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Aspects of the control of breathing in the golden-mantled ground squirrel Webb, Cheryl Lynn 1987

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ASPECTS OF THE CONTROL OF BREATHING IN THE GOLDEN-MANTLED GROUND SQUIRREL By CHERYL LYNN WEBB .Sc. (Hons) U n i v e r s i t y of B r i t i s h Columbia 19 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1987 (c) C h e r y l Lynn Webb 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. The University of British e6Tumbia 1956 Main Mall Vancouver, Canada Department V6T 1Y3 i i ABSTRACT Spermophilus l a t e r a l i s , the golden-mantled ground s q u i r r e l , while euthermic exhibits a strong hypoxic v e n t i l a t o r y response, but a r e l a t i v e l y blunted hypercapnic v e n t i l a t o r y response s i m i l a r to other semi-fossorial mammals. Under res t i n g conditions, c a r o t i d body chemoreceptors provide a tonic excitatory input to the frequency component of v e n t i l a t i o n . Carotid body denervation (CBX) r e s u l t s i n a 40% decrease i n minute v e n t i l a t i o n (V). The o v e r a l l v e n t i l a t o r y response to hypoxia i s unaffected by CBX, although the v e n t i l a t o r y threshold i s s i g n i f i c a n t l y s h i f t e d to lower l e v e l s of i n s p i r e d O 2 . CBX also has l i t t l e e f f e c t on the o v e r a l l response to hypercapnia. Thus, i n S. l a t e r a l i s , i t appears that changes i n the p a r t i a l pressure of O2 ( P 0 2 ) I N T N E blood act c e n t r a l l y , rather than p e r i p h e r a l l y , to play a predominate role i n v e n t i l a t o r y c o n t r o l . Chronic exposure to hypoxia and hypercapnia (CHH, 17% O2 and 4% C O 2 ) does not r e s u l t i n o v e r a l l v e n t i l a t o r y acclimation, with minute v e n t i l a t i o n being s i m i l a r to con t r o l s q u i r r e l s acutely exposed to hypoxic and hypercapnic conditions. In s p i t e of t h i s , CHH exposure does r e s u l t i n adjustments to r e s p i r a t i o n ; frequency i s decreased and t i d a l volume i s elevated compared to control s q u i r r e l s acutely exposed to CHH c o n d i t i o n s . O v e r a l l V s e n s i t i v i t i e s to both hypoxia and hypercapnia are not s i g n i f i c a n t l y a l t e r e d by CHH exposure. I t appears that a c c l i m a t i o n t o c h r o n i c hypoxic and hypercapnic c o n d i t i o n s i n S. l a t e r a l i s may i n c r e a s e a l v e o l a r minute v e n t i l a t i o n r e l a t i v e t o t o t a l minute v e n t i l a t i o n and thus minimize the changes i n a r t e r i a l PO2 and Pco2 during hypoxic and hypercapnic exposure. During entrance i n t o h i b e r n a t i o n , as metabolic r a t e and body temperature d e c l i n e , concomitant decreases i n v e n t i l a t i o n occur. Two p a t t e r n s of r e s p i r a t i o n occur d u r i n g deep h i b e r n a t i o n ; a burst breathing p a t t e r n c h a r a c t e r i z e d by long n o n - v e n t i l a t o r y periods (T^vp) separated by b u r s t s of s e v e r a l breaths and a s i n g l e breath p a t t e r n c h a r a c t e r i z e d by s i n g l e breaths separated by a r e l a t i v e l y short T^vp. In S. l a t e r a l i s during h i b e r n a t i o n at body temperatures between 6° and 10°C, a burst b r e a t h i n g p a t t e r n p r e v a i l s . At s l i g h t l y lower body temperatures, l e s s than 4°C, a s i n g l e breath breathing p a t t e r n p r e v a i l s . Both burst breathing and s i n g l e breath breathing s q u i r r e l s have s i m i l a r o v e r a l l l e v e l s of r e s t i n g minute v e n t i l a t i o n . Burst breathing s q u i r r e l s e x h i b i t a s i g n i f i c a n t r e s p i r a t o r y response to hypoxia ( 3 % O2) and when the decreases i n metabolic r a t e during h i b e r n a t i o n are taken i n t o account ( a i r convection requirement) t h e i r hypoxic s e n s i t i v i t y i s s i m i l a r t o that i n awake S. l a t e r a l i s . In c o n t r a s t , s i n g l e iv breath breathing s q u i r r e l s do not respond to hypoxia at any l e v e l t e s t e d (down to 3% O2). Both burst b r e a t h i n g and s i n g l e breath breathing s q u i r r e l s show l a r g e v e n t i l a t o r y repsonses to hypercapnia. In the burst b r e a t h i n g s t a t e hypercapnic s e n s i t i v i t y i s s i g n i f i c a n t l y higher compared to the s i n g l e breath breathing s t a t e , due t o an augmented frequency response during burst b r e a t h i n g . In both groups of h i b e r n a t i n g s q u i r r e l s v e n t i l a t i o n i s increased d u r i n g hypercapnia s o l e l y by decreases i n the n o n v e n t i l a t o r y p e r i o d . When v e n t i l a t i o n i s standardized f o r the decreases i n metabolic r a t e during h i b e r n a t i o n both burst b r e a t h i n g and s i n g l e breath breathing S. l a t e r l i s e x h i b i t a much higher hypercapnic s e n s i t i v i t y than that seen i n awake S.  l a t e r a l i s . C a r o t i d body denervation has l i t t l e e f f e c t on v e n t i l a t o r y p a t t e r n generation or v e n t i l a t o r y s e n s i t i v i t i e s t o hypoxia and hypercapnia i n h i b e r n a t i n g s q u i r r e l s . I t appears that during h i b e r n a t i o n i n S. l a t e r a l i s , v e n t i l a t i o n i s c o n t r o l l e d p r i m a r i l y by changes i n the p a r t i a l pressure of CO2 (Pc02) i n t n e blood a c t i n g c e n t r a l l y t o s t i m u l a t e v e n t i l a t i o n . The burst breathing p a t t e r n i s produced c e n t r a l l y , as are the r e s p i r a t o r y responses to hypoxia and hypercapnia. Thus, c e n t r a l mechanisms i n v o l v e d w i t h v e n t i l a t o r y c o n t r o l are extremely important i n both the euthermic s t a t e and the h i b e r n a t i n g s t a t e , but the chemical s t i m u l i r e g u l a t i n g v e n t i l a t i o n appear to be fundamentally d i f f e r e n t i n euthermic and h i b e r n a t i n g S. l a t e r a l i s . TABLE OF CONTENTS Abs t r a c t L i s t of Tables L i s t of Figures Acknowledgements I n t r o d u c t i o n M a t e r i a l s and Methods Surgery Summer P r o t o c o l H i b e r n a t i o n P r o t o c o l Results Awake Animals H i b e r n a t i n g Animals D i s c u s s i o n Awake Animals H i b e r n a t i n g Animals General D i s c u s s i o n L i t e r a t u r e C i t e d v i LIST OF TABLES PAGE Table 1 L i s t of n o t a t i o n and u n i t s of r e s p i r a t o r y v a r i a b l e s measured and c a l c u l a t e d i n S. l a t e r a l i s 27 Table 2 R e s t i n g v e n t i l a t o r y v a r i a b l e s i n awake S. l a t e r a l i s 48 Table 3 E f f e c t s of a l t e r a t i o n of i n s p i r e d gas composition on f , and V i n awake S. l a t e r a l i s 56 Table 4 V e n t i l a t o r y and breathing p a t t e r n v a r i a b l e s i n h i b e r n a t i n g S. l a t e r a l i s 72 Table 5 E f f e c t s of decreasing body temperature on v e n t i l a t o r y and breathing p a t t e r n v a r i a b l e s i n h i b e r n a t i n g S. l a t e r a l i s 81 Table 6 E f f e c t s of a l t e r a t i o n of i n s p i r e d gas composition on f , Vt and V i n h i b e r n a t i n g S. l a t e r a l i s 97 v i i LIST OF FIGURES Figur e 1 E f f e c t s of anoxia and hyperoxia on r e s p i r a t o r y frequency and depth before and a f t e r c a r o t i d body denervation i n S. l a t e r a l i s 20 F i g u r e 2 E f f e c t s of intravenous NaCn i n j e c t i o n s on r e s p i r a t o r y frequency and depth before and a f t e r c a r o t i d body denervation i n S. l a t e r a l i s 24 F i g u r e 3 Schematic diagram of whole body plethysmograph arrangement 29 F i g u r e 4 R e l a t i o n s h i p betwen t i d a l volume values obtained using a whole-body plethysmograph method and a pneumotachograph method 34 F i g u r e 5 Schematic diagram of experimental arrangement f o r recording v e n t i l a t i o n i n h i b e r n a t i n g S. l a t e r a l i s 40 F i g u r e 6 Representative records of breathing p a t t e r n during exposure to a i r , hypoxia and hypercapnia i n awake S, l a t e r a l i s 50 V l l l F i g u r e 7 E f f e c t of decreasing F J Q 2 o n V, V*t and f i n awake a i r breathing c o n t r o l and CBX S. l a t e r a l i s F i g u r e 8 E f f e c t of decreasing FJQ2 on V, V«r, and f i n awake CHH c o n t r o l and CBX S. l a t e r a l i s F i g u r e 9 E f f e c t of i n c r e a s i n g F J C O 2 O N V , V T and f i n awake, a i r breathing c o n t r o l and CBX S. l a t e r a l i s F i g u r e 10 E f f e c t of i n c r e a s i n g F J C O 2 O N ^ ' V T and f i n awake, CHH c o n t r o l and CBX S. l a t e r a l i s F i g u r e 11 E f f e c t of decreasing F J Q 2 w i t h a hypercapnic background and i n c r e a s i n g FIC02 wi t h a hyperoxic background on V i n awake a i r breathing c o n t r o l and CBX S. l a t e r a l i s F i g u r e 12 E f f e c t of decreasing F J Q 2 w i t h a hypercapnic background and i n c r e a s i n g FIC02 w i t h a hyperoxic background on V awake CHH c o n t r o l and CBX S. l a t e r a l i s IX Figure 13 Representative records of resting burst breathing pattern i n control and CBX S. l a t e r a l i s during hibernation 74 Figure 14 Single burst of breaths showing a CSR pattern from S. l a t e r a l i s during hibernation 76 Figure 15 Single burst of breaths showing a uniform pattern from S. l a t e r a l i s during hibernation 78 Figure 16 Representative record of breathing pattern t r a n s i t i o n from burst breathing to single breath breathing i n S. l a t e r a l i s during hibernation 83 Figure 17 E f f e c t s of decreasing body temperature on respiratory and breathing pattern variables i n S. l a t e r a l i s during hibernation 85 Figure 18 Bar plo t of e f f e c t of decreasing body temperature on T E i n S . l a t e r a l i s during hibernation 87 X Figure 19 R e l a t i o n s h i p between and r e s p i r a t o r y t i m i n g v a r i a b l e s during burst breathing and s i n g l e breath breathing S. l a t e r a l i s during h i b e r n a t i o n 90 F i g u r e 20 Representative record of breath i n g p a t t e r n during exposure to a i r , hypoxia and hypercapnia i n burst b r e a t h i n g S. l a t e r a l i s during h i b e r n a t i o n 93 F i g u r e 21 Bar p l o t of e f f e c t of decreasing FTO2 and i n c r e a s i n g Fjc02 o n breaths/burst and n o n - v e n t i l a t o r y p e r i o d i n S. l a t e r a l i s during h i b e r n a t i o n 95 F i g u r e 22 E f f e c t of decreasing FJQ2 © N V , V T and f i n burst breathing c o n t r o l and CBX S. l a t e r a l i s d during h i b e r n a t i o n 101 F i g u r e 23 E f f e c t of decreasing FTQ2 o n V » V T A N A " f i n s i n g l e breath breathing c o n t r o l and CBX S. l a t e r a l i s d uring h i b e r n a t i o n 103 F i g u r e 24 E f f e c t of decreasing FJQ2 ° n v» v t a n (* f i n burst breathing c o n t r o l and s i n g l e breath breathing c o n t r o l S. l a t e r a l i s and s i n g l e breath breathing S. columbianus during h i b e r n a t i o n 106 x i F i g u r e 25 E f f e c t of i n c r e a s i n g F J C O 2 o n V ' V T and f i n burst breathing c o n t r o l and CBX S. l a t e r a l i s d uring h i b e r n a t i o n 109 F i g u r e 26 E f f e c t of i n c r e a s i n g F J C Q 2 o n V, V<r and f i n s i n g l e breath breathing c o n t r o l and CBX S. l a t e r a l i s during h i b e r n a t i o n 112 F i g u r e 27 E f f e c t of i n c r e a s i n g F J C O 2 o n V , Vrp and f i n burst breathing c o n t r o l and s i n g l e breath breathing c o n t r o l S. l a t e r a l i s and s i n g l e breath breathing S. columbianus during h i b e r n a t i o n 114 F i g u r e 28 E f f e c t of i n c r e a s i n g F J C Q 2 i n combination w i t h normoxia, hyperoxia and hypoxia on V i n burst breathing and s i n g l e breath breathing S. l a t e r a l i s during h i b e r n a t i o n 117 F i g u r e 29 Representative records of breathing p a t t e r n s during exposure to a i r and a i r p l u s halothane i n burst breathing S. l a t e r a l i s during h i b e r n a t i o n 119 x i i Figure 3 0 Bar p l o t of e f f e c t of exposure t o a i r , acute hypoxia and hypercapnia, and chr o n i c hypoxia and hypercapnia on f , Vrj, V and V A i n awake a i r breathing c o n t r o l and CBX S. l a t e r a l i s 130 F i g u r e 3 1 Bar p l o t showing hypoxic and hypercapnic v e n t i l a t o r y responses of awake S. l a t e r a l i s compared to those of the r a t 134 Figure 32 Representative record of the burst breathing p a t t e r n i n S. l a t e r a l i s d uring h i b e r n a t i o n and Chysemys p i c t a under r e s t i n g c o n d i t i o n s 153 F i g u r e 3 3 Representative records of breathing p a t t e r n changes during decreased body temperatures and during halothane exposure i n S. l a t e r a l i s during h i b e r n a t i o n 155 F i g u r e 34 Comparison of v e n t i l a t o r y repsonses of awake and h i b e r n a t i n g S. l a t e r a l i s t o decreasing F I Q 2 a m * i n c r e a s i n g F J C O 2 167 ACKNOWLEDGEMENTS F i r s t and foremost I would l i k e t o express my a p p r e c i a t i o n t o the one and only Captain Zot, Dr.W.K. Milsom, f o r h i s encouragement, a s s i s t a n c e and f r i e n d s h i p . His endless humour, knowledge and mechanical wizardry have been i n v a l u a b l e through the course of t h i s i n v e s t i g a t i o n . I would l i k e t o thank Dr. Wayne Vogl f o r supp l y i n g the golden-mantled ground s q u i r r e l s , as w e l l as a l l the "boys i n the shop" f o r t h e i r t e c h n i c a l a s s i s t a n c e . I am g r a t e f u l t o Dawn McArthur f o r generous donations of time and t e c h n i c a l a s s i s t a n c e at the beginning of the study. The u n l i m i t e d encouragement and patience from my f r i e n d s and f a m i l y i s g r a t e f u l l y acknowledged. I would p a r t i c u l a r l y l i k e t o thank V e r l e e Webb f o r ty p i n g the t h e s i s . To a l l the members of the Captain Zot crew (plus a few surrogates) Heather, Marianne, S a l l y , Graham, Greg, Mark, Doug and Beth, Thank-you f o r your h e l p , humour and encouragement, p a r t i c u l a r l y during the crunch. G'day. INTRODUCTION Mammalian h i b e r n a t i o n i s a seasonal phenomenon c h a r a c t e r i z e d by a profound decrease i n body temperature and metabolic r a t e . In t h i s way an animal can s e v e r e l y reduce i t s energy expenditure during periods of low food a v a i l a b i l i t y and inclement c o n d i t i o n s (Wang, 1982). This red u c t i o n of energy expenditure i s p a r t i c u l a r l y important f o r s m all mammals. Small animals have a high surface area to volume r a t i o and, t h e r e f o r e , tend to l o s e heat more r e a d i l y to the environment. As a r e s u l t , they must expend more energy to maintain a high body temperature than do l a r g e r mammals. This becomes extremely d i f f i c u l t when food supply i s l i m i t e d . By e n t e r i n g h i b e r n a t i o n and a l l o w i n g i t s body temperature to drop to l e v e l s s l i g h t l y above ambient temperature, a small mammal can reduce i t s energy expenditures by more than 80% over the winter (Wang, 1978). Deep h i b e r n a t i o n i s defined by Lyman (1982) as a s t a t e of dormancy i n which body temperature drops to a p o i n t near ambient temperature, o f t e n as low as 2° to 5°C and rewarming from h i b e r n a t i o n r e q u i r e s only s e l f - g e n e r a t e d heat. This d e f i n i t i o n separates h i b e r n a t i o n from other s t a t e s where metabolic r a t e and body temperature are lowered, such as torpor and hypothermia. Torpor i s a s t a t e of i n a c t i v i t y i n which body temperature d e c l i n e s , but not u s u a l l y below 15-20°C. Torpor i n c l u d e s the d a i l y c y c l e s - 2 -observed i n some b i r d s and small mammals, as w e l l as seasonal torpor observed i n small mammals such as chipmunks and l a r g e r mammals such as bears (Lyman, 1982). Hypothermia i s a s t a t e of depressed metabolic r a t e and lowered body temperature which r e q u i r e s an out s i d e or exogenous heat source f o r rewarming to normal l e v e l s . During h i b e r n a t i o n , metabolic r a t e f a l l s to about 1% to 2% of euthermic l e v e l s (Hammel et a l . , 1968; Wang, 1979) and breathing frequency f a l l s from about 40 to 60 breaths/minute to 1 to 5 breaths/minute (Biorck et a l . , 1956; Holloway and Heath, 1984; Landau and Dawe, 1958; Lyman, 1982; Walker et a l . , 1985). Even w i t h such extreme p h y s i o l o g i c a l changes, h i b e r n a t i n g animals are capable of responding to e x t e r n a l s t i m u l i and environmental changes, implying that h i b e r n a t i o n i s an a c t i v e l y c o n t r o l l e d s t a t e (Lyman, 1982). For example, work done by H e l l e r et a l . (1974) shows that the thermoregulatory system not only f u n c t i o n s during h i b e r n a t i o n but works to maintain a " s e t " body temperature regardless of changes i n ambient temperature. As body temperature f a l l s during entrance i n t o h i b e r n a t i o n , concomitant decreases i n v e n t i l a t i o n occur. Few s t u d i e s have attempted to determine i f the v e n t i l a t o r y system i s a c t i v e l y c o n t r o l l e d during h i b e r n a t i o n and i f the nature of the r e s p i r a t o r y c o n t r o l system i s s i m i l a r to that of the awake euthermic animal. - 3 -V e n t i l a t i o n i n f o s s o r i a l mammals The primary r o l e of r e s p i r a t i o n i s to meet the demands of metabolism by supplying oxygen (O2) to the t i s s u e s and removing , carbon d i o x i d e ( C O 2 ) . The chemical c o n t r o l of r e s p i r a t i o n i n mammals i n v o l v e s a feedback c o n t r o l system which a d j u s t s v e n t i l a t i o n to regula t e blood and e x t r a c e l l u l a r f l u i d p a r t i a l pressures of O2 ( P 0 2 ) a n a " e i t h e r C 0 2 ( P c 0 2 ) o r P H (Cherniack and Longobardo, 1982; Feldman, 1986). In order to regula t e these v a r i a b l e s , animals must monitor these chemical s t i m u l i w i t h receptors which transduce and transmit r e l e v a n t i n f o r m a t i o n to the CNS which, i n t u r n , c o n t r o l s the r e s p i r a t o r y movements that r e g u l a t e PC>2r P c 0 2 a n < 3 P H (Feldman, 1986). In mammals, the two major groups of receptors i n v o l v e d w i t h the chemical r e g u l a t i o n of breathing are found i n the p e r i p h e r a l c i r c u l a t i o n at the s i t e of the c a r o t i d sinus and the a o r t i c arch ( c a r o t i d body chemoreceptors and a o r t i c body chemoreceptors) and i n the c e n t r a l c i r c u l a t i o n at a yet undetermined l o c a t i o n ( c e n t r a l chemoreceptors). I t i s g e n e r a l l y assumed that steady s t a t e v e n t i l a t o r y responses to decreases i n P O 2 (hypoxia) are p r i m a r i l y mediated by c a r o t i d body chemoreceptor inputs to the CNS, whil e responses to increases i n Pco2 (hypercapnia) are p r i m a r i l y c o n t r o l l e d by c e n t r a l chemoreceptor i n p u t s . In most mammals both hypoxia and hypercapnia r e s u l t i n increases i n v e n t i l a t i o n as the c o n t r o l system attempts to re t u r n blood gas te n s i o n to t h e i r appropriate l e v e l s . - 4 -Most h i b e r n a t i n g animals i n h a b i t burrows which serve as p r o t e c t i o n from pr e d a t i o n and from c l i m a t i c extremes. Since gas exchange between the burrow and the atmosphere i s slow, burrow c o n d i t i o n s are o f t e n low i n O2 c o n c e n t r a t i o n (hypoxic) and high i n CO2 c o n c e n t r a t i o n (hypercapnic) (Withers, 1978). The l e v e l s of hypoxia and hypercapnia a t t a i n e d depend on a number of f a c t o r s such as the number of occupants i n the burrow, s o i l p o r o s i t y , s o i l moisture and burrow geometry ( A r i e l i , 1979; Hayward, 1966; Maclean, 1981; Withers, 1978). L i t e r a t u r e values f o r burrow gas composition range from 20% to 8% f o r O2 concentrations (Hayward, 1966; Wi l l i a m s and Rausch, 1973) and from 0% to 13% f o r CO2 concentrations (Williams and Rausch, 1973). Although there i s a wide range of reported compositions f o r burrow gases, average l i t e r a t u r e values are about 17% to 18% fo r O2 c o n c e n t r a t i o n and 3% to 4% f o r CO2 c o n c e n t r a t i o n (Baudinette, 1974; Darden, 1972; Hayward, 1966; McNab, 1966; Studier and P r o c t o r , 1971; Wi l l i a m s and Rausch, 1973). Burrow gases i n the nests of non-hibernating and h i b e r n a t i n g f o s s o r i a l animals during the summer p e r i o d appear to be s i m i l a r . Several s t u d i e s r e p o r t i n g the composition of burrow gases during h i b e r n a t i o n suggest that metabolic r a t e i s so reduced that there i s no d e p l e t i o n of O2 and no b u i l d -up of CO2 except during a r o u s a l and periods of euthermia (Kuhen, 1986; Wi l l i a m s and Rausch, 1973). Thus, during the h i b e r n a t i n g p e r i o d , exposure to hypoxic and hypercapnic c o n d i t i o n s i s s p o r a t i c . In c o n t r a s t , during the summer - 5 -months s e m i - f o s s o r i a l h i b e r n a t o r s spend 65 to 75% of d a y l i g h t hours and 100% of night hours below ground i n the burrow and are, t h e r e f o r e , exposed c h r o n i c a l l y to hypoxic and hypercapnic c o n d i t i o n s . (Scheck and F l e h a r t y , 1980). F b s s o r i a l mammals show c e r t a i n r e s p i r a t o r y adaptations which are b e l i e v e d to be a response to c h r o n i c exposure to the environmental c o n d i t i o n s of the burrow (Boggs et a l . , 1982). F o s s o r i a l and s e m i - f o s s o r i a l species t y p i c a l l y have a higher O2 c a r r y i n g c a p a c i t y i n the blood than do n o n - f o s s o r i a l s p e c i e s . G e n e r a l l y high values f o r hematocrit ( H c t ) , hemoglobin c o n c e n t r a t i o n and red blood c e l l counts have been reported f o r these mammals (Ar et a l . , 1977; Baudinette, 1974; Boggs et a l . , 1982; Chapman and Bennett, 1975). In a d d i t i o n , a l e f t - s h i f t i n the O 2 -hemoglobin d i s s o c i a t i o n curve means that blood becomes f u l l y s a t u r a t e d at lower O2 p a r t i a l pressures, such as those present under burrow c o n d i t i o n s ( B a r t e l s et a l . , 1969; Baudinette, 1974; Boggs et a l . , 1982). These adaptations suggest that f o s s o r i a l mammals may be more t o l e r a n t t o at l e a s t moderate l e v e l s of hypoxia. During r e s t i n g c o n d i t i o n s both metabolic r a t e and minute v e n t i l a t i o n (V) are reduced compared to non-h i b e r n a t o r s of approximately the same s i z e . McNab (1966) and Hudson and Deavers (1973) reported that metabolic rates i n s e v e r a l f o s s o r i a l species were 20% to 60% lower than - 6 -expected on the b a s i s of body weight. In a d d i t i o n , v e n t i l a t o r y responses t o hypoxia and hypercapnia appear t o be s l i g h t l y a l t e r e d i n f o s s o r i a l s p e c i e s compared to non-f o s s o r i a l s p e c i e s ( A r i e l i and Ar, 1979; Boggs and K i l g o r e , 1983; Boggs et a l . , 1984; F a l e s c h i n i and Whitten, 1974; Holloway and Heath, 1984; Schlenker, 1985; Walker et a l . , 1985). A h i g h t o l e r a n c e to hypercapnia i s t y p i c a l of almost a l l f o s s o r i a l mammals and b i r d s (Boggs and K i l g o r e , 1983; Chapin, 1954; Holloway and Heath, 1984; Schlenker, 1985; Walker et a l . , 1985). The t h r e s h o l d f o r a v e n t i l a t o r y response t o i n s p i r e d CO2 appears t o be e l e v a t e d compared t o n o n - f o s s o r i a l mammals, while CO2 s e n s i t i v i t y i s reduced. Darden (1972) suggested, that the observed decrease i n r e s p i r a t o r y s e n s i t i v i t y t o CO2 may r e s u l t from an adjustment i n the s e n s i t i v i t y of the p e r i p h e r a l and c e n t r a l chemorecep-t o r s to a r t e r i a l CO2 t e n s i o n s or an adjustment i n the c e n t r a l i n t e g r a t i o n of in p u t s from chemoreceptors. The change i n CO2 s e n s i t i v i t y may a l s o r e f l e c t d i f f e r e n c e s i n the a b i l i t y of the blood to b u f f e r changes