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Ventilation and diving apnoea in Rana pipiens West, Nigel Hugh 1974

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VENTILATION AND DIVING APNOEA IN RANA PIPIENS  by NIGEL HUGH WEST B.Sc,  University of B r i s t o l , 1969  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department of ZOOLOGY  We accept this thesis as conforming  to the  required standard  THE UNIVERSITY OF BRITISH COLUMBIA July,  1974  In presenting  this thesis i n p a r t i a l f u l f i l m e n t of the requirements for an  advanced degree at the University of B r i t i s h Columbia, I agree that Library s h a l l make i t f r e e l y available for reference  and study.  the  I further  agree that permission for extensive copying of this thesis for s c h o l a r l y purposes may  be granted by the Head of my Department or by h i s  representatives.  It i s understood that copying or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission,  The University of B r i t i s h Columbia Vancouver, Canada  i  ABSTRACT  Two types of v e n t i l a t i o n cycle were recorded i n unanaesthetised but restrained frogs (Rana p i p i e n s ); one concerned with v e n t i l a t i o n of the buccal cavity alone (buccal cycle) and the other with lung v e n t i l a t i o n (lung c y c l e ) . During the former the nares were open and the g l o t t i s closed so that only small pressures were generated by the movement of the buccal f l o o r .  The onset  of a lung v e n t i l a t i o n was s i g n a l l e d by a c t i v i t y i n the laryngeal d i l a t o r muscle and when the g l o t t i s opened lung pressure and volume f e l l while buccal cavity pressure and volume increased. was  A f t e r n a r i a l closure the buccal f l o o r  r a p i d l y raised and gas was forced into the lungs from the buccal c a v i t y .  At peak pressure i n the lungs and buccal cavity the g l o t t i s closed and nares opened, the recovery stroke of the pump being passive.  A i r flow recordings  made at the external nares showed two phases of flow during each buccal cycle, while four phases accompanied each lung v e n t i l a t i o n c y c l e . By p l o t t i n g pressure/volume  loops from the buccal pump an analysis was  made of the mechanical work performed  i n one lung v e n t i l a t i o n cycle, and the  proportion of this work a v a i l a b l e f o r lung i n f l a t i o n a f t e r various losses against viscous and flow r e s i s t i v e forces i n the pump i t s e l f ; while measurement of the areas of t y p i c a l sequences of such loops together with respiratory frequency enabled the mechanical work output o f the pump to be determined f o r frogs ranging i n s i z e from 24 to 86 grams.  Using H i l l ' s c l a s s i c a l equation  for muscle e f f i c i e n c y , i t was possible to estimate mechanical e f f i c i e n c y f o r s i n g l e respiratory cycles by c a l c u l a t i n g the heat of maintenance and heat of shortening of the buccal f l o o r muscles, while simultaneously measuring mechanical work output.  Calculated e f f i c i e n c i e s of lung v e n t i l a t i o n cycles  rose as mechanical work performed  increased from 7.4% at 0.65 gram.cm/cycle to  ii 19.3%  at 2.73  gram.cm/cycle.  Diving apnoea i n Rana pipiens was  induced by the presence of water at the  l e v e l of the external nares, at which point the nares closed, no water entering the buccal cavity during the dive.  Occasional v e n t i l a t i o n cycles occurred  during the dive i n which gas entered the buccal cavity from the lungs, an equal volume then being pumped back into the lungs, but there was v e n t i l a t o r y exchange with the external medium.  no  B i l a t e r a l s e c t i o n of the  trigeminal nerves r e s u l t e d i n an abnormal response to submergence, i n that water entered the buccal cavity, and i n some cases the lungs, while surfacing often did not r e s u l t i n resumption of v e n t i l a t i o n .  Skin mechanoreceptors i n  the region of the external n a r i a l openings serving the ophthalmic branch of the trigeminal were found to be capable of responding  to the minimum stimulus  encountered on submersion, movement of a water meniscus across the n a r i a l region, while a tonic response to hydrostatic pressure occurred i n some preparations.  In c o n t r o l experiments cutaneous mechanoreceptors innervated by  the s p i n a l nerves were shown to have no response to a water meniscus passing across t h e i r receptive f i e l d s , suggesting that they possess higher thresholds than the n a r i a l receptors.  Periods of apnoea could be induced i n a i r i n  Rana pipiens by b i l a t e r a l or u n i l a t e r a l stimulation of the cut p e r i p h e r a l ends of the ophthalmic branch of the trigeminal nerve at threshold voltages as low as 30 mv,  at a frequency of 200 Hz.  Increase i n stimulating voltage r e s u l t e d  i n longer periods of apnoea before v e n t i l a t i o n "broke through", and i n these periods the external nares were closed and buccal pressure was independent of atmospheric pressure.  held  Reduction of the s t i m u l a t i o n frequency  by a f a c t o r of ten a f t e r the i n i t i a t i o n of apnoea, simulating adaptation of the sensory nerves, proved as e f f e c t i v e i n maintaining apnoea as continued stimulation at the o r i g i n a l  frequency.  iii TABLE OF CONTENTS General Introduction  1  Part I. The Mechanical Events Associated with Lung and Buccal V e n t i l a t i o n i n the Frog, Rana pipiens  Part I I .  Introduction  11  Methods  13  The Morphology of the P r i n c i p a l Respiratory Muscles  17  Results  20  Discussion  36  The Mechanical Work and E f f i c i e n c y of V e n t i l a t i o n i n Rana pipiens  Part I I I .  Introduction  44  Methods  46  Results  53  Discussion  72  The I n i t i a t i o n of Diving Apnoea i n Rana pipiens Introduction  76  Methods  78  Results  83  Discussion General Discussion  109 113  Summary  116  L i t e r a t u r e Cited  119  iv LIST OF FIGURES Figure 1.  The s u p e r f i c i a l and deep muscles of the buccal f l o o r of Rana p i p i e n s .  18  Figure 2.  The muscles of the g l o t t i s and larynx of Rana p i p i e n s .  19  Figure 3.  Pressure and volume changes recorded from the buccal c a v i t y .  Figure 4.  21  Pressure and volume changes i n the lungs and buccal cavity, together with a c t i v i t y from the muscles of the r e s p i r a t o r y valves.  Figure 5.  The r e l a t i o n s h i p between lung and buccal pressure i n Rana pipiens.  Figure 6.  22  24  The r e l a t i o n s h i p between n a r i a l closure and g l o t t a l opening i n Rana pipiens.  29  Figure 7.  N a r i a l gas flow during lung and buccal v e n t i l a t i o n .  31  Figure 8.  E l e c t r i c a l a c t i v i t y i n the p r i n c i p a l muscles of the buccal f l o o r during v e n t i l a t i o n .  Figure 9.  32  E l e c t r i c a l a c t i v i t y i n r e s p i r a t o r y muscles during ventilation.  34  Figure 10. Semischematic diagram of lung and buccal pressure and volume changes during lung and buccal v e n t i l a t i o n cycles i n Rana pipiens.  37  Figure 11. The a p p l i c a t i o n of Laplace's law to the buccal f l o o r i n Rana pipiens.  50  Figure 12. Diagrammatic representation of pressure/volume loops i n the buccal c a v i t y and lungs during a lung ventilation cycle.  54  Figure 14.  Sequences of pressure/volume loops recorded from the lungs and buccal c a v i t y of Rana pipiens.  Figure 15.  Regression of mechanical work output on body weight for frogs ranging i n weight from 25 to 87 grams.  Figure 16.  Regression of work appearing  61 i n the lungs on weight  for Rana p i p i e n s . Figure 18.  59  Regression of buccal volume change i n lung and buccal v e n t i l a t i o n on weight.  Figure 17.  58  62  Pressure/volume loop measured from the buccal c a v i t y for one lung v e n t i l a t i n g cycle, together with calculated tension i n the buccal f l o o r over the same c y c l e .  Figure 19.  Terms of the e f f i c i e n c y equation plotted against maximum cycle pressure f o r 12 v e n t i l a t i n g cycles,  Figure 20.  64  65  Calculated mechanical e f f i c i e n c y and t o t a l energy input p l o t t e d against mechanical work output per c y c l e , for 12 v e n t i l a t i n g c y c l e s .  Figure 21.  67  Transverse sections of the snout, i l l u s t r a t i n g the course of the ophthalmic nerve i n Rana p i p i e n s .  80  Figure 22.  Lung and buccal pressure i n Rana pipiens during d i v i n g .  85  Figure 23.  Pressures recorded from the buccal c a v i t y and lungs  Figure 24.  during a dive i n Rana pipiens.  87  Record from a n a r i a l thermistor during submergence.  89  vi  Figure 25.  EMGs recorded from normal and sham-operated  frogs  on submergence.  91  Figure 26. EMGs recorded from denervated frogs on submergence. Figure 27.  92  Afferent nervous a c t i v i t y i n the ophthalmic nerve during d i v i n g .  94  Figure 28. Response of a n a r i a l branch of the ophthalmic nerve to two rates of water flow.  96  Figure 29. Response of a n a r i a l branch of the ophthalmic nerve to punctate stimulation.  97  Figure 30. Tonic a c t i v i t y i n a f i n e n a r i a l branch of the  Figure 31.  ophthalmic nerve.  98  Recordings from cutaneous mechanoreceptors.  99  Figure 32. Response of cutaneous mechanoreceptors  to hydrostatic  pressure.  101  Figure 33. B i l a t e r a l e l e c t r i c a l stimulation of the ophthalmic nerves near the threshold voltage.  102  Figure 34. B i l a t e r a l e l e c t r i c a l stimulation of the ophthalmic nerves i n Rana pipiens.  103  Figure 35. B i l a t e r a l e l e c t r i c a l stimulation of the ophthalmic nerves with a frequency change simulating adaptation. Figure 36. B i l a t e r a l ophthalmic stimulation i n Rana pipiens.  104 106  Figure 37. The r e l a t i o n s h i p between time spent i n apnoea and the stimulating voltage•>  107  vii LIST OF TABLES Table 1.  Calculation of the oxygen cost of v e n t i l a t i o n i n Rana pipiens.  Table 2.  69  Calculation of r e s p i r a t o r y muscle oxygen consumption i n Rana pipiens.  70  viii  ACKNOWLEDGEMENTS  I wish t o thank Dr. David Jones, my s u p e r v i s o r , f o r h i s encouragement and a d v i c e throughout t h e course o f t h i s study.  P a r t o f S e c t i o n I I I was  performed j o i n t l y w i t h Dr. Jones, b u t t h e c o n c l u s i o n s drawn a r e my own and Dr. Jones does n o t n e c e s s a r i l y agree w i t h them. I a l s o w i s h t o thank L o w e l l L a n g i l l e f o r s e v e r a l u s e f u l p e r t a i n i n g to the second p a r t o f t h i s  discussions  thesis.  Thanks a r e due t o my w i f e , J e n i f e r , f o r h e r c o n s t a n t encouragement throughout the study. F i n a n c i a l s u p p o r t was p r o v i d e d by T e a c h i n g A s s i s t a n t s h i p s and a Graduate F e l l o w s h i p from t h e U n i v e r s i t y o f B r i t i s h  Columbia.  1 GENERAL INTRODUCTION  Although apnoea i s a c e n t r a l feature of the periods of submergence undergone by diving vertebrates, much less i s known about the s t i m u l i which i n h i b i t or modify the rhythmic a c t i v i t y of the respiratory centre during diving than about the respiratory and cardiovascular adjustments which occur as a r e s u l t of such stimulation.  Huxley (1913b), working on ducks, proposed  a "postural r e f l e x " on the basis that ducks become apnoeic on assuming c e r t a i n positions i n a i r but the s i g n i f i c a n c e of this observation was lessened when Koppanyi and Kleitman (1927) found that ducks never assume these positions when f r e e l y swimming underwater.  Andersen (1963) presented  evidence that i n the duck apnoea was produced by water immersion o f the area of the beak surrounding the n o s t r i l s .  The response could be abolished by  b i l a t e r a l section of the trigeminal nerve to the beak, i n which case ducks would resume breathing through a tracheal cannula during periods of head submersion.  Butler and Jones (1968) considered however, that the most  important f a c t o r i n i n i t i a t i n g apnoea during diving i n the duck was the contact of water with the respiratory passages at the l e v e l of the g l o t t i s , for they found that ducks continued to breathe ( v i a a tracheal cannula) when water covered the external nares, whereas r a i s i n g the water to the l e v e l of the g l o t t i s caused apnoea.  E l e c t r i c a l stimulation o f the branches o f the  glossopharyngeal nerve serving this area was also found to cause apnoea (Jones and Purves, 1970).  Moreover some doubt has been cast upon the involvement of  the trigeminal nerves as an afferent pathway i n r e f l e x apnoea i n the duck by the recent finding that beak mechanoreceptors were not capable o f a response even when the hydrostatic pressure at the beak surface was raised and lowered between 0 and 50 cm.H 0 simulating diving and emersion (Gregory, 1973). 9  2 Tchobroutsky et a l (1969) have shown that apnoea produced by head immersion i n the newborn lamb, adult r a b b i t , and adult sheep was g l o t t i s was  abolished when the  anaesthetised, or when the superior laryngeal nerves were severed.  They have suggested that a r e f l e x i n i t i a t e d by the contact of the amniotic f l u i d with the airways of the foetus may  be responsible for the i n h i b i t i o n of  respiratory movements i n utero, and that release of the r e f l e x at p a r t u r i t i o n ensures the prompt onset of v e n t i l a t i o n . Reflexes from the area of the nasal mucous membrane have been shown to cause apnoea i n response to mechanical, e l e c t r i c a l and chemical stimulation i n non-diving vertebrates.  Recently Angel1 James and Daly (1972a) have  demonstrated i n dogs that the respiratory responses were diminished d i v i s i o n of the ethmoidal  by  or maxillary branches of the trigeminal and  abolished when both nerves were b i l a t e r a l l y divided. contact with the nasal mucosa was  Water or s a l i n e i n  e f f e c t i v e i n producing apnoea, although  the  l i q u i d had to be i n motion, suggesting that information from mechanoreceptors was  p r i m a r i l y involved. No d e t a i l e d information i s a v a i l a b l e concerning the v e n t i l a t o r y adjust-  ments of amphibians to submergence, although many amphibians have d i v i n g habits and can remain submerged for extremely long periods, Rana esculenta surviving submersion for 2-3 weeks at 14-15°C (Serfaty and Guental, 1943). Indeed the general f i e l d of amphibian v e n t i l a t o r y control has aroused l i t t l e interest.  Smyth (1939), investigated the respiratory response to a l t e r e d  concentrations of 0^ and CO^ i n i n s p i r e d a i r , while De Marneffe-Foulon (1962) studied the e f f e c t s of s h i f t i n g the blood pH on the frequency buccal v e n t i l a t i o n cycles.  of lung and  N e i l et a l (1950), and T a g l i e t t i and Casella  (1966) have demonstrated neurophysiologically that lung s t r e t c h receptors are active during lung i n f l a t i o n and are probably involved i n the  3 r e f l e x termination of lung f i l l i n g .  Some of the mechanoreceptor population  also appear to be s e n s i t i v e to lung d e f l a t i o n ( T a g l i e t t i and C a s e l l a , 1968). More s p e c i f i c a l l y , information on the i n i t i a t i o n of diving apnoea i n anuran amphibians i s l i m i t e d to observations of Lombroso (1913), and Willem (1920) on Rana esculenta.  Lombroso (1913) noted that contact of the external nares with  water was s u f f i c i e n t to i n h i b i t v e n t i l a t i o n , this i n h i b i t i o n being so powerful i n submerged batrachians deprived of access to the surface that death ensued from anoxia before any attempts were made at v e n t i l a t i o n , while Jones (1967) confirmed an observation of Willem (1920) that occasional v e n t i l a t i o n movements i n which gas was moved from the lungs to the buccal c a v i t y and back again occurred during the course of the dive, although  the external nares  remained closed underwater. The increased oxygen consumption that follows a period of submergence i n anurans has been considered to be partly due to the need to replenish the blood oxygen supply and p a r t l y due also to the increased work done by the respiratory muscles i n post dive hyperventilation.  Jones (1967) pointed out  that animals showing bursts of hyperventilation but which were otherwise quiescent consumed a larger amount of oxygen i n these periods, i n d i c a t i n g that the cost of hyperventilation i s high.  An estimate of the oxygen cost of  v e n t i l a t i o n must therefore be obtained before the true oxygen debt a f t e r a dive can be determined.  The energy cost of v e n t i l a t i o n has been studied i n  two main ways; by measuring the energy put into the r e s p i r a t o r y pump, and by measuring the external work performed by the pump.  Oxygen consumption i s a  measure of energy input to the pump, every l i t r e of oxygen consumed producing an average of 4.825 c a l o r i e s . experimental resting V  In man, at l e a s t , i t i s possible to allow an  subject to v e n t i l a t e at a series of minute volumes up from  while measuring oxygen consumption.  I t can then be assumed that  A the respiratory muscles account for the a d d i t i o n a l oxygen consumption above the basal rate, and that this a d d i t i o n a l consumption gives an i n d i c a t i o n of oxygen cost.  L i l j e s t r a n d (1918) f i r s t studied the metabolic cost of  v e n t i l a t i o n i n man,  obtaining a series of minute volumes by both voluntary  hyperventilation and stimulation with CO,, and found that voluntary h y p e r v e n t i l ation had a greater oxygen cost, apparently  due  to tetanus i n muscles usually  unrelated to breathing. A t y p i c a l technique for estimating the oxygen cost and e f f i c i e n c y of a human v e n t i l a t i o n was that of Campbell et a l (1957) who increased V by —  —  E  placing dead space between subjects and a closed c i r c u i t spirometer.  By  measuring oxygen consumption at r e s t and at several l e v e l s of minute volume a curve was  produced from which the oxygen cost of r e s t i n g v e n t i l a t i o n could be  obtained by extrapolation.  A l l recent studies i n man  have shown that the  oxygen cost of r e s t i n g v e n t i l a t i o n i s small and i s almost c e r t a i n l y l e s s than 2% (Campbell et a l , 1970). The e f f i c i e n c y of the r e s p i r a t o r y muscles was  obtained  i n Campbell et a l ' s  (1957) study by measuring the increased oxygen consumption associated with increased mechanical work loads obtained by increasing i n s p i r a t o r y resistance. Oxygen consumption was  converted to i t s mechanical equivalent using  appropriate c a l o r i f i c equivalents.  E f f i c i e n c y was  found to vary from  i n young normal subjects, with e f f i c i e n c y remaining constant subject over the range of imposed workloads. e f f i c i e n c y of breathing i n man  i n any  5-10%  one  Other estimates of the mechanical  have been made by p l o t t i n g pressure-volume  loops while recording oxygen consumption. found e f f i c i e n c i e s of from 1-7%  the  By this method F r i t t s et a l (1959)  i n normal subjects, whereas M i l i c - E m i l i  and P e t i t (1960) found e f f i c i e n c i e s of from 19-25%, which i s i n the range of muscular e f f i c i e n c y .  I t i s s e l f - e v i d e n t that these e f f i c i e n c y  5 determinations i n man are extremely v a r i a b l e , p a r t l y due to unavoidable d i f f i c u l t i e s i n the methods employed.  For example, small amounts of negative  work may be done on the system which would not appear i n the pressure/volume loops, i . e . some muscles may be f o r c i b l y lengthened during t h e i r contraction periods.  Secondly, mechanical work may not be the only parameter which should  be considered as an output of the system; for example, i f a weight i s supported, no mechanical work i s done on the system but the oxygen consumption of the  supporting muscles increases as a function of the weight supported.  An  analogous s i t u a t i o n i n breathing would occur i f the resistance to flow i s increased as i n the case of an airway obstruction. an increase i n oxygen  In this case there may be  consumption with no corresponding increase i n mechanical  work out of the system, as the respiratory muscles would need to contract more f o r c i b l y to produce the same pressure and volume changes. It i s p a r t l y for this reason that the energy cost of g i l l i r r i g a t i o n i n f i s h i s thought to be high, f o r the flow resistance of airways and  gill  curtains depends on the v i s c o s i t y of the medium, and water i s 55 times more viscous than a i r .  Also, i n e r t i a l forces depend on the mass of the medium and  water i s 840 times more dense than a i r ,  although, presumably, i f there i s no  flow reversal i n the system, i n e r t i a l forces w i l l be small. The methods that have been used to determine the energy cost of breathing i n man are very d i f f i c u l t to apply to f i s h .  For example, i t i s d i f f i c u l t  to measure 0^ consumption at d i f f e r e n t l e v e l s of g i l l i r r i g a t i o n i n f i s h , without producing side e f f e c t s , such as increased e x c i t a b i l i t y , so that i t i s unwise to assume that the increased oxygen consumption at elevated l e v e l s of i r r i g a t i o n i s due only to the increased respiratory work, and Cech (1970) considered that the study of Van Dam c r i t i c i s m on these grounds.  Van Dam used C0  9  Cameron  (1938) i s open to  to increase i r r i g a t i o n rate, and  6 assumed that the resultant increase i n oxygen consumption was s o l e l y due to the  increased i r r i g a t i o n , although he noted that 2% (X> produced struggling of  the  f i s h , and presumably increased metabolic oxygen demand.  