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Pharmacological blockade of the cardiovascular dive response : effects on heart rate and diving behaviour… Elliott, Nicole M. 2002

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P H A R M A C O L O G I C A L B L O C K A D E OF THE C A R D I O V A S C U L A R DIVE RESPONSE: EFFECTS ON H E A R T RATE A N D DIVING B E H A V I O U R IN THE H A R B O U R S E A L (PHOCA VITULINA) by NICOLE M . ELLIOTT B.A. , The University of Colorado, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Zoology) We acceDt this thesis as conforming to the-Reouired standard THE UNIVERSITY OF BRITISH C O L U M B I A April 2002 © Nicole M . Elliott, 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ~hbO 10 9f The University of British Columbia Vancouver, Canada Date Apnt 24, 2no2-DE-6 (2/88) Abstract W h i l e d iv ing , harbour seals {Phoca vitulina) manage their oxygen stores through cardiovascular adjustments inc lud ing bradycardia, a concurrent reduct ion i n cardiac output, and peripheral vasoconstr ict ion. A t the surface, post-dive tachycardia faci l itates rapid re loading o f these oxygen stores. A l though harbour seals can tolerate over 20 m i n o f submergence, the major i ty o f their natural d ives are on ly 2 to 6 m in and are usua l ly f o l l owed by surface intervals that are less than 1 m in , so they spend about 80 % of their t ime at sea submerged. G i v en that harbour seals meet their eco log ica l needs through repetit ive short aerobic dives, I was interested i n the funct ional role, i f any, o f the card iovascular d ive response dur ing these short dives. Du r i ng voluntary d iv ing in an 11 m deep tank, the cardiovascular responses to submergence o f f ive captive harbour seals were manipulated us ing speci f ic pharmaco log ica l antagonists, and the effects on d i v i ng behaviour were observed. Ef fects of pharmaco log ica l b lockade on mean heart rate and on heart rate var iab i l i ty were also examined in order to assess the autonomic contro l o f heart rate dur ing voluntary d iv ing . Heart rate was recorded us ing subcutaneous electrodes and a custom data logger wh i l e d i v ing behaviour was monitored us ing a v ideo camera. The muscar in ic b locker methoctramine b locked d iv ing bradycardia; the oc-adrenergic b locker prazos in b locked d i v i ng vasoconstr ict ion; and the (3-adrenergic b locker metopro lo l b locked post-dive tachycardia. M e a n heart rate analysis revealed that d i v ing bradycard ia is pr imar i l y modulated by the vagus, and post-dive tachycardia is due to increased sympathetic st imulat ion o f the heart as we l l as parasympathetic w i thdrawal . Results o f power spectral analysis o f heart rate var iab i l i ty supported the conc lus ion that the parasympathetic nervous system predominates dur ing d iv ing but not dur ing surface intervals. None o f the pharmacolog ica l b lockers had any effect on average d ive or surface interval duration. M e a n dive duration ranged f rom 2.3±0.47 to 2.9±0.10 m in (mean±S.E.M.) for a l l treatments inc lud ing controls, and mean surface interval duration ranged f rom 23 to 29 sec. Seals maintained a h igh percent d ive t ime (83 to 87 %) i n a l l treatments inc lud ing controls. Thus, harbour seals do not need the dive response dur ing short d ives i n order to mainta in an eff ic ient d ive strategy. i i Table of Contents Abstract i i Table o f Contents i i i L i s t o f Tables iv L i s t of F igures v Acknowledgements v i Chapter 1. Ef fect o f pharmaco log ica l b lockade on mean heart rate and d i v i ng behaviour Introduction 1 The d ive response 1 Cont ro l o f the d ive response 2 C lass i f i ca t ion o f dives 5 Rout ine d iv ing behaviour 7 The role o f the d ive response in routine d iv ing 10 Purpose o f this study 11 Mater ia ls and Methods 14 S e a l s . . . . 14 Instrumentation 14 Pre l iminary drug testing 16 D i v i n g experiments 21 Ana l ys i s and statistics 26 Results 28 Pre l im inary drug tests 28 Heart rate dur ing d i v i ng 30 D i v i n g behaviour 34 D iscuss ion 39 Heart rate dur ing d i v i ng 39 D i v i n g behaviour and the role o f the d ive response 44 Chapter 2. Spectral analysis o f heart rate var iabi l i ty Introduct ion 49 Methods 51 Results 54 D i scuss ion 62 Chapter 3. Genera l D i scuss ion and Conc lus i on Heart rate dur ing d iv ing 66 Ro le o f the d ive response dur ing routine d i v i ng 68 Literature C i ted 73 List of Tables Table 1.1. Summary of characteristics of the pharmacological blockers used to inhibit the dive response 23 iv List of Figures F igure 1.1. D i ag ram of some important neural mechanisms invo lved in the cardiovascular response to submersion 4 F igure 1.2. D i ag ram of some important neural mechanisms invo lved in the cardiovascular response f o l l ow ing submersion 6 F igure 1.3. Picture of harbour seal instrumented w i th E C G electrodes, data logger, buckles, neoprene head patch, and neoprene catheter pocket 15 F igure 1.4. D i ag ram of E C G electrode assembly 17 F igure 1.5. D i ag ram of catheter assembly 19 F igure 1.6. Ef fect o f phenylephr ine and isoproterenol on mean heart rate after different doses o f methoctramine, prazosin, and metopro lo l 22 F igure 1.7. Picture o f deep d ive tank and d iagram of the surface o f the tank 24 F igure 1.8. Ef fect o f phenylephr ine and isoproterenol on mean heart rate before and after b lockade w i th methoctramine, prazosin, and metopro lo l 29 F igure 1.9. Ef fect o f pharmaco log ica l b lockade on mean heart rate dur ing dives and post-dive surface intervals 31 F igure 1.10. Heart rate prof i les before, dur ing, and after voluntary dives i n contro l and pharmaco log ica l ly b locked seals 32 F igure 1.11. Heart rate prof i les before, dur ing, and after control dives for adult females versus juven i le males 35 F igure 1.12. Ef fect o f pharmaco log ica l b lockade on d ive and surface interval duration ..' 36 F igure 1.13. Ef fect o f pharmaco log ica l b lockade on the distr ibut ion o f d ive and post-dive surface interval durations 37 F igure 2.1. Instantaneous heart rate and corresponding power spectra (% max peak) for contro l and pharmaco log ica l ly b locked dives i n one seal 55 F igure 2.2. Power density spectra (absolute power) for contro l and treated dives in one seal 56 F igure 2.3. Tota l power of d ive heart rate var iab i l i ty and relative power o f the l ow and h igh frequency components i n control and treated seals 57 F igure 2.4. Instantaneous heart rate and corresponding power spectra for control and treated post-dive surface intervals in one seal 59 F igure 2.5. Tota l power o f surface interval heart rate var iab i l i ty and relative power o f the l ow and h igh frequency components in contro l and treated seals . 60 v Acknowledgements First, I would like to thank my supervisor Dr. David R. Jones for his guidance and encouragement in completing this project. Dave was very patient with me through all of my questions and the difficulties that I faced along the way. His enthusiasm about my work gave me the motivation I needed to continue when I didn't see the end in sight. I would like to thank Russ Andrews for guidance and support as well. Russ dedicated many hours to helping me find a way to record heart rate from the seals and then many more hours when those instruments failed! Despite his busy schedule, he was always eager to answer my questions and point me in the right direction. I would like to thank the other members of the Jones' lab - Amanda Southwood, Kim Borg, and Manuela Gardner - for moral support and for help manipulating the seals. Special thanks go to Amanda for help with the instruments, for extra encouragement, and for giving me inspiration to follow in her footsteps. I owe many thanks to the staff in the Zoology Mechanical Workshop. Bruce Gillespie, Granville Williams, and Don Brandys helped me prepare the dive tank for my experiments and solve any problems I had with the seal facilities. Bruce deserves special thanks for making my project a priority when I needed something fast, and for his brilliant ideas on designing the 'false bottom'. I am extremely grateful to Stephane Lair for assistance with all medical procedures with the seals. Stephane also went above and beyond his duties as a veterinarian by providing medical (and moral!) support during my experiments and by bravely "wrestling" the seals when I needed to attach instruments or inject drugs. I am sincerely grateful to Lisa Skinner who spent many more hours than she should have helping me care for the seals, work on the dive tank, and run experiments. Lisa's time, efforts, and friendship made the completion of my project more pleasant and less frustrating than it could have been. Many other friends, colleagues, and volunteers deserve thanks for helping me over the course of my project. I thank Bill Milsom for his guidance, wisdom and open ears, for lending me instruments for preliminary experiments, and for help drawing diagrams in Corel. I am grateful to Sheila Thornton and Gunna Weingartner for the catheter design and for advice on training and manipulating the seals. I thank Glenn Tattersall for designing Excel templates that were extremely useful in my data analysis and for spending many, many hours with me trying to make sense out of spectral analysis. Jan McPhee showed me how to make the ECG electrodes, and Joe Muise showed me how to work with the Model 8 data logger. I also thank Dave Jones, Amanda Southwood, Bill Milsom, and Peter Hochachka for providing insightful feedback on earlier drafts of this thesis. I thank Jessica Robertson, Angela Grover, Carrie Robb, Amber Bardock, Jody Weir, Michael Pearson, Brian Tagami, and especially Tanya Wilks for help caring for the seals, cleaning pools, working on the dive tank, and running experiments, rain or shine. Finally, I thank Chris Bouchard for wrestling with the seals when I needed help, for opening the gate at South Campus a thousand times, for going with me at 3:00 in the morning to give the seals oral drugs, and for his unconditional support over the last few years especially when I was overwhelmed with work. This work was supported by an NSERCC Research Grant (D.R. Jones). vi Chapter 1. Effect of cardiovascular pharmacological blockade on mean heart rate and diving behaviour Introduction The dive response D i v i n g mammals have a suite o f morpho log ica l and phys io log ica l traits related to their d i v ing behaviour that a l low them to successful ly explo i t aquatic habitats. Seals o f the fami ly Phoc idae are among the greatest divers in terms o f d iv ing capacity, and, although most o f their natural dives are relat ively brief, they exhib i t an extraordinary abi l i ty to surv ive extended periods underwater. The southern elephant seal (Mirounga leonind), for example, typ ica l ly dives for 20 to 30 m in but can remain submerged for as long as 120 m in (H inde l l et al. 1992). F o r over a century, researchers have been t ry ing to understand how d iv ing mammals can sustain their metabol ic needs dur ing extended periods o f oxygen depr ivat ion. Du r i ng submergence, divers meet their oxygen demands by re ly ing on enhanced oxygen stores in their b lood and muscles. Aquat i c mammals have a higher oxygen carry ing capacity than their terrestrial counterparts due to a larger mass-speci f ic b lood vo lume, and phoc id seals have a higher concentrat ion o f p lasma hemoglob in and a higher b lood hematocrit (Lenfant et al. 1970; Snyder, 1983). Furthermore, this hematocrit leve l fluctuates so that dur ing d iv ing , hematocrit is h igh and more oxygen can be stored in the c i rcu lat ion, but when seals are hauled out on land, hematocrit is lower. Seals have enlarged spleens that sequester the extra red b lood cel ls that cou ld potential ly increase b lood v iscos i ty dur ing haul outs (Caste l l in i and Caste l l in i , 1993; Hur fo rd et al. 1996; 1 Thornton et al. 2000). The muscle myoglobin content of diving mammals is also higher than in non-diving species (Castellini and Somero, 1981). Despite increases in oxygen stores in diving mammals, the endogenous stores are insufficient to maintain the pre-dive rate of aerobic metabolism for more than about 20 to 50 % of the maximum breath-hold duration (Scholander et al. 1940). In order to minimize oxygen consumption and conserve these finite oxygen stores for the hypoxia-intolerant brain and heart during extended submergence, cardiovascular adjustments including bradycardia, reduced cardiac output, and peripheral vasoconstriction function to maintain constant arterial blood pressure and redistribute the blood away from most peripheral tissues. As oxygen-limiting conditions develop, overall aerobic metabolism decreases, anaerobic metabolism (by glycolysis and the consumption of high-energy phospate stores) in hypoperfused peripheral tissues leads to an accumulation of byproducts (i.e. lactate and H + ions), and possibly total energy consumption by these peripheral tissues decreases. These cardiovascular and metabolic mechanisms constitute the key components of the classic "diving response" elucidated by the pioneering studies of Scholander, Irving, and their colleagues (Irving et al. 1935; Scholander, 1940; Grinnell etal. 1942). Control of the dive response The responses to diving of apnea, bradycardia, reduced cardiac output and peripheral vasoconstriction are the result of several integrated autonomic reflexes, though they are subject to modification by higher centers of the nervous system. A diagrammatic representation of some important neural mechanisms involved in the dive response is 2 presented in F i g . 1.1. The response is init iated upon submergence by st imulat ion o f fac ia l receptors innervated by the tr igeminal nerves and receptors in the upper respiratory tract (nasal mucosa and larynx) (Dykes, 1974; D r u m m o n d and Jones, 1979). Apnea occurs in the expiratory pos i t ion. The cardiac response is then reinforced throughout the d ive by the cessation of central respiratory dr ive and reduced act iv i ty o f pu lmonary stretch receptors (Dykes, 1974; D rummond and Jones, 1979; Da ly , 1984) together w i th the st imulat ion o f carot id body chemoreceptors by progressive hypox i a and hypercapnia (Da ly et al. 1977). Under normal condit ions, hypox ic and hypercapnic st imulat ion o f chemoreceptors wou ld stimulate breathing, but tr igeminal inputs inhib i t this dr ive dur ing submersion (Daly, 1984). The barostatic ref lex functions in mainta in ing fa i r ly stable arterial b lood pressure dur ing d iv ing , but the inf luence o f the baroreceptors on heart rate and total peripheral resistance is secondary to chemoreceptor, t r igeminal , and respiratory inputs (Jones et al. 1983). Parasympathetic out f low to the heart plays a major role in d i v i ng bradycardia as the vagus releases acety lchol ine onto muscar in ic receptors on the heart (Harr ison and Toml inson , 1960; Murdaugh et al. 1961; Signore and Jones, 1995; B r o w n and Tay lor , 2001). Per ipheral vasoconstr ict ion is brought about by the sympathetic nervous system as a-adrenergic receptors in arterial smooth musc le respond to norepinephrine (Anderson and B l i x , 1974; Signore and Jones, 1995; Ho f fman , 2001). Vasoconst r i c t ion is also mediated by c i rcu lat ing catecholamines (norepinephrine and epinephrine) where b lood f l ow remains suff ic ient to a l low their del ivery (Hance et al. 1982; Hochachka et al. 1995). 