UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Gill blood flow in teleosts Farrell, Anthony Peter 1979

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1979_A1 F37_7.pdf [ 21.3MB ]
Metadata
JSON: 831-1.0094751.json
JSON-LD: 831-1.0094751-ld.json
RDF/XML (Pretty): 831-1.0094751-rdf.xml
RDF/JSON: 831-1.0094751-rdf.json
Turtle: 831-1.0094751-turtle.txt
N-Triples: 831-1.0094751-rdf-ntriples.txt
Original Record: 831-1.0094751-source.json
Full Text
831-1.0094751-fulltext.txt
Citation
831-1.0094751.ris

Full Text

GILL BLOOD FLOW IN TELEOSTS by ANTHONY PETER FARRELL B.Sc, University of Bath, 1974 THESIS SUBMITTED IN PARTIAL FULFILMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE. FACULTY OF GRADUATE STUDIES (Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1979 •(c) Anthony Peter F a r r e l l , 1979 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying pf this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D E - 6 B P 75-51 1 E ABSTRACT U n l i k e the r e s p i r a t o r y organs of a i r b r e a t h i n g v e r t e -b r a t e s where gas exchange i s p e r f u s i o n l i m i t e d , gas t r a n s f e r a c r o s s f i s h g i l l s i s d i f f u s i o n l i m i t e d . F i s h can t h e r e f o r e enhance gas exchange by i n c r e a s i n g the g i l l d i f f u s i n g c a p a c i t y . P r e v i o u s suggestions, i n d i c a t e t h a t f i s h may achieve t h i s by a l t e r i n g the p a t t e r n of g i l l b lood flow t o i n c r e a s e the area of g i l l p e r f u s e d and to reduce the blood-water d i f f u s i o n b a r r i e r . -* To v e r i f y these suggestions an i n v e s t i g a t i o n of the p a t t e r n s of g i l l blood flow, t h e i r r e g u l a t i o n and t h e i r s i g n i f i c a n c e i n gas exchange i n the l i n g cod, Ophiodori elongatus, was undertaken. The c i r c u l a t o r y arrangement i n the g i l l f i l a m e n t of the l i n g cod c o n s i s t s of an a r t e r i o - a r t e r i a l r e s p i r a t o r y network and an a r t e r i p - v e n o u s veriolymphatic system. A l l c a r d i a c output passes through the r e s p i r a t o r y exchange s i t e s , the l a m e l l a e . Blood flow through l a m e l l a e i s d e s c r i b e d by sheet blood flow equations, where flow i s p r o p o r t i o n a l t o the v a s c u l a r sheet t h i c k n e s s (h). The l a m e l l a r v a s c u l a r sheet i s very compliant .and h i n c r e a s e s w i t h transmural p r e s s u r e (AP-, ) . I t i s p r e d i c t -^ . lam ^ ed t h a t i f AP, and flow are r a i s e d , then i n t r a l a m e l l a r shunt-lam in g o f blood flow and a r e d u c t i o n of the blood-water b a r r i e r w i l l r e s u l t , thereby i n c r e a s i n g the g i l l d i f f u s i n g c a p a c i t y ; Not a l l l a m e l l a e appear to be e q u a l l y p e r f u s e d under c e r t a i n i n v i v o c o n d i t i o n s . Furthermore, i f r e s t i n g p e r f u s i o n - i i - . c o n d i t i o n s are simulated, only 67% of the more proximal lamellae are perfused. Thus the t o t a l g i l l area i s not u t i l i s e d at r e s t . To account f o r t h i s s i t u a t i o n i t i s proposed t h a t the c r i t i c a l c l o s i n g pressures a s s o c i a t e d w i t h d i s t a l l a m e l l a r u n i t s are greater than those f o r the proximal lamellae. The a f f e r e n t a r t e r i o l e s were determined to be the major r e s i s t a n c e s i t e i n the g i l l s and they t h e r e f o r e c o n t r o l flow to la m e l l a e . E l e v a t i o n s i n flow and l a m e l l a r input pressure w i l l reduce the l i k e l i h o o d of' c r i t i c a l c l o s i n g and more lamellae w i l l be perfused. Lamellar recruitment increases the g i l l d i f f u s i n g c a p a c i t y . , The demonstrated changes i n flow patterns to and w i t h i n lamellae are e f f e c t e d by elevated flow and input pressures Cor AP^ )• Changes i n c a r d i a c performance and i n the pressure p r o f i l e of the g i l l s a l t e r f l o w and pressure. Cardiac performance i n l i n g cod i s i n f l u e n c e d by i n t r i n s i c , c h o l i n e r g i c and adrenergic c o n t r o l s which a l t e r stroke volume and heart r a t e . The pressure p r o f i l e of the g i l l s can be a l t e r e d by c h o l i h e r g i c a l l y or adre-n e r g i c a l l y mediated changes i n v e s s e l dimensions. The g i l l out-flow a r t e r i e s v a s o c o n s t r i c t i n l o c a l i s e d regions w i t h c h o l i n e r g i c . s t i m u l a t i o n and thereby i n c r e a s i n g g i l l r e s i s t a n c e (Rg) and lamellar' input pressures. A f f e r e n t v e s s e l s apparently d i l a t e with-g-adrenergic s t i m u l a t i o n and thereby lower Rg. Cardiovascular changes are as s o c i a t e d w i t h c o n d i t i o n s of reduced oxygen a v a i l a b i l i t y (hypoxia,) and of increased oxygen demand (s t r u g g l i n g ) i n l i n g cod. The c a r d i o v a s c u l a r changes are such that they a l t e r the p a t t e r n of g i l l blood flow and increase the g i l l d i f f u s i n g c a p a c i t y . Increased oxygen uptake, c a r d i a c output and g i l l v e n t i l a t i o n are a s s o c i a t e d w i t h hypoxia and s t r u g g l i n g . The q u a n t i t a t i v e ' i n c r e a s e s i n c a r d i a c output per se as s o c i a t e d w i t h these c o n d i t i o n s does not f u l l y account f o r the observed increase i n oxygen uptake. I t i s concluded t h a t change i n g i l l d i f f u s i n g c a p a c i t y through a l t e r a t i o n s i n g i l l blood flow patterns are important i n enhancing oxygen uptake across the g i l l s . - i v -•TABLE OF CONTENTS PAGE GENERAL INTRODUCTION ' 1 GENERAL MATERIALS AND METHODS . 6 SECTION I : The morphology and v a s c u l a r pathways of the. g i l l i n l i n g cod. INTRODUCTION . "' ' 16 MATERIALS AND METHODS 21 RESULTS . 3 0 DISCUSSION 74 SECTION II : An examination of g i l l blood flow c h a r a c t e r i s t i c s and g i l l r e s i s t a n c e . and how they change i n v i t r o and i n v i v o . INTRODUCTION 94 MATERIALS AND METHODS 101 RESULTS 108 DISCUSSION 134 SECTION I I I : An examination of the v a s c u l a r r e s i s t a n c e and compliance as they a f f e c t g i l l blood flow using iri v i t r o p r e p a r a t i o n s . INTRODUCTION 151 MATERIALS AND METHODS 155 RESULTS 163 DISCUSSION 176 - v -SECTION IV : A study of g i l l blood flow and i t s r e g u l a t i o n i n Ophiodon,elongatus i n  v i v o . INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION GENERAL DISCUSSION BIBLIOGRAPHY APPENDICIES BIOGRAPHY - v i -ACKNOWLEDGEMENTS . During the course of t h i s work I r e c e i v e d help, advice, encouragement and t e c h n i c a l a s s i s t a n c e from many people. I am g r a t e f u l . I would l i k e to acknowledge the f o l l o w i n g i n p a r t i c u l a r ; Dr.,David R a n d a l l , f o r g i v i n g me the o p p o r t u n i t y to perform t h i s research and f o r p r o v i d i n g s u b t l e guidance throughout my endeavours. Dr. David Jones, whose t e n a c i t y always provided me with an i n c e n t i v e . Dr. Sidney Sobin, f o r the use of the f a c i l i t i e s i n h i s l a b o r a t o r y i n Los Angeles. Dr. David Smith, f o r h i s a s s i s t a n c e i n some of the micropressure experiments. Charles Daxboeck, f o r h i s a s s i s t a n c e i n the oxygen uptake determinations and g i l l p e r f u s i o n s t u d i e s . C o l i n Parkinson, Fergus O'Hara, L a s l o Veto and Susin Crosby of the U.B.C. Zoology Department f o r t h e i r t e c h n i c a l ass i s t a n c e . Gerry Bance and Paul N i t i s h i n of the U n i v e r s i t y of New Brunswick B i o l o g y Department, f o r t h e i r t e c h n i c a l a s s i s t a n c e . Dr. C o l i n M a i l e r of the U n i v e r s i t y of New Brunswick Physics Department, f o r . h i s . h e l p i n developing the equation to d e s c r i b e r e s i s t a n c e i n the a f f e r e n t f i l a m e n t a r t e r y . - v i i -Susan H a r r i s o n , f o r her assistance i n developing and running the programs f o r the POP I I . A l l the post-graduates and post-doctorates i n D.J.R.'s l a b o r a t o r y , f o r t h e i r f r i e n d s h i p and support, as w e l l as i n t e l l e c t u a l interchange. Tony Harmen, f o r the many di s c u s s i o n s with him on the p r o p e r t i e s of blood v e s s e l s . Anna MacDonald, for her typing s k i l l s i n preparing t h i s manuscript. , Miriam, f o r her patience during the w r i t i n g of t h i s t h e s i s . I was supported at U.B.C. by a U.B.C. graduate f e l l o w s h i p and teaching a s s i s t a n t s h i p s . I was able^ to complete ...» -my w r i t i n g while I was a l e c t u r e r i n the U.N.B. Biology Department. The equipment f o r my research was supplied through N.R.C. operating grants to D.J.R. V GENERAL INTRODUCTION - 1 -. In recent years patterns'of blood flow through the g i l l filament and lamellae in teleost f i s h has received some attention. A'number of these studies base their conclusions completely or l a r g e l y on the anatomical arrangements' of blood vessels (e.g. Mott, 1950; Steen and Kruysse, 1964; Hughes and Grimstone, 1965; Richards and Fromm, 1969; Morgan and To v e l l , 1973). Such studies have been valuable, but confusion over preqise vascular arrangements has invalidated some of their conclusions. Furthermore, they generally lacked any consideration of the physical properties of blood vessels, for which considerable l i t e r a t u r e now exists (Bergel, 1972; Attinger and Attinger, 1973, Kaley and Altura, 1977). Blood vessels are not simple r i g i d tubes especially when conducting p u l s a t i l e blood flow. They have a compliance which is dependent in part on the radius/wall thickness r a t i o which may vary with absolute blood pressure and pulse frequency. Thus the resistance of a vessel w i l l vary depending on the conditions of the input anc3 output flow. For instance, vessels w i l l collapse at above ambient pressures, t h e i r ' c r i t i c a l closure pressure', (Burton, 1951; Nicol, et a l . , 1951). The resistance and compliance of vessels are largely passive properties, but they can be influenced neurally or humorally, e.g. adrenergic and/or cholinergic actions. The actions may be tonic e f f e c t s , i . e . to enable the vessel to maintain a given diameter over a range of pressures, or be directed at s p e c i f i c s i t e s e.g. sphincters to control blood pressure and flow to certain regions.. To evaluate the r e l a t i v e importance of these p a s s i v e and a c t i v e v a s c u l a r changes i n f o r m a t i o n on v a s c u l a r i n n e r v a t i o n i s necessary. With the e x c e p t i o n of Gannon's (1972) ex t e n s i v e work on the a d r e n e r g i c i n n e r v a t i o n of t r o u t heart and g i l l s , , t h i s i n f o r m a t i o n is' l a c k i n g . I t i s t h e r e f o r e not s u r p r i s i n g that previous d e s c r i p t i o n s of g i l l blood flow based on anatomy alone- are somewhat l i m i t e d . P h y s i o l o g i c a l s t u d i e s of g i l l blood flow have leaned h e a v i l y on i n v i t r o p e r f u s i o n s t u d i e s (See l a t e r f o r numerous r e f e r e n c e s ) , which date back to the Keys and Bateman (1932) perfused h e a r t - g i l l p r e p a r a t i o n . At best i t i s d i f f i c u l t to mimic i_n v i v o c o n d i t i o n s i n y i j t r o . We a l s o l a c k a d e t a i l e d knowledge of the i n d i v i d u a l importance that each of the vario.us i n vit-ro p e r f u s i o n c o n d i t i o n s has upon p a t t e r n s of flow through the g i l l . Once we e s t a b l i s h t h e i r importance, i t i s l i k e l y that a number of c o n c l u s i o n s from previous i n v i t r o work w i l l need r e i n t e r p r e t a t i p n because of n e g l e c t surrounding the s e l e c t i o n ' of p e r f u s i o n c o n d i t i o n s . In v i v o i n f o r m a t i o n of g i l l blood flow i s not v a s t . Other than the s t u d i e s of Davis (1972) and Booth (1978), which e s t a b l i s h e d that only a p o r t i o n of a l l g i l l l a m ellae were perfused i n t r o u t at r e s t , there has been no study d i r e c t e d towards e s t a b l i s h i n g p a t t e r n s of blood flow through the g i l l . C e r t a i n d e t a i l e d _in v i v o i n v e s t i g a t i o n s of c a r d i o v a s c u l a r f u n c t i o n have been h e l p f u l i n a s c e r t a i n i n g some c h a r a c t e r i s t i c s of pre- and p o s t - b r a n c h i a l blood flow (Jones e t al_. , 1974; Stevens e t aJL. , 1972). Many other i n v i v o s t u d i e s have measured blood p r e s s u r e s and heart r a t e i n t e l e o s t s , but Stevens and Randall (1967b:), Holeton and Randall (1967b), and Kiceniuk and Jones (1977) on l y have estimated values f o r c a r d i a c output f o r r e s t i n g t e l e o s t s . Randall e t a l . (1967) noted at that time t h a t the data on c i r c u l a t i o n and r e s p i r a t i o n i n f i s h were fragmentary; the same might now be s a i d f o r the i n f o r m a t i o n on s p e c i f i c blood-flow p a t t e r n s i n the g i l l s . Why are p a t t e r n s of g i l l blood flow so i n t e r e s t i n g ? Saunders' (1962) observed that i n s e v e r a l t e l e o s t s that O2 uptake ( M 0 2 ) / p r e d i c t e d from t o t a l g i l l area, was nearer to the el e v a t e d l e v e l s measured during swimming r a t h e r than those measured at r e s t . T h i s i s s u r p r i s i n g , s i n c e O2 t r a n s f e r , at the g i l l s i s considered to be a pa s s i v e p r o c e s s . Steen and Kruysse (1964) attempted to e x p l a i n the f i n d i n g s i n terms of anatomical shunts which they developed from Ri e s s ' (1881) o r i g i n a l concept of two blood pathways i n g i l l f i l a m e n t s . . T h e i r choice 'of eel's as an experimental f i s h was unfortunate to e x p l a i n the f i n d i n g s of Saunders s i n c e the e e l g i l l : v a s c u l a t u r e d i f f e r s in a very. important r e s p e c t from the t r o u t and many other f i s h . A l l c a r d i a c output goes through the g i l l s i n t r o u t (Gannon et a l . , 1973) u n l i k e i n e e l s where blood can enter n o n - r e s p i r a t o r y channels and by-pass the l a m e l l a e . As a consequence, a r t e r i a l blood i n e e l s i s f u l l y s a t u r a t e d only during e x e r c i s e , w h i l s t a r t e r i a l blood i n t r o u t i s f u l l y oxygenated at r e s t (Randall e t a l . , 1967). C l e a r l y the e x p l a n a t i o n of Steen and Kruysse cannot be a p p l i e d to the f i n d i n g s of Saunders. Steen and Kruysse, however, made the important observation that alterations in blood flow can a l t e r M 0 2 in eels. In trout the pattern of . g i l l blood flow can change. Only 60% of a l l lamellae are perfused at rest (Booth, 1978); a s i t u a t i o n predicted by Randall et al. (1967) and Morgan and Tovel (1973). Perfusion of more lamellae would increase the surface area for gaseous exchange, which could increase the rate of oxygen transfer. Trout, however, increase M 0 2 by 5 to 10 times during exercise (Saunders, 1962; Stevens and Randall, 1967a; Kiceni.uk and Jones, 1977) and thus lamellar recruitment from 60% to even 100% cannot f u l l y account for this change. Thus Randall et a l . (1967) have suggested that-increased blood volume in.the g i l l respiratory c i r c u l a t i o n , shunting of blood closer to the surface of the g i l l s and a decrease in the thickness of lamellae may a l l be important in a l t e r i n g Mo2• There have also been postulates that flow changes within individual lamellae (intralamellar shunting) (Hughes and . Grimstone, 1965). It i s , therefore, clear.that various patterns of g i l l blood flow must occur and they have fundamental effects on the. rate of M 0 2 in teleosts. However the s p e c i f i c nature of these changes and. t h e i r control is largely - unknown. Thus I w i l l examine various g i l l blood flow patterns, indicating how they are affected or effected by adrenergic, cholinergic or passive mechanisms and suggesting how they may e f f e c t O2 exchange.. This study approached the problem from physiological and morphological standpoints using a number of recently developed techniques in addition to established ones. The experimental f i s h was Ophiodon elongatus (ling cod) because of i t s s u i t a b i l i t y for the in vivo surgery. Nothing is known of the vascular pathways in this f i s h , thus, in Section I, there were examined using h i s t o l o g i c a l and morphological thchniques and 3-D observations of p l a s t i c vascular corrosion casts (Gannon et. a l . , 1973). I used equations to describe the lamellar blood flow after f i r s t determining the vascular dimensions and the passive properties of the vessels with respect to pressure. I also predicted the major g i l l resistance s i t e s and possible flow control s i t e s from the vascular geometry and then tested these predictions using blood pressure measurements in the g i l l filament. I also established the importance of active (humoral or neural), cardiovascular controls in c o n t r o l l i n g g i l l blood flow using in v i t r o and in vivo experiments. The passive properties of the g i l l vessels and how they influence the number of lamellae perfused were examined. In the f i n a l Sect ion,.'the cardiovascular responses of the f i s h to reduced oxygen demand, hypoxia, and to increased oxygen requirements, struggling, were examined and interpreted based on the previous observations and conclusions. The f i n a l outcome was a more complete analysis of blood flow in a teleost which enabled a description of branchial blood flow patterns and their probable mode of regulation. GENERAL MATERIALS AND METHODS Fish supply and holding f a c i l i t i e s S a l i n e s Routine surgery , Experimental aquaria I s o l a t e d perfused holobranch preparation Recording systems In v i v o monitoring - 6 -F i s h supply and h o l d i n g f a c i l i t i e s L ing cod, Ophiodon e l o n g a t u s , were caught by l o c a l fishermen, t r a n s p o r t e d to U.B.C. and held i n l a r g e a q uaria s u p p l i e d with f l o w i n g , aerated water. The water temperature was maintained between 9 and 11°C. The f i s h were fed a maintenance d i e t of l i v e rainbow t r o u t , but were st a r v e d f o r at l e a s t s e v e r a l days p r i o r to an experiment. A t o t a l o f 69 l i n g cod were used i n these experiments with weights ranging from 2.5 to 6.5 kg (average weight =-4.6 kg). Rainbow t r o u t , Salmo g a i r d n e r i , (300 to 600 g) were used i n one s e r i e s of experiments. They were obtained from a l o c a l f i s h hatchery and held i n l a r g e a q u a r i a s u p p l i e d with f l o w i n g , a e r a t e d , d e c h l o r i n a t e d Vancouver tap water a t U.B.C. The water temperature f l u c t u a t e d between 7 and 12°C depending on the season. The t r o u t were fed d a i l y with commercial t r o u t p e l l e t s . A l l experiments were performed at water temperatures between 10 and 11°C. S a l i n e s C o r t l a n d s a l i n e s f o r s a l t w a t e r and freshwater f i s h . (Wolf, 1963) were used i n a l l experiments. The composition i n g . l ~ l of the freshwater f i s h s a l i n e was: 7.25 g NaCl; 0.38g KCl; 0.162 g C a C l 2 ; 0.23 g MgSO 4.7H 20; 1.0 g NaHC0 3; 0.4 g NaH 2P0 4.2H 20 (or 0.36 g NaH 2P0 4.H 20); 1.0 g gl u c o s e . The s a l i n e f o r s a l t w a t e r f i s h c o n t a i n s an a d d i t i o n a l 3.25 g NaCl - .7 -and has an o s m o l a l i t y of 373 m Osm. S a l i n e s used f o r i n v i v o experiments were h e p a r i n i s e d (10 IU.ml--'-')' and the glucose was excluded. Routine s u r g i c a l p r o t o c o l to allow i n v i v o measurements of c a r d i a c output, pre- and p o s t - b r a n c h i a l . blood p r e s s u r e and v e n t i l a t i o n L i n g cod were deeply a n a e s t h e t i s e d (0.02% MS222, trimethane sulphonate), placed v e n t r a l s i d e up i n an o p e r a t i n g s l i n g and cooled with an i c e pack on the body. The g i l l s were c o n t i n u o u s l y i r r i g a t e d with c o o l (8 to 14°C), r e c i r c u l a t i n g seawater c o n t a i n i n g a n a e s t h e t i c (0.005% MS222). A 4-6 cm i n c i s i o n was made i n the s k i n along the i n s i d e of the o p e r c u l a r c a v i t y w a l l i n l i n e with, but s l i g h t l y v e n t r a l to the v e n t r a l a o r t a . The muscle mass, which i s t h i n n e s t i n t h i s area, was c a r e f u l l y teased a s i d e to expose the p e r i c a r d i u m and the more v e n t r a l h y p o b r a n c h i a l a r t e r y . The p e r i c a r d i u m surrounding the v e n t r a l a o r t a was opened and a pneumatic c u f f (Shoukas, 1977) was implanted around the a n t e r i o r end of the v e n t r a l a o r t a . A c u f f - t y p e e l e c t r o m a g n e t i c flow probe ( B i o t r o n i c s ) was s e l e c t e d f o r good f i t on the v e n t r a l a o r t a (10-20% v e s s e l c o n s t r i c t i o n , L a n g i l l e , p e r s . comm.) and was implanted near to the bulbus a r t e r i o s u s . The pneumatic c u f f and the flow probe were, t h e r e f o r e , maximally separated on the v e s s e l . Both c u f f s were anchored to 1 t h e i r adjacent muscle masses with s e v e r a l s i l k thread s u t u r e s . The b e v e l l e d t i p of a n o n - o c c l u s i v e c a t h e t e r - 8 -(50 cm of polyethylene (PE). tubing, PE 60) was introduced upstream into the ventral aorta with the aid of a Medicut cannula (Aloe Medical, St. Louis). A l l catheters were p r e f i l l e d with heparinised s a l i n e . The catheter was secured to the body of the flow probe and to the adjacent muscle mass. The;wound in the muscle mass and in the skin were closed separately with s i l k thread sutures. The dorsal aorta was cannulated via the efferent banchial artery of one of the 4th g i l l arches (Jones et. a_l. , 1974). The catheter, 50 cm of PE 100 tubing, was introduced downstream of the ligated efferent vessel and advanced 2-5 cm towards the dorsal aorta. The vessel was tied around the catheter and the skin i n c i s i o n closed around the catheter which was anchored in place to the tissue. The buccal and opercular c a v i t i e s were also cannulated in some experiments'using methods similar to.those described by Holeton and Randall (1967a) using PE 200 tubing. These surgical procedures routinely lasted approximately one hour and were accomplished with minimal blood loss from the f i s h . •m Recovery of the f i s h from anaesthetic was begun during the f i n a l stages of the surgery by removing the ice pack and commencing g i l l i r r i g a t i o n with fresh saltwater. When strong opercular movements had. began, usually.within 5 minutes, the f i s h was placed in the experimental holding aquarium f i t t e d with a temporary g i l l i r r i g a t i o n tube. Fish were s u f f i c i e n t l y revived aft e r 5 to 10 minutes to swim off the i r r i g a t i o n tube and sustain their own g i l l v e n t i l a t i o n . - 9 -The experimental aquarium (Fig. 1) was a darkened, covered plexiglass box, which limited forward and l a t e r a l movements. The saltwater supply was flow-through, aerated and at 10-11°C, but i t could Lalso be diverted through a gas exchanger to generate hypoxic or hyperoxic water. A constant temperature r e c i r c u l a t i n g system was also in li n e and this was used to re c i r c u l a t e water containing anaesthetics e.g. 0.7% urethane (ethyl carbonate) during in vivo micropressur.e experiments. In experiments where oxygen consumption was measured, the plexiglass box was replaced by an a i r ti g h t , darkened plexiglass cylinder that had a much reduced water dead space. Isolated, perfused 'holobranch preparation The heart and ventral aorta were exposed by an 8-10 cm ventral midline i n c i s i o n in deeply anaesthetised l i n g cod (0.02% MS222). The c i r c u l a t i n g blood was heparinised with an intracardiac i n j e c t i o n of sodium heparin (1,000 IU.kg--'').. The bulbus arteriosus (bulbus). was cannulated occlusively through an i n c i s i o n in the. v e n t r i c l e with a short, heat flared PE 200 catheter. The branchial vessels were cleared of blood with, approximately 200 ml of p r e - f i l t e r e d ( M i l l i p o r e , 0.45H), heparinised saline infused via the. catheter. The best clearing results were obtained when rapid pulsations were applied to the perfusion syringe, and when the g i l l s were completely submerged and i r r i g a t e d with the s a l t water at 10°C during the perfusion. The filament tips and. the small filaments at either end of the - 10 -FIGURE I A schematic diagram of the experimental holding aquarium displaying the three alternative water supplies that were u t i l i s e d . 0= a three way tap which controlled the di r e c t i o n of flow. Gas = gas supply; either a i r , .100% N 2 or 1 0 0 % C>2 • The aquarium was covered with a loose f i t t i n g l i d when the f i s h was inside. 5L gas stripper water supply recirculating reservoir arch were always d i f f i c u l t to clear at this stage. The tips and any unclear filaments were later cleared, either by saline perfusion after the afferent branchial artery was cannulated, or, more commonly, during the i n i t i a l perfusion period of the • t. experiment. Clearing blood, lowered g i l l resistance to perfusion. The whole branchial basket was excised and then individual holobranchs were dissected free. Isolated holobranchs were stored in aerated (95% 0 2; 5% C0 2) saline, on i.ce, u n t i l required for cannulation. The afferent and efferent arch vessels'were exposed at either end of the holobranch and cannulated with blunted and shortened 18 G, thin walled hypodermic needles. In early experiments 0.5 mm lengths of PE 100- were used as catheters.. The arch was kept ice-cooled during cannulations.• The g i l l arch and free filaments d i s t a l to the cannulation s i t e were ligated as near to the catheter as possible. Hence a minimal amount of cut or exposed tissue was perfused and the poorly cleared 'f.ilamehts were excluded from the preparation. It was. estimated that at least 95% of the remaining filaments were clear of blood. These arch l i g a t i o n s also reduced, but did not completely stop, venous and lymphatic outflow which would have otherwise proceeded at an unrestricted rate. The a r t e r i a l perfusion pathway was further isolated by a l s o . l i g a t i n g the hypobranchial artery at the afferent end of the arch. Cannulated arches were stored in. the same ice-cold saline as above and were always used within 3h of their removal from the f i s h . - 12 -R e c o r d i n g Systems B l o o d and w a t e r p r e s s u r e s were d e t e c t e d u s i n g s a l i n e f i l l e d S t a t ham p r e s s u r e t r a n s d u c e r s (P23Db, P23BB and P23V) c o n n e c t e d t o s a l i n e f i l l e d , c a t h e t e r s . F o r m i c r o p r e s s u r e measurements t h e P23Db p r e s s u r e t r a n s d u c e r was o i l f i l l e d . The t r a n s d u c e r s were c a l i b r a t e d a g a i n s t p r e s s u r e s g e n e r a t e d by a s t a t i c w a t e r column b e f o r e t h e e x p e r i m e n t (1 cm = 0 . 0 9 8 k P a s c a l s ) . The t r a n s d u c e r s were a l w a y s b a l a n c e d t o a z e r o s i g n a l a t t h e a p p r o p r i a t e w a t e r o r s a l i n e l e v e l . T h i s b a l a n c e p o i n t was c h e c k e d f r e q u e n t l y t h r o u g h o u t t h e c o u r s e o f an e x p e r i m e n t . The t r a n s d u c e r s f a i t h f u l l y r e p r o d u c e d o s c i l l a t i n g p r e s s u r e s i n t h e r a n g e b e i n g m o n i t o r e d s i n c e t h e f r e q u e n c y r e s p o n s e o f the f l u i d f i l l e d t r a n s d u c e r s and t h e i r a s s o c i a t e d c a t h e t e r s was i n e x c e s s o f 10 Hz, as d e t e r m i n e d by t h e Hanson "pop" t e s t (McDonald, 1960) . P u l s a t i l e v e n t r a l a o r t i c b l o o d f l o w ( c a r d i a c o u t p u t , Q) was m o n i t o r e d w i t h a BL610 f l o w m e ter ( B i o t r o n i x L t d . ) , s e t f o r a 12.5 Hz f r e q u e n c y r e s p o n s e , u s i n g t h e s i g n a l s o f t h e i m p l a n t e d e l e c t r o m a g n e t i c f l o w p r o b e . A zero, f l o w c a l i b r a t i o n s i g n a l was o b t a i n e d i_n v i v o by e i t h e r , b r i e f l y o c c l u d i n g t h e v e n t r a l a o r t a d i s t a l t o t h e f l o w p r o b e by i n f l a t i n g the p n e u m a t i c c u f f , by d i s t u r b i n g t h e f i s h i n t h e aquarium,, o r by an i n t r a v a s c u l a r O a r b a c h o l (K+K) i n j e c t i o n (0.01 t o 0.1 ^ g . m l b l o o d - 1 ) (See l a t e r F i g s . 