2  The oxygen cost  of breathing i n the Tench has been studied by Schumann and P i i p e r (1966) . Highly anaesthetised f i s h were used and respiratory water flow and oxygen uptake were measured.  The cost of breathing i n terms of 0^ uptake was  measured using spontaneous changes i n i r r i g a t i o n rate.  With a respiratory flow  rate j u s t s u f f i c i e n t to supply the 0^ required by the f i s h , i . e .  at resting  i r r i g a t i o n rate, the 0^ cost of breathing was 30% of t o t a l uptake and with a v e n t i l a t i o n rate 3 times higher, 50% of t o t a l 0^ uptake.  Schumann and P i i p e r  (1966) contend that non-ventilatory oxygen demand was constant, the f i s h being i n a "plateau" of anaesthesia, but there i s l i t t l e evidence to support this conclusion (Cameron and Cech, 19 70).  Measurements of the d i f f e r e n t i a l pressure  between the buccal and opercular c a v i t i e s of trout (Hughes and Shelton, 1958) have been used to estimate the mechanical work of g i l l i r r i g a t i o n by Alexander (1967).  Combining these estimates with an assumed mechanical  e f f i c i e n c y of 20%, Alexander (1967) arrived at a figure of 1% f o r the oxygen cost of i r r i g a t i o n . However, he made many assumptions which have been shown to be unwarrantable, such as constant flow through the g i l l s , constant g i l l resistance, and a constant pressure gradient across the g i l l s .  Many of the  objections to the work of Van Dam (1938) and Schumann and P i i p e r (1966) have been avoided by Jones and Schwarzfeld (1974), who changed the work of i r r i g a t i o n i n unanaesthetised trout by a l t e r i n g the pressure head across the g i l l s , and then measuring the r e s u l t i n g changes i n oxygen consumption, enabling the energy cost and e f f i c i e n c y of i r r i g a t i o n to be measured. No studies comparable to those on f i s h and man have yet been made on  7 amphibians.  In this study, however, measurement of the mechanical work of the  buccal pump proved to be f e a s i b l e i n the anuran, Rana pipiens, by the use of pressure/volume loops.  A method for the estimation of mechanical e f f i c i e n c y  i n s i n g l e r e s p i r a t o r y cycles by the determination of respiratory muscle tension and degree of shortening during a r e s p i r a t o r y cycle was also developed i n order that an estimate of the oxygen cost of v e n t i l a t i o n could be made. During preliminary experiments the v e n t i l a t i o n mechanism of Rana pipiens was found to be s u f f i c i e n t l y d i f f e r e n t from published accounts of other anurans (e.g. Sholten, 1942; De Mameffe-Foulon, 1962; De Jongh and Gans, 1969) to warrant further i n v e s t i g a t i o n . Internal gas exchange i n most vertebrates i s brought about by the use of pressure pumps or vacuum pumps, which i n many cases are bimodal, operating i n the pressure mode i n one phase o f the v e n t i l a t o r y cycle, and the suction mode i n another.  In f i s h f o r example,  where more than one pump i s f u n c t i o n a l , the phase r e l a t i o n s h i p s between the pumps must be p r e c i s e l y c o n t r o l l e d to achieve adequate i r r i g a t i o n of the exchange surfaces (Shelton, 1970). Hughes and Shelton (1958) f i r s t used modem techniques  to record  pressures from the buccal and opercular c a v i t i e s of teleosts and showed that a d i f f e r e n t i a l pressure was maintained  from buccal to opercular c a v i t i e s , to  provide f o r water flow through the g i l l resistance.  However, i n periods of  t r a n s i t i o n between the buccal and opercular pumps very low pressure d i f f e r e n t i a l s occur, and as the suction pump takes over from' the pressure pump the d i f f e r e n t i a l may become negative so that flow over the g i l l s may tend to reverse, although probably never does so due to i n e r t i a l forces.  Hughes and  Shelton (1958) point out that the idea of a separate p r e - g i l l and p o s t - g i l l pump i s a s i m p l i f i c a t i o n of the s i t u a t i o n i n that anatomically i t i s d i f f i c u l t to separate the functional parts of each, and that i n many species one pump  8 predominates.  Bottom l i v i n g f i s h seem to depend on a suction-pump mechanism,  and i n some, for example the gurnard, the e x i t of water i s r e s t r i c t e d to a small, d o r s a l l y directed aperture.  The exhalent phase of v e n t i l a t i o n tends to  be increased also, so that water i s ejected rapidly and at high v e l o c i t y .  In  both the t e l e o s t and elasmobranch f l a t f i s h the opercular openings and openings to the g i l l s l i t s can be closed a c t i v e l y , perhaps so that entry of debris into the g i l l chambers i s prevented. to be balanced, although  In pelagic f i s h the pumps appear  the buccal pump predominates i n the horse mackerel  and the opercular pump i n the whiting.  I t appears that i n other pelagic f i s h  active r e s p i r a t o r y movements are not made, but that they r e l y on the current entering the mouth, a kind of ram-jet method of i r r i g a t i o n (Brown and Muir, 19 70).  The mechanism of g i l l i r r i g a t i o n i n elasmobranchs has been shown to be  e s s e n t i a l l y the same as that found i n teleosts (Hughes, 1960), although recently heterogeneity of function w i t h i n the elasmobranch r e s p i r a t o r y mechanism has been emphasized (Hughes, 1973), based on the finding i n the dogfish that water entering the s p i r a c l e , and mouth, exits through the anterior and p o s t e r i o r g i l l s l i t s r e s p e c t i v e l y , and that the r e l a t i v e sizes of the p o s i t i v e and negative pressure phases i n i r r i g a t i o n vary between gill  slits. In modern  lungfish the mechanism of a i r v e n t i l a t i o n consists of a  straightforward adaptation of an aquatic i r r i g a t i o n cycle, with i n s p i r a t i o n being due to the buccal force-pump, while e x p i r a t i o n i s passive and consists of  the release of compressed lung gas which i s aided by the e l a s t i c i t y of the  lung tissue (Bishop and Foxon, 1968;  McMahon, 1969).  As pointed out by  Hughes (1973), the adaptations of modern lungfish to a i r breathing may  be  misleading i f i t i s naively considered that they also apply to the ancestral tetrapods, since there i s evidence that the Dipnoi are a s p e c i a l i z e d group  9 (White, 1966), a point of p a r t i c u l a r i n t e r e s t being that the modern Dipnoi do not possess the dorsal r i b s from which the p l u r a l r i b s of tetrapods  are  derived (Goodrich, 1930). Maximum s p e c i a l i z a t i o n of the buccal force-pump for a i r v e n t i l a t i o n occurs i n frogs (De Jongh and Gans, 1969), the Amphibia being the only modern group i n which the nares rather than the mouth are used as a path f o r a i r entering the buccal c a v i t y .  McMahon (1969) proposed a possible scheme for  the incorporation of the nares into the buccal force-pump mechanism.  He sug-  gested that buccal f i l l i n g must have been d i f f i c u l t i n e a r l y crossopterygians as the gape was  long, necessitating r a i s i n g the head c l e a r of the surface  to obtain a i r . One  s o l u t i o n to the problem, taken by the Dipnoi l i n e ,  was  to reduce the gape; while another s o l u t i o n , taken by the amphibian ancestors, was  to use the nares as a path for buccal f i l l i n g .  Use of the nares also  appears to have conferred on the amphibia the advantage of f i n e r c o n t r o l over inspired a i r , although at the expense of increased resistance to flow. Although this i s an a t t r a c t i v e p o s i b i l i t y , i t appears doubtful that the buccal force-pump was  important  i n the lung v e n t i l a t i o n of ancestral  amphibians, f o r the f o s s i l record shows that they possessed  w e l l developed  r i b s , associated i n modern vertebrates with an a s p i r a t i o n mode of v e n t i l a t i o n (Romer, 1972).  This evidence suggests then that the buccal force-pump of  modern amphibia i s a secondary adaptation associated with r i b reduction, which i t s e l f probably  came about i n response to a comparatively  recent return to a  more aquatic h a b i t , the ancestors of the modern amphibia having the main a t t r i b u t e needed for a s p i r a t i o n , a w e l l developed rib-cage.  Amphibians  s u p e r f i c i a l l y appear to possess a less complex breathing mechanism than that of most f i s h , anurans at least v e n t i l a t i n g by means of a buccal force-pump. However, the addition of v a r i a b l y a c t i v e valves (external nares and  glottis)  10  g r e a t l y i n c r e a s e s i t s f u n c t i o n a l c o m p l e x i t y i f o n l y from the p o i n t of view of  the c o n t r o l l i n g mechanisms i n v o l v e d .  t h e r e has been l i t t l e of  f o r t h i s reason that  agreement between a u t h o r s on the p r e c i s e  the m e c h a n i c a l e v e n t s o f anuran  relationships  ventilation.  This study t h e r e f o r e , n a t u r a l l y the  I t i s perhaps  fell  i n t o three s e c t i o n s .  mechanism o f l u n g and b u c c a l v e n t i l a t i o n was  examined.  I n the  first,  B u c c a l and l u n g  p r e s s u r e s were measured i n f r o g s t h a t were r e s t r a i n e d but u n a n a e s t h e t i s e d and combined  w i t h measurements o f the a s s o c i a t e d volume changes  r e s p i r a t o r y muscle a c t i v i t y  and  to g i v e an o v e r a l l p i c t u r e o f the b r e a t h i n g  mechanism; i n the second s e c t i o n the m e c h a n i c a l work and e f f i c i e n c y o f v e n t i l a t i o n were determined t o g i v e the energy c o s t ; i n the t h i r d experiments were performed for to  to d e t e r m i n e the s t i m u l u s o r s t i m u l i  the i n i t i a t i o n and maintenance such s t i m u l a t i o n .  o f d i v i n g apnoea,  Experiments were a l s o performed  section  responsible  and the s i t e s  sensitive  to determine the  c a p a b i l i t y o f r e c e p t o r s i n t h e s e a r e a s o f r e s p o n d i n g to the s t i m u l i p r e s e n t e d on submersion and attempts were made t o i n i t i a t e p e r i o d s o f apnoea i n a i r by s t i m u l a t i o n o f the a p p r o p r i a t e s e n s o r y n e r v e s .  ii PART 1.  THE MECHANICAL EVENTS ASSOCIATED WITH LUNG AND BUCCAL VENTILATION IN THE FROG» RANA PIPtENS  IOTj^DUGTiON  Although i t has long been recognised that anufari amphibians v e n t i l a t e by means of a buccal force-pump, there i s s u r p r i s i n g l y l i t t l e agreement as to the r e l a t i o n s h i p of the respiratory events iii these animals*  This i s due*, i n  part, to the d i f f i c u l t y of recording the pressure and volume changes within the lungs and buccal cavity along with the associated a c t i v i t y of the nafesj g l o t t i s , and respiratory muscles*  Furthermore, as Fexbn (1964) points out)  many early experiments were made on pithed of anaesthetised animals o f t e n placed on t h e i r backs, so that the r e s u l t s probably do not r e l a t e very c l o s e l y to events i n the Conscious, undistufbed animal, f o r aniifan r e s p i r a t i o n i s notoriously l a b i l e (Gaupp, 1896), V i f t u a l i y a l l studies show the existence of two types of f e s p i f a§ery" movement i n the buccal pump, an ongoing series of movements that v e n t i l a t e only the buccal cavity, interspersed by movements which v e n t i l a t e the lungs (Martin, 1878; Wedenski, 1881}  Wiiiem* 1 9 1 9 , 1920j Shoitefij 1942}  Foulbn, 1962} De Jdngh and Gans, 1969)*  De  Mafneffe=  Dad and Sfivastava (1956) claim a  constant r a t i o between the frequency of buccal and lung v e n t i l a t i o n movements i n Rana t j g t i f l a , but others have found no evidence for t h i s .  On the 6th€f  hand, Gnanamuthu (1936), and Ghefian (1956) held a r a d i c a l l y d i f f e r e n t view of the v e n t i l a t o r y process* claiming that the g l o t t i s never remains closed, and the lungs are v e n t i l a t e d by every buccal cycle*  completely  For instance  Cheriari (1956), working with Rana hexadactyla, found no experimental proof fdf s o l e l y buccal v e n t i l a t i o n , considering that the g l o t t i s never completely  12 closed, although the nares closed twice i n each r e s p i r a t o r y cycle.  (Unfor-  tunately t h e i r experiments were performed with the frogs fastened on t h e i r backs, so that the r e s u l t s were of doubtful value.) Although most authors now  accept the v a l i d i t y of separate lung and buccal  v e n t i l a t i n g cycles, there i s much disagreement on the precise sequence of events during lung v e n t i l a t i o n , i n p a r t i c u l a r the timing of g l o t t a l opening and n a r i a l closure, and whether the e x i t of gas from the lungs on exhalation i s due to e l a s t i c f i b r e s i n the lung walls, or an active c o n t r i b u t i o n from the flank muscles.  Further debate centres around the sequences of lung  i n f l a t i o n and d e f l a t i o n , during which the lungs are i n f l a t e d by a series of lung v e n t i l a t i o n movements of increasing peak pressure, i s o l a t e d from the buccal cavity by the closed g l o t t i s and then deflated again. 1920), Sholten (1942), and De Marneffe-Foulon sequences to be caused by stress, excitement (1969) observed  Willem  (1962) considered  (1919,  these  or pain, but De Jongh and Gans  them i n cannulated but otherwise unrestrained Rana  catesbeiana. The present study was  undertaken to c l a r i f y the breathing mechanism i n  the North American grass frog, Rana pipiens, by recording the pressure and volume changes i n the lungs and buccal cavity during normal breathing i n unanaesthetised  animals.  Volume changes were recorded i n such a way  place no mechanical load on the breathing mechanism.  as to  Records were also taken  of the a c t i v i t y of the main r e s p i r a t o r y muscles during breathing, and of the pattern of flow through the nares during both lung and buccal v e n t i l a t i o n cycles.  13 METHODS  The experiments were performed on 30 grass frogs (Rana p i p i e n s ) , ranging i n weight from 19 to 90 grams, the average weight being 65 grams. experiments were performed at 24°C ±1°C,  A l l the  the frogs being allowed to acclimate  i n tanks for at l e a s t a week at this temperature before use.  A l l operative  techniques were done under s u r g i c a l anaesthesia obtained by immersion i n MS 222 (Sandoz) s o l u t i o n (400 mg/1).  Anaesthesia  occurred within 30 minutes  and the animals remained anaesthetised for a further 30 minutes. A l l recordings were taken from animals at l e a s t 2 hours a f t e r recovery from the anaesthesia. For lung cannulation a small s l i t was made i n the s k i n and body w a l l of the p o s t e r i o r flank, and the t i p of a lung (usually the l e f t ) was exposed.  A  short cannula of f l e x i b l e 2 mm diameter urinary catheter tubing was then inserted i n t o the apex of the lung and tied i n place.  To prevent occlusion  the cannula was cut i n t o a taper, and the t i p was blunted to prevent possible penetration of the lung w a l l (Jones, 1970).  The lungs were usually  found to be i n a semi-inflated state on cannulation.  I f they were collapsed  they were r e - i n f l a t e d by introducing a i r into them v i a the trachea, to f a c i l i t a t e i n s e r t i o n of the cannula.  The s l i t s i n the s k i n and the body w a l l  were closed separately a f t e r cannulation.  Blood loss caused by this  operation was n e g l i g i b l e . The buccal cavity was cannulated flattened to form a flange.  using P.E. 90 tubing with one end  I t was introduced through the l e f t  tympanic  membrane from i n s i d e to outside and held i n p o s i t i o n by a washer of large bore P.E.  tubing which was fed over the cannula and crimped the tympanic membrane  by pressing against the flange.  This prevented  i t from p u l l i n g out o f the  14 tympanum.  Lung pressure was usually recorded by a Hewlett-Packard  model  270 pressure transducer, and buccal pressure by a model 268 BC pressure transducer, although i n a few cases both lung and buccal pressures were recorded by model 268 BC transducers.  The transducers were a i r - f i l l e d and  had a frequency response of 50-70 Hz with damping of less than 0.1 of c r i t i c a l when tested by a Hansen "pop-test".  Following i n s e r t i o n of the cannulae the  frogs were positioned on a cork board and were restrained by pinning, care being taken to p o s i t i o n the pins such that limb c i r c u l a t i o n was not impeded. Changes i n lung volume were measured by means of a Biocom model 991 impedance converter, used as an impedance pneumograph (Geddes and Baker, 1968).  Two fine copper wire electrodes, insulated except f o r 1 mm at the t i p ,  were introduced using a hypodermic needle into the body w a l l , one i n each flank.  Lung volume was c a l i b r a t e d by i n j e c t i n g small volumes (usually 0.1 ml)  of a i r i n steps into the lungs v i a a three-way tap i n the lung cannula.  Both  lungs were i n f l a t e d by this method, as the bronchi connect to each other posterior to the larynx.  I f a l i n e a r response was not obtained on  c a l i b r a t i o n , the positions of the electrodes were adjusted u n t i l the c a l i b r a t i o n was l i n e a r .  The impedance converter was also used i n 2 frogs to  monitor n a r i a l opening by recording impedance mechanographs.  This was  accomplished by i n s e r t i n g fine copper wire electrodes, insulated except f o r the l a s t 1 mm which was bent into a hook, into the medial and l a t e r a l borders of  the external nares and measuring the impedance change across the electrodes.  Decrease i n the measured impedance was considered to represent n a r i a l closure, and an increase i n impedance, n a r i a l opening confirmed by v i s u a l observation). possessed  (this i n fact was  This method of recording n a r i a l movements  the advantage of placing no mechanical load on the nares as would  a s t r a i n gauge type of transducer.  15 Buccal volumes were recorded by means of an E.E.L. 1 inch diameter selinium photocell (Jones, 1970).  The p o s i t i o n of the photocell was  so that a shadow of the buccal f l o o r wa3  adjusted  thrown onto the face of the photocell  by a v a r i a b l e i n t e n s i t y microscope lamp.  Changes i n buccal volume were  measured as changes i n output from the p h o t o c e l l .  Volume changes were  c a l i b r a t e d a f t e r the experiments by withdrawing and r e i n j e c t i n g small volumes of a i r (0.1 ml) i n t o the buccal c a v i t y , the frog having previously been anaesthetised i n p o s i t i o n by i n j e c t i n g 0.25  ml of MS 222 s o l u t i o n i n s a l i n e  (400 g/1) d i r e c t l y i n t o the dorsal lymph sac.  During t h i s c a l i b r a t i o n  procedure the nares were blocked with v a s e l i n e . Gas flow through the nares during lung v e n t i l a t i o n cycles was  investi-  gated i n 2 frogs by means of a bead thermistor connected to a resistance bridge c i r c u i t .  The thermistor, which was  1 mm  i n diameter, was  positioned as  close as possible to one external n a r i a l opening, without making contact with the s k i n .  Exhaled gas produced a d e f l e c t i o n i n the r e s u l t i n g o s c i l l o s c o p e  trace, while inflow deflected the trace i n the opposite d i r e c t i o n .  No  attempt  was made to quantify flow rates, and output at zero flow was established during portions of buccal cycles when the nares were closed. Electromyograms (EMG)  were recorded b i p o l a r l y .  Fine insulated copper wire  electrodes, the l a s t 1 mm of which were bared and bent into a hook shape, were inserted i n t o the appropriate muscle using a fine hypodermic needle. p o s i t i o n of the electrodes was  checked post mortem.  The  When EMGs were recorded  from the g l o t t a l region, the leads were e i t h e r l e d out of the angle of the or through an aperture i n the r i g h t tympanic membrane which was prevent gas  jaw,  sealed to  leakage.  A l l recordings were made on a T e c h n i r i t e TR 722 two channel pen  recorder,  16 a T e c h n i r i t e TR 888 oscilloscope,  eight  channel recorder,  o r a T e l e q u i p r a e n t D 54  A H e w l e t t - P a c k a r d 1201  oscilloscope.  storage  17 THE MORPHOLOGY OF THE PRINCIPAL RESPIRATORY MUSCLES  The muscles of the buccal esculenta  f l o o r have been described  i n d e t a i l f o r Rana  (Ecker, 1889), while descriptions of the g l o t t a l muscles of Rana  pipiens are summarized by Schmidt (1972).  For the purposes of comparison the  terminology used by De Jongh and Gans (1969) w i l l be followed The s u p e r f i c i a l muscles of the buccal  here.  f l o o r , the anterior and posterior  intermandibular and interhyoids, run transversely between the mento-meckalian c a r t i l a g e s and angulars of the lower jaw ( F i g . l a ) .  Between these  transverse  muscle sheets and the tongue l i e two pairs of geniohyoid muscles which run s a g i t t a l l y from the mento-meckalian and anterior angulars to i n s e r t v e n t r a l l y into the posterior processes of the hyoid ( F i g . l b ) . sternohyoid  The large  muscles i n s e r t between the insertions of the geniohyoid muscles  and a r i s e from the coracoid and xiphisternum, thus running i n s i d e the pectoral g i r d l e , while the omohyoids a r i s e from the scapulae and are inserted l a t e r a l l y into the v e n t r a l surface of the hyoid ( F i g . l b ) .  The anterior petrohyoid  muscles ( F i g . 2) a r i s e from the pro-otic bones and are inserted at the l a t e r a l margins of the hyoid c a r t i l a g e . The muscles of the g l o t t i s and larynx  ( F i g . 