3 F igure 1.1. D i ag ram of some neural mechanisms invo lved in the cardiovascular response dur ing submers ion. 1: st imulat ion by water o f t r igeminal or upper respiratory tract receptors leading to ref lex apnea, bradycardia, and selective vasoconstr ict ion. Bradycard ia is modulated by vagal inputs to cardiac muscar in ic receptors. Vasoconstr ic t ion is mediated by sympathetic st imulat ion o f oc-adrenergic receptors i n the arterioles. 2: reduced act ion o f pu lmonary stretch receptors contributes to the bradycardia and vasoconstr ict ion. 3: increasing hypox i a and hypercapnia st imulate arterial chemoreceptors in the carot id bodies wh i ch reinforce the card iovascular response. T r igemina l input inhibits the chemoreceptor dr ive to breathe (1). 4: the cessation o f central inspiratory neuronal dr ive also reinforces the cardiovascular response, as does the d iminut ion in pu lmonary stretch receptor act iv i ty (2). Ar ter ia l baroreceptor pathways are not inc luded. C V M , cardiac vagal motorneurons; S V N , central sympathetic vasomotor neurons; R C , respiratory centers; +, st imulat ion; -, inh ib i t ion. [Mod i f i ed f rom Da ly , 1984.] 4 The post-dive surface interval is characterized by hypervent i lat ion, acceleration o f the heart rate, increased cardiac output and vasodi latat ion. A diagrammatic representation o f the neural events corresponding to the cardiovascular surface response is presented i n F i g . 1.2. D im in i sh i ng inputs f rom tr igeminal receptors and chemoreceptors result in breathing and the wi thdrawal o f neural inputs contro l l ing the d ive response. W i thd rawa l o f sympathetic vasomotor tone results in the vasodi latat ion of peripheral b lood vessels. In muskrats (Ondatra zibethica), post-dive tachycardia is caused by the wi thdrawal o f cardiac vagal tone together w i th increased sympathetic input to P-adrenergic receptors on the heart (Signore and Jones, 1995). Hypervent i la t ion is dr iven by elevated arterial carbon d iox ide levels (Parkos and Wahrenbrock, 1987), and the increase in central inspiratory neuronal act iv i ty, in f lat ion o f the lungs, and feedback f r om pu lmonary stretch afferents reinforce the cardiovascular response to surfacing (Angel l - James et al. 1981; Da ly , 1984). Classification of dives The early research that establ ished the elements of the d i v i ng response was in i t ia l ly done on animals that were forc ib ly submerged (Irving et al. 1935; Scholander, 1940; Gr inne l l et al. 1942). Du r i ng forced submergence, the d ive response is almost a lways profound w i th an extreme bradycardia accompanied by extreme vasoconstr ict ion that redirects b lood f l ow to the brain, eyes, adrenal glands, and heart musc le (Scholander, 1940; Gr inne l l et al. 1942; E isner et al. 1966; Zapo l et al. 1979), so peripheral tissues and organs must rely on loca l oxygen stores or tolerate hypox i c and ischemic condit ions. Later studies i nvo l v i ng mammals trained to d ive on command or vo luntar i ly d i v ing in 5 Figure 1.2. D i ag ram of some neural mechanisms invo lved in the cardiovascular response fo l l ow ing submersion. 1. d imin i sh ing inputs f rom tr igeminal and upper respiratory tract receptors lead to breathing (3) and withdrawal o f both the cardiac vagal tone and sympathetic vasomotor tone. 2. inf lat ion o f the lungs, pu lmonary stretch receptor act iv i ty, and increased central inspiratory neuronal act iv i ty (4) reinforce the cardiovascular response. 3. without tr igeminal inh ib i t ion, arterial hypox ia and hypercapnia stimulate chemoreceptors, ma in ly in the carot id bodies, wh i ch causes hypervent i lat ion and also reinforces the cardiovascular response. 5. w i thdrawal o f t r igeminal input, increased central inspiratory dr ive, pu lmonary stretch receptor act iv ity, and st imulat ion o f chemoreceptors al l result in sympathetic st imulat ion o f cardiac P-adrenergic receptors leading to tachycardia. C V M , cardiac vagal motorneurons; S V N , central sympathetic vasomotor neurons; R C , respiratory centers; C S M , cardiac sympathetic motorneurons; +, st imulat ion; -, inh ib i t ion. Note that C S M may not be independent o f S V N . [Mod i f i ed f rom Da ly , 1984.] 6 the laboratory revealed a less dramatic vers ion o f the classic d ive response (Eisner, 1965; Jones et al. 1973; Fedak, 1986). Fo r example, dur ing forced submersions in harbour seals (Phoca vitulina), heart rate may fa l l to as l ow as 4 beats min" ' whereas heart rate on ly decreases to about 40 beats min" 1 dur ing trained dives (Eisner, 1965). In the last few decades, microprocessor-assisted moni tor ing o f animals d iv ing freely in their natural environment (Kooyman and Campbe l l , 1972; K o o y m a n et al. 1980; H i l l et al. 1987; Thompson and Fedak, 1993; Andrews et al. 1997) has demonstrated that the phys io log ica l and metabol ic responses to submergence are variable and depend upon the condit ions o f the dive. In nature, free-ranging dives typ ica l ly e l ic i t a relat ively modest d i v ing response. However , the response can also be profound when divers must remain submerged for extended periods. Fo r example, very l ow heart rates have been observed when seals were d iv ing under ice, resting at the bottom, or upon sudden reversal o f direct ion dur ing ascent (Eisner et al. 1989; Thompson and Fedak, 1993; Andrews et al. 1997). Routine diving behaviour D i v i n g behaviour is related to how d i v i ng mammals meet their eco log ica l needs (i.e. foraging, loca l explorat ion, predator escape, courtship) w i th in their phys io log ica l l imitat ions. A large proport ion o f the d iv ing phys io logy literature focuses on the abi l i ty to sustain except ional ly long dives; however, marine mammals rarely d ive near their breath-ho ld capacity in nature, and their routine dives are relat ively short ( Boyd and C roxa l l , 1996; But le r and Jones, 1997). Fo r instance, although Wedde l l seals (Leptonychotes weddellii) are capable o f d iv ing under Antarct ic ice for 82 m in (Caste l l in i et al. 1992), 7 less than 10 % of their free-ranging dives exceed 30 m in (Kooyman et al. 1980, 1983; Caste l l in i et al. 1992). S imi la r ly , harbour seals can tolerate over 20 m in o f submergence (Harr ison and Toml inson , 1960; Eguch i and Harvey, 1995), yet the major i ty o f their natural dives are on ly 2 to 6 m in (Fedak et al. 1988; Eguch i and Harvey, 1995; B o w e n et al. 1999). W h i l e the abi l i ty to remain underwater for extended periods is c lear ly benef ic ia l in order to make long exploratory dives, reach distant resources, or deal w i th unexpected pro longed submergence, long dives require more extreme phys io log ica l adjustments wh i ch often necessitate extended periods at the surface to return to the pre-dive state. The correlat ion between dive duration and recovery is related to the concept o f the aerobic dive l im i t ( A D L ) wh i ch is the threshold d ive duration beyond wh i ch a d iver must resort to s ignif icant anaerobic energy contr ibut ions to metabol ism (Kooyman et al. 1983; Kooyman , 1985). Du r i ng "short" dives w i th in the A D L , the d iver has suff ic ient oxygen reserves in the b lood and muscles to accommodate the total energy demand through aerobic pathways. F o l l ow i ng these aerobic dives, surface intervals pr imar i l y funct ion to reload oxygen stores and el iminate accumulated carbon d iox ide. On the other hand, " l o n g " dives beyond the A D L require an increasing rel iance on anaerobic metabo l i sm to meet peripheral energy demands wh i le hemoglob in-bound aerobic stores are conserved for the brain and heart. Such long dives often require extended recovery periods at the surface in order to restore b lood gases as we l l as to replenish g lyco ly t i c fuel reserves and phosphocreatine stores, process anaerobic byproducts, and restore acid-base equ i l i b r ium (Kooyman , 1985; But le r and Jones, 1997). 8 The A D L can be determined empi r i ca l l y by measur ing post-dive b lood lactate, the ma in metabol ite of anaerobiosis, or it can be computed ( c A D L ) f rom the quotient o f oxygen stores and the d iv ing metabol ic rate. B y moni tor ing post-dive b lood lactate concentrations, K o o y m a n et al. (1980) showed that the A D L o f freely d i v i ng Wedde l l seals we igh ing about 400 kg is approximately 26 m in . Beyond this l imit , there was a posi t ive correlat ion between d ive duration and recovery t ime at the surface. Interestingly, on ly about 3% of their free-ranging dives exceeded the A D L , thus indicat ing that the major i ty o f their natural dives are pr imar i l y fueled by aerobic metabo l i sm. A l though the A D L concept has on ly been exper imental ly ver i f ied in free-ranging Wedde l l seals, it is l i ke ly that other seals funct ion s imi lar ly in the w i l d . Regardless o f breath-hold capacity, an eff ic ient d ive strategy should m in im i ze t ime at the surface and max im i ze the proport ion of t ime spent submerged where seals accompl i sh the major i ty o f their eco log ica l tasks. In theory, a series o f short aerobic dives punctuated by br ie f surface intervals should result in more underwater t ime than a few long dives f o l l owed by extended recovery periods at the surface ( Kooyman et al . 1980). In fact, dur ing routine d iv ing in harbour seals, short dives are usual ly fo l l owed by br ief surface intervals o f less than 1 m in duration so that they ef fect ive ly spend 75 to 85 % of their t ime at sea submerged (Fedak et al. 1988). Such an abi l i ty to max im i ze percent d ive t ime depends not on ly on the abi l i ty to maintain aerobic-based metabo l i sm dur ing d i v i ng but also on the rate at wh i ch the oxygen stores are replenished and carbon d iox ide is e l iminated at the surface. A s sum ing that oxygen d i f fus ion rates in the tissues remain constant and that vent i lat ion-perfusion is we l l matched, rapid restoration o f b lood gases between dives is faci l i tated by h igh 9 breathing rates accompanied by post-dive tachycardia and increased c i rcu lat ion to the peripheral tissues (Andrews, 1999). The role of the dive response in routine diving W h i l e the cardiovascular responses to submergence are c lear ly necessary dur ing extended dives in order to conserve f inite oxygen stores for the hypoxia-sensi t ive brain and heart (Irv ing et al. 1941b; But le r and Jones, 1997), the role o f these responses dur ing routine d iv ing is not as obvious. Fo r instance, S ignore and Jones (1995) found that in muskrats, when bradycard ia was abol ished by atropine and/or vasoconstr ict ion was b locked by phentolamine, m a x i m u m underwater surv iva l t ime s ign i f i cant ly decreased, yet muskrats st i l l d ived vo luntar i ly for periods as long as their c A D L . These f indings suggest that either the d ive response is not necessary to sustain short aerobic dives or muskrats re ly on signi f icant anaerobic metabo l i sm dur ing routine d i v ing . O n the contrary, Murdaugh et al. (1961) showed that an atropinized harbour seal drowned after 3 m in o f submersion without bradycardia whereas untreated seals cou ld remain submerged for 15 to 20 m in . Furthermore, the cardiovascular responses to short dives are h igh ly var iable in seals. Jones et al. (1973) found that harbour seals dropped their heart rates to about 40 to 50 % of the pre-dive leve l upon voluntary submergence; however, heart rate dur ing feeding dives was var iable. Wh i l e one seal showed a s igni f icant ly greater bradycardia dur ing feeding dives than other routine dives, another seal actual ly exh ib i ted no change in heart rate i n 20 % of its feeding dives. A l l feeding dives lasted less than 40 sec, so it is l i ke ly that they d id not necessitate any cardiovascular adjustments to conserve oxygen. 10 However , the presence or absence o f bradycard ia dur ing these dives was random and not related to the duration of the d ive. S im i l a r l y , i n Wedde l l seals, there appears to be a negative correlat ion between mean heart rate and d ive duration for dives greater than 20 m in but no correlat ion exists for dives less than 20 m in (H i l l et al. 1987). There is also evidence that bradycardia dur ing short d ives is not necessari ly related to sw imm ing speed or muscular work in seals ( Kooyman and Campbe l l , 1972; Fedak, 1986; W i l l i a m s et al. 1991). Therefore, the var iab i l i ty in the degree of cardiovascular adjustment dur ing routine d iv ing cannot sole ly be expla ined by the d i v i ng metabol ic rate. Purpose of this study Because it is unclear whether the cardiovascular components o f the d iv ing response are necessary dur ing routine d i v i ng and, g iven that harbour seals meet their eco log ica l needs through repetit ive short aerobic dives, I was interested in the funct ional role, i f any, o f the d ive response dur ing these short dives. A l so , g iven the current understanding of the control systems under ly ing the cardiac d ive response, I was interested in the inf luence o f the two branches o f the autonomic nervous system on heart rate dur ing voluntary d iv ing in seals. In the present study, I used pharmaco log ica l b lockers to inhib i t the d ive response in vo luntar i ly d iv ing seals. Spec i f i ca l ly , I used methoctramine to b lock parasympathetic inputs to cardiac muscar in ic receptors i n order to reduce d iv ing bradycardia. Prazos in was used to reduce d iv ing vasoconstr ict ion by b l ock ing sympathetic inputs to a -adrenergic receptors i n the b lood vessels. Me top ro l o l was used to reduce post-dive 11 tachycardia by b lock ing sympathetic inputs to P-adrenergic receptors on the heart dur ing surface intervals. In order to assess the effectiveness o f the pharmaco log ica l b lockade, I monitored heart rate dur ing routine d iv ing . Based on several assumptions, I also used heart rate as an indicator o f the other cardiovascular and metabol ic adjustments occurr ing dur ing d iv ing . Fo r example, d i v ing bradycardia provides indirect evidence for peripheral vasoconstr ict ion and redistr ibut ion of b lood f low, and post-dive tachycardia reflects an increase in cardiac output and c i rculat ion to the periphery. A s sum ing that heart rate is c losely coupled to cardiac output and that stroke vo lume remains constant (or changes systematical ly), changes in heart rate must be accompanied by changes in peripheral vasomotor tone in order to maintain fa ir ly constant arterial b lood pressure. Wh i l e there is some conf l i c t ing evidence regarding stroke vo lume dur ing d iv ing (E isner et al. 1964; Ponganis et al. 1990), numerous investigators have ver i f ied that bradycardia is accompanied by peripheral vasoconstr ict ion (Eisner et al. 1966; K e r e m and Eisner, 1973; Zapo l etal. 1979). Furthermore, in order to assess the autonomic nervous contro l o f heart rate dur ing voluntary d i v i ng in harbour seals, I examined the effects o f pharmaco log ica l b lockade on mean dive and surface interval heart rate. F ina l l y , in order to investigate the role o f the cardiovascular d ive response dur ing voluntary d iv ing , I monitored d ive behaviour wh i l e the d ive response was pharmaco log ica l ly inhib i ted. The research presented in this chapter tests the f o l l ow ing hypotheses: 1) D i v i n g bradycard ia is pr imar i l y modulated by the parasympathetic nervous system. 