16 and 4 0 ) . The l a t t e r two p r o c e d u r e s p r o d u c e d a b r a d y c a r d i a d u r i n g w h i c h v e n t r a l a o r t i c b l o o d f l o w s t o p p e d b r i e f l y . Any m a j o r f l o w p r o b e movement would change the z e r o s i g n a l l e v e l . Such c h a n g e s , w h i l s t a n u i s a n c e , were e a s i l y c o r r e c t e d f o r , . o r a new z e r o s i g n a l was e s t a b l i s h e d u s i n g one o f t h e above t e c h n i q u e s . D r i f t o f the f l o w p r obe s i g n a l d u r i n g , l e n g t h y r e c o r d i n g p e r i o d s was n o t e x t e n s i v e between z e r o c a l i b r a t i o n s and was i g n o r e d . The s i g n a l s from i n d i v i d u a l f l o w p r o b e s were c a l i b r a t e d e i t h e r i n s i t u o r i n . v i t r o w i t h measured s a l i n e p e r f u s i o n s . B e a t t o b e a t h e a r t . r a t e was m o n i t o r e d i n some e x p e r i m e n t s u s i n g a r a t e m e t e r t r i g g e r e d by the p u l s a t i l e b l o o d f l o w s i g n a l . A l l e l e c t r i c a l s i g n a l s were - s u i t a b l y a m p l i f i e d f o r m o n i t o r i n g on c h a r t r e c o r d e r s . I n i t i a l l y a Beckman R f o u r c h a n n e l c h a r t : r e c o r d e r was used t o c o n t i n u o u s l y d i s p l a y r e c o r d s d u r i n g an e x p e r i m e n t . L a t e r a B r u s h 260 s i x c h a n n e l c h a r t r e c o r d e r was used i n c o n j u n c t i o n w i t h a T a n b e r g s e r i e s 115 t a p e r e c o r d e r ( e q u i p p e d w i t h a f l u t t e r c o m p e n s a t i o n , d e v i c e ) . W i t h t h i s l a t t e r s y s t e m a l l s i g n a l s were r e c o r d e d d i r e c t l y o n t o the c h a r t r e c o r d e r and. the b l o o d p r e s s u r e s and b l o o d f l o w s i g n a l s were a l s o s t o r e d d i r e c t l y o n t o m a g n e t i c t a p e . .'The s t o r e d i n f o r m a t i o n was used f o r s u b s e q u e n t a n a l y s e s . , when the s i g n a l s were r e p l a y e d t h r o u g h 5Hz band p a s s f i l t e r s o n t o th e c h a r t r e c o r d e r . The f i l t e r s r e d u c e d b a c k g r o u n d e l e c t r i c a l n o i s e p r o d u c e d by the t a p e r e c o r d e r , but d i d n o t a l t e r the s i g n a l waveform s i g n i f i c a n t l y . Oxygen c o n c e n t r a t i o n was m o n i t o r e d c o n t i n u o u s l y i n t h e i n f l o w i n g w a t e r w i t h a Beckman 0260 0 2 a n a l y s e r t h a t had a u t o m a t i c t e m p e r a t u r e c o m p e n s a t i o n . The s i g n a l s from, the o x y g e n a n a l y s e r were o n l y r e c o r d e d on t h e B r u s h r e c o r d e r d u r i n g h y p o x i c o r h y p e r o x i c e x p o s u r e e x p e r i m e n t s . F o r oxygen u p t a k e e x p e r i m e n t s 14- -the oxygen p a r t i a l pressure, P 0 2 r in water samples was measured with a Radiometer PHM72 system with associated electrodes and water jackets.; The oxygen electrodes were calibrated using zero oxygen solutions (saturated sodium sulphite or N 2 equilibrated seawater) and a i r , or 100% 0 2 equilibrated seawater. In vivo monitoring of blood flow and v e n t i l a t i o n in Ophiodon  elongatus. Q, blood pressures and v e n t i l a t i o n pressures were monitored _in vivo on individual l i n g cod after recovery from routine surgical procedures and acclimation to.the aquarium for 12 to 24 hours. Records were collected for up to 6 days and often continuously for 10 to 15 hours, in any one day. On occasion monitoring was continued over night on'an undisturbed f i s h . Noise was minimised and kept constant (white noise) during experimentation. The f i s h were usually in one of three states. The majority of the time f i s h were "resting" when th'e environment was constant, and this state corresponded to steady state recordings. Fish usually became ."disturbed" when the environment suddenly changed and here the recordings altered in an unpredictable fashion. A "struggle" involved v i s i b l e or audible t a i l movements and i t was accompanied by a p a r t i c u l a r pattern of cardiovascular and respiratory events. Struggling usually followed a disturbance but sometimes i t apparently occurred spontaneously. A l l experiments commenced only when the f i s h were considered to be in a resting state. If the f i s h struggled or were disturbed during the experiment the results 15 were not analysed. An exception to t h i s r u l e was during hypoxic exposure when f i s h o f t e n struggled and in t h i s case the struggle response was analysed s e p a r a t e l y . J - 15a -SECTION I THE MORPHOLOGY AND VASCULAR PATHWAYS OF THE GILLS IN LING COD Gross morphology and morphometries of l i n g cod g i l l s . P l a s t i c corrosion, casting of the g i l l vasculature. The properties of the lamellar c a p i l l a r y bed as revealed by si l i c o n e elastomer casting of the c a p i l l a r i e s under known t r a n s c a p i l l a r y pressure gradients. Predictions on g i l l blood flow. - 16 -• INTRODUCTION In order to interpret physiological experiments concerning blood flow i t is essential to f i r s t establish the nature of the vascular pathways under observation. This has not always been the si t u a t i o n in the past. For instance, a controversy concerning arterio-venous shunts i n . g i l l s was only .recently resolved when the correct vascular networks of g i l l filaments were established (see Steen and Kruysse, 1964; Richards and Fromm, 1969; Gannon et a_l. , 1973; Morgan and Tov e l l , 1973; Vogel et a_l. , 1973, 1974 and 1976; Cameron, 1974; Laurent and Dunel, 1976; Smith, 1976). Three points emerged from this controversy. F i r s t , whilst teleosts a l l have the same basic pattern of g i l l blood vessels, many important species differences e x i s t . Second, some of the techniques used for v i s u a l i s i n g the complex 3-dimensional patterns of vessels were on occasion misleading. The development of methyl methacrylate vascular corrosion casting by Murakami (1971) has, however, allowed 3-dimensional analysis of vascular beds using the scanning electron microscope. Gannon e_t al_. ( 1973) adopted this technique to examine the vasculature in Salmo gairdneri. Since then the g i l l vessels of other teleost species have been examined using a similar technique (Dunel and Laurent, 1977; Olsen, pers. comm.). Lastly/ the wealth.of previous l i t e r a t u r e dating back almost two centuries was somewhat ignored. The anatomical works of Munro (1785), Doellinger (1837) , Hyrtl (1838), Muller ( 1839), - 17 -Riess (1881) on f i s h g i l l v a s c u l a t u r e have now been w e l l reviewed by Smith (1976), Laurent and Dunel (1976).= and Campbell e t a l . ( i n p r e p a r a t i o n ) . I r o n i c a l l y i t i s M u l l e r ' s (1839) major c o n c l u s i o n s that have i n f a c t been v e r i f i e d using modern techniques. Fundamentally, two v a s c u l a r systems e x i s t i n t h e . g i l l s : a r e s p i r a t o r y system with a r t e r i o - a r t e r i a l pathways and a n u t r i t i v e system with a r t e r i o - v e n o u s pathways. Whether the n u t r i t i v e supply i s d e r i v e d only from e f f e r e n t a r t e r i a l v e s s e l s , as i n Salmo g a i r d n e r i , or both a f f e r e n t and e f f e r e n t a r t e r i a l v e s s e l s , as i n A n g u i l l a , i s an important s p e c i e s d i f f e r e n c e s i n c e i n the former s i t u a t i o n a l l the c a r d i a c output must pass through the r e s p i r a t o r y exchange area, the secondary l a m e l l a e . V e s s e l geometry i s h e l p f u l i n e s t a b l i s h i n g t h e o r e t i c a l p a t t e r n s of flow through the v a s c u l a t u r e . V e s s e l r e s i s t a n c e can be c a l c u l a t e d from v e s s e l geometry by assuming P o i s e u i l l e a n flow and knowing c e r t a i n p h y s i o l o g i c a l blood flow v a r i a b l e s . In a p a r a l l e l network of v e s s e l s , such as the g i l l s , r e s i s t a n c e s i t e s are v a l u a b l e i n p r e d i c t i n g flow p a t t e r n s . U n f o r t u n a t e l y , t h e o r e t i c a l , a n a l y s i s of blood flow i n c a p i l l a r y beds where v e s s e l diameters approach v e s s e l l e n g t h , e.g. r e s p i r a t o r y exchange s i t e s , i s d i f f i c u l t using P o i s e u i l l e a n equations (Weibel, 1963). Sheet blood flow equations, however, allow a b e t t e r d e s c r i p t i o n of such c a p i l l a r y blood flow. With sheet blood flow equations, and given the p r o p e r t i e s of the v a s c u l a r bed, " p r e d i c t i o n s of volumes, r e s i s t a n c e s and flow p a t t e r n s can be made r e l a t i v e l y - 1:8 " e a s i l y . S h e e t b l o o d f l o w e q u a t i o n s have been used t o d e s c r i b e a l v e o l a r b l o o d f l o w (Fung and S o b i n , 1969 and 1 9 7 7 ) . The d e r i v e d e q u a t i o n s f o r s h e e t b l o o d f l o w d e s c r i b e t h e c a p i l l a r y s h e e t by i t s t h i c k n e s s ( h ) , i t s a r e a ( e x p r e s s e d as a r a t i o . o f v a s c u l a r a r e a t o t h a t o f t h e t i s s u e ) and i t s v a s c u l a r c o m p l i a n c e . C h a r a c t e r i s t i c a l l y , h i s d e p e n d e n t upon t h e t r a n s m u r a l p r e s s u r e g r a d i e n t ( A P a ^ v ) , b u t t h e p l a n a r d i m e n s i o n s , however, r e m a i n c o n s t a n t . The d e g r e e t o w h i c h h changes i s s e t by o< , t h e c o m p l i a n c e c o e f f i c i e n t . F o r a l v e o l a r c a p i l l a r i e s a i s a t l e a s t an o r d e r o f m a g n i t u d e g r e a t e r t h a n f o r s y s t e m i c c a p i l l a r i e s , i . e . t h e a l v e o l a r v a s c u l a r s h e e t t h i c k n e s s i s v e r y s e n s i t i v e t o t h e t r a n s m u r a l p r e s s u r e . The r e s p i r a t o r y exchange s i t e i n f i s h i s t h e g i l l l a m e l l a e . The l a m e l l a e a r e e r e c t e p i t h e l i a l f o l d s e n c a s i n g a d e nse c a p i l l a r y n e t w o r k . The e p i t h e l i a l f o l d s do n o t s e p a r a t e u nder b l o o d p r e s s u r e b e c a u s e p i l l a r c e l l s e x t e n d between the two e p i t h e l i a l s h e e t s a t r e g u l a r i n t e r v a l s . The p i l l a r c e l l s f o r m the c a p i l l a r y e n d o t h e l i u m . The l a m e l l a e , t h e r e f o r e , show some s t r u c t u r a l s i m i l a r i t i e s w i t h mammalian a l v e o l i w i t h the p i l l a r c e l l s o f l a m e l l a e b e i n g a n a l o g o u s t o t h e a v a s c u l a r p o s t s of a l v e o l i . C a p i l l a r y b l o o d f l o w i n l a m e l l a e m i g h t t h e n be s i m i l a r t o s h e e t f l o w i n a l v e o l i . S c h e i d and P i i p e r (1976) have s u g g e s t e d t h a t " s h e e t f l o w " be a d o p t e d f o r l a m e l l a r f l o w i n e l a s m o b r a n c h f i s h . The l a m e l l a r shape i s c e r t a i n l y s h e e t - l i k e i n e l a s m o b r a n c h s , as i t i s i n t e l e o s t f i s h . Kempton. (1969) p r e f e r r e d t h e t e r m l a m e l l a r s h e e t t o l a m e l l a r c a p i l l a r i e s when describing the dogfish g i l l anatomy. If sheet flow theory were applicable to lamellar blood flow, clear s i m i l a r i t i e s , would exist between blood flow in vertebrate respiratory organs. S i m i l a r i t i e s between mammalian pulmonary and teleost g i l l blood flow are indeed apparent. The d i s t r i b u t i o n of pulmonary blood flow (Permutt et a l . , 1962; West,. 1977), and g i l l blood, flow (Booth, 1978) may a l t e r to meet respiratory oxygen demands. In an upright human only about 2/3rds of the lung is perfused at rest, but during exercise there is increased lung perfusion associated with elevated a r t e r i a l blood pressures' and increased cardiac output. In resting trout only 60% of g i l l lamellae are perfused (Booth, 1978). It is expected that the number of lamellae perfused can be increased given a change in.metabolic demand, e.g. exercise, or in water quality, e.g. hypoxia, since gaseous exchange at the g i l l s is apparently d i f f u s i o n limited (Randall et a l . 1967; Fisher et a l . , 1969; Randall, 1976)., unlike the lungs where perfusion li m i t a t i o n s e x i s t . If ,this were not the case then the increased oxygen delivery (Vg) and removal (Q) from the g i l l which are also associated with these two situations would be pointless. Saunders (1962), Steen and Kruysse (1964) and Hughes and Grimstone (1965) have suggested that the e f f e c t i v e g i l l exchange area can be increased and more lamellae are indeed recruited during hypoxia in trout (Booth, pers. coram). Exercising trout, however, increase oxygen uptake as much as'5 fold (Randall et a_l. , 1967); but lamellar recruitment w i l l only increase oxygen uptake by about 50%. Thus - 2 0 -o t h e r f a c t o r s w h i c h a f f e c t g a s e o u s d i f f u s i o n must c o n t r i b u t e t o i n c r e a s e oxygen u p t a k e . R a n d a l l e t a l . (1967.) c o n c l u d e d t h a t s h u n t i n g o f b l o o d c l o s e r t o t h e s u r f a c e o f the g i l l ' a n d a r e d u c t i o n o f l a m e l l a r t h i c k n e s s m i g h t be o p e r a t i v e d u r i n g h y p o x i a . W i t h a d e t a i l e d knowledge of. t h e p a t t e r n o f f l o w i n g i l l l a m e l l a e i t i s p r e d i c t e d t h a t we w i l l be a b l e t o u n d e r s t a n d how l a m e l l a r f l o w p a t t e r n s may a f f e c t d i f f u s i o n . S h e e t b l o o d f l o w e q u a t i o n s have not been a p p l i e d t o • v t h e t e l e o s t g i l l l a m e l l a e . I e s t a b l i s h e d the p r o p e r t i e s o f .the l a m e l l a r v a s c u l a t u r e i n l i n g cod t o d e t e r m i n e whether s h e e t f l o w e q u a t i o n s c o u l d be a p p l i e d and i n t r a l a m e l l a r f l o w p a t t e r n s c o u l d s u b s e q u e n t l y be p r e d i c t e d from these, e q u a t i o n s . N e i t h e r g i l l v a s c u l a r pathways no r g i l l m o r p h o m e t r i e s have•been s t u d i e d p r e v i o u s l y i n l i n g c o d , so t h e s e s t u d i e s were c a r r i e d o u t h e r e . M e t h y l m e t h a c r y l a t e p l a s t i c v a s c u l a r c o r r o s i o n c a s t i n g and s c a n n i n g e l e c t r o n m i c r o s c o p y were the m a j o r t o o l s used t o d e t e r m i n e v a s c u l a r p a thways. H a v i n g e s t a b l i s h e d v a s c u l a r pathways and g e o m e t r y , p r e d i c t i o n s were made on g i l l b l o o d f l o w u s i n g P o i s e u i l l i a n e q u a t i o n s f o r t h e i n p u t and o u t p u t v e s s e l s and e q u a t i o n s f o r s h e e t b l o o d flow, f o r the g i l l l a m e l l a e . - 21 -MATERIALS AND METHODS G r o s s m o r p h o l o g y and m o r p h o m e t r i e s on f i x e d g i l l t i s s u e In 15 f r e s h l y k i l l e d l i n g cod ( w e i g h t r a n g e 2.7 t o 6.3 kg.) t h e whole b r a n c h i a l b a s k e t was e x c i s e d and f i x e d i n B o u i n ' s f i x a t i v e f o r 12 t o 24 h o u r s . The g i l l s were examined and measured w i t h t h e a i d o f a L e i t z o p e r a t i n g m i c r o s c o p e e i t h e r i m m e d i a t e l y p o s t f i x a t i o n o r a f t e r s t o r a g e i n 70% EtOH.•. A r c h l e n g t h s were measured a t t h e base o f t h e f i l a m e n t s . T h i s l e n g t h a p p r o x i m a t e s t o t h a t o f t h e a r c h a r t e r i e s . The t o t a l number o f f i l a m e n t s were c o u n t e d on e a c h a r c h and t h e mean f i l a m e n t l e n g t h d e t e r m i n e d by m e a s u r i n g the l e n g t h o f e v e r y 10th f i l a m e n t . O n l y t h e p o s t e r i o r h e m i b r a n c h s from t h e 4 a r c h e s on one s i d e o f t h e f i s h were u s u a l l y e xamined, s i n c e i n v e s t i g a t i o n o f t h e whole g i l l a p p a r a t u s showed b i l a t e r a l symmetry e x i s t e d between t h e g i l l a r c h e s and t h a t a n t e r i o r and p o s t e r i o r h e m i b r a n c h s were s i m i l a r . Some g i l l f i l a m e n t s were a l s o examined h i s t o l o g i c a l l y . C omplete f i l a m e n t s were removed from t h e c e n t r a l r e g i o n o f t h e s e c o n d g i l l a r c h and embedded i n p a r a f f i n b l o c k s . H i s t o l o g i c a l 5 y s e r i a l s e c t i o n s were t a k e n i n t h e t r a n s v e r s e , s a g i t t a l and l o n g i t u d i n a l p l a n e s . The s e c t i o n s were mounted on s l i d e s and s t a i n e d w i t h h a e m o t o x y l i n and e o s i n . V a s c u l a r c a s t i n g u s i n g . m e t h y l m e t h a c r y l a t e p l a s t i c The t e c h n i q u e s used were e s s e n t i a l l y t h o s e d e s c r i b e d by Murakami (1971) and m o d i f i e d by Gannon ( 1 9 7 9 ) . The b r a n c h i a l v a s c u l a t u r e was c o m p l e t e l y o r p a r t i a l l y c a s t by i n j e c t i o n _ 22 _ with the p r e - p o l y m e r i s i n g p l a s t i c f l u i d i n 8 f i s h . P r i o r to the i n j e c t i o n the g i l l s were c l e a r e d of blood using h e p a r i n i s e d s a l i n e (see general methods). The p o l y m e r i s i n g p l a s t i c c o c k t a i l , which permitted a 10 to 15 minute working time, was prepared j u s t p r i o r to i n f u s i o n from a k i t ( P o l y s c i e n c e s Inc., Warrington, Pa.). Care was taken to exclude a i r bubbles from the c a t h e t e r when the p l a s t i c i n f u s i o n was s t a r t e d . A l l i n f u s i o n s were made' from a 50 ml s y r i n g e . Up to 10 minutes were taken to i n f u s e 10 t o 20 ml of methyl methacrylate i n t o the g i l l bed to prevent a high c a p i l l a r y p r e ssure developing due to: the r e l a t i v e l y high v i s c o s i t y (50 to 80 cp) of the unpolymerised methyl methacrylate. Excessive c a p i l l a r y p r e s s u r e s would rupture the l a m e l l a r v e s s e l s . A f t e r completing the p e r f u s i o n the c a t h e t e r s were occluded and the g i l l s l e f t submerged and undisturbed f o r two hours w h i l s t the p l a s t i c polymerised and hardened. The t i s s u e was.dissolved from around the c o r r o s i o n c a s t by f i r s t soaking i n warm water (<50°C) o v e r n i g h t and then by d a i l y changes of 20% NaOH s o l u t i o n s . The c a s t was f i n a l l y cleaned i n d i s t i l l e d water i n an u l t r a s o n i c bath and d r i e d with. 100% EtOH. Cast examination and a n a l y s i s The methyl methacrylate c a s t s were f i r s t examined m a c r o s c o p i c a l l y and with a l i g h t microscope. For scanning e l e c t r o n microscopy small areas of i n t e r e s t were c a r e f u l l y d i s s e c t e d from the c a s t , gold coated and examined with a - 03 - . Cambridge Stereoscan microscope. A 10 kV accelerating voltage was used on the microscope to minimise v o l a t i l i s a t i o n of the p l a s t i c cast (Gannon, 1978). Some scanning electron microscopy was performed at the University of New Brunswick, Fredericton using a Cambridge Stereoscan S4-10 and a 5 kV accelerating voltage. The great depth of focus of the scanning electron microscope (SEM)'at high magnifications greatly aided the observation and tracing of vascular networks in the g i l l f ilament. Detailed measurements and weighings were made on a complete orthograde cast of the second g i l l arch of a 4 kg l i n g cod. For every 10th filament the length and the t o t a l number of lamellae and their spacing were recorded (See Plate 5 ) . The diameters of the afferent and efferent filament arteries were measured at various locations a known distance from the base of the filament. Two or three individual lamellae were c a r e f u l l y dissected free at five to eight locations a known distance from the filament base. Only nine filaments were, used, but they were selected to include the complete range of filament lengths found on a 4 kg l i n g cod. The afferent and efferent lamellar a r t e r i o l e s were measured and photomicrographs taken of the indiv i d u a l lamellae for surface area determinations. Filament and lamellar.volumes were estimated by weighing intact filaments and then weighing a l l the lamellae removed from them. Pairs of filaments from both hemibranchs were combined to. increase the accuracy of the weighings. Weights were determined f o r every 10th p a i r of f i l a m e n t s neighbouring those used f o r the morphometric a n a l y s i s d e s c r i b e d ,above. The weights were compared to that of a known volume of methyl methacrylate which had r e c e i v e d the same treatment a f t e r p o l y m e r i s a t i o n . Cast shrinkage was l e s s than 1%. , Types of c a s t s made S e v e r a l modes of p l a s t i c i n j e c t i o n were used. They in v o l v e d orthograde and r e t r o g r a d e p e r f u s i o n s v i a major a r t e r i e s and v e i n s . A. Orthograde a r t e r i a l g i l l p e r f u s i o n s were made v i a the v e n t r a l a o r t i c c a t h e t e r (as above). B. Retrograde a r t e r i a l g i l l p e r f u s i o n s were made v i a an o c c u l s i v e , r e t r o g r a d e d o r s a l a o r t i c c a t h e t e r . T h i s c a t h e t e r (5 cm PE 200 tubing) was implanted i n the v e s s e l through a v e n t r a l body w a l l i n c i s i o n . V i s c e r a l a r t e r i e s were : l i g a t e d to allow p r e f e r e n t i a l r e t r o g r a d e flow to the g i l l s . C. Retrograde venous p e r f u s i o n s were performed v i a the g i l l venous r e t u r n v e s s e l , which i s l o c a t e d i n the p e r i c a r d i a l c a v i t y dorsad to the v e n t r a l a o r t a . An o c c l u s i v e c a t h e t e r was a l s o implanted i n t h i s v e s s e l . D. Double i n f u s i o n s were performed i n which a p a r t i a l orthograde i n f u s i o n was followed by one of the two retrograde i n f u s i o n s . D i f f e r e n t i a l c o l o u r i n g of the p l a s t i c compound d i s t i n g u i s h e d the two i n f u s i o n s . T h i s technique allowed b e t t e r f i l l i n g of the venous and lymphatic networks, s i n c e the a r t e r i a l o u t l e t f o r the r e t r o g r a d e f i l l i n g was e s s e n t i a l l y blocked, and, i n a d d i t i o n , permitted the t r a c i n g of v e s s e l s to t h e i r a f f e r e n t , e f f e r e n t . o r venous o r i g i n s . - 25 -S i l i c o n e elastomer c a s t i n g of l a m e l l a r c a p i l l a r i e s under  constant transmural p r e s s u r e s The s i l i c o n e elastomer m i c r o v a s c u l a r c a s t i n g technique/ as developed f o r the pulmonary c a p i l l a r y bed and f u l l y d e s c r i b e d by Sobin et a l . (1970), was adapted f o r m i c r o v a s c u l a r c a s t i n g i n the f i s h g i l l s . The s i l i c o n e polymers, c a t a l y s t and a c c e l e r a t o r used "were those e s p e c i a l l y s u p p l i e d to Dr. Sobin's l a b o r a t o r y by '.' G. E. C . (Waterford, N.Y.). Ca s t i n g Procedure: Three l i n g cod of s i m i l a r weigh't (3.3, 3.6 and 3.8 kg) were used. I n d i v i d u a l f i s h were l i g h t l y a n a e s t h e t i s e d and prepared with a v e n t r a l a o r t i c c a t h e t e r f o r p e r f u s i o n (see g e n e r a l methods). The g i l l s were submerged and i r r i g a t e d with sea water at a l l times. The pre-polymerised ; s i l i c o n e elastomer was prepared j u s t p r i o r to p e r f u s i o n and had a working time of 20 min. I t was introduced at a constant pressure . head of 70 cm H 2 O without any pre-perf usion with.'saline. '.Thus ' the s i l i c o n e formed an i n t e r f a c e with the h e p a r i n i s e d blood. Once p e r f u s i o n had s t a r t e d , the d o r s a l a o r t a was b i s e c t e d and the elastomer flowed f r e e l y from the v e s s e l . A f t e r about 10 min p e r f u s i o n at a pressure head of 70 cm H^ O,- at l e a s t the proximal two-thirds of a l l c e n t r a l l y l o c a t e d f i l a m e n t s were completely, c l e a r of blood. At t h i s time the pressure head was reduced to c r e a t e a transmural pressure g r a d i e n t of 20 cm H 2 O for. a s e l e c t e d r e g i o n of the arch. T h i s r e g i o n was the c e n t r a l f i l a m e n t s near to the bend of the arch and these f i l a m e n t s l a y - Z<o -p a r a l l e l to the water s u r f a c e . To achieve a transmural pressure of 20 era the p e r f u s i o n head was set at a height of.20 cm H2O above these : f i l a m e n t s . P e r f u s i o n was maintained at t h i s transmural, pressure g r a d i e n t f o r one minute, then through-flow was stopped i n one g i l l arch by clamping the d o r s a l end of the arc h . The s t a t i c pressure g r a d i e n t was maintained f o r 0.5 min before the elastomer was held w i t h i n the arch, at t h i s given transmural p r e s s u r e , by clamping the a f f e r e n t end of the arch as w e l l . Now the p e r f u s i o n pressure was r a i s e d by 10 cm H2O and the p r o t o c o l repeated on a d i f f e r e n t arch. Six g i l l arches were c a s t at d i f f e r e n t pressures f o r each f i s h at 10 cm H2O transmural pressure increments up to 70 cm H 20 i . e . 20, 30, 40, 50, 6.0 and 70 cm H2O. The 4th g i l l arches were not used. .. A f t e r completion of the c a s t i n g the g i l l s were l e f t submerged and undisturbed f o r the 2 hours r e q u i r e d f o r c a t a l y t i c hardening of the elastomer. Tissue: p r e p a r a t i o n : A f t e r the elastomer had hardened the i n t a c t g i l l basket was e x c i s e d and the t i s s u e f i x e d o v e r n i g h t i n a 10% b u f f e r e d f o r m a l i n s o l u t i o n . Four neighbouring f i l a m e n t s from the 'selected area i n each g i l l arch, were d i s s e c t e d f r e e and embedded i n blocks of g e l a t i n . The f i l a m e n t s were a l l between 19. and 21 .mm i n l e n g t h . The r e g i o n 5 mm from the f i l a m e n t base was se c t i o n e d i n a c r y o s t a t . The 10^ and 20^tt t h i c k s e c t i o n s were s t a i n e d with c r e s y l v i o l e t , using cooled s o l u t i o n s mounted on s l i d e s i n g l y c e r o l g e l a t i n and subsequently covered and s e a l e d . The s t a i n was basement membrane s p e c i f i c and s h a r p l y d e f i n e d the - 27 -l a m e l l a r m i c r o v a s c u l a t u r e a g a i n s t the opaque s i l i c o n e which, f i l l e d the v e s s e l s . T h i s d e f i n i t i o n was seen i n the plan view and ( i n c r o s s - s e c t i o n . Measurement techniques: Automated measurements were made from a video screen d i s p l a y i n g microscope images of the se c t i o n e d m a t e r i a l . These measurements were performed i n Dr. Sobin's l a b o r a t o r y i n Los Angeles and the techniques used were those f u l l y d e s c r i b e d by Sobin et a l . (1970). The v a s c u l a r space to t i s s u e r a t i o (VSTR) was determined from the plan s e c t i o n s of the la m e l l a e ( P l a t e 1 ) . In a given m i c r o s c o p i c f i e l d 6 to 12 c l e a r l y v i s i b l e p i l l a r c e l l s o u t l i n e s were traced from the video image onto t r a n s p a r e n t p l a s t i c . The area occupied by the p i l l a r c e l l s was determined and s u b s t r a c t e d from the t o t a l area that they •'occupied ( v a s c u l a r p l u s t i s s u e c e l l area) i . e . the VSTR. Areas of the o u t l i n e s were measured with,an automatic planimeter • (loaned by L a s i c o , Los Angeles, Ca;). The v a s c u l a r sheet, t h i c k n e s s (h) was measured using a s p l i t image' device on the video screen. The diameters of a f f e r e n t and e f f e r e n t a r t e r i e s were a l s o measured i n the same way. Measurements of h were made at a l l transmural p r e s s u r e s , but only l a m e l l a r c r o s s - s e c t i o n s with a l l l a m e l l a r channels f i l l e d : w e r e analysed. VSTR .measurements were made l a r g e l y at. the 20 and 70 cm H2O transmural p r e s s u r e s . A r t e r y diameters were only measured at .20 and.70 cm H 20 transmural p r e s s u r e s . The l a m e l l a r transmural pressure i s here a f t e r r e f e r r e d to as APiam* - 28 -PLATE 1 A p l a n v i e w o f a s e c o n d a r y l a m e l l a f r o m a l i n g cod t h a t had been p e r f u s e d w i t h p o l y m e r i s i n g s i l i c o n e e l a s t o m e r . The t i s s u e was s t a i n e d f o r t h e basement membrane and t h e p i l l a r p e l l s (pc) a r e i n d i c a t e d . The s e c t i o n t h i c k n e s s i s 20V s u c h t h a t t h e l a m e l l a s h e e t i s a l m o s t c o m p l e t e l y i n t a c t . F o r VSTR a n a l y s i s g r o u p s of c l e a r l y v i s i b l e p i l l a r c e l l s were i s o l a t e d , as i n d i c a t e d , and t h e t o t a l a r e a o f the e n c l o s u r e d e t e r m i n e d . The a r e a o f t h e p i l l a r c e l l s e n c l o s e d was combined and s u b s t r a c t e d from th e t o t a l a r e a t o y i e l d t h e VSTR. ( M a g n i f i c a t i o n = 785x: c a l i b r a t i o n b a r = 2 0 y ) . - 28a -A b b r e v i a t i o n s commonly used i n t h e t lpxt m o r p h o l o g i c a l and v a s c u l a r d e s c r i p t i o n s . J ABA . = . a f f e r e n t b r a n c h i i a l a r t e r y AAA = a f f e r e n t a r c h a r t e r y AFA ..