s , responses to lung i n f l a t i o n and s u p e r i o r l a r y n g e a l nerve s t i m u l a t i o n . B r a i n Research 135, 231-254. B e r n t h a l , T. (1938). Chemo-reflex c o n t r o l of v a s c u l a r r e a c t i o n s through the c a r o t i d body. Am. J . P h y s i o l . 121, 1-20. B e r n t h a l , T., W. Greene, J r . and A. M. Revzin. (1951). Role of c a r o t i d chemoreceptors i n hypoxic c a r d i a c a c c e l e r a t i o n . Proc. Soc. exp. B i o l . Med. 76, 121-124. B e r t , P. (1870). Leqons sur l a p h y s i o l o g i e cqmparee de l a r e s p i r a t i o n . B a i l l i e r e , P a r i s . B l i x , A. S. (1975). The importance of asphyxia f o r the development of d i v i n g bradycardia i n ducks. Acta p h y s i o l . scand. 95, 41-45. B l i x , A. S., E. L. Gautvik and H. Refsum. (1974). Aspects of the r e l a t i v e r o l e s of p e r i p h e r a l v a s o c o n s t r i c t i o n and vagal bradycardia i n the establishment of the \" d i v i n g r e f l e x \" i n ducks. Acta p h y s i o l . scand. 90, 289-296. B l i x , A. S., 0. Lundgren and B. Folkow. (1975). The i n i t i a l c a r d i o v a s c u l a r responses i n the d i v i n g duck. Acta p h y s i o l . scand. 94, 539-541. B l i x , A. S., G. Wennergren and B. Folkow. (1976a). Cardiac receptors i n ducks - a l i n k between v a s o c o n s t r i c t i o n and bradycardia during d i v i n g . Acta p h y s i o l . scand. 97, 13-19. B l i x , A. S., A. R e t t e d a l and K.-A. Stokkan. (1976b). On the e l i c i t a t i o n of the d i v i n g responses i n ducks. Acta p h y s i o l . scand. 98, 478-483. Boushey, H. A., P. S. Richardson and J . G. Widdicombe. (1972). R e f l e x e f f e c t s of l a r y n g e a l i r r i t a t i o n on the p a t t e r n of breathing and t o t a l lung r e s i s t a n c e . J . P h y s i o l . (London) 224, 501-513. Boushey, H. A., P. S. Richardson, J . G. Widdicombe and J . C. M. Wise. (1974). The response of l a r y n g e a l a f f e r e n t f i b e r s to mechanical and chemical s t i m u l i . J . P h y s i o l . (London) 240, 153-175. Brodie, T. G. and A. E. R u s s e l l . (1900). On r e f l e x c a r d i a c i n h i b i t i o n . J . P h y s i o l . (London) 26, 92-106. 140 Burne, R. H. (1909). Notes on the v i s c e r a of the walrus, Odoboenus rosmarus. Proc. Zool. Soc. (London), p. 732-738. Burow, C. A. (1838). Ueber das gefasssystem der robben. Arch. Anat. P h y s i o l . Wiss. Med. 1838, pp. 230-258. B u t l e r , P. J . and A. J . Woak.es. (1976). Changes i n heart r a t e and r e s p i r a -tory frequency a s s o c i a t e d w i t h spontaneous submersion of ducks. Biotelemetry I I I . Eds. T. B. F r y e r , H. A. M i l l e r and H. Sandler. Academic P r e s s , N. Y. B u t l e r , P. J . and A. J . Woakes. (1979). Changes i n heart r a t e and r e s p i r a -t o r y frequency during n a t u r a l behaviour of ducks, w i t h p a r t i c u l a r reference to d i v i n g . J . exp. B i o l . 