12 2) Post-dive tachycardia is primarily modulated by the sympathetic nervous system. 3) The cardiovascular components of the dive response, i.e. diving bradycardia and vasoconstriction and post-dive tachycardia, are necessary to maintain short routine dives and surface intervals as well as a high percent dive time in harbour seals. 13 Materials and Methods Seals The research presented in this thesis was conducted on two adult (4 yr old) female and three juven i l e (2 yr old) male harbour seals (Phoca vitulina) ranging in mass f rom 40 to 60 kg. The seals were wi ld-caught at one week to one month o f age f r om a rookery o f f the coast o f B r i t i sh Co lumb ia , under a l icence granted by the Department o f Fisher ies and Oceans. They were housed in the Large W i l d M a m m a l Un i t at the Un ivers i ty o f B r i t i sh Co lumb ia . The i r hous ing consisted o f unheated, f low-through freshwater pools surrounded by large wooden decks for haul-out space. The seals were maintained on a dai ly diet o f herr ing supplemented w i th a v i tamin (Mazu r i V i t a -Zu M a m m a l Tablet, P M I Nutr i t ion International) inserted under the f ish opercu lum. Instrumentation Each seal was anaesthetized us ing 5 % isof lurane (Janssen, Toronto, O N , Canada; induct ion by mask), and after endotracheal intubation, the seal was mainta ined on 1-2 % isoflurane and 98-99 % oxygen. T w o electrocardiogram ( E C G ) electrodes were placed on the dorsal surface of the seal, one above the shoulder blade and one above the pe lv is on opposite sides o f the an imal (F ig . 1.3). Fu r was shaved f rom the areas where inc is ions were to be made, and the exposed sk in was cleaned w i th 70 % alcoho l and an iodine-based solut ion (po lyv iny lpyrro l id ine- iod ine complex 10 %, Iodovet, Roug ier Pharma, M i r abe l , Q C , Canada). Th in-wi re E C G electrodes (28G, shielded, Cooner W i r e Company, Chatsworth, C A , U S A ) were tunneled subcutaneously 9 c m f rom each insert ion site (one cran ia l ly and one caudal ly) w i th a 14G hypodermic needle. E a ch E C G 14 Figure 1.3. Two female harbour seals instrumented with ECG electrodes and one with the data logger. Two ECG electrodes can be seen on the seal on the right, one above the pelvis and one above the shoulder blade. The data logger is attached to the seal by buckles glued to the fur and connected to the ECG electrodes via underwater connectors. The seals have different shapes of neoprene patches on their heads for identification on videotape. They are resting at the surface of the dive tank on top of the false bottom. A neoprene pocket that was used to cover the end of a catheter can be seen on the seal with the data logger. 15 electrode was connected to an external ized waterproof lead and an underwater connector (US I square min i conn, Underwater Systems, Stanton, C A , U S A ) that was glued to a neoprene base w i th 5-min epoxy (Devcon, Ack lands , Vancouver , B C , Canada) (F ig . 1.4). A f te r electrode insert ion, the ampl i f ied E C G was d isp layed on an osc i l loscope to ver i fy that the electrode placement resulted in a clear E C G s ignal . The underwater connector/neoprene base was then glued to the seal 's fur us ing cyanoacrylate adhesive (ZapAGap , R i c hmond R C Supp ly L td . , Del ta, B C , Canada). The electrode insert ion sites were bathed w i th 1 m l Bup ivaca ine hydrochlor ide 25 % (Abbot Laborator ies L td . , Mont rea l , Q C , Canada) to provide post-operative analgesia. A co lored neoprene patch was glued (ZapAGap ) to the seal's fur on its head (F ig. 1.3), for ident i f icat ion on videotape. T w o buckles were glued to the seal 's fur mid-way between the two electrodes (F ig. 1.3) us ing 10-min epoxy (Evercoat Ten Set Epoxy , F ibreglass-Evercoat Co . Inc., C inc innat i , O H ) for the attachment o f an E C G recording instrument. Seals were a l lowed at least 48 hours to recover before experiments. A l l procedures were approved by the A n i m a l Care Commit tee at the Un ivers i ty of Br i t i sh Co lumb ia . The electrodes, neoprene patches, and buckles were either removed upon complet ion o f the experiments by shaving the fur or they fe l l o f f when the seals molted in the late summer or early fa l l . Preliminary drug testing Pre l im inary experiments w i th three seals were done to establ ish the doses o f the pharmacolog ica l b lockers to be used in the d iv ing experiments as we l l as the t ime periods during wh i ch the drugs were most effect ive. Spec i f i c pharmaco log ica l agonists were used 16 Stainless steel conductor Entrance to connector~~ Female underwater connector Water expulsion hole -E p o x y Neoprene base Bioe lectr ic wire, outer shield 1 —j • i -V-W i r e mesh - H o l e in neoprene - T y g o n tubing Solder joint F igure 1.4. D iag ram of E C G electrode assembly. A bioelectr ic wi re (28G) wi th the stainless steel conductor exposed at the end was tunneled in between the seal 's sk in and blubber layer. The insert ion site was exposed in a neoprene hole so that it cou ld be f lushed w i th water and monitored for infect ion. The wire was secured in place by a piece o f tygon tubing just outside of the insertion site and by a wire mesh over lay ing the neoprene hole. The E C G electrode was soldered to the lead of a female underwater connector. The female connector, solder jo int , tygon tubing and wire mesh were secured to a neoprene base and waterproofed w i th a shell o f 5-min epoxy. The neoprene base was glued to the seal's fur. The lead of the data logger was jo ined to the female connector in order to record heart rate dur ing d iv ing . 17 to induce the cardiovascular responses seen dur ing d iv ing in order to assess the doses o f the blockers and effectiveness of b lockade. Under anaesthesia (see above protocol), fur was shaved f rom a smal l area on the lower back where a catheter was to be inserted, and the exposed sk in was c leaned w i th 70 % a lcoho l and an iodine-based solut ion (po lyv iny lpyrro l id ine- iod ine comp lex 10 %, Iodovet). U s i n g a 14G angiocath ( B C Stevens, Vancouver , B C , Canada), approx imate ly 30 c m of P E Micro-renathane tubing (0.050 O.D. x 0.025 I D . , Braintree Sc ient i f i c Inc., Braintree, M A , U S A ) was inserted into the extradural intravertebral ve in , leav ing about 2 c m of tubing outside o f the epidermis. The blunted needle o f a 2 1 G "butter f ly" (w inged needle in fus ion set, Ven isystems, Abbot t Laborator ies Inc., Abbot t Park, IL , U S A ) was inserted in the external ized P E tubing so that the internal tubing was cont inuous w i th the external butterf ly (needle plus butterf ly tubing). The edge o f the P E tubing was sealed around the butterf ly needle us ing tissue adhesive ( 3 M Vetbond, N o . 1469, 3 M A n i m a l Care Products, St. Pau l , M N , U S A ) . A mult isampler rubber hub was attached to the Luer lock end o f the butterf ly tubing. The butterf ly wings were then sewn to a neoprene base that was glued to the seal's fur (ZapAGap ) , and the butterf ly tubing and rubber hub were secured on the seal in a neoprene pocket (F ig . 1.3). A d iagrammatic representation o f the catheter assembly is shown in F i g . 1.5. The catheter was f i l l ed w i th approximately 0.9 m l of heparanized P V P (Po l yv iny l pyro l id ine, S i gma-A l d r i ch Canada Ltd. , 1 g P V P : 12 m l saline, heparin 20 u ml" 1 ) . The catheter was per iod ica l ly f lushed w i th sal ine and ref i l led w i th P V P every one to f ive days. Th is catheter enabled me to easi ly inject the pharmaco log ica l agonists into the extradural ve in and per form pre l iminary drug tests over a per iod o f one to s ix weeks. 18 ck end e base F igure 1.5. D i ag ram of catheter assembly. P E Micro-renathane tubing was inserted into the seal 's extradural intravertebral ve in. The blunted needle o f a w inged needle in fus ion set or "butter f ly" was inserted into the external ized P E tubing. The needle and tubing were secured just above the insertion site in the fo ld o f the neoprene base. The wings o f the butterf ly were sewn to the neoprene base. A mul t i sampler rubber hub was attached to the Lue r l o ck end o f the butterfly tubing, and the entire catheter was f i l l ed w i th hepar in ized P V P ( Po l yv i ny l pyro l id ine). The neoprene base was g lued to the seal 's fur, and the rubber hub and butterf ly tubing were secured in a pocket by g lu ing another strip o f neoprene on top o f the base. 19 Before drug testing, each seal was restricted to a dry enclosure l ined w i th a copper grounding plate to m in im i ze electr ical noise interfer ing w i th the E C G signal. A syr inge attached to the rubber hub was used to draw out the P V P f rom the catheter, and the Luer l ock end o f the butterfly was then attached to a sal ine-f i l led I V l ine ( 1 . 9 m Interl ink System, Baxter Corp. , Toronto, O N , Canada). The catheter was kept patent by cont inuously f lush ing w i th saline ( N a C l 0.9 %, 1 m l min" 1 ) , and the TV l ine served as an extension o f the catheter that enabled me to inject drugs into the extradural ve in w i th m in ima l disturbance to the seals. The seal 's E C G electrodes were connected to a G o u l d Un iversa l ampl i f ier (model 11412301, G o u l d Inc., C leve land , O H , U S A ) to ampl i fy and f i l ter the E C G signal. The signal was then sampled at 222 H z and recorded on a Pent ium computer us ing W indaq (version 1.67, D A T A Q Instruments, Inc., A k r o n , O H , U S A ) . Heart rate was monitored after intravenous (i.v.) inject ion o f the P-adrenergic agonist isoproterenol hydrochlor ide (S i gma-A ld r i ch Canada Ltd. , in saline), or the a -adrenergic agonist 1-phenylephrine hydroch lor ide (S igma-A ld r i ch Canada Ltd. , in saline), or 0.9 % saline alone (control). Based on literature values (Signore and Jones, 1995; Ho f fman 2001), several conservat ive doses of each agonist were in i t ia l ly tested, and the f ina l dose was selected for the desired effect on heart rate (i.e. extreme tachycardia or bradycardia). Ef fects o f the agonists f o l l ow ing administrat ion o f the appropriate antagonist were then determined; antagonists were tested on separate days. Isoproterenol (0.01 j ig kg" 1 i.v.) was used to induce tachycardia and therefore assess the ef f i cacy o f the Pi-adrenergic b locker metopro lo l (Lopressor SR , 24 hr sustained release formulat ion, Novart i s Pharmaceuticals Canada Inc., Canada). Phenylephr ine (0.06 p:g kg-" 1 i.v.) was used to induce both bradycardia and vasoconstr ict ion in order to assess the 20 M2-muscarinic b locker methoctramine (S igma-A ld r i ch Canada Ltd.) and the a i -adrenergic b locker prazos in (Pf izer Inc., Canada), respectively. In one seal, several different doses o f each b locker were tested for b lockade of the agonist- induced response (F ig . 1.6) and for unwanted side effects. The speci f ic doses to be used in d iv ing experiments were ult imately chosen based on the m a x i m u m drug dose causing the desired b lockade (as indicated by heart rate analysis) without any obvious side effects such as tachycardia or lethargy (Table 1.1). These doses were then conf i rmed in two other seals. In al l three seals, the selected doses were tested for b lockade o f the agonist- induced responses at different t ime intervals after administrat ion o f the antagonist (15 m in to 2 hr intervals for up to 6 hr post dose). Based on these results, I estimated the t ime frame dur ing wh i ch d i v i ng experiments wou ld be conducted (Table 1.1). Diving experiments D i v i n g experiments w i th f ive harbour seals were conducted in a 4.5 m diameter x 1 1 m deep freshwater tank (F ig . 1.7) w i th f low-through water f i l tered by a gravel- f i l led canister f i lter. Water temperature ranged f rom 12 to 16°C. The surface o f the d ive tank was covered so that seals were restricted to surfac ing f rom dives in a 2.4 m area and cou ld haul out on the deck space (F ig . 1.7). A "false bo t tom" constructed f rom P V C p ip ing and orange snowfenc ing (F ig . 1.3) rested on the bottom o f the tank. Seals were a l lowed to accl imate to the d ive tank for a per iod of one to two months pr ior to d i v i ng experiments. Du r i ng the course o f d i v ing experiments, two seals were he ld in the d ive tank and experiments were run on one animal at a t ime. 21 180 150 120 90 60 30 0 180 150 H E « 120 90 " 60 H 30 0 180 150 H 120 H 90 60 30 0 P h e n y l e p h r i n e ^ no m u s c a r i n i c ( m e t h o c t r a m i n e ) 0 .02 m g / k g m u s c a r i n i c 0 .12 m g / k g m u s c a r i n i c 0 .23 m g / k g m u s c a r i n i c * * 0 .30 m g / k g m u s c a r i n i c P h e n y l e p h r i n e no a l p h a ( p r a z o s i n ) 0 .14 m g / k g a l p h a 0 .24 m g / k g a l p h a * * I s o p r o t e r e n o l ^ no beta ( m e t o p r o l o l ) 2 m g / k g beta 4 m g / k g be ta** 6 m g / k g beta 60 120 180 240 300 T i m e (sec) 360 420 480 Figure 1.6. E f fect o f intravenous inject ion o f speci f ic agonists on heart rate after different doses of the appropriate antagonist in one seal. Ea ch data point represents the instantaneous heart rate averaged over a 10 sec interval . A r r ows denote the inject ion o f agonists. ** indicates the f ina l doses chosen for d i v i ng experiments based on the desired b lockade and side effects caused by higher doses. A : 1-phenylephrine hydrochlor ide (0.06 |ig kg" 1) alone and after the muscar in ic antagonist methoctramine (subcutaneous). B: phenylephr ine (0.06 (j,g kg" 1) alone and after the a\-adrenergic antagonist prazos in (oral). C: isoproterenol hydrochlor ide (0.01 p:g kg" 1) alone and after the Pi-adrenergic antagonist metopro lo l (oral). Note: not a l l doses tested are shown. 22 cn <N CD 1/3 3 O OH O O CD Ul ti > ii X C T 3 C/3 3 CU M u o cS O '5b o "o o cs cs X D . <D X 4) -u* o cS O 3 CD c/3 CD '0, .1 s X < 2 £ & o OH CD < s cS O B a ' x .2 2 & 3 CD w> oo J ^ 1 3 O X Q U <D C o, rt 3 < X X >/n (N I 3 3 o CO X m I u X Ui X + o l u X p cn u o o un I CN X p cn vn CS u O <D 3 cS •— -u> o o X CD O "o u a, o CD c/3 O % u< a. T3 CD O 3 • a c s o (50 CS JS <D CS X CU OO H - H 3 W - CU oo "3 CU CS 3, M CJ T3 3 C/3 3 cu <U O TD cS o X H i X tj CU <U 3 CU cu X o 3 cS 3 CU 6 u cu OH X (D -o CU o 3 TJ 3 3 O 60 cS CU •3 cS o O 2 60 CU -a >> 3 cS O •o T 5 CJ CU 3 O cj CU rt 60 « u - 3 .E — 60 > S =3 cj ~ CS CU CU 00 u 3 oo 'S O 60 CS CJ (U cu 60 <u .E -a -S is 0 0 oo c3 _ _ 3 E 60 3 >^ -5 <U « g O 3 D • s - a "5 .2 1 o cs s 2 x OO Q ' 2 x ° % 60 > cS bO cj 3 oC 'u <u ~ a. cu oo c 60 § .E ofc: 3 <^  cu * J CS u 11 oo • - H <D X O X* 1-X O, CS Q H •3 CS ' 3 a CS O 0 0 CU U CJ co C cS cS -a "cS 3 cS u 3 O '> cS X 3 60 u 3 s > 5 ol .5 "g 0 0 x .S2 <u| cs o l S CS , (U <u - a 3 o (D 3 CS 3 CJ X 3 X cj cS <ii 3 O 3 s o CS t5 cu cS w 2 SP T3 CU CU X X H H • a §.