= a f f e r e n t f i l a m e n t a r t e r y ALA = a f f e r e n t l a m e l l a r a r t e r i o l e ELA = e f f e r e n t l a m e l l a r " a r t e r i o l e EFA = e f f e r e n t f i l a m e n t a r t e r y EAA • •=• e f f e r e n t a r c h a r t e r y EBA '••'•= e f f e r e n t b r a n c h i a l a r t e r y DA .-.• = • d o r s a l a o r t a CM = c o e l i a c o - m e s e n t e r y a r t e r y CART = f i l a m e n t c a r t i l a g e '.. ' . • • • i, CS = " ' c e n t r a l s i n u s •' . CVL = c e n t r a l v e n o l y m p h a t i c s EVC = e f f e r e n t companion v e s s e l ACV '='•.' a f f e r e n t companion v e s s e l LAM = l a m e l l a r v a s c u l a r s h e e t " l a m e l l a r u n i t " - = LAM p l u s ALA and ELA • b = b a s a l p e r i p h e r a l blood, c h a n n e l o f LAM m = m a r g i n a l p e r i p h e r a l b l o o d c h a n n e l o f LAM a =. a f f e r e n t . e = e f f e r e n t . • a •.•=.' c o m p l i a n c e c o e f f i c i e n t o | LAM v a s c u l a r s h e e t =: mean LAM t h i c k n e s s A P . = l a m e l l a r t r a n s m u r a l p r e s s u r e A P = a l v e o l a r t r a n s m u r a l p r e s s u r e VSTR •= v a s c u l a r s p a c e t o t i s s u e r a t i o _ 30 -RESULTS In general the morphology and major a r t e r i a l pathways of the l i n g cod g i l l s d i f f e r only in d e t a i l from the well established descriptions for other teleosts, notably Salmo  gairdneri. Art emphasis is therefore placed on additional information and contrasting r e s u l t s . To avoid confusion, the terms g i l l filament (= primary lamella) and lamella ( - secondary lamella) are used here. G i l l morphology and morphometries G i l l , arches and filaments: The branchial basket is b i l a t e r a l l y symmetrical. The four g i l l arches (holobranchs) on either side support two arrays of g i l l filaments (hemibranchs). The filaments of each hemibranch of a holobranch i n t e r d i g i t a t e and t h e i r septa are not fused. A number of shorter filaments are located at either end of the arch (Plate 2). The four g i l l arches have d i f f e r e n t lengths (Fig. 2A) and support d i f f e r e n t numbers of filaments (Fig. 2B). G i l l arches 1, 2 and 3 support approximately the same t o t a l length of filaments since g i l l arch .3 supports filaments of greater length on average than arches 1 and 2 (Table T ) . The fourth g i l l arch is reduced in s i z e . The increase in g i l l size (t o t a l filament length) with increasing body w e i g h t i s due largely to longer filaments (Fig. 3) rather than greater filament numbers (Fig. 2B). - 31 -PLATE 2 A. The ge n e r a l arrangement of the g i l l f i l a m e n t s i n the centre of the g i l l arch as re v e a l e d by methyl methacrylate c o r r o s i o n c a s t i n g . White arrows i n d i c a t e the r e c u r r e n t venolymphatic channels of the ar c h . Black arrow i n d i c a t e s e f f e r e n t f i l a m e n t a r t e r y j u n c t i o n with e f f e r e n t arch a r t e r y ( C a l i b r a t i o n marker = 2 cm). B, C and D. Surface views of g i l l l a m e l l a e i n s i t u on the f i l a m e n t a f t e r c r i t i c a l p o i n t d r y i n g . B = center of f i l a m e n t , C = f i l a m e n t t i p and D = f i l a m e n t base and septum (sep). Arrows i n d i c a t e d i r e c t i o n of water flow. (B = 94x, bar =200 ; C and D = 38x, bar = 500y). -32 _ FIGURE 2A The r e l a t i o n s h i p between g i l l a r c h l e n g t h (nun) f o r i n d i v i d u a l g i l l a r c h e s and f i s h w e i g h t . . F o r c l a r i t y i n d i v i d u a l d a t a p o i n t s a r e g i v e n f o r g i l l a r c h e s 1 and 4 o n l y ( s o l i d r e g r e s s i o n l i n e s ) . The r e g r e s s i o n l i n e s f o r g i l l a r c h e s 2 and 3 a r e t h e n o n - c o n t i n u o u s l i n e s . The r e g r e s s i o n l i n e e q u a t i o n s a r e : A r c h 1 9.34 Wt. :+ 107 .1 A r c h 2 9.19 Wt. + 10 4.7 A r c h 3 9.78 Wt. + 86.5 A r c h 4 6.3 8 Wt. + 77.7 FIGURE 2B The r e l a t i o n s h i p between the number o f f i l a m e n t s s u p p o r t e d on one g i l l h e m i b r a n c h o f e a c h o f the 4 a r c h e s and f i s h w e i g h t . The n o t a t i o n used i s t h e same as f o r 2A. The r e g r e s s i o n l i n e e q u a t i o n s a r e : A r c h 1 = 2 . 8 9 Wt. + 243.2 A r c h 2 = 3.26 Wt. + 241.3 A r c h 3 = 4.72 Wt. + 213.8 A r c h 4 = 3.11 Wt. + 186.8 2. (5* (Q tO filaments per hemibranch r o r o O O JL —1- L . ro oo o • i • a a a -i -i -i 3- x r co r o • oo o arch length , mm r o o J L . p Q —\ Q a Q —t o co r o - * 03 TABLE I A summary of the morphometric a n a l y s i s of the g i l l arches and g i l l f i l a m e n t s from 15 l i n g cod (wt range = 4.0 to 6.2 kg; mean = 5.1 ± 0.2 kg). The mean values. standard e r r o r were c a l c u l a t e d from measured values f o r each f i s h • • • (n = number of f i s h ) . The mean f i l a m e n t spacing and the i • ' t o t a l fulament l e n g t h of the g i l l s are based on c a l c u l a t i o n s from the mean values f o r i n d i v i d u a l f i s h . 5 3 a TABLE I Morphometric Arch 1 Arch 2 Arch 3 Arch 4 Arch l e n g t h 155 ± 9 152 ± 9 136 ± 10 113 ± 7 (mm (n = 18) # f i l a m e n t s / h o l b r a n c h 258 ± 7 258 ± 12 238 ± 12 202 ± 6 (n = 18) Mean f i l a m e n t s p a c i n g 0.60 0.59 0.57 0.56 (mm) Mean f i l a m e n t l e n g t h 15.3 ± 0.9 16.4 ± 1.1 16.7 + 0.8 16.9 ± 0.8 (mm) (n = 8) T o t a l f i l a m e n t (m) l e n g t h 0.790 0.846 0.745 0.683 _ 34 _ F I G U R E 3 A) Mean f i l a m e n t length f o r the whole g i l l basket versus f i s h weight. B) T o t a l f i l a m e n t length for. the whole g i l l s t r u c t u r e i n l i n g cod f o r a range of f i s h weights. total length of filaments , m. J O _ l _ a zr mean filament length, mm O • K J o 03 7T L a m e l l a e : These a r e the r e s p i r a t o r y exchange s i t e s . They a r e r e g u l a r l y a r r a n g e d on b o t h s i d e s o f t h e f i l a m e n t s ( P l a t e s 2 and 3 ) , e x t e n d a c r o s s t h e w i d t h o f t h e f i l a m e n t and have a v a r i e t y o f s h apes t h a t r e s e m b l e t r i a n g l e s . The s h o r t e s t edge o f t h e " t r i a n g l e " i s a n t e r i o r and f a c e s t h e w a t e r f l o w ( P l a t e 3 ) . The l a m e l l a e a r e u n s u p p o r t e d e p i t h e l i a l f o l d s t h a t a r e h e l d t o g e t h e r u n d e r t h e s t r a i n o f i n t e r n a l b l o o d p r e s s u r e by r e g u l a r l y s p a c e d p i l l a r c e l l s , w h i c h f o r m t h e e n d o t h e l i a l l i n i n g o f t h e l a m e l l a r c a p i l l a r i e s ( P l a t e 4 ) . A n o t i c a b l e i n t e r s t i t i a l / l y m p h a t i c s p a c e i s l o c a t e d between t h e e p i t h e l i u m and t h e p i l l a r c e l l s . T h i s s p a c e was s w o l l e n c o n s i d e r a b l y i n some p r e p a r a t i o n s . The e p i t h e l i u m i s one c e l l t h i c k e x c e p t a t t h e base o f the. l a m e l l a ( t h e j u n c t i o n w i t h t h e f i l a m e n t ) where i t i s t h i c k e n e d . Some l a m e l l a r c a p i l l a r i e s a r e b u r i e d w i t h i n t h e f i l a m e n t e p i t h e l i u m ( P l a t e 4 ) , and t h i s o c c u r s more e x t e n s i v e l y on t h e e f f e r e n t s i d e o f the l a m e l l a . C l e a r l y t h e b l o o d t o w a t e r e p i t h e l i a l b a r r i e r i s much g r e a t e r i n t h e s e b a s a l r e g i o n s t h a n e l s e w h e r e on t h e l a m e l l a . The l a m e l l a e a r e r e g u l a r l y s p a c e d a l o n g t h e f i l a m e n t l e n g t h and f i l a m e n t s o f d i f f e r e n t l e n g t h s have s i m i l a r l a m e l l a r s p a c i n g s i n t h e 4 kg s p e c i m e n examined ( F i g 4 ) . L a m e l l a e a r e s p a c e d s l i g h t l y c l o s e r t o g e t h e r a t t h e t i p and base o f t h e f i l a m e n t , but t h e s e r e g i o n s a r e l i m i t e d and c o n t r i b u t e l i t t l e t o t h e o v e r a l l l a m e l l a e numbers i n the l o n g f i l a m e n t s o f t h e s e l a r g e f i s h . ' P L A T E i The g e n e r a l r e l i e f o f t h e f i l a m e n t w i t h d o r s a l and v e n t r a l l a m e l l a e as shown by a 2 0 ^ t h i c k h i s t o l o g i c a l c r o s s s e c t i o n . The s e c t i o n was p r e p a r e d f o r VSTR a n a l y s i s . a f a = a f f e r e n t f i l a m e n t a r t e r y ; a l a = a f f e r e n t l a m e l l a r a r t e r y ; c a r t = f i l a m e n t s u p p o r t c a r t i l a g e ; l a m = l a m e l l a r w i t h b = b a s a l b l o o d c h a n n e l and m = m a r g i n a l b l o o d c h a n n e l ; e l a = e f f e r e n t l a m e l l a a r t e r i o l e ; e f a = e f f e r e n t f i l a m e n t a r t e r y and e c v = m a i n e f f e r e n t c o m p a n i o n v e s s e l . a c v = m a i n a f f e r e n t c o m p a n i o n v e s s e l . (120x, b a r = 20U U ) . PLATE 4 H i s t o l o g i c a l s e c t i o n s o f t h e g i l l l a m e l l a (lam) i n -c r o s s s e c t i o n and t h e f i l a m e n t ( f i l ) i n s a g i t t a l s e c t i o n , b = b a s a l l a m e l l a r b l o o d c h a n n e l , m = m a r g i n a l l a m e l l a r b l o o d c h a n n e l , . PC = p i l l a r c e l l , EP = l a m e l l a e p i t h e l i u m and c = c a r t i l a g e . N ote how t h e b a s a l l a m e l l a b l o o d c h a n n e l s a r e b u r i e d i n f i l a m e n t e p i t h e l i u m ( B ) . (lOyU, s e c t i o n s as p r e p a r e d f o r VSTR a n a l y s i s . M a g n i f i c a t i o n s : A = 1 5 5 x , B = 3 5 4 x a n d C = 1 0 4 0 x . C a l i b r a t i o n b a r s : A = 1 0 0 y , B = 20 M and C = 10V) . - 37a -> _ 38 _ F I G U R E 4 The l a m e l l a r spacing determined from vascular casts f o r a complete range of filament lengths from a 4 kg l i n g cod. Filaments s h o r t e r than 8mm were not numerous. and were not examined. 800 6004 CO 0) E o 400 i 200 10 15 Filament Length (mm) To - 3 9 _ V a s c u l a r pathways i n l i n g cod The f o l l o w i n g d e s c r i p t i o n i s a s y n t h e s i s o f the o b s e r v a t i o n s made on p l a s t i c c o r r o s i o n c a s t s , on h i s t o l o g i c a l l y s e c t i o n e d m a t e r i a l and from many d i s s e c t i o n s . The b u l b u s a r t e r i o s u s and v . e n t r a l a o r t a have m u s c u l a r w a l l s ; t h e w a l l o f t h e b u l b u s i s t r a b e c u l a t e d . The v e n t r a l a o r t a i s l o n g (3 cm i n a 4 kg f i s h ) and d i v i d e s i n t o 8 a f f e r e n t b r a n c h i a l a r t e r i e s ( P l a t e 5 ) . G i l l a r c h v e s s e l s : The: a f f e r e n t b r a n c h i a l a r t e r y (ABA) e x t e n d s d o r s a l l y a l o n g t h e a r c h and b i f u r c a t e s t o form t h e a f f e r e n t a r c h a r t e r i e s ( A A A ) ( P l a t e 5 ) . Each AAA i n t u r n forms a p a r a l l e l s e r i e s o f r i g h t a n g l e d b r a n c h e s w h i c h c o r r e s p o n d t o t h e a l t e r n a t i n g a f f e r e n t f i l a m e n t a r t e r i e s o f each h e m i b r a n c h . . The AAA i s somewhat c o m p r e s s e d l a t e r a l l y and t a p e r s a l o n g i t s l e n g t h . The e f f e r e n t v e s s e l s have a s i m i l a r , b u t r e v e r s e d , a r b o r e s c e n t p a t t e r n . The p a r a l l e l s e r i e s o f e f f e r e n t f i l a m e n t a r t e r i e s u n i t e p e r p e n d i c u l a r l y t o an e f f e r e n t a r c h a r t e r y (EAA). The EAAs a r e p a i r e d a t e i t h e r end o f the h o l o b r a n c h (one f o r each h e m i b r a n c h ) , b u t t h e y f o r m a s i n g l e v e s s e l c e n t r a l l y . An e f f e r e n t b r a n c h i a l a r t e r y (EBA) e x t e n d s d o r s a l l y i n e a c h a r c h . The two p o s t e r i o r EBAs u n i t e as do t h e two a n t e r i o r EBAs; t h u s two e f f e r e n t e p i b r a n c h i a l s a r e formed on e i t h e r s i d e o f the ph a r y n x ( P l a t e 6 ) . The e p i b r a n c h i a l a r t e r i e s u n i t e t o form the d o r s a l a o r t a . The f o r m a t i o n o f the d o r s a l a o r t a i s n o t a s i m p l e - 40 -u n i f i c a t i o n o f t h e f o u r e f f e r e n t e p i b r a n c h i a l a r t e r i e s . The e p i b r a n c h i a l s f r o m t h e p o s t e r i o r a r c h e s c e r t a i n l y u n i t e w i t h the d o r s a l a o r t a , b u t a t t h e same t i m e t h e y a p p e a r c o n t i n u o u s w i t h t h e c o e l i a c o - m e s e n t e r i c a r t e r y . The c o n f l u e n c e o f t h e l e f t and r i g h t e p i b r a n c h i a l s f r o m t h e p o s t e r i o r a r c h e s i s p r e d o m i n a n t l y on th e l e f t s i d e o f t h e d o r s a l a o r t a i . e . t h e r i g h t e f f e r e n t e p i b r a n c h i a l a c t u a l l y p a s s e s v e n t r a l l y t o the d o r s a l a o r t a ( P l a t e 6 ) . I n t h e same r e g i o n on t h e r i g h t s i d e o f the d o r s a l a o r t a t h e c o e l i a c o - m e s e n t e r i c a r t e r y i s d e r i v e d . In t h i s l o c a l i s e d r e g i o n , t h e a r t e r i a l w a l l s have deep smooth m u s c l e c o a t s . A g i l l a r c h i n c r o s s s e c t i o n r e v e a l s t h a t t h e EAA l i e s i n between t h e a n t e r i o r l y l o c a t e d b r a n c h i a l a r t e r i e s and t h e more p o s t e r i o r l y l o c a t e d AAA ( F i g . 5 ) . Two m a j o r v e n o l y m p h a t i c c h a n n e l s a r e a l s o l o c a t e d i n t h e a r c h , one i n between t h e AAA and EAA, t h e o t h e r a n t e r i o r t o t h e EAA ( P l a t e 2 ) . The c a r o t i d and a f f e r e n t p s e u d o b r a n c h a r t e r i e s a r e formed f r o m a s i n g l e b r a n c h o f t h e EBA o f the f i r s t g i l l a r c h b e f o r e i t forms t h e e p i b r a n c h i a l a r t e r y . The s i n g l e h y p o b r a n c h i a l a r t e r y i s d e r i v e d f r o m b r a n c h e s o f t h e EAA i n g i l l a r c h e s 2 and 3 and p a s s e s a l o n g t h e p e r i c a r d i a l c a v i t y t o s u p p l y t h e h y p o b r a n c h i a l m u s c u l a t u r e . A r e l a t i v e l y s m a l l b r a n c h from t h e h y p o b r a n c h i a l a r t e r y f o r m s t h e v a s a vasorum o f t h e v e n t r a l a o r t a , the b u l b u s a r t e r i o s u s and p o s s i b l y the v e n t r i c l e . T h e r e was no o t h e r c o r o n a r y s u p p l y t o t h e h e a r t s e e n i n l i n g c o d . 41 -R e s p i r a t o r y n e twork o f t h e g i l l f i l a m e n t A r t e r i a l v e s s e l s : A s i n g l e a f f e r e n t f i l a m e n t a r t e r y (AFA) e x t e n d s w i t h a g r a d u a l t a p e r towards the t i p o f each f i l a m e n t and i s p a r a l l e l t o t h e p o s t e r i o r edge o f t h e f i l a m e n t . The e f f e r e n t f i l a m e n t a r t e r y (EFA) i s s i m i l a r l y a r r a n g e d , b u t on the a n t e r i o r edge o f t h e f i l a m e n t . The EFA i s s l i g h t l y l o n g e r t h a n t h e AFA at i t s base s i n c e i t e x t e n d s o v e r the AAA b e f o r e u n i t i n g w i t h the EAA. A c o n s t r i c t i o n i n t h e c o r r o s i o n c a s t o f the EFA was c o r i s i s t a n t l y s e e n a t the base o f the EFA where i t u n i t e s w i t h t h e AAA ( P l a t e 7 ) . L a m e l l a r c a p i l l a r y beds e x t e n d between t h e AFA and EFA to f o r m a p a r a l l e l , l a d d e r - l i k e network o f v e s s e l s a l o n g t h e f i l a m e n t l e n g t h ( P l a t e 8 ) . . A l a m e l l a r c a p i l l a r y s h e e t and i t s a s s o c i a t e d a r t e r i o l e s a r e termed a " l a m e l l a r . u n i t " ( P l a t e 9 ) . L a m e l l a r u n i t : The n a t u r e o f the a f f e r e n t l a m e l l a r a r t e r i o l e (ALA) i s v a r i a b l e . P r o x i m a l ALAs d i f f e r from d i s t a l ALAs. Some g e n e r a l i s a t i o n s c a n , however, be made. The ALA e x t e n d s a l m o s t c o m p l e t e l y o v e r the w i d t h of the f i l a m e n t s u p p o r t c a r t i l a g e b e f o r e f o r m i n g t h e l a m e l l a r c a p i l l a r i e s ( P l a t e s 3 and 1 0 ) . In d i s t a l r e g i o n s t h e AFA l e n g t h i s r e d u c e d and c o r r e s p o n d s t o the n a r r o w e r f i l a m e n t c a r t i l a g e . I n d i v i d u a l ALAs u s u a l l y s u p p l y i n d i v i d u a l l a m e l l a e on e i t h e r s i d e o f t h e f i l a m e n t . However, some ALAs may d i v i d e a l o n g t h e i r l e n g t h t o s u p p l y two l a m e l l a e on the same s i d e o f t h e f i l a m e n t . The o r i g i n s o f t h e ALA f o r t h e - 4 0 . -d o r s a l and v e n t r a l s i d e s o f the f i l a m e n t a r e q u i t e s e p a r a t e a t t h e base o f t h e f i l a m e n t . However, more d i s t a l l y the ALA o r i g i n s become much c l o s e r and a b o u t m i d - f i l a m e n t the d o r s a l and v e n t r a l ALAs s h a r e a s h o r t , common v e s s e l o r i g i n ( P l a t e 1 1 ) . A n a l y s i s o f p l a s t i c c a s t s a l s o showed t h a t t h e d i a m e t e r o f d i s t a l ALAs was 3 t o 6jx s m a l l e r t h a n p r o x i m a l o n e s . The e f f e r e n t l a m e l l a r a r t e r i o l e s ( E L A ) , by c o n t r a s t , a r e a l w a y s s h o r t and u n d i v i d e d . Even i n i t s s h o r t l e n g t h the ELA has a n o t i c a b l e , " b u l b a r r e g i o n ( P l a t e s 8, 9, 10 and 1 2 ) . B o t h the ALA and ELA have smooth m u s c l e i n t h e i r v e s s e l w a l l s . The v a r i o u s " t r i a n g u l a r " s h apes o f l a m e l l a r c a p i l l a r y s h e e t s and the r e g u l a r l o c a t i o n s o f p i l l a r c e l l s ( h o l e s i n t h e c o r r o s i o n c a s t ) c an to -c l e a r l y be s e e n i n P l a t e 8. The d i s t i n c t p e r i p h e r a l c h a n n e l s o f the l a m e l l a a r e l a r g e r t h a n the c e n t r a l c a p i l l a r i e s . On each l a m e l l a , the o u t s i d e " m a r g i n a l " c h a n n e l i s c o n t i n u o u s w h i l s t t h e l o w e r " b a s a l " c h a n n e l o f t e n t a p e r s t o w a r d s the e f f e r e n t s i d e , • where t h e v e s s e l a l s o l i e s more d e e p l y i n the f i l a m e n t t i s s u e s ' (See P l a t e s 3 and 4). - 43 -Venolymphatic networks of the g i l l f i l a m e n t In a d d i t i o n to the r e s p i r a t o r y network, another v a s c u l a r complex e x i s t s w i t h i n the g i l l f i l a m e n t s : the venolymphatics. No v e s s e l s other than the ALAsare d e r i v e d from the a f f e r e n t f i l a m e n t a r t e r y . Thus the venolymphatic c i r c u l a t i o n i s d e r i v e d t o t a l l y from the e f f e r e n t c i r c u l a t i o n and the formation of lymph. The major venolymphatic v e s s e l s are the a f f e r e n t and e f f e r e n t companion v e s s e l s , which l i e p a r a l l e l to the AFA and EFA r e s p e c t i v e l y , and the l a r g e c e n t r a l sinus that l i e s i n the f i l a m e n t body. These v e s s e l s are a l l i n t e r c o n n e c t e d and have t h i n , non-muscular w a l l s that appear very d i s t e n s i b l e . E f f e r e n t companion v e s s e l s (ECV): A major v e s s e l l i e s p a r a l l e l to and o u t s i d e of the EFA, with which i t also: has short connections ( P l a t e 12). The connections are spaced about every 600u i . e . every 12 lamellae or so, and they have smooth muscle i n t h e i r w a l l s which i s a c o n t i n u a t i o n of that found i n the EFA. S e v e r a l i n t e r - c o n n e c t e d ECVs of a s m a l l e r diameter a l s o extend along the f i l a m e n t length but i n a more meandering f a s h i o n ( P l a t e 12). The s m a l l e r v e s s e l s are connected to the main ECV, and they a l s o have r e g u l a r s h o r t , narrow, lOu diameter i n t e r - c o h n e c t i o n s to the c e n t r a l s i n u s . These are the only i n t e r - c o n n e c t i o n s to the c e n t r a l sinus on the e f f e r e n t s i d e of the f i l a m e n t and they pass around the EFA and i n between the ELAs ( P l a t e 12). Central sinus: A large sinus occupies the center of the filament between the EFA and the filament c a r t i l a g e . The dimensions of the central sinUs were always much greater in retrograde corrosion casts made with retrograde f i l l i n g (Plate 13'). The whole central sinus is connected to the ECV (as described above) and to the afferent companion vessels by.long vessels that extend either side of and the whole width of the filament c a r t i l a g e (Plates. 8 and 11). No di r e c t connections to either the EFA or. the AFA were seen. A small, separate/more diffuse and blind V ending system is also located in the filament body between the central sinus and the lamellar base (Plate 13). . This system i s more pronounced in the region immediately underneath a lamella and i t is connected to the central sinus near to the c a r t i l a g e , It has no other connections, and is consequently thought to represent the lymphatic drainage system of the lamellae. Afferent companion vessels (ACV): These paired vessels lj.e p a r a l l e l to the inside of the AFA, both dorsad and ventrad (Plate 11)-.. As well as their own connections to the central sinus, they themselves are inter-connected by a varicose c a p i l l a r y network around the outside of the.AFA (Plate 11). The ACV was the most poorly cast of the filament vessels and often .only the section in the center of. the filament was f i l l e d , whilst at other times the cast had a segmented appearance along i t s length due to p a r t i a l f i l l i n g at d i f f e r e n t levels along the filament. - 45 - . T h e d r a i n a g e o f t h e f i l a m e n t v e n o l y m p h a t i c s y s t e m , i n t o t h e a r c h a n d s u b s e q u e n t l y f r o m t h e a r c h was n o t e x a m i n e d i n g r e a t d e t a i l . T h e r e a r e no d i r e c t c o n n e c t i o n s o f t h e c e n t r a l s i n u s p e r s e t o t h e a r c h . T h e A C V s a r e u n i t e d w i t h t h e m a j o r v e n o l y m p h a t i c v e s s e l s i n b e t w e e n t h e AAA a n d E F A . A q u i t e d e n s e , i n t e r t w i n i n g n e t w o r k , o f v e s s e l s e x i s t s a t t h e b a s e o f t h e f i l a m e n t a n d i n t h e f i l a m e n t s e p t u m . I t h a s o r i g i n s f r o m t h e E F A b a s e a n d i s o f t e n i n t e r - c o n n e c t e d w i t h n e i g h b o u r i n g f i l a m e n t s ( P l a t e 7),. T h i s a n d t h e E C V s a p p e a r t o c o n n e c t w i t h t h e v e n o l y m p h a t i c v e s s e l s s u r r o u n d i n g t h e a n t e r i o r o f t h e EAA. Some o f t h e v e n o l y m p h a t i c a r c h v e s s e l s , a t l e a s t , w e r e c o n n e c t e d t o t h e s i n u s v e n o s u s b y t h e a n t e r i o r v e n o u s r e t u r n v e s s e l l o c a t e d i n t h e p e r i c a r d i a l c a v i t y , a s i n d i c a t e d b y s u c c e s s f u l r e t r o g r a d e g i l l i n j e c t i o n s v i a t h i s , v e s s e l . PLATE 5 G e n e r a l f o r m and. a r r a n g m e n t o f the a f f e r e n t v e s s e l s t o t h e g i l l s as shown by m e t h y l m e t h a c r y l a t e c o r r o s i o n c a s t s . a) A c a s t o f the b u l b u s a r t e r i o s u s and v e n t r a l a o r t a . b) The a f f e r e n t b r a n c h i a l a r t e r i e s and p a r t o f the v e n t r a l a o r t a . c) The 4 g i l l a r c h e s ( h o l o b r a n c h s ) w i t h f i l a m e n t s _in s i t u . A r c h 2 has e v e r y 1 0 t h f i l a m e n t m i s s i n g t o show s a m p l i n g a r e a s . , d) The a f f e r e n t a r c h a r t e r y w i t h b r a n c h i a l a r t e r y from t h e f i r s t and t h e f o u r t h ( s h o r t e r ) g i l l a r c h e s . Some a f f e r e n t a r t e r i e s a r e p a r t i a l l y c a s t on t h e f o u r t h g i l l a r c h . C a l i b r a t i o n m arker = 2 cm). - 47 -PLATE 6 The g e n e r a l a r r a n g e m e n t o f t h e f o u r (1,2,3,4) e f f e r e n t b r a n c h i a l a r t e r i e s as r e v e a l e d by a m e t h y l m e t h a c r y l a t e c o r r o s i o n c a s t . da = d o r s a l a o r t a ( which was a l m o s t t o t a l l y removed) cm = c o e l i a c o - m e s e n t e r i c a r t e r y . Note t h e c o n f l u e n c e o f t h e b r a n c h i a l a r t e r i e s t o f o rm t h e 2 p a i r s o f e p i b r a n c h i a l s , and a l s o the c o n f l u e n c e o f the e p i b r a n c h i a l s ( s e e t e x t ) . A = d o r s a l v i e w B = v e n t r a l v iew. ( C a l i b r a t i o n b a r = 2 mm). - 48 -FIGURE 5 A s c h e m a t i c l i n e d i a g r a m o f the v a s c u l a r a r r a n g e m e n t i n the g i l l a r c h and p r o x i m a l r e g i o n df a g i l l f i l a m e n t o f a l i n g c o d . L = l y m p h a t i c v e s s e l s ; BA = b r a n c h i a l a r t e r y ; EAA = p a i r e d e f f e r e n t a r c h a r t e r i e s ; AAA = a f f e r e n t a r c h a r t e r y ; e f a = e f f e r e n t f i l a m e n t a r t e r y ; a f a = a f f e r e n t f i l a m e n t a r t e r y ; a c v = a f f e r e n t companion v e s s e l . water flow A R C H C R O S S - S E C T I O N efa acv a fa - 43. -PLATE 7 Two v i e w s o f t h e base o f t h e e f f e r e n t f i l a m e n t a r t e r y ( e f a ) n e a r t o i t s j u n c t i o n w i t h the e f f e r e n t a r c h a r t e r y ( e a a ) . T h e s e d e m o n s t r a t e the c o n s t r i c t i o n ( a r r o w s ) i n the c o r r o s i o n c a s t o f t h e EFAs. Note t h e p l e x u s o f v e n o l y m p h a t i c ( v l ) v e s s e l s a t t h e base o f t h e f i l a m e n t . (A = 78x : B = 50x. C a l i b r a t i o n b a r s = 200u ). - 49a -PLATE 8 M e t h y l m e t h a c r y l a t e c a s t s o f g i l l l a m e l l a e . 1 t o 5 a r e a s e r i e s t o show th e v a r i o u s s hapes and s i z e s o f l a m e l l a e . They p r o g r e s s from a b a s a l f i l a m e n t l o c a t i o n (1) Lo a d i s t a l f i l a m e n t l o c a t i o n (b). a = a f f e r e n t ; e = e f f e r e n t . 6, 7 and 8 a r e the a f f e r e n t , c e n t r a l / m a r g i n a l and e f f e r e n t r e g i o n s o f a l a m e l l a , r e s p e c t i v e l y , i n more d e t a i l ( M a g n i f i c a t i o n s : 1 = 74x, 2 - 64x, 3 = 66x, 4 = 74x, 5 = 61x, 6 - 300x, 7 = 107.6x and 8 = 582x. C a l i b r a t i o n b a r s : 1 t o 5 = 200 y , 6 = 50 , 7 = 10 M and 8 = 2 0 y ) . A b b r e v i a t i o n s a r e e x p l a i n e d i n l e g e n d f o r P l a t e 3. PLATE 9 H i s t o l o g i c a l c r o s s s e c t i o n s Of the g i l l f i l a m e n t i n more d e t a i l t h a n shown i n P l a t e . 