79, 283-300. Cauna, H., K. H. Hinderer and R. T. Wentges. (1969). Sensory receptor organs of the human nasa l r e s p i r a t o r y mucosa. Am. J . Anat. 124, 187-210. Chalmers, G. A. and A. C. M a c N e i l l . (1977). Tyzzer's disease i n w i l d -trapped muskrats i n B r i t i s h Columbia. J . W i l d . Diseases 13, 114-116. Cherniak, N. S., N. H. Edelman and S. L a h i r i . (1970/71). Hypoxia and hypercapnia as r e s p i r a t o r y s t i m u l a n t s and depressants. Resp. P h y s i o l . 11, 113-126. Clausen, G. and A. E r s l a n d . (1968). The r e s p i r a t o r y p r o p e r t i e s of the blood of two d i v i n g rodents, the beaver and the water v o l e . Resp. P h y s i o l . 5, 350-359. Clausen, G. and A. E r s l a n d . (1970/71). Blood 0„ and acid-base changes i n the beaver during submersion. Resp. P h y s i o l . 11, 104-112. Cor d i e r , D. and C. Heymans. (1935). Le centre r e s p i r a t o i r e . P a r i s . C o r r i o l , J . H. and J . J . Rohner. (1968). New f a c t s about bradycardia i n breath h o l d i n g d i v e r s . Rev. Subaq. P h y s i o l . Hyperb. Med. 1, 24-27. Daly, I . deB. and M. deB. Daly. (1959). The E f f e c t s of S t i m u l a t i o n of the C a r o t i d Body Chemoreceptors on the Pulmonary Vascular Resistance i n the Dog: the 'Vagosensory C o n t r o l l e d Perfused L i v i n g Animal' P r e p a r a t i o n . J . P h y s i o l . 148, 201-219. Daly, M. deB. (1972). I n t e r a c t i o n of c a r d i o v a s c u l a r r e f l e x e s . L e c t . Scient. B a s i s Med. 207-332. Daly, M. deB. and M. J . S c o t t . (1958). The e f f e c t s of s t i m u l a t i o n of the c a r o t i d body chemoreceptors on the heart r a t e i n the dog. J . P h y s i o l . (London) 144, 148-166. 141 Daly, M. deB. and M. J . Sc o t t . (1962). An a n a l y s i s of the primary c a r d i o -v a s c u l a r r e f l e x e f f e c t s of s t i m u l a t i o n of the c a r o t i d body chemoreceptors i n the dog. J . P h y s i o l . (London) 162, 555-573. Daly, M. deB. and J . L. Hazzledine. (1963). The e f f e c t s of a r t i c i f i c a l l y induced h y p e r v e n t i l a t i o n on the primary ca r d i a c r e f l e x response to s t i m u l a t i o n of the c a r o t i d bodies i n the dog. J . P h y s i o l . (London) 168, 872-889. Daly, M. deB. and A. Ungar. (1966). Comparison of the r e f l e x responses e l i c i t e d by s t i m u l a t i o n of the se p a r a t e l y perfused c a r o t i d and a o r t i c body chemoreceptors i n the dog. J . P h y s i o l . (London) 182, 379-403. Daly, M. deB., J . L. Hazzledine and A. Ungar. (1967). The r e f l e x e f f e c t s of a l t e r a t i o n s i n lung volume on systemic v a s c u l a r r e s i s t a n c e i n the dog. J . P h y s i o l . (London) 188, 331-351. Daly, M. deB. and B. H. Robinson. (1968). An a n a l y s i s of the r e f l e x systemic v a s o d i l a t o r response e l i c i t e d by lung i n f l a t i o n i n the dog. J . P h y s i o l . (London) 195, 387-406. Daly, M. deB. and J . E. A n g e l l James. (1975). Role of the a r t e r i a l chemo-receptors i n the c o n t r o l of the c a r d i o v a s c u l a r responses to breath-hold d i v i n g . In \"The p e r i p h e r a l chemoreceptors\", Ed. M. J . Purves. Cambridge U n i v e r s i t y Press. Daly, M. deB., R. E i s n e r and J . E. A n g e l l James. (1977). C a r d i o - r e s p i r a t o r y c o n t r o l by the c a r o t i d chemoreceptors during experimental dives i n the s e a l . Am. J . P h y s i o l . 