i n CO2 and hydrogen ions (H+). Few s t u d i e s have examined the hypoxic s e n s i t i v i t y of f o s s o r i a l mammals. These s t u d i e s i n d i c a t e t h a t burrow-d w e l l i n g animals are as s e n s i t i v e or more s e n s i t i v e to hypoxia than are n o n - f o s s o r i a l animals (Boggs and K i l g o r e ; - 7 -1983; Boggs et a l . , 1984; Holloway and Heath, 1984; McArthur, 1986; Walker et a l . , 1985). Thus, whereas a reduced v e n t i l a t o r y s e n s i t i v i t y to hypercapnia i s a common c h a r a c t e r i s t i c of burrow-dwelling mammals a reduced v e n t i l a t o r y s e n s i t i v i t y to hypoxia i s not. I t has been suggested that v e n t i l a t i o n i n awake hi b e r n a t o r s i s c o n t r o l l e d p r i m a r i l y by a r t e r i a l O2 tensions monitored by the p e r i p h e r a l chemoreceptors ( L e i t n e r and Malan, 1973). The adjustments which a l l o w f o r a decreased CO2 s e n s i t i v i t y i n burrow-dwelling animals i s unknown. These adjustments may be a r e s u l t of long term exposure to hypoxic and hypercapnic c o n d i t i o n s during development or they may be g e n e t i c a l l y determined. Mice and r a t s r a i s e d both pre- and p o s t - n a t a l l y under c o n d i t i o n s of c h r o n i c hypercapnia (6% CO2) or chro n i c normocapnia (0% CO2) show no d i f f e r e n c e i n t h e i r CO2 s e n s i t i v i t y . This r e s u l t suggests that CO2 s e n s i t i v i t y i s g e n e t i c a l l y determined rather than developmentally determined ( B i r c h a r d et a l . , 1984). In s p i t e of t h i s evidence, ch r o n i c exposure to a i r may a l t e r r e s p i r a t o r y responses to CO2 and O2 i n f o s s o r i a l s p e c i e s . These adjustments may lead to an ov e r e s t i m a t i o n of the a c t u a l hypercapnic response curve of these animals. In order to ensure that s i g n i f i c a n t m o d i f i c a t i o n s do not occur a f t e r long term a i r exposure i t i s necessary to compare v e n t i l a t o r y s e n s i t i v i t i e s of animals c h r o n i c a l l y exposed to a i r and c h r o n i c a l l y exposed to hypoxic and hypercapnic c o n d i t i o n s . - 8 -R e s p i r a t o r y p a t t e r n s during h i b e r n a t i o n Studies of deep h i b e r n a t i o n r e v e a l the occurrence of two d i s t i n c t v e n t i l a t o r y p a t t e r n s . In some s p e c i e s , such as the marmot and the Columbian ground s q u i r r e l , the r e s p i r a t o r y p a t t e r n c o n s i s t s of s i n g l e breaths i n t e r r u p t e d by breath-hold periods of 1-2 minutes i n length (Endres and T a y l o r , 1930; Malan et a l . , 1973). In other species episodes or bursts of r a p i d , continuous breathing are separated by long v a r i a b l e periods of breath h o l d i n g . This burst breathing p a t t e r n , o f t e n r e f e r r e d to as Cheyne-Stokes R e s p i r a t i o n (CSR), has been observed i n h i b e r n a t i n g hedgehogs, dormice, woodchucks, marmots, golden hamsters, golden-mantled ground s q u i r r e l s and bats ( f o r review see Malan, 1982). Although there i s no d i r e c t evidence to e x p l a i n the occurrence of the d i f f e r e n t p a t t e r n s , f o r the most part the d i f f e r e n c e s i n breathing p a t t e r n have been assumed to be species s p e c i f i c . Several r e s p i r a t o r y s t u d i e s i n v o l v i n g h i b e r n a t i n g species known to show burst b r e a t h i n g , however, have reported the occurrence of a s i n g l e breath p a t t e r n . K r i s t o f f e r s o n and Sovio (1964) reported that the burst breathing p a t t e r n i n the h i b e r n a t i n g hedgehog could be converted i n t o a s i n g l e breath p a t t e r n by decreasing ambient temperature (down to -5°C) or by handling the animal. Pajunen (1974) noted that i n a h i b e r n a t i n g dormouse, - 9 -disturbance caused a d i s r u p t i o n of the burst breathing p a t t e r n , as d i d decreases i n ambient temperature (Pajunen, 1984). Hammel et a l . , (1968), observed a burst breathing p a t t e r n i n the golden-mantled ground s q u i r r e l as long as body temperature, s p e c i f i c a l l y hypothalamic temperature, ranged from 5.5°C t o 10°C. I f hypothalamic temperature dropped below 5.5°C i n response to decreases i n ambient temperature, breathing took on a s i n g l e breath p a t t e r n . These observations suggest that v e n t i l a t o r y p a tterns may be temperature dependent during h i b e r n a t i o n and may a l t e r as a f u n c t i o n of a d i s t u r b e d versus an undisturbed s t a t e . Within a s i n g l e species each p a t t e r n could represent d i f f e r e n t l e v e l s of v e n t i l a t o r y c o n t r o l and v e n t i l a t o r y s e n s i t i v i t y to chemical s t i m u l i . The r o l e of p e r i p h e r a l chemoreceptors during h i b e r n a t i o n To date, s t u d i e s on v e n t i l a t o r y c o n t r o l during h i b e r n a t i o n have been l i m i t e d by the d i f f i c u l t i e s i n o b t a i n i n g v e n t i l a t o r y measures without d i s t u r b i n g the animal. Most s t u d i e s report only changes i n p a t t e r n and frequency during hypoxia and hypercapnia and are t h e r e f o r e , not q u a n t i t a t i v e . In a d d i t i o n , most s t u d i e s of v e n t i l a t o r y responses to chemical s t i m u l i during h i b e r n a t i o n have been concerned with responses to C O 2 . - 10 -CO2 e l i c i t s a f a i r l y strong r e s p i r a t o r y response during h i b e r n a t i o n . The t h r e s h o l d f o r e l i c i t i n g a v e n t i l a t o r y response i s v a r i a b l e , f a l l i n g between 1% and 4% CO2 (Biorch et a l . , 1956; Endres and T a y l o r , 1930; Lyman, 1951, McArthur, 1986). At l e v e l s between 5% and 7% i n s p i r e d CO2, p e r i o d i c breathing becomes continuous (McArthur, 1986; T a h t i , 1975) and long periods of severe hypercapnia o f t e n r e s u l t i n a r o u s a l from h i b e r n a t i o n ( T a h t i , 1975). Most st u d i e s report a strong frequency response to hypercapnia during h i b e r n a t i o n (Endres and T a y l o r , 1930; Lyman, 1951; T a h t i , 1975). Since, i n most non-hibernating animals, hypercapnia i s known to have a strong e f f e c t on V«r, i t i s l i k e l y that s t u d i e s using only frequency as an i n d i c a t i o n of v e n t i l a t o r y s e n s i t i v i t y tend to underestimate the t o t a l CO2 response. McArthur (1986) measured changes i n both t i d a l volume, frequency and minute v e n t i l a t i o n i n response to hypercapnia and found increases of over 500% i n minute v e n t i l a t i o n i n response to severe hypercapnia (7% C O 2 ) . In a d d i t i o n , McArthur (1986) found a s u b s t a n t i a l i n c r e a s e i n Vip during hypercapnic exposure. As e a r l y as the nineteenth century the extreme r e s i s t a n c e of h i b e r n a t o r s to hypoxia and anoxia was recognized ( H a l l , 1836). At l e a s t some of t h i s hypoxic tolerance during h i b e r n a t i o n i s thought to r e s u l t from decreases i n metabolic rate and from changes i n the oxygen-hemoglobin (HbC>2) d i s s o c i a t i o n curve of the blood. - 11 -Adjustments to the curve r e s u l t from a combination of body temperature changes, a s l i g h t r e s p i r a t o r y a c i d o s i s (Bohr e f f e c t ) , and a reduced 2,3 d i p h o s p h o g l y c e r a t e c o n c e n t r a t i o n i n the red blood c e l l s (Endres, 1930; Musacchia and V o l k e r t , 1971). These a l t e r a t i o n s r e s u l t i n a very s t e e p , l e f t -s h i f t e d Hb02 d i s s o c i a t i o n curve, such t h a t the blood remains completely s a t u r a t e d down to l e v e l s o f about 14-25 T o r r (Endres, 1930; Musacchia and V o l k e r t 1971). Thus, h i b e r n a t i n g animals can p o t e n t i a l l y t o l e r a t e extremely low l e v e l s of i n s p i r e d O2 without having any d e s a t u r a t i o n o f the blood. Most s t u d i e s c o n cerning hypoxic t o l e r a n c e r e v e a l t h a t h i b e r n a t i n g animals show l i t t l e or no hypoxic s e n s i t i v i t y ( B i o r c h et a l . , 1956; McArthur, 1986). I t appears that i n some s p e c i e s i n s p i r e d O2 can be reduced t o l e v e l s which cause c e n t r a l v e n t i l a t o r y d e p r e s s i o n and death (1% t o 5% O2) b e f o r e any observed v e n t i l a t o r y s t i m u l a t i o n occurs ( B i o r c h et a l . , 1956; McArthur, 1986). The l a c k of a v e n t i l a t o r y response even d u r i n g severe hypoxia has caused most p r e v i o u s i n v e s t i g a t o r s t o conclude t h a t changes i n O2 p l a y l i t t l e or no r o l e i n v e n t i l a t o r y c o n t r o l d u r i n g h i b e r n a t i o n ( B i o r c h et a l . , 1956; S t e f f e n and R e i d e s e l , 1982; T a h t i , 1975; T a h t i et a l . , 1981). In c o n t r a s t , T a h t i (1975) observed a v e n t i l a t o r y response t o hypoxia a t 16% O2 i n the hedgehog. As O2 l e v e l s decreased, b r e a t h i n g frequency i n c r e a s e d u n t i l , a t 3% O2, b r e a t h i n g became - 12 -continuous. Additionally/ McArthur (1986) noticed increases in minute v e n t i l a t i o n with 3% inspired O2 i n Spermophilus  l a t e r a l i s . It i s possible that the extreme hypoxic tolerance seen i n hibernation i s due, i n part, to adjustments i n the sensory input from the peripheral chemoreceptors or the central integration of the peripheral inputs. The role that c a r o t i d body chemoreceptors play i n v e n t i l a t o r y control during hibernation i s uncertain. Available evidence suggests that peripheral chemoreceptors may play an important role i n the generation of periodic breathing i n euthermic mammals. L a h i r i et a l . (1983) described a strong p o s i t i v e c o r r e l a t i o n between high peripheral chemoreceptor s e n s i t i v i t y and the advent of burst breathing (CSR) i n humans at high a l t i t u d e . In addition, studies on euthermic cats indicate that i n t a c t peripheral chemoreceptors are a prerequisite to the induction of periodic breathing (Cherniak et a l . , 1979). It i s not known i f the respiratory models for the production of periodic breathing can be applied to periodic breathing i n euthermic mammals during hibernation or i f inta c t peripheral chemoreceptors are a prerequisite for the occurrence of burst breathing. It i s evident that there i s information a v a i l a b l e - 13 -on the ven t i l a to ry patterns and v e n t i l a t o r y s e n s i t i v i t i e s to hypoxia and hypercapnia in both f o s s o r i a l mammals and hibernat ing mammals. Few studies have, however, attempted to examine the mechanisms of v e n t i l a t o r y con t ro l i n these mammals. Furthermore, few studies have examined the r e l a t i o n s h i p between ven t i l a to ry cont ro l in an awake f o s s o r i a l mammal and a h ibernat ing f o s s o r i a l mammal. Given the preceeding d iscuss ion the purpose of t h i s study was t h r e e f o l d : (a) to determine i f chronic exposure to hypoxic and hypercapnic cond i t ions , s i m i l a r to those experienced in a burrow environment a f f e c t s v e n t i l a t o r y responses to hypoxia and hypercapnia in awake golden-mantled ground s q u i r r e l s ; (b) to descr ibe the cont r ibut ion of c a r o t i d body chemoreceptors to the resp i ra tory responses of awake and hibernat ing golden-mantled ground s q u i r r e l s and to the production of a burst breathing pat tern in the h ibernat ing animal; and (c) to cor re la te the two reported resp i ra tory patterns seen during hibernat ion to body and ambient temperature and to determine i f the two patterns represent d i f f e r e n t l e v e l s of v e n t i l a t o r y cont ro l i n a s ing le spec ies . - 14 -MATERIALS & METHODS A l l studies were carried out on female golden-mantled ground squirrels (Spermophilus lateralis) obtained from Redding, California. Squirrels were trapped by a supplier in the spring of 1984 and 1985 and were shipped by air to U.B.C. The age of the ground squirrels ranged from f i r s t year juveniles to adults. Squirrels were housed either individually or in pairs in plexiglass cages (45 cm x 25 cm x 20 cm) with wire mesh l i d s . Cages were f i l l e d with wood shavings and cotton wool for nesting material and were cleaned weekly during the summer and monthly during the winter. Purina Lab Chow and water were provided ad libitum during both the summer and the winter. The squirrels' diet was supplemented with fresh f r u i t and vegetables during the summer. From May to October (summer, active season) the ground squirrels were housed in an environmental chamber maintained at an ambient temperature of 20.0 + 0.5°C and a photoperiod of 12 hours light and 12 hours dark (12L:12D). One half of the squirrels used in these experiments were housed in room ai r , while the other half were housed under chronic hypoxic and hypercapnic conditions (CHH animals). Animals chronically exposed to 17-18% O2 and 3-4% - 15 -C O 2 were housed i n p a i r s i n a l a r g e a i r t i g h t p l e x i g l a s s chamber mimicking n a t u r a l burrow c o n d i t i o n s (see i n t r o d u c t i o n ) . During the summer months, when the s q u i r r e l s were a c t i v e , these chronic gas l e v e l s were e s t a b l i s h e d by s e a l i n g the chamber u n t i l the f r a c t i o n a l c o n c e n t r a t i o n of O 2 ( F 0 2 ) dropped and the f r a c t i o n a l c o n c e n t r a t i o n of C O 2 (FQ02) increased to the d e s i r e d l e v e l . This u s u a l l y occurred w i t h i n 2 to 3 hours. These chr o n i c gas l e v e l s were then maintained by a slow, steady flow of a i r through the p l e x i g l a s s chamber. Chamber gases were monitored d a i l y throughout the summer wit h Beckman O 2 (OM-11) and C O 2 (LB-2) gas analysers c a l i b r a t e d d a i l y with room a i r and pre-mixed 5% and 10% C O 2 (Radiometer GMA 2 p r e c i s i o n gas mixing pump). A i r flow rates through the chr o n i c chamber were adjusted to hold the chamber gas composition at the d e s i r e d l e v e l . The ch r o n i c chamber was opened no more than twice per week except during periods of data c o l l e c t i o n . During the h i b e r n a t i o n p e r i o d (November to May), when the metabolic rates of the ground s q u i r r e l s were extremely low, appropriate l e v e l s of F Q 2 and Fc02 were obtained by mixing a i r , N 2 and C O 2 w i t h c a l i b r a t e d flow meters. Chamber gas composition was monitored every second day and the N 2 , C O 2 and a i r flows were adjusted as r e q u i r e d . CHH animals were not s t u d i e d while i n h i b e r n a t i o n , but were maintained under chr o n i c hypoxic and hypercapnic c o n d i t i o n s throughout the w i n t e r . A minimum of 2 months of continuous - 16 -exposure to the hypoxic and hypercapnic c o n d i t i o n s was requ i r e d f o r an experimental animal to be considered as having been c h r o n i c a l l y exposed to these c o n d i t i o n s . Since a l l awake, euthermic s t u d i e s were c a r r i e d out i n the summer of 1985, the m a j o r i t y of the ground s q u i r r e l s i n t h i s experimental group had been exposed to chr o n i c c o n d i t i o n s f o r over 10 months p r i o r to being used i n experiments. SURGERY C a r o t i d Sinus Denervations In one h a l f of both a i r exposed s q u i r r e l s (n=8) and CHH s q u i r r e l s (n=8) the c a r o t i d sinus nerves were se c t i o n e d b i l a t e r a l l y (CBX s q u i r r e l s ) . The c a r o t i d sinus nerve innervates the c a r o t i d body chemoreceptors which are l o c a t e d at the b i f u r c a t i o n of the common c a r o t i d a r t e r y . A l l surgery was performed on ground s q u i r r e l s during the a c t i v e phase of t h e i r y e a r l y c y c l e . In the f a l l of 1984 c a r o t i d sinus denervations were performed on 8 c o n t r o l animals and 8 CHH animals and three a d d i t i o n a l operations were performed the subsequent summer i n order to replace ground s q u i r r e l s which d i d not s u r v i v e the e n t i r e season. - 17 -Ground s q u i r r e l s were a n e s t h e t i z e d w i t h sodium p e n t o b a r b i t a l (Somnotol, MTC pharmacuticals, M i s s i s s a u g a , Canada) d e l i v e r e d i n t r a p e r i t o n e a l ^ (6.5 mg/100 g ) . Once deep r e f l e x e s were a b o l i s h e d , but p r i o r t o any surgery a c o n t r o l t e s t of the v e n t i l a t o r y responses of each animal to s e v e r a l r e s p i r a t o r y s t i m u l i was performed. V e n t i l a t o r y flow was measured wi t h a pneumotacho-graph mask u n i t . A face mask was made using the end of a p l a s t i c s y ringe l i n e d with p l a s t i c i n e . To minimize dead space the mask was molded by i m p r i n t i n g the snout of a dead ground s q u i r r e l i n the p l a s t i c i n e and covering t h i s w i t h expoxy. A small p l e x i g l a s s pneumotachograph was attached t o the end of the mask. T o t a l dead space f o r the u n i t was .15 ml. The pneumotachograph-mask u n i t was placed on the snout of the an e s t h e t i z e d ground s q u i r r e l and he l d secure w i t h adhesive tape. The pneumotachograph was connected to a d i f f e r e n t i a l pressure transducer (Validyne model DP 103-18, Northridge, C a l i f o r n i a ) i n order to measure a i r flow changes across the pneumotachograph membrane. The s i g n a l was a m p l i f i e d to record v e n t i l a t o r y p a t t e r n and frequency (Gould transducer a m p l i f i e r model 13-4615-50 or Gould D.C. a m p l i f i e r model 13-4615-10). C a l i b r a t i o n s of the pneumota-chograph were performed a f t e r each experiment by pumping known volumes of a i r across the pneumotachograph v i a the face mask. Twice a year the pneumotachograph was checked f o r l i n e a r i t y by pumping a l a r g e range of volumes across the membrane. - 18 -Once s t a b l e r e s p i r a t i o n was recorded the i n s p i r e d gas was q u i c k l y changed to e i t h e r 100% O2 (hyperoxia) or 100% N2 (anoxia). The acute changes i n v e n t i l a t o r y frequency and a i r flow during 5-10 breaths of each gas were recorded from the anes t h e t i z e d s q u i r r e l (Figure 1 ) . Responses t o hyperoxia and anoxia were f a s t , u s u a l l y occuring w i t h i n 4-5 breaths, but the magnitude of the responses v a r i e d between animals p o s s i b l y due to d i f f e r e n c e s i n the l e v e l of anesthesia. In ge n e r a l , i n t a c t s q u i r r e l s showed an increase i n v e n t i l a t o r y frequency and an inc r e a s e i n the height of v e n t i l a t o r y d e f l e c t i o n i n response to anoxia and a decrease i n v e n t i l a t o r y frequency i n response to hyperoxia (Figure 1 ) . A f t e r measuring the v e n t i l a t o r y responses to anoxia and hyperoxia, the s q u i r r e l was prepared f o r surgery. Fur was shaved from the upper chest and neck re g i o n , and the s k i n was sprayed w i t h a n t i s e p t i c . A midventral i n c i s i o n , approximately 2 cm long, was made from the base of the jaw to the sternum. On one s i d e of the trachea the common c a r o t i d a r t e r y , the c a r o t i d b i f u r c a t i o n and the base of the i n t e r n a l and e x t e r n a l c a r o t i d a r t e r i e s were l o c a t e d and exposed. The common c a r o t i d a r t e r y was l i g a t e d with s u r g i c a l s i l k and, with the use of a d i s s e c t i n g microscope the c a r o t i d sinus nerve was i s o l a t e d and cut as c l o s e as p o s s i b l e to the glossopharyngeal nerve. The c a r o t i d sinus nerve was then traced back to where i t innervated the - 19 -F i g u r e 1. The e f f e c t s of i n h a l a t i o n of 5 to 10 breaths of 100% O2 (hyperoxia) and 100% N2 (anoxia) on r e s p i r a t o r y frequency and depth both before c a r o t i d body denervation and a f t e r c a r o t i d body denervation i n S. l a t e r a l i s . CAROTID BODY DENERVATION before CBX air |l0aVoO2| air FREO.(MIN") 42 30 24 24 DEFL(MM) 18 10 It 17 after CBX air |100%0 2| FREQ (MIN-) 24 24 DEFL (MM) 19 19 - 21 -c a r o t i d sinus and cut again. The l i g a t u r e was then removed from the c a r o t i d a r t e r y , and any bleeding i n the area was stopped by applying pressure. The same procedure was then repeated on the c o n t r o l a t e r a l c a r o t i d sinus nerve. A f t e r removal of both c a r o t i d sinus nerves the ground s q u i r r e l was f i t t e d w ith the pneumotachograph mask u n i t and the v e n t i l a t o r y responses to hyperoxia and anoxia were measured once again. I f the ground s q u i r r e l showed no immediate response ( w i t h i n 5-10 breaths) to e i t h e r anoxia or hyperoxia the denervation was considered s u c c e s s f u l (Figure 1) and the i n c i s i o n sewn c l o s e d . I f the responses t o anoxia and hyperoxia were not completely a b o l i s h e d another attempt was made to i s o l a t e and s e c t i o n the c a r o t i d sinus nerves. I f c a r o t i d sinus denervation was not complete f o l l o w i n g t h i s second attempt, the animal was not used i n any experiments. Animals were allowed to recover from anesthesia and given Penbritin-250 ( A m p i c i l l i n sodium, USP-Ayerst; 2.5mg/animal) i n t r a m u s c u l a r l y . These animals were allowed to recover f o r at l e a s t 2 months before being used i n any experiments or being induced i n t o h i b e r n a t i o n . Ten months a f t e r surgery the v e n t i l a t o r y responses to anoxia and hyperoxia were measured once more, as p r e v i o u s l y d e s c r i b e d , on CBX s q u i r r e l s . A l l CBX animals showed a reduced or absent v e n t i l a t o r y response to anoxia and hyperoxia as compared to i n t a c t ground s q u i r r e l s . - 22 -At the completion of a l l experiments sodium cyanide (NaCn) t e s t s were c a r r i e d out on s u r v i v i n g CBX c o n t r o l s q u i r r e l s (n=4). Sodium cyanide, i n low c o n c e n t r a t i o n s , binds s p e c i f i c a l l y to a r t e r i a l chemoreceptors, d i s p l a c i n g oxygen and a c t i n g as an acute hypoxic s t i m u l u s . Since NaCn i s q u i c k l y metabolized, i t s a c t i o n s are t r a n s i e n t at low conce n t r a t i o n s . In the absence of a r t e r i a l chemoreceptors, p a r t i c u l a r l y c a r o t i d body chemoreceptors, there should be no v e n t i l a t o r y response to NaCn (Bouverot et a l . , 1973) or a s l i g h t v e n t i l a t o r y response, mediated by the a o r t i c chemoreceptors ( L a h i r i et a l . , 1981). Thus, i n the CBX s q u i r r e l s , there should be l i t t l e or no response to NaCn. Ground s q u i r r e l s were ane s t h e t i z e d as p r e v i o u s l y described and the i n n e r , r i g h t hind l e g shaved and sprayed w i t h a n t i s e p t i c . A 1-2 cm i n c i s i o n was made and the femoral v e i n i s o l a t e d and l i g a t e d . The v e i n was cannulated using P.E. 20 tubi n g . Breathing frequency was measured using e i t h e r a pneumotachograph-mask u n i t , as p r e v i o u s l y d e s c r i b e d or a strain-gauge. The strain-gauge was attached to the sternum of the anesthetized animal and measured r e s p i r a t o r y d e f l e c t i o n of the thorax. The strain-gauge method gave an i n d i c a t i o n of both r e s p i r a t o r y frequency and depth of r e s p i r a t i o n . A c o n t r o l i n j e c t i o n of 0.2 ml s a l i n e i n t o the femoral v e i n was used to ensure that pressure or temperature changes caused by drug i n j e c t i o n d i d not a f f e c t v e n t i l a t i o n (Figure 2). I n j e c t i o n s of 0.08 mg NaCn ( i n 0.2 ml s a l i n e ) - 23 -Fig u r e 2. The e f f e c t s on r e s p i r a t o r y frequency and depth of intravenous i n j e c t i o n s of 0.2 ml s a l i n e and 0.08 mg NaCn or 0.16 mg NaCn (0.2 ml s a l i n e ) i n i n t a c t S. l a t e r a l i s and c a r o t i d body denervated (CBX) S. l a t e r a l i s r e s p e c t i v e l y . 