§ 8 2 S 3 o -0 co a cu X U f_. o r3 cS CS u I CU • a x cS o CU CU 3 E 0 0 2; cu x x cS cS CU X 0 0 3 JB •3 cS <U 3 4-1 cs CU cu Figure 1.7. Freshwater d i v i ng tank (4.5 x 11 m). In the picture, a seal is be ing carr ied to the top o f the d ive tank in a kennel by the crane. The d iagram shows the surface o f the d ive tank. The seals were restricted to surfacing f r om dives in a 2.4 m 2 area and cou ld haul out on the surrounding deck space. A v ideo camera was suspended above the breathing hole for mon i tor ing of voluntary d iv ing behaviour. 24 Heart rate was recorded dur ing d iv ing using a custom-bui l t data logger that consisted of an E C G recorder based on a computer board (Mode l 8; Onset Computer Corp., Bourne, M A , U S A ) interfaced to a compact-f lash memory expans ion board (model C F 8 ; Per ipheral Issues, Mashpee, M A , U S A ) (for details, see Andrews , 1998; Southwood et al. 1999). The data logger was programmed to sample the ampl i f i ed E C G signal at 50 H z and, w i th a memory o f 15 Mbytes , recorded heart rate for 84 h. Be fore d iv ing sessions, seals were brought to the surface of the d ive tank by ra is ing the false bottom on a manual pu l ley system. A t the surface, the data logger was attached to the buckles on the seal and connected to the E C G electrodes v ia underwater connectors (F ig . 1.3). A l l f ive seals received each o f the f o l l ow ing treatments (once) in randomized order: 1) subcutaneous (s.c.) inject ion of the M 2 -se lec t ive muscar in ic antagonist methoctramine (single dose, 0.23 mg kg" 1) 2) oral administrat ion (in a f ish) of the a i-adrenerg ic antagonist prazos in (three doses at 6 hr intervals, 0.24 mg kg" 1 each) 3) oral administrat ion o f the Pi-adrenergic antagonist metopro lo l (two doses at 12 hr intervals, 4 mg kg" 1 each) 4) a combinat ion o f methoctramine (0.23 mg kg"' s.c.) and prazos in (0.24 m g kg" 1 oral) 5) a combinat ion o f methoctramine (0.23 m g kg"' s.c.) and metopro lo l (4 m g kg"' oral) 6) subcutaneous inject ion o f 0.9 % saline (control for a l l methoctramine inject ions) 7) oral administrat ion o f a f i sh without p i l l s (control for prazos in and metopro lo l) . 25 Treatments were done on separate days w i th at least 24 hours between drugs (48 hours f o l l ow ing metoprolo l) . Injections were g iven after the data logger was attached wh i le seals were restricted to a corner on the deck. Ora l p i l l s (prazosin, metopro lo l , or contro l f ish) were g iven on the evening before and the morn ing o f d i v ing experiments (the data logger was already attached). A f te r the seals were released and the false bottom was lowered or after oral p i l l s were administered, voluntary d i v i ng behaviour was recorded us ing a v ideo camera (Lorex, Strategic V i s t a Corp. , Ma r kham, O N , Canada) suspended over the breathing hole i n wh i ch the seals surfaced (F ig . 1.7). Analysis and statistics Data were downloaded to a Pent ium computer f rom the data logger, and us ing the program Acqknow ledge (version 3.01, B I O P A C Systems, Inc., Santa Barbara, C A , U S A ) , R-R intervals were calculated after detecting the Q R S complexes o f the E C G . Instantaneous heart rates were then determined by convert ing inter-beat intervals to beats min" 1 , and mean heart rates for dives and surface intervals were calculated by averaging these values. The first and last 10 sec o f each d ive and the last 3 sec o f each surface interval were exc luded f rom the calculat ion o f means to reduce var iab i l i ty in heart rate caused by the in i t ia l bradycardia be low the heart rate establ ished dur ing the rest o f the dive, cardiac acceleration before surfac ing (anticipatory tachycardia), and cardiac deceleration before submergence (anticipatory bradycardia). Therefore, on ly dives greater than 20 sec and surface intervals greater than 3 sec were analyzed. Fo r each treatment, d iv ing behaviour (i.e. d ive and surface interval durations) was analyzed f rom the videotapes for one hour dur ing wh i ch the b lockade was max ima l . Th i s hour of analysis 26 was in i t ia l ly estimated dur ing pre l iminary drug testing and ul t imately determined by analysis o f heart rate dur ing d i v i ng sessions. Fo r methoctramine-treated groups, d ive behaviour was analyzed approximately 1-2 hr after injections; for prazosin, 1.25-2.25 hr after the third oral dose; and for metopro lo l , 4-5 hr after the second oral dose (Table 1.1). Contro ls for each group were analyzed to match these t ime periods. D i v i n g heart rate data reported in the text inc lude on ly values f r om these t ime periods as we l l . A l l values for heart rate and d ive behaviour g iven in the text represent grand means + S . E .M . (N=5) for each treatment. The means for each group were compared us ing one-way repeated measures analysis o f variance ( A N O V A ) and Tukey mult ip le compar ison tests. Di f ferences were considered signif icant when P<0.05. A l l statistical analyses were performed using SigmaStat software (vers ion 2.0, Jandel Sc ient i f i c , San Rafae l , C A , U S A ) . 27 Results Preliminary drug tests Pre l im inary drug testing was necessary to establ ish the doses of the b lockers used in d i v ing experiments (F ig . 1.6, see Mater ia ls and Methods) and also to determine the t ime frame in wh i ch those doses were most effective. The a-adrenergic agonist phenylephr ine was used to assess the M 2 muscar in ic antagonist methoctramine and the OC|-adrenergic antagonist prazos in. Phenylephr ine (0.06 pig kg" 1 i.v.) alone reduced heart mean rate by approximate ly 80 % (F ig . 1.8A,B) and typ ica l ly to instantaneous heart rates as l ow as 9 beats min" 1 regardless o f the in i t ia l rate. Th is vagal ly mediated bradycardia is a barostatic ref lex due to phenylephr ine- induced vasoconstr ict ion ra is ing arterial b lood pressure. Subcutaneous inject ion o f methoctramine (0.23 mg kg" 1) b locked this response so that phenylephrine decreased heart rate on ly by about 16 % 0.5 hr after inject ion (F ig . 1.8A). A t this dose, partial b lockade o f the phenyleprhine- induced bradycard ia was apparent approximate ly 15 m in fo l l ow ing inject ion, peaking at around 30 to 45 m in and gradual ly tapering o f f over 2.5 hr (Table 1.1). H igher doses o f methoctramine caused a substantial degree o f tachycardia and were therefore not selected for d i v i ng experiments (F ig . 1.6A). F o l l ow i ng oral administrat ion o f prazos in (0.24 mg kg" 1 ), phenylephr ine on ly caused a sl ight decrease i n heart rate (29 % decrease 1.25 hr after oral dose) compared to phenylephrine alone (F ig . 1.8B), and this was l i ke ly due to (Xi-blockade o f vasoconstr ict ion. A t this dose, a partial b lockade was apparent approximately 45 m i n to 1 hr after the oral dose was g iven and gradual ly tapered o f f over 3 hr (Table 1.1). W h i l e a higher dose o f prazos in may have inh ib i ted the phenylephr ine- induced bradycard ia by a 28 1 2 0 -\ 9 0 H Phenylephrine^/ no muscarinic (methoctramine) 0.50 hr post muscarinic 1.75 hr post muscarinic Phenylephrine^ no alpha (prazosin) .25 hr post alpha 3.50 hr post alpha Isoproterenol \j/ C —•— no beta (metoprolol)^' —»— 3.50 hr post beta —"— 5.25 hr post beta - 1 2 0 - 6 0 6 0 1 2 0 Time (sec) 1 8 0 2 4 0 3 0 0 Figure 1.8. E f fect o f intravenous inject ion o f speci f ic agonists on heart rate before and after b lockade w i th the appropriate antagonist i n one seal. Ea ch data point represents the mean instantaneous heart rate for a 10 sec interval. A r r ows denote the inject ion o f agonists. A : 1-phenylephrine hydrochlor ide (0.06 jug kg" 1) alone and after the muscar in ic antagonist methoctramine (0.23 m g kg" 1 s . c ) . B: phenylephrine (0.06 f ig kg" 1) alone and after the oti-adrenergic antagonist prazos in (0.24 mg kg" 1 oral). C: isoproterenol hydrochlor ide (0.01 p:g kg" 1) alone and after the pi-adrenergic antagonist metopro lo l (4 mg kg" 1 oral). Cont ro l sal ine injections caused no s ignif icant effect on heart rate. 29 greater degree, a 100 % blockade was un l i ke ly because the oral drug was formulated for the treatment of chronic hypertension in humans. A l so , g iven the potential adverse effects o f overdos ing (e.g. hypotension, loss of consciousness, b lockade o f 0C2 receptors), I was hesitant to test higher doses when the selected dose was already we l l above the dose range prescr ibed for humans (Ho f fman, 2001). F ina l l y , the P-adrenergic agonist isoproterenol was used to determine the dose and effectiveness o f the Pi-adrenergic antagonist metopro lo l . Isoproterenol (0.01 p:g kg" 1 i.v.) alone caused instantaneous heart rate to increase to 180 beats min" 1 , so mean heart rate increased by approximate ly 127 % (F ig . 1.6C, 1.8C). A f te r P-blockade w i th metopro lo l (4 m g kg" 1 oral), isoproterenol increased heart rate on ly by 53 % 3.5 hr after the oral dose (F ig. 1.8C). A suff ic ient b lockade was evident about 3 hr f o l l ow ing oral administrat ion and cont inued unt i l at least 6 hr post dose (Table 1.1). A higher dose o f metopro lo l caused lethargy and was therefore not selected for d iv ing experiments (F ig . 1.6C). Heart rate during diving The effects o f the pharmaco log ica l b lockers on d ive and post-dive surface interval heart rate are presented i n F i g . 1.9, and d i v i ng heart rate prof i les are shown in F i g . 1.10. In the contro l groups, mean d ive heart rate ranged f rom 47+3 beats min" 1 to 49+4 beats -min" 1 , and mean surface interval heart rate ranged f rom 133±3 beats min" 1 to 138±4 beats min" 1 (F ig . 1.9). Du r i ng a typica l d ive bout in control seals, heart rate dropped immediate ly upon d iv ing to about 17 % o f the pre-dive surface rate w i th in 5 to 10 sec o f the dive. Heart rate then increased to about 35 % of the pre-dive rate w i th in 30 to 40 sec o f the in i t iat ion o f the d ive and remained at this level unt i l about 10 to 20 sec before 30 180 150 120 90 60 30 Surface D i v e muscarinic alpha beta musc+alpha musc+beta 180 150 120 H 90 H 60 -\ 30 B Surface D i v e muscarinic alpha beta musc+alpha musc+beta Figure 1.9. Mean heart rate (±S.E.M.) during post-dive surface intervals (A) and dives (B) in control and pharmacologically blocked harbour seals (N=5). Heart rate data from control groups were combined to give the mean heart rates indicated by the horizontal lines, although statistical analyses were performed on controls for each data set. * indicates values significantly different from the control group within dive states (P<0.05). For each treatment, the dive heart rate was significantly lower than the surface interval heart rate. Alpha: seals treated with oc-adrenergic blocker; beta: P-adrenergic blocker; musc+alpha: muscarinic and a-adrenergic blockers combined; musc+beta: muscarinic and P-adrenergic blockers combined. 31 180 150 120 90 60 30 Xi II 180 150 120 90 60 30 oral controls combined alpha blocker beta blocker — i 1 1 1 - i 1 1 1 1 1 - / / • -20 -10 0 10 20 30 40 80 90 100 110 120 130 140 150 ~7 / V A B -H--20 10 0 10 20 30 " 4 0 8 0 ~ injected controls combined, muscarinic blocker musc+alpha blockers musc+beta blockers 90 100 10 120 130 140 150 Time (sec) F igure 1.10. Heart rate prof i les before, dur ing and after voluntary dives i n a-adrenergic-and P-adrenergic-blocked harbour seals (A ) and in muscar inic-, muscar in ic p lus a -adrenergic- and muscar in ic p lus P-adrenergic-blocked seals (B) . Cont ro l data for the two oral drugs (A) and for the three injected groups (B) were combined, although statistical analyses were performed on controls for each data set. A r r ows denote the beg inn ing and end o f the dive. Each data point represents the mean heart rate for the preceding 5 sec interval. F o r each treatment, mean heart rates dur ing two dives (approximately 120 sec) were averaged for each an imal . Data f r om a l l seals (N=5) were then comb ined to give the means (+S.E.M) i l lustrated. The data were normal i zed so that dives o f different lengths ended at the same t ime. 32 surfacing when it increased rap id ly so that pre-dive levels were reached upon or w i th in 5 sec o f surfac ing (F ig . 1.10). In the a - and (3-adrenergic-blocked groups, the heart rate prof i les fo l l owed a s imi lar pattern as in the contro l group ( init ia l drop, sl ight increase to a steady leve l , pre-surfacing increase to surface levels). In the three muscar in ic- injected groups, heart rate decreased to a lesser degree so that the extreme in i t ia l drop and steep increase 10 to 20 sec before surfacing were not pronounced (F ig . 1.10). In the muscar in ic-b locked group, mean d ive heart rate was s igni f icant ly higher than in the control group (110±3 beats min" 1 versus 49±4 beats min" 1) wh i le mean surface heart rate was not s igni f icant ly different f r om the contro l group (137+3 beats min" 1 versus 138±4 beats min" 1) (F ig . 1.9). D i v e heart rate in a-adrenergic-b locked animals was s igni f icant ly higher than in control seals (64±3 beats min" 1 versus 47±3 beats min" 1 ) , but surface rates were not s igni f icant ly different (121+5 beats min" 1 versus 133±3 beats min-" 1) (F ig . 1.9). A f te r (3-adrenergic b lockade, d ive heart rate was not s igni f icant ly different f r om the contro l group (42±3 beats min" 1 versus 48±3 beats min" 1 ) , but surface heart rate was s igni f icant ly lower (98+1 beats min" 1 versus 137±3 beats min" 1) (F ig . 1.9). In the muscar in ic- plus a-adrenergic-blocked group, d ive heart rate was s igni f icant ly higher than in control seals (109+3 beats min" 1 versus 49±4 beats min" 1 ) , but surface rates were not s igni f icant ly different (136±3 beats min" 1 versus 133±3 beats min" 1 ) (F ig . 1.9). D i v e heart rate after muscar in ic plus p-adrenergic b lockade was s igni f icant ly higher than in the contro l (88±1 beats min" 1 versus 49+4 beats min" 1 ) , and surface heart rate was also s igni f icant ly lower (111+2 beats min" 1 versus 138±4 beats min" 1 ) (F ig . 1.9). In each treatment condi t ion, the dive heart rate was s igni f icant ly lower than the surface heart rate. 33 A n interesting f ind ing was differences in the in i t ia l d ive heart rate between the two adult females and the three juven i le males. In the females, heart rate dropped immediate ly upon d iv ing to about 13 % of their pre-dive rate and then increased over the next 20 sec to a steady leve l at about 3 1 % of the surface rate. However , the juven i le males d id not d isp lay an extreme in i t ia l bradycardia but rather decreased their heart rate immediate ly to a steady leve l at approximately 39 % of their pre-dive rate (F ig 1.11). The mean heart rates (averaged over 5 sec periods) for the first 15 sec of dives were s igni f icant ly lower in females than in males; however, mean heart rates for the remainder of dives were not different (F ig . 