323, H508-H516. Davidson, N. S., S. Goldner and D. I . McCloskey. (1976). R e s p i r a t o r y modu-l a t i o n of baroreceptor and chemoreceptor r e f l e x e s a f f e c t i n g heart r a t e and c a r d i a c vagal e f f e r e n t nerve a c t i v i t y . J . P h y s i o l . (London) 259, 523-530. Davies, A. and M. Roumy. (1977). The i n s p i r a t o r y augmenting e f f e c t of lung i r r i t a n t receptor a c t i v i t y . J . P h y s i o l . (London) 275, 14P. D i l l , D. B. and H. T. Edwards. (1931). R e s p i r a t i o n and metabolism i n a young c r o c o d i l e (Crocodylus acutus, C u v i e r ) . Copeia 1, 1-3. i Douglas, W. W. and J . L. Malcolm. (1955). The e f f e c t of l o c a l i z e d c o o l i n g on conduction i n cat nerves. J . P h y s i o l . (London) 130, 53-71. Dykes, R. W. (1974a). Factors r e l a t e d to the dive r e f l e x i n harbor s e a l s : R e s p i r a t i o n , immersion bradycardia, and the l a b i l i t y of heart r a t e . Can. J . P h y s i o l . Pharmacol. 52, 248-258. Dykes, R. W. (1974b). Factors r e l a t e d to the dive r e f l e x i n harbor s e a l s : Sensory c o n t r i b u t i o n s from the t r i g e m i n a l r e g i o n . Can. J . P h y s i o l . Pharmacol. 52, 259-265. 142 Ebbecke, U. (1944). Ubersichten. Der g e s i c h t s r e f l e x des trigeminus a l s warmeschutzreflex (wind - und w e t t e r r e f l e x ) des kopfes. K l i n . Wschr. 23, 141-145. E l i a s s e n , E. (1960a). Cardiovascular responses to submersion asphyxia i n avian d i v e r s . Arbok Univer. Bergen - Mat. Natur. S e r i e 2, 1-100. E l i a s s e n , E. (1960b). Cardiovascular pressures i n the h i b e r n a t i n g hedgehog w i t h s p e c i a l regard to the pressure changes during a r o u s a l . Arbok Univer. Bergen - Mat. Natur. S e r i e 6, 1-27. E i s n e r , R. (1965). Heart r a t e response i n forced versus t r a i n e d experimental dives i n pinnipeds. Hvalradets S k r i f t e r 48, 24-29. E i s n e r , R. (1969). Cardiovascular adjustments to d i v i n g . In \"The Biology of Marine Mammals\". Ed. H. T. Andersen. Academic P r e s s , N. Y. E i s n e r , R., D. L. F r a n k l i n , R. L. van C i t t e r s and D. V. Kenney. (1966a). Cardiovascular defense against asphyxia. Science 153, 941-949. E i s n e r , R., D. W. Kenney and K. Burgess. (1966b). D i v i n g bradycardia i n the t r a i n e d d o l p h i n . Nature 212, 407-408. E i s n e r , R., D. D. Hammond,and H. R. Parker. (1969). C i r c u l a t o r y responses to asphyxia i n pregnant and f e t a l animals: A comparative study of Weddell s e a l s and sheep. Yal e J . B i o l . Med. 42, 202-217. E i s n e r , R., J . T. Shurley, D. D. Hammond and R. E. Brooks. (1970). Cerebral tolerance to hypoxemia i n asphyxiated Weddell s e a l s . Resp. P h y s i o l . 9, 287-297. E i s n e r , R., J . E. A n g e l l James and M. deB. Daly. (1977). C a r o t i d body chemoreceptor.reflexes and t h e i r i n t e r a c t i o n s i n the s e a l . Am. J . P h y s i o l . 