0.2ml saline t CONTROL 0.2 ml saline CBX iiiiniiiiiii i i 20 s e c ^6mg NaCn (.2 ml) •MPN0NI - 25 -i n c o n t r o l animals and 0.16 mg NaCn ( i n 0.2 ml s a l i n e ) i n CBX animals were then made and the v e n t i l a t o r y responses of the animals recorded. In a l l c o n t r o l animals an immediate and pronounced frequency response was e l i c i t e d by the NaCn i n j e c t i o n (Figure 2). In c o n t r a s t , none of the CBX animals e x h i b i t e d a v e n t i l a t o r y response to NaCn i n j e c t i o n s . I t was assumed, from these r e s u l t s , that a l l the CBX animals remained denervated throughout the e n t i r e study. Body Temperature Implants P r i o r to h i b e r n a t i o n , a l l experimental s q u i r r e l s were c h r o n i c a l l y implanted w i t h e l e c t r o d e s f o r the measurement of core body temperature (Tfc,). The ground s q u i r r e l s were an e s t h e t i z e d as p r e v i o u s l y d e s c r i b e d . Once deep r e f l e x e s were a b o l i s h e d , f u r was shaved from the abdomen and the s k i n sprayed with a n t i s e p t i c . An i n c i s i o n was made through the s k i n and then through the body w a l l . A thermistor (Fenwall E l e c t r o n i c s , Massachusetts, U.S.A.) coated w i t h epoxy and p a r a f f i n wax was placed i n the abdominal c a v i t y and the inner i n c i s i o n c l o s e d . A double flanged p l e x i g l a s s button c o n t a i n i n g connector p i n s attached to the thermistor was sewn i n t o the outer i n c i s i o n a l l o w i n g the outer s k i n to be c l o s e d . P o s t - o p e r a t i v e l y the ground s q u i r r e l was given P e n b r i t i n i n t r a m u s c u l a r l y as p r e v i o u s l y d escribed. The thermistor s i g n a l was checked on a d i g i t a l d i s p l a y monitor. C a l i b r a t i o n s of each t h e r m i s t o r - 26 -were performed, p r i o r to surgery, i n a water bath over a range of approximately 1°C to 40°C. SUMMER PROTOCOL Measurement of V e n t i l a t i o n Table 1 presents a summary of r e s p i r a t o r y v a r i a b l e s measured and nota t i o n s made during the present study. In the awake ground s q u i r r e l , v e n t i l a t i o n was measured using whole body plethysmograph as described by E p s t e i n and Ep s t e i n (1980) and modified by Jacky (1978, 1980). A modified flow-through plethysmograph allowed constant f l u s h i n g of the chamber to reduce O 2 d e p l e t i o n and C O 2 accumulation (Figure 3 ) . The flow-through method a l s o allowed f o r r a p i d changes i n i n s p i r e d gas mixtures without d i s t u r b i n g the ground s q u i r r e l . Two i d e n t i c a l chambers were used; an animal chamber and a reference chamber (Figure 3 ) . Both chambers (23 cm x 14 cm x 13 cm) had an i n f l o w and outflow port (.9 cm i n diameter) f o r the d e l i v e r y of a i r and various gas mixtures. The top of each chamber was f i t t e d w i t h p o r t s to a l l o w each chamber to be connected to both a d i f f e r e n t i a l pressure transducer to measure r e s p i r a t i o n and a manometer to maintain equal pressures i n the two chambers. The animal chamber had an a d d i t i o n a l port f o r a therm i s t o r to measure chamber temperature ( T c ) . In a d d i t i o n , the animal chamber had a removable l i d which was f i t t e d w i t h - 27 -TABLE 1. L i s t of notation and units of respiratory variables measured and calculated i n the golden-mantled ground s q u i r r e l IS. l a t e r a l i s ) . NOTATION RESPIRATORY VARIABLE UNITS V Minute V e n t i l a t i o n ml /lOOg/min VT T i d a l Volume ml/lOOg £ Respiratory frequency breaths/mln T I Inspiratory time sec T E Expiratory time sec TTOT Total breath duration sec TE' Length of end inspiratory pause sec TNVP Length of nonventilatory period (interburst) sec TVP Length of breathing episode sec V02 Oxygen consumption ml O2/100g/hr VC02 CO2 production ml CO2/100g/hr Tl/TTOT Duty cycle -V/VQ2 A i r convection requirement ml air/ml 02 - 28 -F i g u r e 3. Schematic diagram of whole body plethysmograph arrangement used to measure v e n t i l a t i o n i n awake S. l a t e r a l i s . See t e x t f o r e x p l a n a t i o n . CALIBRATION PUMP REFRIGERATOR n FLOWMETER BALANCE MANOMETER MATCHED INLET RESISTANCE THERMISTOR ANIMAL CHAMBER u REFERENCE CHAMBER CO. ft O, ANALYZER u — s DFFERENTIAL 'PRESSURE TRANSDUCER INTEGRATER ft CHART RECORDER - 30 -four l a t c h e s and l i n e d w i t h neoprene to ensure an a i r t i g h t s e a l . The e n t i r e plethysmograph system was placed i n a dark r e f r i g e r a t o r i n order to minimize pressure disturbances from the room and disturbances to the animal. Using the plethysmograph method (Drorbaugh and Fenn, 1955) pressure changes i n the animal chamber r e l a t i v e to the reference chamber are created by the warming and humidifying of i n s p i r e d a i r during normal r e s p i r a t i o n . This pressure change i s considered to be p r o p o r t i o n a l to t i d a l volume (VT) and can be measured, as i n our system, by a d i f f e r e n t i a l pressure transducer (Validyne model DP103-18, Northridge, C a l i f o r n i a ) . C a l i b r a t i o n s of the system were performed at the end of each experiment w i t h the animal present i n the animal chamber. Known volumes of a i r were pumped i n t o the chamber at a frequency s i m i l a r to that of the animal's b r e a t h i n g . The c a l i b r a t i o n volume was chosen to produce a pressure d e l f e c t i o n at l e a s t 10 times as great as the pressure d e f l e c t i o n produced by the animal breathing (Jacky, 1978). A c c o r d i n g l y , f o r c a l i b r a t i o n s , the system gain was reduced by a f a c t o r of 10. C a l i b r a t i o n measurements were made wh i l e the a i r flow rate through the system was v a r i e d to ensure that the rate of a i r flow through the system had no e f f e c t on the - 31 -pressure d e f l e c t i o n s . In a d d i t i o n , measurements were made with and without the animal i n the chamber to determine whether the presence of an animal i n the chamber had any e f f e c t on the c a l i b r a t i o n pressure d e f l e c t i o n s . Expired volume i s c a l c u l a t e d using the formula; VE = P M x V c a i x T A ( P B - P CH2Q)  Peal [ T A ( P B - P CH20)-T C(PB " PAH20)] Drorbaugh and Fenn, 1955. where P M i s the measured pressure d e f l e c t i o n , P c a i i s the c a l i b r a t e d pressure d e f l e c t i o n , V c a i i s 1/10 the c a l i b r a t i n g volume, T c i s the temperature of the animal chamber (°K), PCH20 i s t n e water vapour pressure at T c, T A i s the body temperature of the animal and PAH20 i - s t n e water vapour pressure at T A (Jacky, 1978). E p s t e i n and E p s t e i n (1978) concluded that t h i s formula can le a d to an underestimation of V>r i f the assumption that expired gas returns t o ambient temperature and humidity before a subsequent i n s p i r a t i o n i s not met. Epst e i n and E p s t e i n (1978) th e r e f o r e proposed that e x p i r e d gas should be considered to be at nasal c o n d i t i o n s i n order to o b t a i n accurate V>P measurements. Jacky (1980) d e r i v e d a c o r r e c t i o n forumla to r e t r o s p e c t i v e l y c o r r e c t t i d a l volume - 32 -estimates produced by the Drorbaugh and Fenn formula; V E A C O R = 1 " ( T i / T T 0 T ) ( 1 - G A / G N ) where G A represents the pressure and temperature changes from a l v e o l a r to chamber c o n d i t i o n s (as i n the Drorbaugh and Fenn formula), G N represents the pressure and temperature changes from a l v e o l a r to nasal c o n d i t i o n s , Tj i s i n s p i r a t o r y time and T ^ O T i - s t o t a l breath d u r a t i o n . These two forumlas were used i n the c a l c u l a t i o n of V^. Simultaneous measurements of Vrp i n a n e s t h e t i z e d s q u i r r e l s using both the plethysmograph and pneumotachograph mask u n i t i n d i c a t e d that the two measurements were i n c l o s e agreement (Figure 4). Experimental P r o t o c o l A l l experiments were performed from 0700 to 1800 hours i n a s i x week pe r i o d i n J u l y and August 1985. At the s t a r t of each experiment a ground s q u i r r e l was placed i n the animal chamber and l e f t undisturbed on the c o n t r o l gas ( e i t h e r a i r or 17% O2 and 4% CO2 f o r c o n t r o l and C H H animals r e s p e c t i v e l y ) f o r a minimum of 1 hour to achieve r e s t i n g c o n d i t i o n s . T o t a l gas flow through the chamber was approximately 1 l i t r e per minute. This gas flow minimized the metabolic build-up of CO2 and d e p l e t i o n of O2 i n the animal chamber. R e s p i r a t o r y frequency was recorded at - 33 -F i g u r e 4. The r e l a t i o n s h i p between t i d a l volume values c a l c u l a t e d using the whole body plethysmograph and measured using a pneumotachograph i n two a n e s t h e t i z e d ground s q u i r r e l s exposed t o v a r y i n g l e v e l s of C O 2 . The dashed l i n e represents the l i n e of e q u a l i t y . See t e x t f o r d e s c r i p t i o n of methods. 22-2IH 1.8« o • / / / / / O O VT (ml) 1 6 1 plethysmograph o o o oo o / / o o /f o / . / / . . / oo o 8° / / * / 1.0-ID 12 U 1.6 1B VT (ml) pneumotachograph 2.0 22 - 35 -moderate chart speed (2.5 mm/sec) over a 2 minute p e r i o d . At the end of the i n i t i a l c o n t r o l gas exposure a breath i n g t r a c e , c o n s i s t i n g of a minimum of 15 breaths at high chart speed (lOmm/sec), was recorded to al l o w measurement of r e s p i r a t o r y pressure d e f l e c t i o n and r e s p i r a t o r y t i m i n g v a r i a b l e s . In a d d i t i o n , T c was recorded f o r each gas exposure. The ground s q u i r r e l was then exposed to v a r i o u s gas mixtures (see below) i n a random order and a l t e r n a t i n g w i t h the c o n t r o l gas. The chambers were f u l l y f l u s h e d w i t h gas mixtures i n about 5 minutes and the ground s q u i r r e l was maintained on the gas f o r an a d d i t i o n a l 15 minutes i n order to achieve a steady s t a t e v e n t i l a t o r y response. Breathing t r a c e s were then recorded as p r e v i o u s l y d e s c r i b e d . Traces were only recorded i f the animal was awake and nonactive. Test gas mixtures were as f o l l o w s ; hypoxia: 17, 12 and 8% f r a c t i o n a l i n s p i r e d O2 (F102) hypercapnia: 2, 4 and 6% f r a c t i o n a l i n s p i r e d CO2 ( FIC02) hypoxia/hypercapnia: 4% FJCO-2 i n 17r 12 and 8% FI02 hypoxia/Hypercapnia: 50% FJQ2 i n 0, 2 4 and 6% FIC02« Gas mixtures were produced using flow meters to mix 100% N2, CO2 and/or O2 with room a i r . Gas concentrations were continuously monitored with Beckman O2 and CO2 gas ana l y s e r s . Inflow and outflow gases of the animal chamber - 36 -were monitored to ensure complete chamber f l u s h i n g and adequate chamber flow. Barometric pressure (PB) w^S recorded at the beginning and end of each experiment. I f Pg v a r i e d over the course of the experiment an averaged value was used i n c a l c u l a t i o n s . Nasal temperature (Tjj) was measured on 3 occassions over the summer by i n s e r t i n g a c a l i b r a t e d thermistor bead approximately .5cm i n t o the n o s t r i l . TJJ measurements of 32°C i n the awake ground s q u i r r e l are i n agreement wi t h measurements p r e v i o u s l y reported i n our l a b (McArthur, 1986) and reported by others (Schmid, 1976; Jacky, 1980; Fleming et a l . , 1 9 8 3 ) . A l l ground s q u i r r e l s were weighed at the end of the experiment. On average experiments l a s t e d 6 to 8 hours. Data A n a l y s i s The high speed r e s p i r a t o r y t r a c e s f o r each gas exposure were analysed f o r i n s p i r e d pressure d e f l e c t i o n . T i d a l volume (V^) was c a l c u l a t e d using the formula of Drorbaugh and Fenn (1955) as modified by Jacky ( 1 9 8 0 ) . The measurements of i n s p i r a t o r y time (Tj) and t o t a l breath d u r a t i o n (T^OT) used i n V<p c a l c u l a t i o n s were obtained from work done by McArthur (1986) on S. l a t e r a l i s . Values of Tj and T<TOT obtained i n t h i s study were checked against those of McArthur to ensure they were not s i g n i f i c a n t l y d i f f e r e n t . Frequency was c a l c u l a t e d by counting breaths i n at l e a s t s i x - 37 -ten second segments of steady s t a t e v e n t i l a t i o n . Each segment was m u l t i p l i e d by 6 to give breaths per minute. Mean values f o r f and V-p were c a l c u l a t e d f o r each i n d i v i d u a l s q u i r r e l and these mean values were used t o c a l c u l a t e o v e r a l l mean values f o r each experimental group. Minute v e n t i l a t i o n (V) was c a l c u l a t e d as the product of Vrj and f and expressed i n ml/min/lOOg f o r each s q u i r r e l and group means were c a l c u l a t e d . For the c a l c u l a t i o n of a i r convection requirement V/VQ2 i - n Figure 31 a value of 1.54 ml 02/nil/min/lOOg was used f o r VQ-2 values; t h i s was estimated from date on awake S. l a t e r a l i s ( H e l l e r , 1978) and S.  r i c h a r d s o n i i (Wang, 1978). Changes i n v e n t i l a t i o n w i t h i n experimental groups and between experimental groups i n response to gas mixtures were analysed using a s i n g l e c l a s s a n a l y s i s of variance (ANOVA) or a poin t to poi n t 1-way ANOVA. Trends were considered to be s i g n i f i c a n t l y d i f f e r e n t at P<.05 l e v e l unless otherwise s t a t e d . HIBERNATION PROTOCOL Animals were induced i n t o h i b e r n a t i o n i n e a r l y November i n two consecutive years. Over a 10 day p e r i o d photoperiod was decreased from 12L:12D to 2L:22D and temperature was decreased from 20°C to 5°C. Most ground - 38 -s q u i r r e l s entered h i b e r n a t i o n before the i n d u c t i o n p e r i o d was complete and a l l s q u i r r e l s were h i b e r n a t i n g w i t h i n one month of i n d u c t i o n . Experiments were not s t a r t e d u n t i l 1 month a f t e r i n i t i a l i n d u c t i o n i n t o h i b e r n a t i o n . Handling of the ground s q u i r r e l s Was minimized except during experimental p e r i o d s . Hibernation was terminated i n May: photoperiod and temperature were g r a d u a l l y returned t o summer l e v e l s . Experimental P r o t o c o l V e n t i l a t i o n during h i b e r n a t i o n was measured using the pneumotachograph-mask u n i t described e a r l i e r (Figure 5 ) . Only v e n t i l a t o r y responses of a i r c o n t r o l and a i r CBX s q u i r r e l s were s t u d i e d during h i b e r n a t i o n . H i b e r n a t i n g animals were placed i n a flow-through animal chamber ( p r e v i o u s l y described) s i t u a t e d i n a 500 cub i c i n c h r e f r i g e r a t o r . Temperature was c o n t r o l l e d by an e x t e r n a l r h e o s t a t . Gas exposure experiments were performed at two ambient temperatures; 6.0 ± 0.1°C and 2.4 ± 0.1°C. The movement of the s q u i r r e l s from the environment chamber i n t o the l a b o r a t o r y r e f r i g e r a t o r i n v a r i a b l y caused a r o u s a l from h i b e r n a t i o n . A f t e r a r o u s a l the s q u i r r e l was l e f t f o r a minimum of 24 hours to allo w re-entry i n t o h i b e r n a t i o n . A f t e r t h i s time the pneumotachograph-mask u n i t (as p r e v i o u s l y described) was secured to the snout and Tg leads connected when p o s s i b l e . This procedure sometimes i n i t i a t e d 39 -Figu r e 5. Schematic diagram showing the experimental arrangement used to record v e n t i l a t i o n during h i b e r n a t i o n i n S. l a t e r a l i s . See t e x t f o r d e s c r i p t i o n . refrigerator — i r to gas analyzers amplifier integrator 1 — 1 / r — 1 / « • ' c h a r t recorder differential pressure transducer o to gas analyzers face-mask + pneumotachograph - 41 -a r o u s a l , but u s u a l l y a f t e r some signs of disturbance ( i . e . movement and increased v e n t i l a t i o n ) the ground s q u i r r e l r e -entered deep h i b e r n a t i o n . Once h i b e r n a t i n g the ground s q u i r r e l was not d i s t u r b e d unless necessary and i n some cases animals wearing a mask would remain h i b e r n a t i n g f o r up to 7 days. When steady s t a t e v e n t i l a t i o n was achieved at 6.0 ± 0.1°C, a c o n t r o l r e s p i r a t o r y t r a c e was recorded from the animal while breathing a i r f o r 2 to 3 hours i n the burst breathing s t a t e . Chart recorder speed during t h i s p e r i o d was f a s t enough f o r the a n a l y s i s of v e n t i l a t o r y p a t t e r n (.25 mm/sec). Chart recorder speed was increased (2.0 mm/sec) and the i n t e g r a t e d s i g n a l recorded to o b t a i n b r e a t h i n g records f o r a n a l y s i s of V<r and r e s p i r a t o r y t i m i n g components. In burst breathing animals a minimum of one bu r s t , approximately 20 breaths, was recorded at high speed. A i r f l o w through the chamber was maintained at approximately 1 l i t r e / m i n u t e f o r a l l gases. A f t e r a i r c o n t r o l t r a c e s were obtained various gas mixtures (see below) were introduced i n a random order. Ground s q u i r r e l s were exposed to a i r a f t e r every t h i r d exposure to a t e s t gas. During burst b r e a t h i n g , when n o n v e n t i l a t o r y periods could l a s t f o r 30 to 40 minutes, gas exposures l a s t e d from 1.5 hours to 2 hours u n t i l a steady s t a t e response was achieved. Long a i r exposures were required a f t e r s t r e s s f u l gases ( i . e . 8% FiC02) an<* t n e animal was o f t e n l e f t overnight to recover. At the end of - 42 -each gas exposure, v e n t i l a t o r y t r a c e s were obtained as p r e v i o u s l y d e s c r i b e d . T c and Tb ( i f a v a i l a b l e ) were recorded f o r each gas mixture. Test gases f o r h i b e r n a t i n g animals were as f o l l o w s ; hypoxia: 10, 5 and 3% FJQ2 hypercapnia: 2, 4, 6 and 8% FJCQ 2 hypoxia/hypercapnia: 5% FJQ2 i n 21, 6 and 8% F J C Q 2 hyperoxia/hypercapnia: 50% FJO2 i n O, 2, 4, 6 and 8% F I C 0 2 Gases were created and d e l i v e r e d by mixing 100% N 2 , C O 2 and/or O2 w i t h a i r using c a l i b r a t e d flow meters. Gas composition was continuously monitored using Beckman O 2 and CG*2 gas a n a l y s e r s . Both i n f l o w and outflow gas concentrations were monitored to ensure proper f l u s h i n g of the animal chamber. T o t a l experiment time was approximately 30 hours, but animals were o f t e n maintained overnight on a i r and the experiment continued the next day. I f the animal aroused during the experiment, i t was l e f t i n the animal chamber u n t i l i t re-entered h i b e r n a t i o n and the experiment was then continued. At the end of each experiment the weight of the ground s q u i r r e l was recorded and c a l i b r a t i o n s f o r t i d a l volume measurements were performed. C a l i b r a t i o n s i n v o l v e d pumping known volumes of a i r across the pneumotachograph with a syringe and recording the i n t e g r a t e d a i r f l o w s i g n a l . - 43 -The t r a n s i t i o n from a burst breathing s t a t e to s i n g l e breath breathing s t a t e was examined simply by al l o w i n g animals to enter deep h i b e r n a t i o n at an ambient temperature of 6.9 + 0.1°C. Ambient temperature was then reduced by about 4°C to 1.8 ± 0.1°C. V e n t i l a t i o n was monitored as body temperature decreased p a s s i v e l y by a s i m i l a r magnitude, about 4°C. These experiments were c a r r i e d out s e p a r a t e l y from gas response experiments. Hibe r n a t i n g ground s q u i r r e l s were f i t t e d w i t h a pneumotachograph and Tg leads and allowed to reach a steady s t a t e breathing p a t t e r n . Records of the burst b r e a t h i n g p a t t e r n and frequency were long, u s u a l l y 2 to 3 hours. Chart recorder speed was increased at the end of t h i s p e r i o d to record V<r and r e s p i r a t o r y timing components (as p r e v i o u s l y d e s c r i b e d ) . Chamber temperature was then decreased and the new steady s t a t e temperature was reached w i t h i n 10 minutes. A short time l a t e r the body temperature of the s q u i r r e l began to d e c l i n e . The changes i n breath i n g p a t t e r n were monitored continuously at low chart recorder speed and every 15 minutes chart recorder speed was increased b r i e f l y to measure V j and r e s p i r a t o r y t i m i n g components. Tg was recorded, when p o s s i b l e , every 15 minutes. At the end of each experiment the weight of the ground s q u i r r e l was recorded, and c a l i b r a t i o n s of the pneumotachograph were performed as p r e v i o u s l y d e s c r i b e d . - 44 -Measurements of oxygen consumption (V02) and CQ2 production (Vco2) were obtained from h i b e r n a t i n g s q u i r r e l s at ambient temperatures of approximately 6°C and 1°C. H i b e r n a t i n g animals were placed i n an a i r t i g h t container f i t t e d w i t h two s y r i n g e s . A f t e r a two hour p e r i o d , i f a r o u s a l had not been i n i t i a t e d , gas samples were e x t r a c t e d from the c o n t a i n e r . Changes i n O2 and CO2 concentrations were measured wi t h Beckman O2 and CO2 gas a n a l y s e r s . VQ2 and VQC2 were c a l c u l a t e d by s u b t r a c t i n g the f i n a l O2 and CO2 concentrations from the i n i t i a l c o n c entrations and m u l t i p l y i n g the d i f f e r e n c e by the t o t a l volume to g i v e ml O2 consumed and ml CO2 produced over a known p e r i o d . S i n g l e breath breathing experiments were performed at ambient temperatures of 2.4 ± 0.1°C. S q u i r r e l s were maintained i n the r e f r i g e r a t o r , as p r e v i o u s l y d e s c r i b e d , u n t i l steady s t a t e r e s p i r a t i o n was achieved w i t h the pneumotachograph - mask u n i t i n p l a c e . P r o t o c o l f o r t h i s set of experiments was i d e n t i c a l to that used f o r burst b r e a t h i n g , except that gas exposures tended to be s h o r t e r (40 to 60 minutes i n length) because n o n v e n t i l a t o r y periods were l e s s than a minute during s i n g l e breath b r e a t h i n g . At the end of the experiment the animal's weight was recorded and c a l i b r a t i o n s f o r performed as p r e v i o u s l y d e s c r i b e d . In order to observe the e f f e c t s of a n e s t h e t i c on the burst breathing p a t t e r n during h i b e r n a t i o n , four - 45 -l a t e r a l i s , h i b e r n a t i n g at about 6°Cf were exposed to vaporous halothane. S q u i r r e l s were maintained i n the experimental set-up r wearing the penumotachograph-mask u n i t u n t i l steady s t a t e burst breathing was obtained. Only measurements of v e n t i l a t o r y p a t t e r n were recorded. Vaporized halothane was d e l i v e r e d to the animal chamber by passing the i n f l o w a i r through a v a p o r i z e r c a l i b r a t e d to d e l i v e r a n e s t h e t i c i n volumes percent ( v o l . % ) . Each burst breathing s q u i r r e l was exposed to g r a d u a l l y i n c r e a s i n g l e v e l s of halothane, u n t i l a change i n the burst b r e a t h i n g p a t t e r n was observed, at about 3 v o l . %. The s q u i r r e l was maintained on t h i s l e v e l of halothane f o r approximately 30 minutes, to ensure steady s t a t e r e s p i r a t i o n . Halothane was then removed from the i n f l o w gas and the animal was maintained on a i r u n t i l a burst breathing s t a t e had been achieved again. Data A n a l y s i s Breathing t r a c e s from both burst breathing and s i n g l e breath breathing animals were analysed f o r f , VIJ, T I ' TTOT a n& T E ' Burst breathing t r a c e s were f u r t h e r analysed f o r burst frequency (B/min), breaths per burst (b/B), burst d u r a t i o n ( T y p ) , i n t r a b u r s t end i n s p i r a t o r y pause (Tg') and length of the n o n v e n t i l a t o r y p e r i o d ( T f l v p ) . For the a n a l y s i s of v e n t i l a t o r y p a t t e r n and o v e r a l l f a - 46 -minimum of 40 minutes of recording f o r burst breathing animals and 20 minutes of recording f o r s i n g l e breath breathing animals was used. For the a n a l y s i s of other r e s p i r a t o r y v a r i a b l e s a minimum of 10-15 breaths (or 1 burst) was used. Minute v e n t i l a t i o n (V ) was c a l c u l a t e d from the product of f and V"p. O v e r a l l means f o r experimental groups were c a l c u l a t e d as p r e v i o u s l y d e s c r i b e d . S i n g l e breath data c o l l e c t e d during the win t e r s of 1984 and 1985 were compared using a one-way ANOVA and were not s i g n i f i c a n t l y d i f f e r e n t . The v e n t i l a t o r y responses to va r i o u s gas mixtures were t h e r e f o r e combined f o r these groups. S t a t i s t i c a l a n a l y s i s , both w i t h i n and between groups was performed as p r e v i o u s l y described. RESULTS AWAKE ANIMALS Re s t i n g V e n t i l a t i o n The r e s t i n g v e n t i l a t o r y p a t t e r n of a i r breathing S.  l a t e r a l i s i s shown i n Figure 6. Table 2 summarizes r e s p i r a t o r y v a r i a b l e s f o r a l l four experimental groups of s q u i r r e l s while breathing a i r . At r e s t , at an ambient temperature (T c) of 22 to 25°C a l l s q u i r r e l s show a s i m i l a r continuous breathing p a t t e r n . In both a i r breathing and chro n i c hypoxic and hypercapnic exposed (CHH) s q u i r r e l s c a r o t i d body denervation (CBX) r e s u l t s i n a decrease i n r e s t i n g V compared to i n t a c t animals (Table 1 ). In a i r breathing s q u i r r e l s CBX reduces f , such that V i s approximately 35% lower than i n i n t a c t animals. CHH CBX s q u i r r e l s show a s l i g h t decrease i n V<p wi t h l i t t l e change i n f such that o v e r a l l V i s only s l i g h t l y decreased compared to CHH c o n t r o l s q u i r r e l s . Minute v e n t i l a t o r y responses to hypoxia and hypercapni are s i m i l a r during acute and chro n i c exposure. The p a t t e r n of v e n t i l a t o r y response i s s l i g h t l y d i f f e r e n t i n the two groups. Animals respond to acute exposure to hypoxia and hypercapnia w i t h l a r g e r increases i n f and - 48 -TABLE 2. R e s t i n g v e n t i l a t o r y v a r i a b l e s d u r i n g a i r exposure i n awake golden-mantled ground s q u i r r e l s IS^ l a t e r a l i s ) . A l l values are mean +, standard e r r o r . See Table 1 f o r e x p l a n a t i o n of symbols. AIR CHRONIC INTACT CBX INTACT CBX n 8 7 6 5 Mass 162 + 6 174 + 3 168 + 8 161 + 12 (grams) Tc 2 5 . 