2) have been divided into  c o n s t r i c t o r s and d i l a t o r s on purely morphological grounds. g l o t t i s are formed by the arytenoid  The l i p s of the  c a r t i l a g e s , into the l a t e r a l apices of  which are inserted the laryngeal d i l a t o r muscles, which o r i g i n a t e l a t e r a l l y on the hyoid.  There are three groups of muscles which have been  described  as c o n s t r i c t o r s ; the anterior, posterior and external laryngeal c o n s t r i c t o r s . The external c o n s t r i c t o r s surround the arytenoid  c a r t i l a g e s v e n t r a l l y while  the anterior and posterior c o n s t r i c t o r s are dorsal i n p o s i t i o n ( F i g . 2 ) .  18  Figure 1.  (a)  The s u p e r f i c i a l muscles of the buccal f l o o r .  1, pectoral g i r d l e ; 2, interhyoid muscle; 3, posterior intermandibular; 4, angular of lower jaw; 5,  connective  tissue; 6, anterior intermandibular muscle (under connective (b)  tissue).  The deeper muscles of the buccal f l o o r .  1, sternohyoid muscle; 2, omohyoid; 3, medial  geniohyoid;  4, l a t e r a l geniohyoid; 5, angular of lower jaw; 6, a n t e r i o r intermandibular muscle.  18a  a  19  Figure 2.  The muscles of the g l o t t i s and larynx. 1, process of  cricoid  c a r t i l a g e ; 2, p o s t e r i o r laryngeal  c o n s t r i c t o r ; 3, a n t e r i o r laryngeal c o n s t r i c t o r ; 4, laryngeal d i l a t o r ; 5, arytenoid c a r t i l a g e ; 6, external laryngeal c o n s t r i c t o r ; 7, anterior petrohyoid muscle; 8„ hyoid c a r t i l a g e .  19a  Dorsa I  Ventral  20 RESULTS  (a)  Pressure and volume changes i n the lungs and buccal cavity. It was  possible to distinguish  two d i s t i n c t types of breathing movements  i n Rana pipiens; v e n t i l a t i o n movements which did not involve the active p a r t i c i p a t i o n of the g l o t t i s and nares  (buccal c y c l e s ) , and movements i n  which large p o s i t i v e pressures were generated by t h e i r coordinated a c t i v i t y (lung v e n t i l a t i o n c y c l e s ) .  The pressures developed during buccal v e n t i l a t i o n  cycles, with the g l o t t i s closed and the nares open, ranged i n amplitude ±0.1 to ±0.5 cm R^O  i n i n d i v i d u a l frogs.  The volume changes of the buccal  cavity associated with buccal v e n t i l a t i o n averaged 0.56 and were approximately  from  ml i n a 50 gram frog,  2/3 of the magnitude of the volume changes which  occurred during lung v e n t i l a t i o n ( F i g . 3a).  They were simple i n form,  consisting of a f a l l to a minimum volume, followed by a volume increase at almost the same rate as the f a l l .  The volume changes occurred at the same  frequency as the associated pressure changes, but were not i n phase with them; the pressure maxima preceding the volume minima by 80-120 m sees i n i n d i v i d u a l frogs. The pressure and volume changes which occurred i n the buccal pump during lung v e n t i l a t i o n were more complex and of greater amplitude occurring during buccal v e n t i l a t i o n .  than  As shown i n Figure 4 , a  those  simultaneous  f a l l i n lung pressure and lung volume occurred as the g l o t t i s opened and lung gas flowed into the buccal cavity. and there was  Coincident with t h i s buccal pressure rose,  also a small increase i n buccal volume.  Buccal volume at t h i s  point always increased by an amount smaller than the volume decrease of the lungs, suggesting that the nares were at l e a s t p a r t i a l l y open at t h i s point i n the cycle, and that part of the gas contained i n the buccal cavity passed  21  Figure 3.  (a)  Pressure and volume changes recorded from the buccal  cavity. (b)  (Increase i n buccal volume i s up on the trace.)  Slow speed recording to show sequences of lung i n f l a t i o n  i n Rana pipiens. buccal volume. (c)  Top trace, buccal pressure; lower trace, (Increase i n volume i s up on the trace.)  Pressures recorded from the lungs and buccal c a v i t y .  Top trace, lung pressure; lower trace, buccal pressure.  22  Figure A.  Pressure and volume changes i n the lungs and buccal cavity, together with a c t i v i t y from the muscles of the respiratory valves. (a)  E.M.G. from n a r i a l closer, M. l a t e r a l i s narium.  (b)  E.M.G. from laryngeal d i l a t o r muscles, M. d i l a t a t o r  laryngls. (c)  Lung volume, increase i n volume i s up on the trace.  (d)  Lung pressure.  (e)  Buccal volume, decrease i n volume i s up on the trace.  (f)  Buccal pressure.  22a  m.d.  0-5 ml.  5  cms.H O  (  ,  r I _  a  oL  0-5 ml.  ms.H 0 2  u^u^^vT""^  ^  J~i  r-\  r-i  f—i  Time (sec.)  1-\  /—v_  i  r—\  r—v_  23 through them.  Lung and buccal pressure e q u i l i b r a t e d at the end of this phase  and often produced a marked step or i n f l e x i o n i n the buccal pressure (Fig.  3).  curve  In some frogs the pressure i n the system stayed constant for up to  160 m sees or even f e l l s l i g h t l y , presumably due to gas leakage through the nares.  The nares then closed and a large decrease i n buccal volume occurred  which caused a rapid elevation i n buccal pressure. at  The r i s e i n lung pressure  this point closely p a r a l l e l e d the r i s e i n buccal pressure, and as  i l l u s t r a t e d by Figure 5a and b, no evidence was pressure across the g l o t t i s i n this phase.  found f o r a l a r g e d i f f e r e n t i a l  The increase i n lung pressure  associated with a s i m i l a r increase i n lung volume. pressure the lungs maintained  the new  At peak j o i n t  was  lung/buccal  pressure l e v e l due to g l o t t a l closure,  while buccal pressure f e l l back to atmospheric,  t y p i c a l l y i n 50-60 m sees,  and undershot to give a pressure i n the buccal cavity of -0.25  to -0.4  cm  H^O  before slowly returning to atmospheric pressure, at which point the buccal v e n t i l a t i o n s r e s t a r t e d . The attainment  of maximum buccal pressure s l i g h t l y  preceded the point of minimum buccal volume i n lung v e n t i l a t i o n , and i t s f a l l to atmospheric pressure occurred either just before or at the point of minimum buccal volume.  In e i t h e r case the f a l l to atmospheric pressure  nearly isovolumetric because of i t s extreme r a p i d i t y (50-60 m sees).  was  It  appeared to be due to two factors, f i r s t l y the opening of the nares synchronously  with g l o t t a l closure at the point of peak buccal pressure, and  secondly the simultaneous  cessation of a c t i v i t y of the r e s p i r a t o r y muscles of  the buccal f l o o r with the r e s u l t that buccal f l o o r tension r a p i d l y f e l l to zero.  Buccal pressure therefore also f e l l rapidly and e q u i l i b r a t e d through  the open nares with atmospheric pressure.  The slow buccal volume increase  (250-300 m sees) which occurred once minimum buccal volume was  attained then  served to drive buccal pressure below atmospheric pressure, u n t i l f i n a l l y  the  24  Figure 5.  The relationship between lung and buccal pressure during (a) lung i n f l a t i o n ; (b) lung d e f l a t i o n . pressure; lower trace, buccal pressure.  Top trace, lung  25 buccal pressure again e q u i l i b r a t e d with atmospheric  pressure, at which  point the next respiratory cycle commenced. Lung v e n t i l a t i o n cycles i n the frogs investigated d i d not occur randomly, but i n w e l l defined sequences of 9-30 such movements interspersed with buccal v e n t i l a t i o n cycles, the sequences being separated from each other by pauses during which the lungs remained i n f l a t e d and only buccal v e n t i l a t i o n cycles were performed.  Figure 3b and c i l l u s t r a t e s a series of such sequences,  while Figure 5a and b shows the d e t a i l s of one such sequence of lung i n f l a t i o n and d e f l a t i o n .  During the l a s t few lung v e n t i l a t i o n cycles of a sequence the  pressures developed by the buccal pump increased i n a way so that each cycle produced a lung pressure 10 to 20 percent greater than that achieved by the previous c y c l e , and at the end of a sequence the lungs were f u l l y at a pressure of 4-5 cm  inflated  H^O and remained i n f l a t e d through the period of  buccal v e n t i l a t i o n (Figure 3c). Lung d e f l a t i o n was usually brought about by the f i r s t few lung v e n t i l a t i o n cycles of the subsequent sequence, each of which attained a lower peak pressure than the preceding one, u n t i l a r e l a t i v e l y constant lung pressure of 1.5 to 2 cm H^O obtained which was maintained p r i o r to the next lung i n f l a t i o n sequence.  Lung d e f l a t i o n proved  to be more v a r i a b l e than i n f l a t i o n , on some occasions the lungs were deflated on the f i r s t lung v e n t i l a t i o n c y c l e a f t e r a period of i n f l a t i o n , while on other occasions this occurred over two or three cycles (compare Figures 3c and 5a and b ) . The frequency with which lung i n f l a t i o n occurred v a r i e d according to the condition of the frog and i n frogs which were obviously disturbed lung i n f l a t i o n cycles were suppressed.  In the majority of frogs studied however  the lungs were i n f l a t e d approximately i n f l a t e d f o r 10 to 20 seconds.  once per minute and remained f u l l y  Although  the lungs were i s o l a t e d from the  26 buccal cavity by the closed g l o t t i s during periods of lung i n f l a t i o n small amplitude pressure fluctuations s t i l l occurred i n the lungs (Figs. 3c, 5a and b).  These small amplitude  fluctuations varied i n s i z e with the magnitude of  the buccal o s c i l l a t i o n s ( F i g . 3a) and occurred at the same frequency as the buccal v e n t i l a t i o n cycles.  They could have been caused either mechanically  (due to the muscle insertions of the buccal f l o o r p u l l i n g on the flanks) or by pressure changes of the buccal cavity being transmitted i n d i r e c t l y through the tissues to the thoraco-abdominal  cavity, i n which the lungs l i e .  The small pressure peaks recorded from the lungs lagged behind the pressure peaks of the preceding buccal cycles by 350-400 m sees so this transmittance of  pressure was  a r e l a t i v e l y slow process which tends to favour the notion  of  i n d i r e c t transmittance.  Further long term pressure changes occurred i n  the lungs during the i n f l a t e d period i n many frogs.  Figure 3c i l l u s t r a t e s  a t y p i c a l example i n which lung pressure gradually rose and then f e l l , while i n some cases there was i n f l a t e d phase.  a gradual f a l l i n lung pressure throughout  the  A simple c a l c u l a t i o n indicates that a long term (20 sec)  fall  in pressure i s u n l i k e l y to indicate uneven respiratory exchange r a t i o s i n the lung, since the volume change was  generally greater than that which  would be predicted from the metabolic rate of the frogs used.  I t seems most  probable that these changes were due to small postural changes i n the frogs concerned,  although the frogs were restrained throughout  the experiments,  or  to changes i n the tonic a c t i v i t y of the pulmonary smooth muscle, c o n t r o l l e d perhaps by the l e v e l of interpulmonary (b)  CO^  (Kobayasi et a l , 1961) .  The nares and g l o t t i s The external nares and g l o t t i s provide the valves of the buccal pump.  The positions of the external nares were monitored by recording EMG's from  27  the M. l a t e r a l i s narium, which occupies the space between the anterior portion of the maxillary bone and the ascending process of the premaxilla (Ecker, 1889).  Muscle potentials recorded from this region were coincident with  n a r i a l closure as could be deduced from the buccal pressure traces.  Glottal  opening was monitored by recording EMG's of high amplitude from the laryngeal d i l a t o r y muscles,  the M. d i l a t a t o r  l a r y n g i s , which are inserted l a t e r a l l y on  the arytenoid c a r t i l a g e s which form the cartilaginous walls of the v e r t i c a l slit-like glottis. Figure 4 i l l u s t r a t e s the r e l a t i o n s h i p between e l e c t r i c a l a c t i v i t y i n these muscles and the pressure and volume traces from both the buccal cavity and the lungs.  No a c t i v i t y was recorded i n either set of muscles during  buccal v e n t i l a t i o n ,  the nares remaining open, while the g l o t t i s was closed.  In lung v e n t i l a t i o n  however, the onset of a c t i v i t y i n the laryngeal d i l a t o r  muscles e i t h e r s l i g h t l y preceded or was synchronous with the beginning of the i n i t i a l f a l l i n lung pressure and volume, and a c t i v i t y continued u n t i l peak lung/buccal pressure was reached.  Throughout this period the g l o t t i s  remained open and the lungs retained the new pressure l e v e l due to g l o t t a l closure.  No a c t i v i t y could be found i n those muscles anatomically described  as g l o t t a l c l o s e r s , i . e . the anterior, posterior and external laryngeal c o n s t r i c t o r s , although several attempts were made to record from them. I t appears that o r d i n a r i l y  i n Rana pipiens the g l o t t i s i s held open by muscular  a c t i v i t y but that g l o t t a l closure occurs passively.  The g l o t t i s was always  found to be closed i n curarised (0.1 mg/10 g wt d-tubocurarine chloride) frogs i n d i c a t i n g that the inherent e l a s t i c i t y of the laryngeal c a r t i l a g e s and t h e i r connections was s u f f i c i e n t to maintain g l o t t a l closure.  Furthermore,  i f air  was pumped into the lungs of these frogs v i a a cannula, the g l o t t i s was able to support lung pressures of 10-15 cm H O before opening.  This pressure i s  28 2-3 times greater than that developed i n f l a t i o n cycles.  across the g l o t t i s during normal lung  In 3 pithed frogs stimulation of the M. d i l a t a t o r  laryngis (1.5 v, 4 Hz, 10 m sec impulses)  resulted i n g l o t t a l opening.  Immediate passive closure occurred when stimulation ceased. The onset of a c t i v i t y i n the M, l a t e r a l i s narium s i g n a l l e d n a r i a l closure which occurred at the s t a r t of the phase of a c t i v e e l e v a t i o n of buccal pressure, t y p i c a l l y 50-60 m sees a f t e r g l o t t a l opening, and a c t i v i t y continued u n t i l peak, buccal/lung pressure was reached, at which point i t stopped, i n d i c a t i n g n a r i a l opening.  No a c t i v i t y could be recorded from the  M. d i l a t o r narium which by i t s anatomical p o s i t i o n has been considered to be a n a r i a l opener (Ecker, 1889), suggesting that e l a s t i c forces are s u f f i c i e n t for n a r i a l opening.  The nares were always found to be open i n curarised  frogs, confirming that no muscle a c t i v i t y i s required for opening.  Figure 6  i l l u s t r a t e s the relationship between the impedance mechanograph of the external nares, and the associated buccal pressure trace. that the s t a r t of n a r i a l displacement  The records show  coincided with the beginning of the  rapid r i s e i n buccal pressure which i s due to the active contraction of the muscles of the buccal f l o o r .  The nares presumably closed very soon a f t e r the  beginning of n a r i a l displacement  and remained closed up to the point of the  minimum impedance measured, which was coincident with the point of peak buccal pressure.  During the f a l l i n g phase of buccal pressure impedance across the  nares increased, i n d i c a t i n g n a r i a l opening; maximum impedance i n d i c a t i n g maximum n a r i a l opening occurred at the point when the buccal pressure trace had f a l l e n back to zero.  No n a r i a l displacements  were recorded during buccal  v e n t i l a t i o n cycles, the nares remaining open. Figure 7 i l l u s t r a t e s flow at the nares during buccal v e n t i l a t i o n c y c l e s , followed by the f i r s t phase of a lung v e n t i l a t i o n c y c l e .  Flow was biphasic  29  Figure 6.  The r e l a t i o n s h i p between n a r i a l closure and g l o t t a l opening. Top trace, impedance mechanograph of nares.  Middle trace,  E.M.G. from g l o t t a l openers, M. d i l a t a t o r l a r y n g i s . trace, buccal pressure.  Lower  30 during the buccal cycles, consisting of an i n i t i a l  outflow of buccal gas, as  the volume of the buccal cavity decreased, followed by an inflow as the buccal f l o o r f e l l passively.  Between the two phases of flow there was a short  period (100 m sees) of n e g l i g i b l e flow, while the duration of the e n t i r e c y c l e was approximately 1 second. v e n t i l a t i o n cycles.  Figure 7b shows n a r i a l a i r flow during 2 lung  During the f i r s t  phase of each cycle, A-B, there was a  major outflow of buccal gas through the nares.  This i s due to g l o t t a l  opening, which allowed lung gas to flow into the buccal cavity r a i s i n g i t s pressure above atmospheric,  so that an outflow of gas occurred v i a the nares.  At the s t a r t of the next phase, B-C, n a r i a l closure occurred, and there was no n a r i a l flow, although buccal pressure was being r a p i d l y elevated by the muscles of the buccal pump.  N a r i a l opening at peak buccal pressure C,  coincident with g l o t t a l c l o s i n g , resulted i n a second phase of outflow of buccal gas through the nares, as buccal pressure f e l l back to atmospheric at D.  Once buccal pressure f e l l below atmospheric however, flow r e v e r s a l  occurred at the nares and a i r entered, r e f i l l i n g the expanding buccal cavity (D-A) (c)  preparatory to the next v e n t i l a t i o n c y c l e , The a c t i v i t y of the p r i n c i p l e respiratory muscles Muscle a c t i v i t y during the buccal cycles occurred only during the phase  of  the c y c l e i n which pressure rose and none was present during the f a l l i n g  pressure phase.  EMG's were recorded from the p o s t e r i o r intermandibular  muscle almost immediately  buccal pressure rose above atmospheric, but usually  stopped before peak pressure was reached.  On the other hand, a c t i v i t y i n  the medial and l a t e r a l geniohyoid muscles continued to the point of peak pressure (Figure 8a and b ) . Figure 9a i l l u s t r a t e s the a c t i v i t y recorded from the sternohyoid muscle and the a n t e r i o r petrohyoid, together with a trace of pressure.  No a c t i v i t y  31  Figure 7.  N a r i a l gas flow during (a) four buccal v e n t i l a t i o n cycles; (b) two lung v e n t i l a t i o n cycles. i n schematic diagram, Figure 10.  A-D'  correspond  to phases  3 1 9  LSI  d m  1  I1IJ  MO|JUI  MOj^no  DOS I  I  1  I, MO|j4no  32  Figure 8.  (a)  Top trace, buccal pressure.  posterior intermandibular muscle, pressure.  Lower trace, a c t i v i t y i n (b)  Top trace, buccal  Lower trace, a c t i v i t y i n medial geniohyoid muscle.  32a  b  Isec  33 was present i n e i t h e r of these muscles during the buccal cycles, but both showed strong a c t i v i t y during lung v e n t i l a t i o n c y c l e s .  The sternohyoid,  which i s inserted a n t e r i o r l y to the hyoid and attached p o s t e r i o r l y to the pectoral g i r d l e was the f i r s t muscle to show a c t i v i t y during a lung i n f l a t i o n cycle.  A c t i v i t y i n this muscle started 15-20 m sees before the g l o t t i s  opened, as judged by the accompanying pressure trace and continued u n t i l lung and buccal pressures had e q u i l i b r a t e d .  A second burst of a c t i v i t y was  sometimes recorded during the r i s i n g phase of buccal pressure, e s p e c i a l l y when a high peak pressure was generated.  From the p o s i t i o n of the sternohyoid  i t has been proposed that i t s contraction serves to distend the p o s t e r i o r portion of the buccal cavity, increasing buccal volume (Das and Srivastava, 1957;  De Jongh and Gans, 1969).  This suggests that the volume increase of  the buccal cavity which occurred i n the f i r s t phase of the lung v e n t i l a t i o n cycle did not r e s u l t s o l e l y from gas leaving the lungs, but also had an active component, although the s i g n i f i c a n c e of the second burst of a c t i v i t y observed i n some cycles during the r i s i n g phase of lung pressure i s obscure. During the rapid e l e v a t i o n of buccal pressure, i n lung v e n t i l a t i o n cycles, EMG a c t i v i t y was recorded from the a n t e r i o r petrohyoid muscle, the medial and l a t e r a l geniohyoid muscles, the p o s t e r i o r intermandibular muscle, the omohyoid, and the interhyoid ( F i g . 9b), a l l of which commenced f i r i n g at the beginning of  the r a p i d l y r i s i n g phase of buccal pressure, a f t e r lung and buccal  pressures had e q u i l i b r a t e d and a c t i v i t y continued u n t i l or s l i g h t l y a f t e r peak buccal pressure was reached.  The amplitude of the potentials recorded  from the p o s t e r i o r intermandibular muscle during lung v e n t i l a t i o n cycles was always very much l a r g e r than that of those obtained during buccal v e n t i l a t i o n cycles, i n d i c a t i n g that greater numbers of motor units were involved i n lung v e n t i l a t i o n cycles ( F i g . 8 ) . This was also generally true f o r the l a t e r a l  34  Figure 9.  (a)  Top trace, a c t i v i t y i n sternohyoid muscle.  trace, a c t i v i t y i n anterior petrohyoid muscle.  Middle Lower trace,  buccal pressure. (b)  Top trace, a c t i v i t y i n omohyoid muscle.  a c t i v i t y i n interhyoid muscle.  Middle trace,  Lower trace, buccal pressure.  34a  time, sec  35 and m e d i a l g e n i o h y o i d  muscles, a l t h o u g h  EMG  b u c c a l v e n t i l a t i o n movements ( F i g . 8b).  No  occur  i n the  relatively  amplitude increased during muscular a c t i v i t y was  f a l l i n g p r e s s u r e phase which f o l l o w e d peak p r e s s u r e ;  slow i n c r e a s e i n b u c c a l volume which o c c u r r e d  t h e r e f o r e presumably r e s u l t e d from g r a v i t a t i o n a l and  during  elastic  large  found t o the  these  forces.  