3. A - t h e a f f e r e n t s i d e , B and C t h e e f f e r e n t s i d e o f t h e f i l a m e n t . (A and C = 155x, b a r = 100u : B = 650x, b a r = 20u ). A b b r e v i a t i o n s a r e e x p l a i n e d i n l e g e n d f o r P l a t e 3. - 52 -PLATE 10 A methyl methacrylate c o r r o s i o n c a s t of a g i l l f i l a m e n t r e v e a l i n g the g e n e r a l arrangement of the v a s c u l a t u r e . Some l a m e l l a e have been removed to r e v e a l the s t r u c t u r e of the venolymphatics ( v l ) and c e n t r a l s i n u s (cs) which l i e i n between the opposing l a m e l l a e . In C note the stubs of the ECV to CS connecting v e s s e l s because the ECV was removed. ( M a g n i f i c a t i o n s : A - 67x, B - 121x and C = 188x. C a l i b r a t i o n s bars A = 200 y , B = 100 y and C = 50 y ) . A b b r e v i a t i o n s are e x p l a i n e d i n legend of P l a t e ' 3 i - 53 -PLATE 11 A d e t a i l e d v i e w , o f the c o r r o s i o n c a s t s o f the v e s s e l s on t h e a f f e r e n t s i d e of the f i l a m e n t . A/ A g e n e r a l v i e w . Note t h e p a r a l l e l s e r i e s o f c e n t r a l . v e n o l y m p h a t i c v e s s e l s c o n n e c t i n g t h e acv. t o t h e c s ( 73x, b a r = 20 0 u ) . B/ C l o s e up v i e w o f t h e o r i g i n o f t h e a f f e r e n t l a m e l l a r a r t e r i o l e s ( a l a ) i n t h e p r o x i m a l r e g i o n o f t h e AFA . Note t h e d o r s a l and v e n t r a l v e s s e l s have s e p a r a t e o r i g i n s and the s l i g h t t a p e r o f the v e s s e l towards i t s base (460x, b a r = 20 p ) . C/ As B above, b u t i n t h e d i s t a l r e g i o n o f t h e AFA. Note t h e s i n g l e p o i n t o f o r i g i n f o r d o r s a l and v e n t r a l ALAs. D/ A f r a c t u r e s e c t i o n ' o f t h e f i l a m e n t c a s t t o r e v e a l t h e d o r s a l and v e n t r a l a r r a n g e m e n t o f the p a i r e d ACVs. .(146x, b a r = lOOu ). E / The d i f f u s e network o f v e s s e l s i n t e r c o n n e c t i n g t h e p a i r o f main ACVs. T h i s was d i s s e c t e d o u t f r o m t h e AFA, w h i c h would n o r m a l l y l i e i n s i d e (392x, b a r = 20y ). - 54 -PLATE 12 A detailed view of the corrosion casts of the vessels on the efferent side of the filament. A/ A s i d e view of the. EFA to demonstrate the p a r a l l e l alignment of the main ECV and the meandering nature of smaller ECVs (200x, bar = 50y ). B/ A dorsal view of the EFA to show the nature and spacing pf the EFA to ECV connections. Note the indentations on the EFA are continued on this connection,' unlike the section of the ECV in A (146x, bar = 100y ). C/ Dorsal view of the filament to show the small vessels between the ECV and the central sinus (cs). Note these vessels: i n t e r d i g i t a t e between the ELAs (331x, bar = 50y ). - 55 -PL'ATE 13 A d e t a i l e d view of the 'corrosion c a s t s of the v e s s e l s i n the c e n t r a l r e g i o n of v the f i l a m e n t . The high degree of f i l l i n g of the c e n t r a l binus f i l l i n g was achieved by r e t r o g r a d e c a s t i n g procedures. \ A/ A f r a c t u r e s e c t i o n of the f i l a m e n t to demonstrate the la r g e p r o p o r t i o n s that the c e n t r a l s i n u s (cs) can a t t a i n (141x, bar = 100 y ) . • B/ A view of the d i f f u s e network of c e n t r a l venolymphatics ( c v l ) which l i e i n between and separate from the lamella. (lam) and the c e n t r a l s i n u s . (See a l s o P l a t e 11 A). Note the bas a l l a m e l l a r channel and the more d e f i n e d nature of the c v l that i s underneath (258x, bar = 50u ) . C/ A more d e t a i l e d view of the base of the l a m e l l a and the c v l than i n B (1132x, bar = 10U ). - 55a -- 56 -Lamellar c a s t s with s i l i c o n e elastomer over a range.of  s t a t i c transmural pressure g r a d i e n t s The v a s c u l a r space to t i s s u e r a t i o (VSTR) f o r lamellae 5 mm from the base of the f i l a m e n t d i d not change over the 20 to 70 cm transmural pressure range and has a mean value of 88.1% ± 0.3 (n=65 f o r 3 f i s h at 4 transmural pressures) (Fig.6) The p i l l a r c e l l shape changed somewhat depending upon i t s l o c a t i o n i n the l a m e l l a r sheet (Table I I , P l a t e 14) but the VSTR was not a f f e c t e d . 1,609 i n d i v i d u a l measurements of the l a m e l l a r v a s c u l a r sheet t h i c k n e s s were made. The mean sheet t h i c k n e s s , h, in c r e a s e d l i n e r a l y with Ap-^am w i t h i n the pressure . range 30 to 60 cm H2O ( F i g . 7 ) . The compliance c o e f f i c i e n t ( a ) from t h i s : . r e l a t i o n s h i p i s 0.07U .cm H 2 0 - 1 . The a. value was much * reduced at A P l a m v a l u e s i n excess of 60 cm H 20. At A P l a m values of 20 cm H2O a l l l a m e l l a r channels were not • f i l l e d i n some l a m e l l a e ( P l a t e 15), but only, lamellae with a l l ' channels f i l l e d were included i n the analysis.. No l a m e l l a channel was seen with a t h i c k n e s s l e s s than 5.2y. The sheet t h i c k n e s s i s non-uniform with r e s p e c t to the l a m e l l a r height ( F i g . 8 ) . There i s a p r o g r e s s i v e t h i c k e n i n g of the v a s c u l a r sheet at i n c r e a s i n g d i s t a n c e s from the l a m e l l a r basal .channel, which i s true at a l l the transmural pressures examined ( F i g . 8 ) . For a n a l y s i s , the l a m e l l a r channels were separated . i n t o 3 equal areas c o n t a i n i n g 4 to 8 l a m e l l a r channels, but the two most basal and the two most marginal channels were t r e a t e d - 57 -separately: hence the 5 lamellar regions in Figures 8 and 9 . The basal region includes some channels which l i e within the filament epithelium (Plates 4 and 1 5 ) . For each region the a value was dependent upon the absolute pressure, but for a given A P i a m , the a value varied between the 5 regions analysed (Fig. 9 ) . At a low AP-j_am the a value was generally-greatest in d i s t a l regions, but very low in the basal region. Thus the vascular sheet thickness increases p r e f e r e n t i a l l y in d i s t a l regions of the lamella when AP j a m is increased from 20 to.50 cm H 2 0 . Over a 20 to 70 cm H 2 0 A P l a m change, the increase in the absolute values for vascular thickness was about the same in a l l the five regions examined (Fig. 8 ) . In some sections the filament a r t e r i e s were measured in cross-section. The diameter measurements at the two extreme pressures (20 and 70 cm H 2 O ) in the three -fish examined were not s i g n i f i c a n t l y d i f f e r e n t (Table I I ) . FIGURE (. Mean v a l u e s f o r t h e l a m e 1 J a r v a s c u l a r t i s s u e suace r a t i o , VSTR, i n t h r e e f i s h (0 = 3.3 k g , A = 3.6 k g , O = 3.8 kg w e i g h t ) o v e r a w i d e r a n g e o f t r a n s m u r a l p r e s s u r e s , A P j _ a m . V e r t i c a l b a r s r e p r e s e n t s t a n d a r d e r r o r s o f e a c h p o i n t ( o n l y t h e maximum r a n g e i s i n d i c a t e d ) and t h e number o f o b s e r v a t i o n s f o r e a c h p o i n t i s i n d i c a t e d a l o n g s i d e . The h o r i z o n t a l l i n e i s t h e mean v a l u e o f 8 8 . 1 % f o r a l l t h e VSTR v a l u e s . 1 - . 59 -TABLE I I A. . The v a r i a t i o n i n p i l l a r c e l l c r o s s s e c t i o n a l a r e a e x p r e s s e d as p l a n a r a r e a . The p i l l a r c e l l s were g r o u p e d a c c o r d i n g t o t h r e e l o c a t i o n s on e a c h l a m e l l a e : a f f e r e n t , c e n t r a l and e f f e r e n t . B. The mean d i a m e t e r s o f the a f f e r e n t and e f f e r e n t f i l a m e n t a r t e r i e s f r o m s i l i c o n e e l a s t o m e r c a s t s a t two t r a n s m u r a l p r e s s u r e s o f 20 and 70 cm H2O. The measurements were made f r o m h i s t o l o g i c a l s e c t i o n s t a k e n 5.0 mm from t h e base o f f i l a m e n t s 10 t o 20 mm l o n g t a k e n from 3 l i n g c o d . The. measurements a t e a c h p r e s s u r e a r e n o t s i g n i f i c a n t l y d i f f e r e n t . - 59a -TABLE IIA P i l l a r c e l l l o c a t i o n area ( y 2 ) ± s.e (n) A f f e r e n t 18.4 ± 1.1 (16) C e n t r a l 23.0 ± 0.8 (29)* E f f e r e n t 21.6 ± 1.2 (14)* * I n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e (95 % C.I.) from the mean val u e f o r the a f f e r e n t l o c a t i o n . TABLE IIB Transmural p r e s s u r e , cm H 0 A r t e r y diameter, y , ± s.e.(n) a f f e r e n t f i l a m e n t e f f e r e n t f i l a m e n t 20 218 ±2.6 (59) . . 2 0 5 + 4 . 1 (30) 70 214 ±5.8 (62) 198 ± 4.3 (62) - 60 -PLATE 14 '• H i s t o l o g i c a l p l a n views of g i l l l a m e l l a e as used for. VSTR a n a l y s i s . Note the r e g u l a r arrangement of p i l l a r c e l l s (pc) , but that the p a t t e r n and s i z e of the p i l l a r c e l l s v a r i e s with the r e g i o n of the l a m e l l a s e l e c t e d A = c e n t r a l , B = a f f e r e n t r e g i o n of the l a m e l l a . (725x, bar = 20u ). - 60a -- b l FIGURE 7 Mean values ± s.e. of l a m e l l a r vascular sheet thickness measurements, h, at d i f f e r e n t transmural pressures, A P l a m . The re g r e s s i o n l i n e i s f o r h values f o r A P l a m values between 30 and 60 cm II2O only and i s given by h = 8.31 + 0.07 A p i a m For an explanation of the other symbols see the legend of Figure 6. - 61a -- 52 F I G U R E '6 Mean l a m e l l a r vascular sheet t h i c k n e s s , h, f o r 5 la m e l l a r regions at 3 d i f f e r e n t transmural pressures, 2U, and 70 cm H 2 O . Note how c l o s e l y the l i n e s f o r 20 and 70 cm H 20 p a r a l l e l each other, while at 50 cm H 20 the shape i s much d i f f e r e n t . (See d i s c u s s i o n f o r explanation) • = basal 2 channels o = basal area • = marginal 2 channels A = c e n t r a l area • = d i s t a l area V e r t i c a l bars represent ± s.e. 18 Lamellar central free base area lamellar edge - 6 3 FIGURE '.) * Changes i n mean l a m e l l a r vascular sheet l a m e l l a r t h i c k n e s s , h, v/ith transmural pressure, APj_ a m, f o r d i f f e r e n t regions of the l a m e l l a . This demonstrates the v a r i a t i o n of the compliance c o e f f i c i e n t , a , with the region of the filament and absolute pressure, since a represents the gradient of each l i n e . V e r t i c a l bar represent ± s.e. 1 basal channels marginal channels distal area central area basal area 1 0 3 0 5 0 7 0 AP , c m H 2 0 - 64 P L A T E 15 H i s t o l o g i c a l c r o s s s e c t i o n s o f g i l l l a m e l l a e t o s how t h e s i z e o f t h e b l o o d c h a n n e l s . T h e v e s s e l s ' w e r e c a s t u n d e r d i f f e r e n t t r a n s m u r a l p r e s s u r e s ( A P i a m ) , A = 20 cm H 2 0 B = 40 cm H 2 0 a n d C = 60 cm H 2 0 . N o t e t h a t some v e s s e l s a r e c o l l a p s e d ( a r r o w s ) a t 20 cm H 2 0 a n d t h a t t h e v e s s e l s a r e l a r g e a t h i g h e r A P j _ a m . ( 7 2 5 x , b a r = 20 u ) . - 64a -- 65 -The geometry of the b r a n c h i a l v e s s e l s The b r a n c h i a l v a s c u l a t u r e i s a complex network. These r e s u l t s are based on s e l e c t e d measurements which I b e l i e v e are r e p r e s e n t a t i v e samples that can be e x t r a p o l a t e d to the whole g i l l . The f i l a m e n t was considered as a u n i t with v a r i a b l e length S i m i l a r l y , l a m e l l a r u n i t s were considered to vary with r e s p e c t to t h e i r p o s i t i o n on a f i l a m e n t and the o v e r a l l l e n g t h of the f i l a m e n t on which they were found. The geometry of the g i l l v a s c u l a t u r e i s summarised i n T a b l e . I I I . The data are drawn l a r g e l y from measurements made on one c o r r o s i o n cast of a 4 kg l i n g cod. The l a m e l l a r v a s c u l a r sheet t h i c k n e s s data are drawn from the s i l i c o n e c a s t i n g experiments. Lamellar s u r f a c e area: The pl a n a r v a s c u l a r s u r f a c e area of i n d i v i d u a l l a m e l l a e i s g r e a t e r on longer f i l a m e n t s ( F i g . 10) and the t o t a l l a m e l l a r s u r f a c e area i n c r e a s e s with f i l a m e n t length ( F i g . 11) The area of i n d i v i d u a l l amellae i s a l s o dependent upon ^ / i t s ^ p o s i t i o n on the f i l a m e n t . However, the l a m e l l a r area i s almost l i n e a r l y d i s t r i b u t e d along the f i l a m e n t l e n g t h , r e g a r d l e s s of the absolute f i l a m e n t length ( F i g . 1 2). Even' though f i g u r e 12 d i s p l a y s a s l i g h t l y "S" shaped r e l a t i o n s h i p , 60% of the t o t a l l a m e l l a r area i s found on the proximal 60% of the f i l a m e n t . The t o t a l g i l l l a m e l l a r v a s c u l a r s u r f a c e area f o r a 4 kg l i n g cod i s 1.38 m2 or 345 mm2.g~i. Th i s value assumes a t o t a l number of f i l a m e n t s of 3760 (Fig.. 2); a mean - 66, -f i l a m e n t l e n g t h f o r the g i l l o f 14.4 mm ( F i g . 3) and a t o t a l l a m e l l a r s u r f a c e a r e a f o r a 14.4 mm f i l a m e n t o f 367 mm^  ( F i g . 1 1 ) . T h i s a r e a v a l u e i s b a s e d on p l a n a r s u r f a c e a r e a s , b u t t h e p e r i p h e r a l c h a n n e l s a r e , however, r o u n d e d . The e f f e c t o f the p e r i p h e r a l v e s s e l c u r v a t u r e was e s t i m a t e d by t r a c i n g t h e o u t s i d e o f t h e v e s s e l s i n P l a t e 15, w h i c h i s r e p r e s e n t a t i v e o f a m i d - l a m e l l a r c r o s s - s e c t i o n . The p l a n a r s u r f a c e a r e a was i n c r e a s e d by 6%, b u t t h i s p e r c e n t a g e w o u l d . c l e a r l y v a r y f r o m l a m e l l a t o l a m e l l a , s i n c e the o v e r a l l shape o f l a m e l l a e v a r i e s . L a m e l l a r volume: T h i s was c a l c u l a t e d f r o m t h e w e i g h t s o f c o r r o s i o n c a s t s . The t o t a l l a m e l l a r u n i t volume f o r a f i l a m e n t i n c r e a s e s p r o p o r t i o n a t e l y w i t h t h e f i l a m e n t volume and i s 63% o f t h e l a t t e r ( F i g . 1 3 ) . Both volumes i n c r e a s e e x p o n e n t i a l l y w i t h f i l a m e n t l e n g t h . The f i l a m e n t volume combines l a m e l l a r , a r t e r i a l and v e n o l y m p h a t i c v o l u m e s . T h e . t o t a l volume o f a l l the l a m e l l a r u n i t s o f a 4 kg l i n g cod was 3.34 m l . T h i s v a l u e was d e r i v e d f r o m f i g u r e 13 where the t o t a l l a m e l l a r volume f o r a 14.4 mm f i l a m e n t = 0.9 ^ x l ( F i g . 1 3 ) . L a m e l l a r volume was a l s o e s t i m a t e d f r o m t h e i r g e o m e t r y . T o t a l l a m e l l a r volume i n t h i s c a s e was 4.85 ml ( T a b l e I V ) . Assuming o n l y 60% o f l a m e l l a e a r e p e r f u s e d a t r e s t ( S e c t i o n I I I ) and a r e s t i n g c a r d i a c o u t p u t o f 0.8 m l . s e c - 1 ( S e c t i o n I I ) , t h e t r a n s i t time o f b l o o d t h r o u g h the l a m e l l a e i s c a l c u l a t e d t o be between 2.5 and 3.6 s e c o n d s , f o r t h e e s t i m a t e d and weighed l a m e l l a volumes r e s p e c t i v e l y . - 67 -A f f e r e n t f i l a m e n t a r t e r y : The d i a m e t e r o f the base of the AFA i n c r e a s e s l i n e a r l y on f i l a m e n t s o f i n c r e a s i n g l e n g t h , but a d o u b l i n g i n d i a m e t e r i s a c c o m p a n i e d by a q u a d r u p l i n g o f t h e l e n g t h ( F i g . 1 4 a ) . The AFA a r t e r y a l s o t a p e r s a l o n g i t s l e n g t h . The r e l a t i o n s h i p between t h e % d e c r e a s e i n AFA d i a m e t e r a l o n g t h e f i l a m e n t l e n g t h i s d i s p l a y e d i n F i g . 14b. The r e l a t i o n s h i p i s n o n - l i n e a r . T h e r e i s a l m o s t no t a p e r o v e r th e p r o x i m a l 20 t o 30% o f the f i l a m e n t . A t a l o c a t i o n 60% a l o n g t h e f i l a m e n t l e n g t h t h e AFA has t a p e r e d by 10-30% ( F i g . 1 4 b ) . - 6 8 TABLE I I I The geometry of the g i l l v e s s e l s i n a 4 kg l i n g c od, Ophiodon e l o n g a t u s • The d a t a were d e r i v e d from measurements made on a methyl m e t h a c r y l a t e c o r r o s i o n c a s t of the b r a n c h i a l b a s k e t . F i l a m e n t numbers were counted and l a m e l l a r numbers were e x t r a p o l a t e d f o r a mean f i l a m e n t l e n g t h of 14.4 mm u s i n g f i g u r e 4. TABLE I I I V e s s e l Diameter Number ' (y)  Length T o t a l X sectianal,. Volume (cm) area ( cm ) (ml) V e n t r a l Aorta 400 0 3.0 0.126 0.378 A f f e r e n t B r a n c h i a l A r t e r y 2500 5.0 0.393 1.97 A f f e r e n t Arch A r t e r y 700 16 7.5 0.0616 0.462 A f f e r e n t Filament A r t e r y 200 3760 1.44 1.18 1.70 A f f e r e n t Lamellar A r t e r i o l e 20 1.94 x 10 0.045 6.10 0.274 Lamella 60% 100% 10 10 1.16 x 10 0.1 1.94 x 10 6 0.1 29.0 48 .5 2.9 4.85 E f f e r e n t Lamellar A r t e r i o l e 28 1.94 x 10 6 0.009 1.19 0.010 E f f e r e n t Filament A r t e r y 190 3760 1.5 1.07 1.61 E f f e r e n t Arch A r t e r y 70 0 16" 7.5 0.0616 0.462 A f f e r e n t B r a n c h i a l A r t e r y 2000 5.0 0.251 1.25 - b9 FIGURE_10 The p l a n a r v a s c u l a r s u r f a c e a r e a o f l a m e l l a e f o r a range o f f i l a m e n t l e n g t h s . Each p o i n t r e p r e s e n t s a d e t e r m i n a t i o n f o r an i n d i v i d u a l l a m e l l a a t a measured d i s t a n c e f r o m the f i l a m e n t b a s e . The f i l a m e n t s were t a k e n from t h e 2nd g i l l a r c h o f 4 kg l i n g c o d . N.B. The e f f e c t o f m a r g i n a l v e s s e l c u r v a t u r e would be t o i n c r e a s e s u r f a c e a r e a v a l u e s by a b o u t 6% (See P . 6 7 ) . Distance from base of filament (mm) - 7 u F I G U R E 11 The t o t a l l a m e l l a r vascular surface area (planar) for the complete range of filament lengths found on a 4 kg l i n g cod. This r e l a t i o n s h i p was e s t a b l i s h e d using an i n t e g r a t i o n of the r e l a t i o n s h i p s showing i n F i g . 7 , plus other s i m i l a r r e l a t i o n s h i p s not included i n that graph. The l i n e was f i t t e d by eye. N.B. The e f f e c t of the marginal v e s s e l curvature would be to increase the areas by approximately 6%. FIGURE 12 R e s u l t s of l a m e l l a r surface areas from F i g . 10 and others (n • = 9 f i l a m e n t s of d i f f e r e n t l e n g t h s ) , that are expressed as percentages. Note the s l i g h t "S" shape r e l a t i o n s h i p , but t h a t 60% of the t o t a l l a m e l l a r area i s found i n the proximal 60% of the f i l a m e n t l e n g t h . Data were taken from a l l four f i l a m e n t s , which show a s i m i l a r r e l a t i o n s h i p f o r % area to % l e n g t h . J£TOTAL LAMELLAR SURFACE AREA o I 3 X < V - s-U -- 72 -F I G U R E 13 The e x p o n e n t i a l r e l a t i o n s h i p between t o t a l f i l a m e n t volume and t o t a l l a m e l l a r u n i t volume w i t h r e s p e c t t o f i l a m e n t l e n g t h . The r e s u l t s a r e f rom w e i g h t s o f p l a s t i c c a s t s and e a c h p o i n t r e p r e s e n t s a d i f f e r e n t f i l a m e n t . L e n g t h r e f e r s t o t h e combined l e n g t h s o f two f i l a m e n t s w i t h e q u a l l e n g t h t h a t o p p o s e d each o t h e r on t h e a n t e r i o r and p o s t e r i o r h e m i b r a n c h s (See M a t e r i a l s and M e t h o d s ) . The l i n e s were f i t t e d by e y e . 16 14-12-10-8 6-Q • Filament volume • • Lamella volume only 10 "20 30 40" Total length of f i laments (mm) - 73 -F I G U R E 14 ' : ' The geometry of the afferent filament artery from vascular corrosion casts. A/ The diameters of the base of vessels versus their length (n = 17 filaments), to show that shorter filaments have narrower afferent filament arteries at the i r base. B/ The afferent filament artery tapers along i t s length in a non-linear fashion.. Over the proximal 20 to 30% of i t s length there is l i t t l e or no taper. The amount the vessel has tapered by the position 60% along i t s length is indicated by the broken l i n e , i.e. there has been only a 10 to 30% decrease in i t s dimensions. - 7 3 a -- 74. -DISCUSSION The p a t t e r n of r e s p i r a t o r y v a s c u l a r pathways i n l i n g cod g i l l s d i f f e r s l i t t l e from those p r e v i o u s l y d e s c r i b e d f o r other t e l e o s t s (See I n t r o d u c t i o n f o r r e f e r e n c e s ) . Ling cod, i f anything, have a l e s s complex v e s s e l arrangement than other t e l e o s t s . For i n s t a n c e , i n l i n g cod the AFA i s simply tapered and round i n c r o s s - s e c t i o n , whereas ampullae are present i n carp and t r o u t ( H y r t l , 1838; R i e s s , 1881; Campbell et a_l. , i n prepar-a t i o n ) . Furthermore, there are no connections between the AFA and c e n t r a l sinus which have been p r e v i o u s l y reported f o r A n g u i l l a a n g u i l l a (Steen and Kruysse, 1964; Dunel and Laurent, 1977). These d i f f e r e n c e s h i g h l i g h t some of the sp e c i e s v a r i a t i o n amongst t e l e o s t s . A s t r i k i n g d i f f e r e n c e between l i n g cod and t r o u t i s i n the nature of the venolymphatic arrangement. In t r o u t s m a l l , meandering c a p i l l a r i e s connect the EFA with both the ECV and the c e n t r a l sinus (Campbell et a l . , i n p r e p a r a t i o n ; Vogel et a1. , 1976; Vogel, 19 78). By c o n t r a s t Ophiodon have r e g u l a r but s h o r t EFA to ECV connections, which have a l a r g e r diameter and a l s o possess a smooth muscle coat. My f i n d i n g s support the gene r a l o b s e r v a t i o n that marine f i s h have b e t t e r developed venolymphatic network than freshwater f i s h (Steen and Kruysse, 19 64) . A simple f u n c t i o n a l summary of the v a s c u l a r flow through l i n g cod g i l l s i s now proposed. (See P l a t e 16 f o r a composite diagram of the v a s c u l a r pathways i n the g i l l f i l a m e n t ) . A l l the c a r d i a c output passes through the g i l l l a m e l l a e . At the - 75 PLATE 16 A s c h e m a t i c c r o s s s e c t i o n through the g i l l f i l a m e n t to demonstrate the arrangement of the v a s c u l a t u r e c o n t a i n e d w i t h i n . Blue r e p r e s e n t s venous blood i n the r e s p i r a t o r y v a s c u l a t u r e and red r e p r e s e n t s a r t e r i a l b lood i n the same system. The v e n o l y m p h a t i c s are i n y e l l o w . The f i l a m e n t support c a r t i l a g e i s c o l o r e d green. L A M E L L A E F F E R E N T F I L A M E N T A R T E R Y c o m p a n i o n vessels efferent lamellar arteriole pillar ce l l s afferent lamellar arteriole A F F E R E N T F I L A M E N T A R T E R Y c o m p a n i o n v e s s e l • WATER FLOW - 7b - . • • • . . l a m e l l a e t h e r e w i l l be some lymph f o r m a t i o n which d r a i n s v i a e x t r a c e l l u l a r c h a n n e l s between the p i l l a r and e p i t h e l i a l c e l l s i n t o t h e c e n t r a l s i n u s . O x y g e n a t e d b l o o d e n t e r s t h e . e f f e r e n t f i l a m e n t a r t e r y and t h e n c e t o t h e e f f e r e n t , a r c h a r t e r i e s , . The e f f e r e n t f l o w f r o m the g i l l a r c h e s may t a k e one o f s e v e r a l r o u t e s d e p e n d i n g on w h i c h a r c h i s c o n s i d e r e d . In a l l a r c h e s f l o w can p a s s i n t o the d o r s a l a o r t a , , w i t h l e s s e r amounts p a s s i n g t o the head r e g i o n ( i n c l u d i n g t h e p s e u d o b r a n c h s ) and i n t o the h y p o b r a n c h i a l s u p p l y . A p o r t i o n o f the o x y g e n a t e d b l o o d i n t h e e f f e r e n t f i l a m e n t a r t e r y a l s o e n t e r s t h e main e f f e r e n t companion v e s s e l s . T h i s o x y g e n a t e d b l o o d i s d i s t r i b u t e d t o the f i l a m e n t , and a r c h t i s s u e s and r e t u r n s d i r e c t l y t o the s i n u s v e n o s u s . The c e n t r a l s i n u s i s d r a i n e d v i a the a f f e r e n t companion v e s s e l s . The v e n o l y m p h a t i c s y s t e m r e t u r n s d i r e c t l y t o the h e a r t . How f l o w i s g e n e r a t e d i n t h e a f f e r e n t companion v e s s e l i s p r o b l e m a t i c s i n c e t h e r e a r e a t l e a s t t h r e e h i g h r e s i s t a n c e ( i . e . narrow) c h a n n e l s between t h e . e f f e r e n t f i l a m e n t a r t e r y and ACV. L i k e mammalian l y m p h a t i c s , f l o w i s p r o b a b l y d r i v e n by a r t e r i a l p u l s a t i o n s and f i l a m e n t movements and i t i s a i d e d by n o n - r e t u r n v a l v e s . V e n o - l y m p h a t i c f l o w i s d i s c u s s e d i n more d e t a i l i n S e c t i o n I I . L a m e l l a r b l o o d f l o w Two i n d e p e n d e n t r e l a t i o n s h i p s were d e r i v e d f o r the l a m e l l a r v a s c u l a r s h e e t . . F i r s t , mean l a m e l l a r v a s c u l a r s h e e t . t h i c k n e s s (h) v a r i e s d i r e c t l y w i t h the t r a n s m u r a l p r e s s u r e ( A P i a m ) over a t y p i c a l i n v i v o pressure range (h - 8.36 + 's • 0.07 A p l a m ) . Second, v a s c u l a r space to t i s s u e r a t i o (VSTR) does not vary with A p l a m . By d e r i v i n g these equations •. i t i s now evident that the l a m e l l a r v a s c u l a r sheet i s q u i t e compliant but, at the same time, a n i s o t r o p i c . In a d d i t i o n , the l a m e l l a r v a s c u l a r sheet behaves in a manner p r e d i c t e d by sheet blood flow theory (Fung and Sobin, 1969). The s i m i l a r i t i e s between c a p i l l a r y s t r u c t u r e and blood flow i n the lungs and g i l l s are now confirmed. In f a c t the two v a s c u l a r beds appear more a l i k e than p r e v i o u s l y thought s i n c e blood flow i n both i s best d e s c r i b e d as a sheet flow. The 88.1% VSTR f o r l i n g cod compares w e l l with the 91% f o r mammalian a l v e o l i (Sobin et. al_. , 1970). A r a t i o , of 80% was determined f o r f r o g s by Maloney and C a s t l e (1969) u s i n g - d i f f e r e n t methods. The p o s s i b i l i t y now e x i s t s that sheet blood flow i s found i n the r e s p i r a t o r y c i r c u l a t i o n of a l l v e r t e b r a t e s . The compliance of the l a m e l l a r v a s c u l a r sheet i s high ( c t = 0 . 0 7 H .cm h^O--1-)', and i s comparable to the a l v e o l a r sheet in. greyhound lungs (21 to 31 kg), where a = 0.079y .cm H 2 0 _ 1 when A p l a m = 25 cm H 20. The cat a l v e o l a r sheet i s even more compliant (a = ,0.21-y .cm H 2 0 - 1 ) . The h value f o r the lamellae of l i n g cod (about 3.5 kg) i s g r e a t e r than that f o r lung a l v e o l i of cats (3 to 5 kg) (Sobin e t ' . a l . , 1972 ). There i s a l s o l e s s r e s i s t a n c e to blood flow i n the b r a n c h i a l c i r c u l a t i o n than i n the pulmonary or other systemic v a s c u l a r beds. These two f a c t s are no doubt r e l a t e d , s i n c e r e s i s t a n c e i s i n v e r s e l y p r o p o r t i o n a l to h 4 . A g i l l l a m e l l a has a non-uniform v a s c u l a r sheet t h i c k n e s s and the g r e a t e s t h values are a s s o c i a t e d with d i s t a l l a m e l l a r r e g i o n s . Since ot values f o r each region of the l a m e l l a are dependent upon the absolute A.Pi a m , h does not i n c r e a s e u n i f o r m l y from r e g i o n to r e g i o n as A P i a m r i s e s . T h i s s i t u a t i o n i s more complex than that f o r lungs, where h i s uniform f o r a given a l v e o l u s . The a value has a constant value i n cat lungs, but does however vary with A P a^ v i n greyhound lungs: at 25 cm H 20, a = 0.079U .cm H 2 0 - 1 and: at 10 cm H 20, a= 0.12 y.cm H 2 0 _ 1 ( G l a z i e r et a l . , 1969). The non-unifOrm p r o p e r t i e s , of the g i l l l a m ellae have some important consequences with r e s p e c t to i n t r a l a m e l l a r blood flow p a t t e r n s . C o n s i d e r i n g only p a s s i v e d i s t r i b u t i o n s , of flow, blood flow w i l l always be g r e a t e s t i n c e n t r a l and d i s t a l regions of the l a m e l l a e s i n c e h i s g r e a t e s t here f o r a l l A p l a m , ( F i g . 