232, H517-H525. E r r i n g t o n , P. L. (1963). Muskrat po p u l a t i o n s . Iowa State U n i v e r s i t y Press. F e i g l , E. and B. Folkow. (1963). Cardiovascular responses i n \" d i v i n g \" and during b r a i n s t i m u l a t i o n i n ducks. Acta p h y s i o l . scand. 57, 99-110. Ferrante, F. L. and H. M. F r a n k e l . (1971). Cardiovascular responses of anaesthetized n u t r i a and cats during apnea. Am. J . P h y s i o l . 221, 251-254. Folkow, B. and E. H. Ru b i n s t e i n . (1965). Behavioural and autonomic patt e r n s evoked by s t i m u l a t i o n of the l a t e r a l hypothalmic area i n the c a t . Acta p h y s i o l . scand. 65, 292-299. Folkow, B., B. Lisander and B. Oberg. (1971). Aspects of the c a r d i o v a s c u l a r nervous c o n t r o l i n a mammalian d i v e r (Myocaster coypus). Acta p h y s i o l . scand. 82, 439-446. 143 Francois-Franck, Ch.-A. (1889). C o n t r i b u t i o n a 1'etude exp£rimentale des nevroses r e f l e x e s d ' o r i g i n e nasale. Arch. P h y s i o l . 21, 538-555. Gandevia, S. C., D. I . McCloskey and E. K. P o t t e r . (1978a). I n h i b i t i o n of baroreceptor and chemoreceptor r e f l e x e s on heart r a t e by a f f e r e n t s from the lungs. J . P h y s i o l . (London) 276, 369-381. Gandevia, S. C , D. I . McCloskey and E. K. P o t t e r . (1978b). R e f l e x brady-c a r d i a o c c u r r i n g i n response to d i v i n g , nasopharyngeal s t i m u l a t i o n and o c c u l a r pressure, and i t s m o d i f i c a t i o n by r e s p i r a t i o n and swallowing. J . P h y s i o l . (London) 276, 383-394. Glogowska, M., P. S. Richardson, J . G. Widdicombe and A. J . Winning. (1972). The r o l e of the vagus nerves, p e r i p h e r a l chemoreceptors and other a f f e r e n t pathways i n the genesis of augmented breaths i n cats and r a b b i t s . Resp. P h y s i o l . 16, 179-196. Hainsworth, R. (1974). C i r c u l a t o r y responses from lung i n f l a t i o n i n anaesthetized dogs. Am. J . P h y s i o l . 226, 247-255. Hainsworth, R., L. Jacobs and J . H. Comroe, J r . (1973). A f f e r e n t lung denervation by b r i e f i n h a l a t i o n of steam. J . Appl. P h y s i o l . 34, 708-714. H a l l , F. G., D. B. D i l l and E. S. G. Barron. (1936). Comparative physiology i n high a l t i t u d e s . J . C e l l . Comp. P h y s i o l . 8, 301-313. Hammond, D. D., R. E i s n e r , G. Simison and R. Hubbard. (1969). Submersion bradycardia i n the newborn elephant s e a l Mirounga a n g u s t i r o s t r i s . Am. J . P h y s i o l . 216, 220-222. Harding, R., P. Johnson and M. E. M c C l e l l a n d . (1976). Water receptors i n the l a r y n x of the lamb. J . P h y s i o l . (London) 256, 104-105P. Harding, R., P. Johnson and M. E. McClelland. (1978). L i q u i d - s e n s i t i v e l a r y n g e a l receptors i n the developing sheep, cat and monkey. J . P h y s i o l . (London) 277, 409-422. H a r r i s o n , R. J . (1960). Experiments w i t h d i v i n g s e a l s . Nature 188, 1068-1070. H a r r i s o n , R. J . and J . D. W. Tomlinson. (1960). Normal and experimental d i v i n g i n the common seal.(Phoca v i t u l i n a ) . E x t r a . Mamm. 24, 386-399. H a r r i s o n , R. J . and G. L. Kooyman. (1971). D i v i n g i n Marine Mammals. Oxford Biology Reader No. 6. Oxford Univ. Press. H a r r i s o n , R. J . , S. H. Ridgeway and P. L. Joyce. (1972). Telemetry^of heart r a t e i n d i v i n g s e a l s . Nature 237, 280. 