phases  36 DISCUSSION  The present study has permitted a reappraisal of the mechanism of anuran v e n t i l a t i o n , as w e l l as providing new  information on the volume changes of  the buccal cavity and lungs associated with the v e n t i l a t o r y c y c l e s , which i s necessary  for a more complete understanding  of the r e s p i r a t o r y events.  A  d e s c r i p t i o n of the sequence of events occurring during a t y p i c a l lung v e n t i l a t i o n cycle w i l l c l a r i f y the r e l a t i o n s h i p s between them, and w i l l an overview of the mechanism to be made ( F i g . 10).  enable  The beginning of the f i r s t  phase of a lung v e n t i l a t i o n cycle (A-B) occurs, whether the preceding c y c l e was  a buccal or a lung v e n t i l a t i o n cycle, when buccal pressure i s at  atmospheric.  The beginning of this phase i s indicated by (1) a  simultaneous  f a l l i n lung pressure and volume due to opening of the g l o t t i s ; (2) an increase i n buccal pressure; (3) an increase i n buccal volume (which i s never as great as the associated decrease i n lung volume); (4) e l e c t r i c a l a c t i v i t y i n the M. d i l a t a t o r l a r y n g i s , which opens the g l o t t i s ; and (5) a c t i v i t y i n the M. steraohyoideus, contraction of which distends the p o s t e r i o r portion of the buccal cavity (Das and Srivastava, 1957;  De Jongh and Gans, 1969).  t i o n of the lung and buccal pressure traces i s accomplished of the second phase of the lung v e n t i l a t i o n cycle (B-C). phase i s marked by (1) n a r i a l closure, indicated by EMG  Equilibra-  before the s t a r t  The s t a r t of t h i s a c t i v i t y i n the  M. l a t e r a l i s narium, and also by impedance mechanographs of the external nares; (2) EMG  a c t i v i t y i n the main r e s p i r a t o r y muscles of the buccal f l o o r ;  (3) a rapid decrease i n the volume of the buccal cavity; (4) a rapid r i s e i n the buccal pressure driven by the decrease i n buccal volume; and (5) a simultaneous  r i s e i n lung pressure and volume, as the buccal cavity and lungs  are i n communication through the open g l o t t i s .  The t h i r d phase of the cycle  37  Figure 10.  Semischematic diagram of lung and buccal pressure and volume changes i n a buccal and a lung v e n t i l a t i o n  cycle.  Up on trace i s an increase i n lung volume, decrease i n buccal volume. investigated. I l l  , muscular a c t i v i t y i n a l l frogs 1  during t h i s phase.  , muscular a c t i v i t y i n some frogs  37 a  lung vol  0-5ml.  •buccal  l a r y n g e a l n a r i a l  d i l a t o r  c o n s t r i c t o r  m e d i a l  g e n i o h y o i d  l a t e r a l  g e n i o h y o i d  p o s t ,  i n t e r m a n d i b u l a r  s t e r n o h y o i d a n t .  p e t r o h y o i d  o m o h y o i d i n t e r h y o i d  A  B C  if  __-—-!  lung p.  cms.H 0 2  buccal p  0-5 s e c .  38 (C-D)  i s commenced by, (1) the end of a c t i v i t y i n the M. d i l a t a t o r l a r y n g i s ,  i n d i c a t i n g closure of the g l o t t i s , and i s o l a t i o n of the lungs from the buccal cavity; (2) n a r i a l opening; (3) a rapid f a l l of buccal pressure to  atmospheric;  (4) an associated, but much slower increase i n buccal volume which drives buccal pressure considerably below atmospheric (D'-D"), before e q u i l i b r a t i o n occurs through the open nares.  At this point the next v e n t i l a t i o n cycle,  whether buccal or lung, commences. The volume decrease of the buccal force pump ( F i g . 10, B-C), and  the  action of i t s valves i n lung v e n t i l a t i o n are due to active muscular contraction, while the subsequent increase i n buccal volume i s driven by g r a v i t i a t i o n a l and i n e r t i a l forces.  The most simple c o n t r o l mechanism which can be suggested  for  the lung v e n t i l a t i o n cycle i s the sequential e x c i t a t i o n of the valves and respiratory muscles neurones.  by means of a group of s e l f - e x c i t i n g r e s p i r a t o r y motor  In t h i s respect i t i s i n t e r e s t i n g that discharges have been recorded  from the frog medulla associated with the increasing pressure phase of lung v e n t i l a t i o n cycles (Ito and Watanabe, 1962;  Jones, 1970),  This r e s p i r a t o r y sequence resembles that of Rana catesbeiana (De Jongh and Gans, 1969), but d i f f e r s i n several important respects.  In Rana pipiens  the buccal cycle preceding a lung v e n t i l a t i o n cycle never shows an exaggerated negative pressure phase and cannot be considered the f i r s t phase of the lung v e n t i l a t i o n cycle; nor i s there e l e c t r i c a l a c t i v i t y of the M. stemohyoideus during this phase as reported f o r Rana catesbeiana.  A c t i v i t y i n the M.  stemohyoideus i n Rana pipiens, commenced at A, Figure 10, and throughout the phase A-B, when buccal volume was  continued  increasing, but no  activity  occurred before t h i s phase. As soon as buccal and lung pressure e q u i l i b r a t e , they are both r a p i d l y raised by the decrease i n volume of the buccal cavity.  No large pressure  39 d i f f e r e n t i a l was recorded between the lungs and buccal c a v i t y during  this  phase (Willem, 1919, 1920; De Marneffe-Foulon, 1962), although a s i g n i f i c a n t pressure d i f f e r e n t i a l has been demonstrated i n Rana catesbeiana Gans, 1969).  (De Jongh and  This i s puzzling, considering the large s i z e and evidently low  flow resistance of the open g l o t t i s , although De Jongh and Gans speculate that i t may be "due to the post contraction relaxation of the smooth muscles of the lung w a l l s " . The opening of the g l o t t i s , which i n i t i a t e s the pressure  events of the  lung v e n t i l a t i o n c y c l e , i s due to the contraction of the M. d i l a t a t o r l a r y n g i s , while closure i s normally passive.  EMG a c t i v i t y continued  i n the  M. d i l a t a t o r laryngis throughout the period of time when the g l o t t i s was open, as deduced from the pressure record, but no a c t i v i t y could be recorded the positions of those muscles described anatomically ing  normal breathing.  from  as g l o t t a l closers dur-  Schmidt (1972) supports the view that closure o f the  g l o t t i s can occur passively i n Rana pipiens, although he showed that the cons t r i c t o r muscles are a c t i v e during c a l l i n g , and suggests that t h e i r function may be to reduce the duration of the c a l l t r i l l s . E a r l i e r electromyographic studies throw l i t t l e l i g h t on the subject because the authors f a i l to d i s c r i minate between buccal and lung v e n t i l a t i n g cycles (Kato, 1951; Oka, 1957). Shinkai and N a r i t a (1957), however, found large spike discharges  from the M.  d i l a t a t o r laryngis during lung v e n t i l a t i o n cycles, but f a i l e d to f i n d a c t i v i t y in laryngeal c o n s t r i c t o r s .  In Rana catesbeiana  the s i t u a t i o n appears to be  d i f f e r e n t i n that e l e c t r i c a l a c t i v i t y i n the M. d i l a t a t o r laryngis does not continue throughout the period when the g l o t t i s i s open, but only occurs on the onset of g l o t t a l opening.  Closure of the g l o t t i s i s coincident with  a c t i v i t y from the region of the anterior laryngeal c o n s t r i c t o r muscles (De Jongh and Gans, 1969).  Since i n R. catesbeiana  the g l o t t i s i s opened a c t i v e l y ,  AO remains open without tonic muscular a c t i v i t y i n the d i l a t o r s , and i s then closed by active c o n s t r i c t o r a c t i v i t y , then some form of c l i c k - s t o p mechanism must be involved.  In this study i t has been demonstrated that passive  closure  of the g l o t t i s follows stimulation of the M. d i l a t a t o r laryngis muscles, and that the inherent e l a s t i c i t y of the laryngeal c a r t i l a g e s i s s u f f i c i e n t to maintain closure of the g l o t t i s even i f there i s a large pressure between the lungs and the buccal cavity.  gradient  I f the laryngeal muscles are  removed and the g l o t t i s i s then forced open, immediate passive closure of the g l o t t i s w i l l s t i l l occur (Schmidt, 1972). N a r i a l closure i n Rana pipiens was  monitored by measuring the impedance  change across the external n a r i a l opening and by recording the a c t i v i t y of the M. l a t e r a l i s narium (Ecker, 1889), which continued  throughout the period  of n a r i a l closure, as deduced from the pressure records.  Electrical activity  i n these muscles, coincident with n a r i a l closure has been previously demonstrated (Jones, 1970), and s t r i a t e d muscle f i b r e s have been observed i n the area of the nasal c a r t i l a g e s (Shinkai and N a r i t a , 1957). evidence was  No  concrete  found for the frequently proposed mechanism of n a r i a l closure by  the i n d i r e c t action of the muscles of the lower jaw on the premaxilla (Gaupp, 1896;  Willem, 1919,  1920;  De Jongh and Gans, 1969), although manual  elevation of the premaxilla c e r t a i n l y does cause external n a r i a l closure i n anaesthetised  animals.  The claim of Baglioni (1900) that the a n t e r i o r  processes of the hyoid close the i n t e r n a l nares appears to be without foundation. Flow measurements, recorded  at the external nares, demonstrate the  existence i n Rana pipiens of two phases of flow during buccal v e n t i l a t i o n cycles and of four phases of flow during the lung v e n t i l a t i o n c y c l e . i n i t i a l major outflow of buccal gas, which occurs i n phase A-B  The  of the lung  41 v e n t i l a t i o n cycle ( F i g . 10) must be associated with a s i m i l a r outflow as lung gas e x i t s .  During B-C,  there i s no flow at the nares, as the nares are  closed, but flow through the g l o t t i s must reverse as the lungs are i n f l a t e d , u n t i l the g l o t t i s closes at C.  The four phases of flow at the nares are  therefore associated with two phases at the g l o t t i s , f o r each v e n t i l a t i o n cycle.  The pattern of n a r i a l flow reported here i s e s s e n t i a l l y s i m i l a r to  that deduced from pressure records f o r Rana catesbeiana (De Jongh and Gans, 1969), although no evidence was  found f o r a "major inflow" of a i r preceding  a lung v e n t i l a t i o n c y c l e . Lung i n f l a t i o n sequences occurred i n a l l the frogs studied, the lungs remaining i n f l a t e d f o r 10 to 20 seconds.  Although the sequences were noted  by early workers (Martin, 1878; Wedenski, 1881), they generally have been considered to be symptomatic of stress or pain brought about by securing the frog (Willem, 1919,  1920;  Scholten, 1942;  De Marneffe-Foulon, 1962),  Their  existence has however been demonstrated i n cannulated but unrestrained Rana catesbeiana, and also i n unrestrained Rana pipiens and Rana temporaria i n which buccal movements were recorded by means of a photocell (De Jongh and Gans, 1969; West, 1969, unpublished).  In a series of lung v e n t i l a t i o n  cycles during which lung pressure and volume i s maintained at a steady l e v e l , the net outflow from the lungs during phase A-B of each cycle i s balanced by the inflow during phase B-C the same.  ( F i g . 10), and the volume of the lungs remains  In lung i n f l a t i o n sequences the outflow i s suppressed by shortening  the time a v a i l a b l e f o r outflow, n a r i a l closure and decrease i n buccal volume occurring soon a f t e r opening of the g l o t t i s .  In many of the frogs i n v e s t i -  gated the l a s t one or two lung v e n t i l a t i o n cycles of an i n f l a t i o n sequence showed a decrease i n buccal volume before opening of the g l o t t i s so that the outflow of gas from the lung was  reduced to a minimum or eliminated.  These  42 movements were very rapid, and often accompanied by postural movements of the frog.  They approximate to "pure f i l l i n g movements", i n which gas flows only  into the lung (Wedenski, 1881;  Scholten, 1942).  i n f l a t i o n period i s accomplished  Lung d e f l a t i o n following an  by lengthening the outflow phase A-B ( F i g .  10) s u f f i c i e n t l y to allow a major f a l l i n lung volume and pressure. of  The rate  outflow i s obviously also dependent on the previously established pressure  l e v e l i n the lungs, so that this period may not be s i g n i f i c a n t l y longer than i n v e n t i l a t i o n cycles which merely maintain lung pressure and volume. lungs are then r e i n f l a t e d to a lower pressure l e v e l during phase  The  B-C.  Occasionally d e f l a t i o n occurs by means of a series of rapid cycles i n which the increase i n pressure during phases B-C i s very small.  These cycles  approximate to the "pure emptying movements" described by Wedenski (1881) and Scholten (1942).  As pointed out by De Jongh and Gans (1969), the existence  of  sequences of lung i n f l a t i o n and d e f l a t i o n i n anurans means that the  concept  of  t i d a l volume i s of doubtful value when applied to these animals unless the  type of cycle i s s p e c i f i e d , as the volume of gas pumped into the lungs i n any cycle w i l l depend on whether the lung i s being i n f l a t e d or d e f l a t e d . Hutchinson at  eit a l (1967) found a wide range of t i d a l volumes i n Rana pipiens  25°C. The i n i t i a l  emptying of lung gas into the buccal cavity during a lung  v e n t i l a t i o n cycle means that some mixed gas i s almost c e r t a i n l y returned to the lungs (Gans et a l , 1969), the gas volume passing into the buccal cavity from the lungs during A-B t o t a l buccal volume.  ( F i g . 10) being t y p i c a l l y 30 to 50% of  I t would be more e f f i c i e n t to empty the lungs,  completely r e f i l l the buccal cavity with a i r , and then pump this into the lungs i f a c q u i s i t i o n of oxygen was  at a premium, eliminating the p o s s i b i l i t y  A3 of mixed gas being returned to the lungs.  Jones (1972) found that PaC0  2  fell  from 9.A8 mmHg i n normally breathing frogs (Rana esculenta) to 7.77 mmHg i n frogs a r t i f i c i a l l y v e n t i l a t e d to the same volume (although a r t e r i a l pH and PaC>2 did not change s i g n i f i c a n t l y ) suggesting that lung v e n t i l a t i o n with mixed gas may serve to maintain and regulate blood PCO^ i n adult anuran amphibians. acid  ^CO^ *  So  n  ^ weakly ionized i n s o l u t i o n and behaves as a weak  i n the blood, but i t s conjugate base HCO^  as a p r i n c i p a l b u f f e r f o r H  i s strong enough to serve  and may be used therefore to control body f l u i d  pll i n the face of, f o r example, the combined respiratory and metabolic acidosis which occurs during submergence i n frogs (Jones, 1972).  Ventilatory control  of blood PCC>2 may be e s p e c i a l l y s i g n i f i c a n t i n frogs i n view of the fact that the skin may represent a surface f o r CO^ exchange which i s not capable o f precise regulation i n the face, f o r example, of changes i n temperature. Poikilotherms appear to d i r e c t t h e i r regulation of acid-base balance towards a s t a b i l i z a t i o n of OH /H r a t i o i n the face o f changes i n ambient +  temperature  (Howell e t a l , 1970; Rahn and Baumgardner, 1972), and the data of Reeves (1972) suggests that b u l l f r o g s could regulate v e n t i l a t i o n i n order to maintain a stable C0„ content of the blood plasma over a wide range of body  temperatures.  AA PART I I .  THE  MECHANICAL WORK AND  EFFICIENCY OF  VENTILATION IN RANA PIPIENS  INTRODUCTION  No attempt has  previously been made to study the detailed mechanics  and  dynamics of v e n t i l a t i o n i n an amphibian, or to measure the mechanical work involved i n breathing.  Furthermore only one  series of experiments has been  performed which provides information on the oxygen cost of v e n t i l a t i o n i n anurans (Jones, 1972), although i t has been stated  that lung v e n t i l a t i o n i s  the most important s i n g l e factor i n t h e i r respiratory  exchange (Hutchinson  et a l , 1968). The elevating  energy cost of v e n t i l a t i o n i n horse and the v e n t i l a t i o n rate with C ^  i n man  stimulation  has been measured by  or v o l u n t a r i l y ,  and  measuring the associated increase i n oxygen consumption (Zuntz and Hagemann, 1898,  c i t e d from L i l j e s t r a n d , 1918;  v e n t i l a t i o n volume due  O t i s , 1954).  Spontaneous increases i n  to hypoxia have been u t i l i z e d i n f i s h (Van  Schumann and P i i p e r , 1966).  Extrapolation  an estimate of the energy cost of breathing at r e s t .  then been used to give The  r e s u l t s of these  studies suggest that the cost of r e s t i n g v e n t i l a t i o n i n man  i s less than  2 percent, while i n f i s h i t i s from 5 to 20 percent (Cameron and Shelton, 1970).  apply to amphibians, however, due unpredictable v e n t i l a t o r y  Cech,  This technique i s d i f f i c u l t  (Smyth, 1939).  Also i t i s  that s i g n i f i c a n t changes i n cutaneous gas uptake could occur due i n the number of open skin c a p i l l a r i e s i n response to C0„  1970;  to  to the l a b i l i t y of v e n t i l a t i o n and  response to CO^  1938;  of the increase i n oxygen  consumption back to the resting v e n t i l a t i o n rate has  Campbell et a l , 1970;  Dam,  the possible  to a r i s e  i f hypercapnia  45 was  used to stimulate breathing (Poczopko, 1956). An a l t e r n a t i v e method of determining energy cost i s to c a l c u l a t e the  mechanical work performed by the respiratory pump which may then be combined with an assumed or experimentally determined value f o r the mechanical e f f i c i e n c y of the pump to give the cost of v e n t i l a t i o n .  The mechanical work  of v e n t i l a t i o n i n man has been estimated by measuring t i d a l volume o r flow, and interoesophageal pressure, and constructing pressure-volume E m i l i and P e t i t , 1960; Tenny and Reese, 1968).  Alexander  loops ( M i l i c -  (1967) has  calculated the mechanical work done i n v e n t i l a t i o n i n f i s h on the basis of d i f f e r e n t i a l buccal and opercular pressures and mean flow rates, while Jones and Schwarzfeld (1974) have used pressure and flow measurements i n trout to measure the mechanical work of v e n t i l a t i o n , which, combined with measurements of oxygen consumption under various applied pressure heads, gave mechanical e f f i c i e n c y and thus the oxygen cost of v e n t i l a t i o n . Consequently  i n the present experiments,  the mechanical work of  v e n t i l a t i o n i n restrained but unanaesthetised Rana pipiens was measured and an attempt was made to determine experimentally the mechanical e f f i c i e n c y of the buccal pump.  These values have been used to give an estimate of the  energy cost of v e n t i l a t i o n , and of the metabolic rate of the respiratory muscles.  46 METHODS  The experiments were carried out on t h i r t y - f o u r Rana pipiens weighing between 20 and 90 g.  The frogs were held i n tanks at room  temperature  for at l e a s t a week before the s t a r t of the experiments which were performed at 24°C ± 1°C. In order to measure the mechanical work output of the buccal pump, buccal pressure was  recorded by means of a cannula inserted through the tympanic  membrane, and volume by means of a photo c e l l , as described i n the previous section.  Care was  taken to ensure that the volume responses were l i n e a r  over the p h y s i o l o g i c a l range, and i f non-linear responses were obtained the photocell p o s i t i o n was  adjusted u n t i l l i n e a r i t y was achieved.  and volume changes were displayed on a Hewlett-Packard scope operated i n X-Y mode to produce pressure-volume  1201A  The pressure storage o s c i l l o -  loops (Jones, 1970). By  operating the o s c i l l o s c o p e at maximum persistance i t was p o s s i b l e to display up to eight of such loops on the storage surface at one time, which were then photographed by a Hewlett-Packard  o s c i l l o s c o p e camera.  The pressure and  volume traces were recorded simultaneously on a T e c h n i r i t e 2 channel pen recorder run at 1 mm per sec i n order to measure v e n t i l a t i o n frequency. Sequences of loops which were stored on the o s c i l l o s c o p e were marked by means of the recorder event marker. To measure mechanical work appearing i n the lungs during lung i n f l a t i o n sequences, lung pressure was  recorded i n 4 frogs by means of a cannula of  urinary catheter tubing as described In Section 1, while changes i n lung volume were measured by means of the Biocom 991 impedance converter used as an impedance pneumograph.  L i n e a r i t y of the system proved to be ±5 percent.  47 Several lung i n f l a t i o n sequences were recorded for each frog, together with the frequency of lung i n f l a t i o n . Data from 5 frogs whose weights ranged from 72 to 87.5 g were used to estimate the mechanical e f f i c i e n c y of i n d i v i d u a l v e n t i l a t i o n c y c l e s . The mechanical e f f i c i e n c y of the buccal pump for one v e n t i l a t i o n cycle was considered to be given by the following modification of H i l l ' s (1939) equation for muscular e f f i c i e n c y . (i)  Mechanical E f f i c i e n c y % = W X 100 W + a.x +  J Fdt  Where W = mechanical work measured from the buccal pump i n one v e n t i l a t i o n cycle (gram.cm) a.x = the heat of shortening of the muscles of the buccal f l o o r for the cycle (gram.cm)  J^&t - maintenance heat (the heat production associated with the force exerted by the muscles of the buccal f l o o r throughout the v e n t i l a t i o n cycle) (gram.cm) In order to measure mechanical work performed i n s i n g l e v e n t i l a t i o n cycles buccal pressure was recorded on the T e c h n i r i t e 2 channel pen recorder run at 25 or 50 mm per sec, while the volume changes of the buccal cavity were simultaneously filmed i n side-view on 16 mm T r i X r e v e r s a l f i l m using a Bolex cine camera running at a nominal 24 or 32 frames per sec.  