7 + 0 . 5 2 5 . 9 + 0 . 7 2 5 . 4 + 0 . 9 2 2 . 9 + 0 . 6 (°C) £ 4 2 + 3 2 8 + 1 3 7 + 4 4 3 + 3 (breaths/rnin) VT 0 . 8 + 0 . 0 5 0 . 7 + 0 . 0 4 1 . 1 + 0 . 1 0 . 9 + 0 . 1 (ml/lOOg) $ 3 3 + 2 2 1 + 2 4 5 + 4 3 7 + 4 (ml/lOOg/min) - 49 -F i g u r e 6 . Representative breathing t r a c e s recorded by the whole body plethysmograph i n S. l a t e r a l i s at room temperature exposed t o a i r (A), 8% i n s p i r e d O2 (B) and 6% i n s p i r e d CO2 (C). - 50 -- 51 -smaller increases i n V"t than are observed i n CHH s q u i r r e l s . Animals c h r o n i c a l l y exposed t o hypoxia and hypercapnia show an elevated r e s t i n g V whi l e exposed to a i r i n both i n t a c t and CBX groups compared to a i r breathing c o n t r o l and CBX s q u i r r e l s r e s p e c t i v e l y . In CHH c o n t r o l s an increase of 35% i n V over a i r breathing c o n t r o l s , i s achieved through increases i n Vip. In CHH CBX s q u i r r e l s i ncreases of 76% i n V comapred to a i r breath i n g CBX s q u i r r e l s are mediated by increases i n V^ and f. (Table 1 ) . Response to Hypoxia The e f f e c t s of decreasing F J Q 2 on v e n t i l a t i o n i n a i r breathing and CHH S. l a t e r a l i s i s shown i n Figure 7 and Figure 8 . A l l four groups show a strong v e n t i l a t o r y response to hypoxia. A i r breathing i n t a c t S. l a t e r a l i s respond to severe hypoxia ( 8 % O 2 ) w i t h a 140% increases i n V over normoxic l e v e l s . The hypoxic response i s mediated s o l e l y through increases i n f (Table 3). Severe hypoxia ( 8 % O 2 ) a l s o causes a s i g n i f i c a n t decrease i n V^. V e n t i l a t o r y response threshhold f o r hypoxia f a l l s between 17% and 21% O 2 i n i n t a c t s q u i r r e l s . - 52 -Fi g u r e 7. E f f e c t of decreasing FJO2 o n minute v e n t i l a t i o n , t i d a l volume and frequency i n awake a i r breathing S. l a t e r a l i s ( • ) and awake a i r breathing CBX S.  l a t e r a l i s ( O ) • A l l values are mean ± standard e r r o r f o r 7 t o 8 animals. - 53 10 15 20 •a 5 10 15 20 - 54 -Fi g u r e 8. E f f e c t of decreasing F102 on minute v e n t i l a t i o n , t i d a l volume and frequency i n awake CHH S.  l a t e r a l i s ( • ) and awake CHH CBX S. l a t e r a l i s ( O ) • A l l values are mean ± standard e r r o r f o r 6 animals. - 56 -TABLE 3. Effects of alteration in inspired gas composition on the frequency (f, breaths/minute), t idal volume (Vj, ml/lOOg) and minute ventilation (V, ml/lOOg/min) of awake golden-mantled ground squirrels IS. lateral is ) . A l l values are mean £ standard error. AIR CHRONIC GAS INTACT CBX INTACT CBX n 8 7 6 6 AIR f 42 3 28 £ 1 37 +_ 4 43 £ 3 V T 0.8 £ 0. 05 0.7 £ 0. 04 1.1 £ 0.1 0.9 £ 0. 1 • V 33 2 21 £ 2 45 4 37 £ 4 17% 02 f 71 9 33 £ 5 38 +_ 2 32 £ 7 0% C02 v? 0.8 £ 0. 07 0.7 £ 0. 07 1.4 £ 0.2 1.1 £ 0. 1 V 56 +_ 6 24 £ 3 53 £ 7 35 £ 9 12% 02 f 105 +_ 9 67 £ 8 80 £ 5 70 £ 7 0% C02 VT 0.6 +_ 0. 04 0.6 +_ 0. 09 1.0 £ 0.1 0.8 £ 0. 05 V 68 +_ 5 39 £ 5 79 £ 4 58 £ 5 8% 02 f 131 ± 5 109 +_ 9 130 £ 9 99 £ 9 0% C02 VT 0.6 £ 0. 04 0.6 +_ 0. 07 0.9 £ 0.1 0.8 £ 0. 03 V 80 + 6 62 +_ 7 119 £ 14 75 £ 5 21% 02 f 40 £ 7 33 +_ 4 37 £ 4 46 £ 8 2% C02 v T 0.8 £ 0. 07 0.9 0. 06 1.1 £ 0.09 1.0 £ 0. 05 V 32 £ 4 28 3 39 £ 5 45 £ 5 - 57 -TABLE 3 cont... 21% 02 f 43 +_ 5 37 + 6 46 ± 4 39 +_ 6 4% C02 v T 1.0 +_ 0. 07 0.9 ± 0.07 1.4 ± 0.1 1.2 +_ 0.1 V 40 +_ 5 33 4 60 ± 6 51 +. 12 21% 02 f 47 +_ 6 49 +_ 9 47 4 52 +_ 5 6% C02 v T 1.1 +_ 0. 06 1.3 + 0.08 1.3 0.07 1.3 +_ 0.1 V 49 +_ 5 62 ± 10 61 ± 4 67 +_ 11 50% 02 £ 27 +_ 5 41 + 6 36 3 31 +. 7 0% CO 2 v T 0.8 +_ 0. 09 1.1 0.09 1.6 +_ 0.09 1.1 +_ 0.1 V 21 +_ 3 50 +_ 7 51 + 12 33 +_ 6 50% 02 £ 43 +_ 5 29 +_ 2 41 +_ 3 45 + 16 2% C02 VT 0.8 +_ 0. 1 0.9 +. 0.07 1.4 +_ 0.03 1.1 +_ 0.1 V 31 +_ 6 28 +_ 3 50 +_ 3 58 11 50% 02 f 36 +_ 5 37 +_ 3 45 +_ 3 33 + 6 4% C02 VT 0.9 0. 11 1.2 + 0.12 1.3 +_ 0.09 1.3 0.12 V 34 + 4 43 6 60 +_ 5 41 +_ 8 50% 02 £ 53 i 7 46 +_ 9 43 +_ 11 41 +_ 6 6% CO2 VT 1.1 +_ 0. 08 1.3 i 0.11 1.6 +_ 0.10 1.4 +. 0.16 V 50 +_ 4 61 13 69 +_ 10 62 ±_ 12 17% 02 £ 65 +, 7 47 i 5 47 i 5 42 +_ 6 4% C02 VT 1.0 +_ 0. 09 0.9 + 0.13 1.4 +_ 0.05 1.2 +_ 0.06 V 59 +_ 3 38 +_ 3 64 +_ 3 52 +. 8 12% 02 £ 62 +_ 4 48 9 50 +_ 5 57 +_ 9 4% C02 VT 0.9 +_ 0. 07 1.0 0.11 1-4 ± 0.09 1.1 +_ 0.08 V 60 +_ 8 52 +_ 8 70 10 60 +_ 14 8% 02 £ 105 +_ 8 86 +_ 9 98 + 11 72 +_ 9 4% C02 VT 0.9 +_ 0. 07 0 .8 +_ 0.07 1.2 0.12 1.0 + c 1.11 V 95 +_ 4 64 +, 8 110 + 10 68 +_ 8 - 58 -C a r o t i d body denervation, i n both a i r breathing and CHH s q u i r r e l s , r e s u l t s i n a downward s h i f t of the e n t i r e v e n t i l a t o r y response curve with l i t t l e change i n the o v e r a l l v e n t i l a t o r y s e n s i t i v i t y compared to i n t a c t c o n t r o l s i n each r e s p e c t i v e group (Figures 7 and 8). The downshifted response curve i s a r e s u l t of lower r e s p i r a t o r y frequencies at a l l l e v e l s of O2 i n a i r breathing CBX s q u i r r e l s and lower t i d a l volumes at a l l l e v e l s of O2 i n CHH CBX s q u i r r e l s . As i n i n t a c t s q u i r r e l s , a l l CBX animals responds to hypoxia through increases i n f. In a d d i t i o n , CBX causes a s l i g h t l e f t s h i f t i n the hypoxic response t h r e s h o l d down to between 12% and 17% O2 i n both a i r breathing and CHH groups. Chronic exposure to hypoxia and hypercapnia leads to an upward s h i f t i n the hypoxic response curve i n both i n t a c t and CBX s q u i r r e l s compared to r e s p e c t i v e a i r breathing groups (Figures 7 and 8) r e s u l t i n g from a maintained increase i n V<r at a l l l e v e l s of i n s p i r e d 02- The o v e r a l l magnitude of the v e n t i l a t o r y response to 8% O2 i s s l i g h t l y e l e v a t e d i n CHH c o n t r o l animals compared to a i r breathing c o n t r o l s q u i r r e l s . In c o n t r a s t , CHH exposure does not a l t e r the s e n s i t i v i t y of CBX s q u i r r e l s to hypoxia. Both groups of CHH s q u i r r e l s respond to hypoxia s o l e l y by increases i n f and both groups show s l i g h t decreases i n V-y during severe hypoxia. CHH exposure does not a l t e r hypoxic v e n t i l a t o r y t h r e s h o l d . - 59 -Response to Hypercapnia Fig u r e 9 and Figure 1 0 show the v e n t i l a t o r y responses of a i r breathing and CHH S. l a t e r a l i s to increa s e s i n FTC02* a 1 1 four groups e x h i b i t an increase i n V i n response t o hypercapnia. I n t a c t a i r breathing S. l a t e r a l i s responds t o 6% CO2 w i t h a comparatively moderate 5 8 % increase i n V . The increase i n V i s acheived p r i m a r i l y through s i g n i f i c a n t i ncreases i n V"T w i t h only s l i g h t increases i n f. The threshhold f o r the v e n t i l a t o r y response f a l l s between 2% and 4% C0 2. At low l e v e l s of C02r CBX s q u i r r e l s show a downshifted v e n t i l a t o r y response curve compared to i n t a c t animals, but as l e v e l s of hypercapnia increase to 6% CO2 there i s no s i g n i f i c a n t d i f f e r e n c e between absolute V values. The increased s e n s i t i v i t y to hypercapnia i n CBX s q u i r r e l s i s due to an elevated frequency response than i s seen i n i n t a c t s q u i r r e l s , both a i r breathing CBX and CHH CBX s q u i r r e l s increase V through more or l e s s equal increases i n V<r and f. Hypercapnic response thresholds are s l i g h t l y l e f t s h i f t e d by c a r o t i d body denervations, such that V increases i n response to hypercapnia at l e v e l s below 2% C0 2. - 60 -F i g u r e 9. E f f e c t of i n c r e a s i n g FJCO2 o n minute v e n t i l a t i o n , t i d a l volume and frequency i n awake a i r b r e a t h i n g S. l a t e r a l i s ( • ) and awake a i r breathing CBX S.  l a t e r a l i s ( O ) . A l l values are mean ± standard e r r o r f o r 7 to 8 animals. - 61 -MINUTE VENTILATION TIDAL VOLUME (ml/min/100g) (ml/100 g) FREQUENCY (min1) 0 2 * 6 'CO-0 2 4 - 62 -F i g u r e 10. E f f e c t of i n c r e a s i n g FJCO2 o n minute v e n t i l a t i o n , t i d a l volume and frequency i n awake CHH S.  l a t e r a l i s ( • ) and awake CHH CBX S. l a t e r a l i s ( O ) • A l l values are mean ± standard e r r o r f o r 6 animals. - 63 100+ F. r/o) •co2 - 64 -Chronic exposure to hypoxia and hypercapnia has l i t t l e e f f e c t on o v e r a l l s e n s i t i v i t y to hypercapnia. CHH exposure does r e s u l t i n an o v e r a l l e levated V response curve due to a maintained increase i n at a l l l e v e l s of C O 2 . Neither the hypercapnic response threshhold nor the p a t t e r n of v e n t i l a t o r y response was a l t e r e d by CHH exposure. Responses to hyperoxia, hyperoxic hypercapnia and hypoxic  hypercapnia The e f f e c t s of hyperoxia, hyperoxic hypercapnia and hypoxic hypercapnia on v e n t i l a t o r y responses are given i n Table 3. Figure 11 and Figure 12 shows the V responses of a l l four experimental groups to combined gas mixtures. In a i r breathing c o n t r o l s q u i r r e l s hyperoxia causes a 36% decrease i n V, which r e s u l t s s o l e l y from a s i g n i f i c a n t decrease i n f. In c o n t r a s t , hyperoxia causes a 53% increase i n V i n a i r breathing CBX animals. Adjustments i n V are produced p r i m a r i l y by increases i n V<r i n CBX s q u i r r e l s . In CHH c o n t r o l animals hyperoxia causes a s l i g h t i n crease i n V, produced by an increase i n Vr whereas i n CHH CBX s q u i r r e l s hyperoxia r e s u l t s i n no o v e r a l l change i n V. G e n e r a l l y the v e n t i l a t o r y responses to hyperoxia, p a r t i c u l a r l y i n CHH animals, are v a r i a b l e (Table 3). - 65 -O v e r a l l v e n t i l a t o r y responses to hypercapnia are not s i g n i f i c a n t l y a l t e r e d by the a d d i t i o n of 50% O2 i n any group of S . l a t e r a l i s (Figure 11 and Figure 12). O v e r a l l V responses to hypercapnia remained low r e l a t i v e to hypoxic responses. In a i r breathing c o n t r o l and CHH c o n t r o l s q u i r r e l s , although a hyperoxic background has no s i g n i f i c a n t e f f e c t on the o v e r a l l v e n t i l a t o r y response, v e n t i l a t o r y threshhold f o r CO2 response i s s h i f t e d s l i g h t l y t o the r i g h t i n both groups. As i n normoxia CHH c o n t r o l and CHH CBX s q u i r r e l s maintain an elevated hypercapnic response curve r e l a t i v e to a i r breathing groups mediated by an increased V"T at a l l l e v e l s of hypercapnia. In a l l four groups increases i n V are s t i l l caused p r i m a r i l y by increases i n Vrp. During acute exposure to 17% O2 and 4% CO2 (CHH c o n d i t i o n s ) a i r breathing s q u i r r e l s and CHH s q u i r r e l s e x h i b i t s i m i l a r V responses. The p a t t e r n of v e n t i l a t o r y response i s s l i g h t l y d i f f e r e n t i n the two groups, w i t h CHH s q u i r r e l s having a s i g n i f i c a n t l y higher and s i g n i f i c a n t l y lower f compared to a i r breathing c o n t r o l s q u i r r e l s . I n t a c t CBX s q u i r r e l s respond to acute exposure of 17% O2 and 4% O2 w i t h smaller increases i n V and V-r comapred to CHH CBX s q u i r r e l s . In g e n e r a l , a constant hypercapnic background (4% CO2) has l i t t l e e f f e c t on the o v e r a l l v e n t i l a t o r y response curve to hypoxia (Figure 11). O v e r a l l s e n s i t i v i t y to - 66 -Figure 11. E f f e c t of decreasing FJQ2 w i t h a hypercapnic background (A, 4% CO2) and i n c r e a s i n g FJCC-2 w i t n a hyperoxic background (B, 50% O2) on minute v e n t i l a t i o n i n awake a i r breat h i n g CBX S.  l a t e r a l i s ( • ) and awake a i r brea t h i n g CBX S.  l a t e r a l i s ( O ) . A l l values are mean ± standard e r r o r f o r 7 t o 8 animals. - 68 -F i g u r e 12. E f f e c t of decreasing Fi02 w i t h a hypercapnic background (A, 4% C O 2 ) and i n c r e a s i n g Fico2 w i t h a hyperoxic background (B, 50% O 2 ) on minute v e n t i l a t i o n i n awake CHH S. l a t e r a l i s ( • ) and awake CHH CBX S. l a t e r a l i s ( O ) • A l l values are mean ± standard e r r o r f o r 6 animals. - 70 -hypoxia i s s l i g h t l y reduced i n a l l groups, except a i r breathing c o n t r o l s q u i r r e l s . At moderate l e v e l s of hypoxia (21%, 17% and 12%) the hypercapnic background causes an increase i n V due to an elevated VIJ>. Vrp remains el e v a t e d i n a l l groups at a l l l e v e l s of hypercapnic hypoxia r e l a t i v e to normocapnic hypoxia. A hypercapnic background a l s o reduced the o v e r a l l frequency response to hypoxia by 10% to 17% i n a l l experimental groups. Thus, although absolute values of V were not reduced at 8% 0 2 / hypercapnia r e s u l t s i n an elevated V«r at a l l l e v e l s of O 2 and a blunted f response. A i r b reathing CBX s q u i r r e l s show a blunted hypoxic response during hypercapnia compared to a i r breathing c o n t r o l s q u i r r e l s (Figure 11). A i r breathing CBX s q u i r r e l s i ncrease V 60% during hypercapnic hypoxia (8% 0 2 / 4% C O 2 ) wh i l e i n c o n t r o l s q u i r r e l s increase V 150%. The decrease i n s e n s i t i v i t y i n CBX s q u i r r e l s i s due to a reduced Vp and f response. CHH CBX s q u i r r e l s e x h i b i t e d a blunted hypoxic response compared to i n t a c t CHH c o n t r o l s , s i m i l a r to that seen during normocapnia. HIBERNATING ANIMALS Resting V e n t i l a t i o n At an ambient temperature (Tc) of 6°C and a body temperature of 7°C golden-mantled ground s q u i r r e l s e x h i b i t a - 71 -burst breathing p a t t e r n . Figure 13 i l l u s t r a t e s a t y p i c a l burst breathing p a t t e r n under r e s t i n g c o n d i t i o n s . Both c o n t r o l and CBX S. l a t e r a l i s show s i m i l a r b u r s t i n g p a t t e r n s (Figure 13). Corresponding r e s t i n g v e n t i l a t o r y v a r i a b l e s are presented i n Table 4. The burst breathing p a t t e r n c o n s i s t s of a s e r i e s of r a p i d breaths f o l l o w e d by a long breath hold p e r i o d . I n d i v i d u a l b u r s t s are v a r i a b l e not only i n the number of breaths they c o n t a i n , but a l s o i n V<j>, f and tim i n g components w i t h i n the b u r s t . Several bursts were analysed, breath by breath, t o determine i f they resembled a Cheyne-Stokes b u r s t i n g p a t t e r n . Figure 14 i l l u s t r a t e s a burst which has f e a t u r e s t y p i c a l of a CSR p a t t e r n . Tj and T^ do not change over the course of the b u r s t , but there i s a waxing and waning of both frequency (measured by Tg) and V p . Figure 15 represents a much more uniform b u r s t , i n which there i s l i t t l e change i n T j , Tg, T E and Vrn of i n d i v i d u a l breaths through the b u r s t . V a r i a b i l i t y of the burst breathing p a t t e r n i s high both between d i f f e r e n t s q u i r r e l s , and i n an i n d i v i d u a l s q u i r r e l during a s i n g l e h i b e r n a t i o n bout. The number of breaths/burst (b/B) ranges from 2 or 3 to over 100 breaths/burst (x = 19 b/B i n c o n t r o l s ) , while T^ v p ranges from l e s s than 1 minute to over 40 minutes (x = 509 sec i n c o n t r o l s ) . In s p i t e of the l a r g e v a r i a b i l i t y i n p a t t e r n , - 72 -TABLE 4. Resting v e n t i l a t o r y v a r i a b l e s i n h i b e r n a t i n g golden-mantled ground s q u i r r e l s (S_. l a t e r a l i s ) . A l l values are mean £ standard e r r o r . See Table 1 f o r e x p l a n a t i o n of symbols. BURST BREATHING SINGLE BREATH BREATHING CONTROL CBX CONTROL CBX n 7 6 9 10 Hass (grams) 169 £ 7 179 +_ 9 155 £ 7 162 £ 5 T C <°C) 6.3 £ 0.1 5.7 +_ 0.1 2.5 £ 0.2 2.4 £ 0.2 T b (°C) 7.4 £ 7.2 +_ - in=4) Breaths/burst 19 +_ 2 18 + 4 - -TVP , , (sec) 84 +_ 9 79 21 - -™ V P (sec) 690 +_ 118 503 136 - -i i (sec) - - 23 £ 3 30 £ 4 f (breaths/min) 1.7 £ 0.2 2.2 £ 0.2 2.6 £ 0.2 2.1 £ 0.2 VT 1 (ml/lOOg) 0.67 £ 0.06 0.46 £ 0.02 0.63 £ 0.04 0 .71 £ 0.05 V (ml/min/lOOg) 1.1 + 0.08 1.0 £ 0.08 1.6 £ 0.1 1.4 £ 0.2 (m?/hr/100g) 2.0 0.1 2.0 £ 0.1 combined = 2.3 £ 0.1 Yml^hr/lOOg) 1.6 0.1 1.7 £ 0.1 combined - 2.1 £ 0.1 TTOT , (sec) 2.5 ± 0.08 2.3 £ 0.09 5.5 £ 0.01 5.5 £ 0.2 Tl 1 (sec) 1.0 0.03 1.0 £ 0.04 2.2 £ 0 . 1 2.3 £ 0 . 1 TE fc (sec) 1.5 +_ 0.03 1.3 £ 0.07 3.3 £ 0.1 3.2 £ 0.1 - 73 -F i g u r e 13. Representative records of r e s t i n g burst breathing p a t t e r n s i n h i b e r n a t i n g c o n t r o l and CBX S.  l a t e r a l i s at a T c of about 6°C. BURST BREATHING DURING HIBERNATION control CBX - 75 -Figu r e 14. A s i n g l e b u r st of breathing showing a Cheyne-Stokes p a t t e r n from S. l a t e r a l i s h i b e r n a t i n g at a T c of about 6°C. The bottom panel represents a breath by breath a n a l y s i s of i n s p i r a t o r y time ( T i ) , e x p i r a t o r y time ( T E ) / end i n s p i r a t o r y time (TE /) and t i d a l volume ( V ? ) . Note the changes i n r e s p i r a t o r y frequency and depth through the b u r s t . - 76 -BURST BREATHING PATTERNS Cheyne-Stokes 10 sec 2D 15 ID J5 T| feec) 2D 15 ID J5 Trr (sec) ttD 8.0 60 40 2D TE' (sec) to .8 J6 A 2 VT (ml/100g) 10 15 20 25 1 5 10 15 20 25 BREATH NUMBER - 77 -Figure 15. A s i n g l e burst of breaths from S. l a t e r a l i s h i b e r n a t i n g at a T c of about 6°C. The bottom panel represents a breath-by-breath a n a l y s i s of i n s p i r a t o r y time ( T j ) , e x p i r a t o r y time ( T E ) , end i n s p i r a t o r y pause (TE') and t i d a l volume (V«r). Note there i s l i t t l e change i n r e s p i r a t o r y frequency and depth through the b u r s t . - 78 -BURST BREATHING PATTERNS Regular i » 10 sec Tj (sec) 7g (sec) 20 15 10 .5 20 15 10 5' T£ (sec) V T (ml/100g) 80 60 2D mriTTTTlTITTOT 1 10 20 to JB J6 A 1 5 t) 15 20 BREATH NUMBER - 79 -o v e r a l l r e s t i n g frequency remains f a i r l y constant (Table 4). V e n t i l a t o r y frequency i s s l i g h t l y lower, while Vp i s s i g n i f i c a n t l y higher i n c o n t r o l s q u i r r e l s compared to CBX s q u i r r e l s . This r e s u l t s i n a s l i g h t l y higher r e s t i n g V i n c o n t r o l s q u i r r e l s (Table 4). During burst breathing T^OT i s s i m i l a r i n both c o n t r o l and CBX s q u i r r e l s . The r a t i o of T j to i s .7 f o r both groups. The duty c y c l e (the r a t i o of Tj to T ^ O T ) i s a l s o s i m i l a r i n both groups being, on average, 0.4 (Table 5). T^oT' T I A N ^ T E do not change s i g n i f i c a n t l y i n response to any gas mixture. Changes i n frequency are achieved s o l e l y by changes i n the end i n s p i r a t o r y pause (T^ or T ^ v p ) . Oxygen consumption (VQ2) was not measured simultaneously w i t h v e n t i l a t i o n and i t has been assumed that VQ2 values obtained are accurate f o r animals i n deep h i b e r n a t i o n at a T c of 6 to 7°C. VQ2 values f o r c o n t r o l and CBX s q u i r r e l s are not s t a t i s t i c a l l y d i f f e r e n t , the mean f o r both i n t a c t and CBX s q u i r r e l s i s 2.0 ml/hour/lOOg. During h i b e r n a t i o n s i n g l e breath breathing occurs i n S. l a t e r a l i s at T c of 2.5°C and Tb of about 4.9°C. Under normoxic and normocapnic c o n d i t i o n s the r e s p i r a t o r y p a t t e r n c o n s i s t s of s i n g l e breaths f o l l o w e d by an end i n s p i r a t o r y pause ( T E ) ranging from 10 to 60 seconds i n length (Figure 16). V e n t i l a t o r y f f o r c o n t r o l and CBX s q u i r r e l s i s - 80 -s i m i l a r , as i s V-j (Table 4). Thus, o v e r a l l V i s not d i f f e r e n t between c o n t r o l and CBX ground s q u i r r e l s . In a d d i t i o n , VQ2 values are not s i g n i f i c a n t l y d i f f e r e n t , being, on average, 2.2 ml/hour/lOOg. R e s p i r a t o r y timing v a r i a b l e s f o r c o n t r o l and CBX S.  l a t e r a l i s are not s i g n i f i c a n t l y d i f f e r e n t (Table 4). The ti m i n g v a r i a b l e d i d not change s i g n i f i c a n t l y i n response to any gas mixture. In approximately 15% to 20% of experiments i n v o l v i n g s i n g l e breath breathing s q u i r r e l s double i n s p i r a t i o n s were observed at one time or another (Figure 33). The double i n s p i r a t i o n s seldom continued over the course of the e n t i r e experiment and there was no apparent reason f o r t h e i r occurrence. T r a n s i t i o n Data Since breathing patterns of c o n t r o l and CBX s q u i r r e l s under normoxic c o n d i t i o n s are not s i g n i f i c a n t l y d i f f e r e n t , t r a n s i t i o n data represents combined values c o l l e c t e d from c o n t r o l and CBX s q u i r r e l s . Table 5 presents the changes which occur i n s e v e r a l r e s p i r a t o r y v a r i a b l e s during the t r a n s i t i o n from burst breathing (0% t r a n s i t i o n ) to s i n g l e breath breathing (100% t r a n s i t i o n ) . The e n t i r e t r a n s i t i o n r e q u i r e s between 3 to 4 hours to complete f o r most animals. As chamber temperature dropped from 6.9°C to 1.8°C body temperature underwent a s i m i l a r magnitude drop - 81 -TABLE 5. E f f e c t s of decreasing body temperature (Tfc,) on r e s p i r a t o r y v a r i a b l e s i n golden-mantled ground s q u i r r e l s (S. l a t e r a l i s ) . A l l values are mean £ standard e r r o r . See Table 1 or t e x t for- e x p l a n a t i o n of symbols. 0% 33% 66% 100% TRANSITION TRANSITION TRANSITION TRANSITION (burst breathing) ( s i n g l e breath) n 12 12 11 11 T c (°C) 6.9 +_ 0. 1 - - 1.8 £ 0. 1 T D (°C, n=4) 8.6 +_ 0. 2 6.2 £ 0. 4 4.8 £ 0. 5 4.4 £ 0. 5 T r a n s i t i o n Time (hours) 0 1.1 £ 0. 4 2.2 £ 0. 4 3.4 £ 0. 2 B r e a t h s / b u r s t 20.6 +_ 1. 9 3.1 £ 0. 2 1.4 £ 0. 06 1.0 T V P (sec) 89 + 7 12.4 £ 2. 0 9.3 ± 3. 6 5.5 £ 0. 4 Bursts/min 0.09 0. 01 0.5 £ 0. 03 1.0 £ 0. 06 2.0 £ 0. 2 f (breaths/min) 1.7 +_ 0. 1 1.6 £ 0. 09 1.6 £ 0. 1 2.0 £ 0. 1 V T 1 (ml/lOOg) 0.80 + 0. 03 0.65 £ 0. 04 0.78 £ 0. 05 0.74 £ 0. 05 V (ml/lOOg/min) 1.4 0. 1 1.4 £ 0. 1 1.3 £ 0. 1 1.5 £ 0. 1 TTOT , . (sec) 2.4 +_ 0. 2 4.0 £ 0. 1 4.7 £ 0. 1 5l 5 £ 0. 1 T T A (sec) 1.0 £ 0. 03 1.6 £ 0. 06 1.9 £ 0. 04 2.2 £ 0. 04 TE . . (sec) 1.5 0. 05 2.4 £ 0. 06 2.8 £ 0. 08 3.3 £ 0. 