144 Heistad, D. D. and R. C. Wheeler. (1970). Simulated d i v i n g during hypoxia i n man. J . Appl. P h y s i o l . 28, 652-656. Hempleman, H. V. and A. P. M. Lockwood. (1978). The Physiology of D i v i n g i n Man arid Other Animals. Oxford B i o l o g y Reader S e r i e s No. 99. Oxford U n i v e r s i t y Press. Hering, H. E. (1933). Arch. s c i . B i o l . N a p o l i V o l . 28. Proc. X l V t h I n t . P h y s i o l . Cong. Heymans, C. (1929). Uber d i e p h y s i o l o g i e und pharmacologie des herz-vagus-zentrums. Ergebn. P h y s i o l . 28, 244-311. Hochachka, P. W., J . F i e l d s and T. Mustafa. (1973). Animal l i f e without oxygen: B a s i c biochemical mechanisms. Am. Zool. 13, 543-555. Hochachka, P. W. and K. B. Storey. (1975). Metabolic consequences of d i v i n g i n animals and man. Science 187, 613-621. Huang, T. F. and Y. I . Peng. (1976). Role of the chemoreceptor i n d i v i n g bradycardia i n the r a t . Jap. J . P h y s i o l . 26, 395-401. Hunter, J . (1787). Trans. Roy. Soc. (London) 77, 331. Huxley, F. M. (1913). On the r e f l e x nature of apnoea i n the duck i n d i v i n g . I I . R e f l e x p o s t u r a l apnoea. Quart. J . Exp. P h y s i o l . 6, 159-182. I r v i n g , L. (1937). The r e s p i r a t i o n of beaver. J . C e l l . Cotnp. P h y s i o l . 9, 437-451. I r v i n g , L. (1938). The i n s e n s i t i v i t y of d i v i n g animals to CO2. Am. J . P h y s i o l . 124, 729-734. I r v i n g , L. (1939). R e s p i r a t i o n i n d i v i n g mammals. P h y s i o l . Rev. 19, 112-134. I r v i n g , L., P. F. Scholander and S. W. G r i n n e l l . (1941b). S i g n i f i c a n c e of the heart r a t e to the d i v i n g a b i l i t y of s e a l s . J . C e l l . Comp. P h y s i o l . 18, 283-297. I r v i n g , L., P. F. Scholander and S. W. G r i n n e l l . (1942). The r e g u l a t i o n of a r t e r i a l blood pressure i n the s e a l during d i v i n g . Am. J . P h y s i o l . 135, 557-566. James, L. S. (1962). Biochemical a l t e r a t i o n s observed i n the neonate. In \" P e r i n a t a l Pharmacology\". Ed. C. D. May. Ross L a b o r a t o r i e s , Columbus, Ohio. Johansen, K. (1959). Heart a c t i v i t y during experimental d i v i n g of snakes. Am. J . P h y s i o l . 197, 604-606. 145 Jones, D. R. (1966). Factors a f f e c t i n g the recovery from d i v i n g bradycardia i n the f r o g . J . Exp. B i o l . 44, 397-411. Jones, D. R. (1973). Systemic a r t e r i a l baroreceptors i n ducks and the consequences of t h e i r denervation on some c a r d i o v a s c u l a r responses to d i v i n g . J . P h y s i o l . (London) 234, 499-518. Jones, D. R., H. D. F i s h e r , S. McTaggart and N. H. West. (1973). Heart r a t e during breath-holding and d i v i n g i n the u n r e s t r a i n e d harbor s e a l (Phoca v i t u l i n a r i c h a r d i ) . Can. J . Z o o l . 