To enable  extreme close-ups to be taken a short extension tube was placed between the lens and camera, while a mirror included i n the frame displayed a front-view of the buccal cavity.  The frog was illuminated by two photofloods which were  l i t only when f i l m i n g was taking place.  The f i l m frames were synchronised  with the pressure records by i n c l u d i n g a r o t a t i n g 1 cm diameter disc divided into a black and white sector i n the camera f i e l d , which closed a switch i n  48 the event marker c i r c u i t of the pen recorder once per r e v o l u t i o n . switch connected to the second channel of the pen recorder was filming commenced, and a time s i g n a l was  A further  depressed when  also displayed on this  channel.  Following a sequence of filming volume changes i n the buccal c a v i t y were c a l i b r a t e d by withdrawing a i r from the buccal cavity (with the nares and g l o t t i s blocked) i n 0.2 ml steps and filming each step.  Tracings of the  l a t e r a l view of the buccal cavity were then made on constant density graph paper, the tracings weighed to e s t a b l i s h the weight d i f f e r e n c e represented the 0.2 ml c a l i b r a t i o n steps, and a c a l i b r a t i o n curve was p h y s i o l o g i c a l range there was percent.  drawn.  by  Over the  a l i n e a r increase of weight with volume ± 5  Buccal volume was measured from the cine frames at every frame  during the chosen cycle by tracing the l a t e r a l view of the buccal c a v i t y onto constant density graph paper, weighing the tracings on a Sartorious milligram balance and reading volume from the c a l i b r a t i o n curve. each point was  Buccal pressure at  read from the synchronised pressure traces and plotted against  volume, to give a volume-pressure loop, the area of which represented  the  mechanical work performed i n the buccal cavity during the i n d i v i d u a l c y c l e . The heat of maintenance of muscle i n which twitch summation, or  tetanus  i s occurring i s the sum of the heats of a c t i v a t i o n of the muscle f i b r e s  due  to successive s t i m u l i , and i s proportional to the force exerted by a muscle and the time for which i t i s maintained  (^Fdt).  In order to estimate  heat of maintenance of the muscles of the buccal f l o o r , i t was  first  to determine tension i n the f l o o r throughout the r e s p i r a t o r y c y c l e .  necessary This  accomplished by the a p p l i c a t i o n of Laplace's law to the buccal f l o o r . two major r a d i i of curvature of the buccal f l o o r ( l o n g i t u d i n a l and  the  was  The  transverse)  were traced from the f i l m frames each time buccal pressure and volume were  49 recorded f o r the mechanical work p l o t and substituted together with buccal pressure at that time i n t o : (ii)  P  T  (1 + 1 ) R  l  *2  2 grams per cm  grams per cm  cm  cm  which i s the general form of the Law of Laplace (Figure 11). This gave the tension i n the buccal f l o o r at the point of i n t e r s e c t i o n of the two r a d i i (Burton, 1957). throughout  The two measured r a d i i of curvature were taken to be constant  the f l o o r so that f o r a given buccal pressure, tension produced  by the l o n g i t u d i n a l and transverse muscles of the buccal f l o o r would be the same at any point on the f l o o r .  The p r i n c i p a l respiratory-muscles (medial  and l a t e r a l geniohyoids, intermandibulars and interhyoids) were assumed to be attached uniformly to the borders of the buccal f l o o r , and to l i e along the r a d i i of curvature.  To estimate the t o t a l force exerted by these muscles i n  order to produce a given buccal f l o o r tension, the buccal f l o o r was considered to approximate a square i n plan view, the length of one side being the distance from jaw-bone to jaw-bone at the angle of the jaw, the muscles being uniformly attached along the sides of the square.  For the production of a  given f l o o r tension the t o t a l force on the muscle attachments was thus: (iii)  F grams  m  T  x  abed  grams/cm  (Figure 11)  cm  and the force produced by the muscles, T x 1/2 abed cm.  Force, F, was  calculated each time pressure and volume were recorded and the i n t e g r a l of force.time was plotted graphically as the area under the curve f o r each r e s piratory movement investigated. Only the area under that part of the curve during which the respiratory muscles were active (Section 1) was used i n the calculations and the heat (gram.cm) associated with this value of force.time  50  Figure 11.  Tracings from f i l m frames of 80 g Rana pipiens i l l u s t r a t i n g the 2 major r a d i i of curvature of the buccal f l o o r , and the a p p l i c a t i o n of Laplace's law to the f l o o r .  51 was  read from a c a l i b r a t i o n curve constructed from data of Fales (19 72) on  the heat production of Rana pipiens s k e l e t a l muscle under various conditions of force.time. The heat of shortening of amphibian muscle i s a.x, where a i s a constant 2 with a mean value of 400 grams/cm (Abbot, 1951)  cross-sectional area f o r amphibian muscle  and x i s the shortening (cm) of the muscle.  The shortening of  the l o n g i t u d i n a l and transverse buccal f l o o r muscles was measured externally from the f i l m frames as the difference between the circumferential distance between a and b, a and d (Figure 1) at the maximum and minimum r a d i i of curvature during the v e n t i l a t i o n c y c l e .  The cross-sectional area of the  appropriate muscles (medial and l a t e r a l geniohyoids, intermandibulars and interhyoids) was measured a f t e r sectioning and s t a i n i n g i n haemotoxylin eosin.  and  No corrections were made f o r muscle shrinkage i n f i x a t i o n .  The heat of recovery, l i b e r a t e d a f t e r muscular contraction, has been found to be almost exactly equal to the i n i t i a l heat (consisting of the sum of the heat of shortening and the heat of maintenance) l i b e r a t e d during the contraction, and must be included i n the e f f i c i e n c y equation (1) i n order to give a true measure of muscular e f f i c i e n c y .  I t was  therefore necessary to  multiply a.x (heat of shortening) i n the denominator of the equation ( i ) by a factor of 2, although the values of maintenance heat were not doubled as the values determined by Fales (1972) included the associated heat of recovery. The mass of the respiratory muscles was measured i n three frogs by weighing  the frog and then d i s s e c t i n g out the muscles used i n v e n t i l a t i o n a f t e r  double p i t h i n g .  These muscles were the medial and l a t e r a l geniohyoids, the  intermandibulars, the interhyoids, the sternohyoids, anterior petrohyoids and omohyoids (Part I ) .  Once dissected out, the muscles were dropped  52 i n t o a p r e v i o u s l y weighed  c o n t a i n e r o f amphibian s a l i n e , which was  i n o r d e r t o o b t a i n the wet weight o f the m u s c l e s . as a p e r c e n t a g e o f body w e i g h t .  T h i s was  reweighed  then e x p r e s s e d  53 RESULTS  (a)  The mechanical work appearing i n the buccal cavity and lungs i n one i n f l a t i o n cycle. Figure 12a and b i s a diagrammatic representation of the pressure  and volume events i n the buccal cavity and lungs respectively, during a lung v e n t i l a t i o n cycle.  The cycle commences at A (12a), P (12b) when the  g l o t t i s opens, and lung pressure and volume f a l l while buccal pressure and volume simultaneously r i s e (A to B, P to Q). The nares close at the end of this phase so that a common pressure l e v e l i s attained at the end of i t . Active e l e v a t i o n of the buccal f l o o r then occurs i n the phase B to C, Q to R, which i s terminated when the g l o t t i s shuts at C, R.  The nares open simul-  taneously, so that although the lungs maintain the new pressure l e v e l , buccal pressure drops r a p i d l y to zero and overshoots before the loop i s completed. The t o t a l amount of work p o t e n t i a l l y a v a i l a b l e f o r lung i n f l a t i o n i s represented by area F B C D A F. The amount of work performed i n the cycle by the buccal pump i s represented by area A B C D E A.  Area B D B, however,  i s performed against viscous forces i n the pump i t s e l f and i s l o s t as heat, while area D E A D represents work performed against f l o w - r e s i s t i v e forces i n drawing a i r through the nares i n order to r e f i l l the pump. as heat.  I t , too, i s l o s t  Area B F A represents a contribution from the previous lung  i n f l a t i o n cycle.  On g l o t t a l opening the lungs release the energy stored by  e l a s t i c s t r e t c h during the previous cycle; area P Q U T represents the t o t a l work done by the lungs, while B F A i s the f r a c t i o n contributed to lung i n f l a t i o n i n the next c y c l e . i n expelling lung gas through  effectively  The remainder i s done  the p a r t i a l l y open nares, and i s l o s t as heat.  Therefore the net amount of work a v a i l a b l e f o r lung i n f l a t i o n i n the cycle  54  Figure 12.  (a)  Diagrammatic representation of pressure and volume  changes i n the buccal cavity i n a lung v e n t i l a t i o n cycle, (b)  Pressure and volume changes i n the lungs during the  same cycle.  55 i s represented by area F B C D A F-B D B and the net work done i n the lungs i s represented by area Q P R S T U Q. The decrease i n buccal volume i s mirrored by a s i m i l a r volume increase i n the lungs when the two are i n contact through the open g l o t t i s (B to C, Q to R).  Furthermore i n Rana pipiens there i s no large d i f f e r e n t i a l pressure  across the g l o t t i s (Section 1).  Therefore F B C D A F-B D B^=Q  P R S T U Q,  l i t t l e work being l o s t as heat i n overcoming flow resistance through  the  g l o t t i s , which must therefore be a pathway of low resistance compared to the nares. The magnitude of the viscous losses i n the pump was  investigated by  passively i n f l a t i n g and d e f l a t i n g the buccal cavity of curarised frogs (0.1 per 10 grams weight) with sealed nares (Figure 13a and b) .  mg.  The loop travels  clockwise and the area of the loop over the appropriate pressure range represents the amount of work performed against viscous forces i n the pump, which proved to be 5.4-6.5 percent of t o t a l buccal work i n the frogs investigated. The p o s s i b i l i t y that a c t i v e flank muscle contraction aided lung d e f l a t i o n (Section 1) was  further investigated by comparing normal lung i n f l a t i o n cycles  with cycles obtained i n the same frog a f t e r c u r a r i s a t i o n of s k e l e t a l muscle by i n f l a t i n g and d e f l a t i n g the lungs v i a a cannula inserted into the t i p of one lung.  The area of the loops obtained were the same to w i t h i n 5 percent  (Figure 13c and d).  This provides further support f o r the view that lung  d e f l a t i o n does not involve the s k e l e t a l muscle of the flanks, although i t does not preclude the p o s s i b i l i t y that the tonic a c t i o n of smooth muscle i n the lung wall may  a s s i s t e l a s t i c i t y i n expelling lung gas.  56  Figure 13.  (a) Pressure/volume loops from the buccal pump of an 80 g frog during 2 lung and 2 buccal v e n t i l a t i o n c y c l e s . (b)  Pressure/volume loop  from the buccal cavity of the  same frog with sealed nares, i l l u s t r a t i n g work performed against viscous forces i n the pump. (c)  Pressure/volume loop recorded from the lungs during a  lung i n f l a t i o n sequence. (d)  Pressure/volume loop obtained from the same frog by  i n f l a t i o n and d e f l a t i o n of the lungs a f t e r c u r a r i s a t i o n .  57 (b)  Mechanical work output of the buccal pump. Figure 14a shows a t y p i c a l lung i n f l a t i o n sequence recorded from the  lungs, while 14b and c shows a t y p i c a l sequence of P-V loops recorded from 3 the buccal cavity.  Pressure was measured i n cm H^O and volume i n cm . The  values of work calculated from the areas of the pressure volume loops were therefore i n gram.cm.  The mechanical work performed i n the buccal cavity per  hour was a r r i v e d at by c a l c u l a t i n g the mean amount of work performed i n a sequence of lung v e n t i l a t i o n cycles and multiplying by the frequency of such sequences then adding to this figure the mean mechanical work performed i n a buccal v e n t i l a t i o n loop, m u l t i p l i e d by the o v e r a l l frequency of buccal ventilation.  Measurement of mechanical work appearing i n the lungs during a  t y p i c a l i n f l a t i o n sequence, m u l t i p l i e d by the frequency of such sequences enabled c a l c u l a t i o n of the amount of work appearing i n lung i n f l a t i o n sequences per hour.  The data obtained from these experiments was used to c a l c u l a t e the  regression of mechanical work on body weight.  The regression l i n e s were f i t t e d  by the l e a s t squares method and were plotted by a computer. Figure 15 i l l u s t r a t e s the regression of mechanical work performed by the buccal pump per hour on body weight, f o r frogs ranging i n weight from 24.5 to 86.5 g.  For these frogs, i f M = Mechanical work performed per hour and  W = the body weight i n grams, then: log M • log k + b l o g W The exponent b i s equal to the slope of the plot of l°8^g mechanical work against 1°S^Q weight, while the k value was obtained from the y intercept. Therefore: log M - 1.47 + 1.199 l o g W  or  M - 3.39 X 10"V-*  199  58  Figure 14.  (a)  Pressure/volume loop recorded from the lungs during a  t y p i c a l lung i n f l a t i o n sequence. (b)  Sequence of pressure/volume loops recorded from the  buccal cavity of a 24.5 (c)  g Rana p i p i e n s .  Simultaneous l i n e a r recording of pressure and volume.  1, event channel (7 lung v e n t i l a t i o n loops occurring before event marker are shown i n (b)); 2, buccal pressure; 3, buccal volume, up on the trace represents increase i n volume; 4, time, 10 sec marker.  58a  59  Figure 15.  Regression of mechanical work output of the buccal pump on weight f o r Rana pipiens ranging i n weight from 24.5 to 86.5 g.  The interrupted l i n e s indicate one standard error  of the mean.  60 The regression of mean buccal volume change during v e n t i l a t i o n on mass was also investigated (Figure 16) f o r frogs i n the same s i z e range.  If V =  mean change i n the buccal volume, then: i n lung v e n t i l a t i o n , log V = 1.154 + 1.216 l o g W or V = 7.015 X 10~  3  W * 1  216  i n buccal v e n t i l a t i o n , log V - 1.253 + 1.175 l o g W or V - 5.585 X 10~  3  W  1 , 1 7 5  Mean buccal pressure change i n the lung v e n t i l a t i o n cycles d i d not vary s i g n i f i c a n t l y between d i f f e r e n t sizes of frog.  I t therefore appears that f o r  a given r e s p i r a t o r y frequency, the mean volume change i n lung v e n t i l a t i o n cycles i s the main factor i n determining  the mechanical work rate of the  buccal pump i n d i f f e r e n t weights of frog, and therefore the regressions of mechanical work and mean buccal volume change on weight have s i m i l a r exponents. For mechanical work appearing i n the lungs per hour during lung i n f l a t i o n sequences, p l o t t e d against body weight (Figure 17): log M = "3.681 + 0.6451 l o g W or M - 2.09 X 10"  4  W°-  6 4 5 1  Thus only a f r a c t i o n of the t o t a l work done on the lungs by the buccal forcepump appears i n the lungs during sequences of lung i n f l a t i o n and d e f l a t i o n .  61  Figure 16.  Regression of buccal volume change i n lung and buccal v e n t i l a t i o n cycles on weight f o r Rana pipiens at 25 C. 8  F i l l e d c i r c l e s , lung v e n t i l a t i n g cycles; open c i r c l e s , buccal v e n t i l a t i n g c y c l e s .  Buccal volume change in ventilation movements  62  Figure 17.  Regression of work appearing i n the lungs during  lung  i n f l a t i o n cycles on weight f o r Rana pipiens a t 25°C. The interrupted l i n e s i n d i c a t e one standard e r r o r of the mean.  62a  63 (c)  The mechanical e f f i c i e n c y of the buccal pump. Pressure-volume measurements of i n d i v i d u a l respiratory cycles obtained  by the method described, produced the same type of pressure-volume loops as that obtained by X-Y p l o t t i n g on the o s c i l l o s c o p e , or by the method of Jones (1970).  Figure 18a i l l u s t r a t e s a t y p i c a l loop, the area of which gave the  mechanical work done by the buccal pump i n one lung v e n t i l a t i o n ; Figure 18b shows the calculated tension produced i n the buccal f l o o r for the same breathing cycle plotted against time, together with the corresponding pressures.  buccal  The muscles of the buccal f l o o r are only active between A and B  (Section 1) consequently value of force.time.  only the area above A-B was  A good c o r r e l a t i o n was  used to determine the  seen between tension i n the buccal  f l o o r and buccal pressure at that time i n a l l the frogs investigated, suggesting that the time i n t e g r a l of the pressure change over the appropriate part of the breathing cycle i s a good index of the average r e s p i r a t o r y force, as pointed out by McGregor and Becklake (1961), Figure 19 i l l u s t r a t e s the i n d i v i d u a l terms of the e f f i c i e n c y plotted against the peak pressure per cycle, which was  equation  considered to be an  i n d i c a t i o n of muscular load, for the 12 cycles investigated.  The heat of  shortening of the buccal f l o o r muscles a.x, i s r e l a t i v e l y constant i n a l l cycles, the d i f f e r i n g amounts of mechanical work performed i n d i f f e r e n t cycles depending l a r g e l y on the buccal pressure achieved through the c y c l e , which i n turn depends upon force generated i n the buccal f l o o r .  The heat of maintenance  associated with force.time i s r e l a t i v e l y small compared to the heat of shortening of the respiratory muscles, although i t increases with an increase i n the maximum cycle pressure,, Figure 20 shows the calculated mechanical e f f i c i e n c i e s of 9 lung and 3  64  Figure 18.  (a)  Pressure/volume loop measured from the buccal cavity  of Rana pipiens.  The loop cycles anti-clockwise from zero  pressure. (b)  Calculated tension i n the buccal f l o o r , together with  the corresponding buccal pressure (continuous l i n e ) f o r the same c y c l e .  65  Figure 19.  Terms of the e f f i c i e n c y equation plotted against maximum cycle pressure f o r 12 i n d i v i d u a l v e n t i l a t i n g cycles, together with calculated t o t a l energy input per c y c l e . Open c i r c l e s , buccal v e n t i l a t i n g cycles; f i l l e d lung v e n t i l a t i n g cycles.  circles,  20  r  65a  Total energy 15  E  10  E o O  Mechanical work  0 * 0  o-o <  E o  6  Heat of maintenance  0  1  o-o  i  0  a  E &  o o  o  •  •  e Heat of shortening  -ft Maximum cycle pressure  cm HO  66 buccal v e n t i l a t i n g cycles, plotted against the t o t a l mechanical work performed i n each c y c l e .  E f f i c i e n c y of lung v e n t i l a t i o n increased with mechanical work  performed per cycle from 7.4 percent at 0.65 gram.cm to a maximum of 19.3 percent at 2.73 gram.cm.  The mechanical e f f i c i e n c y of the three buccal  v e n t i l a t i o n s investigated proved to be low, although e f f i c i e n c y increased i n the high amplitude buccal movement i n which a r e l a t i v e l y large amount of mechanical work was performed.  Calculated e f f i c i e n c y increased with increased  mechanical work performed i n v e n t i l a t i n g cycles because t o t a l volume change varies very l i t t l e i n a l l lung v e n t i l a t i n g cycles (Figure 14a and b), and hence the measured amount of shortening of the medial and l a t e r a l geniohyoids i s v i r t u a l l y constant.  This i s r e f l e c t e d In the value of a.x, the heat of  shortening, i n the denominator of the e f f i c i e n c y equation, which i s of r e l a t i v e l y large magnitude compared to the values f o r mechanical work and the force.time equivalent.  Therefore, i f mechanical work i s doubled, the  numerator of the f r a c t i o n i s doubled, while the denominator  i s increased only  s l i g h t l y , and the value f o r e f f i c i e n c y almost doubles. The calculated t o t a l energy, or mechanical equivalent of the oxygen consumed f o r each respiratory cycle studied i s also p l o t t e d i n Figures 19 and 20.  Total energy per lung v e n t i l a t i n g cycle increased s t e a d i l y as the t o t a l  mechanical work performed each cycle increased, i n s p i t e of the accompanying increase i n mechanical e f f i c i e n c y .  The low e f f i c i e n c y of the lung v e n t i l a -  t i o n movement which generated the high peak pressure of 9.2 cm h^O was due to the r e l a t i v e l y large magnitude of the heat of maintenance  i n the denomina-  tor of the e f f i c i e n c y equation. Although the value for mechanical work i n the low amplitude buccal v e n t i l a t i n g cycles i s low, mechanical e f f i c i e n c y i s also low, 0.4 percent-0.5 percent, so that the calculated t o t a l energy f o r these  67  Figure 2 0 .  Calculated mechanical e f f i c i e n c y and t o t a l energy input plotted against mechanical work f o r 12 v e n t i l a t i n g  cycles.  Open c i r c l e s , e f f i c i e n c y of buccal v e n t i l a t i n g  cycles;  f i l l e d c i r c l e s , e f f i c i e n c y of lung v e n t i l a t i n g  cycles;  t r i a n g l e s , t o t a l energy input per cycle.  