08 T I / T E 0.67 0.67 0.68 0.67 Duty Cycle T T I ? T T O T ) 0.42 0.40 0.40 0.40 Time a c t i v e l y b r e a t h i n g 7% 11% 13% 19% V T / T I 0.8 0.5 0.4 0.3 - 82 -Figure 16. Representative record of the breathing p a t t e r n t r a n s i t i o n from burst breathing at a T c of 7°C to s i n g l e breath breathing at a T c of 2°C i n S.  l a t e r a l i s during h i b e r n a t i o n . golden mantled ground squirrel •Wr 0 12 3 min. 'A ' ° - 84 -F i g u r e 17. Bar p l o t showing the e f f e c t s of decreasing ambient and body temperature on r e s p i r a t o r y v a r i a b l e s ; breaths per b u r s t , b u r s t s per minutes, o v e r a l l frequency, t i d a l volume, minute v e n t i l a t i o n , t o t a l breath d u r a t i o n ( T T Q T ) i n s p i r a t o r y time ( T j ) , e x p i r a t o r y time (TE) and the r a t i o of T J / T E . 0% t r a n s i t i o n represents burst breathing at a Tb of 8.6 ± 0.2°C and 100% t r a n s i t i o n represents s i n g l e breath b r e a t h i n g at a Tb of 4.4 ± 0.5°C. For 0% and 33% t r a n s i t i o n n=12 and f o r 66% and 100% t r a n s i t i o n n = l l . The v e r t i c a l l i n e on each bar represents one standard e r r o r of the mean. 33 66 100 TTOT (sec) MINUTE VENTILATION (ml/min/100g) T, (sec) - m] •XTS)!*' 1 4 0 33 66 100 T E (sec) 66 100 33 66 WO 33 66 100 % TRANSITION - 86 -Fig u r e 18. Bar p l o t of the e f f e c t s of decreasing ambient and body temperature on the end i n s p i r a t o r y pause (Tg) i n S. l a t e r a l i s during the t r a n s i t i o n from burst breathing to s i n g l e breath b r e a t h i n g . The graph represents t o t a l values f o r 11 t o 12 s q u i r r e l s . - 87 -BURST BREATHING V8.6±.1 TA=6.9±3 33% TRANSITION TB=&2±.2 TA=18±J SECONDS - 88 -from 8.6°C to 4.4°C. Although i s maintained s l i g h t l y above T C i t does decrease p a s s i v e l y as T C i s lowered. Figure 16 and Figure 17 i l l u s t r a t e the changes i n r e s p i r a t o r y p a t t e r n which occur during the t r a n s i t i o n from burst breathing to s i n g l e breath breathing. As f a l l s , the number of breaths/burst decreases u n t i l steady s t a t e s i n g l e breath breathing i s reached (Figure 16). During t h i s time Tflvp decreases so that the bursts become c l o s e r together. These changes i n T^vp (or TE') during the t r a n s i t i o n are f u r t h e r i l l u s t r a t e d i n Figure 18. During burst breathing i n t r a b u r s t T E i s very short and i n t e r b u r s t T E ( TNVP) i s very long. As the t r a n s i t i o n proceeds the i n t r a b u r s t Tg" lengthens as the breaths w i t h i n a burst g r a d u a l l y get f a r t h e r apart. I n t e r b u r s t T E shortens as the burs t s get c l o s e r together. This trend continues u n t i l steady s t a t e s i n g l e breath breathing i s achieved and " i n t r a b u r s t " and " i n t e r b u r s t " T E are i n d i s t i n g u i s h a b l e (Figure 18). Despite l a r g e changes i n r e s p i r a t o r y p a t t e r n , f remains r e l a t i v e l y constant throughout the t r a n s i t i o n (Figure 17). Conversely, V<r decreases s l i g h t l y during the breathing p a t t e r n t r a n s i t i o n . O v e r a l l V does not change throughout the t r a n s i t i o n . - 89 -Figur e 19. R e l a t i o n s h i p between t i d a l volume (Vj) and i n s p i r a t o r y time and t o t a l breath d u r a t i o n (Tj and T>TOT r e s p e c t i v e l y ) i n the burst breathing ( • ) and s i n g l e breath breathing ( 0 ) S.  l a t e r a l i s during h i b e r n a t i o n . Note the slope represents the r e l a t i o n s h i p of V T / T T . A l l r e s p i r a t o r y values represents mean standard e r r o r f o r 11 to 12 animals. Volume (ml/100g) Time (sec) - 91 -TTOT increases s t e a d i l y through the t r a n s i t i o n from 2.4 seconds during burst breathing to 5.5 seconds during s i n g l e breath breathing (Figure 19). P r o p o r t i o n a l increases i n T i and T^ occur such that Tj/Tg and T J / T T O T (the duty c y c l e ) do not change through the t r a n s i t i o n . The increase i n T<TQT with no increase i n v e n t i l a t o r y f r e s u l t s i n an increase i n the t o t a l time spent a c t i v e l y breathing i n s i n g l e breath breathing s q u i r r e l s . S i n g l e breath breathing animals spend approximately 18% to 24% of time a c t i v e l y b reathing whereas burst breathing s q u i r r e l s only spend 7% of time a c t i v e l y breathing (Table 5). Given that V<r does not change but Tj increases the r a t i o of V T / T J . decrease by a f a c t o r of 2 i n s i n g l e breath breathing animals r e l a t i v e to burst breathing animals (Table 5). Since V T / T J i s a measure of v e n t i l a t o r y d r i v e to breath, the decrease i n t h i s r a t i o i n d i c a t e s that the v e n t i l a t o r y d r i v e has decreased i n s i n g l e breath breathing s q u i r r e l s . Response to hypoxia Hypoxia has l i t t l e e f f e c t on r e s p i r a t i o n i n burst breathing s q u i r r e l s u n t i l severe l e v e l s are reached (3% FI02)« Figure 20 i l l u s t r a t e s t y p i c a l p a t t e r n changes which occur i n response to severe hypoxia. Both c o n t r o l and CBX - 92 -F i g u r e 20. Representative b r e a t h i n g t r a c e s recorded by pneumotachograph^ i n S. l a t e r a l i s during h i b e r n a t i o n at a T c of about 6°C exposed t o a i r (A), 3% i n s p i r e d 0 2 (B) and 8% i n s p i r e d CO2 (C). GOLDEN-MANTLED GROUND SQUIRREL BURST BREATHING A) air 1 min - 94 -Fig u r e 21. Bar graph showing the e f f e c t s of changing F J Q 2 and Fico2 o n breaths per burst and average non-v e n t i l a t o r y p e r i o d i n i n t a c t and CBX S. l a t e r a l i s d uring h i b e r n a t i o n a t a T c of about 6°C. A l l values are mean ± standard e r r o r f o r 5 to 7 animals. Dashed l i n e s represent i n t a c t (n=2) and CBX (n=l) animals who continued t o show a b u r s t i n g p a t t e r n at 6% CO2 and 8% CO2. See t e x t f o r e x p l a n a t i o n . = i n t a c t S. l a t e r a l i s = CBX S. l a t e r a l i s BREATHS PER BURST continuous 40 30 20 10 I At or air AVERAGE NON-VENTILATORY PERIOD (sec) 800 600 400 200 1 ::i TV? «-l -air 10 "'0: 5 (%) air F'co2 » - 96 -s q u i r r e l s r e t a i n a b u r s t i n g p a t t e r n even at 3% 0 2 (Figure 2 1 ) . In i n t a c t s q u i r r e l s as F J Q 2 decreases the number of breaths/burst (b/B) decreases as does TNVP (Figure 2 1 ) . These changes r e s u l t i n an o v e r a l l increase i n f (Figure 2 2 , Table 6 ). CBX s q u i r r e l s show a s i m i l a r decrease i n T^vp i n response to hypoxia, but show l i t t l e change i n the number of breaths/burst (Figure 13). The o v e r a l l V response to hypoxia i n c o n t r o l and CBX burst breathing s q u i r r e l s i s p l o t t e d i n Figure 2 2 . The magnitude of the v e n t i l a t o r y response i s s i m i l a r i n both c o n t r o l and CBX animals. In c o n t r o l s q u i r r e l s hypoxic v e n t i l a t o r y threshhold occurs between 2 1 % and 1 0 % 02f below which the response curve r i s e s s t e e p l y . CBX s q u i r r e l s e x h i b i t a s i g n i f i c a n t l e f t s h i f t i n the hypoxic response t h r e s h o l d w i t h l i t t l e or no increase i n V u n t i l F 1 0 2 f a l l s below 5% 0 2. In both groups v e n t i l a t o r y responses to hypoxia are mediated e x c l u s i v e l y by increases i n f. The t i d a l volume of CBX s q u i r r e l s remains s i g n i f i c a n t l y lower than c o n t r o l s over the range of hypoxic gases, r e s u l t i n g i n a downshifted response curve. During s i n g l e breath breathing n e i t h e r c o n t r o l nor CBX ground s q u i r r e l s e x h i b i t a s i g n i f i c a n t v e n t i l a t o r y response t o hypoxia, even at severe l e v e l s (3% O 2 ) . F igure 23 i l l u s t r a t e s the v e n t i l a t o r y responses to hypoxia during s i n g l e breath br e a t h i n g . Although at moderate l e v e l s of - 97 -T A B L E 6 . E f f e c t s o f a l t e r a t i o n s o f i n s p i r e d g a s c o m p o s i t i o n o n t h e f r e q u e n c y ( f , b r e a t h s / m i n u t e ) , t i d a l v o l u m e ( V j , m l / l O O g ) , a n d m i n u t e v e n t i l a t i o n ( V , m l / l O O g / m i n ) i n h i b e r n a t i n g g o l d e n - m a n t l e d g r o u n d s q u i r r e l s ( 5 . l a t e r a l i s ) . A l l v a l u e s a r e mean +_ s t a n d a r d e r r o r . N u m b e r s i n p a r e n t h e s e s r e p r e s e n t s a m p l e s i z e . BURST BREATHING SINGLE BREATH BREATHING CONTROL CBX CONTROL CBX AIR f 1.7 + 0 . 2 2.2 + 0. 2 2.6 + 0. 2 2.1 + 0. 2 v T 0.67 + 0.08 0.46 + 0. 02 0.63 + 0. 04 0.71 + 0. 05 • V 1.1 +0 .1 1.0 + 0. 1 1.6 + 0. 1 1.4 + 0. 2 (7) (7) (10) (10) 10% 02 f 2.5 + 0.4 2.3 + 0. 2 2.7 + 0. 4 1.8 + 0. 3 0% C02 v T 0.66 + 0.07 0.50 + 0. 02 0.73 + 0. 04 0.77 + 0. 06 V 1.6 + 0 . 3 1.1 + 0. 1 1.9 + 0. 3 1.2 + 0. 2 (5) (6) (8) (9) 5% 02 f 3.2 +0 .4 2.5 + 0. 2 3.4 + 0. 5 2.0 + 0. 3 0% C02 VT 0.66 + 0.06 0.41 + 0. 02 0.60 + 0. 04 0.63 + 0. 06 V 2.1 + 0 . 2 1.0 + 0. 1 1.8 + 0. 1 1.0 + 0. 1 (6) (6) (8) (9) 3% 02 f 4.5 + 0.4 5.4 + 0. 7 2.2 + 0. 4 2.2 + 0. 3 0% C02 V T 0.64 + 0.08 0.46 + 0. 05 0.64 + 0. 04 0.56 + 0. 04 V 2.4 +0 .4 2.4 + 0. 3 1.5 + 0. 3 1.1 + o .2 (6) (5) (7) (7) 21% 02 f 2.9 + 0.4 3.2 + 0. 5 3.4 + 0. 4 2.6 + 0. 3 2% C02 V T 0.71 £ 0.10 0.52 + 0. 06 0.66 + 0. 04 0.74 + 0. 05 V 1.9 + 0 . 2 1.7 + 0. 3 2.2 + 0. 3 1.8 + 0. 2 (6) (6) (8) (10) - 98 -TABLE 6 cont... 21% 02 f 6.4 + 0. 6 7.1 + 1. 3 4.8 + 0.4 3.8 + 0. 2 4% C02 V T 0.75 + 0. 09 0.52 + 0. 04 0.77 + 0.06 0.89 + 0. 05 V 4.6 + 0. 8 3.6 + 0. 5 3.6 + 0.3 3.1 + 0. 3 (6) (6) (8) (10) 21% 02 f 10.4 + 1. 5 9.9 + 1. 5 5.0 + 0.5 4.3 + 0. 4 6% C02 VT 0.77 + 0. 08 0.60 + 0. 02 0.96 + 0.03 0.88 +0 . 07 V 7.6 + 1. 4 5.9 + 1. 0 4.8 + 0.6 4.4 + 0. 6 (6) (6) (8) (10) 21% 02 f 10.4 + 1. 4 9.8 + 1. 4 5.2 + 0.4 5.6 + 0. 4 8% C02 V T 0.90 + 0. 08 0.81 + 0. 06 1.00 + 0.11 1.10 + 0. 13 V 9.3 + 1. 4 8.0 + 1. 3 4.9 + 0.4 6.2 + 0. 9 (6) (7) (5) (5) 50% 02 f 1.9 + 0. 2 2.3 + 0. 2 2.9 + 0.2 2.5 + 0. 3 0% C O 2 v T 0.66 ± 0. 04 0.52 + 0. 06 0.63 + 0.05 0.71 + 0. 05 V 1.3 + 0. 1 1.2 + 0. 1 1.7 +0 .2 1.7 + 0. 3 (6) (6) (8) (10) 50% 02 £ 2.8 + 0. 3 3.7 + 0. 8 3.2 +0 .2 2.6 + 0. 3 2% C02 V T 0.64 + 0. 04 0.52 + 0. 01 0.73 + 0.05 0.75 + 0. 05 V 1.9 + 0. 2 1.9 + 0. 4 2.3 +0 .2 1.8 + 0. 2 (6) (6) (8) (10) 50% 02 f 5.5 + 0. 5 6.9 + 1. 0 4.7 +0 .3 3.6 + 0. 2 4% C02 V T 0.73 + 0. 05 0.54 + 0. 04 0.83 + 0.07 0.87 + 0. 06 • V 3.9 + 0. 3 3.7 + 0. 5 4.0 + 0.4 3.1 + 0. 3 (5) (6) (7) (10) 50% 02 f 11.3 + 1. 1 10.5 + 1. 7 5.0 + 0.'3 4.9 + 0. 4 6% C O 2 V T 0.88 + 0. 08 0.64 + 0. 05 0.91 + 0.08 1.0 + 0. 08 V 9.7 + 1. 2 6.6 + 1. 3 4.4 + 0.5 5.3 + 0. 6 (6) (6) (8) (8) - 99 -TABLE 6 cont... 50% 02 f 10.4 £ 1. 9 10.8 £ 1. 8 6 .1 + 0 . 8 5 .0 + 0 . 2 8% C02 V T 0.87 £ 0 . 1 0.77 + 0 . 07 1 .1 + 0 . 13 1.3 + 0 . 19 V 8.9 £ 1. 0 8 .2 £ 1. 5 6 .0 + 0 . 6 6 .1 + 0 . 8 (6) (6) (4) (5) 5% 02 f 5 .0 £ 0 . 6 5.5 £ 1. 0 4.6 + 0 . 3 3.5 + 0 . 2 4% C02 v T 0.76 £ 0 . 08 0.57 £ 0 . 04 0.80 + 0 . 03 0.84 + 0 . 06 • V 3.8 £ 0 . 5 3 .0 £ 0 . 4 3.6 + 0 . 3 2.9 + 0 . 3 (6) (6) (8) (10) 5% 02 £ 11.3 £ 1. 1 10.4 £ 1. 7 4.4 + 0 . 2 4.9 + 0 . 3 6% C02 VT 0.76 £ 0 . 04 0.59 + 0 . 03 0.94 + 0 . 05 0.97 + 0 . 12 V 8 .2 + 0 . 7 6 .2 + 1. 3 4 . 2 + 0 . 3 5 .1 £ 0 . 8 (6) (5) (5) (5) 5% 02 f 9.3 + 1. 2 10.8 £ 1. 7 5 .0 + 0 . 2 5.3 £ 0 . 3 8% CO2 V T 1 .0 + 0 . 1 0.70 £ 0 . 05 0.96 + 0 . 07 1 . 0 £ 0 . 14 V 8.4 £ 1. 1 7.3 £ 0 . 9 4.7 + 0 . 2 5.6 £ 0 . 8 (6) (6) (5) (5) - 100 -F i g u r e 22. E f f e c t of decreasing FJQ2 o n minute v e n t i l a t i o n , t i d a l volume and frequency i n burst breathing i n t a c t S. l a t e r a l i s ( • ) and burst b r e a t h i n g CBX S. l a t e r a l i s ( A ) during h i b e r n a t i o n at T c of about 6°C. A l l values are mean ± standard e r r o r f o r 5 t o 7 animals. 101 -MINUTE VENTILATION (ml/min/100g) TIDAL VOLUME (ml/100 g) 101 10 15 20 5 10 15 20 (%) - 1 0 2 -Figure 2 3 . E f f e c t of decreasing F J Q 2 on minute v e n t i l a t i o n , t i d a l volume, and frequency i n s i n g l e breath breathing i n t a c t S. l a t e r a l i s ( • ) and s i n g l e breath breathing CBX S. l a t e r a l i s ( A ) during h i b e r n a t i o n a T c of about 2°C. A l l values are mean ± standard e r r o r f o r 7 to 1 0 animals. - 103 -MINUTE VENTILATION (ml/min/100g) TIDAL VOLUME (ml/100 g) • FREQUENCY (min1) i 10 15 20 5 10 15 20 - 104 -hypoxia (10% and 5% O2) c o n t r o l s q u i r r e l s e x h i b i t a s l i g h t increase i n V, at severe l e v e l s (3% O2) V i s not s i g n i f i c a n t l y d i f f e r e n t from normoxic l e v e l s . CBX s q u i r r e l s do not respond to hypoxia at moderate l e v e l s , but show a 21% decrease i n V at 3% O2 mediated by a decrease i n V T (Figure 23). Figure 24 compares the v e n t i l a t o r y responses of burst breathing and s i n g l e breath breathing c o n t r o l S.  l a t e r a l i s to hypoxia. V e n t i l a t o r y responses of Spermophilus  columbianus, a species of ground s q u i r r e l which only e x h i b i t s a s i n g l e breath p a t t e r n , have been i n c l u d e d f o r comparison (McArthur, 1986). Burst breathing S. l a t e r a l i s e x h i b i t a moderate v e n t i l a t o r y response to severe hypoxia, while s i n g l e breath breathing S. l a t e r a l i s show no s i g n i f i c a n t v e n t i l a t o r y response at any l e v e l of hypoxia t e s t e d . S i m i l a r to the hypoxic v e n t i l a t o r y response of s i n g l e breath S. l a t e r a l i s , S. columbianus does not show a v e n t i l a t o r y response to hypoxia, even at severe l e v e l s . Response to Hypercapnia Burst breathing c o n t r o l and CBX S. l a t e r a l i s show a strong v e n t i l a t o r y response to hypercapnia. In most s q u i r r e l s r e s p i r a t i o n becomes continuous at high l e v e l s of FIC02 (Figure 20). Figure 21 i l l u s t r a t e s the changes i n - 105 -Fig u r e 24. E f f e c t of decreasing FTQ2 o n minute v e n t i l a t i o n , t i d a l volume, and frequency i n burst b r e a t h i n g S.  l a t e r a l i s ( • ) h i b e r n a t i n g at a T c of about 7°C, s i n g l e breath b r e a t h i n g S. l a t e r a l i s ( A ) h i b e r n a t i n g a t a T c of about 2°C, and s i n g l e breath breathing Spermophilus columbianus ( • ) h i b e r n a t i n g at a T c of about 6°C (McArthur, 1986). A l l values are mean ± standard e r r o r f o r 5 t o 10 animals. - 106 -15 20 -, 107 -r e s p i r a t o r y p a t t e r n with i n c r e a s i n g FJCO2« Below 4% CO2 the number of breaths/burst remains r e l a t i v e l y constant w h i l e T N V P becomes shorter (Figure 21). In both groups, above 4% CG"2 breathing becomes continuous. Several animals (n=2 f o r c o n t r o l s and n=l f o r CBX) maintain a burst breathing p a t t e r n up to 8% CO2. In these animals, at 6% and 8% C02r the number of breaths/burst i s small (x = 6 b/B) and T^vp i s short (x = 29 sec) (Figure 21). Minute v e n t i l a t i o n at 8% CQ2 i n c r e a s e d , on average, 745% and 700% over r e s t i n g l e v e l s i n burst breathing c o n t r o l and CBX s q u i r r e l s r e s p e c t i v e l y (Table 5). Both groups show l i t t l e change i n v e n t i l a t i o n at 2% CO2 and a more or l e s s l i n e a r increase i n V above 2% CO2 (Figure 25). Increases i n both V<r and f c o n t r i b u t e to the o v e r a l l r e s p i r a t o r y response. Below 6% CO2 f increases s u b s t a n t i a l l y , whereas V<r increases only s l i g h t l y . Increases i n f are achieved by decreases i n T^vp. Above 6% CO2 f no longer i n c r e a s e s , but lar g e increases i n V T c o n t r i b u t e to the r i s e i n V . This trend i s s i m i l a r i n both c o n t r o l and CBX s q u i r r e l s . CBX s q u i r r e l s maintain a lower V T than the c o n t r o l s q u i r r e l s throughout the hypercapnic exposure (Figure 25). Thus at most l e v e l s of hypercapnia the CBX s q u i r r e l s have a s l i g h t l y downshifted response curve. - 108 -F i g u r e 25. E f f e c t of i n c r e a s i n g FTCO2 o n minute v e n t i l a t i o n , t i d a l volume, and frequency i n burst breathing i n t a c t S. l a t e r a l i s ( • ) and burst breathing CBX S. l a t e r a l i s ( 0 ) h i b e r n a t i n g at a T c of about 7 ° C . A l l values are mean ± standard e r r o r f o r 5 to 7 animals. - 109 -TIDAL VOLUME (ml/100 g) 'CO-- 110 -In s i n g l e breath breathing S. l a t e r a l i s both c o n t r o l and CBX s q u i r r e l s e x h i b i t s i m i l a r responses t o hypercapnia (Figure 26). In both groups V increases o n l y s l i g h t l y between 0% and 2% C O 2 and above 2% C O 2 V i n c r e a s e s i n a l i n e a r f a s h i o n . Increases i n both f and Vp c o n t r i b u t e to the changes i n V . T i d a l volume increases s t e a d i l y over the range of hypercapnic gases (Figure 26) while f does not increase above 4% C O 2 . Although s i n g l e breath breathing S. l a t e r a l i s shows a strong hypercapnic response, i t i s blunted compared t o burst breathing S. l a t e r a l i s (Figure 27). At 8% C O 2 both burst breathing and s i n g l e breath breathing s q u i r r e l s e x h i b i t continuous r e s p i r a t i o n . Burst breathing s q u i r r e l s increase V more than 700% above normocapnic c o n d i t i o n s at 8% CG"2r whereas s i n g l e breath breathing s q u i r r e l s increase V* only 200% to 300% over normocapnic c o n d i t i o n s at 8% C O 2 . Both burst breathing and s i n g l e breath breathing S.  l a t e r a l i s show s i m i l a r strong increase i n V T , but the s i n g l e breath breathing s q u i r r e l s show a blunted frequency response compared to burst breathing s q u i r r e l s at 8% C O 2 (Figure 27). S. columbianus has a s l i g h t l y higher v e n t i l a t o r y s e n s i t i v i t y to hypercapnia than s i n g l e breath breathing S.  l a t e r a l i s , but a blunted V . response r e l a t i v e to burst breathing S. l a t e r a l i s (Figure 26, McArthur, 1986). This r e l a t i v e y blunted hypercapnic s e n s i t i v i t y i s due to a low frequency response i n S. columbianus. - I l l -F i g u r e 2 6 . E f f e c t of i n c r e a s i n g F T C O 2 on minute v e n t i l a t i o n , t i d a l volume, and frequency i n s i n g l e breath br e a t h i n g i n t a c t S. l a t e r a l i s ( • ) and s i n g l e breath breathing CBX S. l a t e r a l i s ( 0 ) h i b e r n a t i n g at a T c of about 2°C. A l l values are mean ± standard e r r o r f o r 5 to 10 s q u i r r e l s . - 112 -TIDAL VOLUME (ml/100 g) i MINUTE VENTILATION (ml/min/100g) 1 • • FREQUENCY (min1) / t - V i : r • i i i i 1 ^ : 0 2 * 6 8 'CO* 0 2 * 6 6 (%) - 113 -Figur e 27. E f f e c t of i n c r e a s i n g FTCQ-2 o n rainute v e n t i l a t i o n , t i d a l volume, and frequency i n burst b r e a t h i n g S.  l a t e r a l i s ( • ) h i b e r n a t i n g at a T c of about 6°C, s i n g l e breath breathing S. l a t e r a l i s ( A ) h i b e r n a t i n g at a T c of about 2°C and s i n g l e breath breathing S. columbianus ( • ) h i b e r n a t i n g a T c of about 6°C (McArthur, 1986). A l l values are means ± standard e r r o r f o r 5 t o 10 animals. - 114 -MINUTE VENTILATION (ml/min/100g) 101 'CO. (%) - 115 -Responses to hyperoxia, hyperoxic hypercapnia and hypoxic  hypercapnia Hyperoxia alone has l i t t l e e f f e c t on r e s p i r a t i o n i n burst breathing c o n t r o l s q u i r r e l s (Table 6 ) . In burst breathing CBX s q u i r r e l s hyperoxia causes a s l i g h t i n c r e a s e i n V corresponding to a s l i g h t increase i n V^. In s i n g l e breath breathing c o n t r o l and CBX s q u i r r e l s hyperoxia does not cause any s i g n i f i c a n t changes i n v e n t i l a t i o n (Table 6). The hyperoxic response i n s i n g l e breath CBX animals i s mediated by a small increase i n f. Figure 28 i l l u s t r a t e s V responses t o i n c r e a s i n g hypercapnia with a normoxic, hyperoxic or hypoxic background i n burst breathing c o n t r o l s q u i r r e l s . The v a r i o u s 0 2 backgrounds have l i t t l e e f f e c t on the hypoxic response curve i n burst breathing c o n t r o l or CBX S. l a t e r a l i s (Table 6). V e n t i l a t o r y t h r e s h o l d and the o v e r a l l hypercapnic V. response are unaltered by the v a r i o u s oxygen backgrounds. In s i n g l e breath breathing animals hyperoxia and hypoxia a l s o have no e f f e c t on the hypercapnic response t h r e s h o l d or on the o v e r a l l V response to hypercapnia i n e i t h e r c o n t r o l (Figure 28) or CBX s q u i r r e l s (Table 6). The c o n t r i b u t i o n s of f and V^ to the increases i n v e n t i l a t i o n - 116 -F i g u r e 2 8 . E f f e c t of i n c r e a s i n g FTCG-2 * n combination w i t h 2 1 % O2 ( O ) r 50% O2 (A) and 5% O2 (•) on minute v e n t i l a t i o n i n burst breathing S. l a t e r a l i s h i b e r n a t i n g at a T a of about 7°C (A) and s i n g l e breath breathing S. l a t e r a l i s h i b e r n a t i n g at a T a of about 2°C (B). - 118 -F i g u r e 29. Representative records of breathing p a t t e r n s i n h i b e r n a t i n g S. l a t e r a l i s at T c of about 7°C during exposure to a i r (A) and exposure to a i r p l u s 3 v o l % halothane (B). - 119 -CO 120 -are not s i g n i f i c a n t l y d i f f e r e n t from those seen during normoxia. E f f e c t of A n e s t h e t i c s Four burst breathing golden mantled ground s q u i r r e l s h i b e r n a t i n g at 6.5°C were exposed to v a r y i n g l e v e l s of vaporized halothan a n e s t h e t i c . At l e v e l s of about 2.5 to 3 v o l % halothane the normal burst breathing p a t t e r n i s converted to a s i n g l e breath p a t t e r n (Figure 29). Further increases i n halothane concentrations above 3.5 v o l % cause a l o s s of r e s p i r a t o r y a c t i v i t y . Removal of the a n e s t h e t i c r e s u l t e d i n a gradual r e t u r n to a b u r s t i n g p a t t e r n . - 121 -DISCUSSION AWAKE ANIMALS At room temperature awake golden-mantled ground s q u i r r e l s breathe continuously. Acute exposure to hypoxia produces a strong v e n t i l a t o r y response, while acute exposure to hypercapnia produces a comparatively blunted v e n t i l a t o r y response. C a r o t i d body denervation r e s u l t s i n a r e d u c t i o n i n r e s t i n g v e n t i l a t i o n and a s l i g h t reduction i n v e n t i l a t o r y s e n s i t i v i t y to hypercapnia. Chronic exposure to hypoxic and hypercapnic c o n d i t i o n s (CHH) r e s u l t s i n a maintained increase i n minute v e n t i l a t i o n through changes i n both t i d a l volume and frequency but r e s p i r a t o r y s e n s i t i v i t y t o hypoxia and hypercapnia i s u n a l t e r e d . Methodology The whole body plethysmograph has been used i n many r e s p i r a t o r y s t u d i e s i n v o l v i n g small mammals ( B i r c h a r d et a l . , 1984; Blake and Banchero, 1985; Darden, 1972; Holloway and Heath, 1984; L a i et a l . , 1981; Walker et a l . , 1985). This method allows f o r measurement of both r e s p i r a t o r y p a t t e r n and t i d a l volume i n unanesthetized, u n r e s t r a i n e d animals. Both a n e s t h e t i c s and devices used t o measure r e s p i r a t i o n such as face masks and neck s e a l s , can produce undesirable a l t e r a t i o n s i n normal v e n t i l a t i o n ( G a u t i e r , 1976; G i l b e r t et a l . , 1972; Fleming et a l . , 1983). In - 122 -a d d i t i o n , the experimental set-up can be placed i n a c l o s e d area so that changes i n gas mixtures and measurements of T c and Tb can be performed without t a c t i l e or v i s u a l contact with the animal. The accuracy of the c a l c u l a t e d t i d a l volume using the plethysmograph method depends on the r e l i a b i l i t y of measurements of independent v a r i a b l e s such as Pg, TQ and p a r t i c u l a r l y Tb and TJJ (Malan, 1973). D i r e c t measurements of body and nasal temperature were not taken during the course of the experiments. Tb and TJJ of a c t i v e s q u i r r e l s were assumed to be r e l a t i v e l y constant at 37°C and 32°C r e s p e c t i v e l y regardless of changes i n ambient temperature, breathing frequency or gas exposure. Exposure to severe l e v e l s of hypoxia and hypercapnia have been reported to cause decreases i n body temperature' ( F a l e s c h i n i and Whitten, 1975; Jennings, 1979, L i a et a l . , 1975). For example, F a l e s c h i n i and Whitten (1975) reported a 3°C drop i n body temperature i