1.11). The more pronounced in i t ia l bradycardia i n females was apparent in the control , a - and (3-blocked groups. Diving behaviour F i g . 1.12 shows the effect of pharmaco log ica l b lockade on mean dive and post-d ive surface interval duration. Ef fects on the distr ibutions of d ive and surface interval durations are shown in F i g . 1.13. Average d ive duration in contro l seals ranged f rom 2.61+0.32 m in to 2.83+0.49 m in , and average surface interval duration ranged f r om 0.40±0.04 m in to 0.43±0.04 m in (F ig. 1.12). None o f the treatments had any signif icant effect on mean dive durat ion (2.34±0.47 m in for the muscar in ic group; 2.40+0.27 m in for the a-adrenergic group; 2.80±0.39 m in for the P-adrenergic group; 2.67±0.45 m in for the muscar in ic plus a-adrenergic group; 2.6710.47 m in for the muscar in ic plus P-adrenergic group) (F ig . 1.12). In fact, seals made voluntary dives as long as 8.12 m i n without a surface tachycardia, 6.72 m in when bradycardia was b locked, 4.72 m in when vasoconstr ict ion was b locked, and 4.93 m in when bradycardia and vasoconstr ict ion were 34 -20 -10 0 10 20 30 40 80 90 100 110 120 130 140 150 T ime (sec) F igure 1.11. Heart rate prof i les before, dur ing and after control dives for two adult female versus three juven i le male harbour seals. A r r ows denote the beginn ing and end o f the dive. Each data point represents the mean instantaneous heart rate for the preceding 5 s interval . * indicates values that are s ign i f i cant ly different between males and females (P<0.05). Fo r each group, mean heart rates dur ing s ix dives (approximately 120 s) were averaged for each seal. Data were then comb ined to g ive the means (+S.E.M) i l lustrated. The data were normal i zed so that dives of different lengths ended at the same time. 35 1.00 0.75 0.50 0.25 H .2 0.00 muscarinic alpha beta musc+alpha musc+beta control E O O blocked B muscarinic alpha beta musc+alpha musc+beta Figure 1.12. Mean surface interval (A) and dive (B) durations (+S.E.M.) in control and pharmacologically blocked harbour seals (N=5). Note that the time scales for each graph are different. Alpha: seals treated with a-adrenergic blocker; beta: P-adrenergic blocker; musc+alpha: muscarinic and a-adrenergic blockers combined; musc+beta: muscarinic and P-adrenergic blockers combined. There were no significant differences between treatment groups and the controls. 36 Dives Surface intervals 50 40 30 20 10 0 control m uscarin ic ? ?° ^  ^  ^  <r <r ^  50 40 30 20 10 0 5 0 40 30 20 10 0 control musc + alpha control musc + beta Time (sec) 70 60 50 40 30 20 10 0 s° 0? , ^ >° ? ,fe* b * s N -V s IX s S N ^ so ^ ^ ^ & ^ N S ' -|S ">V * V S V 1 No ^ ^ „J5 ^ £ g N N' n>' ^ ' *>' SS' 1 s * ^ ^ ^ «,« bo teo N s ' a v * , v t>' b v 1 >? -v* >° to* S S -V s -vN 0 S b V ^ Time (sec) Figure 1.13. Dis t r ibut ion of d ive durations and distr ibut ion o f surface interval durations in control and pharmaco log ica l ly b locked harbour seals (N=5). H is tograms represent the total number o f dives analyzed for a l l animals under the various treatment condit ions. A l pha: seals treated w i th a-adrenergic b locker; beta: p-adrenergic b locker; musc+alpha: muscar in ic and a-adrenergic b lockers combined; musc+beta: muscar in ic and P-adrenergic b lockers combined. 37 b locked (F ig . 1.13). Furthermore, there was no signif icant change in mean surface interval duration after b lockade (0.40+0.04 m in for the muscar in ic group; 0.47+0.04 m in for the a-adrenergic group; 0.44±0.02 m in for the P-adrenergic group; 0.48±0.02 m in for the muscar in ic plus a-adrenergic group; 0.44±0.04 m in for the muscar in ic plus P-adrenergic group) (F ig . 1.12). Pharmaco log ica l b lockade d id not s ign i f i cant ly change the distr ibut ion o f d ive and surface interval durations (F ig . 1.13). There was no effect o f b lockade on percentage of t ime spent submerged dur ing d iv ing sessions. In control seals, mean percent d ive t ime ranged f rom 86±1 % to 87+1 %, and in treated seals, percent d ive t ime ranged f rom 83±2 % to 85+2 %. 38 Discussion Heart rate during diving D i v i n g induced a marked decrease in heart rate to about 35 % of the surface leve l in control seals. Prev ious studies have also shown a s imi lar drop in heart rate, to 25-50 % of surface levels, dur ing voluntary dives in harbour seals (Pasche and K r og , 1980; Jones et al. 1973; Fedak et al. 1988). D i v i n g bradycard ia was present in a l l control dives regardless o f d ive duration. In fact, seals d isp layed bradycardia even when they d ipped their heads under water for periods less than 20 sec. Th i s is in contrast to the f indings o f Jones et al. (1973) who showed that one harbour seal d id not d isp lay a bradycardia dur ing some short feeding dives. Post-dive tachycardia was present dur ing al l control surface intervals regardless o f surface interval duration. Rest ing heart rate was not formal ly recorded f r om these seals dur ing d iv ing sessions since d iv ing was continuous, but dur ing periods o f "rest" on land in pre l iminary experiments, heart rate was approximate ly 75 beats min" 1 . In control seals, d iv ing heart rate was always be low this leve l and surface heart rate was always we l l above it. The two adult (4 yr old) females showed s igni f icant ly lower d ive heart rates for the first 15 sec of dives than the three juven i l e (2 y r old) males. These differences in the in i t ia l d ive heart rate cannot be attributed to body mass since the mean weights for males and females were not s igni f icant ly different; however, gender and/or age may p lay a role. N o studies have been done to investigate the role o f gender in the cardiac d ive response, but there is evidence that the cardiovascular responses to breath-holding change w i th development i n seals. Previous studies have shown that older seals develop a more profound d iv ing bradycardia more rapid ly than younger seals (Harr ison and Toml inson , 39 1960), and they d isp lay lower heart rates dur ing sleep-associated apneas on land (Caste l l in i et al. 1994). The development o f cardiorespiratory contro l is also associated w i th greater breath-hold tolerance (Harr ison and Toml inson , 1960; Caste l l in i et al. 1994). Fo r al l seals, the effects o f the b lockers on heart rate were in agreement w i th their pharmaco log ica l action on the autonomic nervous system. Methoct ramine is a polymethylene tetraamine compound that is h igh ly selective for M 2 subtype muscar in ic receptors wh i ch are predominant ly found in the heart in many terrestrial mammals (Hammer and Giachett i , 1983; G i ra ldo et al. 1988; Me lch io r re , 1988; Hendr ix and Rob inson, 1997). Therefore, methoctramine reduced d iv ing bradycard ia most l i ke l y by inh ib i t ing the action o f acety lchol ine on cardiac M 2 receptors in the seals. Furthermore, methoctramine should have also reduced d iv ing vasoconstr ict ion as a result o f the barostatic ref lex funct ion ing to mainta in constant b lood pressure wh i le d i v ing heart rate was h igh. Me top ro l o l is a (31-selective adrenergic antagonist that b locks the act ion o f noradrenal ine on P i receptors wh i ch are predominant ly found in the myoca rd ium in humans (Pr ichard and Tom l i n son , 1986; Ho f fman , 2001). Therefore, metopro lo l reduced post-dive surface tachycardia most l i ke ly by b lock ing sympathetic inputs to cardiac P i adrenoceptors in the seals. Prazos in is a h igh ly selective 0Ci-adrenergic antagonist w i th an af f in i ty for 0C| receptors about 1000-fold greater than for a 2 receptors (Davey, 1980; Ho f fman , 2001). In humans and other terrestrial mammals , b lockade o f (Xi receptors inhibits vasoconstr ict ion induced by catecholamines so that vasodi lat ion occurs in arterioles. The fa l l in peripheral vascular resistance leads to decreases in arterial b lood pressure and venous return to the heart, and, as a result of the barostatic ref lex, sl ight increases in heart rate and cardiac output (Davey, 1980; Saeed et al. 1982; Ho f fman , 40 2001). Prazos in caused a slight but s ignif icant increase in d iv ing heart rate probably due to a i-adrenerg ic b lockade causing peripheral vasodi latat ion in the seals. Unfortunately, I cou ld not moni tor b lood f l ow and arterial b lood pressure dur ing dives; however, the increase in d ive heart rate after administrat ion o f prazos in and the lack o f a marked effect o f the a-adrenergic agonist phenylephrine in prazosin-treated animals suggest that the a -blockade was indeed effect ive dur ing d iv ing experiments. The effects o f cardiovascular pharmaco log ica l b lockade reflect the dynamic inf luence o f the two branches of the autonomic nervous system on heart rate dur ing d iv ing . Because mean surface heart rate was unchanged by muscar in ic b lockade but s igni f icant ly lower after P-adrenergic b lockade, post-dive tachycardia is attributed to increased sympathetic st imulat ion of the heart as we l l as vagal w i thdrawal at the surface. Mean d ive heart rate after muscar in ic b lockade was s igni f icant ly higher than the dive heart rate in contro l seals whereas d ive heart rate f o l l ow ing P-blockade was not s igni f icant ly different; therefore, the parasympathetic nervous system is the pr imary modulator o f bradycardia dur ing voluntary d iv ing . However , the role o f the sympathetic system dur ing d i v i ng is not as straightforward. Heart rate dur ing dives was s igni f icant ly lower than dur ing surface intervals in muscar in ic-b locked seals suggesting that an increased level o f sympathetic st imulat ion at the surface is w i thdrawn dur ing submergence. Sympathet ic inputs to the heart may not be wi thdrawn complete ly, though, because d i v i ng heart rate after p-adrenergic plus muscar in ic b lockade was s igni f icant ly lower than after muscar in ic b lockade alone. However , P-blockade alone d id not s igni f icant ly l ower d iv ing heart rate. 41 One possible explanat ion for these discrepancies i n d ive heart rates is that the two d iv is ions o f the autonomic nervous system interact asymmetr ica l ly so that the parasympathetic system dominates the sympathetic system when vagal out f low to the heart is max ima l . In other words, sympathetic tone persists dur ing d iv ing but is not expressed because the vagus modulates heart rate by means of an accentuated antagonism. Accentuated antagonism has also been observed in d i v ing muskrats (Signore and Jones, 1995), and is the result o f a cho l inerg ica l ly mediated insensit iv i ty o f cardiac cel ls to adrenergic st imulat ion (K imu ra et al . 1985; Signore and Jones, 1995). Such a response dur ing d i v i ng wou ld exp la in why harbour seals develop a bradycardia despite increases in c i rcu lat ing catecholamines (Hance et al. 1982; Hochachka et al. 1995). It wou ld also faci l itate the rapid swi tch ing between dive and surface states because the effect ive response to changes in sympathetic act ivat ion occurs more s l ow ly than changes due to parasympathetic act iv i ty (Fur i l l a and Jones, 1987; Japundzic et al. 1990). A puzz l i ng result is that the d ive heart rate was s igni f icant ly l ower than the surface heart rate in muscar in ic- plus p-adrenergic-blocked seals. S imultaneous b lockade o f parasympathetic and sympathetic out f low to the heart should reveal the aneural or intr insic heart rate. In double b locked seals, d ive heart rate was 88 beats min" 1 whereas surface heart rate was 111 beats min" 1 . Th is f ind ing suggests that either b lockade was not complete, or there is a non-muscar in ic, non-Pi-adrenergic factor af fect ing heart rate dur ing d iv ing in harbour seals. One poss ib i l i ty is that the surface tachycardia may be caused by st imulat ion of cardiac p 2 receptors by c i rcu lat ing adrenaline. I chose a p r selective antagonist in order to avo id effects on p 2 receptors in vascular and bronch ia l smooth muscle. A l s o , sympathetic st imulat ion of the heart in humans is k nown to occur 42 pr imar i ly v i a Pi receptors, although it is uncertain to what extent act ivat ion o f cardiac p 2 receptors contributes to increases in heart rate (Hof fman, 2001). It is l i ke ly that p 2 receptors p lay a larger role in cardiac responses in seals. On several occasions, seals d isp layed a decrease in heart rate 1 to 3 seconds before submergence. Ant ic ipatory bradycard ia has prev ious ly been observed in seals (Jones et al. 1973; Fedak, 1986) and dolphins (Eisner, 1969). Ant i c ipatory bradycardia may be useful i n the sense that oxygen conservat ion dur ing d iv ing is proport ional to the rapidity w i th wh i ch the d ive response develops (Irv ing et al. 1941a). Such a response also provides some evidence that higher centers are i nvo lved in the neural contro l o f the d ive response i f bradycard ia can be init iated without st imulat ion of the tr igeminal or upper respiratory tract receptors. In fact, there are several studies that show that marine mammals can mod i f y their responses to d i v i ng as a result o f learning and experience. E isner (1969) reported that in dolphins trained to d ive on command, heart rate began to decrease immediate ly upon presentation o f the command st imulus. In harbour seals, the cardiovascular responses to forced dives can be habituated such that the forced d ive response becomes more moderate wi th training (Jobsis et al. 2001). S imi la r ly , Fedak (1986) reported that experienced gray seals showed an anticipatory response to any indicat ion that a forced d ive was about to occur. Furthermore, pre-surfacing tachycardia was seen in a l l contro l dives and was unaffected by a - or P-adrenergic b lockade but reduced in methoctramine-injected animals, suggesting that it is caused by the wi thdrawal o f vagal inputs. Card iac acceleration before surfacing has been reported in both seals (Jones et al. 1973; Fedak et al. 1988; Thompson and Fedak, 1993; Andrews et al. 1997) and muskrats (Signore and 43 Jones, 1995). B y restoring c i rcu lat ion to tissues that may have been hypoperfused dur ing the dive, pre-surfacing tachycardia should further reduce the oxygen content o f the b lood, thereby max im i z i ng oxygen uptake at the beg inn ing o f the surface interval (Thompson and Fedak, 1993). Diving behaviour and the role of the dive response Previous studies reveal that harbour seals in the w i l d typ ica l ly d ive for 2 to 6 m in w i th surface intervals last ing less than 1 m in , so they spend 75 to 85 % o f their t ime at sea submerged (Fedak et al. 1988; Eguch i and Harvey, 1995; B o w e n et al. 1999). The data presented in this thesis agree w i th literature values. In contro l seals, d ive durat ion ranged f rom 23 sec to 5.4 m in and the mean duration was 2.7 m in ; surface intervals ranged f rom 4 sec to 1.4 m in , and mean surface interval duration was 25 sec. Du r i ng contro l d iv ing sessions, seals spent approximate ly 86 % o f their t ime submerged. Pharmaco log ica l b lockade o f d i v i ng bradycardia and vasoconstr ict ion d id not signi f icant ly affect routine d