I  Mechanical efficiency % O  1  I  O  A  .  O  >  O  O  O  »  O  *i9  S  5  >  O  3  C  J  68 cycles i s only s l i g h t l y lower than that calculated for  low-amplitude  lung-ventilatory cycles. The mean mechanical e f f i c i e n c y of the respiratory cycles analysed was 10.6 percent, but this i s almost c e r t a i n l y higher than the o v e r a l l e f f i c i e n c y of v e n t i l a t i o n , for i n an undisturbed frog r e l a t i v e l y  mechanical  inefficient  low amplitude lung v e n t i l a t i o n cycles and buccal cycles predominate. o v e r a l l mechanical e f f i c i e n c y was  The  therefore taken to be 8 percent, the  e f f i c i e n c y of low amplitude lung v e n t i l a t i o n cycles.  This value, together  with a value for the mechanical work of v e n t i l a t i o n (Section a) enables an estimate to be made of the oxygen co3t of breathing i f the data of Hutchinson et a l (1968) are used f o r the t o t a l oxygen consumption and R.Q. pipiens at 25°C.  Hutchinson et a l . (1968) found an R.Q.  pipiens at 25°C.  This i s equivalent to a c a l o r i f i c output of 4.71 x 10  (Cal/ml 0^).  This value was  of 0.73  of Rana f o r Rana  used i n Table 1 i n which the oxygen cost of  v e n t i l a t i o n i s calculated for 20 and 50 g frogs at 25°C f o r maximum, minimum and mean, and assumed o v e r a l l values of mechanical e f f i c i e n c y . oxygen cost of v e n t i l a t i o n for a resting frog proved to be 5 percent, and a l l values are within the range 2-7 (d)  The  approximately  percent,  Measurements of muscle mass and c a l c u l a t i o n of respiratory muscle metabolism. Results of dissections of 3 Rana pipiens with a mean weight of 63.6 g  (range, 60.5  to 68.2 g) gave an average of 0.92  percent (0.58 g), as  the proportion of t o t a l body weight of the respiratory muscles, the range of the measurements being from 0.81  to 0.98  percent.  breathing of frogs i n this weight range was (Section a ) .  The mechanical work of  found to be 0.50  Joules per hour  The oxygen consumption of the respiratory muscles, expressed i n  the standard way  as ml 0,/100 g/min i s calculated i n Table 2 f o r the  TABLE 1 For 50 g Rana pipiens at 25°C Mechanical work of breathing  0.41 Joules per hour  (Section a)  T o t a l oxygen consumption  5.0 ml 0  (Hutchinson, Whitford and Kohl, 1968)  2  per hour  100 Joules per hour Efficiency percent  Total energy input to buccal pump  Oxygen cost percent  Max. observed e f f i c i o n c y of lung v e n t i l a t i o n  19.5%  2.1 Joules/hour  2.1%  Min. observed e f f i c i e n c y of lung v e n t i l a t i o n  5.9%  6.9 Joules/hour  6.9%  Mean observed e f f i c i e n c y of lung v e n t i l a t i o n  10.6%  3.9  Joules/hour  3.9%  5.1 Joules/hour  5.1%  8.0%  Overall e f f i c i e n c y of lung and buccal v e n t i l a t i o n For 20 g Rana pipiens a t 25°C Mechanical work of breathing  0.22  Joules per hour  T o t a l oxygen consumption  2.8 ml 0  2  per hour  (Section a) (Hutchinson, Whitford and Kohl, 1968)  56 Joules per hour Efficiency percent  1.9%  5.9%  3.7 Joules/hour  6.8%  10.6%  2.1 Joules/hour  3.7%  2.8  4.9%  19.5%  Min. observed e f f i c i e n c y of lung v e n t i l a t i o n Mean observed e f f i c i e n c y of lung v e n t i l a t i o n  8.0%  1.1  Oxygen cost percent  Joules/hour  Max. observed e f f i c i e n c y of lung v e n t i l a t i o n  O v e r a l l e f f i c i e n c y of lung and buccal v e n t i l a t i o n  Total energy input to buccal pump  Joules/hour  70  TABLE 2  Mechanical work output (Joules/hour)  E f f i c i e n c y of buccal pump (percent)  0.50  Overall e f f i c i e n c y  Calculated work input ( t o t a l energy) (Joules/hour)  Oxygen equivalent of work input (ml 02/hour)  VO2 respiratory muscle (ml 0 /100g /min 2  19.5%  2.6  0.13  0.37  5.9%  8.5  0.42  1.21  8.0%  6.3  0.31  0.89  71 maximum, minimum and o v e r a l l e f f i c i e n c i e s of v e n t i l a t i o n , assuming 1 ml 0 s 19.56 Joules (Weast, 1968).  72 DISCUSSION  No previous studies have been performed on the mechanical work output o f the amphibian buccal pump, but i t i s possible to compare respiratory work i n Rana pipiens and a mammal of s i m i l a r s i z e by using data of C r o s f i l and Widdicombe (1961) on the mouse, which indicates that 0.025 Joules per gram per hour i s the work output i n normal breathing.  This i s approximately 3 times  the value of the respiratory work output i n the frogs studied i n the present investigation. The method described for estimating the mechanical e f f i c i e n c y gives r i s e to a range of e f f i c i e n c i e s of from 5.9 to 19.5 percent f o r lung v e n t i l a t i o n with an o v e r a l l e f f i c i e n c y value of 8 percent. There are no comparable figures for mechanical e f f i c i e n c y i n amphibians although e f f i c i e n c y i n f i s h i s probably very low, about 1-4 percent at rest (Jones, 1971; Jones and Schwarzf e l d , 1974).  Mechanical e f f i c i e n c y i n man has been found to range from 5.5  to 8.6 percent ( L i l j e s t r a n d , 1918; O t i s , 1954; Campbell e t a l , 1957; Cherniak, 1959; F r i t t s and Cournand, 1956) although values of 19 to 25 percent have been found by comparing mechanical work, determined by pressure/flow measurement, with t o t a l energy required f o r breathing ( M i l i c - E m i l i and P e t i t , 1960). H i l l ' s equation f o r muscular e f f i c i e n c y applies to an i s o t o n i c muscle contraction. of  How w e l l i s this condition f u l f i l l e d i n the respiratory muscles  the buccal f l o o r of a frog during a respiratory cycle?  At the s t a r t of the  cycle the buccal f l o o r muscles cannot begin to shorten u n t i l the muscular force exceeds the load, which i s a function of the pressure generated w i t h i n the buccal cavity and this i s therefore i n i t i a l l y small.  Load increases with  muscular shortening, which reduces buccal volume, u n t i l at peak pressure  73 muscular force equals load on the muscles, and shortening stops.  As the  muscles shorten rapidly (0.2-0.3 seconds) the force exerted by them i s probably never greatly i n excess of load.  The muscle contraction occurring i n a  respiratory cycle i s therefore s i m i l a r to an isometric contraction i n which a muscle shortens under load, except i n this case load increases through contraction instead of remaining  the  constant.  The values of maintenance heat given by Fales (1972) and used here were determined  i n amphibian muscle i n isometric twitch and tetanus.  conditions 1 gram.sec  B  1 gram.cm at low values of force.time.  For both The fundamental  mechanism of contraction remains the same however i n both i s o t o n i c and  isomet-  r i c contractions, so that i t seems reasonable that the heat generated f o r a given value of force.time should not be s i g n i f i c a n t l y d i f f e r e n t i n either condition.  The estimation of muscle shortening by external measurement i s  probably a reasonable r e f l e c t i o n of the shortening of the l a t e r a l and medial geniohyoids and the intermandibular and interhyoid muscles.  I t f a i l s to take  into account the shortening of the sternohyoids, omohyoids and petrohyoids which also contribute to the v e n t i l a t i o n cycle.  The calculated value of the  heat of shortening i s almost c e r t a i n l y therefore a conservative estimate of i t s true value, which would tend to r e s u l t i n high values f o r the  mechanical  e f f i c i e n c y of the buccal pump, i n turn reducing the calculated energy cost of ventilation.  Jones (1972) found that curarised, a r t i f i c i a l l y v e n t i l a t e d Rana  esculenta at 24°C showed an average reduction i n oxygen consumption of 16 percent compared with normally breathing animals.  Individual frogs showed  reductions between 1 and 35 percent however, and i n some frogs s t r i a t e d muscle other than respiratory muscle must have been curarised, so that i t would be unwise to assume that 16 percent represented the energy cost of breathing.  74 The energy cost of 5 percent calculated f o r man 1959;  reported here i s at l e a s t double that  at rest ( L i l j e s t r a n d , 1918;  F r i t t s et a l , 1959;  Cournand et a l , 1954;  M i l i c - E m i l i and P e t i t , 1960;  Cherniak,  Campbell et a l , 1970),  although a good deal less than energy cost of v e n t i l a t i o n i n f i s h , which i s generally reported to be 8 to 20 percent at rest (Van Dam, and Cech, 1970;  1938;  of the o v e r a l l oxygen consumption  Schumann and P i i p e r , 1966;  Jones and Schwarzfeld, 1974).  Alexander, 1967;  Cameron  This large cost i n f i s h  ap-  pears to be due i n part to the high v i s c o s i t y and density of water ( v i s c o s i t y 100  times and density 1000  times that of a i r at 18°C)  suggesting  that the work  done against viscous and e l a s t i c forces developed i n the i r r i g a t i n g system, which i s independent of the properties of the medium, i s probably r e l a t i v e l y unimportant i n determining the oxygen cost of v e n t i l a t i o n compared to the amount of work done against flow r e s i s t i v e forces, which depend on the v i s c o s i ty and density of the medium.  The amount of work performed against  flow  resistance i n the frogs investigated proved to be small which i s consistent with the favourable physical properties of a i r as a r e s p i r a t o r y medium. Only a small proportion of the t o t a l body weight i n Rana pipiens i s made up of respiratory muscle, and d i f f e r e n t estimates of the oxygen cost of breathing would obviously a l t e r the calculated metabolic rate of these muscles. The calculated value of respiratory muscle oxygen consumption, VO29 0.89 100 g/min may  O2/  be compared with the oxygen consumption of the whole frog at  25°C which i s 0.17  ml O /100 g/min (Hutchinson e t a l , 1968). 2  muscles at r e s t are therefore metabolising metabolism.  ml  The r e s p i r a t o r y  at 5 times the rate of o v e r a l l  This estimate of respiratory muscle metabolism appears  reasonable, although as yet there i s no comparable data for other amphibians. Values of VO^ for some muscles i n f i s h are somewhat higher, being 2.50 6.60  and  ml O /100 g/min for Tuna red muscle and Carp white muscle, r e s p e c t i v e l y o  7 5 at rest (Cameron and Cech, 1970).  In man the basal metabolic rate f o r  muscle Is estimated to be 0.2 ml O2/IOO g/min, while Landis and Pappenheimer (1963) estimate the oxygen consumption of a c t i v e l y contracting muscle i n man at 10 ml 0-/100 g/min.  76 PART I I I . THE INITIATION OF DIVING APNOEA IN RANA PIPIENS  INTRODUCTION  A l l air-breathing vertebrates are forced into an apnoeic condition on submersion i n water.  The i n i t i a t i o n and maintenance of apnoea during submer-  sion have not been greatly investigated, although there i s a growing amount of evidence that sensory information from the area around the n o s t r i l s , the nasal cavity, and i n some cases the region of the g l o t t i s and upper respiratory t r a c t i s involved (Huxley, 1913a; Lombroso, 1913; Andersen, 1963a; Cohn et a l , 1968; Butler and Jones, 1968; Jones and Purves, 1970; Angell James and Daly, 1972a and b; Drummond and Jones, 1972). In s p i t e of the fact that amphibians are more completely adapted to a semiaquatic existence than other diving vertebrates, there i s l i t t l e  Informa-  t i o n a v a i l a b l e concerning the stimulus which causes i n h i b i t i o n of v e n t i l a t i o n during periods of submergence, or the s i t e s s e n s i t i v e to such stimulation. Willem (1920) thought that external n a r i a l closure and apnoea on submersion i n Rana esculenta was r e f l e x , due to the wetting of the snout, and that the occasional release of lung gas underwater  through the nares involved the  temporary overriding of this r e f l e x i n h i b i t i o n .  Spurway and Haldane (1953)  considered that the presence of water at the snout provides an i n h i b i t o r y stimulus to v e n t i l a t i o n i n newts and that resumption of v e n t i l a t i o n observed on surfacing of the snout was due to "the cessation of i n h i b i t o r y sensory s t i m u l i rather than because of any p o s i t i v e sensory s t i m u l i from the a i r " . Certainly the presence of atmospheric 0_ or C0_ does not appear to be involved  77 i n stimulating the resumption of v e n t i l a t i o n on surfacing i n amphibians, for i f frogs (Rana temporaria) emerge from water into an atmosphere of nitrogen they s t i l l resume v e n t i l a t i o n (Jones, 1966). The purpose of the present i n v e s t i g a t i o n was  to determine the sensory  areas important i n the i n i t i a t i o n and maintenance of diving apnoea i n the frog Rana pipiens, to record from the nerves serving these areas during simulated dives, and to attempt to i n i t i a t e apnoea i n the frogs by e l e c t r i c a l of these nerves, i n order to e s t a b l i s h their role during d i v i n g .  stimulation  78 METHODS  The experiments were performed on 65 Rana pipiens with a weight range of from 50 to 85 g, although animals i n the 75-85 g weight range were used exclusively i n the nerve stimulation experiments.  A l l the experiments were  performed at an a i r temperature of 24°C. Experiments involving submergence of normal animals and animals a f t e r section of the ophthalmic nerves, were carried out i n a perspex tank of 3 l i t r e s capacity.  The tank was connected to a large water reservoir so that  the water l e v e l could be raised and lowered at w i l l .  Water temperature was  maintained at 20°C except i n those experiments i n which a bead thermistor was placed i n the nasal cavity of the frog, when i t was maintained at 14°C.  The  animals were anaesthetised by immersion i n MS 222 (Sandoz) (300-400 mg/1.), positioned on a cork board and restrained by pinning (Jones, 1970).  During  normal dives buccal pressure and volume were recorded by the methods described i n Section 1.  Struggling made t h i s method impractical f o r recording  r e s p i r a t i o n i n frogs i n which the ophthalmic nerves were cut.  Consequently,  i n these frogs EMG a c t i v i t y was recorded from the posterior intermandibular muscle and the larynx i n order to monitor v e n t i l a t i o n . Section of the ophthalmic branch of c r a n i a l nerve V at the l e v e l of the nasal cavity was accomplished under deep MS 222 anaesthesia. The frog was placed on i t s back, and the jaws were held apart. the  Incisions were then made i n  f l o o r of the nasal cavity, from the i n t e r n a l n a r i a l openings to the  midline.  The c a r t i l a g e s forming the f l o o r were then r e f l e c t e d back to the  midline, exposing the nasal c a v i t i e s .  The main branches of the ophthalmic  nerve enter each nasal cavity through foramina i n the sphenethmoid  cartilage,  79 and then across the c a v i t i e s between the c a r t i l a g e and the o l f a c t o r y epithelium (Figure 21a, b ) , before passing through the s k u l l to supply the skin of the external n a r i a l region and the snout (Ecker, 1889).  In the  operated animals they were cut b i l a t e r a l l y close to the point of emergence from the sphenethmoid  c a r t i l a g e while f o r sham operations they were merely  i d e n t i f i e d through s l i t s made i n the o l f a c t o r y epithelium p a r a l l e l to t h e i r courses.  The nasal c a r t i l a g e s were then returned to p o s i t i o n and, i n the  larger frogs, held i n place by 2 s i l k s t i t c h e s .  Both experimental and sham  operated animals were allowed to recover from the anaesthesia and l e f t f o r 24 hours i n a large holding tank before being used i n an experiment, and only those frogs which appeared to respire normally a f t e r the operation were used. Blood loss during the operation was small and the s u r v i v a l was good i n both operated and sham operated frogs. Recordings of afferent nervous a c t i v i t y were made from the ophthalmic branches of V at the l e v e l of the nasal cavity.  The nerves were sectioned  c e n t r a l l y i n double-pithed frogs and electroneurograms monitored using a p a i r of f i n e , s i l v e r wire hook electrodes under mineral o i l .  Those branches with  t h e i r receptive f i e l d s round the external nares were chosen f o r the recordings. The response to a water meniscus moving over the nares and to water flow was investigated by placing the frogs i n the 3 l i t r e tank, i n which the water l e v e l could be raised and lowered.  The frogs were pinned on t h e i r backs with  the lower jaw pinned back, and the external n a r i a l openings were plugged to prevent water entering them underneath the mineral o i l and thus grounding the electrodes.  An attempt was also made to subject the n a r i a l region to water  pressure by sewing a membrane around the snout and connecting this to a water column.  However, this proved impractical due to the d i f f i c u l t y of membrane  attachment to the frog.  Therefore, i n order to determine the e f f e c t s of  80  Figure 21a and b.  Transverse sections of the snout of a 20 g frog, i l l u s t r a t i n g the course of the ophthalmic nerve. 1, nasal c a r t i l a g e ; 2, ophthalmic branches; 3, nasal mucous epithelium.  80a  81 pressure some t r i a l s were carried out i n which the n a r i a l region was submerged under 4-6 mm of mercury, which was retained around the nares by a modelling clay dam, while i n others the entire frog was submerged under mineral o i l  (S.G. 0.87). Recordings were made from the cutaneous branches of the second, t h i r d , and fourth s p i n a l nerves i n order to determine the responses of cutaneous nerves serving other areas of the s k i n to pressure and the movement of a water meniscus. In order to approximate the conditions obtaining at the snout, where the s k i n i s f i r m l y connected to the underlying c a r t i l a g e , flaps of skin containing the receptive f i e l d s were placed on a ground glass d i s c to which hydrostatic pressure was applied.  The f i b r e s serving a receptive f i e l d were  led out through a small hole i n the centre of the d i s c . Nervous a c t i v i t y was amplified and f i l t e r e d by means of a Tetronix 122 preamplifier and displayed on a Tetronix 502A o s c i l l o s c o p e , being simultaneousl y recorded by means of a Hewlett-Packard 3900C tape recorder.  Suitable  signals were l a t e r photographed on playback by means of a Grass o s c i l l o s c o p e camera, or e l s e played into a Brush penrecorder at reduced speed.  In some  experiments a ratemeter was used to determine f i r i n g r a t e , and some data was analysed as Time Interval Histograms by means of a D i g i t a l Lab 8E computer. In those experiments which involved e l e c t r i c a l stimulation of the ophthalmic branch of V, the nerves were exposed b i l a t e r a l l y at the l e v e l of the orbit.  I t was necessary to remove the eyes under deep MS 222 anaesthesia i n  order to accomplish nerve stimulation.  I n i t i a l l y the n i c t i t a t i n g membrane was  removed and the eye muscles were cut close to the eyeball, and the optic nerve, artery and vein were t i g h t l y l i g a t u r e d . to the l i g a t u r e and the eye removed.  The optic s t a l k was then cut d i s t a l l y  The ophthalmic branch of V, which runs  82  between the cranium and the e y e b a l l , below the superior rectus muscle but above a l l the other eye muscles (Ecker, 1889), was then c a r e f u l l y freed from i t s connective tissue sheath and associated blood v e s s e l s . operation was n e g l i g i b l e .  Blood loss i n the  Before complete recovery from the anaesthesia the  frogs were secured i n a head holder, which r i g i d l y fixed the head i n r e l a t i o n to the stimulating electrodes, but allowed respiratory movements to occur normally (Jones, 1970).  Complete recovery from the anaesthesia usually  occurred i n 30-40 minutes and v e n t i l a t i o n restarted spontaneously.  Before  the frog had completely recovered the nerves were placed on the stimulating electrodes under mineral o i l , and the d i s t a l ends of the nerves were severed at the anterior end of the o r b i t .  Fine hook electrodes were used, the  stimulating pulses being provided from a Grass model S4G stimulator, and displayed on a Tetronix 502A o s c i l l o s c o p e .  Unipolar s t i m u l a t i o n was used at  50-1000 Hz 0,4-4 msec duration and 30 mv to 5 v i n t e n s i t y .  Ventilation  movements were monitored by recording buccal pressure, which was displayed on a Hewlett-Packard  4 channel pen recorder, while periods of nerve  stimulation were indicated by means of the event channel.  The p o s i t i o n of the  nares was observed by means of a binocular dissecting microscope.  Care was  taken to keep the s k i n of the animals moist throughout the course of these experiments.  83 RESULTS  (a)  Preliminary experiments Several types of preliminary experiments were performed on Rana pipiens  to determine the s i t e i n i t i a t i n g apnoea on immersion i n water.  In the f i r s t  of these the water l e v e l was gradually raised, and the e f f e c t s on v e n t i l a t i o n were noted.  In a t o t a l of 30 t r i a l s on 5 frogs v e n t i l a t i o n movements ceased  when the water reached the l e v e l of the external nares i n 27 t e s t s .  In three  experiments temporary apnoea occurred when water came i n contact with the buccal f l o o r , but normal v e n t i l a t i o n movements were resumed a f t e r 10-15 seconds, and continued u n t i l the water had r i s e n to the l e v e l of the external nares when they stopped. When the frogs were surfaced a f t e r periods of submergence which varied from 2 to 15 minutes, resumption of breathing occurred immediately the water l e v e l f e l l below the l e v e l of the external nares i n 23 cases.  