51, 671-680. Jones, D. R. and N. H. West. (1978). The c o n t r i b u t i o n of a r t e r i a l chemo-receptors and baroreceptors to d i v i n g r e f l e x e s i n b i r d s . In \" R e s p i r a t o r y Function i n B i r d s , . A d u l t and Embryonic\". Ed. J . P i i p e r . S p r inger-Verlag, B e r l i n . Kawakami, Y., B. H. Natelson and A. B. DuBois. (1967). Cardiovascular e f f e c t s of face immersion and f a c t o r s a f f e c t i n g d i v i n g r e f l e x i n man. J . Appl. P h y s i o l . 23, 964-970. Knowleton, G. C. and M. G. Larrabee. (1946). A u n i t a r y a n a l y s i s of pulmonary volume re c e p t o r s . Am. J . P h y s i o l . 147., 100-114. Kooyman, G. L. (1973). R e s p i r a t o r y Adaptations i n Marine Mammals. Amer. Zool. 13, 457-468. Kooyman, G. L. and H. T. Andersen. (1969). In \"The B i o l o g y of Marine Mammals.\" Ed. H. T. Andersen. Academic Press. Kooyman, G. L., D. H. Kerem, W. B. Campbell and J . J . Wright. (1971). Pulmonary f u n c t i o n i n f r e e l y d i v i n g Weddell s e a l s , Leptonychotes w e d d e l l i . Resp. P h y s i o l . 12, 271-282. Kooyman, G. L. and W. B. Campbell. (1972). Heart r a t e s i n f r e e l y d i v i n g Weddell s e a l s , Leptonychotes w e d d e l l i . Comp. Biochem. P h y s i o l . 43A, 31-36. Koppanyi, T. and M. S. Dooley. (1928). The cause of c a r d i a c slowing accompanying p o s t u r a l apnea i n the duck. Am. J . P h y s i o l . 85, 313-323. Koppanyi, T. and M. S. Dooley. (1929). Submergence and p o s t u r a l apnea i n the muskrat. Am. J . P h y s i o l . 88, 592-595. Kratchmer, F. (1870). liber r e f l e x e von der nasenschleim-haut auf athmung und k r e i s l a u f . Sber. Akad. Wiss. Wein. 62, 147-170. Larrabee, M. G. and G. C. Knowleton. (1946). E x c i t a t i o n and i n h i b i t i o n of phrenic motoneurones by i n f l a t i o n of the lungs. Am. J . P h y s i o l . 147, 90-99. 146 L e i t n e r , L.-M., M. Roumy and M. J . M i l l e r . (1974). Motor responses t r i g g e r e d by d i v i n g and by mechanical s t i m u l a t i o n of the n o s t r i l s and of the g l o t t i s of the duck. Resp. P h y s i o l . 21, 385-392. L e i v e s t a d , H. (1960). The e f f e c t of prolonged submersion on the metabolism and the heart r a t e i n the toad. Srbok Univ. Bergen 5, 1-15. . Le i v e s t a d , H., H. Andersen and P. F. Scholander. (1957). P h y s i o l o g i c a l response to a i r exposure i n c o d f i s h . Science 126, 505. Levy, M. N., H. DeGeest and H. Zieske. (1966). E f f e c t s of r e s p i r a t o r y center a c t i v i t y on the heart. C i r c . Res. 18, 67-78. L i n , Y. C. and D. G. Baker. (1975). Cardiac output and i t s d i s t r i b u t i o n during d i v i n g i n the r a t . Am. J . P h y s i o l . 228, 733-737. Lisand e r , B. Unpublished observations. Lombroso, U. (1913). Uber d i e reflexhemmung der herzens wahrend der r e f l e k t o r i s c h e n atmungshemmung b e i verschiedenen t i e r e n . Z e i t s c h r . B i o l . 61, 517-538. Lord, R. Unpublished observations. M i l l s , J . E., H. S e l l i c k and J . G. Widdicombe. (1970). E p i t h e l i a l i r r i t a n t r eceptors i n the lungs. In \"Breathing: Hering-Breuer Centenary Symposium\". Ed. R. P o r t e r . Ciba Foundation Symposium. J . and A. C h u r c h i l l . London. McEwan, E. H., N. A i t c h i s o n and P. Whitehead. (1974). Energy metabolism of o i l e d muskrats. Can. J . Zool. 52, 1057-1062. Moore, T. 0., Y. C. L i n , D. A. L a l l y and S. K. Hong. (1972). E f f e c t s of temperature, immersion, and ambient pressure on human apneic bradycardia. J . Appl. P h y s i o l . 