ive duration. Th is is in contrast to the f indings o f Mu rdaugh et al. (1961) who showed that one seal d ied after 3 m in o f submersion when bradycard ia was abol ished by atropine. However , that part icular seal was tethered to a rope harness dur ing d iv ing and was probably h igh ly stressed and perhaps not surfac ing long enough to reoxygenate suff ic ient ly. The dose o f atropine g iven (0.09 mg kg" 1) was also s imi la r to the lethal dose reported for chi ldren (B rown and Tay lor , 2001). On the other hand, the seals in my study had enough onboard oxygen to mainta in routine dives without the oxygen-conserv ing d ive response. No t on ly d id chol inerg ic and adrenergic b lockade prevent a restrict ion of b lood f l ow (and oxygen del ivery) to the periphery, but oc-44 adrenergic b lockade should have also prevented splenic contract ion and thus the inject ion of more oxygenated red b lood cel ls into the c i rcu lat ion dur ing d i v i ng (Hochachka et al. 1995; Hur fo rd et al. 1996). Aga i n , b lockade of this response d id not affect d ive behaviour, suggesting it is not necessary for the performance o f short dives. Pharmaco log ica l b lockade of d i v ing bradycardia, vasoconstr ict ion, and post-dive tachycardia d id not s igni f icant ly affect routine surface interval duration either. Therefore, the seals in this study were able to mainta in routine d ive t imes without oxygen conservat ion and yet prevent an oxygen debt large enough to require extra t ime at the surface. Fo r Wedde l l seals, dives that i nvo lve an increasing rel iance on anaerobic metabo l i sm usual ly necessitate extended surface intervals to replenish g lyco ly t i c fuel reserves, process anaerobic byproducts, and restore b lood and tissue p H (Kooyman et al. 1980). Unfortunately, I d id not measure post-dive b lood lactate levels, but the seals d id not surface or haul out on the deck for extended recovery periods even two hours after max ima l blockade; therefore, it is l i ke ly that they also avoided signif icant anaerobic energy contr ibut ions to d iv ing metabol ism. Furthermore, assuming that (3-blocked seals d id not fu l l y reload their oxygen stores at the surface, their oxygen reservoir was st i l l large enough to enable continuous d iv ing (and some dives as long as 8.1 min) . Seals also maintained a h igh percent d ive t ime (approximately 84 %) in a l l treatments; thus, the cardiovascular d ive response was not necessary to mainta in an "e f f i c ient" d ive strategy dur ing short d i v ing sessions. A l though I d id not measure oxygen stores or the metabol ic rate dur ing d i v i ng ( D M R ) , mean dive durations were a l l w i th in estimates o f the aerobic d i v ing l im i t ( c A D L = o x y g e n s to res/DMR) for harbour seals. Spec i f i ca l ly , i f total body oxygen stores 45 in the harbour seal equal 57 m l 0 2 kg" 1 (assuming 50 % desaturation o f arterial b lood and 85 % desaturation o f venous b lood; Dav i s et al. 1991), and i f the D M R is equal to the resting metabol ic rate ( R M R ) o f 7.3 m l 0 2 min" 1 kg" 1 (Davis et al. 1991), then the c A D L should be 7.8 m in . In fact, R M R is essential ly the metabol ic rate when no oxygen-conserv ing mechanisms are being ut i l i zed; therefore, harbour seals are theoretical ly capable o f d iv ing for up to 7.8 m in without the d ive response ( i f they use al l o f their avai lable oxygen stores). It fo l l ows that any oxygen-conserv ing mechan ism cou ld potential ly increase this aerobic l im i t , or alternatively, any phys io log ica l response result ing in higher oxygen demands such as exercise or stress cou ld potent ia l ly decrease it. Dav i s et al. (1985) showed that harbour seals sw imming in a f lume at 1.4 m sec"1 increased their oxygen consumpt ion two times over resting. E ven i f the D M R is equal to twice the R M R , then the c A D L should be 3.9 m in . Because mean d ive durations in contro l and treated seals ranged f rom 2.3 to 2.8 m in , a l l dives were most l i ke ly aerobic in nature. In a s imi lar study, S ignore and Jones (1995) found that after pharmaco log ica l b lockade o f the d ive response, muskrats st i l l d ived voluntar i ly for periods as long as their c A D L , but m a x i m u m underwater surv iva l t ime s igni f icant ly decreased. A l t hough I d id not measure the seals' d i v i ng capacity after b lockade, I expect that b lockade o f the d ive response in harbour seals should l im i t m a x i m u m dive duration and also extend recovery t ime at the surface for dives beyond the c A D L . Aga i n , i f seals are capable o f d i v i ng for up to 7.8 m in without any oxygen-conserv ing mechanisms (depending on the D M R ) , then it fo l lows that any dives beyond that l im i t wou ld either require some degree o f a cardiovascular d ive response and some degree o f metabol ic suppression or, alternatively, 46 an increasing rel iance upon anaerobic metabo l i sm to meet energy demands. The longest dive recorded f rom a free-div ing harbour seal was 19 m in (Eguch i and Harvey, 1995), so harbour seals are certainly capable o f d i v i ng beyond their c A D L , though they do so infrequently. A l t hough the harbour seals in this study cou ld per form a series o f short aerobic dives without the cardiovascular d ive response, contro l seals consistently d isp layed a cardiac response dur ing d iv ing , suggesting that it is at least useful . A relat ively moderate degree o f bradycard ia and peripheral vasoconstr ict ion is probably ut i l i zed dur ing such short d ives to l im i t the deplet ion o f b lood oxygen by peripheral organs and part icular ly by the muscles, thereby reserving oxygen stores for the brain and heart in case of emergencies (i.e. unplanned extension o f submergence). W h i l e some supplementation o f the musc le oxygen store cou ld delay the onset of anaerobic metabo l i sm (Davis and Kanatous, 1999; Jobsis et al. 2001), unrestricted b lood f l ow to the muscles wou ld l im i t aerobic d ive capacity. Because o f the greater af f in i ty o f myog lob in for oxygen compared to hemoglob in, blood-borne oxygen wou l d qu i ck ly di f fuse into the active muscles and render the loca l myog lob in-bound oxygen store unavai lable for use. Dav i s and Kanatous (1999) developed a numer ica l mode l that describes the potential importance o f the d ive response in op t im iz ing the use of b lood and muscle oxygen stores dur ing dives i nvo l v i ng different levels o f muscular exert ion. They found that b lood and musc le oxygen stores must be consumed simultaneously but that cardiac output and muscle perfus ion must be reduced be low resting levels in order to max im i ze the A D L over a range o f D M R (2-9 m l oxygen min" 1 kg" 1 ) . Furthermore, Jobsis et al. (2001) found that dur ing trained submersions o f harbour seals, increased musc le b lood f l ow was accompanied by a 47 reduct ion in myog lob in desaturation, suggesting a higher rate of oxygen extract ion f r om the b lood. Nevertheless, musc le perfus ion dur ing submersion was s igni f icant ly reduced f rom resting values. Free ly d i v i ng Wedde l l seals have also been shown to mainta in musc le b lood f l ow at a reduced rate (Guyton et al. 1995). A l though post-dive tachycardia was also not necessary to sustain a series o f short aerobic dives punctuated by short surface intervals, control seals consistently d isp layed h igh heart rates at the surface. In between short dives, surface tachycardia faci l itates the restoration o f b lood gases and oxygen stores to pre-dive levels (Thompson and Fedak, 1993; Andrews et al. 1997). W h i l e seals are able to d ive cont inuously without this degree o f tachycardia, d i v ing w i th a larger reservoir o f oxygen wou ld a l low for greater f l ex ib i l i t y in behaviour in that a "safety ma rg i n " wou ld be avai lable i f the d ive must be extended. 48 Chapter 2. Spectral analysis of heart rate variability Introduction W h i l e previous research has determined the nervous pathways i nvo lved in the cardiovascular responses to submergence (see Chapter 1), few studies have investigated the autonomic contro l o f heart rate dur ing voluntary d i v i ng in mammals (Murdaugh et al. 1961; Signore and Jones, 1995). Furthermore, these studies o f voluntary d i v i ng mammals have on ly examined the effect of autonomic b lockade on mean heart rate rather than on heart rate var iab i l i ty when, i n fact, beat-to-beat variat ions in the cardiac per iod are caused by the dynamic balance between cardiac vagal and sympathetic regulat ion (Ma l l i an i , 1999). Therefore, analysis o f cardiac interval var iab i l i ty i n seals cou ld potent ial ly provide specif ic quantitative in format ion about the sympathovagal balance modulat ing heart rate dur ing d i v ing . Th i s var iab i l i ty can be descr ibed as the sum of nonrandom osci l latory components in the heart rate signal def ined by their frequency and ampl i tude us ing power spectral analysis. Exper iments that have b locked sympathetic and parasympathetic inputs to the sinoatrial node in humans and dogs have demonstrated by spectral analysis that f luctuations in instantaneous heart rate greater than 0.03 H z are caused by changing levels o f the efferent act iv i ty o f these inputs and that each o f the autonomic branches mediate heart rate osci l lat ions in different frequency bands (Akse l rod et al. 1981; Pomeranz et al. 1985; Pagani et al. 1986). Fo r example, both the parasympathetic and sympathetic branches inf luence instantaneous heart rate var iab i l i ty at lower frequencies (0.05-0.15 H z ) whereas the parasympathetic system predominant ly inf luences osci l lat ions 49 at higher frequencies (0.15-1.00 Hz) that are often associated with breathing. The relationship between the power associated with low frequency spectral components and the power of high frequency components is thought to reflect the state of the sympathovagal balance modulating sinus node pacemaker activity (Pagani et al. 1986; Malliani, 1999). Although I did not originally intend to examine heart rate variability, I decided to apply the concepts of power spectral analysis to the instantaneous heart rate data I had collected from parasympathetic- and sympathetic-blocked seals. In this chapter, I examine the effects of pharmacological blockade on heart rate variability using power spectral analysis in order to assess the balance between vagal and sympathetic modulation of heart rate during diving. The following hypotheses are tested: 1) Heart rate oscillations in the frequency range of 0.151 to 1.000 Hz are modulated by the parasympathetic nervous system and are associated with breathing. 2) Heart rate oscillations in the frequency range of 0.051 to 0.150 Hz are mediated by the parasympathetic and the sympathetic branches. 50 Methods Real- t ime series o f the instantaneous heart rate data discussed in Chapter 1 were analyzed us ing power spectral analysis. Spectral analysis can be used to describe heart rate var iab i l i ty as the sum of the osc i l latory components in the heart rate signal def ined by their frequency and ampl itude. The power o f ind iv idua l frequency components is equal to ampl i tude 2 , and the total power in the spectrum is proport ional to the total variance o f the R-R intervals i n the heart rate s ignal . Fo r each treatment group (muscar in ic, a-adrenergic, P-adrenergic, muscar in ic plus a-adrenergic, muscar in ic plus P-adrenergic b lockade) three dives f r om each seal (N=5) ranging f rom 100 to 460 sec were chosen for the analysis. The first and last 10 sec o f each d ive were exc luded f rom the analysis to reduce var iab i l i ty in heart rate caused by the in i t ia l bradycardia be low the heart rate establ ished dur ing the rest o f the d ive and by cardiac acceleration - 1 0 sec before surfac ing (anticipatory tachycardia). Because seals d id not haul out on deck for any length o f t ime dur ing d i v i ng sessions and because post-d ive surface intervals rarely exceeded 30 sec (wh ich is not an adequate per iod o f t ime to assess nonrandom var iab i l i ty w i th spectral analysis), several surface intervals at least 20 sec long were combined to give one surface breathing t ime series (100 to 125 sec) for each seal (N=4) for each treatment. Though the - 2 0 sec junct ions may have introduced some art i f ic ia l l ow frequency var iab i l i ty (<0.05 H z ) to the heart rate s ignal , much o f the power in that frequency range was f i l tered out dur ing the analysis. The last 3 sec of each surface interval were exc luded f rom the analysis to reduce var iab i l i ty i n heart rate caused by cardiac deceleration before submergence (anticipatory bradycardia). On l y four seals were used for the surface interval data because one seal 's surface intervals were 51 frequently punctuated by short apneas (d ipp ing its head underwater) that were associated w i th decreases in instantaneous heart rate. R-R intervals were calculated as descr ibed in Chapter 1. The R-R interval t ime series ( in ms) were l inear ly interpolated at 4 H z , and the low-frequency basel ine trend was removed by app ly ing a mov ing fourth-order po lynomia l funct ion. The power spectra were estimated by app ly ing the Fast Four ier T rans form (FFT) a lgor i thm to the smoothed t ime series. The output o f the F F T was corrected to give the power of ind iv idua l osc i l latory components at their respective frequencies in s~ Hz" (i.e. power density). The frequency resolut ion of the ind iv idua l power spectra was 0.0156 H z or 0.94 cyc les m in" 1 . Fo r each o f the power density spectra, the frequency at wh i ch the m a x i m u m power occurred and that peak power value (PP) were noted. Tota l power for each spectrum was determined by ca lcu lat ing the area under the power density curve. Based on literature values, the power spectra were then d iv ided into two frequency components o f interest: l ow frequency ( L F ) at 0.051-0.150 H z and high frequency (HF) at 0.151-1.000 H z . V e r y l ow frequency components ( V L F , 0-0.05 Hz ) were not considered as these can on ly be accurately assessed us ing long periods of data, are often inf luenced by noise, and were most ly f i l tered out by the analysis. The power of each frequency component ( L F and H F ) was calculated as area under the power density spectrum w i th in the respective frequency band and expressed as a percentage o f the total power. Rat ios o f L F power to H F power in normal i zed units (nu) were also determined. Va lues in the text are g iven as grand means (N=5 for dives, N=4 for surface intervals) + S . E .M . The means for each treatment group were compared w i th in d ive states (dive or surface interval) us ing one-way repeated measures analysis o f var iance ( A N O V A ) and 52 Dunnett multiple comparison tests. Differences were considered significant when P<0.05. All statistical analyses were performed using SigmaStat software (version 2.0, Jandel Scientific, San Rafael, CA, USA). 53 Results F i g . 2.1 shows the R-R intervals and the corresponding power density spectra, expressed as a percentage o f the peak power value, for one d ive f rom each treatment. F i g . 