In 3 t r i a l s  breathing did not resume u n t i l a f t e r a l a g of 10 to 30 seconds, while i n the remaining 4 t r i a l s breathing d i d not r e s t a r t u n t i l the water l e v e l had f a l l e n below the l e v e l of the buccal f l o o r .  The rate of t o t a l submersion and emersion  i n the t r i a l s varied from 15 seconds to 2 minutes. Three frogs, blinded by section of the o p t i c nerves, performed i n the same way as the sighted animals.  Furthermore, no v a r i a t i o n s i n v e n t i l a t i o n rate  could be induced i n frogs which were placed i n a large beaker i n a tank i n which the water l e v e l was raised and lowered.  In these experiments the water  surface passed acro3s the frogs' v i s u a l f i e l d , although the frogs never came into contact with the water. I t was possible however, that the presence of water on the surface of the  84  eyes or tympanic membrane, both on a l e v e l with the external nares, could be important i n the development of apnoea.  To test this hypothesis  were made with three frogs i n which the frogs were secured  20  trials  to a v e r t i c a l cork  block, head up, so that the tympanic membranes, tympanic membranes and eyes, and f i n a l l y the tympanic membranes, eyes and external nares could be submerged by r a i s i n g the water l e v e l .  In no t r i a l could apnoea be induced by  wetting  the tympanic membranes, or the tympanic membranes and eyes; only when the water meniscus was  at the l e v e l of the external nares did apnoea occur.  These preliminary t r i a l s strongly suggested that water at the l e v e l of the external nares r e f l e x l y induced apnoea i n Rana pipiens, water at the l e v e l of the eyes and tympanic membranes having no e f f e c t on v e n t i l a t i o n , (b)  Diving i n normal animals. Figure 22a and b i l l u s t r a t e s the pressure and volume changes recorded  from  the buccal c a v i t y of frogs during the course of 2 dives of d i f f e r i n g duration (the base l i n e change i n the volume trace on submersion and emersion i s due the water meniscus moving past the face of the p h o t o c e l l ) . reached the l e v e l of the external nares i n 10-20  to  The water surface  seconds, at which point  respiratory movements ceased and the nares closed.  In both cases the nares  closed during a period of buccal v e n t i l a t i o n when the lungs were i n f l a t e d and i s o l a t e d from the g l o t t i s .  A few seconds a f t e r n a r i a l immersion i n each dive  bubbles of gas were released from the nares, bubble release being preceded by a rapid increase i n buccal pressure  (Figure 22a and b ) .  In cases where  animals were submerged with the lungs f u l l this gas represented lung contents, and i t s release was  accompanied by contraction of the flanks,  increasing endopulmonary pressure and decreasing lung volume. however, i t represented  part of the  In others,  loss of some of the buccal gas (Figure 23a),  and  85  Figure 22a and b.  Two dives of d i f f e r i n g duration.  Top trace of each  p a i r , buccal pressure; lower trace, buccal volume. Increase i n volume i s up on trace.  The arrows  indicate submersion and surfacing.  0 indicates l o s s  of gas through the nares.  86  resulted i n the buccal f l o o r being pressed close to the roof of the buccal cavity during the dive due to i t s reduced gas volume.  In the e a r l y part of  the period of submersion both lung and buccal pressure slowly increased i n response to the Increase In hydrostatic pressure as the water rose, u n t i l both had increased by 2-3  cm H^O  which corresponded to the height of the  water column above the f r o g . Throughout the course of the dive two types of pressure events were recorded from the buccal c a v i t y and lungs.  In the f i r s t  (Figure 23b),  glottal  opening resulted i n a simultaneous f a l l i n lung pressure and increase i n buccal pressure, followed by e q u i l i b r a t i o n of lung and buccal pressures. buccal pressure rose faster than i n the corresponding  phase A-B  The  of a lung  v e n t i l a t i o n cycle (Section 1) and the e q u i l i b r a t i o n pressure was  higher,  because i n this case there was no loss of gas through the nares, which were closed underwater .  Buccal volume increased i n this phase due to the i n f l u x  of lung gas i n t o the buccal c a v i t y through the open g l o t t i s (Figure 23b). After e q u i l i b r a t i o n , decrease i n buccal volume raised the pressure i n the system u n t i l the o r i g i n a l lung pressure was  attained, at which point the  g l o t t i s closed, i s o l a t i n g the lungs once more. muscle was  The posterior intermandibular  active during this phase, as presumably were the other muscles  involved i n lung v e n t i l a t i o n cycles.  The cessation of muscular a c t i v i t y i n  the buccal f l o o r muscles then allowed buccal pressure to f a l l back to the pressure due to the column of water above the buccal c a v i t y .  The  frequency  of these underwater lung v e n t i l a t i o n cycles was very v a r i a b l e , ranging from 1-10  per minute i n i n d i v i d u a l frogs and often t h e i r frequency  the course of the period of submergence (Figure 22b). maintained  increased during  Lung pressure  was  at a f a i r l y constant l e v e l throughout the period of submergence,  87  Figure 23.  Pressures recorded from the buccal cavity and lungs during a dive,  a, submersion; b, 10 minutes a f t e r submersion;  c, surfacing. pressure.  Top trace, event marker.  Lower t r a c e , buccal pressure.  Middle trace, lung  E  0  L  v'  [.'"!////J/J//J/jJiJiJ^jiJ;i  l  88 although buccal pressure often f e l l s l i g h t l y , together with a s l i g h t increase i n buccal volume throughout the course of the longer dives (Figure 22b), suggesting that i t must have i n i t i a l l y been held s l i g h t l y higher than the hydrostatic pressure by tone i n the muscles of the buccal f l o o r .  The period  of apnoea caused by submersion ended when the water l e v e l f e l l past the l e v e l of the external nares.  Emersion i n i t i a t e d a burst of lung v e n t i l a t i o n  cycles of high pressure, followed by an increased frequency  of v e n t i l a t i o n  (Figure 23c), most of the cycles being of the lung v e n t i l a t i o n type (Jones and Shelton, 1964). A second type of pressure event was recorded from the lungs, and sometimes the buccal cavity, during dives.  This consisted of a low amplitude  f l u c t u a t i o n i n the pressure record which occurred at a frequency  of 40-50  per minute (Figure 23a, b, c ) , and was probably due to the volume changes of the cardiac cycle being transmitted v i a the thoraco-abdominal cavity to the closed buccal cavity and lungs.  This was never noted during periods of a i r  breathing when i t was masked by the pressure changes associated with buccal v e n t i l a t i o n and the i n d i r e c t transmission of these pressure changes to the lungs.  The appearance o f this f l u c t u a t i o n i n the buccal pressure trace  appeared to depend on buccal volume.  I t was frequently observed i n those  dives i n which part of the buccal gas was i n i t i a l l y l o s t through the nares and buccal volume was low, i n which case the volume changes of the cardiac cycle presumably produced s i g n i f i c a n t pressure fluctuations In the buccal cavity. No water could be found on inspection of the buccal and nasal c a v i t i e s of normal frogs on emergence.  Figure 24 i l l u s t r a t e s the response of a bead  thermistor i n the nasal cavity of a frog during immersion i n water 10°C colder than the ambient temperature.  No temperature drop was observed on  89  Figure 24.  Output from a thermistor located i n the n a r i a l c a v i t y . Arrow indicates the point of n a r i a l submergence.  oo  2°C ex.*"  30 sec.  90  n a r i a l submergence, the d e f l e c t i o n of the trace a f t e r submergence being  due  to the release of gas bubbles through the nares. (c)  Denervation experiments Rhythmic e l e c t r i c a l a c t i v i t y associated with v e n t i l a t i o n recorded  the larynx and the posterior intermandibular  from  muscles c eased immediately water  covered the external nares i n normal and sham operated  frogs, while resumption  of v e n t i l a t i o n r a p i d l y followed n a r i a l emergence (Figure 25a).  In the course  of these experiments, during which the frogs underwent r e p e t i t i v e 15-minute dives, evidence was  found for laryngeal c o n s t r i c t o r muscle a c t i v i t y , i n the  form of a spindle of low amplitude muscle spikes occurring immediately a f t e r the burst of a c t i v i t y i n the laryngeal d i l a t o r muscles.  On some occasions  laryngeal c o n s t r i c t o r a c t i v i t y occurred, accompanied by a short burst of a c t i v i t y i n the posterior intermandibular  muscle a few seconds a f t e r  submergence i n both normal and sham operated muscular a c t i v i t y was  frogs (Figure 25b) .  This  associated with an elevation of the buccal f l o o r , and  the release of buccal gas through the nares. B i l a t e r a l s e c t i o n of the ophthalmic branch of the trigeminal nerve at the l e v e l of the nasal cavity resulted i n an a l t e r e d response to submergence and emergence.  Instead of a cessation of e l e c t r i c a l a c t i v i t y , sustained  tonic a c t i v i t y started i n both the laryngeal c o n s t r i c t o r muscles and also i n the posterior intermandibular of the nares (Figure 26a).  muscles one to two seconds a f t e r submergence  This was  accompanied by intense struggling on the  part of frog, and e l e v a t i o n of the f l o o r of the buccal cavity to the roof of the buccal cavity, giving the buccal f l o o r a concave appearance. time a i r was was  At the same  l o s t from the nares, and i n some frogs the mouth gaped and  l o s t from the side of the mouth.  The  tonic e l e c t r i c a l a c t i v i t y  gas  recorded  91  Figure 25.  EMGs recorded from the posterior intermandibular and laryngeal d i l a t o r muscles on submergence and surfacing, a, normal animal; b, sham operated animal.  91a  brynx -ft,  hi  '  1  *h  •>  i  brynx  -4—it»—w<—b.—&  mip  Jj^^^Jd—Jt,—^ I  — t — e *  I  «b  te>  J&*_A<  M  mip  mm*  brynx-  mip  I  25  I  I  lsec.  *  <%  A  & -  92  Figure 26.  EMGs recorded from the posterior intermandibular and laryngeal d i l a t o r muscles. a)  denervated frog.  b)  normal frog,  Arrow indicates n a r i a l submergence.  Arrow indicates i n j e c t i o n of 0.2 ml  water into buccal c a v i t y .  92a  93 from the muscles died down i n one to two minutes, and the frogs became quiescent.  N a r i a l emergence did not i n i t i a t e the resumption of normal r e s p i r a -  t i o n i n these denervated frogs.  Respiration i n two of the f i v e frogs restarted  i n the second and t h i r d minute respectively a f t e r emergence, while i n the remaining three frogs, r e s p i r a t i o n had not resumed by the f i f t h minute.  On  i n v e s t i g a t i o n , 0.02 to 0.1 ml of water was removed by means of a 1 ml hypodermic from the buccal cavity of 4 of the f i v e frogs, while water was found i n the lungs of the three frogs that did not resume r e s p i r a t i o n .  Water  was never found i n the buccal c a v i t i e s of normal frogs a f t e r dives. I n j e c t i o n of 0.2 ml of water into the buccal cavity v i a a cannula i n the tympanic membrane i n normal frogs produced tonic a c t i v i t y i n both sets of muscles and struggling s i m i l a r to that observed i n denervated frogs (Figure 26b), suggesting that this i s a response to the presence of water i n the buccal c a v i t y . (d)  Recordings from the external branch of the ophthalmic nerve. When the external nares were submerged i n water at room temperature,  mechanoreceptor a c t i v i t y i n branches of the ophthalmic nerve serving the skin around the nares was i n i t i a t e d .  A c t i v i t y was greatest during the time when  the water surface was moving over the external nares, and gradually adapted afterwards (Figure 27a).  Emersion of the nares resulted i n a s i m i l a r burst  of spike a c t i v i t y , the units involved being those of large and intermediate spike height (Figure 27b) .  The closeness of the recording s i t e s to the nares  l i m i t e d the depth of water that could be used to 5-6 mm,  and under these  conditions adaptation occurred i n approximately f i v e seconds on submersion, and i n approximately ten seconds on emersion.  Once adaptation had occurred  a f t e r water submersion, a c t i v i t y could be restarted by causing water flow over  94  Figure 27.  Afferent nervous a c t i v i t y i n a n a r i a l branch of the ophthalmic nerve. a)  N a r i a l submersion.  Meniscus s t a r t s to t r a v e l across  external nares at arrow. b)  N a r i a l emmersion.  Meniscus s t a r t s to t r a v e l across  external nares at arrow.  94a  95  the nares.  Figure 28a ( i ) and  two rates of water flow.  ( i i ) i l l u s t r a t e s the response of the nerve to  Water was  delivered from 1 mm  I.D.  1 cm from the external nares which were at a depth of 6 mm. was  tubing positioned The flow rate  0.2 ml per second i n Figure 28a ( i ) and 0.5 ml per second i n Figure 28a  (ii).  In order to simulate the e f f e c t of pressure at greater depths mercury  or mineral o i l was  used.  Under a pressure of 1.4  Figure 28b i l l u s t r a t e s a t r i a l using mercury. cm I^O  the nerve f i r e d at a frequency  a f t e r 15 seconds stimulation and had completely (Figure 28b  (i)) .  of the nerve was  At a simulated depth of 5.6  of 20  Hz  adapted a f t e r 60 seconds cm ^ 0  the adaptation rate  considerably slower, and the nerve s t i l l  frequency of 20 Hz a f t e r 4 minutes stimulation (Figure 28b  f i r e d at an impulse (ii) , ( i i i ) ,  (iv)).  The response obtained when a mineral o i l meniscus moved across the external nares was  generally not as great as the response obtained with a water  meniscus, possibly because of the lower density of the o i l (Figure 29b). The units involved were mechanoreceptors of intermediate spike height (Figure 29a and c ) , which adapted r a p i d l y once the meniscus had r i s e n above the l e v e l of the nares.  Tonic a c t i v i t y i n the nerve had generally increased  over the control l e v e l i n a i r by the time the nares were under 1 cm o i l (0.87 cm ^ 0  pressure), but no further increase i n frequency  applied pressures of 2 and 3 cm h^O  (Figure 30a, b, c and d).  occurred under The  tonically  active f i b r e s were those of small and intermediate spike height. Control recordings of mechanoreceptors serving the cutaneous branches of the second, t h i r d and fourth s p i n a l nerves showed that they responded to suddenly applied steady in  a s i m i l a r way  (rectangular) stimulation of their receptive  to those serving the ophthalmic nerve (Figure 31a, b, c ) ,  but no response to the passage of a water meniscus was preparations.  fields  obtained i n 12  Response to an increase i n hydrostatic pressures to 10 cm  H,0  96  Figure 28.  a.  Response of a n a r i a l branch of the ophthalmic nerve to water flow over the nares a f t e r adaptation to submersion has occurred,  i , 0.2 ml/sec; i i ,  0.5 ml/sec.  Top  trace, event marker, b.  Tonic a c t i v i t y i n a n a r i a l branch of the ophthalmic nerve i n response to pressure applied at the nares. i,  1.4 cm H^O  5.6 cm H 0 2  H 0, o  9  4 min.  pressure, 20 sec a f t e r a p p l i c a t i o n ;  20 sec; i i i ,  5.6 cm H 0, 2  ii,  90 sec; i v , 5.6  cm  96a  l a c  ill  111"'  I  i i mi  Mj i[n  i iniill  i|  liillii | i ! i  MII  ^WP^il^ilPtrtW^ill^^PIBtitliil^lM^MIIrtt^^ipwIflllUW^WIi.M^IIII^  | l«> l| II II lliliiji i |)i)i )i ln |i M i m m K|l  '* )l  4  •> '•' I  *  J$«e.  fciQM  ifi n|ii il t f i I  |  |i|i|nlii|  1,1  »  >»M,II>H^  ni mn  mi i i  iinj  (mi |.ii p..ii|>M H»i>llh.l>| l  l  IHniHi lib | i Hi In I Mil  l  I ) (hi). |H  ti I|I It t iii i i j| 11 il i t i ii iii |i Mn il i |i 111 I|I> nM  97  Figure 29.  a.  Response of a n a r i a l branch of the ophthalmic nerve to punctate stimulation i n a i r , with time i n t e r v a l h i s t o gram of discharge frequency.  b.  Response of same branch to the passage of a mineral o i l meniscus across the nares.  c.  Response to punctate stimulation a f t e r 10 min under 1 cm mineral o i l *  Top  trace  s  event marker; middle t r a c e , neurogram^ bottom  t r a c e , time=  97a  98  Figure 30.  a.  Ongoing a c t i v i t y i n a f i n e n a r i a l branch of the ophthalmic nerve i n a i r .  b.  Tonic a c t i v i t y under 1 cm o i l (S.G. 0.87),.  c.  Tonic a c t i v i t y under 2 cm o i l .  d.  Tonic a c t i v i t y under 3 cm o i l .  98a  T  '"'""nWtTTT\UUUrrTUfTUWfTTUfTUmMTUHIim^  0 25 sec  " milium n  m i II i II  i  IIIIIIIIIIIIIIIIII)  II m i n i m i n m u i i m i n H i  0 25sec  TTmTnTTTTfHi-TfnriirTn.nmMiiiiriTriTrrTiiTniTinirniiMniiiMiifriinfiiiMiiiiiiitiniiitininHiTtriitrni  025 sec  *HM  i  'in  i  '  I  Time  I  0-23 sec  IMI  i  i  I  ,  , „ „  i  |  I,,,  99  Figure 31.  Recordings branch of  of mechanoreceptors the  3rd s p i n a l  serving  the  dorsal  nerve.  a.  Rectangular  punctate  stimulation in  b.  Rectangular  punctate  s t i m u l a t i o n under a  pressure c.  of  10  cutaneous  air. hydrostatic  cm I ^ O .  Few f i b r e p r e p a r a t i o n i n  air.  Top t r a c e , e v e n t m a r k e r ; m i d d l e t r a c e , neurogram; b o t t o m trace,  time.  C-25sec  100 i n 2 cm R^O  increments  followed by a sudden f a l l to atmospheric pressure  limited to a few f i b r e s responding  to the i n i t i a l increment and to the  i n pressure (Figure 32), with no increase i n tonic a c t i v i t y at any  was  fall  pressure  l e v e l , and no response to the water meniscus, (e)  Nerve stimulation B i l a t e r a l stimulation of the ophthalmic nerves at the l e v e l of the o r b i t  with 1-4 msec pulses proved e f f e c t i v e i n i n i t i a t i n g periods of apnoea with a latency of less than 30 msec i n most frogs. of stimulation was  The most e f f e c t i v e  frequency  from 250-500 Hz while the threshold voltage was  30-300 mv.  Frogs In apnoea became very quiescent and v i r t u a l l y no s t r u g g l i n g occurred. The apnoeic periods did not continue i n d e f i n i t e l y .  Short periods of apnoea  of 10 to 20 seconds duration, produced by stimulation at a voltage j u s t above the threshold voltage were terminated by the resumption of v e n t i l a t i o n , even though the stimulation s t i l l continued.  The nares remained open i n these  short periods of apnoea, and buccal pressure remained at atmospheric (Figure 33a and b ) .  A s l i g h t increase i n the stimulating voltage r e s u l t e d i n n a r i a l  closure occurring at the s t a r t of the period of apnoea, buccal pressure being held s l i g h t l y above atmospheric u n t i l the nares opened once more, often midway through the apnoeic period (Figure 33c and d).  Further increase i n  the stimulating voltage produced longer periods of apnoea with closed nares. On n a r i a l closure buccal pressure was  i n i t i a l l y maintained  above atmospheric„  presumably due to tone i n the buccal f l o o r muscles, although i t f e l l slowly through the apnoeic period (Figures 34, 35).  Buccal volume was  low during the  apnoeic periods, the buccal f l o o r being elevated, and often small pressure fluctuations r e f l e c t i n g the heartbeat were present i n the buccal pressure trace (Figures 34,  35).  101  Figure 32.  Response of mechanoreceptors  serving the dorsal cutaneous  branch of the 3rd s p i n a l nerve to hydrostatic pressure.  Top  t r a c e , event marker; middle t r a c e , neurogram;  trace,  time.  bottom  CN  6 0)  102  Figure 33.  B i l a t e r a l e l e c t r i c a l stimulation of the ophthalmic nerves i n the o r b i t . a and b, 30 c, 60 d, 150  200 pps, 4 m sec pulses,  mv.  mv. mv.  Top trace, event marker; middle trace, buccal lower trace, time (sec).  pressure;  102a  103  Figure 34.  B i l a t e r a l e l e c t r i c a l stimulation of the ophthalmic nerves. 300 mv,  200 pps, 4 m sec pulses.  middle trace, buccal pressure;  Top trace, event marker;  lower trace, time (sec).  <u  104  Figure 35.  B i l a t e r a l e l e c t r i c a l stimulation of the ophthalmic nerves. 300 mv,  200 pps,  frequency was  4 m sec pulses.  changed.  trace, buccal pressure;  Top  At arrow stimulating  trace, event marker; middle  lower trace, time (sec).  105 Pressure events resembling underwater lung v e n t i l a t i o n cycles occurred with increasing frequency throughout  the longer periods of apnoea, often  threatening to "break through" the apnoea (Figure 35). not occur during these cycles and i f buccal pressure was never f e l l to the atmospheric  value.  N a r i a l opening did above atmospheric i t  These cycles appeared s i m i l a r i n every  respect to those occurring during submersion. N a r i a l opening and the resumption  of normal v e n t i l a t i o n cycles at the  end of a stimulation period was not immediate (Figure 34) but occurred a f t e r a lag of about 10 seconds.  Even a f t e r periods of apnoea as short as  3-5  minutes the frequency of v e n t i l a t i o n increased greatly over the pre-apnoeic frequency and i n i t i a l l y consisted e n t i r e l y of lung v e n t i l a t i o n cycles. Periods of apnoea of " i n d e f i n i t e " duration were obtained i n 2 frogs. was  Apnoea  considered to be i n d e f i n i t e i f a period of apnoea, broken only by  dive-type v e n t i l a t i o n movements, was  induced for longer than 15 minutes, and  only ended a f t e r the stimulation stopped.  Figure 36 i l l u s t r a t e s one such  t r i a l , which continued f o r 21 minutes before the stimulation was The normal respiratory pattern was  stopped.  