33, 36-41. Murdaugh, J r . , H. V., J . C. Seabury and W. L. M i t c h e l l . (1961). E l e c t r o -cardiogram of the d i v i n g s e a l . C i r c . Res. 9, 958-961. Murdaugh, J r . , J . V., C. E. Cross, J . E. M i l l e n , J . B. Gee and E. D. Robin. (1968). D i s s o c i a t i o n of bradycardia and a r t e r i a l c o n s t r i c t i o n during d i v i n g i n the s e a l Phoca v i t u l i n a . Science 162, 364-365. Nahas, G. G. (1956). Heart r a t e during short periods of apnea i n c u r a r i z e d dogs. Am. J . P h y s i o l . 187, 302-306. Orr, J . B. and A. Watson. (1913). Study of the r e s p i r a t o r y mechanism i n the duck. J . P h y s i o l . (London) 46, 337-348. Ott, N. T. and J . T. Shephard. (1971). Vasodepressor r e f l e x from lung i n f l a t i o n i n the r a b b i t . Am. J . P h y s i o l . 221, 889-895. 147 Parker, H. R. and M. J . Purves. (1967). Some e f f e c t s of maternal hyperoxia on the blood gas tensions and v a s c u l a r pressures i n the f e t a l sheep. Quart. J . Exp. P h y s i o l . 52, 205-221. Patton, N. D. (1913). The r e l a t i v e i n f l u e n c e of the l a b y r i n t h i n e and c e r v i c a l elements i n the production of p o s t u r a l apnea i n the duck. Quart. J . Exp. P h y s i o l . .6, 197-207. P i c k w e l l , G. V. and E. Douglas.- (1968). P h y s i o l o g i c a l Responses to Asphyxia i n Animals, I n c l u d i n g Man. In \"Experiments i n Physiology and Biochemistry\". Volume 1. Ed. G. A. Kerkut. Academic P r e s s , London and New York. R e i t e , 0. B., J . Krog and K. Johansen. (1963). Development of bradycardia during submersion of the duck. Nature 200, 684-685. Reynolds, S. R. M. (1962). Nature of f e t a l adaptation to the u t e r i n e environment: A problem of sensory d e p r i v a t i o n . Am. J . Obstet. Gynecol. 83, 800-808. Ri c h e t , C. (1899). De l a r e s i s t a n c e des canards a l'asphyxie. J . P h y s i o l . P a t h o l . Gen. 1, 641-650. Robin, E. D., H. V. Murdaugh J r . , W. Pyron, E. Weiss and P. Soteres. (1963). Adaptation to d i v i n g i n the harbor s e a l - gas exchange and v e n t i l -atory response to C0 2. Am. J . P h y s i o l . 205, 1175-1177. Robinson, D. (1939). The muscle hemoglobin as an oxygen s t o r e i n d i v i n g . Science 90, 276-277. S a t c h e l l , G. H. (1961). The response of the dog f i s h to anoxia. J . Exp. B i o l . 38, 531-543. Scholander, P. F. (1940). Experimental i n v e s t i g a t i o n s on the r e s p i r a t o r y f u n c t i o n i n d i v i n g mammals and b i r d s . Hvalr§d. S k r i f t . 22, 1-131. Scholander, P. F. (1960). Experimental s t u d i e s on asphyxia i n animals. In \"Oxygen Supply to the Foetus\". Eds. J . Walker and A. C. T u r n b u l l . Oxford. B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s . Scholander, P. F. (1963). The master switch of l i f e . S c i . Am. 209, 92-106. Scholander, P. F., L. I r v i n g and S. W. G r i n n e l l . (1942a). Aerobic and anaerobic changes, i n s e a l muscles during d i v i n g . J . B i o l . Chem. 142, 431-440. Scholander, P. F., L. I r v i n g and S. W. G r i n n e l l . (1942b). On the temperature and metabolism of the s e a l during d i v i n g . J . C e l l . Comp. P h y s i o l . 