2.2 shows the power density spectra for the dives i n F i g . 2.1 in absolute power units. The total power o f the dive heart rate var iab i l i ty i n each treatment, expressed as logio power, is presented in F i g . 2.3A, and the contr ibut ion o f L F and H F components to the total power is presented in F i g . 2.3B. The power spectra of the dive R-R interval t ime series exhib i t one ma in component (max imum power peak) at approximately 0.09 H z or 5.4 cyc les min" 1 , meaning that a predominant osc i l la t ion in heart rate occurs every 11.1 sec (F ig . 2.1, 2.2). In some spectra, several smal ler spectral components are apparent between 0.1 and 0.45 H z (10 to 2.2 sec periods) (F ig . 2.1, 2.2), but the frequencies at wh i ch they occur are inconsistent for dives w i th in treatment groups. The frequency at wh i ch the m a x i m u m power peak occurred is s imi la r among treatments. The mean total power o f the d ive heart rate var iabi l i ty, i.e. the total var iance in the R-R intervals, was s igni f icant ly decreased f rom the control group in the muscar in ic-, muscar in ic plus a-adrenergic, and muscar in ic plus p-adrenergic-blocked groups (660.7 ±243.0 s 2 for the control group; 24.0±19.6 s 2 for the muscar in ic group; 7.211.8 s 2 for the muscar in ic plus a-adrenergic group; 9.9+6.3 s 2 for the muscar in ic plus P-adrenergic group) (F ig . 2.3A). The total power was not s igni f icant ly different f r om the contro l in the a-adrenergic- and P-adrenergic-blocked groups (158.1157.4 s 2 for the a-adrenergic group; 864.11326.9 s 2 f o r the P-adrenergic group) (F ig . 2.3A). In a l l treatments, the total power for dives was compr ised most ly o f L F power, though H F components contr ibuted as we l l . The mean relative power o f the L F 54 rt o X ! u rt rt 60 50 40 30 20 10 80 70 60 50 40 140 130 120 I 10 100 90 140 130 120 110 100 90 120 110 100 90 80 70 0 30 60 90 120150180 Time (sec) 100 80 60 40 20 0 100 80 60 40 20 rt 0 to 100 <U X, bo 80 60 4 -o 40 20 0 C <u 100 -o 80 (U 60 o C - 40 <L> _> 20 "S 0 100 80 60 40 20 0 100 80 60 40 20 0 PP = 2,960 sVHz HR = 39 bpm A Control PP = 426 s /Hz HR = 62 bpm B Ot-Adrenergic PP = 5,989 s'/Hz HR = 39 bpm C P-Adrenergic PP = 4 s /Hz HR = 109 bpm D Muscarinic PP = 23 s7Hz HR = 114 bpm E Muscarinic + a-Adrenergic PP = 3 s7Hz HR = 95 bpm F Muscarinic + P-Adrenergic 0.0 0.1 0.2 0.3 0.4 0.5 H z Figure 2.1. Instantaneous heart rate (R-R intervals) and corresponding power spectra for control and pharmacologically blocked dives in one seal. 200 seconds of each dive are shown. Power density is expressed as a percentage of the maximum power value for each spectrum. PP indicates the maximum power value corresponding to the 100 % peak. HR indicates the mean heart rate for the dive in beats min"1 (bpm). 55 6000000 control alpha-adrenergic beta-adrenergic muscarinic musc+alpha musc+beta 5000000 3000000 2000000 1000000 A Hz Figure 2.2. Absolute power density spectra for the control and pharmacologically blocked dives shown in Fig. 2.1 (N=l). The smaller graph shows the same spectra but zoomed in to a smaller power scale. Musc+alpha: muscarinic and a-adrenergic blockers combined; musc+beta: muscarinic and P-adrenergic blockers combined. 56 Figure 2.3. M e a n login total power (±S.E.M.) o f d ive heart rate var iab i l i ty (A ) and relative power (±S.E.M.) o f the L F and H F components (B) in control and pharmaco log ica l ly b l o cked seals (N=5). L F refers to l ow frequency components in the power spectrum f r om 0.051 to 0.150 H z , and H F refers to h igh frequency components f rom 0.151 to 1.000 H z . The mean L F power to H F power ratio is g iven above the relative power bars. * indicates values s igni f icant ly dif ferent f r om the contro l group ( P O . 0 5 ) . A l pha : a-adrenergic blocker; beta: p-adrenergic b locker; musc+alpha: muscar in ic and a-adrenergic b lockers combined; musc+beta: muscar in i c and P-adrenergic b lockers comb ined . 57 component ranged f rom 63.0±7.2 % to 73.0±4.9 %, and the mean relative power o f the H F component ranged f rom 20.5±4.3 % to 33.1+6.0 % for a l l treatments inc lud ing the contro l (F ig . 2.3B). The mean L F / H F ratio ranged f rom 2.37±1.55 nu to 4.89±2.17 nu (F ig. 2.3B). The power o f the ind iv idua l L F and H F components and the L F / H F ratios for dives were not s igni f icant ly altered by pharmaco log ica l b lockade. F i g . 2.4 shows the R-R intervals and the corresponding power density spectra for one surface interval f rom each treatment group. The total power o f the surface heart rate var iab i l i ty in each group, expressed as logio power, is presented in F i g . 2.5A, and the contr ibut ion o f L F and H F components to the total power is presented in F i g . 2.5B. In the R-R interval t ime series, an osci l latory component occurr ing at 0.05 H z (20 sec periods) is v is ib le (F ig . 2.4). Th is is an art i f ic ia l osc i l la t ion caused by the j o in ing o f data segments to lengthen the surface interval t ime series. Mos t o f the V L F noise was f i l tered out o f the t ime series before app ly ing the F F T and d id not contribute s igni f icant ly to the power spectra. The surface interval power spectra exhib i t more spectral components i n the H F frequencies than was seen in the dives, although, w i th the except ion of the m a x i m u m peak, the frequencies at wh i ch they occur are inconsistent for surface intervals w i th in treatment groups. Fo r al l groups except the p-blocked group, the m a x i m u m power peak occurred between 0.06 and 0.09 H z (3.6 to 5.4 cyc les min" 1 , 16.7 to 11.1 sec periods) (F ig . 2.4). In the P-adrenergic group, the m a x i m u m power occurred at 0.45 H z or 27 cycles min" 1 (heart rate osc i l lat ion occurr ing every 2.2 sec), though other h igh-power peaks also occurred (inconsistently) between 0.08 and 0.20 H z and between 0.30 and 0.40 H z (F ig. 2.4). 58 c E C/3 c5 CU cd <u BC 100 140 130 120 110 100 140 130 120 110 100 30 60 90 Time (sec) a cu Cu CU bo c cu -a cu a o a. cu > CU Pi 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 1 PP = 8 s7Hz HR = 128 bpm A Control PP = 11 s7Hz HR = 126 bpm B a-Adrenergic PP= 12s/Hz HR = 95 bpm C P-Adrenergic PP = 2 s7Hz HR = 130 bpm D Muscarinic PP = 4 s7Hz HR = 129 bpm 0.0 E Muscarinic + a-Adrenergic PP = 3 s7Hz HR= 120 bpm F Muscarinic + W V A , , M P-Adrenergic 0.2 0.4 0.6 0.8 Hz Figure 2.4. Instantaneous heart rate (R-R intervals) and corresponding power spectra for control and pharmacologically blocked post-dive surface intervals in one seal. 100 seconds of each surface interval time series (comprised of individual surface intervals 20 sec or greater) are shown. Power density is expressed as a percentage of the maximum power value for each spectrum. PP indicates the maximum power value corresponding to the 100 % peak. H R indicates the mean heart rate for the surface interval in beats min" 1 (bpm). 59 control muscarinic alpha beta musc+alphanusc+beta control muscarinic alpha beta musc+alphanusc+beta F igure 2.5. M e a n login total power (±S.E.M.) o f post-dive surface interval heart rate var iab i l i ty (A) and relative power (±S.E.M.) o f the L F and H F components (B) in contro l and pharmaco log ica l ly b locked seals (N=5). L F refers to l ow frequency components i n the power spectrum f rom 0.051 to 0.150 H z , and H F refers to h igh frequency components f r om 0.151 to 1.000 H z . The mean L F power to H F power ratio is g iven above the relative power bars, i n d i c a t e s values s igni f icant ly different f rom the contro l group (P<0.05). In B, * refers to the relative H F power. A l pha : a-adrenergic b locker; beta: (3-adrenergic blocker; musc+alpha: muscar in ic and a-adrenergic b lockers combined; musc+beta: muscar in ic and P-adrenergic b lockers combined. 60 The mean total power o f the surface heart rate var iab i l i ty was s ign i f i cant ly higher 2 2 in (3-adrenergic-blocked seals compared to control seals (25.0+.6.6 s versus 2.3 ±0.8 s') (F ig . 2.5A). The mean total power in the a-adrenergic and muscar in ic- injected groups was not s igni f icant ly different f rom the control group (9.1±1.9 s for the a-adrenergic group; 4.7±3.3 s 2 for the muscar in ic group; 1.7±4.6 s 2 for the muscar in ic plus a-adrenergic group; 2.6±0.9 s 2 for the muscar in ic plus P-adrenergic group) (F ig . 2.5A). The mean total power i n each group was lower for the surface intervals than for the dives. In general, H F power contr ibuted more to the total power for surface intervals than for dives. In a l l treatment groups except for the P-adrenergic group, the total power for surface intervals was compr ised equal ly o f L F and H F power. The mean relative power o f the H F component ranged f rom 43.4±4.1 % to 79.3±5.7 % for a l l groups and was s igni f icant ly higher in the P-adrenergic-blocked group than in the control group (79.3+5.7 % versus 51.2+6.7 %) (F ig . 2.5B). The muscar in ic and a-adrenergic b lockers d id not s igni f icant ly affect relative H F power. The mean relative power o f the surface L F component ranged f rom 18.0±5.7 % to 45.4+7.0 % for a l l groups and was not s igni f icant ly changed by pharmaco log ica l b lockade (F ig. 2.5B). The mean L F / H F ratio ranged f r om 0.25+0.10 nu to 1.12+0.28 nu and was not s igni f icant ly changed by blockade (F ig . 2.5B). Fo r a l l groups, the L F / H F ratios were lower in the surface intervals than in the dives. 61 Discussion Power spectral analysis can be used to quant i fy nonrandom osc i l latory components in the heart rate var iab i l i ty s ignal (real t ime representation o f R-R intervals). These osci l lat ions are most ly attributed to cardiovascular ref lexes related to c i rcu latory control and reflect the dynamic balance between cardiac vagal and sympathetic regulat ion. Therefore, spectral analysis o f heart rate can prov ide quantitative in format ion about the autonomic nervous control of the heart. Research on humans and dogs has ident i f ied three typ ica l peaks in the heart rate power spectrum: a peak centered near 0.04 H z thought to be related to peripheral vasomotor regulat ion and thermoregulat ion, a peak near 0.10 H z considered important in arterial b lood pressure regulat ion, and a peak at the respiratory frequency corresponding to respiratory sinus arrhythmia (the change in heart rate associated w i th the inspiratory and expiratory phases o f venti lat ion) (Preiss et al. 1975; Akse l r od et al. 1981; H i r s ch and B i shop , 1981; Pomeranz et al. 1985). Furthermore, experiments us ing autonomic b lockers have shown that the two autonomic branches mediate heart rate f luctuations in different frequency bands. In the h igh frequency range o f 0.15 to 1.00 H z (HF) , heart rate is modulated sole ly by the vagus, whereas in the low frequency range o f 0.05 to 0.15 H z ( LF ) , heart rate f luctuations are jo in t l y mediated by the parasympathetic and sympathetic branches (Akse l rod et al. 1981; Pomeranz et al. 1985; Pagani et al. 1986). In the present study, I used power spectral analysis in an attempt to further assess the sympathovagal modulat ion o f heart rate in d i v ing seals. Fo r dives, the total power i n the heart rate s ignal was s igni f icant ly reduced by muscar in ic b lockade but unchanged by (3-adrenergic b lockade; therefore, heart rate var iab i l i ty dur ing d iv ing is attributed to 62 parasympathetic modulat ion of heart rate rather than sympathetic modulat ion. Bo th the L F and H F components were decreased by parasympathetic b lockade since the ratio o f L F power to H F power was unchanged. A dist inct spectral component associated w i th the m a x i m u m power value was apparent near 0.09 H z in al l o f the d ive heart rate power spectra. W h i l e the power o f this peak was reduced by parasympathetic b lockade, the frequency at wh i ch it occurred d id not shift for different treatments, suggesting that b lockade was incomplete. However , a L F osc i l la t ion near 0.09 H z was also apparent in the power spectra for post-dive surface intervals across treatments. Th i s heart rate osc i l lat ion every 11.1 sec is near the 0.10 H z peak seen in heart rate and b lood pressure power spectra for humans and dogs and, therefore, may reflect a baroreceptor response to arterial b lood pressure f luctuations. However , I cannot con f i rm this without ana lyz ing osci l lat ions i n arterial b lood pressure dur ing d iv ing . Fo r this and many other reasons, it certainly wou ld have been useful to measure b l ood pressure in the seals! The total power in the surface interval heart rate var iab i l i ty s ignal was lower than dur ing dives and was unaffected by muscar in ic b lockade, thus ind icat ing that parasympathetic inputs to the heart that cause h igh heart rate var iab i l i ty and bradycard ia dur ing d iv ing are wi thdrawn at the surface. The H F components contr ibuted more to the total power dur ing surface intervals than dur ing dives. In humans and dogs, these H F components are mediated solely by the vagus and reflect respiratory act iv i ty (Akse l rod et al. 1981; Pomeranz et al. 1985; Pagani et al. 1986); therefore, the frequency shift in spectral power dur ing surface intervals cou ld be due to respiratory modulat ion o f cardiac vagal act iv ity. However , the total surface power and the H F power were not reduced by muscar in ic b lockade. Furthermore, the L F power d id not decrease s ign i f i cant ly in 63 P-blocked seals dur ing dives or surface intervals, though the L F component is considered to be a general marker o f sympathetic excitat ion for dogs and humans (Pagani et al. 1986). Another puzz l i ng result is that after P-adrenergic b lockade, the m a x i m u m power peak for surface interval spectra shifted f rom the L F band to around 0.45 H z in the H F band. A l s o , the total power of the surface heart rate var iab i l i ty and the relative H F power were s igni f icant ly increased after p-blockade. Th i s is in contrast to previous experiments on humans and dogs that have shown that sympathetic b lockade on ly decreases L F power and conc luded that the sympathetic system does not inf luence H F f luctuations in heart rate (Akse l rod et al. 1981; Pomeranz et al. 1985). Furthermore, the shifts in peak power frequency and total power seen after P-blockade were not evident in the muscar in ic- plus P-blocked group. A possible explanat ion for these discrepancies in H F power cou ld be that b lockade o f the sympathetic inputs to the heart that mediate post-dive tachycardia somehow al lows for a greater expression o f the parasympathetic tone and respiratory sinus arrhythmia (wh ich wou ld be inhib i ted by muscar in ic b lockade in the muscar in ic-plus P-blocked group). In fact, a recent study on heart rate var iab i l i ty in humans reported that P-adrenergic b lockade enhanced respiratory sinus arrhythmia over a range o f breathing frequencies (Tay lor et al. 2001). Unfortunately, I d id not col lect data on breathing frequency at the surface and cou ld not ver i fy any speci f ic spectral peaks corresponding to respiratory sinus arrhythmia, but the peak heart rate osc i l la t ion at 0.45 H z or 27 cycles min" 1 seen in the p-blocked group is in the range o f surface interval breathing frequencies for elephant seals (Andrews et al. 2000). 