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The initiation of and recovery from diving bradycardia in the muskrat Drummond, Peter Charles Patterson 1980

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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 L<f>v<&; 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. 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