n S.  l a t e r a l i s during a 30 minute hypoxic exposure. In a d d i t i o n i t i s p o s s i b l e that during severe hypoxia or hypercapnia when r e s p i r a t o r y frequency i s extremely h i g h , i n s p i r e d and expired a i r are not f u l l y s aturated or desaturated across the nasal passages. These a l t e r a t i o n s may have l e d to an underestimation of V<i>. - 123 -Changes i n r e s p i r a t o r y timing v a r i a b l e s can a l s o increase the e r r o r f a c t o r i n t i d a l volume measurements w i t h t h i s technique by up to 30% (Jacky, 1980). Although comprehensive measurements of T T , T^ and TTOT were not taken i n t h i s study, values were a v a i l a b l e from the study by McArthur (1986) i n S. l a t e r a l i s during exposure to s i m i l a r hypoxic and hypercapnic gases. Despite these p o t e n t i a l sources .of e r r o r i n the plethysmograph method used i n the present study, simultaneous measurements of V<j> made using both the plethysmograph and pneumotachograph methods were s i m i l a r (Figure 4). Resting V e n t i l a t i o n I n t a c t c o n t r o l animals Values obtained f o r r e s t i n g minute v e n t i l a t i o n i n S. l a t e r a l i s are about 50% smaller than V values p r e d i c t e d by S t a h l (1967) using s c a l i n g equations f o r a l l mammals. T i d a l volume measurements i n the present study are about 0.8 ml/lOOg. This value f a l l s w i t h i n the range p r e d i c t e d by S t a h l (1967) f o r r a t s (0.4 to 1.0 ml/lOOg). Frequency values, however, f a l l s l i g h t l y below the range of 45 to 100 breaths/minute p r e d i c t e d by S t a h l (1967) and w e l l below the reported values of 97 to 115 breaths/minute f o r 250 gram r a t s ( S t a h l , 1967). - 124 -Low r e s t i n g values f o r minute v e n t i l a t i o n are commonly observed i n f o s s o r i a l and s e m i - f o s s o r i a l species ( A r i e l i and Ar, 1979; Darden, 1972, Holloway and Heath, 1984; Schlencher, 1985; Walker et a l . , 1985). In most st u d i e s the reduction i n V i s a t t r i b u t e d p r i m a r i l y to decreases i n r e s t i n g v e n t i l a t o r y frequency. The e f f e c t of a f o s s o r i a l environment on VT i s more v a r i a b l e . Walker et a l . (1985) and A r i e l i and Ar (1979) found VT values i n semi-f o s s o r i a l and f o s s o r i a l animals which are higher than V T values i n comparable s i z e d r a t s . In c o n t r a s t , Schlenker (1985) reported VT values i n the Djungarian hamster which are smaller than VT values of s i m i l a r s i z e d mice. These r e s u l t s suggest there may be no e f f e c t of burrow-dwelling on VT« The combination of a r e l a t i v e l y s m a l l b r e a t h i n g frequency and l a r g e VT commonly observed i n many f o s s o r i a l mammals may r e s u l t i n a more e f f e c t i v e a l v e o l a r v e n t i l a t i o n than i s observed i n other mammals (Tenney and Bogg, 1986). Previous measurements of VT i n S. l a t e r a l i s performed i n our l a b o r a t o r y using s i m i l a r plethysmograph techinques y i e l d e d VT values about 60% smaller than those obtained i n the present study (McArthur, 1986). This discrepancy i n VT measurements may r e f l e c t d i f f e r e n c e s i n the s p e c i f i c plethysmograph used, d i f f e r e n c e s i n the measurement of pressure d e f l e c t i o n s or d i f f e r e n c e s i n the c a l i b r a t i o n of the pressure d e f l e c t i o n s caused by r e s p i r a t i o n . Resting frequency i s about 40% higher than - 125 -those found by McArthur (1986) suggesting that r e s t i n g frequency may not have been achieved i n the previous study. These changes i n breathing p a t t e r n r e s u l t i n r e s t i n g V values which are 50% smaller than those reported i n the present study. These d i s c r e p e n c i e s i n f , V-j and V values may be due to d i f f e r e n c e s i n methodology or i n the p h y s i o l o g i c a l s t a t e of the s q u i r r e l s during the study. P a n t i n g , which allows f u r r e d animals to d i s s i p a t e heat, r e s u l t s i n a r a p i d , shallow breathing p a t t e r n s i m i l a r to those reported by McArthur (1986). This may be one f a c t o r c o n t r i b u t i n g to d i f f e r e n c e s i n breathing p a t t e r n and o v e r a l l V i n the two s t u d i e s . CBX Animals C a r o t i d body denervations r e s u l t i n a r e l a t i v e h y p o v e n t i l a t i o n under r e s t i n g c o n d i t i o n s . A 40% r e d u c t i o n i n minute v e n t i l a t i o n i s achieved through decreases i n r e s p i r a t o r y frequency with no s i g n i f i c a n t changes i n V^. Q u a l i t a t i v e l y s i m i l a r decreases i n V a f t e r CBX have been observed i n a l a r g e v a r i e t y of animals (Bisgard et a l . , 1980; Bouverot and Bureau, 1975; Bouverot et a l . , 1973; Fordyce and Tenney, 1984; F o r s t e r et a l . , 1981; M i l l e r and Tenney, 1975), although a few s t u d i e s have reported no change i n V a f t e r CBX (Watt et a l . , 1942) or only acute decreases i n V which r e t u r n to preoperative l e v e l s over time (Bisgard et a l . , 1980; Smith and M i l l s , 1980). The h y p o v e n t i l a t i o n which o f t e n r e s u l t s from CBX i s accompanied by an increase i n PaC02 U P t o l e v e l s which are known to - 126 -normally act as a v e n t i l a t o r y stimulus (Bouverot and Bureau, 1975; Bouverot et a l . , 1973; M i l l e r and Tenney, 1975). The increase i n PaC02 a n <^ ^ t s e f f e c t s on v e n t i l a t i o n does not compensate f o r the l o s s of c a r o t i d body chemoreceptors and t h e i r i n f l u e n c e on r e s t i n g v e n t i l a t i o n ( M i l l e r and Tenney, 1975). Thus, i t appears that i n S. l a t e r a l i s c a r o t i d body chemoreceptors are important i n the maintenance of normal l e v e l s of v e n t i l a t i o n . Under normoxic and normocapnic co n d i t i o n s c a r o t i d body chemoreceptors provide a t o n i c e x c i t a t o r y i n f l u e n c e on c e n t r a l r e s p i r a t o r y mechanisms, p a r t i c u l a r l y those i n f l u e n c i n g breathing frequency. CHH Animals The o v e r a l l v e n t i l a t o r y response during both chr o n i c and acute exposure to hypoxia and hypercapnia i s s i m i l a r , but the v e n t i l a t o r y p a t t e r n i s s l i g h t l y a l t e r e d during CHH exposure. CHH exposure r e s u l t s i n an el e v a t e d V under normoxic and normocapnic c o n d i t i o n s , due to a maintained increase i n V<r. Although there are no stu d i e s which have examined the v e n t i l a t o r y a c c l i m a t i o n to chronic hypoxia and hypercapnia together i n other species of mammal, there are many st u d i e s which have looked at v e n t i l a t o r y responses to chronic hypoxia and hypercapnia s e p a r a t e l y . - 127 -Mammals, other than humans, exposed to long term hypoxia g e n e r a l l y e x h i b i t a sustained increase i n V which approximates the l e v e l of v e n t i l a t i o n achieved during acute hypoxic exposure (Fordyce and Tenney, 1984; Bouverot et a l . , 1973; F o r s t e r et a l . , 1981; Mortola et a l . , 1986). Most of these s t u d i e s report that the elevated V i s produced by sustained increases i n both Vrp and f (Fordyce and Tenney, 1984; Bouverot et a l . , 1973, Mortola et a l . , 1986). Increases i n Vt a s s o c i a t e d with long term hypoxic exposure lead to more e f f i c i e n t pulmonary v e n t i l a t i o n r e l a t i v e to the acute hypoxic response. Dempsey and F o r s t e r (1982) suggest that the mechanism behind these long term changes during hypoxia could r e s u l t from changes i n the e x c i t a b i l i t y of medullary r e s p i r a t o r y neurons or changes i n the suprapontine or b r a i n stem mechanisms. In g e n e r a l , changes to v e n t i l a t i o n during chronic hypoxia appear to i n v o l v e complex adjustments to r e s p i r a t o r y mechanisms (Dempsey and F o r s t e r , 1982). R e l a t i v e l y few s t u d i e s have looked at V e n t i l a t o r y a c c l i m a t i o n to c h r o n i c hypercapnia. Most evidence i n d i c a t e s that during c h r o n i c hypercapnia v e n t i l a t i o n i n c r e a s e s and remains elevated throughout the chronic exposure (Dempsey and F o r s t e r , 1982). The increases i n v e n t i l a t i o n during chronic CO2 exposure equal or exceed the i n c r e a s e s i n v e n t i l a t i o n during acute CO2 exposure (Dempsey and F o r s t e r ; 1982). S i m i l a r p a t t e r n changes to those observed i n the - 128 -present study i n response to chronic hypercapnia have been reported both i n man (Schaefer et a l . , 1966) and l a b o r a t o r y animals (Dodd and Milsom, 1987; L a i et a l . , 1981). In these st u d i e s c h r o n i c hypercapnia caused sustained increases i n a l v e o l a r minute v e n t i l a t i o n ( V A ) , while dead space (Vrj) i n i t i a l l y increased and subsequently returned to normal l e v e l s over time. In S. l a t e r a l i s , assuming that there i s no change i n dead space during exposure to e i t h e r c h r o n i c or acute hypoxia and hypercapnia, V A increases during i n i t i a l (acute) exposure and then i s f u r t h e r elevated a f t e r c h r o n i c exposure to hypoxia and hypercapnia p r i m i a r i l y by f u r t h e r increases i n VT (Figure 3 0 ) . V A has been c a l c u l a t e d assuming that under r e s t i n g c o n d i t i o n s the r a t i o of VQ to V T i s approximately .33 ( S t a h l , 1966; Tenney and Boggs, 1986) and that t h i s r a t i o i s unaltered by gas exposure. Increases i n V A during CHH exposure are s i m i l a r i n both i n t a c t and CBX s q u i r r e l s . Increases i n V A serve to reduce the a l v e o l a r PQ2 and Pco2 t o a r t e r i a l PQ2 a n d PC02 d i f f e r e n c e s (Tenney and Boggs, 1986). These changes increase 0*2 l o a d i n g and CO2 unloading i n the lungs and thus minimize changes from normal l e v e l s of a r t e r i a l PQ-2 a n <3 Pc02« During c h r o n i c hypercapnia both plasma and c e r e b r a l s p i n a l f l u i d H + c o n c e n t r a t i o n r e t u r n to near normal l e v e l s , making i t u n l i k e l y that CO2 mediated changes i n H + concentration are re s p o n s i b l e f o r the hypernea (Dempsey and F o r s t e r , 1982). Most evidence to date - 1 2 9 -F i g u r e 3 0 . Bar p l o t showing changes i n frequecy, t i d a l volume, minute v e n t i l a t i o n and a l v e o l a r minute v e n t i l a t i o n during exposure t o a i r , acute hypoxia and hypercapnia (AHH, 1 7 % 0 2 and 4% CO2) and c h r o n i c hypoxia and hypercapnia (CHH, 1 7 % O2 and 4% CO2). A l v e o l a r v e n t i l a t i o n was c a l c u l a t e d from the equation: V A = ( V T - V D) x f where VQ was assumed to be 3 3 % of normal r e s t i n g V T (Tenney and Boggs, 1 9 8 6 ) and i t was assumed th a t Vr> d i d not change w i t h exosure t o d i f f e r e n t gas mixtures. • - i n t a c t S. l a t e r a l i s CBX S. l a t e r a l i s - 130 -60T 40 204 FREQUENCY (min1) u 10 6+ TIDAL VOLUME (ml/100g) MINUTE VENTILATION (ml/m in/100 g) 60 AO 20 ALVEOLAR VENTILATION (mt/min/IOOg) AIR AHH GAS EXPOSURE CHH 131 -suggests that the mechanisms underlying r e s p i r a t o r y p a t t e r n changes during chronic CO2 exposure could i n v o l v e a d i s e q u i l i b r i u m between c e r e b r a l s p i n a l f l u i d and i n t e r s t i t i a l s p i n a l f l u i d at the s i t e of i n t e r c r a n i a l chemoreceptors, a change i n the c o n t r i b u t i o n of pulmonary CO2 sensing mechanisms or a change i n chemoreceptor a c t i v i t y which d i r e c t l y or i n d i r e c t l y a l t e r s medullary r e s p i r a t o r y neuron f u n c t i o n i n g (Dempsey and F o r s t e r , 1982). In g e n e r a l , a c c l i m a t i o n to both chr o n i c hypoxia and hypercapnia i n v o l v e s complex changes i n r e g u l a t i o n of breathing p a t t e r n and v e n t i l a t i o n . Response to Hypoxia I n t a c t C o n t r o l Animals I n t a c t S. l a t e r a l i s show a strong v e n t i l a t o r y reponse to decreases i n i n s p i r e d O 2 . Increases i n V during hypoxia are achieved s o l e l y by increases i n f (Figure 7). T i d a l volume during severe hypoxia a c t u a l l y decreases. Two s t u d i e s on v e n t i l a t o r y responses i n s e m i - f o s s o r i a l golden hamsters a l s o report hypoxic h y p e r v e n t i l a t i o n (Holloway and Heath, 1984; Walker et a l . , 1985). Increases e x c l u s i v e l y i n frequency i n response to hypoxia have a l s o been reported i n s e v e r a l n o n - f o s s o r i a l species such as the r a t (Cragg and Drysdale, 1983) and the cat (Holloway and Heath, 1984). In c o n t r a s t , increases i n V mediated by increases i n both and f have been observed i n the f o s s o r i a l mole r a t ( A r i e l i and Ar, 1979) the Djungarian hamster (Schlencher, 1985), the - 1 3 2 -golden-mantled ground s q u i r r e l (McArthur, 1 9 8 6 ) and the r a t (Holloway and Heath, 1 9 8 4 ; Maskrey et a l . , 1 9 8 1 ; Walker et a l . , 1 9 8 5 ) . Thus, the r e l a t i v e c o n t r i b u t i o n of and f to the hypoxic v e n t i l a t o r y response appears to be study s p e c i f i c , p o s s i b l y r e s u l t i n g from d i f f e r e n t methods used to measure r e s p i r a t i o n i n the var i o u s s t u d i e s . In ge n e r a l , l a r g e increases i n frequency i n response to hypoxia are common to most mammals. The o v e r a l l v e n t i l a t o r y response of S. l a t e r a l i s to hypoxia at 8% FTC-2 i s a 1 4 0 % increase i n V . Increases of s i m i l a r magnitude i n V i n response to hypoxia have been observed i n both s e m i - f o s s o r i a l species (Holloway and Heath, 1 9 8 4 ; McArthur, 1 9 8 6 ; Schlenker, 1 9 8 5 ; Walker et a l . , 1 9 8 5 ) and n o n - f o s s o r i a l species such as mice and r a t s (Holloway and Heath, 1 9 8 4 ; Schlenker, 1 9 8 5 ; Walker et a l . , 1 9 8 5 ) . The hypoxic s e n s i t i v i t y of S. l a t e r a l i s and other s e m i - f o s s o r i a l mammals i s equi v a l e n t t o , or greater than, hypoxia s e n s i t i v i t y i n n o n - f o s s o r i a l s p e c i e s . This c o n c l u s i o n has been confirmed i n s t u d i e s d i r e c t l y comparing hypoxic v e n t i l a t o r y responses from s i m i l a r s i z e d s e m i - f o s s o r i a l and n o n - f o s s o r i a l species (Holloway and Heath, 1 9 7 9 ; Schlenker, 1 9 8 4 ; Walker et a l . , 1 9 8 5 ) . Figure 3 1 i l l u s t r a t e s the hypoxic v e n t i l a t o r y response of a s e m i - f o s s o r i a l s p e c i e s , the golden-mantled ground s q u i r r e l ( t h i s study) to that of the white r a t (Pappenheimer, 1 9 7 7 ) . - 133 -Fi g u r e 31. Bar p l o t showing the percent change from normoxic and normocapnic c o n d i t i o n s i n frequency ( f ) , t i d a l volume (V^) and minute v e n t i l a t i o n (V) i n response t o hypoxia (10% 0*2) and hypercapnia (5% CO2) i n awake golden-mantled ground s q u i r r e l s compared to the white r a t . Rat v e n t i l a t i o n data taken from Pappenheimer (1977). = golden-mantled ground s q u i r r e l s = white r a t - 134 -200 160 120 80 40 -40 HYPOXIA (10% 02) rl D 120+ 80 40 0 -40 HYPERCAPNIA (5% C02) T L f Mrf1) (ml/IOOg) (ml/min/IOOg) - 135 -CBX Animals C a r o t i d body denervations do not g r e a t l y a f f e c t the o v e r a l l magnitude of the hypoxic v e n t i l a t o r y response of e i t h e r a i r breathing or CHH animals. Although CBX s q u i r r e l s e x h i b i t a lower V at a l l l e v e l s of FJQ-2 due t o a lower r e s p i r a t o r y frequency, i n t a c t and CBX groups e x h i b i t s i m i l a r increases i n V . CBX i n both a i r breathing and CHH animals, however, does r e s u l t i n a l e f t s h i f t i n the v e n t i l a t o r y response t h r e s h o l d , Many previous s t u d i e s have examined the r o l e of c a r o t i d body chemoreceptors during acute v e n t i l a t o r y response to hypoxia. I t i s c l a s s i c a l l y assumed that CBX produces a long term decrease i n hypoxic s e n s i t i v i t y . Decreases i n v e n t i l a t i o n i n response to severe hypoxia a f t e r CBX are the r e s u l t of low Pa02 l e v e l s causing c e n t r a l depression of v e n t i l a t i o n (Fordyce and Tenney, 1984). Loss or r e d u c t i o n of v e n t i l a t o r y s t i m u l a t i o n during hypoxia i n c a r o t i d body denervated animals has been observed i n goats (Tenney and Brooks, 1966), r a t s (Chioccho et a l . , 1984; Sapru and K r i e g e r , 1977), dogs (Bouverot et a l . , 1973) and cats (South and M i l l s , 1980; Fordyce and Tenney, 1982). In view of these r e s u l t s , v e n t i l a t o r y responses to hypoxia i n i n t a c t animals have been assumed to r e f l e c t a balance between f a c i l i t o r y c a r o t i d body chemoreceptor input and the d i r e c t c e n t r a l depression caused by low c e n t r a l P 0 2 (Gau t i e r , 1976). - 136 -Mover and Beecher (1942) observed that i n CBX cats hypoxia produced l a r g e increases i n f and small decreases i n V>p. S i m i l a r r e s u l t s have s i n c e been obtained i n ca t s ( M i l l e r and Tenney, 1975; Sorenson and Mines; 1970) and i n dogs (Davenport et a l . , 1947; Watt et a l . , 1943). In general most of these s t u d i e s report that hypoxia does depress v e n t i l a t i o n i n t i a l l y , but then s t i m u l a t e s v e n t i l a t i o n a f t e r a long p e r i o d of l a t e n c y . The present study supports the idea that a f t e r c a r o t i d body denervation hypoxia can act c e n t r a l l y to s t i m u l a t e r e s p i r a t o r y frequency and at severe l e v e l s may i n h i b i t V^. I t i s a l s o p o s s i b l e that the reduced i s p u r e l y a f u n c t i o n of high frequency r e s p i r a t i o n . CHH Animals In S. l a t e r a l i s c hronic exposure to hypoxia and hypercapnia r e s u l t s i n an upward s h i f t of the e n t i r e hypoxic response curve. There i s l i t t l e change i n o v e r a l l hypoxic s e n s i t i v i t y i n CHH CBX animals, and there i s a s l i g h t increase i n s e n s i t i v i t y i n the i n t a c t CHH animals. Studies of c h r o n i c hypoxia suggest that there i s l i t t l e a l t e r a t i o n i n v e n t i l a t o r y s e n s i t i v i t y to hypoxia (Dempsey and F o r s t e r , 1982), but c h r o n i c hypoxia does d i s p l a c e the e n t i r e v e n t i l a t o r y response curve upward ( L a h i r i et a l . , 1971; L a h i r i et a l . , 1983; Mortola et a l . , 1986). Few s t u d i e s have looked at the e f f e c t s of chronic hypercapnia on hypoxic s e n s i t i v i t y . Falchuk et a l . (1966) found that 48 hours of hypercapnic exposure d i d not e f f e c t the hypoxic response at 137 -comparable PAC02 l e v e l s . In general the c e n t r a l adjustments which r e s u l t i n a high V T i n CHH animals are a f f e c t e d by acute hypoxia i n a s i m i l a r f a s h i o n as a i r breathing s q u i r r e l s exposed to acute hypoxia. A d d i t i o n a l l y , CHH CBX s q u i r r e l s do not e x h i b i t an elevated hypoxic s e n s i t i v i t y , suggesting that p e r i p h e r a l chemoreceptors may be i n v o l v e d i n the increased hypoxic s e n s i t i v i t y i n CHH c o n t r o l s q u i r r e l s . The combination of 4% CO2 w i t h i n c r e a s i n g l e v e l s of hypoxia causes a s l i g h t e l e v a t i o n i n the absolute V at each l e v e l of Fj.02 i n a l l groups of animals. The s l i g h t l y i ncreased V r e s u l t s from the e f f e c t of hypercapnia on VIJ . Hypercapnia a l s o caused a s l i g h t decrease i n the o v e r a l l frequency response at 8% O2. As i n t h i s study, Maskrey et a l . (1981) found that a 4% hypercapnic background d i d not s i g n i f i c a n t l y a l t e r the o v e r a l l response to 10% O2 i n r a t s . In a d d i t i o n , hypercapnia had a s l i g h t negative i n t e r a c t i v e e f f e c t on frequency which p e r s i s t e d a f t e r c a r o t i d sinus denervation (Maskrey et a l . , 1981). I t appears that hypoxia and hypercapnia i n t e r a c t c e n t r a l l y to decrease the frequency response normally a s s o c i a t e d w i t h hypoxia alone. In c o n t r a s t , a l a r g e amount of l i t e r a t u r e suggests that p o s i t i v e hypoxic and hypercapnic i n t e r a c t i o n s occur p r i m a r i l y at the c a r o t i d body chemoreceptors and r e s u l t i n incre a s e s i n the slope of the hypoxic resonse curve (Dempsey and F o r s t e r , 1982; Falchuk et a l . , 1966; F i t z g e r a l d and L a h i r i , 1986). I f c a r o t i d body chemoreceptors are the s i t e - 138 -of hypoxic and hypercapnic i n t e r a c t i o n s , given the small c o n t r i b u t i o n of c a r o t i d body chemoreceptors to hypoxic and hypercapnic responses i n S. l a t e r a l i s , i t i s not s u r p r i s i n g that i n t e r a c t i o n s appear to be small and to be c e n t r a l l y produced. Response to Hyperoxia Hyperoxia alone causes a decrease i n V i n i n t a c t S.  l a t e r a l i s . The decrease i n V i s mediated s o l e l y through a red u c t i o n i n frequency. A s i m i l a r decrease i n v e n t i a l t i o n has been observed i n dogs and r a b b i t s (Bouverot et a l . , 1973; Watt et a l . , 1943). I n s p i r a t i o n of 50% 0 2 r e s u l t s i n an increase i n Pa02' a n <^ p r o v i d i n g that a low P a o 2 n a s a s t i m u l a t o r y r o l e on v e n t i l a t i o n during normoxia, hyperoxia should decrease the chemoreceptor hypoxic d r i v e . The hypoxic d r i v e during normoxia i n S. l a t e r a l i s appears to be mediated e n t i r e l y by c a r o t i d body chemoreceptors, s i n c e CBX and hyperoxia both produce s i m i l a r adjustments i n V ( M i l l e r and Tenney, 1975). Although during normoxia CBX lowers V, during hyperoxia CBX animals e x h i b i t an elevated V. These increases i n V are mediated e x c l u s i v e l y through increases i n V<p. M i l l e r and Tenney (1975) found s i m i l a r r e s u l t s i n CBX ca t s and suggested that O2 acts to s t i m u l a t e c e n t r a l regions of the b r a i n concerned with r e s p i r a t o r y t i d a l volume c o n t r o l . Thus i n the absence of c a r o t i d bodies these - 139 -regions are r e l a t i v e l y depressed during normoxia. M i l l e r and Tenney (1975) a l s o suggested that the region s e n s i t i v e to hyperoxia may be w i t h i n the b r a i n stem s t r u c t u r e and that the discharge a c t i v i t y may be l i m i t e d i n p a r t by the a v a i l a b i l i t y of 0*2. Response to hypercapnia I n t a c t C o n t r o l Animals In S. l a t e r a l i s the r e l a t i o n s h i p between V and FIC02 i s more or l e s s l i n e a r . V r i s e s p r i m a r i l y through increases i n and small but s i g n i f i c a n t increases i n f. S i m i l a r response p a t t e r n s to hypercapnia have been found i n a number of animals i n c l u d i n g the l a b o r a t o r y r a t (Maskrey et a l . , 1981), the golden hamster (Holloway and Heath, 1984; Walker et a l . , 1985) and the burrowing owl (Boggs and K i l g o r e , 1983). Other s t u d i e s report more or l e s s equal increases i n and f ( A r i e l i and Ar, 1977; Darden, 1977; Holloway and Heath, 1984; Schlenker, 1985). Thus, the r e l a t i v e c o n t r i b u t i o n of V<p and f to the hypercapnic v e n t i l a t o r y response i s v a r i a b l e and appears to be species s p e c i f i c . The o v e r a l l magnitude of the hypercapnic v e n t i l a t o r y response i s lower i n S. l a t e r a l i s than most n o n - f o s s o r i a l mammals. Blunted hypercapnic s e n s i t i v i t y appears to be a common adaptation t o f o s s o r i a l or semi-- 1 4 0 -f o s s o r i a l e x i s t a n c e (Boggs et a l . y 1984) and has been documented i n a v a r i e t y of species i n c l u d i n g the burrowing owl (Boggs and K i l g o r e , 1 9 8 2 ) , the pocket gopher (Darden, 1972), the mole r a t ( A r i e l i and Ar, 1979), the marmot ( L e i t n e r and Malan, 1973), the golden-mantled and Columbian ground s q u i r r e l s (McArthur, 1986), and the golden hamster (Holloway and Heath; 1 9 8 4 ; Walker et a l . , 1985). A blunted hypercapnic response has not been observed i n the semi-f o s s o r i