8 ) . The e f f e c t of r a i s i n g blood pressure w i l l be to a l t e r i n t r a l a m e l l a r flow p a t t e r n s (Table IV). F l u c t u a t i o n s i n ' b l o o d ' pressure w i l l a l t e r A P i a m f s i n c e blood pressures are about 50 times those p r e s s u r e s a s s o c i a t e d with normal g i l l v e n t i l a t i o n . In Table IV the. p r o p o r t i o n of blood flow to the basal areas i s compared with the remainder of the l a m e l l a . These c a l c u l a t i o n s * show that i f A P i a m i s increased and flow ( i . e . Q) i s r a i s e d , then blood flow i s shunted p r e f e r e n t i a l l y to the c e n t r a l and d i s t a l r e g i o n s . I n t r a l a m e l l a r shunting of blood flow can. t h e r e f o r e occur a u t o m a t i c a l l y as a r e s u l t of e l e v a t e d Q and blood - 79 -p r e s s u r e s . . I t can now be s e e n how . e l e v a t e d Q. and b l o o d p r e s s u r e s can i n c r e a s e the d i f f u s i n g c a p a c i t y o f f i s h . F i r s t t h r o u g h i n t r a l a m e l l a r s h u n t i n g o f b l o o d t o r e g i o n s o f the l a m e l l a a s s o c i a t e d w i t h r e d u c e d d i f f u s i o n d i s t a n c e s . Gaseous exchange i s d i f f u s i o n l i m i t e d and t h e d i f f u s i o n b a r r i e r s a r e w e l l documented (Hughes and P e r r y , 1976; P i i p e r e t a_l. , 19 7 7 ) . Between the b l o o d and e n v i r o n m e n t t h e r e a r e t h r e e b a r r i e r s . One o f t h e s e , t h e e p i t h e l i a l t h i c k n e s s , c l e a r l y i n c r e a s e s s i g n i f i c a n t l y a t the base o f t h e l a m e l l a , p r e s u m a b l y as s u p p o r t f o r t h e l a m e l l a f o l d . F u r t h e r m o r e , up t o 5 l a m e l l a r c h a n n e l s may be b u r i e d i n t h e f i l a m e n t e p i t h e l i u m ( P l a t e s 4 and 1 5 ) . The d i f f u s i o n d i s t a n c e s a r e t h u s much g r e a t e r i n b a s a l r e g i o n s t h a n i n d i s t a l r e g i o n s . I n t r a l a m e l l a r s h u n t i n g : i s t h e r e f o r e d i r e c t e d away from r e g i o n s o f h i g h d i f f u s i o n d i s t a n c e s and hence the o v e r a l l g i l l d i f f u s i o n c a p a c i t y w i l l be r a i s e d . A l t h o u g h th e e s t i m a t e d q u a n t i t y o f b l o o d s h u n t e d may a p p e a r s m a l l ( T a b l e I V ) , t h e i n c r e a s e s i n d i f f u s i n g c a p a c i t y w i l l be more p r o n o u n c e d . The m a r g i n a l c h a n n e l has a much l a r g e r ' f r e e ' s u r f a c e a r e a t h a n o t h e r l a m e l l a r c h a n n e l s ( t h e c u r v a t u r e o f t h e m a r g i n a l c h a n n e l i n c r e a s e s the p l a n a r s u r f a c e a r e a o f t h e l a m e l l a by a t l e a s t 6 % ) . S e c o n d l y , when A P ^ a m i s r a i s e d and h i n c r e a s e s . , the d i f f u s i o n b a r r i e r t h i c k n e s s must d e c r e a s e by. a C o r r e s p o n d i n g amount b e c a u s e t h e VSTR does not change. The VSTR i s a measure o f t h e p i l l a r c e l l s i z e i n r e l a t i o n t o the v a s c u l a r s h e e t d i m e n s i o n s . S i n c e the p i l l a r c e l l d i m e n s i o n s do not change w i t h A p ^ a m , t h e n th e d i f f u s i o n b a r r i e r must a l t e r t o accommodate - 80 T A B L E JV A s u m m a r y o f t h e c h a n g e s i n f l o w t h a t c a n o c c u r w i t h o u t a n y c h a n g e i n r e s i s t a n c e when A ^ i a m ^ s r a i s e d . T h e b a s a l r e g i o n s a r e c o m p a r e d w i t h t h e r e m a i n d e r o f t h e l a m e l l a . F l o w was a s s u m e d t o be p r o p o r t i o n a l t o t h e f o u r t h p o w e r o f t h e v a s c u l a r s h e e t t h i c k n e s s , a n d h v a l u e s w e r e t a k e f r o m f i g u r e 3 . T h e s e c o n d c o l u m n s u m m a r i s e s how t h e s e f l o w c h a n g e s a r e r e g i o n a l l y d i s t r i b u t e d . - 80a -T A B L E I V increase i n D i s t r i b u t i o n absolute flow of flow % •AP, ,cm H„0 lam . 2 20 50 70 20 50 70 Basal reg ions 0 52 122 40 31 36 C e n t r a l , d i s t a l and marginal regions 0 115 156 60 69 64 increases, i n v a s c u l a r sheet t h i c k n e s s . T h i r d l y , o v e r a l l l a m e l l a r blood volume i s increased with e l e v a t e d ^Piam a n c J Q* T h i s i n \ i t s - e l f i n c r e a s e s the l a m e l l a r d i f f u s i n g c a p a c i t y . Danzer et. a l . (1968) c o n s i d e r the incr e a s e d aveolar. c a p i l l a r y volume the major reason f o r an el e v a t e d d i f f u s i n g c a p a c i t y of the human lung during e x e r c i s e . Although they c o n s i d e r changes i n membrane t h i c k n e s s of l i t t l e importance i n t h i s r e s p e c t , lung membranes are extremely t h i n . The l a m e l l a r d i f f u s i o n b a r r i e r , however, may be 10 f o l d that of the a l v e o l i and i n f i s h , membrane changes c l e a r l y have a g r e a t e r p o t e n t i a l to a l t e r d i f f u s i o n c a p a c i t a n c e , as presented above. These three mechanisms which increase g i l l d i f f u s i n g c a p a c i t y with e l e v a t e d Q and blood pressure may, t h e r e f o r e , enable e x e r c i s i n g f i s h to in c r e a s e i n oxygen uptake 5 f o l d , when g i l l s u r f a c e area i s only increased by 50%. The r e l a t i v e importance of each of the three mechanisms must s t i l l be e s t a b l i s h e d . C l e a r l y i n t r a l a m e l l a r shunting can occur p a s s i v e l y as a r e s u l t of changes i n AP]_ a m and flow to the l a m e l l a . Whether i n t r a l a m e l l a r shunting i s always a p a s s i v e (automatic) phenomenon needs to be d i s c u s s e d . I n t r a l a m e l l a r shunting has been suggested i n the past based on v i s u a l o b s e r v a t i o n s of l a m e l l a r flow (Steen and Kruysse, 1966; Richards and Fromm, 1969) and that p i l l a r c e l l s might be c o n t r a c t i l e and as a r e s u l t ' a c t i v e l y ' c o n t r o l l a m e l l a r blood flow ( B e t t e x - G a l l a n d and Hughes, 1973). This c o n c l u s i o n was based on the o r i e n t a t i o n of t h i n f i l a m e n t s i n p i l l a r c e l l s before and a f t e r ATP treatment (Hughes and Grimstone, 1965; - 82 - . . r • B e t t e x - G a l l a n d and Hughes, 1 9 7 3 ) . T h a t p i l l a r c e l l s c o n t r a c t has o f t e n been p o s t u l a t e d t o e x p l a i n i n c r e a s e d g i l l r e s i s t a n c e , but t h e a c t u a l l i k e l i h o o d o f i n v i v o p i l l a r c e l l c o n t r a c t i o n was q u e s t i o n e d by R i c h a r d s and Fromm ( 1 9 6 9 ) . I t i s a l s o q u e s t i o n e d h e r e . T h e r e i s no d i r e c t e v i d e n c e t h a t h umoral o r n e u r a l i n f l u e n c e s a f f e c t p i l l a r c e l l c o n t r a c t i l i t y i n v i v o and any f u t u r e s t u d i e s must show t h a t any changes i n l a m e l l a r d i m e n s i o n s and f l o w c a n n o t be a c c o u n t e d f o r by changes i n A P i a m . A c e t y l c h o l i n e has been c i t e d ( s p e c u l a t i o n , n o t d i r e c t o b s e r v a t i o n ) as h a v i n g a c o n t r a c t i l e e f f e c t on p i l l a r c e l l s ( S t e e n and K r u y s s e , 1 9 6 6 ) , b u t t h i s i s u n l i k e l y t o be o f i m p o r t a n c e i n v i v o b e c a u s e the p i l l a r c e l l s a r e n o t . i n n e r v a t e d (Hughes and G r i m s t o n e , 19'65; Newstead, 1965 and 1967; Gannon, 1 9 7 2 ) . A c e t y c h o l i n e r e l e a s e d i n t o t h e c i r c u l a t i o n i s r a p i d l y d e s t r o y e d by c h o l i n e s t e r a s e . O t h e r f a c t o r s l i k e oxygen l e v e l o r t h e l e v e l o f c i r c u l a t i n g c a t e c h o l a m i n e s may a f f e c t p i l l a r c e l l t e n s i o n , b u t t h i s has y e t t o be d e m o n s t r a t e d i n f i s h . A t p r e s e n t t h e r e i s no r e a s o n t o c o n c l u d e t h a t changes i n i n t r a l a m e l l a r f l o w a r e a n y t h i n g b u t p a s s i v e ( a u t o m a t i c ) r e s p o n s e s t o changes i n t r a n s m u r a l p r e s s u r e . L a m e l l a r b l o o d c h a n n e l s d i s p l a y t h e phenomenon, o f c r i t i c a l c l o s u r e a t low APi a m . .. The c o n c e p t o f c r i t i c a l c l o s u r e o f s m a l l v e s s e l s was f i r s t e s t a b l i s h e d by B u r t o n (1951) and N i c o l e t a l . ( 1 9 5 1 ) . I t has s i n c e been o b s e r v e d i n m e s e n t e r i c v e s s e l s by Lamport and Baez ( 1 9 6 2 ) , c o n t e s t e d . o n - 83 -t h e o r e t i c a l grounds by Peterson (1962) and demonstrated by Fung and Sobin (1972) i n pulmonary a l v e o l i . At a AP^ a m of about 20 cm H 20 many l a m e l l a e were observed with no s i l i c o n e i n some basa l and c e n t r a l channels of otherwise f i l l e d l a m e l l a e ( P l a t e 8). A l l l a m e l l a r channels had been p r e v i o u s l y exposed to a higher APj_ a m d u r i n g the p r e l i m i n a r y s i l i c o n e p e r f u s i o n and u n f i l l e d channels were never seen at a APY_am exceeding 30 cm H 20. These u n f i l l e d channels must have c o l l a p s e d at the low A P i a m . Wood (1974a) examined the c r i t i c a l c l o s i n g pressure f o r t r o u t g i l l s _in v i t r o , but i n c o r r e c t l y considered the d i f f e r e n c e s between input and output p r e s s u r e s i n s t e a d of AP^ a i n. Re-analysis of h i s r e s u l t s r e v e a l s that many g i l l p r e p a r a t i o n s d i s p l a y e d c r i t i c a l c l o s i n g at pressures of 15 to 25 H 20 f o r A P i a m , which i s i n agreement with iny work. Because v e s s e l s can c o l l a p s e at pre s s u r e s above ambient e x p l a i n s my - o b s e r v a t i o n that no v e s s e l s were open below h = 5.2u. S i m i l a r o b s e r v a t i o n s have been made f o r the mesentery and a l v e o l i . C r i t i c a l c l o s i n g p r e s s u r e s are a s s o c i a t e d with v e s s e l s not being open below 5u (mesentery; Lamport and Baez, 1962) and 2.5u ( a l v e o l i ; Fung and Sobin, 1972). Not a l l l a m e l l a e , nor a l l l a m e l l a r channels c o l l a p s e d at the o v e r a l l AP-^am of 20 cm H 20 ( F i g . 15). Thus not a l l v e s s e l s have the same c r i t i c a l c l o s i n g p r e s s u r e . The sigmoid curve of Figure 7 can now be e x p l a i n e d . There i s no sharp cut o f f at h•= 5.2u f o r two reasons. F i r s t , h i s a mean value of a non-uniformly t h i c k v a s c u l a r sheet. Second, only - 84 -c o m p l e t e l y f i l l e d l a m e l l a e were a n a l y s e d a t low AP-|_am and t h u s l a m e l l a e w i t h low c r i t i c a l c l o s i n g p r e s s u r e s were s e l e c t e d . A t h i g h a A P i a r a , . i n c r e a s e s i n h a r e l i m i t e d by t h e f i n i t e c u r v a t u r e o f t h e l a m e l l a r s h e e t (Fung and S o b i n , 1972). Whether c r i t i c a l c l o s u r e o c c u r s i n v i v o i s s p e c u l a t i v e , u n l i k e the mammalian s i t u a t i o n . C o l l a p s e d l a m e l l a r v a s c u l a r c h a n n e l s would be a d v a n t a g e o u s i n some s i t u a t i o n s e.g. o s m o t i c exchange w i t h the w a t e r c o u l d be m i n i m i s e d o r when l a m e l l a e were damaged o r p a r a s i t i s e d . T h a t t h e l a m e l l a r v a s c u l a r s h e e t can c o l l a p s e would a p p e a r t o be an i m p o r t a n t r e a s o n f o r m a i n t a i n i n g d o r s a l a o r t i c p r e s s u r e i n v i v o , and i n e x p e r i m e n t a l s i t u a t i o n s " i n v i t r o " . In c o n c l u s i o n , , s h e e t b l o o d f l o w t h e o r y can be a p p l i e d t o l a m e l l a r b l o o d f l o w i n f i s h g i l l s . I n t r a l a m e l l a r s h u n t i n g can o c c u r p a s s i v e l y as a c o n s e q u e n c e o f p r e s s u r e c h a n g e s . The. i m p o r t a n c e o f i n t r a l a m e l l a r s h u n t i n g i n t e l e o s t s i s t h a t t h e d i f f u s i o n c a p a c i t y f o r r e s p i r a t o r y exchange can be v a r i e d . _ 8 5 -i c t i o n s on s i t e s o f g i l l r e s i s t a n c e |; I t i s p o s s i b l e t o c a l c u l a t e t h e r e s i s t a n c e t o f l o w i n the t j i l l s and d e t e r m i n e s i t e s o f high; r e s i s t a n c e w i t h i n the g i l l v a s c u l a r n e t w o r k , based on t h e p r e v i o u s l y d e s c r i b e d m o r p h o l o g y and v e s s e l g e o m e t r y o f the g i l l s . S i t e s o f h i g h r e s i s t a n c e w i l l be i n f l u e n t i a l , i n t h e p a t t e r n o f b l o o d f l o w t h r o u g h t h e g i l l s . Fung and S o b i n (1977) have summarised the e q u a t i o n s t h e y d e v e l o p e d f o r d e t e r m i n i n g r e s i s t a n c e i n s h e e t f l o w s y s t e m s and t h e s e e q u a t i o n s can be a p p l i e d t o t h e l a m e l l a e . The e q u a t i o n s I have a d a p t e d a r e as f o l l o w s : L a m e l l a r r e s i s t a n c e , R i a m / i s g i v e n b y Rlam - 3 2 U , . , 2 , , 3 ( 1 ) h + h h + h h + h a a v a v v where h a and h v a r e t h e s h e e t t h i c k n e s s e s a t t h e a f f e r e n t and e f f e r e n t ends o f the l a m e l l a . and C = 4 n k £ L 2 a . ( 2 ) SA where . h= k i n e m a t i c v i s c o s i t y , k and f a r e c o n s t a n t s d e t e r m i n e d by the g e o m e t r y o f t h e p i l l a r c a l l s , 1. i s t h e a v e r a g e l e n g t h o f t h e l a m e l l a r c a p i l l a r y c h a n n e l s , a i s the c o m p l i a n c e c o e f f i c i e n t , S i s t h e VSTR v a l u e and A i s the l a m e l l a r a r e a . Flow, Q, i s g i v e n by Mean sheet t h i c k n e s s , h, i s gi.v-en by h = h Q + a A P l a m (4) where h Q - 8. 3 1 y and ^ = 0.07 y . c r n ^ O - ! f o r l i n g cod. Blood flow i n the remaining g i l l v e s s e l s , the a f f e r e n t and e f f e r e n t a r t e r i e s and a r t e r i o l e s , i s probably P o i s e u i l l i a n and can t h e r e f o r e be d e s c r i b e d by P o i s e u i l l e ' s equations. P o i s e u i l l e ' s equation s t a t e s that flow, Q, = AP r 4 IT 8L where AP i s the pressure drop along a v e s s e l of length L and of r a d i u s r . T h i s was v e r i f i e d by c a l c u l a t i n g 'a' f o r these . v e s s e l s . 'a' was l e s s than 0.8 where 'a' =\~2TUAand w = angular n frequency (McDonald., 1960). The r e l a t i o n s h i p between Q and r e s i s t a n c e i s complex because the v a s c u l a r system i s compliant. If input p r e s s u r e . i n c r e a s e s Q w i l l r i s e , but as the v e s s e l s are compliant they expand. So r e s i s t a n c e f a l l s as pressure r i s e s , s i n c e r e s i s t a n c e i s i n v e r s e l y p r o p o r t i o n a l to the f o u r t h power of the- v e s s e l r a d i u s . Thus i n t h i s a n a l y s i s I have assumed a constant c a r d i a c output - 44 m l . m i n - 1 (See s e c t i o n II) and a constant input pressure., where A P l a m = 45 cm H^O, to permit the c a l c u l a t i o n of pressure drops along v e s s e l s i n the g i l l s . At constant pressure and flow the g i l l r e s i s t a n c e i s i n v e r s e l y r e l a t e d to the pressure drop. In the case of the l a m e l l a e , r e s i s t a n c e and flow were c a l c u l a t e d d i r e c t l y from the dimensions of the g i l l s . . . ' 4 -The pressure drop between the v e n t r a l and d o r s a l aortae _ 87 -i s 13 cm H 20 _in v i v o (see s e c t i o n I I ) . T h i s value represents the sum of the pressure drops i n - % n d i v i d u a l v e s s e l s of the g i l l s . Below I have made a p a r t i a l account f o r t h i s pressure drop by making c a l c u l a t i o n s f o r the v e s s e l s of the g i l l f i l a m e n t o n l y . D e t a i l e d i n f o r m a t i o n of the values^used i n the c a l c u l a t i o n s ' i s • t \ presented i n Appendix I. The l a m e l l a e : In the equations d e a l i n g with sheet flow (1,2,3 and 4) o n l y . R i a m and h v are unknowns.• Assuming 60% l a m e l l a r p e r f u s i o n , h v was c a l c u l a t e d to be 11.44 u- and R l a m t o b e 7 , 6 5 x 1 0 3 cm H 20.min.ml - 1. Thus 10 . APlam °n the e f f e r e n t s i d e of the l a m e l l a was 44.71 cm H 20. Therefore the pressure drop across the lam e l l a e i s very s m a l l , 0.29 cm H 20 (45 to 44.71 cm H 20); From equation (1) the e f f e c t of r a i s i n g A P l a m from 45 to 65 cm H 20 on l a m e l l a r blood volume was c a l c u l a t e d . The hi would i n c r e a s e from i 11.44 to 12.86u , which represents a 12% in c r e a s e i n c a p i l l a r y • ' i i . • f ' . volume. A f f e r e n t f i l a m e n t a r t e r y : These v e s s e l s have, many le n g t h s and r a d i i i n any g i l l bed. As a r e s u l t not a l l f i l a m e n t s r e c e i v e the same amount of flow. As an AFA has a r t e r i o l e branches along i t s l e n g t h , flow w i l l d i m i n i s h along the l e n g t h . Furthermore the v e s s e l tapers along i t s l e n g t h . T h i s v a r i a b i l i t y hampers c a l c u l a t i o n ' o f AP f o r the AFAs. I t was t h e r e f o r e assumed that flow to any f i l a m e n t was dependent upon the number of lamellae 88 _ i t s u p p o r t e d . B l o o d f l o w t o any f i l a m e n t i s t h e n p r o p o r t i o n a l t o i t s l e n g t h s i n c e l a m e l l a e a r e e v e n l y s p a c e d ( F i g . 1 2 ) . V e s s e l r a d i u s i s a l s o d i r e c t l y r e l a t e d t o f i l a m e n t l e n g t h ( F i g . 1 4 ) . A r e p r e s e n t a t i v e AFA ( o r f i l a m e n t ) was s e l e c t e d t o r e p r e s e n t t h e g i l l bed and t h i s v e s s e l had a l e n g t h e q u i v a l e n t t o the mean f i l a m e n t l e n g t h f o r t h e whole g i l l . F o r a 4 kg l i n g cod t h e AFA was 14.4 mm i n l e n g t h and had.a 100 y r a d i u s . F u r t h e r m o r e t h e e f f e c t s o f v e s s e l t a p e r and f l o w l o s s t o b r a n c h i n g a r t e r i o l e s on v e s s e l r e s i s t a n c e n u l l i f y e a ch o t h e r , , a s s u m i n g 60% l a m e l l a r p e r f u s i o n ( A p p e n d i x I ) . The r e p r e s e n t a t i v e AFA used i n t h e s e c a l c u l a t i o n s was t h e r e f o r e c o n s i d e r e d t o ' b e o f a u n i f o r m d i a m e t e r . AP = 2.1 cm H^O f o r t h e a f f e r e n t f i l a m e n t a r t e r i e s u n der t h e above c o n d i t i o n s . The r e s i s t a n c e o f t h e r e p r e s e n t a t i v e AFA i s 1.84 x 1 0 2 cm H 2 0 . m i n . m l - 1 . The l a m e l l a r a r t e r i o l e s : The e f f e r e n t l a m e l l a r a r t e r i o l e s have a r e l a t i v e l y u n i f o r m l e n g t h and r a n g e o f d i a m e t e r s on any f i l a m e n t . Because o f the b u l b a r s w e l l i n g i n t h e ELA, i t s r a d i u s can ra n g e from 11 t o 1 5 y . By a s s u m i n g t h e m i n i m u m : r a d i u s f o r t h e v e s s e l an o v e r e s t i m a t e o f the p r e s s u r e d r o p was d e t e r m i n e d t o be 0.15 cm H 20 w i t h 60% o f l a m e l l a e p e r f u s e d . The r e s i s t a n c e o f t h e v e s s e l i s 3.95 x 10-^ cm H^O.min . m l - 1 . The a f f e r e n t l a m e l l a r a r t e r i o l e s have complex v a r i a t i o n s i n t h e i r g e o m e t r y . V e s s e l l e n g t h c an be as much as 7 0 0 u i n \ p r o x i m a l l o c a t i o n s and as s h o r t as 1 2 0 y d i s t a l l y . D i s t a l - S9 - . a r t e r i o l e s have d i a m e t e r s up t o 6 u n a r r o w e r t h a n p r o x i m a l v e s s e l s and some ALAs have a n a r r o w e r r e g i o n a t t h e i r p o i n t o f o r i g i n f r o m t h e : AFA ( P l a t e .11). In a d d i t i o n f l o w c a n n o t be e s t i m a t e d a c c u r a t e l y b e c a u s e o f b i f u r c a t i o n s i n the v e s s e l . These b i f u r c a t i o n s may s u p p l y two l a m e l l a e on the same s i d e o f t h e f i l a m e n t , o r , more commonly and i n d i s t a l r e g i o n s , t h e y may s u p p l y a p a i r o f d o r s a l and v e n t r a l l a m e l l a e . The v a r i a t i o n s i n ALA g e o m e t r y and f l o w p r e v e n t a g e n e r a l c a l c u l a t i o n o f AP and , r e s i s t a n c e , as p r e s e n t e d above. I n s t e a d t h e c a l c u l a t i o n s were made u s i n g r e p r e s e n t a t i v e d i m e n s i o n s o f ALAs from p r o x i m a l , c e n t r a l and d i s t a l l o c a t i o n s .(see A p p e n d i x I ) . The v a l u e s f o r t h e s e c a l c u l a t i o n s a r e p r e s e n t e d i n T a b l e V, a l o n g w i t h v a l u e s , f o r t h e o t h e r f i l a m e n t v e s s e l s as c a l c u l a t e d above. The c a l c u l a t e d r e s i s t a n c e s and p r e s s u r e d r o p s f o r ALAs w i l l be u n d e r e s t i m a t e s s i n c e i t was.assumed one ALA s u p p l i e d one l a m e l l a and t h e v e s s e l s had u n i f o r m d i a m e t e r s . T h i s u n d e r e s t i m a t i o n would be g r e a t e s t f o r the d i s t a l ALAs s i n c e t h e y were a l l . b i f u r c a t e d . In t h e g i l l s the ALAs a r e c l e a r l y the m a j o r r e s i s t a n c e s i t e and t h e m a j o r p r e s s u r e d r o p o c c u r s h e r e ( T a b l e V ) . The ALAs, t h e r e f o r e , have an i d e a l g eometry t o c o n t r o l i n p u t p r e s s u r e s t o t h e l a m e l l a e and t h u s d e t e r m i n e i n t r a l a m e l l a r f l o w p a t t e r n s . A 25% r e d u c t i o n i n the. d i a m e t e r . o f a d i s t a l ALA would i n c r e a s e r e s i s t a n c e to.4.98 x 10^ cm H2O.min.ml - 1 and AP t o a b o u t 15 cm H-20. g i v e n no o t h e r c h a n g e s . Thus c r i t i c a l - 90 TABLE V A summary of the c a l c u l a t i o n s of v a s c u l a r r e s i s t a n c e , pressure drop across v e s s e l s (AP) and blood t r a n s i t times i n g i l l f i l a m e n t v e s s e l s . Three c a l c u l a t i o n s were made f o r a f f e r e n t l a m e l l a r a r t e r i l e s . i n proximal, c e n t r a l and d i s t a l l o c a t i o n s of the f i l a m e n t because t h e i r geometry i s d i f f e r e n t f o r v a r i o u s regions of the f i l a m e n t . See t e x t and Appendix I f o r d e t a i l s of the c a l c u l a t i o n s . - 90a -TABLE V R e s i s t a n c e _^' (cm H^O.min.ml ) AP (cm H 20) B lood t r a n s i t time(sec) A f f e r e n t f i l a m e n t a r t e r y 1.84 X 10 2 2.1 2.1 A f f e r e n t l a m e l l a r a r t e r i o l e s Proximal C e n t r a l 1.27 1.21 X X 10 5 10 5 4.8 4.6 ) }0.34 ) ) D i s t a l 1.33 X 105 5.0 Lamella 7.65 X IO 3 0.29 2.5 to 3.6 E f f e r e n t f i l a m e n t a r t e r y 3.95 X 10 3 0.15 0.01 - 91 -c l o s u r e p r e s s u r e f o r l a m e l l a r c h a n n e l s and i n d e e d f o r the whole g i l l (Wood, 1974a) would be a t t a i n e d . F i n e c o n t r o l o f ALA d i a m e t e r might be a c h i e v e d n e u r a l l y . ALAs r e c e i v e a d r e n e r g i c i n n e r v a t i o n i n o t h e r t e l e o s t s , but t h i s has y e t t o be e s t a b l i s h e d f o r Ophipdoni. An a f f e r e n t a r t e r i o l e c o n t r o l s i t e o f c a p i l l a r y b l o o d p r e s s u r e i s c o n s i s t e n t w i t h f i n d i n g s f o r o t h e r m i c r o c i r c u l a t o r y s y s t e m s (see S e c t i o n I I ) . Morgan and T o v e l l (1973) c o n c l u d e d t h a t t h e a f f e r e n t f i l a m e n t a r t e r y r e s t r i c t e d : f l o w t o d i s t a l l a m e l l a e because, i t s t a p e r , r e d u c e d l a m e l l a r i n p u t p r e s s u r e s . They, however, d i d n o t c o n s i d e r t h a t AFA b r a n c h i n g r e d u c e s f l o w a l o n g t h e AFA. My f i n d i n g s l e n d no s u p p o r t t o t h e i r c o n c l u s i o n o r t h a t AFAs c o n t r o l i n p u t p r e s s u r e s o r f l o w t o l a m e l l a e . A g r e a t e r t h a n 50% r e d u c t i o n o f v e s s e l d i a m e t e r o v e r th e whole v e s s e l l e n g t h i s needed t o d e v e l o p a s u f f i c i e n t p r e s s u r e d r o p t h a t c r i t i c a l c l o s u r e p r e s s u r e s a r e r e a c h e d : i n t h e l a m e l l a e . A r t e r i a l v e s s e l s r a r e l y a l t e r t h e i r d i a m e t e r more t h a n 50% ( P e t e r s e n , 1 9 62). Of t h e 13 cm H 2 0 p r e s s u r e d r o p a c r o s s t h e b r a n c h i a l v e s s e l s in. v i v o / mY c a l c u l a t i o n s - , reve.al t h a t 9.6 em H 2 0 o r more i s due t o v a s c u l a r r e s i s t a n c e i n t h e f i l a m e n t . I assume h e r e t h a t t h e p r e s s u r e d r o p i n t h e EFA i s s i m i l a r t o t h a t d e t e r m i n e d f o r t h e AFA. ' The s m a l l p r e s s u r e d r o p a c r o s s the l a m e l l a e may be s u r p r i s i n g t o some a u t h o r s who p r e v i o u s l y c o n s i d e r e d t h a t the c a p i l l a r y bed r e g u l a t e d g i l l b l o o d f l o w p a t t e r n s . The c a l c u l a t i o n s p r e s e n t e d h e r e f o r l a m e l l a r r e s i s t a n c e do n o t s u p p o r t t h i s c o n t e n t i o n . W i t h such a s m a l l AP a c r o s s l a m e l l a e , a h i g h p r e s s u r e a s s o c i a t e d w i t h v e n t i l a t i o n 1 ( t h e o t h e r component of the transmural pressure) could have important effects on intralamellar flow patterns. This may be why v e n t i l a t i o n -perfusion synchrony occurs in Ophiodon during stress when ve n t i l a t i o n pressures r i s e up to 5 cm H 20. Ventilation-perfusion synchrony occurs in other f i s h during s t r e s s f u l conditions (Randall,. 1967; Hughes, 1972; Roberts, 1975) . Blood t r a n s i t times through filament vessels are also-shown in Table V. They were calculated using a constant flow and .the vessel volumes presented in Table IV. .Lamellar'.'transit time represents' the. residence time of red. blood c e l l s at the gas exchange s i t e . The time of 2.5 sec is higher than, but of the same order as the 1 sec t r a n s i t time in mammalian lungs (West, . 1977). In mammalian lungs the degree of gaseous' exchange is determined by v e n t i l a t i o n and perfusion rates (perfusion limited) since the alveolar d i f f u s i o n barriers are so thin. If the alveolar b a r r i e r is thickened by disease, gas exchange i s . severely reduced i.e . the system approaches d i f f u s i o n l i m i t a t i o n s . Considering that lamellar d i f f u s i o n distances are 10 times greater than in a l v e o l i , but that red blood c e l l residence time is only 2 to 3 times greater, i t is possible that lamellar gaseous exchange is d i f f u s i o n limited in lin g cod. .Fisher et a l . (1969) and Randall. (1976) have concluded that lamellar gaseous exchange is d i f f u s i o n limited for other teleosts . In summary, I have.demonstrated, in this section, that a l l cardiac output passes through the lamellae in l i n g cod. The venolymphatic flow i n the g i l l f i l a m e n t s must be d e r i v e d e n t i r e l y from e f f e r e n t v e s s e l s and lymph formation. I p r e d i c t e d from c a l c u l a t i o n s based on v e s s e l geometry, that ALAs.are the major r e s i s t a n c e s i t e i n the g i l l s and consequently determine the l a m e l l a r i n p u t . p r e s s u r e and A P i a m . I n t r a l a m e l l a r blood flow p a t t e r n s can now be p r e d i c t e d from M?]