64 The ratios o f L F to H F power dur ing both dives and surface intervals were unchanged by autonomic b lockade (wi th in each d ive state). Th i s suggests that the relative powers o f the two components do not accurately reflect the state o f cardiac sympathovagal balance or autonomic tone. Whereas vagal and sympathetic act iv ity are important i n produc ing cardiovascular osc i l lat ions in part icular frequency bands at least i n humans and dogs, other factors such as the abi l i ty o f the vasculature to respond to sympathetic act iv ity, respiratory capacity, the central nervous system, and other ref lex pathways also contribute to var iab i l i ty (Malpas, 2002). G i v en the potential inf luence o f nonautonomic pathways on heart rate var iabi l i ty, it may be d i f f i cu l t to def in i t ive ly relate changes in the power o f an osc i l lat ion as due to a change in under ly ing autonomic control . Furthermore, wh i l e the dist inct ion between the L F and H F domains (0.15 Hz ) has been approximated for humans and dogs, it may not be appl icable to d i v i ng seals. G i v e n that the frequencies o f heart rate osc i l lat ions are subject to conduct ion times for the afferent and efferent signals (Malpas, 2002), it is possible that the autonomic pathways contro l l ing heart rate in seals may affect f luctuations in frequency ranges different f rom humans and dogs. 65 Chapter 3. General Discussion and Conclusion In Chapter 1, I examined mean diving heart rate after pharmacological blockade of the cardiovascular components of the dive response in order to assess the autonomic control of heart rate during voluntary diving in harbour seals. I attempted to further tease apart the dynamic balance between the two autonomic branches and the influence of that balance on diving heart rate by performing power spectral analysis of heart rate variability in Chapter 2. Furthermore, I investigated the role of these cardiovascular responses during 'routine' diving, in Chapter 1, in order to address the question as to whether the dive response is necessary for the performance of short dives by harbour seals. Heart rate during voluntary diving Based on the first two hypotheses presented in Chapter 1, I predicted that muscarinic blockade would inhibit diving bradycardia and that p-adrenergic blockade would inhibit post-dive tachycardia. Through analysis of mean diving heart rate after blockade, I have confirmed these predictions. The research discussed in Chapter 1 indicates that diving bradycardia is primarily modulated by the vagus, and post-dive tachycardia is due to increased sympathetic stimulation of the heart as well as parasympathetic withdrawal. I have also shown that pre-surfacing tachycardia, which may facilitate rapid oxygen loading at the surface, is caused by parasympathetic withdrawal alone. In addition, I have presented some evidence that parasympathetic outflow to the heart prevents the expression of persistent sympathetic tone during diving 66 by means of an accentuated antagonism. Th i s accentuated antagonism wou ld exp la in the development of d i v ing bradycardia despite increases in c i rcu lat ing catecholamines and the rapid development o f post-dive tachycardia although cardiac responses to sympathetic act ivat ion are s lower than responses to parasympathetic act ivat ion. Furthermore, this study has conf i rmed that some o f the receptors invo lved in the cardiovascular d ive response in seals are s imi lar to receptor subtypes found in terrestrial mammals . Spec i f i ca l ly , d i v i ng bradycardia occurs v i a M2 subtype cardiac muscar in ic receptors, and post-dive tachycardia occurs v ia p i and poss ib ly P2 adrenoceptors on the heart. Based on the hypotheses presented in Chapter 2, I predicted that on ly the R-R interval t ime series or heart rate s ignal dur ing surface intervals (and not dur ing dives) wou ld exhib i t s ignif icant h igh frequency osc i l latory components since they are associated w i th breathing in humans and dogs (Pagani, 1986; Pomeranz et al. 1985). I also predicted that muscar in ic b lockade wou ld reduce h igh and l ow frequency osci l lat ions in the heart rate s ignal and that adrenergic b lockade wou l d reduce l ow frequency components. Interestingly, both surface interval and d ive heart rates exhib i ted osci l lat ions in the h igh frequency range, though h igh frequency components contributed more to the total power o f the s ignal dur ing surface intervals than dives. Therefore, h igh frequency osci l lat ions may reflect but are not sole ly caused by respiratory modulat ion o f heart rate. Furthermore, muscar in ic b lockade d id indeed reduce both l ow and h igh frequency components but on ly dur ing dives. The conc lus ion that the parasympathetic system pr imar i ly modulates heart rate osc i l lat ions at a l l frequencies dur ing dives but not s igni f icant ly dur ing surface intervals is consistent w i th the results o f Chapter 1. O n the other hand, p-adrenergic b lockade d id not affect l ow frequency osc i l lat ions in d ive or 67 surface interval heart rates as expected. However , p-blockade d id increase the power of h igh frequency osci l lat ions in the surface interval heart rate s ignal, thus suggesting that h igh frequency components may reflect both vagal and sympathetic modulat ion of heart rate. In contrast to the accentuated antagonism occurr ing dur ing d iv ing , sympathetic neural act iv i ty may somehow constrain heart rate var iab i l i ty produced by parasympathetic modulat ion when seals are at the surface. Role of the cardiovascular dive response during routine diving Based on the third hypothesis presented in Chapter 1, I predicted that pharmacolog ica l b lockade o f the cardiovascular components o f the d ive response wou l d alter routine d iv ing behaviour; speci f ica l ly, d ive duration and overa l l percent d ive t ime wou ld decrease and surface interval duration wou l d increase. However , the results of this study were not consistent wi th these predict ions since seals were able to mainta in routine d ive and post-dive surface interval durations as we l l as a h igh percent d ive t ime when the oxygen-conserv ing d ive response was inhib i ted. In Chapter 1, I 've discussed some possible explanations as to why seals d isp layed a dive response dur ing a l l control d ives when it was not necessary. The cardiovascular responses to submergence are probably hard-wired so that dur ing short d ives i n nature, a moderate d ive response functions to conserve b lood oxygen stores for the brain and heart in case the seal 's return to the surface is delayed. W h i l e the cardiovascular d ive response was c lear ly not necessary for the performance o f short dives in captive harbour seals, the s igni f icance o f the d ive response to seals per forming routine dives in the w i l d is unknown. A l though the seals in this study 68 exhibi ted d ive and surface interval durations s imi lar to those typ ica l ly observed in free-d i v i ng harbour seals (Fedak et al. 1988; Eguch i and Harvey, 1995; B o w e n et al. 1999), I am not certain that their metabol ic rates were s imi lar . W h i l e there is some evidence that free d i v i ng seals do not expend energy at rates substantial ly higher than resting (Caste l l in i et al. 1992), seals foraging in nature wou l d l i ke ly incur greater metabol ic costs than captive seals, for example, costs associated w i th exercise at higher s w i m speeds to pursue prey and costs associated w i th digest ion. The d ive response may be more central to an economic ut i l izat ion of oxygen dur ing dives that are characterized by h igh oxygen demands. Wi thout any redistr ibution o f b lood f low, exerc is ing muscles wou ld qu i ck ly reduce the oxygen content o f the b lood, thereby reducing aerobic stores avai lable for the brain and heart and ult imately l im i t i ng d ive capacity. Nevertheless, seals i n this study cou ld per form dives up to 4.9 m in without restr ict ing b lood f l ow to the periphery, ind icat ing that onboard oxygen stores were suff ic ient to meet energy demands by a l l tissues. Furthermore, the lack of an increase in surface interval duration indicates that the seals d id not require signif icant anaerobic metabo l i sm to meet peripheral energy demands wh i le conserv ing b lood oxygen stores for hypoxia- intolerant tissues. Wh i l e I cannot rule out the poss ib i l i ty that changes in arterial b lood gases and p H may have led to a decrease in aerobic metabol ic rate o f peripheral tissues, a s imi lar decrease may occur dur ing free dives as a result of reduced cardiac output and tissue perfusion (But ler and Jones, 1997). The idea that short, aerobic dives may require l itt le cardiovascular adjustment f rom resting condit ions compared to longer dives that require a more extreme response was in i t ia l ly proposed by K o o y m a n (1985). Later, H i l l et al. (1987) showed that, in freely d iv ing Wedde l l seals, mean dive heart rate was negatively correlated w i th d ive duration 69 for dives greater than 20 min , but there was no correlat ion for dives less than 20 m in . O n the other hand, more pelagic species l i ke the elephant seal seem to ut i l i ze a different strategy. Andrews et al. (1997) showed that elephant seals regulate their heart rate in proport ion w i th d ive duration in a curv i l inear fashion, so these seals gradual ly and cont inual ly lower their heart rate as the d ive progresses. E lephant seals also exhib i t much longer, cont inuous d i v i ng bouts compared to coastal seals l ike the harbour seal, and they d ive for periods beyond their c A D L more often (Le Boeu f et al. 1988; H i nde l l et al. 1992). Th i s impress ive d iv ing abi l i ty is thought to be associated w i th a decrease in overa l l d i v i ng metabo l i sm wh i ch may in fact be l inked to the d ive response i tsel f (Le Boeu f et al. 1988; Hochachka , 1992; Andrews et al. 1997). If the reduct ion in oxygen del ivery does somehow init iate metabol ic suppression in hypoperfused tissues and i f a decl ine in metabol ic rate does enable cont inuous and pro longed d iv ing , then b lockade o f the d ive response in elephant seals might y ie ld different results, i.e. a change in their d i v i ng behaviour. The conc lus ion that the d ive response is not necessary for " rout ine" d i v ing in harbour seals is also consistent w i th the hypothesis that the cardiovascular components of the d ive response are not adaptations to d i v ing (Hochachka and Mot t i shaw, 1998; Mot t i shaw et al. 1999). In a study o f the evolut ion o f the d ive response in pinnipeds (seals and sea l ions), Hochachka and Mot t i shaw (1998) found that m a x i m u m bradycardia (i.e. lowest heart rate observed dur ing d iv ing) across p inn iped species d id not correlate w i th the m a x i m u m dive duration observed in the w i l d , and they proposed that apnea, bradycardia, and vasoconstr ict ion were not adaptations to d i v ing per se but rather ancestral traits that have been conserved throughout p inn iped phylogenet ic history 70 (Mott i shaw et al. 1999). A n important point to be made is that a correlat ion might exist between heart rates dur ing voluntary d i v i ng and aerobic d ive durations across p inn iped species. Moreover , a compar ison o f heart rates and d ive durations between pinnipeds and a c lose ly related sister species cou ld y ie ld different results, i.e. that bradycardia is an adaptation to d i v ing in pinnipeds. However , it is p lausible that the components o f the d ive response may have evo lved in response to selective pressures in a b io log i ca l setting other than d iv ing . In fact, some vers ion o f the d i v i ng response is present in almost a l l air-breathing vertebrates, inc lud ing humans, dur ing breath-holding (Scholander, 1940; Scholander, 1963); thus, the d ive response may have originated in vertebrates as a defensive response to breath-holding or some other stress (Jones and M i l s o m , 1982; Mot t i shaw et al. 1999). Th is defense ref lex was most l i ke ly further developed for d iv ing , poss ib ly under posit ive selection pressures, in some ancestral species o f the p inn iped l ineage and, in its extreme form, is absolutely cruc ia l to the management o f oxygen stores dur ing pro longed dives (Irving et al. 1941b; But le r and Jones, 1997). Fo r seals in nature, extended dives may be important when they are searching for prey at depth or escaping f rom predators near the surface. However , long dives near breath-hold capacity are relat ively infrequent and routine dives are comparat ive ly br ie f (Boyd and C roxa l l , 1996; But le r and Jones, 1997). Th is is often attributed to the fact that a series o f short aerobic dives punctuated by short surface intervals results in a greater percentage o f t ime spent submerged and hence a greater potential for increased foraging t ime compared to several long dives that require extended recovery periods (Kooyman , 1985; Fedak; 1986; Thompson and Fedak, 1993). Interestingly, the captive seals in this study consistently d isp layed a h igh percent d ive t ime dur ing d i v i ng experiments, 71 suggesting that opt imal foraging t ime is not necessari ly the dr iv ing force beh ind an "ef f i c ient" d ive strategy. A l o n g the same l ines, i f estimates of the A D L (3.9 m i n for metabol ic rates two-fo ld greater than resting) are correct, the seals i n this study and perhaps seals i n the w i l d often surface before they reach their aerobic l imi ts . W h y not remain submerged unt i l oxygen stores are nearly exhausted? Based on breath-by-breath measurements o f end-tidal oxygen and carbon d iox ide concentrations dur ing surface intervals in the harbour porpoise, Bout i l i e r et al. (2001) recently proposed that surface interval durations are governed by the readjustment o f carbon d iox ide stores rather than oxygen stores. Perhaps the accumulat ion o f carbon d iox ide and the result ing increase in tissue and b lood p H also dictate the end to an aerobic dive. A l though seals can tolerate much higher arterial carbon d iox ide tensions compared to terrestrial mammals (Ke rem and Eisner, 1973), a study o f harp and hooded seals indeed showed that d ive duration decreased s igni f icant ly w i th increasing alveolar carbon d iox ide tension (Pasche, 1976). In conc lus ion, through pharmaco log ica l b lockade o f the cardiovascular components of the d iv ing response, I have shown that the d ive response is not necessary to sustain serial short aerobic dives i n captive harbour seals. Nevertheless, seals consistently d isp layed a cardiac response dur ing al l contro l d ives regardless o f d ive duration. W h i l e they may not be necessary, cardiovascular adjustments are probably ut i l i zed dur ing short dives in order to max im i ze aerobic d ive capacity and to conserve oxygen for emergencies. 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