a l Djungerian hamster (Schlenker, 1985). Figure 30 i l l u s t r a t e s t y p i c a l hypercapnic V repsonses i n a semi-f o s s o r i a l s p e c i e s , the golden-mantled ground s q u i r r e l (the present study) and a n o n - f o s s o r i a l s p e c i e s , the white r a t (Pappenheimer, 1977). The o v e r a l l reduction i n s e n s i t i v i t y at a l l l e v e l s of CO2 could r e s u l t from decreases i n chemoreceptor s e n s i t i v i t y (Darden, 1 9 7 2 ; Holloway and Heath, 1 9 8 4 ) , low VQ to V"T r a t i o s or increases i n the b u f f e r i n g c a p a c i t y of the blood. As mentioned p r e v i o u s l y , s e m i - f o s s o r i a l s p e c i e s , i n c l u d i n g S. l a t e r a l i s , tend to breathe w i t h a small frequency and l a r g e t i d a l volume ( A r i e l i and Ar, 1 9 7 9 ; Darden, 1972) or a reduced VD (Darden, 1972). These v e n t i l a t o r y adjustments r e s u l t i n a higher V A r e l a t i v e to n o n - f o s s o r i a l mammals. Changes i n i n s p i r e d CO2 may r e s u l t i n s m aller changes i n a r t e r i a l Pco2' a n ^ t h i s may c o n t r i b u t e to the reduced hypercapnic response curve (Tenney and Boggs, 1986). Changes i n b u f f e r i n g c a p a c i t y may occur through - 141 -increases i n blood b u f f e r s such as bicarbonate or through r e n a l adjustments (Boggs et a l . , 1984; Chapman and Bennett, 1975; Falchuk et a l . , 1966). Hyperoxia has l i t t l e e f f e c t on the o v e r a l l hypercapnic response curve. In both i n t a c t and CBX animals hyperoxia alone s i g n i f i c a n t l y a l t e r s V from normoxic l e v e l s . The a d d i t i o n of 2% CC»2 to hyperoxia returns V to l e v e l s which are not s i g n i f i c a n t l y d i f f e r e n t from those seen i n normoxia at 2% C O 2 . Thus, i n i n t a c t s q u i r r e l s hyperoxia alone may depress p e r i p h e r a l chemoreceptor i n p u t , but t h i s depression i s abo l i s h e d w i t h increases i n C O 2 . S i m i l a r l y i n CBX animals the increases i n V<r r e s u l t i n g from hyperoxia are ab o l i s h e d with the a d d i t i o n of low l e v e l s of C O 2 . In ge n e r a l , i t appears that the p e r i p h e r a l and c e n t r a l mechanisms i n v o l v e d i n the hyperoxic response are s e n s i t i v e to and overridden by increases i n C 0 2 . CBX Animals C a r o t i d body denervations do not s i g n i f i c a n t l y a l t e r the p a t t e r n of v e n t i l a t o r y response to hypercapnia. A s l i g h t i n c rease i n o v e r a l l hypercapnic s e n s i t i v i t y i n d i c a t e s that p e r i p h e r a l chemoreceptors may have a s l i g h t i n h i b i t o r y e f f e c t on c e n t r a l hypercapnic s e n s i t i v i t y . Maskrey et a l . (1981) found that CBX i n r a t s caused a s l i g h t decrease i n hypercapnic s e n s i t i v i t y through decreases i n both and f responses. Berchenbosch et a l . , (1979) found that c a r o t i d - 142 -body chemoreceptors c o n t r i b u t e d about 40% to hypercapnic v e n t i l a t o r y responses during hyperoxia. Other s t u d i e s have reported reduced hypercapnic s e n s i t i v i t y a f t e r CBX although the magnitude of the reduction i s extremely v a r i a b l e ( F i t z g e r a l d and L a h i r i , 1986). The r o l e of c a r o t i d body chemoreceptors i n hypercapnic responses remains u n c e r t a i n (Dempsey and F o r s t e r , 1982), but r e s u l t s from the present study suggest that c a r o t i d body chemoreceptors do not play an important r o l e i n v e n t i l a t o r y responses to CO2 i n the golden-mantled ground s q u i r r e l . I t i s p o s s i b l e that the r e l a t i v e y s m a ll r o l e of p e r i p h e r a l chemoreceptors i n hypercapnic responses i n S. l a t e r a l i s c o n t r i b u t e s to the o v e r a l l blunted hypercapnic s e n s i t i v i t y . CHH Animals Chronic exposure to hypoxia and hypercapnia has l i t t l e e f f e c t on the o v e r a l l response to hypercapnia compared to a i r breathing S. l a t e r a l i s . I n t a c t CHH animals e x h i b i t an el e v a t e d V"T at a l l l e v e l s of hypercapnia r e l a t i v e to a i r breathing c o n t r o l s q u i r r e l s . Chronic hypoxia alone does not appear to a l t e r c a r o t i d chemoreceptor responses to hypercapnia ( L a h i r i et a l . , 1983). Chronic hypercapnia r e s u l t s i n a s h i f t i n the hypercapnic response t h r e s h o l d to higher l e v e l s of Pc02' but has l i t t l e e f f e c t on v e n t i l a t o r y s e n s i t i v i t y i n humans (Falchuk et a l . , 1966; K e l l o g g , 1960). R e s u l t s of the - 143 -present study i n d i c a t i n g that a c c l i m a t i o n to CHH c o n d i t i o n s has l i t t l e e f f e c t on hypercapnic s e n s i t i v i t y i s supported by previous s t u d i e s on a c c l i m a t i o n to both chronic hypoxia and c h r o n i c hypercapnia (Dempsey and F o r s t e r , 1982, Jenning and Chen, 1975; Schaefer et a l . , 1963). Most s t u d i e s on f o s s o r i a l and s e m i - f o s s o r i a l mammals i n v o l v e maintaining animals outside of burrow c o n d i t i o n s f o r long periods of time before measuring O2 and CO2 s e n s i t i v i t i e s . These s t u d i e s g e n e r a l l y assume that no " d e a c c l i m a t i z a t i o n " occurs over t h i s p e r i o d with respect to v e n t i l a t o r y c o n t r o l (Boggs et a l . , 1982). D e a c c l i m a t i z a t i o n has been documented i n humans and other mammals upon r e t u r n to a i r a f t e r long term hypoxic exposure ( L a h i r i et a l . 1976) and long term hypercapnic exposure (Jennings and Chen, 1976). For example, L a h i r i et a l . (1982) found that even a f t e r being r a i s e d at high a l t i t u d e (hypoxic c o n d i t i o n s ) , d e a c c l i m a t i z a t i o n occurred i n humans moved to sea l e v e l (normoxic c o n d i t i o n s ) . I f the reduced CO2 s e n s i t i v i t y t y p i c a l of burrow-dwelling animals i s a r e s u l t of long term exposure to hypoxia and hypercapnia, i t i s p o s s i b l e that d e a c c l i m a t i z a t i o n could occur. B i r c h a r d et a l . (1984) exposed p e r i n a t a l r a t s to c h r o n i c hypercapnic c o n d i t i o n s to determine i f m o d i f i c a t i o n to C0"2 v e n t i l a t o r y s e n s i t i v i t y could be caused by prolonged exposure during development. No changes i n the b u f f e r base - 144 -or i n the responses to hypercapnia could be detected between r a t s r a i s e d under hypercapnic c o n d i t i o n s or those r a i s e d under normocapnic c o n d i t i o n s . B i r c h a r d et a l . (1986) concluded that a l t e r a t i o n s i n the hypercapnic response curve may be g e n e t i c i n o r i g i n rather than developmental. Farber et a l . (1972), working on developing opposums, concluded that c h r o n i c hypercapnia during development d i d not a f f e c t a d u l t r e s p i r a t o r y responses to C O 2 . In a d d i t i o n , l a b o r a t o r y r a i s e d hamsters s t i l l r e t a i n a reduced hypercapnic s e n s i t i v i t y , implying the d i f f e r e n c e s between f o s s o r i a l and n o n - f o s s o r i a l species represents a g e n e t i c a l l y determined c h a r a c t e r i s t i c ( A r i e l and Ar, 1979; B i r c h a r d et a l . , 1984; Boggs et a l . , 1984). R e s u l t s from the present study support the idea that C O 2 s e n s i t i v i t i e s are g e n e t i c a l l y determined. HIBERNATING ANIMALS The major f i n d i n g of t h i s p o r t i o n of the study i s that during h i b e r n a t i o n the r e s p i r a t o r y p a t t e r n of S.  l a t e r a l i s i s temperature s e n s i t i v e . Two patterns of steady s t a t e r e s p i r a t i o n are observed; at r e l a t i v e l y high ambient and body temperatures a burst breathing p a t t e r n i s observed, and at lower ambient and body temperatures a s i n g l e breath brea t h i n g p a t t e r n i s observed. Large changes i n r e s p i r a t o r y p a t t e r n do not g r e a t l y a l t e r o v e r a l l l e v e l s of r e s t i n g v e n t i l a t i o n or v e n t i l a t o r y responses to hypoxia or - 145 -hypercapnia. G e n e r a l l y , h i b e r n a t i n g S. l a t e r a l i s e x h i b i t a g r e a t l y reduced v e n t i l a t o r y s e n s i t i v i t y to hypoxia and a comparatively high v e n t i l a t o r y s e n s i t i v i t y to hypercapnia. C a r o t i d body chemoreceptors are not important i n determining r e s t i n g v e n t i l a t i o n or o v e r a l l v e n t i l a t o r y responses to hypoxia or hypercapnia. Methodology Q u a n t i t a t i v e measurements of r e s p i r a t i o n during h i b e r n a t i o n have been hampered by the s e n s i t i v i t y of h i b e r n a t i n g animals to handling ( S t e f f e n and R i e d e s e l , 1984). T i d a l volume has seldom been measured si n c e the methods employed to measure V"T o f t e n cause a r o u s a l (Lyman, 1982). In a d d i t i o n , p e r i o d i c or burst breathing p a t t e r n s are known to be s e n s i t i v e to disturbance ( K r i s t o f f e r s o n and Sovio, 1964; Pajunen, 1970; Pembrey and P i t t s , 1899). The pneumotachograph-mask u n i t used i n the present study to measure breathing p a t t e r n and t i d a l volume d i d not appear to d i s t u r b the h i b e r n a t i n g animal. S q u i r r e l s could be maintained i n h i b e r n a t i o n f o r up to 7 days while wearing the mask. In a d d i t i o n , r e s p i r a t o r y p a t t e r n and r e s p i r a t o r y values observed during h i b e r n a t i o n at ambient temperatures of about 6°C are comparable to p r e v i o u s l y reported values f o r S. l a t e r a l i s (Hammel et a l . , 1968; McArthur, 1986; S t e f f e n and R e i d e s e l , 1982). - 146 -Several s t u d i e s have observed a high v a r i a b i l i t y i n breathing at constant ambient temperatures, p a r t i c u l a r l y i n burst breathing h i b e r n a t o r s . Lyman (1951) working with h i b e r n a t i n g hamsters and golden-mantled ground s q u i r r e l s found that frequency could vary as much as 25% from the mean. Malan et a l . (1973) found high v a r i a b i l i t y i n f even when there was no measurable change i n oxygen consumption or ambient temperature. Thus i t appears that frequency can f l u c t u a t e without changes i n the depth of h i b e r n a t i o n . Pajunen (1984) noted that the p a t t e r n of p e r i o d i c breathing during h i b e r n a t i o n changed over the h i b e r n a t i o n season. In a d d i t i o n , i r r i t a b i l i t y to e x t e r n a l s t i m u l i increased during a bout of h i b e r n a t i o n (Lyman, 1982). I t i s p o s s i b l e that the amount of time spent i n "deep" h i b e r n a t i o n , both s e a s o n a l l y and during an i n d i v i d u a l h i b e r n a t i o n bout, may e f f e c t r e s p i r a t o r y p a t t e r n and r e s p i r a t o r y s e n s i t i v i t y to hypoxia and hypercapnia. During the present study an attempt was made to reduce p o s s i b l e v a r i a b i l i t y i n r e s p i r a t o r y responses r e s u l t i n g from seasonal v a r i a b i l i t y and p r o g r e s s i v e i r r i t a b i l i t y . Although experiments examining the v e n t i l a t o r y responses to various gases were c a r r i e d out over a 4 to 5 month p e r i o d , no responses were t e s t e d u n t i l animals had been i n deep h i b e r n a t i o n f o r at l e a s t 1 month. Throughout the h i b e r n a t i n g season s q u i r r e l s undergo p e r i o d i c - 147 -arousals approximately every 7 to 8 days. In order to reduce the p o s s i b l e e f f e c t s of pr o g r e s s i v e i r r i t a b i l i t y through a h i b e r n a t i o n bout animals used i n experiments had always entered h i b e r n a t i o n only 1 to 2 days p r i o r to the study. Responses to gas s t i m u l i appeared to be r e l a t i v e l y constant both w i t h i n an experimental groups and w i t h i n an i n d i v i d u a l animal. R e s p i r a t o r y P a t t e r n The occurrence of two very d i f f e r e n t i n t e r m i t t e n t breathing p a t t e r n s i n h i b e r n a t i n g animals has been observed f o r many years (Pembrey and P i t t s , 1899). The two p a t t e r n s , burst and s i n g l e breath b r e a t h i n g , have g e n e r a l l y been assumed to be species s p e c i f i c (Malan, 1982). Observations that changes i n the burst breathing p a t t e r n are the f i r s t s i g n that an animal has been d i s t u r b e d (Pajunen, 1970) have been used to e x p l a i n the occurrence of the two d i f f e r e n t breathing p a t t e r n s i n one h i b e r n a t i n g s p e c i e s . Several authors have noted the importance of ambient and body temperature on r e s p i r a t o r y p a t t e r n (Hammel et a l . , 1968); K r i s t o f f e r s o n and Sovio, 1966; Pajunen, 1984). The "optimum" h i b e r n a t i o n temperature has been determined f o r most hi b e r n a t o r s at between 4°C to 7°C (Kayser, 1961, i n Lyman, 1982). Consequently, most s t u d i e s maintain h i b e r n a t i n g animals i n t h i s temperature range. - 148 -Increases i n ambient temperature above t h i s range (Ta=10°C) r e s u l t i n decreases i n the breath-hold length ( K r i s t o f f e r s o n and Sovio, 1969; Pajunen, 1984). Decreases i n ambient temperature down to 0°C cause r e s p i r a t i o n i n the dormouse to breathe i n a s i n g l e breath breathing p a t t e r n f o r long periods of time, up to 17 hours, i n t e r s p e r s e d with short breath hold periods (Pajunen, 1984). During the f i r s t one-h a l f to two-thirds of these breathing periods frequency was very low, l e s s than 1 breath/minute (Pajunen, 1984). In a d d i t i o n , p r e l i m i n a r y experiments suggested that at s l i g h t l y lower ambient temperatures (-2°C) the burst breathing p a t t e r n becomes a continuous s i n g l e breath p a t t e r n (Pajunen, 1984). K r i s t o f f e r s o n and Sovio (1964) working w i t h hedgehogs found that i n "deeply hypothermic" animals ( T a = -5°C) r e s p i r a t i o n a l s o took on a s i n g l e breath p a t t e r n . Hammel et a l . (1968) reported that above hypothalamic temperatures of 5.5°C S. l a t e r a l i s e x h i b i t a burst breathing p a t t e r n . At s l i g h t l y lower hypothalamic temperatures (3.5°C) breathing became i n c r e a s i n g l y regular and l o s t i t s p e r i o d i c p a t t e r n . Hammel et a l . (1968) suggested that the change i n r e s p i r a t o r y p a t t e r n observed i n S. l a t e r a l i s as body temperature d e c l i n e s could be i n d i c a t i v e of a t r a n s i t i o n a l s t a t e l e a d i n g to ar o u s a l should ambient temperature continue to decrease. R e s u l t s from the present study do not suggest that the s i n g l e breath breathing i s a t r a n s i t i o n a l s t a t e preceeding a r o u s a l . S. l a t e r a l i s can be maintained f o r - 149 -s e v e r a l days at lower body temperatures with steady s t a t e v e n t i l a t i o n . In a d d i t i o n , measurements of o v e r a l l minute v e n t i l a t i o n are not s i g n i f i c a n t l y d i f f e r e n t between the two s t a t e s . The observation that the burst breathing p a t t e r n i s extremely temperature s e n s i t i v e i n d i c a t e s the importance of measurement of ambient and body temperatures during r e s p i r a t o r y s t u d i e s . The l e v e l or depth of h i b e r n a t i o n does not s i g n i f i c a n t l y change during the t r a n s i t i o n from burst breathing to s i n g l e breath breathing. O v e r a l l minute v e n t i l a t i o n , V^, and f remain constant w h i l e oxygen consumption increases s l i g h t l y . At an ambient temperature of 2°C S. l a t e r a l i s maintain only a small temperature gradient between T D and T a, th e r e f o r e no a d d i t i o n a l energy f o r maintenance of T 5 should be r e q u i r e d . Thus, the r e d u c t i o n i n T D should r e s u l t i n a f u r t h e r decrease i n t i s s u e metabolism and, i f anything, a reduction i n VQ2* Large increases i n r e s p i r a t o r y timing components occur during s i n g l e breath breathing. No o v e r a l l changes occur i n the Tj/Tg r a t i o or i n the duty c y c l e during the t r a n s i t i o n from burst breathing to s i n g l e breath breathing. This suggests that there i s no l o s s of the i n t e g r i t y of the r e s p i r a t o r y system. The increase i n breath d u r a t i o n could r e s u l t from the d i r e c t e f f e c t s of reduced body temperature on c e n t r a l mechanisms or i n d i r e c t e f f e c t s on pulmonary - 150 -mechanics or both. The decrease i n V T / T J i n the s i n g l e breath breathing s t a t e i m p l i e s that the v e n t i l a t o r y d r i v e to breathe i s reduced. A two-fold increase i n the time spent a c t i v e l y breathing i s seen during s i n g l e breath b r e a t h i n g . This i m p l i e s that the work or energy r e q u i r e d f o r r e s p i r a t i o n has a l s o increased. The suggestion that r e s p i r a t o r y work increases during s i n g l e breath breathing i s supported by the small but s i g n i f i c a n t increase i n VQ 2 . The t r a n s i t i o n between patterns occurs i n the presence or absence of c a r o t i d body chemoreceptors i m p l i c a t i n g the CNS as the s i t e mediating the p a t t e r n changes. L i t t l e i s known about CNS s t r u c t u r e s i n v o l v e d i n the production of burst breathing. Some work has been done i n an attempt to e l u c i d a t e the c e n t r a l s t r u c t u r e s i n v o l v e d i n the production of burst breathing p a t t e r n s i n c r o c o d i l e s (Naifeh et a l . , 1971a, 1971b). Naifeh et a l . (1971b) found that b r a i n stem l e s i o n s i n the r o s t r a l medulla above the nucleus laminarus r e s u l t e d i n the conversion of a p e r i o d i c b u r s t i n g p a t t e r n i n t o a s i n g l e breath breathing p a t t e r n . Thus, the b u r s t i n g p a t t e r n i n c r o c o d i l e s appears to be c o n t r o l l e d by higher CNS c e n t e r s . Naifeh et a l . (1971a) a l s o used a n e s t h e t i c s to look at the c e n t r a l production of the b u r s t i n g p a t t e r n i n c r o c o d i l e s . Both chloroform and p e n t o b a r b i t a l , at low l e v e l s , produced a r e g u l a r , s i n g l e breath p a t t e r n . The r e g u l a r i t y of the s i n g l e breath p a t t e r n and the length of time the p a t t e r n could be sustained lead - 151 -to the suggestion that the s i n g l e breath p a t t e r n may be the b a s i c p a t t e r n of c r o c o d i l i a n r e s p i r a t i o n when uninfluenced by other CNS centres. S i m i l a r e f f e c t s of anaesthetics on the burst breathing p a t t e r n of Chrysemys p i c t a (the western painted t u r t l e ) have been observed (Milsom and Webb, unpub-l i s h e d o b s e r v a t i o n s ) . The burst breathing p a t t e r n of S.  l a t e r a l i s seen during h i b e r n a t i o n i s very s i m i l a r to that seen i n Chrysemys p i c t a (Figure 32) and p r e l i m i n a r y observa-t i o n s suggest that a n e s t h e t i c s a l s o have profound e f f e c t s on the burst breathing p a t t e r n seen i n S. l a t e r a l i s . Exposure to a n e s t h e t i c s transforms the burst breathing p a t t e r n observed at an ambient temperature of 6°C i n t o a regular breathing p a t t e r n c o n s i s t i n g of s i n g l e or double breaths followed by a v e n t i l a t o r y pause (Figure 33). The p a t t e r n i s s i m i l a r to the s i n g l e breath p a t t e r n of S. l a t e r a l i s h i b e r n a t i n g at 2°C. During a n e s t h e t i c exposure i n d i v i d u a l breaths were o f t e n c h a r a c t e r i z e d by double i n s p i r a t i o n s s i m i l a r to those observed i n about 20% of s i n g l e breath breathing animals at reduced body temperatures. Figure 33 i l l u s t r a t e s the double i n s p i r a t i o n s i n both s i n g l e breath breathing groups. Removal of the a n e s t h e t i c leads to a gradual r e t u r n t o a burst breathing p a t t e r n i n S. l a t e r a l i s . Thus, a n e s t h e t i c s ( i n S. l a t e r a l i s , t u r t l e s and c r o c o d i l e s ) , reduced body temperature ( i n S. l a t e r a l i s ) , and b r a i n stem l e s i o n s ( i n c r o c o d i l e s ) a l l w i l l convert burst - 1 5 2 -Fig u r e 32. Representative record of the burst breathing p a t t e r n observed i n S. l a t e r a l i s (the golden-mantled ground s q u i r r e l ) during h i b e r n a t i o n at a Tb of about 7°C and C. p i c t a (the western paint e d t u r t l e ) under r e s t i n g c o n d i t i o n s at a Tb of about 22°G. A) Spej^philus lateralis 1min B) Chrysemys picta 1 min - 154 -Figure 3 3 . Representative records of the e f f e c t s of decreasing ambient temperature from 7°C t o 2°C on b r e a t h i n g p a t t e r n i n S. l a t e r a l i s during h i b e r n a t i o n (A) and the e f f e c t s of a n e s t h e t i c exposure (halothane) on the burst breathing p a t t e r n of S. l a t e r a l i s (B) during h i b e r n a t i o n at a T a of 7°C. Note that both decreased ambient temperature and a n e s t h e t i c exposure can r e s u l t i n double i n s p i r a t i o n s during s i n g l e breath b r e a t h i n g . - 155 -A) EFFECTS OF AMBIENT TEMPERATURE a) air Ta=7°C — * \ min b) air To=20C 1min -^v N v -10 sec B) EFFECTS OF HALOTHANE a) air 1^7 °C 1min b) air + halothan Ta=7°C -HH H4WH4I i) i || i n •< I—H-H+ "imiri — — l/V* ^ / ^ to sec i= inspiration e s expiration - 156 -breathi n g to a s i n g l e breath breathing p a t t e r n . I t i s conceivable that a l l of these treatments act to block the i n f l u e n c e of higher b r a i n centres on the b a s i c r e s p i r a t o r y p a t t e r n produced i n the b r a i n stem region (Feldman, 1986). In S. l a t e r a l i s at T D of about 6-7°C, spontaneous e l e c t r i c a l a c t i v i t y can s t i l l be recorded from the CNS (Strumwasser, 1959). Indeed, the maintained e l e c t r i c a l a c t i v i t y i n the CNS at very low T D may be r e l a t e d to s p e c i f i c m o d i f i c a t i o n s of the b r a i n t i s s u e during h i b e r n a t i o n ( A l o i a , 1979). In s p i t e of p o s s i b l e c e l l u l a r m o d i f i c a t i o n s which ensure neural f i r i n g , a body temperature much below 4 to 5°C must be nearing the lower l i m i t s of neural f u n c t i o n . I t i s p o s s i b l e that the small drop i n T D from about 8°C down to about 4°C causes i n h i b i t i o n of neural t r a n s m i s s i o n . Some p e r i p h e r a l neurons are known to be more s e n s i t i v e to changes i n temperature than others (Ruch, 1973). S i m i l a r d i f f e r e n c e s i n temperature s e n s i t i v i t y may be found i n the CNS neurons, and the areas i n v o l v e d w i t h the production of a burst breathing p a t t e r n may no longer f u n c t i o n at reduced body temperatures. This suggestion i s supported by the obse r v a t i o n that both a n e s t h e t i c s and c o l d have r e v e r s i b l e e f f e c t s on breathing p a t t e r n . The burst breathing p a t t e r n observed during h i b e r n a t i o n i s o f t e n r e f e r r e d to as Cheyne-Stokes R e s p i r a t i o n (CSR). True CSR i n mammals i s g e n e r a l l y a s s o c i a t e d w i t h p a t h o l o g i c a l c o n d i t i o n s (Lyman, 1982). The - 157 -CSR p a t t e r n by d e f i n i t i o n c o n s i s t s of a gradual increase i n frequency and depth of breathing, followed by a gradual decrease i n frequency and depth. During h i b e r n a t i o n , any one burst may con t a i n elements common to CSR, but j u s t as o f t e n r e s p i r a t i o n s t a r t s and stops with l i t t l e change i n the depth or r a t e of r e s p i r a t i o n (Figure 15; Lyman, 1982). Thus breathing p a t t e r n observed during h i b e r n a t i o n i s not a true CSR p a t t e r n , and the term burst breathing i s a simpler and more accurate term f o r t h i s type of r e s p i r a t i o n . The production and c o n t r o l of p e r i o d i c patterns i n euthermic mammals has been the t o p i c of many c l i n i c a l and experimental s t u d i e s . Several t h e o r i e s on the production of p e r i o d i c breathing have been