_am/ since l a m e l l a r flow, i s best d e s c r i b e d by sheet blood flow equations. The v a s c u l a r sheet of a l a m e l l a i s compliant, but s i n c e i t s compliance v a r i e s r e g i o n a l l y , i n t r a l a m e l l a r shunting of blood flow can occur a u t o m a t i c a l l y as P-iain a n c ^ Q change. When r e s t i n g blood pressures are e l e v a t e d , i n t r a l a m e l l a r shunting occurs away from the base of the l a m e l l a , towards regions of reduced e p i t h l i a l t h i c k n e s s . Since gaseous exchange i s a p p a r e n t l y d i f f u s i o n l i m i t e d i n l i n g c o d / . i n t r a l a m e l l a r shunting enhances gaseous exchange. Lamellae have a c r i t i c a l c l o s u r e pressure and whether they are perfused or not i s determined l a r g e l y by the input p r e s s u r e . The r e s i s t a n c e a s s o c i a t e d , w i t h ' d i s t a l l a m e l l a e i s g r e a t e r than f o r proximal l a m e l l a e . Thus d i s t a l l a m e l l a e are l e s s l i k e l y to be perfused. If d i s t a l l a m e l l a e were not normally perfused i n v i v o , then e l e v a t e d v e n t r a l a o r t i c blood pressures i n l i n g cod would a l t e r the p a t t e r n of g i l l blood flow i n two ways: a) recruitment of d i s t a l l a m e l l a e , b) i n t r a l a m e l l a r blood flow changes. Both flow a l t e r a t i o n s , as pointed out, enhance 0 2 t r a n s f e r . The p r e d i c t i o n s O u t l i n e d i n t h i s S e c t i o n are now t e s t e d e x p e r i m e n t a l l y i n vivo, and in v i t r o i n subsequent s e c t i o n s . - 93a -SECTION I I AN EXAMINATION OF GI L L BLOOD FLOW CHARACTERISTICS AND GILL RESISTANCE AND HOW THEY CHANGE IN VITRO AND IN VIVO B l o o d f l o w t o t h e g i l l s i n r e s t i n g l i n g c o d . M i c r o p r e s s u r e measurements in v i v o , a n a e s t h e t i s e d 1 i n g cod M i c r o p r e s s u r e measurements i n i s o l a t e d , p e r f u s e d h o l o b r a n c h s and t h e e f f e c t b f a d r e n e r g i c and c h o l i n e r g i c d r u g s on g i l l r e s i s t a n c e The i n v i v o e f f e c t s o f i n t r a v a s c u l a r a d m i n i s t r a t i o n o f a d r e n e r g i c and c h o l i n e r g i c d r u g s on g i l l r e s i s t a n c e and g i l l b l o o d f l o w i n r e s t i n g l i n g c o d . -54 -INTRODUCTION In r e c e n t years mammalian m i c r o c i r c u l a t i o n s have become b e t t e r understood as a r e s u l t of d i r e c t s t u d i e s of blood flow and blood pressure i n small a r t e r i e s , a r t e r i o l e s and c a p i l l a r i e s . Blood p r e s s u r e s can be measured i n small v e s s e l s by. micropuncture; a technique pioneered by Landis (1934). Landis measured average c a p i l l a r y blood micropressures with p i p e t s of 8 to 15 p. t i p diameter. Only r e c e n t l y , however, have accu r a t e , p u l s a t i l e micropressure measurements been made i n small blood v e s s e l s (Weiderhielm e t a l . , 1964). Weiderhielm e j t a l . developed a high f i d e l i t y , automatic, s e r v o n u l l i n g device which used p i p e t s as small as 0.1 t i p diameter. Weiderhielm 1s device has si n c e been r e f i n e d both e l e c t r o n i c a l l y and me c h a n i c a l l y ( I n t a g l i e t t a et. a l . , 1970; I n t a g l i e t t a , 1973). The major i n s i g h t gained from micropressure measurements i s that major pressure drops (major s i t e s of r e s i s t a n c e to flow) can be l o c a t e d . Furthermore, flow d i r e c t i o n s can be assigned from pressure measurements w i t h i n a system, given the v a s c u l a r arrangement as w e l l as input and output p e r f u s i o n p r e s s u r e s . M i c r o p r e s s u r e s t u d i e s have been l i m i t e d p r i m a r i l y to mammalian systemic v a s c u l a r beds that were e a s i l y a c c e s s i b l e , v i s i b l e and r e l a t i v e l y s t a b l e or f r e e from movement e.g. the bat wing (Weiderhielm and Weston, 1973), the r a b b i t mesentery and omentum (Zweifach and Lipowsky, 1977). From these and many other s t u d i e s i t i s now recognized that c a p i l l a r y blood flow i s p u l s a t i l e w i t h a p r e s s u r e p u l s e o f the o r d e r 1 - 2 cm H2O. A g e n e r a l f i n d i n g i s t h a t t h e m a j o r s i t e o f v a s c u l a r r e s i s t a n c e i s l o c a t e d i n p r e c a p i l l a r y a r t e r i o l e s ; a c o n c l u s i o n a l s o made by L a n d i s ( 1 9 3 4 ) . Thus t h e a r t e r i o l a r r e s i s t a n c e r e d u c e s c a p i l l a r y b l o o d p r e s s u r e t o a b o u t 20 mm Hg, which r e p r e s e n t s o n l y 15 t o 20% o f t h e c e n t r a l s y s t e m i c b l o o d p r e s s u r e . Furthermore,, i t i s now h e l d t h a t t h e i n t r a c a p i l l a r y p r e s s u r e i s l a r g e l y r e g u l a t e d a t i n p u t s i t e s o r p r e c a p i l l a r y s p h i n c t e r s w h i c h may be l o c a t e d , i n t h e a r t e r i o l e ( N i c o l l , 1 9 7 1). T h i s c o n c e p t i s s u p p o r t e d by t h e work o f F r o n e k and Z w e i f a c h ( 1 9 7 5 ) . They examined the e f f e c t o f * v e s s e l d i l a t i o n on m i c r o p r e s s u r e s by a d m i n i s t e r i n g i s o p r e n a l i n e and d e m o n s t r a t e d t h a t a r t e r i o l e s as s m a l l as 20y would d i l a t e and p r o d u c e m a j o r r e s i s t a n c e c h a n g e s . L u n d v a l l and J a r h . u l t (1974) a l s o r e p o r t a d r e n e r g i c d i l a t i o n o f p r e c a p i l l a r y s p h i n c t e r s . I f a r t e r i o l e s a r e r e g u l a t i n g c a p i l l a r y p r e s s u r e , t h e y w i l l a l s o c o n t r o l f l o w t o the c a p i l l a r y bed. S o b i n e t a l . (1977) have c o n c l u d e d t h a t smooth m u s c l e i n pu l m o n a r y a r t e r i o l e s o'f, f e t a l and new b o r n mammals i s c o n c e r n e d only, w i t h pulmonary b l o o d f l o w r e g u l a t i o n . I have a l s o been a b l e t o w i t n e s s s l o w a r t e r i o l e p u l s a t i o n s o f myogenic o r i g i n i n mammalian m e s e n t e r i c v e s s e l s ( t h r o u g h t h e c o u r t e s y o f Dr. I n t a g l i e t t a and co-workers)'. The a r t e r i o l e c o n t r a c t i o n s would s t o p b l o o d f l o w w i t h o u t c o m p l e t e v e s s e l c o n s t r i c t i o n and d i v e r t , f l o w t o a n e a r b y b r a n c h . R e g u l a t i o n o f i n t r a c a p i l l a r y b l o o d p r e s s u r e i s c l e a r l y o f p r i m e i m p o r t a n c e t o mammals s i n c e i t s e t s the t r a n s c a p i l l a r y blood pressure. This pressure w i l l determine tra.nscapillary f l u i d exchange i . e . the rate of lymph formation (St a r l i n g , 1896; Landis 1934; Weiderhielm, 1968), and, in the lungs at.least, both the d i s t r i b u t i o n of blood flow and c a p i l l a r y blood volume (Fung and Sobin, 1977). Intr.acapillary pressure w i l l be largely determined by the a r t e r i o l e resistance.. Do similar mechanisms for regulating intralamellar pressures exist in fish? The branchial vascular flow dynamics in teleosts d i f f e r in a number of important respects from the pulmonary.and systemic c i r c u l a t i o n in mammals. The branchial vascular bed reduces the central blood pressure by only 25 - 30% in most teleosts, including Ophiodon (Randall, 1970; Stevens et a l . , 1974). The intralamellar blood pressures are therefore comparatively high: generally at least 30 cm.^O which is the outflow blood pressure measured in the dorsal aorta. Lamellar c a p i l l a r y blood pressure should also be quite p u l s a t i l e ; this again is predicted from the p u l s a t i l i t y of dorsal.aortic blood pressure. I predicted in Section I that the major resistance to g i l l blood flow was the afferent lamellar a r t e r i o l e . If this i s true, ALA resistance would determine the p r e f e r e n t i a l perfusion of proximal lamellae and the A P i a m which affects intralamellar blood flow and the rate of lymph formation. Clearly, i t is important to confirm that ALA is the major resistance s i t e in the dynamic s i t u a t i o n . The micropressure system, as developed for mammalian investigations, was therefore used to examine pressure within g i l l filament vessels and 97 -e s t a b l i s h the major r e s i s t a n c e s i t e . . A l t e r a t i o n s i n v a s c u l a r dimensions are r e f l e c t e d i n measurements Of g i l l r e s i s t a n c e . G i l l r e s i s t a n c e (Rg) may. be a l t e r e d by d i r e c t v a s c u l a r a c t i o n s of hormones, through neural a c t i v i t y or a myogenic feedback system. The i s o l a t e d perfused g i l l or holobrahch has been used e x t e n s i v e l y i n the past to study, the e f f e c t s of a d r e n e r g i c and c h o l i n e r g i c drugs on Rg. I t i s c l e a r that i n t r a v a s c u l a r i n f u s i o n of a d r e n a l i n e reduces Rg i n  v i t r o , however a c o n t r o v e r s y surrounds the exact nature of the a d r e n e r g i c response (Krawkow, 1913; Mott, 1951; Randall and Stevens, 1967; Richards and Fromm, 1969; Randall et al. 1972 ; Wood, 1974a). In t r o u t , at l e a s t , i t appears there are 0 6 - a d r e n e r g i c c o h s t r i c t o r y and ^ - a d r e n e r g i c d i l a t o r y .receptors i n the g i l l s (Wood, 1975). The exact s i t e of these r e c e p t o r s has yet to be l o c a l i s e d but i t i s known that a d r e n e r g i c f i b r e s innervate a f f e r e n t l a m e l l a r a r t e r i o l e s (Gannon, 1972). Catecholamine i n f u s i o n w i l l a l s o reduce flow i n t o the ' , venolymphatics i n t r o u t ( G i r a r d and Payan, 1976) p o s s i b l y through o£-adrehergic v a s o c o n s t r i c t i o n of connecting v e s s e l s (Dunel and Laurent, 1977). A c e t y l c h o l i n e i n f u s i o n Ln v i t r o markedly i n c r e a s e s g i l l r e s i s t a n c e (Ostlund and Fange, 1962; Wood, 1975). Attempts to l o c a l i s e , the s i t e of a c e t y l c h o l i n e a c t i o n i n the g i l l were not s u c c e s s f u l (Steen and .Kruysse, 1964; Richards and Fromm, 1969; Klaverkamp and Dyer, 1974) u n t i l q u i t e r e c e n t l y (Smith, 1977; Dunel and Laurent, 1977). Smith provided good experimental evidence that a c e t y l c h o l i n e causes a major v a s o c o n s t r i c t i o n at - 98 -the base of the EFA i n t r o u t ; a s i t e which probably corresponds to a marked c o n s t r i c t i o n seen i n p l a s t i c v a s c u l a r c o r r o s i o n c a s t s of t r o u t EFAs (Campbell e t a_l. , i n p r e p a r a t i o n ; Smith, 1976) and other t e l e o s t s (Dunel and Laurent, 1977). G i l l blood flow and changes i n g i l l r e s i s t a n c e have not been examined e x t e n s i v e l y _in v i v o • S t u d i e s such as Stevens e_t a l . (1972), Jones e t a l . (1974) and Chan and Chow (1976) have examined blood flow i n t e l e o s t s i n some d e t a i l , but not b r a n c h i a l blood flow s p e c i f i c a l l y . Stevens et. a l . . (1972), working with Ophiodon concentrated l a r g e l y on c a r d i a c f u n c t i o n and demonstrated t h a t . t h e heart r a t e was under vagal c o n t r o l . I t v a r i e d widely with water temperature and.had the major i n f l u e n c e on Q. A l s o , i n c r e a s e d vagal a c t i v i t y was a s s o c i a t e d with e x e r c i s e and with d i s t u r b i n g the f i s h . They showed an a - a d r e n e r g i c systemic v a s o c o n s t r i c t i o n , but concluded that the a s s o c i a t e d c a r d i a c responses were r e f l e x o g e n i c . Jones and co-workers (1974) compared a h y d r a u l i c c i r c u l a t o r y model with measured c a r d i o v a s c u l a r v a r i a b l e s from Gadus morhua and examined the importance of v a s c u l a r compliance i n p u l s a t i l e blood flow.. They concluded b r a n c h i a l blood flow was both continuous and p u l s a t i l e . They a l s o d i s c u s s e d the s i g n i f i c a n c e of matching str o k e volume with g i l l blood volume and concluded stroke volume r a r e l y exceeded g i l l blood volume. Chan and Chow (1976) made an . ext e n s i v e i n v e s t i g a t i o n of the pharmacological e f f e c t s on c a r d i o v a s c u l a r f u n c t i o n i n A n g u i l l a japonica. using i n t r a v a s c u l a r - 99 -i n j e c t i o n s o f most known v a s o a c t i v e a g e n t s . They measured a v a r i e t y o f a r t e r i a l and venous b l o o d p r e s s u r e s , but were u n a b l e t o c a l i b r a t e t h e i r D o p p l e r f l o w m e ter t o a l l o w a c c u r a t e measurement o f Q c h a n g e s . A number o f c a r d i o v a s c u l a r i n v e s t i g a t i o n s have a l s o been made on d o g f i s h . The works o f S a t c h e l l ( 1 9 6 2 ) , Kent and P e i r c e (1975 and .1978 ) and t h e r e c e n t e x p e r i m e n t a l s e r i e s by B u t l e r and T a y l o r (197 5) and T a y l o r e t a l . (1977) have been p a r t i c u l a r l y u s e f u l s i n c e t h e y h i g h l i g h t t h e r e m a r k a b l e s i m i l a r i t i e s i n c a r d i o v a s c u l a r r e s p o n s e s between t e l e o s t s and e l a s m o b r a n c h s . F o r example, t h e v a g a l l y m e d i a t e d b r a d y c a r d i a i n r e s p o n s e t o e n v i r o n m e n t a l h y p o x i a has been examined e x t e n s i v e l y i n b o t h f i s h g r o u p s . (See S m i t h and J o n e s (1978.) and Daxboeck and H o l e t o n (19 78) f o r r e f e r e n c e s ) . V a g a l i n n e r v a t i o n o f t h e t e l e o s t h e a r t i s w e l l e s t a b l i s h e d . The c a r d i a c vagus c a r r i e s l a r g e l y c h o l i n e r g i c f i b r e s and a n e g a t i v e c h r o n o t r o p i c v a g a l t o n e e x i s t s d u r i n g r e s t i n g s t a t e s i n a number o f t e l e o s t s , but not i n Salmo  g a i r d n e r i ( R a n d a l l , 1970) . A d r e n e r g i c c a r d i a c e x c i t a t i o n i s a l s o e v i d e n t i n some t e l e o s t s and b o t h p o s i t i v e i n o t r o p i c and c h r o n o t r o p i c e f f e c t s a r e b e l i e v e d t o be m e d i a t e d by g - r e c e p t o r s i n the h e a r t (Gannon and B u r n s t o c k , 1969; R a n d a l l , 1970; Chan and Chow, 1976; Holmgren, 1 9 7 7 ) . A d r e n e r g i c c a r d i a c s t i m u l a t i o n may be due t o endogenous c a t e c h o l a m i n e l e v e l s ( W a h l q v i s t and N i l s s o n , 1977) o r v i a v a g a l s t i m u l a t i o n (Gannon and B u r n s t o c k , 1969) . V a g a l s t i m u l a t i o n a p p e a r s more complex t h a n a s i m p l e n e g a t i v e - 100 -c h r o n o t r o p i c e f f e c t s i n c e i n t r o u t , at l e a s t , the vagus a l s o c a r r i e s some ad r e n e r g i c e x c i t a t o r y f i b r e s (Yamauchi and Burnstock, 1968; Gannon and Burnstock, 1969). Gannon (1972) and Cobb and Santer (1973) both document a small c a r d i a c a c c e l e r a t i o n a f t e r vagal s t i m u l a t i o n of the heart was.stopped. Kulaev (1958) and Rudinov (1959), as reported by Chan and Chow (1976), found that the e f f e c t of s t i m u l a t i n g reduced numbers of vagal f i b r e s , (produced by s e c t i o n i n g vagal branches one by one) was to change the e l i c i t e d b r a d y c a r d i a to a t a c h y c a r d i a . Previous i n v e s t i g a t i o n s have, t h e r e f o r e , provided i n f o r m a t i o n on c o n t r o l of c a r d i a c a c t i v i t y in v i v o and on general blood flow i n t e l e o s t s . The i n v e s t i g a t i o n of g i l l blood flow has not, however, been d e t a i l e d enough f o r an a n a l y s i s of how '> pa t t e r n s of g i l l blood flow change. For t h i s reason pre- and-p o s t - b r a n c h i a l blood p r e s s u r e s were monitored along with Q and heart r a t e i n r e s t i n g Ophiodon. V e n t i l a t i o n r a t e and .amplitudes were a l s o monitored. Micropressure measurements were made i n fi l a m e n t v e s s e l s i n a n a e s t h e t i s e d f i s h and i n i s o l a t e d g i l l arch p r e p a r a t i o n s to l o c a l i s e the major s i t e ' o f g i l l r e s i s t a n c e . , The e f f e c t s of adr e n e r g i c and c h o l i n e r g i c a g o n i s t drugs on g i l l r e s i s t a n c e and blood flow were a l s o examined by i n j e c t i o n s i n t o the v e n t r a l a o r t a of r e s t i n g l i n g cod and drug i n f u s i o n s i n t o i s o l a t e d g i l l arch p r e p a r a t i o n s . Thus i n v i v o and i n v i t r o experiments were c a r r i e d out to a) confirm that the ALA was the major r e s i s t a n c e s i t e to g i l l blood flow, b) e s t a b l i s h whether v a s c u l a r changes a l t e r the balance of v e s s e l resistances, s u f f i c i e n t l y to a l t e r g i l l blood flow p a t t e r n s . - 101 -. MATERIALS -AND METHODS . . I n t r a v a s c u l a r a d m i n i s t r a t i o n o f a d r e n e r g i c and c h o l i n e r g i c  a g o n i s t s i n v i v o A d r e n e r g i c and c h o l i n e r g i c a g o n i s t d r u g s were i n j e c t e d i n t r a v a s c u l a r l y i j n v i v o v i a i n d w e l l i n g c a t h e t e r s i n r e s t i n g f i s h . An a t t e m p t was made t o o b s e r v e any e f f e c t s on t h e g i l l v a s c u l a t u r e . Thus the a g o n i s t s were a d m i n i s t e r e d t h r o u g h t h e v e n t r a l a o r t i c c a t h e t e r and would p a s s t h r o u g h th e g i l l s b e f o r e r e a c h i n g t h e h e a r t o r s y s t e m i c C i r c u l a t i o n . A l l d r u g i n j e c t i o n s were made i n c o n c e n t r a t e d form, u s i n g a 0.1 t o 0.25 ml c a r r i e r volume o f h e p a r a n i s e d s a l i n e . The f i n a l b l o o d c o n c e n t r a t i o n s (g.ml--'-) o f the d r u g s , based on a 5% b l o o d volume, were 1 x 1 0 ~ 7 ACH ( O - a c e t y l c h o l ' i n e c h l o r i d e , B.D.H.); 1 x 1 0 ~ 7 NAD ( L - a r t e r e n o l b i t a r t r a t e , S i g m a ) ; 1 x 1 0 ~ 8 t o 1 0 - 7 ISOP ( D L - i s o p r o t e r e n o l HCI, S i g m a ) ; 1 x 1 0 ~ 8 t o l O ^ 7 CARB ( e a r b a c h o l , K + K) and 1 x 1 0 ~ 6 t o I O " 5 ATROP ( a t r o p i n e a l k a l o i d , N.B.C.). These c o n c e n t r a t i o n s were s e l e c t e d a f t e r t r i a l s w h i c h d e t e r m i n e d t h e minimum dosage r e q u i r e d t o p r o d u c e an o b s e r v a b l e r e s p o n s e . H i g h e r c o n c e n t r a t i o n s o f t e n promoted s t r u g g l i n g o f t h e f i s h . ACH and NAD a r e n a t u r a l l y o c c u r i n g n e u r o - t r a n s m i t t e r s and b o t h a r e m e t a b o l i s e d w i t h i n s e v e r a l m i n u t e s . ISOP and CARB, however, a r e s y n t h e t i c a g o n i s t s which a r e . s l o w l y m e t a b o l i s e d i n a p e r i o d o f up t o 1 h r . R e c o v e r y p e r i o d s were a l l o w e d between s u c c e s s i v e d r u g i n j e c t i o n s . The - 10 2 -recovery p e r i o d was 1 hr f o l l o w i n g NAD or ACH and 2 to 3 hr or ove r n i g h t f o l l o w i n g ISOP or CARB a d m i n i s t r a t i o n . Recovery p e r i o d s were r i g o r o u s l y adhered to and were prolonged i f there was any doubt concerning the f i s h ' s recovery. ATROP i s known to produce a prolonged muscarine c h o l i n e r g i c blockade i n other f i s h ( R a n d a l l , 1970). Hence, a f t e r ATROP treatment i n l i n g cod, f u r t h e r experiments were considered to be on f i s h with a c h o l i n e r g i c blockade. Micropressure measurements i n the g i l l filament, v a s c u l a t u r e These measurements were made on i s o l a t e d perfused holobranchs and iri v i v o on a n a e s t h e t i s e d or n a r c o t i s e d , r e s t r a i n e d f i s h . I s o l a t e d perfused holobranch p r o t o c o l : F i v e holobranchs were prepared as p r e v i o u s l y d e s c r i b e d (See general methods). The whole b r a n c h i a l basket was c l e a r e d i n s i t u using s a l i n e d e l i v e r e d from a Harvard p u l s a t i l e i n f u s i o n pump, but only the f i r s t or second holobranchs were used. A p u l s a t i l e i n f l o w , (Qi) of 1.25 ml. min-i . Kg -; 1 was maintained throughout the experiment. The s a l i n e was not p r e f i l t e r e d and i t was gased.with carbogen. Drugs f o r ad r e n e r g i c or c h o l i n e r g i c s t i m u l a t i o n of the holobranch v a s c u l a t u r e were introduced by way of a 3-way tap, which- allowed a 30 to 60 sec p e r f u s i o n with s a l i n e c o n t a i n i n g an a g o n i s t . The a g o n i s t drug c o n c e n t r a t i o n s ( g.ml - 1) of p e r f u s a t e were I O - 6 to I O - 7 NAD and I O - 7 to I O - 8 f o r ISOP and ACH. - 1G3 -Input p e r f u s i o n pressure (P^) and output p e r f u s i o n pressure (P Q) were monitored, which allowed c a l c u l a t i o n of o v e r a l l g i l l r e s i s t a n c e (Rg) = (P^-P 0)/Q^. The outflow (0 o ) from the e f f e r e n t arch a r t e r y cannula was measured p e r i o d i c a l l y under c o n t r o l c o n d i t i o n s , but always during drug p e r f u s i o n s . In v i v o procedure: These experiments were performed on 10 l i n g cod that had been r o u t i n e l y prepared f o r blood flow and blood pressure measurements (See gen e r a l methods). The animal was immobilised p r i o r to the experiment with e i t h e r 2% urethane added to the aquarium or with an i n t r a p e r i t o n e a l i n j e c t i o n of curare (3 mg.Kg - 1). The immobilised f i s h was supported i n a body holder, with i t s g i l l s i r r i g a t e d , and was r o t a t e d onto one s i d e . T h e . g i l l f i l a m e n t s were made a c c e s s i b l e f o r micropuneture by removing p a r t of one operculum. I n d i v i d u a l f i l a m e n t s were s t a b i l i s e d f o r micropuneture by a h o l d i n g device manoeuvered around them with a micromanipulator. The h o l d i n g device c o n s i s t e d of a len g t h of PE 260 t u b i n g , which f i t t e d f r e e l y i around the f i l a m e n t . I t had many windows cut out to allow f r e e access f o r water and f o r the entry of m i c r o p i p e t s . - 10 4 -M i c r o p r e s s u r e i n s t r u m e n t p r e p a r a t i o n and measurement p r o t o c o l The m i c r o p r e s s u r e s y s t e m , i t s c a l i b r a t i o n and the a n a l y s i s o f the measurements a r e d i s c u s s e d i n d e t a i l i n A p p e n d i x I I . The p r i n c i p l e s o f t h e m i c r o p r e s s u r e s y s t e m and i t s o p e r a t i o n f o l l o w e d the s e r v o n u l l i n g t e c h n i q u e s of W e i d e r h i e l m e t  a l . (1964) and I n t a g l i e t t a ( 1 9 7 3 ) . A m i c r o p r e s s u r e 4 s y s t e m (I.P.M., San D i e g o ) s e r v o n u l l i n g d e v i c e was used i n c o n j u n c t i o n w i t h b e v e l l e d m i c r o p i p e t s . I n d i v i d u a l m i c r o p i p e t s were made from s c r u p u l o u s l y c l e a n e d b o r o s i l i c a t e g l a s s c a p i l l a r y t u b i n g (0.1 mm O.D. and 0.08. mm ID; C o r n i n g , New York) on a v e r t i c a l p i p e t p u l l e r (model 700 C, D a v i d Kopf I n s t r u m e n t s , T u j u n g a , C a . ) . The m i c r o p i p e t t i p d i a m e t e r s were r e g u l a r l y 2 t o 4V . Micropipets were f i l l e d w i t h t r i p l e f i l t e r e d (0.45V, M i l l i p o r e ) IM N a C l . The t i p was f i l l e d o v e r n i g h t by c a p i l l a r i t y and t h e stem o f t h e m i c r o p i p e t was b a c k - f i l l e d from a s y r i n g e . Any a i r b u b b l e s i n th e stem were a l w a y s removed a t t h i s s t a g e by c a r e f u l l y , f l i c k i n g t h e p i p e t stem. The t i p s were r e m a r k a b l y h a r d y t o t h i s p r o c e d u r e and t h e c l e a n l i n e s s o f t h e i n s i d e o f the stem was r e f l e c t e d i n th e e ase w i t h w h i c h a i r b u b b l e s were removed. P i p e t b e v e l l i n g g r e a t l y f a c i l i t a t e d m i c r o p u n e t u r e , t h e r e f o r e each p i p e t was s h a r p e n e d on one o r , o c c a s i o n a l l y , two s i d e s w i t h a BV-10 m i c r o p i p e t b e v e l l e r ( S u t t e r I n s t r u m e n t Co., Los A n g e l e s ) . The m i c r o p r e s s u r e s y s t e m was c a l i b r a t e d a g a i n s t p r e s s u r e s from a s t a t i c column o f w a t e r . The f r e q u e n c y r e s p o n s e o f the s y s t e m , was a l w a y s i n e x c e s s o f 10Hz and was t e s t e d u s i n g the 'pop' t e s t - 105 -(McDonald., 1960) i n a s p e c i a l l y d e s i g n e d chamber i s s u e d by t h e m a n u f a c t u r e r s (IPM, San D i e g o ) f o r t h i s p u r p o s e . The m i c r o p r e s s u r e s y s t e m was n u l l e d a t ' t h e s e a w a t e r o r s a l i n e s u r f a c e i n a l l e x p e r i m e n t s . The m i c r o p i p e t was manoeuvered w i t h a manual N a r a s h i g e v a r i a b l e a n g l e m i c r o m a n i p u l a t o r and i t s p r o g r e s s was f o l l o w e d w i t h a L e i t z o p e r a t i n g m i c r o s c o p e . In v i v o m i c r o p u n c t u r e s were made on b o t h a f f e r e n t and e f f e r e n t s i d e s o f g i l l f i l a m e n t s l o c a t e d n e a r t o t h e bed i n t h e a r c h on t h e f i r s t and s e c o n d g i l l h o l o b r a n c h s . M i c r o p u n c t u r e s were made up t o 3 h r s a f t e r t h e f i s h was i m m o b i l i s e d , b u t e x p e r i m e n t s were t e r m i n a t e d when t h e f i s h e i t h e r began v o l u n t a r y movements (2 t o 3 h o u r s ) o r Q became u n s t a b l e and d e c l i n e d , as was t h e c a s e w i t h c u r a r i s e d f i s h a f t e r 1 t o 2 h r . B l o o d p r e s s u r e s and Q were m o n i t o r e d c o n t i n u o u s l y t h r o u g h o u t the e x p e r i m e n t . I_n v i t r o m i c r o p u n c t u r e s were made i n t h e same r e g i o n s o f t h e i s o l a t e d g i l l h o l o b r a n c h and t h e e x p e r i m e n t s . l a s t e d 2 t o 3 h r s . A n a l y s i s . V e n t r a l a o r t i c (VA) and d o r s a l a o r t i c (DA) p r e s s u r e s a r e d e s c r i b e d by t h e i r mean.and p u l s e p r e s s u r e s where p u l s e = s y s t o l e - d i a s t o l e and mean = d i a s t o l e + 1/3 p u l s e . A l l p r e s s u r e s a r e e x p r e s s e d i n cm H 2 0 u n l e s s s t a t e d o t h e r w i s e (1 cm H 2 0 = 0.098 k P a s c a l s ) . S t r o k e volume (ml) was d e t e r m i n e d from the a r e a under t h e p u l s a t i l e f l o w r e c o r d i n g . H i g h c h a r t s p e e d s , 5 mm.sec - 1, were used t o i m p r o v e . t h e a c c u r a c y o f a r e a measurements. Be a t t o b e a t h e a r t irate was d e t e r m i n e d f r o m t h e - 106 -flow r e c o r d and i s expressed i n b e a t s . m i n - 1 . C a r d i a c output (ml •min--'-) , Q, i s the product of stroke volume and heart r a t e . Q and stroke volume are expressed per kg f i s h weight (ml.Kg"-'- and ml .min--'- .Kg--'- r e s p e c t i v e l y ) . G i l l r e s i s t a n c e , Rg, i s d e f i n e d as VA mean - DA mean/Q and systemic " r e s i s t a n c e , Rs, i s d e f i n e d as DA mean/Q. The r e s i s t a n c e u n i t s are cm H2O .min. Kg .ml--'-. The Rg, t h e r e f o r e , assumes the : major r e s i s t a n c e i n the l a m e l l a r u n i t s where a l l Q passes ( S e c t i o n I ) . The Rs assumes venous r e t u r n t o the heart i s at ambient pressure (see R a n d a l l , 1968; R a n d a l l , 1970). The c a r d i o v a s c u l a r data were compiled, i n i t i a l l y graphed and s t a t i s t i c a l l y analysed using a PDP 11 computer. The grouped data were expressed as mean values ± standard e r r o r (s.e.) from n " i n d i v i d u a l " o b s e r v a t i o n s on m f i s h , unless otherwise s t a t e d . An " i n d i v i d u a l " o b s e r v a t i o n r e p r e s e n t s s i n g l e measurement f o r any given v a r i a b l e . A 95% confidence l i m i t was used as a t e s t of s i g n i f i c a n c e with the Student ' t'1 t e s t . G r a p h i c a l treatment of data . In a l l graphs d i s p l a y i n g rn v i v o data the s i n g l e p o i n t s are " i n d i v i d u a l " values wherever p o s s i b l e i . e . there may be an o v e r l a p of " i n d i v i d u a l " values and thus some p o i n t s may represent more than one " i n d i v i d u a l " v a l u e . The mean r e s t i n g value of the v a r i a b l e being graphed i s i n d i c a t e d by the broken h o r i z o n t a l l i n e . The standard e r r o r f o r the mean r e s t i n g value i s represented by v e r t i c a l bars, but o f t e n the standard e r r o r - 107 -l i e s w i t h i n the a c t u a l p o i n t . The mean r e s t i n g value i s used as a p o i n t of r e f e r e n c e , s i n c e no s t a t i s t i c a l i n f e r e n c e can be made from t h i s type of g r a p h i c a l treatment. The graphs are presented to i l l u s t r a t e the trends f o r q u a l i t a t i v e changes with time (or oxygen content, S e c t i o n IV). Q u a n t i t a t i v e changes are d e a l t with i n the t a b l e s , where s t a t i s t i c a l comparisons are made. - 108 -RESULTS B r a n c h i a l blood flow and r e s p i r a t i o n i n unanaesthetised r e s t i n g  l i n g cod The r e s t i n g s t a t e F i s h were observed i n the sea at depths up to 30 m. They r e s t on the sea f l o o r propped on t h e i r l a r g e p e c t o r a l f i n s . Each v e n t i l a t i o n was p e r c e p t a b l e only by s l i g h t o p e r c u l a r movements that were separated by s e v e r a l seconds. In the h o l d i n g a q u a r i a f i s h adopted a s i m i l a r r e s t i n g behaviour, spending long p e r i o d s s t a t i o n a r y on the bottom. In the experimental aquarium bu c c a l c a v i t y movements during v e n t i l a t i o n i n r e s t i n g f i s h were ha r d l y p e r c e p t a b l e to the eye. The mouth remained s l i g h t l y open but was sealed d u r i n g b u c c a l compression ( F i g . 15) by the buccal f l a p . The p e r i o d of reduced p r e s s u r e i n the o p e r c u l a r c a v i t y was v i s u a l l y c o r r e l a t e d to o p e r c u l a r abduction. The d i f f e r e n t i a l pressure g r a d i e n t across the g i l l s i s s m a l l , l e s s than 0.4 cm H^O at i t s h i g h e s t and i s at ambient pressure during the r e s p i r a t o r y pause p r i o r to i n h a l a t i o n ( F i g . 1 5 ) . The v e n t i l a t i o n r a t e s ranged from 6 to 16.min"1 with a mean value = 11.9 ± 0.4 i • (n = 60 f o r 8 f i s h ) . Heart rate o f t e n f l u c t u a t e d from beat to beat, but Q, was unchanged. Heart r a t e s between f i s h ranged from 22 to 35 b e a t s . m i n - 1 which was a g r e a t e r v a r i a t i o n than the. beat to beat f l u c t u a t i o n s . V e n t r a l a o r t i c blood flow was continuous throughout the c a r d i a c c y c l e at r e s t i n g heart r a t e s ; The d i a s t o l i c p o r t i o n of flow was not steady and ~ 109 -c h a r a c t e r i s t i c a l l y d e c l i n e d with time ( F i g . 