regained 2 minutes a f t e r the end of  stimulation. In an attempt to simulate the s i t u a t i o n during a normal dive, where presumably impulse frequency i n the trigeminal nerve peaks on submersion and emersion while adaptation occurs during the dive, the stimulating frequency was  reduced by an order of magnitude shortly a f t e r the i n i t i a t i o n  of apnoea, then b r i e f l y pulsed at the o r i g i n a l frequency j u s t before the end of stimulation i n some t r i a l s .  This method of stimulation proved to be as  e f f e c t i v e i n producing apnoea as maintaining the o r i g i n a l frequency the course of the "dive" (Figure 35).  throughout  E f f o r t s were also made to simulate the  106  Figure 36.  B i l a t e r a l e l e c t r i c a l stimulation of the ophthalmic nerves. 2 V, 300 pps, 2 m sec pulses,  a, 0 min; b, 7 min; c, 21 min.  At arrow stimulating frequency was changed.  Top trace,  event marker; middle trace, buccal pressure; lower trace, time (sec).  106 a  107  Figure 37.  Relationship of time i n apnoea to stimulus voltage.  200 pps,  4 m sec pulses. a.  open bars,, frog i n a i r ; s o l i d bars, submerged to nares.  b.  open bars, b i l a t e r a l ophthalmic stimulation; s o l i d bars,  u n i l a t e r a l stimulation. appropriate.  Standard errors are shown where  107 a  a  ICXH  I  80H  604  4<H  100  4  300  200  400  millivolts  4 0 H  o 41 o  C CL  30H  o  «  2 0 H  10H  100  T  200 millivolts  r 300  108 e f f e c t s of water flow past the external nares by gating the stimulating pulses so that t h e i r frequency varied randomly between 0 and the maximum frequency of stimulation throughout the period of stimulation. however, proved less e f f e c t i v e than the above and was  This method,  discontinued.  In order to t e s t whether s i t e s other than the external nares were important for the i n i t i a t i o n and maintenance of apnoea, the duration of apnoea was  compared i n t r i a l s i n which the frog was  submerged to the l e v e l of the  external nares with that induced when the frog was  completely out of water.  No consistent difference i n the length of the apnoeic period was d i s c e r n i b l e i n the two nerve was  conditions (Figure 37a), although stimulation of one ophthalmic less e f f e c t i v e i n maintaining  (Figure 37b).  apnoea than b i l a t e r a l stimulation  B i l a t e r a l stimulation of the cutaneous branches of the dorsal  branches of the second, t h i r d and fourth s p i n a l nerves performed as a control produced no changes i n the respiratory pattern u n t i l the voltage was to the point where struggling occurred.  increased  B i l a t e r a l stimulation of the  abdominal cutaneous branch of the v e n t r a l branch of the t h i r d s p i n a l nerve at 1-2 v, 250 Hz,  3 msec pulses i n h i b i t e d lung v e n t i l a t i o n cycles i n 2 of the 5  frogs, although they reappeared before the end of the period of stimulation. Sato (1954) produced a s i m i l a r e f f e c t i n Rana nigromaculata by l i g h t l y c l i p p i n g the abdomen*  109  DISCUSSION  The r e s u l t s i n d i c a t e that diving apnoea i n Rana pipiens i s r e f l e x l y i n i t i a t e d by the contact of water with the external nares, and that water does not normally enter the nasal c a v i t i e s or the buccal c a v i t y .  The nares are  closed during a dive, but occasional respiratory movements occur i n some frogs i n which gas i s moved from the lungs to the buccal cavity and back again.  These r e s u l t s agree with Lombroso's (1913) early observations that  apnoea i s induced by contact of the n o s t r i l s with water, and that there i s no intake or exhalation of water during the dive, although movements of the buccal f l o o r may occur at i n t e r v a l s . B i l a t e r a l section of the ophthalmic branch of the trigeminal nerve whose sensory f i b r e s serve the n a r i a l region, resulted i n a f a i l u r e to close the nares on submergence and the entry of water into the buccal c a v i t y , and i n some cases the lungs.  The entry of water into the buccal c a v i t y caused  sustained tonic a c t i v i t y i n the laryngeal c o n s t r i c t o r muscles, the posterior intermandibular muscle of the buccal f l o o r , and probably other buccal f l o o r muscles, as w e l l as struggling on the part of the frog.  Zotterman (1949)  suggested that the tongue water receptor of the frog may " r e f l e x l y contribute i n keeping the mouth of the frog closed as well as to i n h i b i t the respiratory movements when under water".  Although water does not normally enter the mouth  on immersion i t seems l i k e l y that sensory information from tongue water receptors i s responsible f o r the responses observed i n denervated frogs. These appear to be designed  to c l e a r the buccal cavity of water by reducing  buccal volume, and to prevent water entering the airways and lungs by laryngeal c o n s t r i c t o r muscle a c t i v i t y .  110 B i l a t e r a l e l e c t r i c a l stimulation of the ophthalmic branches of the trigeminal nerve caused periods of apnoea i n Rana pipiens.  Stimulation near  the threshold voltage resulted i n b r i e f periods of apnoea during which the external nares remained open and buccal pressure was  at atmospheric.  i n voltage induced longer periods of apnoea with closed nares.  Increase  Electrical,  mechanical or chemical stimulation of the nose were e a r l y known to cause r e f l e x apnoea and bradycardia i n several mammalian species (Brodie and Russel, 1900;  Lombroso, 1913; A l l e n , 1928a).  More recently, Angell James and  Daly  (1972a) produced periods of apnoea of 10 to AO seconds duration i n dogs by drawing tap water or s a l i n e s o l u t i o n over the nasal mucosa.  The l i q u i d had to  be i n motion to i n i t i a t e apnoea, suggesting that information from nasal mechanoreceptors was  of prime importance.  Recordings made from the ophthalmic branch of the trigeminal nerve show that skin mechanoreceptors i n the region of the external nares are able to respond to the movement of a water meniscus across t h e i r receptive f i e l d s i n a simulated dive.  Adaptation to pressures of 5 to 6 cm H^O  occurred i n a matter  of minutes, but once adapted the mechanoreceptors s t i l l responded to water flow.  Gregory (1973), working on ducks, could f i n d no response from beak  mechanoreceptors served by the ophthalmic nerve to simulated d i v i n g .  In the  units he investigated there was no response even when the hydrostatic pressure at the surface of the beak was  raised and lowered between 0 and 50 cm  although he points out that they may present.  1^0,  not have been the most s e n s i t i v e units  The units involved i n the frog appear to be s i m i l a r to Catton's  (1958) type a and b f i b r e s , producing r e s p e c t i v e l y large fast adapting spikes, and smaller r e l a t i v e l y slowly adapting spikes with a lower threshold. According to Catton (1958), these spikes are propagated i n myelinated while the receptors appear to be free nerve endings.  fibres,  The snouts of two frogs  Ill were s e r i a l l y sectioned, and stained i n Glee's s i l v e r s t a i n , but no  specialised  endings could be found i n the region of the external nares. In some preparations studied, complete adaptation to a stimulus of a few cm R^O  occurred i n a matter of minutes, while i n others a pressure of less  than 1 cm H^O  produced tonic a c t i v i t y , although the frequency did not increase  with increased pressure.  If trigeminal input i s an important  factor i n  i n h i b i t i n g rhythmic a c t i v i t y i n the respiratory centre during immersion, overriding chemoreceptive input for example, i t i s probably throughout the dive. hour or more?  How  maintained  then do frogs maintain apnoea i n dives l a s t i n g an  I t i s f e a s i b l e that i n the f i e l d movement of the frog, or  currents i n the body of water could bring about continuing spike a c t i v i t y i n response to flow.  Furthermore increase i n pressure, due to deep d i v i n g would  provide a greater stimulus i n t e n s i t y than those investigated, possibly slowing adaptation and causing the recruitment of less s e n s i t i v e u n i t s .  However,  frogs secured on boards and submerged i n a few centimetres of water f o r periods of an hour maintain apnoea and spontaneously (Jones, 1967).  resume v e n t i l a t i o n when emerged  It i s possible that the mechanoreceptors could become s e n s i t i s -  ed during the course of a free dive, enhancing the a f f e r e n t input to the C.N.S.  I t has been demonstrated i n Rana pipiens that stimulation of the f i r s t  sympathetic  ganglion r e s u l t s i n a sympathetic  e f f e r e n t response i n cutaneous  branches of the trigeminal nerve which e l i c i t s an a f f e r e n t mechanoreceptor discharge (Chernetski, 1964a).  In cats, i n j e c t i o n of strychnine into the s p i n a l  trigeminal nucleus makes head regions hypersensitive to touch (King and Barnett, 1957).  The adjustment of mechanoreceptor e x c i t a b i l i t y appears to be under  sympathetic  adrenergic c o n t r o l , a p p l i c a t i o n of epinephrine to Pacinian  corpuscles increasing the amplitude  and r i s e rate of the generator p o t e n t i a l  112 (Lowenstein  and Altamirano-Orrego,  1956).  The p o s s i b i l i t y of such a sympa-  thetic enhancement of trigeminal receptor input on immersion does not seem unreasonable i n frogs, where there are close morphological the c r a n i a l nerves and the sympathetic  r e l a t i o n s between  system (Chernetski, 1964b), although i t  was not observable i n the double-pithed preparations used i n these experiments. Uyperpnoea occurred a f t e r short dives and also a f t e r short periods of apnoea induced by trigeminal stimulation.  In mammals hyperpnoea i s probably  a response to the increase i n a r t e r i a l P  which occurs i n the apnoeic 2  period (Angell James and Daly, 1969), while i n the frog the f a l l i n blood pressure which occurs during long periods of submersion may stimulate hyperpnoea on surfacing (Jones, 1967).  also help  De Marneffe-Foulon  (1962)  found that endopulmonary pressure can c o n t r o l the v e n t i l a t i o n rate i n the frog, a f a l l i n pressure stimulating v e n t i l a t i o n , and i t has been suggested this may to  that  explain post-dive hyperpnoea i f lung pressure f a l l s on submersion due  the release of gas bubbles at the s t a r t of the dive (Jones, 1966).  However, lung pressure i n v a r i a b l y r i s e s when a frog i s submerged even though lung volume may  be low, the lungs acting as simple closed hydrostats.  It  seems more l i k e l y then that the increase i n v e n t i l a t o r y drive a f t e r short periods of apnoea i s due to the e f f e c t of the absence of rhythmic input from lung mechanoreceptors during the apnoeic period, and to the d i r e c t influence of  the release of trigeminal i n h i b i t i o n on the r e s p i r a t o r y centre, although  nothing i s known of the central mechanisms involved.  113 GENERAL D I S C U S S I O N  Although aquatic  i t appears  mode o f l i f e  a force-pump  that  modern Anurans have p r o b a b l y  than t h e i r  Palaezoic  ancestors  for a i r v e n t i l a t i o n apparently  (Foxon,  arose early  reverted  to a  1964),  use of  i n phylogeny:  more  The  modern l u n g f i s h Protopterus a e t h i o p i c u s f o l l o w s a s i m i l a r sequence d u r i n g a i r breathing into  i n which the buccal  the buccal  c a v i t y expands  cavity before  the lungs  buccal  gas i s  then forced  into  refill  them.  I t has been  suggested  cavity could serve  to clear  at  are deflated  the l u n g s by b u c c a l that  i t of water  preliminary i n f l a t i o n of the buccal specific  and a i r e n t e r s  Gans perfected  (McMahon, 1969) .  i n lung f i s h ,  (1970)  considered  i n frogs  i n order  the v o c a l cords  c a v i t y c o u l d compensate  that  the buccal  that  v o c a l i z a t i o n , an important  organisation,  pump.  but  habitat.  their  developed r i b s ,  which c o u l d have been  t o an a q u a t i c  habit,  seems,  1972).  such  however,  b y a n a s p i r a t i o n pump  to t h e i r  E a r l y a m p h i b i a w e r e p r e s u m a b l y more  reversion  (Romer,  is  i n modern  arguing that  It  explanation  may b e r e l a t e d  factor  f o r the r e t e n t i o n reversion  terrestrial  of  to an  i n habit,  used i n s u c t i o n - p u m p i n g  and the subsequent  reduction i n rib  s i z e may h a v e l e d t o t h e r e n e w e d d o m i n a n c e o f t h e b u c c a l pump i n m o d e r n forms  in  f o r c e - p u m p was r e t a i n e d a n d  be f a c i l i t a t e d ,  A more l i k e l y  t h e b u c c a l pump i n m o d e m a n u r a n s  showing w e l l  f o r changes  that  s i n k i n g when t h e a n i m a l  c o u l d be v i b r a t e d as e f f i c i e n t l y  by a b u c c a l p r e s s u r e  aquatic  buccal  or a l t e r n a t i v e l y ,  an i m p o r t a n t mechanism i s u n l i k e l y t o be r e s t r u c t u r e d .  as  the  to  surface.  anuran b e h a v i o u r and s o c i a l  that  Mixed  force-pumping i n order  a s p i r a t i o n of a i r into  g r a v i t y o n l u n g d e f l a t i o n and so p r e v e n t  the water  t h r o u g h t h e mouth  114  The need for apnoea to be r e l i a b l y induced as the frog i s obvious.  i n a diving vertebrate such  Many mammalian physiologists have deduced a voluntary  component i n the responses to submergence (Irving e_t a l , ' 1941) , but this i s hardly l i k e l y to be true for the Amphibia, and recent work has suggested that even i n the harbor seal the responses may i n fact be conditioned (Jones et a l , 1973).  Angell James and Daly  (1972b) noted that receptors situated i n  the nasal passages themselves are hardly l i k e l y to be involved i n the respiratory and cardiovascular adjustments to diving i n the s e a l , as the s e a l , l i k e the frog, closes i t s n o s t r i l s on submergence.  Jones et^ a l  (1973) pointed out that there i s nothing i n the l i t e r a t u r e to suggest that the s e a l i n fact possesses such receptors, although  the recent studies of  Dykes (pers. comm.) suggest that mechanoreceptors on the skin of the head and p a r t i c u l a r l y at the base of the vibrassae may have a r o l e to play, p a r t i c u l a r l y i n the cardiovascular responses to submersion.  Although i t i s c l e a r that  stimulation of trigeminal mechanoreceptors can bring about apnoea i n Rana pipiens i t i s u n l i k e l y that there i s a d i r e c t chronotropic e f f e c t on the heart from peripheral receptors, as the generation of diving bradycardia i s a slow process i n the frog, the two main factors i n f l u e n c i n g heart rate during periods of submergence being the shortage of oxygen and the cessation of v e n t i l a t i o n (Jones and Shelton, 1964). Jones (1967) demonstrated that to evaluate the true oxygen debt at the end of a dive i t i s necessary  to know the oxygen cost of v e n t i l a t i o n i n the  frog, because hyperpnoea follows periods of submergence i n anurans, and the work output of the buccal pump must be above the pre-dive l e v e l , the increased oxygen consumption of the pump being included i n the increase i n oxygen consumption which follows a dive.  In R. pipiens lung v e n t i l a t i o n rate was  115  double the pre-dive rate i n the f i r s t ten minutes of a 60 min dive i n aerated water, while the t o t a l oxygen consumption was  26% more than pre-dive.  Assuming that the oxygen coat of v e n t i l a t i o n pre-dive Is 5% of the r e s t i n g metabolism (Part I I ) , the combination of switching to high amplitude lung v e n t i l a t i o n cycles requiring perhaps double the energy input per cycle, and the increased respiratory frequency  could w e l l cause the oxygen cost of  hyperventilation to r i s e to 15-20% of the t o t a l pre-dive oxygen consumption. In Rana pipiens no oxygen debt appears to b u i l d up during a dive, as much extra oxygen being consumed on emergence from 100% oxygenated water as on emergence from aerated water.  In both cases hyperpnoea occurs a f t e r the  dive and i t appears f e a s i b l e that the cost of hyperpnoea could be a major factor i n the extra post-dive oxygen consumption shown by frogs.  116 SUMMARY  1.  The lungs are v e n t i l a t e d i n Rana pipiens by means of buccal force  pump mechanism.  Pressure i n the buccal cavity fluctuated around  atmospheric,  but lung pressure was never allowed to f a l l to atmospheric pressure. 2.  Two d i s t i n c t types of pressure events occurred i n the buccal cavity;  buccal v e n t i l a t i o n cycles i n which the g l o t t i s remained closed and the external  nares were open, and lung v e n t i l a t i o n cycles which involved the sequential  p a r t i c i p a t i o n of the nares and g l o t t i s as w e l l as the r e s p i r a t o r y muscles of the buccal f l o o r .  No evidence was found f o r a s u b s t a n t i a l d i f f e r e n t i a l  pressure across the g l o t t i s , or f o r the a c t i v e p a r t i c i p a t i o n of the flank muscles during lung v e n t i l a t i o n c y c l e s . 3.  Lung and buccal v e n t i l a t i o n cycles were not randomly interspersed.  Lung v e n t i l a t i o n cycles occurred i n regular sequences towards the end of which each cycle reached a pressure peak some 10-20 percent higher than the preceding  cycle, u n t i l the lungs were f u l l y i n f l a t e d .  These sequences were  separated by periods during which the f i l l e d lungs were i s o l a t e d from the buccal cavity by the closed g l o t t i s . During these periods only buccal v e n t i l a t i o n cycles occurred.  Lung d e f l a t i o n was accomplished  during the f i r s t few  cycles of the subsequent lung v e n t i l a t i o n sequence. 4.  Gas flow recorded at the nares was found to be biphasic during buccal  v e n t i l a t i o n c y c l e s , but to consist of 4 phases during lung v e n t i l a t i o n c y c l e s . 5.  The suggestion i s made that the i n i t i a l emptying o f lung gas i n t o the  buccal cavity on lung v e n t i l a t i o n and the r e a s p i r a t i o n of mixed gas may be s i g n i f i c a n t i n the maintenance of blood P ^  i n Rana p i p i e n s .  117 6.  An analysis was made of the mechanical work done by the buccal pump  during one lung v e n t i l a t i o n cycle, and the proportion of this work a v a i l a b l e for i n f l a t i o n of the lungs a f t e r various losses against viscous and flow r e s i s t i v e forces i n the pump i t s e l f . 7.  The mechanical work of v e n t i l a t i o n was estimated i n restrained but  unanaesthetised Rana pipiens by recording the areas of representative sequences of pressure/volume  loops recorded from the buccal pump, together  with the r e s p i r a t i o n frequency.  Mechanical work of v e n t i l a t i o n proved to be  0.5 Joules per hour f o r frogs with a mean weight of 64 grams. 8.  The mean mechanical e f f i c i e n c y of the buccal pump was  10.6 percent.  calculated at  The calculated e f f i c i e n c i e s of i n d i v i d u a l lung v e n t i l a t i o n  cycles increased as the mechanical work done i n a cycle increased from percent at 0.65  gram.cm per cycle to 19.3 percent at 2.73  a f t e r which e f f i c i e n c y 9.  By combining  7.4  gram.cm per cycle,  fell. data on the mechanical work of v e n t i l a t i o n and e f f i c i e n -  cy with data i n the l i t e r a t u r e on the oxygen consumption of Rana pipiens i t was possible to estimate the oxygen cost of v e n t i l a t i o n at 5 percent. respiratory muscles make up 0.92 t i o n of these muscles, V0~, was  percent of body weight.  The  The oxygen consump-  calculated at 0.89 ml 0- per 100 gram per  minute. 10.  Upon submersion,  apnoea i n Rana pipiens was not induced u n t i l the  water surface had reached the l e v e l of the external nares.  During the period  of submersion the nares were closed and buccal pressure was  elevated due to  the hydrostatic pressure of the head of water above the buccal cavity. period of apnoea was  The  punctuated In some frogs by v e n t i l a t i o n movements i n  which lung gas entered the buccal c a v i t y .  The nares remained closed. V e n t i -  l a t i o n spontaneously restarted at the end of a period of submersion as soon as  118 the water l e v e l f e l l below the external nares. 11.  Water did not normally enter the buccal cavity during periods of  submersion, and there was no tonic e l e c t r i c a l a c t i v i t y i n the buccal f l o o r muscles during the dive.  Denervation of the region of the external nares,  by b i l a t e r a l s e c t i o n of the ophthalmic branch of the trigeminal nerve ( c r a n i a l nerve V) resulted i n intake of water into the buccal cavity and i n some cases the lungs, together with intense tonic a c t i v i t y i n the muscles of the buccal f l o o r , which elevated i t towards the roof o f the buccal c a v i t y .  Surfacing  did not r e s u l t i n resumption of v e n t i l a t i o n i n these frogs. 12.  Mechanoreceptors i n the region of the external nares served by the  ophthalmic branch of the trigeminal were found to be capable of responding to the movement of a water meniscus across the snout.  Adaptation to pressures  of 6 cm H^O occurred i n a matter of 4-5 minutes i n some preparations, while i n others a long term increase i n tonic a c t i v i t y occurred.  Mechanoreceptors  s t i l l responded to water flow a f t e r complete adaptation to pressure had occurred. 13.  B i l a t e r a l stimulation of the ophthalmic branch at the l e v e l of the  o r b i t caused periods of apnoea i n Rana pipiens.  Thresholds varied from 30 to  300 mv. at 200 p.p.s., 4 msec, pulses, i n i n d i v i d u a l frogs.  At voltages near  the threshold the apnoeic periods occurred with the nares open.  Increase i n  voltage resulted i n longer periods of apnoea i n which the nares were closed and buccal pressure was independent of atmospheric pressure.  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