proposed (Cherniak and Longobardo, 1973; Longobardo et a l . , 1960), but only one model attempts to account f o r the occurrence of a l l types of p e r i o d i c breathing (Khoo et a l . , 1982). This model suggests that i n the euthermic awake s t a t e i n mammals the r e s p i r a t o r y system i s r e l a t i v e l y s t a b l e . Decreases i n the s t a b i l i t y of the r e s p i r a t o r y system r e s u l t i n the production of p e r i o d i c p a t t e r n s of r e s p i r a t i o n . Decreased s t a b i l i t y i n the r e s p i r a t o r y system can be acheived by abnormally long c i r c u l a t o r y d e l a y s , changes i n p e r i p h e r a l and c e n t r a l chemoreceptor d r i v e and decreases i n t o t a l body or lung CO2 and O2 s t o r e s (Khoo et a l . , 1982; Cherniak and Longobardo, 1973). Although changes i n a l l of these f a c t o r s probably c o n t r i b u t e to an increased r e s p i r a t o r y i n s t a b i l i t y during - 158 -h i b e r n a t i o n , to what degree, i f any, they promote a p e r i o d i c p a t t e r n i s u n c e r t a i n . In view of t h i s l i m i t e d i n f o r m a t i o n i t i s d i f f i c u l t to apply any models i n v o l v i n g production of p e r i o d i c breathing i n euthermic mammals to the p e r i o d i c breathing seen during h i b e r n a t i o n . The e f f e c t s of reductions of body temperature and metabolism on r e s p i r a t o r y patterns i n e i t h e r h i b e r n a t i n g or non-hibernating mammals has not been w e l l s t u d i e d . Cherniak et a l . (1979) induced p e r i o d i c breathing i n anaesthetized cats by c o o l i n g the v e n t r a l medullary surface. In a d d i t i o n , i t has been suggested that p e r i o d i c breathing i n r e p t i l e s may be a s s o c i a t e d with reduced metabolic rates and O2 requirements and that burst breathing p a t t e r n s may be a st r a t e g y to reduce the cost of breathing (Milsom, 1984). I t i s p o s s i b l e that changes i n body temperature and metabolic r a t e could c o n t r i b u t e to the mechanisms concerned with the production of burst breathing patterns during h i b e r n a t i o n . Resting V e n t i l a t i o n The r e s p i r a t o r y values measured f o r v e n t i l a t i o n during normoxia i n S. l a t e r a l i s are s i m i l a r to those reported i n s t u d i e s by Hammel et a l . (1968) and S t e f f e n and Re i d e s e l (1984). N o n - v e n t i l a t o r y periods i n the present study l a s t e d , on average, 11 to 12 minutes compared to 8 minutes reported by S t e f f e n and Ri e d e s e l (1984) and about 10 - 159 -minutes reported by Hammel et a l . (1968). N o n v e n t i l a t o r y periods i n each of these s t u d i e s are about two to three times longer than those reported by McArthur (1986) f o r S.  l a t e r a l i s . O v e r a l l v e n t i l a t o r y frequency i n burst breathing and s i n g l e breath breathing S. l a t e r a l i s i s w i t h i n the range of .5 to 3.0 breaths/minute reported f o r most h i b e r n a t i n g animals ( K r i s t o f f e r s o n and Sovio, 1966; Landau and Dawe, 1958, Lyman, 1941; Malan et a l . , 1973; Pajunen, 1984; S t e f f e n and R i e d e s e l , 1984). T i d a l volume measurements i n S. l a t e r a l i s are s l i g h t l y lower than those measured by S t e f f e n and R e i d e s e l (1984), 1.2ml/100g, but s l i g h t l y higher than those measured by McArthur (1986), 0.5 ml/lOOg. V a r i a t i o n s i n ; r e s p i r a t o r y v a r i a b l e s may be r e l a t e d to d i f f e r e n c e s i n ambient temperatures used i n d i f f e r e n t s t u d i e s . Oxygen consumption measurements of about 2.0 ml 02/100g/hour f o r both s i n g l e breath and burst breathing s q u i r r e l s were a l s o i n the range of 2.1 to 3.5 ml/lOOg/hour p r e v i o u s l y reported f o r S. l a t e r a l i s (Hammel et a l . , 1968; McArthur, 1986; S t e f f e n and R i e d e s e l , 1984). C a r o t i d body denervations do not r e s u l t i n s i g n i f i c a n t changes i n normoxic minute v e n t i l a t i o n i n burst b r e a t h i n g or s i n g l e breath breathing S. l a t e r a l i s . In burst breathing s q u i r r e l s CBX caused a reduction i n Vi> i n a i r and during a l l gas exposures. This suggests that c a r o t i d body chemoreceptors may be important i n determining base l i n e t i d a l volume during burst breathing. O v e r a l l , c a r o t i d body - 160 -chemoreceptors do not pl a y an important r o l e i n determining breathing p a t t e r n or l e v e l s of v e n t i l a t i o n i n a i r . This c o n c l u s i o n i s f u r t h e r supported by the observation that hyperoxia, i n i n t a c t animals, has no e f f e c t on r e s p i r a t i o n . I f c a r o t i d body chemoreceptors are a c t i v e during normoxia, hyperoxia would decrease the e x i s t i n g v e n t i l a t o r y d r i v e by decreasing p e r i p h e r a l chemoreceptor input and t h e r e f o r e reduce V. Response to hypoxia V e n t i l a t o r y responses to decreases i n i n s p i r e d 0 2 are low i n a l l groups of S. l a t e r a l i s . Burst breathing s q u i r r e l s show a moderate increase i n V at 5% O2 and a 100% increase i n V at 3% O 2 . The increase i n V i s achieved s o l e l y by decreases i n T^yp l e a d i n g to increases i n f. The observed hypoxic h y p e r v e n t i l a t i o n i s s i m i l a r to observations i n other s t u d i e s which report moderate v e n t i l a t o r y s t i m u l a t i o n by hypoxia. T a h t i et a l . (1975) observed v e n t i l a t o r y s t i m u l a t i o n below 16% O2 i n the hedgehog, wi t h breathing becoming continuous at 1.7% to 3% O 2 . McArthur (1986) a l s o observed a doubling i n V at 3% O2 i n burst breathing S. l a t e r a l i s . In c o n t r a s t , during s i n g l e breath b r e a t h i n g , S. l a t e r a l i s do not show an increase i n V at any l e v e l of Fj .02 used i n t h i s experiment. S i m i l a r l y McArthur (1986) found no increase i n V i n the s i n g l e breath breathing S. columbianus at 3% F J O 2 « - 161 -C a r o t i d body denervations do not s i g n i f i c a n t l y a l t e r the o v e r a l l v e n t i l a t o r y response to hypoxia i n e i t h e r burst breathing or s i n g l e breath breathing s q u i r r e l s . A s i g n i f i c a n t l e f t - s h i f t i n the response t h r e s h o l d at 5% O2 i n burst breathing S. l a t e r a l i s i n d i c a t e s that c a r o t i d body chemoreceptors may be involved i n the hypoxic frequency response at r e l a t i v e l y moderate l e v e l s of hypoxia. The frequency response a s s o c i a t e d with severe l e v e l s of hypoxia i n burst breathing s q u i r r e l s appears to be c e n t r a l l y produced. The low hypoxic s e n s i t i v i t y i n hi b e r n a t o r s i s not s u r p r i s i n g i n view of the e f f e c t s of temperature on metabolic r a t e and the hemoglobin -02 (HbC>2) d i s s o c i a t i o n curve. As an animal enters h i b e r n a t i o n , metabolic r a t e drops to about 1% to 2% that of a euthermic animal (Malan, 1982; Wang, 1978). The d e c l i n e i n metabolic r a t e g r e a t l y reduces the t i s s u e demand f o r O 2 . As Tb drops there i s a la r g e l e f t - s h i f t i n the Hb02 d i s s o c i a t i o n curve. Several s t u d i e s i n d i c a t e that the p a r t i a l pressure at which the hemoglobin i s 50% saturated with O2 at a body temperature of 5°C i s l e s s than 10 Torr (Clausen and E r s l a n d , 1968; Musacchia and V o l k e r , 1971). This p a r t i a l pressure corresponds to i n s p i r e d O2 l e v e l s of about 1%. Thus, a r t e r i a l blood remains saturated with O2 down to very low p a r t i a l pressures. - 162 -L e v e l s of i n s p i r e d 0 2 required to cause desatur-a t i o n of the blood are u n l i k e l y to be found under n a t u r a l c o n d i t i o n s . In a d d i t i o n , even during long breath hold p e r i o d s , l e v e l s of P a02 a r e u n l i k e l y to f a l l to l e v e l s r e q u i r e d to cause d e s a t u r a t i o n of the blood. S t e f f e n and R i e d e s e l (1982) report that e n d - t i d a l PQ2 a t the end of a breath-hold p e r i o d was about 90-95 Torr. S i m i l a r a r t e r i a l p a r t i a l pressures of between 88 and 120 Torr have been reported at end breath-hold (Musacchia and V o l k e r t , 1971; T a h t i , 1978 i n S t e f f e n and R i e d e s e l 1984). These l e v e l s of PQ2 a r e n o t l i k e l y to r e s u l t i n l e s s than 100% s a t u r a t i o n and i t i s , t h e r e f o r e , u n l i k e l y that PQ2 a c t s as an important chemical stimulus to breathe during h i b e r n a t i o n . The d i f f e r e n c e s i n the hypoxic s e n s i t i v i t y between burst breathing and s i n g l e breath breathing S. l a t e r a l i s may r e s u l t from s l i g h t l y reduced body temperatures i n the s i n g l e breath breathing s q u i r r e l s . I t i s conceivable that at a T D of 7° to 8 ° C blood i s not f u l l y s aturated at 3% to 5% F T G-2' thus causing a s l i g h t v e n t i l a t o r y response. A decrease i n T D to about 4 ° C may f u r t h e r l e f t - s h i f t the HbC>2 d i s s o c i a t i o n curve such that blood remains f u l l y s aturated even at 3% F I 0 2 -Response to Hypercapnia Both burst breathing and s i n g l e breath breathing S. l a t e r a l i s e x h i b i t a r e l a t i v e l y high s e n s i t i v i t y to hypercapnia. The p a t t e r n of hypercapnic v e n t i l a t o r y response i s s i m i l a r i n both groups, c o n s i s t i n g of a l a r g e frequency response and a s l i g h t response. Increases i n frequency are achieved by changes i n T^vp with no adjustment to the d u r a t i o n of i n d i v i d u a l breaths. The o v e r a l l v e n t i l a t o r y response to hypercapnia appears to be q u a l i t a t i v e l y s i m i l a r to responses reported f o r hedgehogs (Biorch et a l . , 1956; T a h t i , 1975), the golden hamster (Lyman, 1951), the marmot (Endres and T a y l o r , 1930) and the 1 3 - l i n e d , Columbian and golden-mantled ground s q u i r r e l s (Lyman, 1951; McArthur, 1986). Most s t u d i e s report that l e v e l s of 1% to 3% C O 2 s t i m u l a t e r e s p i r a t i o n through decreases i n T^yp and that r e s p i r a t i o n i s continuous above 5% C O 2 (Biork et a l . , 1956; Lyman, 1951; McArthur, 1986; T a h t i , 1975). The magnitude of the frequency response v a r i e s g r e a t l y , from a 200% increase i n frequency reported f o r golden hamsters at 5% C O 2 (Lyman, 1951) to a 550% inc r e a s e i n frequency reported i n the present study at 6% CG"2. T i d a l volume responses have not been measured i n most r e s p i r a t o r y s t u d i e s , but V<r has been shown to increase between 35% and 60% during hypercapnia by McArthur (1986) and i n the present study. During s i n g l e breath breathing S. l a t e r a l i s show a lower o v e r a l l s e n s i t i v i t y to hypercapnia than during burst - 164 -brea t h i n g . The decreased s e n s i t i v i t y i s a r e s u l t of a smaller o v e r a l l increase i n the breathing frequency. At 6% C O 2 both groups breathe continuously and f u r t h e r increases i n C O 2 do not r e s u l t i n f u r t h e r increases i n f. I t appears that i n each group maximum f i s determined by the d u r a t i o n of TIJOT* During s i n g l e breath breathing the breath d u r a t i o n i s about two times that during burst breathing (5.5 seconds and 2.5 seconds r e s p e c t i v e l y ) , and ther e f o r e the maximum breathing frequency i s about one h a l f that during burst breathing (5.2 breaths/minute and 10.4 breaths/minute). A s i m i l a r trend can be seen i n the Columbian ground s q u i r r e l which a l s o breathes with a s i n g l e breath p a t t e r n ; t o t a l breath d u r a t i o n i s 6.5 seconds and maximum breathing frequency under s i m i l a r hypercapnic c o n d i t i o n s i s about 4.5 breaths/minute (McArthur, 1986). CBX does not s i g n i f i c a n t l y a l t e r o v e r a l l v e n t i l a t o r y responses i n e i t h e r burst breathing or s i n g l e breath breathing S. l a t e r a l i s . Although burst breathing CBX s q u i r r e l s had s l i g h t l y reduced t i d a l volumes at a l l l e v e l s of C O 2 , hypercapnia s t i l l acted c e n t r a l l y to produce increases i n V<T> Thus, the major e f f e c t of hypercapnia was a c e n t r a l s t i m u l a t i o n of v e n t i l a t o r y frequency and t i d a l volume. Although there i s a great deal of v a r i a b i l i t y i n the a c t u a l magnitude of the hypercapnic response i n - 165 -hi b e r n a t i n g animals, i t i s evident that hypercapnic s e n s i t i v i t y i s high r e l a t i v e to hypoxic s e n s i t i v i t y . Observations of a high CO2 s e n s i t i v i t y imply that the length of the breath hold p e r i o d may be r e l a t e d to l e v e l s of e i t h e r a r t e r i a l Pc02 o r P H (McArthur, 1986; S t e f f e n and R i e d e s e l , 1984; T a h t i , 1982). I t has been suggested that v e n t i l a t i o n i n burst breathing animals i s i n i t i a t e d by a r i s e i n PaC02 or f a l l i n pH beyond a c r i t i c a l or thr e s h o l d l e v e l of P aC02 or pH. Increases i n the i n s p i r e d CO2 f r a c t i o n a l t e r P aco2 or pH such that the th r e s h o l d f o r r e s p i r a t i o n i s reached more q u i c k l y over the breath hold p e r i o d and T^ v p becomes shorter (McArthur, 1986). Therefore, during h i b e r n a t i o n PCO2 or pH act as the primary chemical stimulus i n c o n t r o l l i n g breathing (McArthur, 1986; T a h t i , 1982) and s p e c i f i c a l l y breath hold d u r a t i o n . GENERAL DISCUSSION The chemical c o n t r o l of breathing i n awake S.  l a t e r a l i s appears to be fundamentally d i f f e r e n t from the chemical c o n t r o l of breathing during h i b e r n a t i o n . Figure 34 i l l u s t r a t e s the d i f f e r e n c e i n r e s p i r a t o r y s e n s i t i v i t i e s to hypoxia and hypercapnia i n the two s t a t e s . A i r convection requirement takes i n t o account the d i f f e r e n c e s i n metabolic r a t e between h i b e r n a t i o n and euthermia, and th e r e f o r e can be used to compare v e n t i l a t o r y responses i n the two s t a t e s . A i r convection requirements represents the number of mis. of - 166 -F i g u r e 34. Comparison of the e f f e c t s of decreasing F J Q 2 ( a ) and i n c r e a s i n g F J C O 2 ( B) o n a ^ r convection requirement (V /V02) * n awake S. l a t e r a l i s ( • ) , and during burst breathing i n S. l a t e r a l i s ( O ) h i b e r n a t i n g at T c of about 7°C and during s i n g l e breath breathing i n S. l a t e r a l i s ( • ) h i b e r n a t i n g at a T c of about 2°C. - 168 -a i r which are moved per ml. of 0 2 which i s consumed. Awake euthermic S. l a t e r a l i s show a strong hypoxic v e n t i l a t o r y response and a comparatively blunted hypercapnic v e n t i l a t o r y response (Figure 34). As with most h i b e r n a t i n g s p e c i e s , S. l a t e r a l i s are a l s o s e m i - f o s s o r i a l and normally l i v e i n chr o n i c hypoxic and hypercapnic c o n d i t i o n s (Boggs et a l . , 1984). Many of the v e n t i l a t o r y adaptations observed i n f o s s o r i a l species appear to minimize r e s p i r a t o r y work while ensuring adequate d e l i v e r y of 0 2 to the t i s s u e s (Boggs et a l . , 1984). Resting v e n t i l a t i o n i s low compared to p r e d i c t e d values based on body weight (Boggs et a l . , 1984). The r e l a t i v e l y high hypoxic s e n s i t i v i t y ensures that under hypoxic c o n d i t i o n s there i s adequate pulmonary v e n t i l a t i o n . Reduced s e n s i t i v i t y to C02r on the other hand, may be a means to reduce the work of breathing s i n c e i n an environment of high CO2 increases i n v e n t i l a t i o n would not serve to reduce CO2 i n the blood much f u r t h e r (Boggs et a l . , 1982). The r e l a t i v e l y low frequency to t i d a l volume r e l a t i o n s h i p may enhance a l v e o l a r v e n t i l a t i o n and makes O2 l o a d i n g and CO2 unloading i n the lungs more e f f e c t i v e . This adjustment may a l s o serve to decrease v e n t i l a t o r y s e n s i t i v i t y to increases i n i n s p i r e d CO2. In a d d i t i o n , reduced s e n s i t i v i t y to CO2 may be due to an increase i n the l e v e l s of b u f f e r base i n the body (Tenney, 1954), or i t may be an adaptation at the c e l l u l a r l e v e l i n the c e n t r a l chemoreceptors (Darden, 1972; Tenney and Boggs, 1986). 169 -Chronic exposure to hypoxia and hypercapnia does not a l t e r the CO2 s e n s i t i v i t y of golden-mantled ground s q u i r r e l s . This suggests that the reduced responsiveness to hypercapnia i n v o l v e s long term adaptations or g e n e t i c adaptations through m o d i f i c a t i o n s to the b u f f e r i n g c a p a c i t i e s of the blood and r e n a l system, the e f f i c i e n c y of a l v e o l a r v e n t i l a t i o n under r e s t i n g c o n d i t i o n s , or the chemoreceptor c h a r a c t e r i s t i c s . C a r o t i d body chemoreceptors i n s e v e r a l mammals c o n t r i b u t e up to 50% to the acute hypercapnic response. The observation that i n S. l a t e r a l i s p e r i p h e r a l chemoreceptors do not c o n t r i b u t e to the hypercapnic response suggests a p o s s i b l e mechanism f o r the decrease i n o v e r a l l hypercapnic s e n s i t i v i t y . Chronic exposure to hypoxia and hypercapnia does r e s u l t i n m o d i f i c a t i o n of the frequency and t i d a l volume components of r e s p i r a t i o n which may f u r t h e r increase O2 l o a d i n g and CO2 unloading i n the lungs under borrow gas c o n d i t i o n s as described above. Although p e r i p h e r a l chemoreceptors do play an e x c i t a t o r y r o l e during r e s t i n g v e n t i l a t i o n and during moderate l e v e l s of hypoxia, they are not necessary f o r the production of a hypoxic h y p e r v e n t i l a t i o n . Hypoxic v e n t i l a t o r y responses, mediated s o l e l y by increases i n frequency, can be produced c e n t r a l l y i n S. l a t e r a l i s . Only a l i m i t e d number of s t u d i e s have reported c e n t r a l e x c i t a t i o n - 170 -i n response to hypoxia. Tenney and M i l l e r (1975) suggest that the s i t e r e s p o n s i b l e f o r hypoxic s e n s i t i v i t y may not be i n the b r a i n stem region but rather i n a higher b r a i n centre such as the diencephalon. I t i s unknown i f low p e r i p h e r a l c h e m o s e n s i t i v i t y and high c e n t r a l hypoxic s e n s i t i v i t y are common to other f o s s o r i a l s p e c i e s . I t i s a l s o unclear what the adaptive s i g n i f i c a n c e of adjustments i n the r e l a t i v e r o l e s of p e r i p h e r a l and c e n t r a l chemoreceptor s e n s i t i v i t i e s compared to n o n - f o s s o r i a l mammals may be. As S. l a t e r a l i s enter h i b e r n a t i o n at 5°C continuous breathing i s converted i n t o a p e r i o d i c p a t t e r n . The reduced v e n t i l a t i o n during h i b e r n a t i o n does not imply a l o s s of v e n t i l a t o r y c o n t r o l as suggested by Hammel et a l . (1968) and Lyman (1972). Figure 34 i l l u s t r a t e s that v e n t i l a t o r y s e n s i t i v i t y to hypercapnia i s at l e a s t 3 times that of the awake animal. The hypoxic s e n s i t i v i t y during h i b e r n a t i o n at a T^ of 8°C i s s i m i l a r to that of the awake animal once reductions i n metabolic r a t e are accounted f o r (Figure 34). This i m p l i e s that decreased t i s s u e demand f o r O2 may be the major f a c t o r i n the high hypoxic t o l e r a n c e t y p i c a l of h i b e r n a t i n g s p e c i e s . The decrease i n hypoxic s e n s i t i v i t y i n ground s q u i r r e l s h i b e r n a t i n g at 2°C could r e s u l t from a f u r t h e r l e f t s h i f t i n the HbC>2 d i s s o c i a t i o n curve. A l t e r n a t i v e l y , i f hypoxic chemosensitive areas are l o c a t e d - 171 -i n higher b r a i n c e n t r e s , such as the diencephalon, i t i s p o s s i b l e that nerve t r a n s m i s s i o n i n or from these areas i s temperature s e n s i t i v e . Therefore, as i n the production of a burst breathing p a t t e r n , decreased body temperature may block input from these areas. I t i s i n t e r e s t i n g to note that i n euthermic animals the c e n t r a l l y produced hypoxic h y p e r v e n t i l a t i o n reported by s e v e r a l authors can be abo l i s h e d by a n e s t h e t i c s (Moyer and Beecher, 1942; M i l l e r and Tenney, 1975). I t i s unclear whether the s i t e of c e n t r a l hypoxic s e n s i t i v i t y i n the h i b e r n a t i n g animal i s s i m i l a r to that seen i n the euthermic animal. The r e l a t i v e hypercapnic response i s much greater during h i b e r n a t i o n than i n euthermia (Figure 34). The l e v e l s of CO2 reported f o r burrows during the summer p e r i o d , about 3% to 4% CO2, would not s t i m u l a t e r e s p i r a t i o n s i g n i f i c a n t l y i n the euthermic animal. Levels of 3% to 4% CO2 during h i b e r n a t i o n would d r a m a t i c a l l y increase r e s p i r a t i o n . In the w i n t e r , when metabolic rate i s extremely low, however, s e v e r a l s t u d i e s report that burrow c o n d i t i o n s are no longer hypoxic and hypercapnic except during periods of a r o u s a l and euthermia (Kuhen, 1986; Wi l l i a m s and Rausch, 1973). Thus, the r e l a t i v e l y high CO2 s e n s i t i v i t y during h i b e r n a t i o n would not r e s u l t i n elevated v e n t i l a t i o n . - 172 -Current data suggests that CO2 i s important i n v e n t i l a t o r y c o n t r o l and that f l u c u a t i o n s i n Pco2 o r P H m a Y determine the length of the breath hold p e r i o d i n burst breathing animals (McArthur, 1986; S t e f f e n and Ri e s e d e l 1984; T a h t i , 1982). I t i s d i f f i c u l t to apply t h i s mechanism of v e n t i l a t o r y c o n t r o l to the s i n g l e breath breathing. During burst breathing the breath hold p e r i o d i s , on average, 10 minutes long. During t h i s time even at low metabolic r a t e s a r t e r i a l Pco2 could change enough to i n i t i a t e r e s p i r a t i o n . During s i n g l e breath breathing i n S. l a t e r a l i s breath hold periods are only about 40 seconds long. This breath hold p e r i o d i s not long enough to a l l o w s u b s t a n t i a l changes i n Pco2« Undoubtedly e i t h e r the th r e s h o l d which i n t i a t e s r e s p i r a t i o n or the stimulus which i n i t i a t e s r e s p i r a t i o n has changed at the lower temperatures. I t i s evident that long term o v e r a l l minute v e n t i l a t i o n i s maintained constant d e s p i t e a considerable change i n v e n t i l a t o r y p a t t e r n . What the mechanism f o r such p r e c i s e v e n t i l a t o r y c o n t r o l i s remains unknown, although i t i s e v i d e n t l y centered i n the CNS. Whether there i s an adaptive s i g n i f i c a n c e to the two observed r e s p i r a t o r y p a t t e r n s during h i b e r n a t i o n i s a l s o unknown. Burrow or hibernaculum temperatures have been reported to reach l e v e l s f a r below those used i n most l a b o r a t o r i e s (Maclean, 1981; W i l l i a m s and Rausch, 1973). H i b e r n a t i n g S. l a t e r a l i s could t h e r e f o r e be exposed to - 1 7 3 -temperatures which would produce s i n g l e breath bre a t h i n g . I t i s evident that the r e s p i r a t o r y p a t t e r n s observed during h i b e r n a t i o n are produced i n p a r t , by c e n t r a l mechanisms. The burst breathing p a t t e r n may i n v o l v e i n f l u e n c e s of higher CNS areas on the b a s i c r e s p i r a t o r y p a t t e r n produced by the b r a i n stem, and the t r a n s i t i o n between the two patterns i n v o l v e s the d i r e c t e f f e c t s of temperature on neural t r a n s m i s s i o n i n the CNS. Studies i n v o l v i n g s e l e c t i v e CNS c o o l i n g during h i b e r n a t i o n • are r e q u i r e d to c l a r i f y the r o l e of the CNS i n p a t t e r n generation during h i b e r n a t i o n . 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