16). Thus at lower heart r a t e s , end d i a s t o l i c flow and pressure were reduced. F l u c t u a t i o n s i n VA pressure were o f t e n g r e a t e r than t h o s e . i n DA mean p r e s s u r e . Between 24% and 30% of VA mean pressure was l o s t to the g i l l bed r e s i s t a n c e ^ A Pg mean/VA mean) and the v e n t r a l * ',5. a o r t i c pressure pulse (VA pulse) was damped by about 60% ( APg ' pulse/VA pulse) (Table V I ) . '-• - 11.0 -TABLE VI ' A/ Measured c a r d i o v a s c u l a r v a r i a b l e s i n r e s t i n g l i n g cod. Data i s d e r i v e d from r e s t i n g values p r i o r to a l l treatments i n 8 f i s h . Values expressed as mean values ± standard e r r o r f o r the 103 o b s e r v a t i o n s . VA mean, VA p u l s e , DA mean and DA pulse are the mean and pulse pressures i n the v e n t r a l a o r t a and d o r s a l a o r t a expressed I n cm H^O. B/ Summary of c a l c u l a t i o n s based on the v a r i a b l e s measured i n Table VIA. Q = SV x HR, APg = VA mean - DA mean, APg pulse = VA pul s e - DA p u l s e . Rg = APg/Q, Rs .= DA A b b r e v i a t i o n s are e x p l a i n e d above. - 110a -TABLE VI A/ Heart r a t e beats . r a i n ~ l Stroke Volume ml.kg-1 VA mean VA p u l s e DA mean DA pulse B/ C a r d i a c output, Q. ml .min-l-kg"1 Rg Rs A Pg APg p u l s e 29.8 ± 0.4 0.37 ± 0.008 52.6 ± 0.4 12.4 ± 0.2 39.6 ± 0.3 6.0 ± 0 . 2 10.91 ± 0.22 1.24 ± 0.04 3.86 ± 0.12 13.1 ± 0.4 6.4 ± 0.2 - I l l -F I G U R E 15 T y p i c a l r e c o r d s f o r s i m u l t a n e o u s r e c o r d i n g s o f r e s p i r a t o r y and c a r d i o v a s c u l a r v a r i a b l e s . T h e r e i s a r e s p i r a t o r y p a u s e i n t h e o p e r c u l a r ( o . p . ) and b u c c a l c a v i t i e s ( b . p . ) as i n d i c a t e d by t h e p e r i o d o f a m b i e n t p r e s s u r e (atm.) i n b o t h c a v i t i e s . B u c c a l and o p e r c u l a r v e n t i l a t i o n p r e s s u r e s and d o r s a l a o r t a b l o o d p r e s s u r e (DA) a r e a l l m e a s u r e d i n cm a b o v e a m b i e n t p r e s s u r e . P u l s a t i l e f l o w i n t h e v e n t r a l a o r t a . ( Q , m l . m i n - ! ) i l l u s t r a t e s t h e c o n t i n u o u s d i a s t o l i c f l o w . H e a r t r a t e (h . r . , b e a t s .min--'-) i s shown as an o n - l i n e b e a t t o b e a t r e c o r d . - I l i a -b.p. +2-atm. -J I w L L L — A im„X «*Xnh<~A* +2-1444-Ar—V—-K—V-V—-A—V-DA so c m h beats 8 0 - 1 J i. m m 1 I o-J - 112 -FIGURE 16 . A t y p i c a l r e c o r d o f the c a r d i o v a s c u l a r v a r i a b l e s i n a r e s t i n g l i n g c o d . Note the c o n t i n u o u s d i a s t o l i c f l o w and t h e e f f e c t o f b r e a t h i n g on t h e f l o w r e c o r d i n g d u r i n g d i a s t o l e . Boo! r e p r e s e n t s a s h o c k ( b a n g i n g ) . Note the z e r o f l o w d u r i n g d i a s t o l e . A l l a b b r e v i a t i o n s a r e e x p l a i n e d i n l e g e n d f o r F i g . 15. VA i s v e n t r a l a o r t i c p r e s s u r e . - 112a -B O O ! - 1 1 3 -Observations on a n a e s t h e t i s e d l i n g cod There was o f t e n a marked change i n the i n t e n s i t y of the red c o l o r a t i o n to the g i l l f i l a m e n t s along t h e i r l e n g t h , u s u a l l y o c c u r i n g about midway along the f i l a m e n t . The d i s t a l f i l a m e n t a l regions were l i g h t e r i n c o l o u r than the proximal h a l f of the f i l a m e n t and t h i s d i f f e r e n c e was mdre pronounced on the a f f e r e n t r a t h e r than the e f f e r e n t s i d e of the f i l a m e n t . These c o l o u r d i f f e r e n c e s were a t t r i b u t e d to d i f f e r e n t degrees of blood oxygenation, with the l i g h t e r regions corresponding to g r e a t e r oxygenation. The o p e r c u l a r and f i l a m e n t muscles were not always completely immobilised by a n a e s t h e s i a . Large o p e r c u l a r movements were a b o l i s h e d , but s m a l l e r , rhythmic movements o f t e n p e r s i s t e d . The f i l a m e n t s a l s o d i s p l a y e d s m a l l , a n t e r i o - p o s t e r i o r o s c i l l a t o r y movements, which were enhanced i f g i l l i r r i g a t i o n was stopped. The f i l a m e n t s a l s o had very s l i g h t p u l s a t i l e movements which were synchronised with the blood p u l s e . T h i s p u l s a t i l i t y was e a s i l y seen when the pressure s e n s i t i v e m i c r o p i p e t touched the f i l a m e n t , and i n f a c t the p u l s a t i l i t y caused a r t i f a c t s during some micropressure measurements . During urethane ana e s t h e s i a Q was maintained c l o s e to r e s t i n g l e v e l s , even though heart r a t e was reduced (Table VII, c f Table V I ) . Rg was e l e v a t e d but Rs was reduced (Table V I I ) . Both r e s i s t a n c e values are a s s o c i a t e d with a reduced DA p r e s s u r e . - 1 1 4 .-I f g i l l i r r i g a t i o n f l o w w a s r e d u c e d o r s t o p p e d , t h e n t h e f i s h w o u l d d e v e l o p a n i m m e d i a t e b r a d y c a r d i a . N o r m a l h e a r t r a t e s w o u l d r e t u r n w h e n g i l l i r r i g a t i o n w a s r e s t o r e d t o t h e n o r m a l f l o w r a t e s . -115 -TABLE V I I • Mean v a l u e s ± s . e . f o r t h e c a r d i o v a s c u l a r v a r i a b l e s r e c o r d e d f r o m 5 l i n g cod under deep u r e t h a n e a n a e s t h e s i a d u r i n g . t h e 9 m i c r o p r e s s u r e r e c o r d i n g s t h a t were made. Mean b l o o d p r e s s u r e s a r e p r e s e n t e d and Rg = VA mean - DA mean/Q and Rs = DA mean/Q i n cm H2O.min.kg.ml - 1. - 115a -TABLE VII Car d i a c output, Q. 10.7 ± 0.3 (ml .min"1. kg"1) Heart r a t e 20.5 ± 1.2 (beats .min"1) V e n t r a l a o r t i c (VA) .49.2 ± 1.8 p r e s s u r e (cm. H2O) D o r s a l a o r t i c (DA) 22.8 ± 0 . 7 p r e s s u r e (cm. H2O) G i l l r e s i s t a n c e , Rg 2.48. ± 0.09 Systemic ..resistance, Rs 2.14 ± 0.04 - 116 . -G i l l f i l a m e n t m i c r o p r e s s u r e measurements ..' '• I n v i v o l i n g :cod: N i n e r e c o r d i n g s o f p u l s a t i l e m i c r o p r e s s u r e s were made i n 5 a n a e s t h e t i s e d l i n g cod a t v a r i o u s l o c a t i o n s a l o n g t h e a f f e r e n t and e f f e r e n t s i d e s of. t h e f i l a m e n t l e n g t h . Two r e c o r d i n g s , one a f f e r e n t and one e f f e r e n t , were l e s s t h a n t h e DA p r e s s u r e ( F i g . 1 7 ) . F o r t h e r e m a i n i n g measurements, the m i c r o p r e s s u r e s a s s o c i a t e d w i t h the a f f e r e n t s i d e r e c o r d i n g s , w e r e a l l g r e a t e r t h a n t h o s e on t h e e f f e r e n t s i d e . The a f f e r e n t s i d e i r e c o r d s were 70 t o 95% o f t h e VA p r e s s u r e , b u t the two e f f e r e n t r e c o r d s were o n l y s l i g h t l y above the DA p r e s s u r e . These two g r o u p s c l e a r l y r e p r e s e n t b l o o d p r e s s u r e s i n t h e a f f e r e n t and e f f e r e n t r e s p i r a t o r y network." . S i x o t h e r m i c r o p r e s s u r e measurements.were made on c u r a r i s e d f i s h , b u t were n o t i n c l u d e d i n t h e a n a l y s i s b e c a u s e e i t h e r Q was much r e d u c e d , o r t h e DA c a t h e t e r was no l o n g e r p a t e n t . Oh one o c c a s i o n a c u r a r i s e d f i s h . d i s p l a y e d art , i n t e r e s t i n g r e s p o n s e when the f i l a m e n t was t o u c h e d by the a d v a n c i n g m i c r o p i p e t . B o t h VA and DA p r e s s u r e s would i m m e d i a t e l y r i s e by 2 t o 3 cm H 20 f o r a b o u t a 1 m i n u t e p e r i o d , p r e s u m a b l y r e p r e s e n t i n g a t o u c h r e f l e x . I s o l a t e d p e r f u s e d h o l o b r a n c h s : I n v i t r o the m i c r o p i p e t s were l e s s p r o n e t o b l o c k a g e and movement compared t o _in v i v o b e c a u s e t h e p r e p a r a t i o n was more e a s i l y m a n i p u l a t e d and more s t a b l e . As a r e s u l t b e t t e r q u a l i t y r e c o r d i n g s were o b t a i n e d ( F i g . 1 7 ) , - 1J 7 -t h e r e was a h i g h e r r a t i o o f s u c c e s s f u l m i c r o p u n c t u r e s and m i c r o p r e s s u r e s c o u l d be r e c o r d e d f o r up t o s e v e r a l m i n u t e s , u n l i k e i n v i v o where the r e c o r d i n g t i m e was s e v e r a l s e c o n d s . The 22 m i c r o p r e s s u r e r e c o r d s t h a t were made ^Ln v i t r o a r e summarised i n F i g . 17. T h r e e c a t e g o r i e s o f p r e s s u r e , s i m i l a r t o t h e i r i v i v o r e c o r d i n g s , a r e e v i d e n t . By c o m p a r i s o n , however, the e f f e r e n t m i c r o p r e s s u r e s and o u t p u t p r e s s u r e s a r e r e d u c e d due t o th e h i g h o v e r a l l g i l l r e s i s t a n c e o f the p r e p a r a t i o n (Rg = 6.16± 0.33, n = 20). The a f f e r e n t m i c r o p r e s s u r e s were a l s o c o m p a r a t i v e l y r e d u c e d and c l e a r l y r e f l e c t an e l e v a t e d r e s i s t a n c e t o f l o w i n v e s s e l s a f f e r e n t t o the g i l l l a m e l l a e . ACH p e r f u s i o n f o r 30 t o 60 s e c c a u s e d Rg t o r i s e i n 90% o f the 24 t r i a l s , w i t h an accompanying r i s e i n Pj_. Q Q was r e d u c e d and sometimes c e a s e d d u r i n g the peak ACH r e s p o n s e . M i c r o p r e s s u r e r e c o r d i n g s a f t e r ACH a d m i n i s t r a t i o n were u s u a l l y i n v a l i d a t e d by f i l a m e n t movements a s s o c i a t e d w i t h t h e r i s i n g Rg t h a t d i s l o d g e d t h e m i c r o p i p e t . In two i n s t a n c e s , however, the e f f e r e n t m i c r o p r e s s u r e r o s e a f t e r ACH p e r f u s i o n ( F i g . 16b and F i g . 18). On 5 o t h e r o c c a s i o n s the e f f e r e n t m i c r o p r e s s u r e was a l s o r i s i n g b e f o r e t h e p i p e t became d i s l o d g e d . No s u c c e s s f u l c o n t i n u o u s m i c r o p r e s s u r e r e c o r d i n g s were made d u r i n g a d r e n e r g i c i n f u s i o n . F i l a m e n t movements a g a i n a c c o m p a n i e d g i l l r e s i s t a n c e c h a n g e s . Rg d e c r e a s e d w i t h a d r e n e r g i c s t i m u l a t i o n , n = 5 f o r NAD and n = 6 f o r ISOP, o r d i d not change, n = 2 f o r ISOP. NAD p e r f u s i o n c a u s e d a marked f a l l i n P]_ and r i s e i n P Q . The p r e s s u r e c h a n g es a c c o m p a n y i n g ISOP t r e a t m e n t were s i m i l a r , b u t v e r y s m a l l . _ lie -FIGURE 17 A summary o f t h e m i c r o p r e s s u r e measurements made i n the a f f e r e n t and e f f e r e n t s i d e s of the g i l l f i l a m e n t i n a n a e s t h e t i s e d l i n g cod (IN VIVO) and i n i s o l a t e d p e r f u s e d h o l o b r a n c h s f r o m l i n g cod (_IN VITRO) . Each m i c r o p r e s s u r e p o i n t r e p r e s e n t s an a v e r a g e v a l u e f o r a s i n g l e m i c r o p u n c t u r e . a) lH v i v o m i c r o p r e s s u r e s a r e e x p r e s s e d as a p e r c e n t a g e o f the v e n t r a l a o r t i c mean b l o o d p r e s s u r e (va) t h a t was measured a t t h e same t i m e . The a v e r a g e v a l u e f o r a l l d o r s a l a o r t i c mean b l o o d p r e s s u r e s (da) i s s i m i l a r l y e x p r e s s e d w i t h t h e s t a n d a r d e r r o r i n d i c a t e d by the v e r t i c a l b a r s (n = 9 f o r 5 f i s h ) . b) In v i t r o m i c r o p r e s s u r e s a r e e x p r e s s e d as p e r c e n t a g e s o f t h e i n p u t mean p r e s s u r e . The v e r t i c a l b a r s i n d i c a t e t h e s t a n d a r d e r r o r o f the a v e r a g e v a l u e f o r a l l o u t p u t mean p r e s s u r e s (n = 2 2 ) . The r a n g e s o f m i c r o p r e s s u r e s w hich a r e g r e a t e r t h a n da o r o u t p u t p r e s s u r e i s e n c l o s e d by s o l i d l i n e s . P u l s a t i l e m i c r o p r e s s u r e s t h a t were below t h i s range a r e i n d i c a t e d by V. The m i c r o p r e s s u r e r e c o r d e d a f t e r ACH a d m i n i s t r a t i o n i s i n d i c a t e d by o. - 118a -IN VIVO 100 D Q_ c — « / > »- 2> O D wo O </> > f i -Q. 50 CO O U J 3 to CO U J o£ Q. 0 va afferent efferent micropressures IN V ITRO input afferent efferent micropressures output - 119 _ FIGURE 18 E x a m ples o f t y p i c a l m i c r o p r e s s u r e r e c o r d i n g s . a) an i n v i v o r e c o r d o f p u l s a t i l e p r e s s u r e f r o m t h e a f f e r e n t v e n o l y m p h a t i c s w i t h a c c o m p a n y i n g v e n t r a l and d o r s a l a o r t i c b l o o d p r e s s u r e s , cm 1^0, and c a r d i a c o u t p u t , Q, m l . m i n - 1 . Note the f i l a m e n t movement a r t i f a c t s i n t h e m i c r o p r e s s u r e r e c o r d a f t e r t h e 4 t h p u l s e . b) an _in v i t r o r e c o r d o f an a f f e r e n t f i l a m e n t m i c r o p r e s s u r e w i t h i n p u t and o u t p u t p e r f u s i o n p r e s s u r e s . c) an i_n v i t r o r e c o r d o f e f f e r e n t m i c r o p r e s s u r e and the e f f e c t o f ACH p e r f u s i o n (1 x 1 0 - 7 g.ml -- 1- f o r 30 s e c ) . The d e c l i n e i n P Q was a ccompanied by a n e a r z e r o o u t f l o w . A l l p r e s s u r e s a r e i n cm H 20. A, IN VIVO. $OC 1 1 - i r ? i i r > t i i ' M i i I I 50 dorsal aorta 25 H 150-1 o...:.UJJOJL all pressures in c m H 2 0 B, Ui VITRO 50 afferent | MtoMMtktittl AAAAAAAA/ /"^ ^ filament **c iiiimuiiiiiuiiiiiiiiunii I I I I I I H I I I I I - J — « — t — i — r 100-1 0 J 10 AWMVJ ,/\^ output, P0 7 5 j efferent -J filament ACH 1 . . . H ' . " A ' * ' * " V A ' . . . . . . ( V A ' . W A V A I V A ' - -JIIHII.IIIIIIIIH.'I.IIII i,iiii,iiiiiiii,.iu,iiiiiii,iui,„iiii,,i;ii.,iil„,»,.,i,,. u,,i» ,^n„„i,uii,im„i;. 100 input, Pj J 0 15 4 output, P0 - izo .-The e f f e c t of i n t r a v a s c u l a r i n j e c t i o n of ad r e n e r g i c a g o n i s t s  i n r e s t i n g l i n g cod, NORADRENALINE (NAD): The response to NAD a d m i n i s t r a t i o n i n t o the v e n t r a l a o r t a was r a p i d and began a f t e r 20 to 30 sec of the i n j e c t i o n . The response was r e p r o d u c i b l e with blood pressures and blood flow i n c r e a s i n g simultaneously (Fig*,. 19). At the peak p r e s s o r response to NAD, the mean. Q was el e v a t e d 42% above the r e s t i n g v a l u e . The p a t t e r n of VA blood flow during the c a r d i a c c y c l e was a l s o changed since both s y s t o l i c and d i a s t o l i c flows i n c r e a s e d . S p e c i f i c a l l y , peak s y s t o l i c flow was g r e a t e r and d i a s t o l i c flow r a t e remained almost constant, u n l i k e i t s normal d e c l i n e with time during r e s t ( F i g . 19). These changes r e s u l t e d i n the mean stroke volume increasing, by 34% . A f t e r NAD i n j e c t i o n Rg decreased by 30% of the mean r e s t i n g value (Table V I I I , F i g . 20). In i n d i v i d u a l f i s h Rs e i t h e r d i d not change or increased as a result' of the ' i n j e c t i o n . O v e r a l l , there was no s i g n i f i c a n t change in. mean Rs values (Table VII) . There was an i n c r e a s e i n mean blood pressures but the pressure drop across the g i l l s d i d not change. VA and DA pulse pressures a l s o i n c r e a s e d . However, there was a much g r e a t e r damping of the pressure pulse by the g i l l s , A Pg p u l s e , i n d i c a t i n g an i n c r e a s e i n the t o t a l g i l l compliance (Table VIII) . V e n t i l a t i o n c h a r a c t e r i s t i c a l l y changed a f t e r a NAD i n j e c t i o n . There was a n o t i c e a b l e pause i n v e n t i l a t i o n which c o i n c i d e d with the r i s e i n Q and VA mean pressure ( F i g . 19). _ 121 -FIGURE 1.9 The e f f e c t of a n o r a d r e n a l i n e i n j e c t i o n (NAD) i n t o the v e n t r a l a o r t a of a l i n g cod on c a r d i o v a s c u l a r and r e s p i r a t o r y v a r i a b l e s that were monitored s i m u l t a n e o u s l y . C a r d i a c output (Q), m l . m i n - 1 , i n c r e a s e s due to s y s t o l i c and d i a s t o l i c flow changes. (Compare expanded t r a c e s 1, . • r e s t i n g , and 2, peak response). In t h i s f i s h there was a l s o an i n c r e a s e d heart r a t e . Note a l s o the synchronous r i s e i n VA and DA pres s u r e s (cm H 20) . From the pressure measurements i n the o p e r c u l a r c a v i t y (op) (cm H 20) the b r i e f r e s p i r a t o r y pause at the onset of the p r e s s o r response can be seen. - 122 -TABLE VIII A/ The e f f e c t of a no r a d r e n a l i n e (NAD) i n j e c t i o n i n t o the v e n t r a l a o r t a i n 8 l i n g cod (12 o b s e r v a t i o n s ) on measured c a r d i o v a s c u l a r v a r i a b l e s . The r e s t and peak response values are compared s t a t i s t i c a l l y , * s i g n i f i c a n t d i f f e r e n c e . The a b b r e v i a t i o n s are e x p l a i n e d i n Table VI. B/ The a f f e c t of a n o r a d r e n a l i n e i n j e c t i o n i n t o the v e n t r a l a o r t a on c a r d i o v a s c u l a r v a r i a b l e s c a l c u l a t e d from those measured i n Table VII A above. - 122a -A/ B/ T A B L E V I I I Rest(n=22) Peak(n=12) Heart Rate beats.min" 1 30.6 ± 1.0 32.9 ± 2.2 Stroke Volume ml.kg- 1 0.356 ± 0.014 0.477 ± 0.039* VA mean 52.6 ± 0.8 74.7 ± 2.6* VA p u l s e 12.2 ± 0.5 24.3 ± 1.8* DA mean 39.4 ± 0.8 60.9 ± 2.0* DA pu l s e 6.1 ±0.4 12.5 ± 1.2* Rest Peak Car d i a c output, Q ml.min - 1.kg" 1 10.7 ± 0.4 15.2 ± 1.1' Rg 1.26 ± 0.07 0.88 ± 0.06* Rs 3.81 ± 0.22 4.35 ± 0.37 APg ' 13.3 ± 0.7 13.8 ± 1.4 APg pu l s e 5.0 ± 0.4 11.8 ± 1.0* - 123 ~ . FIGURE 20 A summary o f t h e a n a l y s i s o f t h e e f f e c t o f n o r a d r e n a l i n e i n j e c t i o n (NAD) i n t o the v e n t r a l a o r t a i n 8 f i s h upon c a r d i a c o u t p u t , Q, s t r o k e volume, S.V. and g i l l r e s i s t a n c e , Rg. Time ( s e c ) i s measured from the c o m p l e t i o n Of t h e i n j e c t i o n . - 123a -r rest value ± $.e. 2 OH Q 1 ml.min".' Kg' — i — NAD • 50 150 250 350 0-8H S V . 0 4-| ml.Kg"' A* t A * y II NAD 50 150 250 350 Rg 1-6 H 0-8' cm H20.min.Kg.ml* B D B 0 ° ° 3 B • • • D • • • T 50 NAD 50 150 TIME AFTER'NAD'INJECTION, sec. 350 - 124 -ISOPRENALINE ( I S O P ) : ISOP i n j e c t e d i n t o the v e n t r a l a o r t a p r o d u c e d , i n g e n e r a l , s m a l l i n i t i a l c hanges d u r i n g t h e f i r s t 100 s e c . The Q was e l e v a t e d s l i g h t l y due t o an i n c r e a s e d s t r o k e volume ( F i g . 21, T a b l e I X ) . B o t h Rg and Rs were r e d u c e d s i g n i f i c a n t l y d u r i n g t h e i n i t i a l 100 s e c ( T a b l e I X ) . D u r i n g t h e 100 s e c a f t e r ISOP i n j e c t i o n t h e p r e s s u r e d r o p a c r o s s t h e g i l l s ( A Pg mean) was n o t a l t e r e d , b u t t h e r e was an i n c r e a s e d damping o f t h e p r e s s u r e p u l s e , APg p u l s e ( T a b l e I X ) . The b i p h a s i c r e s p o n s e t o ISOP i n j e c t i o n s was c h a r a c t e r i s e d by a s i g n i f i c a n t e l e v a t i o n o f h e a r t r a t e a f t e r t = 100 s e c ( T a b l e IX, F i g s . 21 and 22) and Q r e t u r n i n g t o a r e s t v a l u e . Rg and Rs were b o t h a t v a l u e s i n between t h o s e o f r e s t and t h e i r r e d u c e d v a l u e s when t < 100 s e c . - 125 -r FIGURE 21 A summary of the a n a l y s i s of the e f f e c t of i s o p r e n a l i n e (ISOP) i n j e c t i o n i n t o the v e n t r a l a o r t a of 8 f i s h upon c a r d i a c output (Q), heart rate ( h r ) , stroke volume (sv) and g i l l r e s i s t a n c e (Rg). Time (sec) i s measured from the completion of the i n j e c t i o n . - 125a -= rest value 1 i.e. 2 0 H Q 1 0 4 ml. min"'Kg"' • • • • j». » 1 • • s • I I ISOP 50 "1— 150 1 250 — ! — 350 450 0.8 H SV 0 . 4 ml.Kg-' • • • • • • • • • ISOP 50 —T— 150 250 350 450 50 H H R 25 beats, min"1 o 0 O rP O O O ° o 8 " °..rf»o o o 00 O o o ISOP 50 150 250 o o 1 350 450 Rg 3 -, 2 J 1 T cm.H2O.min.Kg.mr1 A : 1 - — 1 _ _ — • » T » » » * 1 I ISOP 50 — I — 150 —|— 250 — T — 350 —I 450 TIME A F T E R ' I S O P ' INJECTION, sec . _ 126 _ TABLE IX A/ The e f f e c t of an i s o p r e n a l i n e (ISOP) i n j e c t i o n i n t o the v e n t r a l a o r t a i n 8 l i n g cod (10 o b s e r v a t i o n s ) on measured c a r d i o v a s c u l a r v a r i a b l e s . The r e s t values are compared with a l l values when t <100 sec and t> 100 sec a f t e r i n j e c t i o n . * denotes s t a t i s t i c a l d i f f e r e n c e . The a b b r e v i a t i o n s are explained i n Table.VI. B/ The e f f e c t of a i s o p r e n a l i n e i n j e c t i o n i n t o the v e n t r a l a o r t a on c a r d i o v a s c u l a r v a r i a b l e s c a l c u l a t e d from those measured i n Table IX A above. - izba -TABLE IX A/ Heart Rate b e a t s . m i n - 1 Rest(n=18) t<100sec(h=17) 't>100sec(n=22) 30.2 ± 0.9 29.7 ± 0.8 34.9 ± 1.9* Stroke Volume , - i ml.kg" 0.352 ± 0.023 0.401 ± 0.022* 0.313 ± 0 . 0 2 3 VA mean 50.8 + 0.7 47.5 ± 1.1* 44.1 ± 0.8' VA p u l s e 13.1 ± 0.4 14.7 ± 0.9* 11.7 ± 0.8' DA mean DA p u l s e 39.4 ± 0.9 6.2 ± 0.3 36.3 ± 1.2* 6.6 ± 0.5 34.4 ± 0.8* 5.3 ± 0.5* B/ C a r d i a c output, Q ml.min-.kg"""1 Rest t<100sec t>100sec 1 0 . 5 + 0 . 6 11.8 ± 0 . 7 * 10.6 ± 0 . 8 Rg 1.19 ± 0.13 0.92 ± 1.10* 1.00 ± 0.12 Rs 4.13 ± 0 . 3 8 3.34 ± 0 . 3 0 ' 3.63 ± 0.32 APg pulse 11.5 ± 0.8 6.9 ± 0.4 11.1 ± 1.2 ; . l ± 0.6" 9.7 ± 0.9V 6.4 ± 0.6 - 127 -FIGURE 22 . The e f f e c t of an i s o p r e n a l i n e (ISOP) i n j e c t i o n i n t o the v e n t r a l a o r t a of a l i n g cod on c a r d i o v a s c u l a r and r e s p i r a t o r y v a r i a b l e s which were monitored s i m u l t a n e o u s l y . Note the b i p h a s i c response of c a r d i a c output (Q, ml.min - 1-) and heart r a t e ( h . r . , beats.min~l) dur i n g p e r i o d s '2' and '3' as compared to r e s t , p e r i o d '1'. Expanded t r a c e s Of p e r i o d '1', '2' and '3' are a l s o shown. In p e r i o d '2' heart r a t e c h a r a c t e r i s t i c a l l y decreases and str o k e volume i n c r e a s e s , w h i l s t i n p e r i o d '3' heart r a t e i n c r e a s e s and stroke volume i s decreased. During these responses DA and VA mean pressures (cm R^O) were reduced, but pulse p r e s s u r e s are e l e v a t e d during p e r i o d '2'. V e n t i l a t i o n i s i n d i c a t e d by o p e r c u l a r p r e s s u r e s (cm H2O) and does not change. - 12 8 -The e f f e c t of i n t r a v a s c u l a r a d m i n i s t r a t i o n of c h o l i n e r g i c agents  i n r e s t i n g l i n g cod ACH i n j e c t i o n i n t o the v e n t r a l a o r t a produced a peak . response w i t h i n 50 t o 60 sec of i n j e c t i o n ( F i g s . 23 and 24). The extent of the response to ACH i n j e c t i o n v a r i e d from f i s h to f i s h . The Q and stroke volume both i n c r e a s e d s i g n i f i c a n t l y , but the mean heart r a t e d i d not change (Table X). The peak s y s t o l i c flow was e l e v a t e d and the d i a s f o l i c . f l o w d i d not d e c l i n e with time. Therefore the shape of the v e n t r a l a o r t i c flow pulse was.changed to a p a t t e r n resembling that seen during the peak response to a NAD i n j e c t i o n . There was marked i n c r e a s e i n Rg, but Rs d i d not change (Table X). During the peak response VA and DA pulse pressures were e l e v a t e d , and APg pulse was a l s o . i n c r e a s e d . V e n t i l a t i o n r a t e s were in c r e a s e d a f t e r an ACH i n j e c t i o n . Carbachol (CARB) i n j e c t i o n s produced peak responses i n blood flow and pre s s u r e s i m i l a r to the ACH responses. However, CARB, which remains longer i n the c i r c u l a t i o n , reduced heart r a t e d r a m a t i c a l l y , to 12 to 15 b e a t s . m i n - 1 a f t e r about 90 to 120 sec. The Q was also, reduced below r e s t i n g values at t h i s time. One hour a f t e r an a t r o p i n e (ATROP) i n j e c t i o n heart r a t e had g r a d u a l l y r i s e n to a steady s t a t e value of about 45 beats, min--*- (n=3 f i s h ) . There were no beat to beat heart r a t e f l u c t u a t i o n s and t h i s peak h e a r t r a t e was sus t a i n e d f o r at l e a s t 2 h r s . (This was the f i n a l experiment before s a c r i f i c i n g the f i s h ) . . The'Q'was'not changed by ATROP i n j e c t i o n . Ninety minutes a f t e r the ATROP i n j e c t i o n a CARB i n j e c t i o n i n t o the d o r s a l a o r t a - 129 -had no e f f e c t on heart r a t e , Q or Rg, u n l i k e the u n a t r o p i n i s e d f i s h . The Rs however decreased by 15% about 30 sec a f t e r the . i n j e c t i o n with corresponding r e d u c t i o n s i n VA and DA mean pressu r e s ( F i g . 25). V e n t i l a t i o n r a t e was increased s l i g h t l y above the pre-CARB i n j e c t i o n r a t e . - 1 3 0 -FIGURE 23 . .. . The a f f e c t of an a c e t y l c h o l i n e injection'(ACH) i n t o the v e n t r a l a o r t a of 2 d i f f e r e n t l i n g cod (a) and ( b ) . Note the v a r i a b i l i t y i n the peak pres s o r response between f i s h . In (a) the c a r d i a c output (Q), ml.min -!, changes are small with heart r a t e ( h r ) , beats.min - 1-, i n c r e a s i n g s l i g h t l y , i f anything. DA mean pre s s u r e , cm H 20, was depressed and the o p e r c u l a r pressure (op) record i n d i c a t e s that v e n t i l a t i o n r a t e i n c r e a s e d . In (b) only c a r d i a c output and VA and DA pressure are shown, with areas '1' and '2' d i s p l a y e d with an expanded time s c a l e . The p r e s s o r response of ACH i s evident with DA pressures also- i n c r e a s i n g s l i g h t l y . Q increased markedly through increased stroke volume, with £lood flow being e l e v a t e d during both the s y s t o l i c and d i a s t o l i c p e r i o d s . - 131 -TABLE X A/ The e f f e c t of an a c e t y l c h o l i n e (ACH) i n j e c t i o n i n t o the v e n t r a l a o r t a i n 8 l i n g cod (11 o b s e r v a t i o n s ) on measured c a r d i o v a s c u l a r v a r i a b l e s . The r e s t and peak response values are compared s t a t i s t i c a l l y , * s i g n i f i c a n t d i f f e r e n c e . The a b b r e v i a t i o n s are e x p l a i n e d i n Table VI. B/ The e f f e c t of an a c e t y l c h o l i n e i n j e c t i o n i n t o the v e n t r a l a o r t a on c a r d i o v a s c u l a r v a r i a b l e s c a l c u l a t e d from those measured i n Table X A above. 131a -TABLE X A/ Rest(n=30) Peak(n=ll) Heart Rate beats .min - 1 29.1 ± 0.7 29.1 ± 1.2 Stroke Volume ml. kg VA mean VA p u l s e DA mean DA p u l s e 0.400 ± 0.016 0.440 ± 0 . 0 2 0 * 52.7 ± 0.6 12.4 ± 0.4 39.7 ± 0.6 6.0 ± 0 . 3 63.7 ± 2.0* 22.4 ± 2.3* 42.3 ± 1.4* 7.9 ± 0 . 6 B/ C a r d i a c output, Q ml .min"1. k g - 1 Rg Rs A Pg A Pg p u l s e Rest 11.5 ± 0.1 1.16 ±0.08 3.60 ±0.16 13.2 ± 0.9 6.4 ±0.3 Peak 12.7 ± 0 . 6 * 1.71 ± 0.19' 3 .25 ± 0.23 22.2 ±2.2' 14.5 ±2.1' - 132 -. FIGURE.24 A summary of the a n a l y s i s of the e f f e c t of a c e t y l c h o l i n e (ACH) i n j e c t i o n i n t o the v e n t r a l a o r t a of 8 l i n g cod upon c a r d i a c output (Q), g i l l r e s i s t a n c e (Rg) and the drop i n mean pressure over the g i l l s (APg). Time (sec) i s measured from the time of completion of the i n j e c t i o n . - 132a -= rest value ± s.e. 20 Q ml.min.