19, 67-78. Selye, H. (1949). Textbook of Endocrinology. Second e d i t i o n . Acta Endo-c r i n o l . , Inc., Montreal. 148 Snyder, C. D. (1915). A study of the causes of r e s p i r a t o r y change of heart r a t e . Am. J . P h y s i o l . 37, 104-117. Song, S. H., W. K. Lee, Y. A. Chung and S. K. Hong. (1969). Mechanism of apneic bradycardia i n man. J . Appl. P h y s i o l . 27, 323-327. Storey, A. T. and P. Johnson. (1975). Laryngeal water receptors i n i t i a t i n g apnea i n the lamb. Exp. Neurol. 47, 42-55. Str^mme, S. B. and A. S. B l i x . (1976). I n d i r e c t evidence f o r a r t e r i a l chemoreceptor r e f l e x f a c i l i t a t i o n by face immersion i n man. A v i a t . Space and Environ. Med. 47, 597-599. Tchobroutsky, C , C. Merlet and P. Rey. (1969). The d i v i n g r e f l e x i n r a b b i t , sheep and newborn lamb and i t s a f f e r e n t pathways. Resp. P h y s i o l . 8, 108-117. Thornton, R., C. Gordon and J . H. Ferguson. (1978). Role of thermal s t i m u l i i n the d i v i n g response of the muskrat (Ondatra z i b e t h i c a ) . Comp. Biochem. P h y s i o l . 61A, 369-370. Traube, L. (1865). Gesammelte b e i t r a g e . Z e i t s c h r . Med. Wiss., No. 56. Van C i t t e r s , R. L., 0. A. Smith, N. W. Watson and D. L. F r a n k l i n . (1965). F i e l d study of d i v i n g responses i n the northern elephant s e a l . Hvalr9dets S k r i f t e r 48, 15-23. Vincent, S. and T. Cameron. (1920). A note on the i n h i b i t o r y r e s p i r a t o r y r e f l e x i n the f r o g and some other animals. J . Comp. Neurol. 31, 283-292. West, N. H. and D. R. Jones. (1976). The i n i t i a t i o n of d i v i n g apnoea i n the f r o g , Rana p i p i e n s . J . Exp. B i o l . 64, 25-38. Whayne, J r . , T. F. and T. K i l l i p I I I . (1967). Simulated d i v i n g i n man: Comparison of f a c i a l s t i m u l i and response i n arrhythmia. J . Appl. P h y s i o l . 22, 800-807. White, F. N. and G. Ross. (1966). C i r c u l a t o r y changes during experimental d i v i n g i n the t u r t l e . Am. J . P h y s i o l . 211, 15-18. White, S. W. and R. J . McRitchie. (1973). Nasopharyngeal r e f l e x e s : I n t e g r a t i v e a n a l y s i s of evoked r e s p i r a t o r y and c a r d i o v a s c u l a r e f f e c t s . Aust. J . Exp. B i o l . Med. S c i . 51, 17-31. White, S. W., R. J . McRitchie and D. L. F r a n k l i n . (1974). Autonomic c a r d i o -v a s c u l a r e f f e c t s of n a s a l i n h a l a t i o n of c i g a r e t t e smoke i n the r a b b i t . Aust. J . Exp. B i o l . Med. S c i . 52, 111-126. Yonce, L. R. and B. Folkow. (1970). The i n t e g r a t i o n of the c a r d i o v a s c u l a r response to d i v i n g . Am. Heart Journ. 79, 1-4. "@en ; edm:hasType "Thesis/Dissertation"@en ; edm:isShownAt "10.14288/1.0095200"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Zoology"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. 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