^Kg"' • • 9 _ Q9 • 3 — s — ACH 50 150 250 Rg 1 M c m H jO.min.Kg.mr1 a • a i • ACH i 50 • a • 150 250 35-I APg 15 H cm H 2 0 1 I a i •R«. I • ACH 50 150 250 TIME AFTER'ACH' INJECTION , sec _ 133 _ FIGURE 25 The e f f e c t of a ca r b a c h o l i n j e c t i o n (CARB) i n t o the d o r s a l a o r t a of a p r e v i o u s l y a t r o p i n i s e d l i n g cod. Heart r a t e (hr, b e a t s . m i n - 1 ) and c a r d i a c output (Q, ml.min - 1) do not change, but v e n t r a l (VA) and d o r s a l (DA) a o r t i c p r e s s u r e s are reduced. V e n t i l a t i o n r a t e i n c r e a s e s , as i n d i c a t e d by the b u c c a l p ressure (b.p., cm H2O) r e c o r d . The expanded t r a c e i s a demonstration o f . t h e zero flow c a l i b r a t i o n using a pneumatic c u f f . The c u f f was i n f l a t e d over the time p e r i o d i n d i c a t e d by the t h i c k h o r i z o n t a l bar on the Q r e c o r d . - 133a --.134 -DISCUSSION The r e s t i n g l i n g cod A d e t a i l e d in v i v o examiraation of c a r d i o v a s c u l a r v a r i a b l e s p e r t a i n i n g to b r a n c h i a l blood flow was made with Ophiodon elongatus using simultaneous measurements of pre- and p o s t - b r a n c h i a l blood p r e s s u r e s , and Q. The f i n d i n g s of the present study are g e n e r a l l y c o n s i s t e n t with other in v i v o s t u d i e s i n t e l e o s t s (Stevens e t a l . , 1972: Jones et a l . , 1974; Chan and Chow, 1976). V e n t r a l a o r t i c flow and, t h e r e f o r e , g i l l blood flow are continuous even during diastolfe due to the Wind-kessel e f f e c t of the l a r g e bulbus a r t e r i o s u s . Blood p r e s s u r e s and Q have s i m i l a r values to those measured (or c a l c u l a t e d ) f o r other t e l e o s t s . A Q value of 5.9 m l . m i n - 1 . K g - 1 was reported by Stevens e_t a l . (1972) f o r sm a l l e r specimens of Ophiodon (1 to 4 kg). They a l s o r e p o r t e d a heart r a t e of 47 beats .min--'- and a v e n t i l a t i o n r a t e of 26.8 b r e a t h s . m i n - 1 . Such high r a t e s and low Q values were only found i n d i s t u r b e d or traumatised f i s h i n my study. A s l i g h t l y higher water temperature (13°C) was used by Stevens e t a l . , which may e x p l a i n , i n p a r t , the high c a r d i a c and r e s p i r a t o r y r a t e s . Q i n r e s t i n g Gadus morhua i s 52 ml.min--'- (5 f i s h weighing 2 t o 3 kg) (Jones et a l . , 1974) . Although no mean weight was given f o r Gadus, i t i s apparent that Q i s g r e a t e r i n Gadus than i n Ophiodon. The i n t e r s p e c i f i c d i f f e r e n c e i n Q i s expected s i n c e f r e e swimming f i s h w i l l have higher Q than bottom d w e l l i n g f i s h . Trout f o r i n s t a n c e , have - 135 -a s i m i l a r r e s t i n g Q to Gadus of about 20 ml.min -'-.kg 1 (as c a l c u l a t e d by the F i c k p r i n c i p l e , Stevens and R a n d a l l , 1967b). In the r e s t i n g l i n g cod the heart r a t e v a r i e s from beat to to beat, but Q changes l i t t l e s i n c e there are compensatory changes i n s t r o k e volume,. These o b s e r v a t i o n s can be e x p l a i n e d i n terms of S t a r l i n g ' s Law of the h e a r t . According to S t a r l i n g ' s law, stroke volume can be a l t e r e d i n t r i n s i c a l l y and i s r e l a t e d to the degree o f s t r e t c h i n g of a t r i a l , muscle f i b e r s when the chamber i s f i l l e d . Greater a t r i a l f i l l i n g w i l l produce higher stroke volumes and g r e a t e r a t r i a l f i l l i n g can be achieved by a reduced heart r a t e , e l e v a t e d venous p r e s s u r e s , or both. The general a p p l i c a b i l i t y and the importance of S t a r l i n g ' s law of the heart i n r e s t i n g t e l e o s t s , has been d i s c u s s e d by Randall (1970) . Recently, Short and co-workers (1977) made an e x t e n s i v e study of c a r d i a c a c t i v i t y i n d o g f i s h and e s t a b l i s h e d the importance of S t a r l i n g ' s law i n these elasmobranchs. The APg mean represents only a 25% to 30% l o s s of VA p r e s s u r e , which suggests that the g i l l s have an unusually low r e s i s t a n c e t o flow f o r a c a p i l l a r y network compared to mammalian . m i c r o c i r c u l a t i o n s . The reason f o r t h i s may be due to l a m e l l a r sheet flow s i n c e h i s high, almost double that found f o r cat pulmonary m i c r o c i r c u l a t i o n (See S e c t i o n I ) . The pressure p u l s e , i s , however, damped by 60%, which i s not at a l l s u r p r i s i n g given the compliant nature of the m i c r o v a s c u l a r bed ( S e c t i o n s I and I I I ) . The importance of systemic compliance oh damping of the DA - 136 -pulse pressure (Jones e t , a l . , 1974) was. not examined here. V e n t i l a t i o n i n l i n g cod d i d not appear to d i f f e r i n any major r e s p e c t from pr e v i o u s d e s c r i p t i o n s of t e l e o s t v e n t i l a t i o n p a t t e r n s ( B a l l i n t i j n and Hughes, 1963; Shelton, 1970), although it.was not examined thoroughly i n t h i s study. Ophiodon have buccal and o p e r c u l a r v e n t i l a t i o n pumps. The buccal pumping and the o p e r c u l a r s u c t i o n phases generated the g r e a t e s t h y d r o s t a t i c pressure changes, but r e s t i n g v e n t i l a t o r y pressures never exceeded 1 cm H 20. V e n t i l a t i o n r a t e s could be as low as 6 to 7 b r e a t h s . m i n - 1 i n some f i s h that had been l e f t undisturbed f o r many hours. The mean v e n t i l a t i o n r a t e (12 breaths .min--'-) i s low compared with the t r o u t , f o r i n s t a n c e , where the r a t e i s commonly 60 breath.min --'-. The q u i e t v e n t i l a t o r y p a t t e r n of Ophiodon c o r r e l a t e s w e l l with i t s benthic h a b i t a t (Shelton, 1972). Micropressure measurements and g i l l r e s i s t a n c e T h i s study a p p l i e d the micropressure techniques to both r e s p i r a t o r y v e s s e l s and the f i s h m i c r o c i r c u l a t i o n f o r the f i r s t time. I t s a p p l i c a t i o n was c l e a r l y r e s t r i c t e d by movement a r t i f a c t s and the i n a b i l i t y to see the p i p e t i n s i d e the v e s s e l s . In v i v o the s e n s i t i v i t y of the g i l l to, i r r i g a t i o n , the i n s t a b i l i t y of the g i l l f i l a m e n t s w i t h i n the water c u r r e n t and the incomplete response of the g i l l f i l a m e n t and o p e r c u l a r muscles to an a e s t h e s i a were unresolveable problems. Consequently many of the micropuneture r e c o r d i n g s were not used because of - 137 -u n c e r t a i n t y concerning t h e i r v a l i d i t y (Appendix I I ) . The i n  v i t r o p r e p a r a t i o n was t e c h n i c a l l y more s u i t a b l e , but possessed s l i g h t l y d i f f e r e n t p e r f u s i o n c h a r a c t e r i s t i c s compared to i n  v i v o . The f i l a m e n t movements a s s o c i a t e d with Rg changes f o l l o w i n g drug i n f u s i o n were unfortunate, and the g i l l l a m e l l a e were f a r too f l e x i b l e f o r micropuncture measurements. Despite the above l i m i t a t i o n s the high f i d e l i t y of some i n v i t r o micropressure r e c o r d i n g s ( F i g . 18b) was indeed encouraging. A g e n e r a l p a t t e r n f o r the pressure d i s t r i b u t i o n w i t h i n the f i l a m e n t v a s c u l a t u r e was e s t a b l i s h e d both i n * v i v o and i_n * v i t r o . T h i s p a t t e r n shows that the major r e s i s t a n c e to flow r e s i d e s w i t h i n the l a m e l l a r u n i t and that the combined r e s i s t a n c e of the a f f e r e n t b r a n c h i a l a r t e r i e s i s s m a l l . These c o n c l u s i o n s help s u b s t a n t i a t e p r e d i c t i o n s made i n S e c t i o n . I . The absolute pressure l o s s i n the b r a n c h i a l a r t e r i e s and i n the l a m e l l a r u n i t cannot be e s t a b l i s h e d with c e r t a i n t y s i n c e the micropressure measurements may not have been made 'from, only the f i l a m e n t a r t e r i e s , i . e . some measurements could have been made i n l a m e l l a r a r t e r i o l e s . Nevertheless a good i d e a of the p o s s i b l e range of r e s i s t a n c e s can be deduced from f i g u r e 17. In the a n a e s t h e t i s e d f i s h VA blood pressure was reduced by 56% due to the t o t a l g i l l r e s i s t a n c e . As l i t t l e as 1.0% of Rg was due to the r e s i s t a n c e of the a f f e r e n t b r a n c h i a l a r t e r i e s , which agrees with that reported i n S e c t i o n I where AFA r e s i s t a n c e was c a l c u l a t e d as 16% of Rg. The l a m e l l a r u n i t , on average, accounted f o r over h a l f , (58%) of Rg (a value d e r i v e d by comparing the ~ 138 -average of a l l a f f e r e n t and a l l e f f e r e n t m i c r o p r e s s u r e s ) . S i m i l a r c o n c l u s i o n s to these can be made i n v i t r o . In t h i s case the l a m e l l a r u n i t was on average 44% of Rg (a maximum value of 73%). I t i s a l s o c l e a r that a f f e r e n t and e f f e r e n t a r t e r y r e s i s t a n c e s were i n c r e a s e d i n i s o l a t e d holobranch p r e p a r a t i o n s , which probably accounts f o r the o v e r a l l r e d u c t i o n i n the l a m e l l a r component to o v e r a l l Rg. The a f f e r e n t a r t e r i o l e i s c l e a r l y the major r e s i s t a n c e i n the l a m e l l a r u n i t and the hypothesis that the major s i t e of g i l l r e s i s t a n c e i s the a f f e r e n t l a m e l l a r a r t e r i o l e s has now r e c e i v e d some experimental support. In Ophiodon i t appears that the proximal f i l a m e n t i s more g r e a t l y perfused than d i s t a l f i l a m e n t under c e r t a i n _in v i v o c o n d i t i o n s , assuming that the light, c o l o r a t i o n of d i s t a l l a m e l l a e i n a n a e s t h e t i s e d f i s h ' was due to slower f l o w i n g , f u l l y oxygenated blood (See S e c t i o n I I I a l s o ) . A s i m i l a r , t e n t a t i v e c o n c l u s i o n was a l s o made by Davis (1972) who made v i s u a l o b s e r v a t i o n s and took, i n f r a red f i l m s of t r o u t g i l l s . Blood flow c o n t r o l at the ALA e x p l a i n s 'the d i f f e r e n c e between proximal and d i s t a l l a m e l l a r p e r f u s i o n s i n c e d i s t a l ALAs have a higher r e s i s t a n c e to flow due to t h e i r geometry ( S e c t i o n I ) . Davis (1972).also concluded that an a d r e n a l i n i n j e c t i o n i n c r e a s e d the p e r f u s i o n of the f i l a m e n t t i p as w e l l as the f i l a m e n t as a whole. Increased p e r f u s i o n of the t i p r e g i o n would i n v o l v e l a m e l l a r r e c r u i t m e n t . However, the mode.by which ALAs would c o n t r o l l a m e l l a r recruitment i s s t i l l s p e c u l a t i v e s i n c e l a m e l l a r recruitment could occur a u t o m a t i c a l l y with i n c r e a s e d blood p r e s s u r e , blood flow or both. In a d d i t i o n - 139 -there may be autonomic c o n t r o l of ALA size,, s i n c e in t r o u t the v e s s e l s have a d r e n e r g i c i n n e r v a t i o n . Randall (1976). p o s t u l a t e d 8- adrenergic: d i l a t i o n of d i s t a l ALAs to increase l a m e l l a r p e r f u s i o n . In a c t u a l i t y a combination of these two'controls i s expected. Drug i n j e c t i o n s t u d i e s The response to any drug i n j e c t i o n i n v i v o is.complex and i t s i n t e r p r e t a t i o n i s , t h e r e f o r e , d i f f i c u l t and to some degree s p e c u l a t i v e . Drugs i n j e c t e d i n t o the v e n t r a l a o r t a pass through the g i l l i n about 15 to 20 seconds ( S e c t i o n I ) . Thus, b r a n c h i a l v a s c u l a r responses to such i n j e c t i o n s should have a time l a g of t h i s o r d e r . I analysed peak r e s p o n s e s , # b u t peak responses developed over a p e r i o d of 30 to 60. sec. Thus by combining a l l peak responses with up to 100 sec delay a f t e r the i n j e c t i o n I i n c l u d e d a l l responses that were b r a n c h i a l i n o r i g i n . In v i t r o b r a n c h i a l v a s o d i l a t i o n and v a s o c o n s t r i c t i o n are e a s i l y demonstrable with drug i n f u s i o n s s i n c e i s constant. The demonstrated b r a n c h i a l v a s o a c t i v e e f f e c t s of a g o n i s t s drugs i n  v i t r o were a l s o u t i l i s e d i n the a n a l y s i s of i n v i v o responses to the same a g o n i s t s . Lag times f o r a response were h e l p f u l i n e v a l u a t i n g the s i t e s of drug a c t i o n as i n d i c a t e d by the ob s e r v a t i o n s on the c a r d i a c e f f e c t s of s y n t h e t i c a g o n i s t drugs i n j e c t e d i n t o the v e n t r a l a o r t a . The i n o t r o p i c e f f e c t of ISOP on the heart and the negative c h r o n o t r o p i c e f f e c t of CARB both occured about 2 minutes a f t e r i n j e c t i o n . T h i s time lag i s - 14 0 -c o n s i s t a n t with estimated of c i r c u l a t i o n times f o r t e l e o s t s (Davis, 1970). In v i v o d i s t i n c t i o n must be made between d i r e c t a g o n i s t a c t i o n s and r e f l e x o g e n i c (hemostatic?) mechanisms. Randall and Stevens (1967), f o r i n s t a n c e , noted r e f l e x o g e n i c vagal s t i m u l a t i o n of the heart a f t e r a d r e n a l i n e i n j e c t i o n s i n t r o u t . Lutz and Wyman (1932) repo r t e d that an inc r e a s e d VA pressure i n d o g f i s h e l i c i t e d a v a g a l l y mediated b r a d y c a r d i a , as d i d Mott (1951) with e e l s . Furthermore, S a t c h e l ! (1962) reported that a bra d y c a r d i a was evoked during hypoxia when VA pressure a l s o i n c r e a s e d . I w i l l attempt to make my d i s t i n c t i o n by using known va s o a c t i v e a c t i o n s from i n v i t r o responses and time lag of the response. For example, a NAD i n j e c t i o n i n Ophiodon had the d i r e c t a g o n i s t e f f e c t of reducing Rg, but the a s s o c i a t e d i n c r e a s e i n Q must be r e f l e x o g e n i c s i n c e the time lag of the response pre c l u d e s r e c i r c u l a t o r y e f f e c t s of NAD on the heart. Under the steady s t a t e p u l s a t i l e p e r f u s i o n c o n d i t i o n s of the i n v i t r o experiments ACH markedly Increased Rg. In v i v o , ACH i n j e c t e d i n t o the v e n t r a l a o r t a a l s o caused a marked b r a n c h i a l v a s o c o n s t r i c t i o n i n l i n g cod s i n c e Rg rose markedly (Table X). The presence of a c h o l i n e r g i c a l l y mediated b r a n c h i a l v a s o c o n s t r i c t i o n i s i n agreement with p r e v i o u s i_n v i t r o and i n v i v o i n v e s t i g a t i o n s i n t e l e o s t s p e c i e s (See I n t r o d u c t i o n ) . A s p e c i f i c s i t e of b r a n c h i a l c h o l i n e r g i c v a s o c o n s t r i c t i o n has been hypothesised (Smith, 1977; Dunel and Laurent, 1977), which i s l o c a t e d at the base of the e f f e r e n t - 141 -f i l a m e n t a r t e r y . -A number of o b s e r v a t i o n s i n my study are c o n s i s t a n t with t h e i r h y p o t h e s i s . F i r s t l y , there are v a s c u l a r c o n s t r i c t i o n s at the base of the EFA i n l i n g cod ( S e c t i o n I) s i m i l a r to those observed in other t e l e o s t s . Secondly, ACH i n f u s i o n in v i t r o r e s u l t e d i n flow being d i v e r t e d from normal e f f e r e n t pathways, since outflow v i a the e f f e r e n t a r t e r y decreased markedly with i n f l o w unchanged; ah o b s e r v a t i o n a l s o recorded by Smith (1977). The o b s e r v a t i o n can be e x p l a i n e d by Increased venolymphatic flow due to c h o l i n e r g i c c o n s t r i c t i o n at the EFA base, because EFA to venolymphatics connections are l o c a t e d upstream to t h i s s i t e . An a l t e r n a t i v e arguement i s t h a t a general c h o l i n e r g i c c o n s t r i c t i o n i n a l l a r t e r i e s e l e v a t e s Rg, s i m i l a r to the a c t i o n demonstrated on s t r i p s of l a r g e b r a n c h i a l a r t e r i e s (Klaverkamp and Dyer, 1974). Smooth muscle a l s o surrounds the s h o r t v e s s e l s connecting the EFA to the ECV and, t h e r e f o r e , these v e s s e l s would a l s o be c o n s t r i c t e d , thus preventing venolymphatic flow i f the major response was a general c h o l i n e r g i c v a s o c o n s t r i c t i o n . Thus, while some general c h o l i n e r g i c v a s o c o n s t r i c t i o n may occur, i t cannot account f o r the observed r e d u c t i o n i n e f f e r e n t a r t e r y outflow. L a s t l y , the e f f e r e n t micropressures c o n s i s t e n t l y rose a f t e r ACH treatment i n the i s o l a t e d holobranch p r e p a r a t i o n s . Since Q Q f e l l at the same time, the c h o l i n e r g i c c o n s t r i c t i o n s i t e must be downstream of the pressure measurements. The s i t e could e i t h e r be at the base of the EFA, again., or w i t h i n the e f f e r e n t arch a r t e r y . A g e n e r a l response i n the EAA can be argued a g a i n s t because i t would r e q u i r e e x t e n s i v e c h o l i n e r g i c i n n e r v a t i o n of the - 142 -EAA or c i r c u l a t i n g l e v e l s of a c e t l y c h o l i n e , The p o s s i b i l i t y of hormonal ACH a c t i o n s i n t e l e o s t s has been d i s c u s s e d and r e j e c t e d (Wood, 1975), but nothing i s known about the d i s t r i b u t i o n of c h o l i n e r g i c f i b r e s i n the g i l l s . Whether the s i t e of c h o l i n e r g i c a c t i o n i s the base of the EFA or not w i l l have to await l o c a l i s a t i o n of c h o l i n e r g i c t e r m i n a l s . The o b s e r v a t i o n that g i l l . f i l a m e n t s move with ACH i n f u s i o n i n v i t r o may be important i n producing Rg changes. The base of the EFA l i e s c l o s e to the c a r t i l a g e which surrounds the g i l l a r ch. I t i s p o s s i b l e that the base of the EFA. could be p h y s i c a l l y c o n s t r i c t e d by the c a r t i l a g e when the arch hemibranchs are spread out. Whenever perfused holobranchs were being set up, the o r i e n t a t i o n of the arch bend, which a f f e c t e d the spread of hemibranchs, was important s i n c e some manipulations would r a i s e Pj_ independent of other f a c t o r s . Smith (1976) has a c t u a l l y noted the d i f f i c u l t y of i n f u s i n g v i s c o u s f l u i d s past the base of the e f f e r e n t f i l a m e n t a r t e r y i n t r o u t . Thus f i l a m e n t movements may be important in, the Rg e l e v a t i o n a f t e r ACH a d m i n i s t r a t i o n . What i s c e r t a i n i s that b r a n c h i a l v a s o c o n s t r i c t i o n i s l o c a l i s e d to the e f f e r e n t g i l l a r t e r i e s i n l i n g cod and w i l l r e s u l t i n e l e v a t e d EFA i n t r a v a s c u l a r pressure unless flow i s reduced. Consequently, i n t r a l a m e l l a r pressure ( A p i a m ) ; and l a m e l l a r input p r e s s u r e s w i l l a l s o r i s e i n the whole f i l a m e n t . These e l e v a t e d p r e s s u r e s i n i t i a t e l a m e l l a r r e c r u i t m e n t , i n t r a l a m e l l a r shunting and i n c r e a s e d l a m e l l a r blood volume - 143 .-a u t o m a t i c a l l y . I t i s postuleited, t h e r e f o r e , that c h o l i n e r g i c v a s o c o n s t r i c t i o n at the base of the f i l a m e n t i n i t i a t e s changes i n blood flow of" the whole f i l a m e n t . Input flow must be maintained or e l e v a t e d i n face of the increased Rg, however, f 6 r these flow p a t t e r n changes to be e f f e c t e d . In. v i v o a f t e r the VA i n j e c t i o n of ACH, Q i n c r e a s e d r e f l e x o g e n i c a l l y , which e f f e c t e d the l a m e l l a r r e c r u i t m e n t , l a m e l l a r volume changes and i n t r a l a m e l l a r shunting i n i t i a t e d by b r a n c h i a l v a s o c o n s t r i c t i o n . The ACH i n j e c t i o n was a l s o a s s o c i a t e d with e l e v a t e d v e n t i l a t i o n r a t e s and pumping pressures ( F i g . 23) which i n c r e a s e g i l l v e n t i l a t i o n (Vgj. Vagal and c h o l i n e r g i c neurones have been i m p l i c a t e d with r e s p i r a t o r y c o n t r o l (Hughes and Shelton, 1962; Roberts, 1975), but my experiments provide no evidence f o r mechanism, behind the i n c r e a s e i n Vg. Increased Vg, however, i n c r e a s e s 0 2 d e l i v e r y to the g i l l s which i s fundamental to enhancing 0 2 t r a n s f e r i n f i s h . I, t h e r e f o r e , concluded that the b r a n c h i a l a c t i o n s of-ACH. are a l l geared towards i n i t i a t i n g c o n d i t i o n s f o r increased 0 2 t r a n s f e r , which are e f f e c t e d by i n c r e a s e d Q ( r e f l e x o g e n i c ) and Vg. The heart i n l i n g cod has c h o l i n e r g i c r e c e p t o r s which reduce heart r a t e i f . s t i m u l a t e d - note the e f f e c t s of CARB i n j e c t i o n s . The heart a l s o r e c e i v e s c h o l i n e r g i c tone s i n c e a t r o p i n e i n j e c t i o n s r e s u l t e d i n a sustained t a c h y c a r d i a : a r e s u l t which agrees with previous f i n d i n g s f o r Ophiodon (Stevens e_t a l . , 1972) , carp ( R a n d a l l , 1968) and Gadus morhua (Holmgren, 1977). The r e s t i n g t r o u t l a c k s vagal tone (Stevens and R a n d a l l , • * - .14 4 -1967a). I t i s i n t e r e s t i n g that no heart rate higher than the a t r o p i n i s e d r a t e was ever observed i n my experiments.. And that the c h a r a c t e r i s t i c beat to beat heart r a t e f l u c t u a t i o n s were a b o l i s h e d b y , a t r o p i n e . C h o l i n e r g i c tone i s probably mediated v i a vagal c a r d i a c i n n e r v a t i o n as i n other f i s h . Consequently, i f the gen e r a l c h o l i n e r g i c nerve a c t i v i t y increased i n l i n g cod, then a bra d y c a r d i a and an e f f e r e n t b r a n c h i a l v a s o c o n s t r i c t o n could a l l be expected to f o l l o w immediately. Decreased c h o l i n e r g i c a c t i v i t y would i n c r e a s e heart r a t e and Cj, i f stroke volume, was maintained. • I t i s c l e a r that i n t r a v a s c u l a r i n f u s i o n of catecholamines reduces Rg i n v i t r o and i n v i v o i n l i n g cod. My fi r j d i n g s agree with previous r e s u l t s f o r catecholamine i n j e c t i o n s i n t e l e o s t s both i n v i v o and i n v i t r o . ISOP, a pure f> - adr e n e r g i c a g o n i s t , produced only a small Rg change iri v i t r o . In t r o u t g i l l s ^ - a d r e n e r g i c d i l a t i o n produces more marked Rg •reductions (Wood, 1975). Why i s there t h i s d i f f e r e n c e ? When comparing r e s u l t s of d i f f e r e n t experiments one must co n s i d e r the v a s c u l a r p r o p e r t i e s of the i n v i t r o p r e p a r a t i o n and the p e r f u s i o n c o n d i t i o n s . Wood c o n t r o l l e d Input pressures with reduced i n f l o w r a t e s . I used normal Inflow r a t e s and input pressures were high. I suggest that under the c o n d i t i o n s of my i n v i t r o p r e p a r a t i o n the v e s s e l s had l i t t l e or no v a s c u l a r tone and only small •' d i l a t o r y responses were p o s s i b l e . V a s c u l a r tone i s d e f i n e d as the a b i l i t y of a v e s s e l w a l l to maintain i t s diameter over a range of i n t e r n a l p r e s s u r e s . - 1 4 5 : -NAD, a potent a- and 6 - adrenergic agonist, produced a greater reduction In Rg than did ISOP in my experiemnts. There are two possible explanations for t h i s . F i r s t NAD could be a -d i l a t o r y and $ - d i l a t o r y in Ophiodon. Since only, a -constrictory receptors have been found previously in teleost g i l l vasculature (Wood, 1975), this explanation is u n l i k e l y . An a -constrictory and 8 - d i l a t o r y action is the other p o s s i b i l i t y , but how can a - c o n s t r i c t i o n decrease g i l l resistance? In g i l l s 0 1 - c o n s t r i c t i o n has only small, effects on o v e r a l l perfusion pressures (Wood, 1975). Therefore vessel diameters are unlikely to be altered greatly by ct - c o n s t r i c t i o n . I f , ins tead, . the a -c o n s t r i c t i o n e f f e c t is. to s t i f f e n afferent a r t e r i a l vessel walls, i.e. increase vascular tone, the pressure p r o f i l e in the g i l l s would be altered. Pulse pressures would be larger which would raise lamellar input pressures. Higher input pressures would overcome c r i t i c a l closure pressures and r e c r u i t lamellae, in addition to increasing the lamellar volume. Both changes reduce Rg with constant conditions. The hypothesis of a -c o n s t r i c t i o n conferring branchial vascular tone is given support by comparing vascular, properties i h vivo and in v i t r o . G i l l arch, preparations are noted for their lack of vascular tone, high compliance and high resistance to flow (Wood, 1974b; vi s u a l observations; Section I I I ) . The findings of Payan and Girard (1977) can also be interpreted to suggest either a lack of or a reduced vascular tone in v i t r o since phentolamine and propanolol ( a - and 8 - adrenergic antagonists, respectively) had l i t t l e - 146 -a f f e e t . o n g i l l p e r f u s i o n pressures or flow by themselves. In v i v o , g i l l r e s i s t a n c e i s low (Table VI) and g i l l v a s c u l a r tone does e x i s t (Wood, 1974b, Helgason and N i l s s o n , 1973) c o n t r a r y to the suggestions of Randall and Stevens (1967). In r e s t i n g f i s h b r a n c h i a l v a s c u l a r tone may i n f a c t be due to c i r c u l a t i n g catecholamines which are at s u f f i c i e n t l e v e l s to e l i c i t t o n i c v a s c u l a r e f f e c t s (Nakano and Tomlinson, 1967; Mazeaud e t a l . : ( 1977). I t i s concluded that the b r a n c h i a l v a s c u l a t u r e i n Ophiodon possess 6 - d i l a t o r y and a - c o n s t r i c t o r y r e c e p t o r s . In r e s t i n g l i n g cod VA i n j e c t i o n s of NAD and of ISOP produced d i f f e r e n t responses. NAD had a marked p r e s s o r e f f e c t , but ISOP produced a depressor e f f e c t . One e x p l a n a t i o n f o r t h i s i s t h a t .the systemic c i r c u l a t i o n possesses a, - a d r e n e r g i c r e c e p t o r s , which produce a very potent v a s o c o n s t r i c t i o n (Stevens and R a n d a l l , 1967; Wood and Shel t o n , 1975; Stevens et a l . , 1974). The c o e l i a c a r t e r y i s one s p e c i f i c s i t e of a d r e n e r g i c v a s o c o n s t r i c t i o n i n t e l e o s t s (Holmgren, 1978)* NAD i n j e c t e d i n t o the v e n t r a l a o r t a i n my experiments increased Rs, i n most l i n g cod. In the remainder, Rs was not changed by NAD i n j e c t i o n and i n these the p r e s s o r response was a r e s u l t of Q being e l e v a t e d . NAD a d m i n i s t r a t i o n i n l i n g cod i n a d d i t i o n to b r a n c h i a l v a s o d i l a t i o n w i l l b r i n g about l a m e l l a r recruitment and i n t r a l a m e l l a r shunting because of i t s . p r e s s o r e f f e c t s and e l e v a t i o n of Q. Lamellar recruitment can a p p a r e n t l y be q u a l i t a t i v e l y assessed by changes i n pulse pressures s i n c e the - 147 •-l a m e l l a e are complaint v a s c u l a r sheets ( S e c t i o n 1). P e r f u s i o n of a d d i t i o n a l complaint v a s c u l a r beds i n p a r a l l e l r a i s e s . t h e o v e r a l l g i l l compliance arid i n c r e a s e the damping of the pressure pulse across the g i l l s . The APg pulse was increased with a d m i n i s t r a t i o n of catecholamines i n t o r e s t i n g l i n g cod. Notably, the g r e a t e s t i n c r e a s e i n APg pulse occured with NAD a d m i n i s t r a t i o n , which c o r r e l a t e s w e l l with the NAD p r e s s o r response that g r e a t l y enhanced l a m e l l a r r e c r u i t m e n t . ' Cardiac output increased r e f l e x o g e n i c a l l y i n response • • • .1 ' • to catecholamine i n j e c t i o n s i n t o the v e n t r a l a o r t a . .The e l e v a t e d Q was a r e s u l t of i n c r e a s e d stroke volume, as there was l i t t l e change i n heart r a t e , Pg mean, however, was r e l a t i v e l y unchanged as a r e s u l t of Rg and Q changes. In other words the changes i n Q and Rg were compensatory or a u t o m a t i c a l l y matched. A decrease i n Rg can be due to l a m e l l a r recruitment and/or v a s o d i l a t i o n . L a m e l l a r • r e c r u i t m e n t w i l l i n c r ease f i l a m e n t blood volume the g r e a t e s t , s i n c e the lamellae c o n t a i n the maj'ority of the blood ( F i g . 13, S e c t i o n I ) . The r e c r u i t e d l a m e l l a e w i l l thus be l a r g e l y r e s p o n s i b l e f o r accommodating the. i n c r e a s e d Q. I, t h e r e f o r e , p o s t u l a t e that the increased Q c l o s e l y matches l a m e l l a r r e c r u i t m e n t , i n which case increased Rg only r e f l e c t s the b r a n c h i a l v a s o d i l a t i o n . Thus the NAD i n j e c t i o n s produced g r e a t e r l a m e l l a r r e c r u i t m e n t and b r a n c h i a l v a s o d i l a t i o n than,ISOP s i n c e the i n c r e a s e s i n input p r e s s u r e s , Q and APg pulse were g r e a t e r . C l e a r l y changes i n b r a n c h i a l blood flow p a t t e r n s occur i n response to catecholamines through increase input pressure - 14 b .-and Q. • The venolymphatics L i t t l e i s known concerning flow i n the venolymphatic system of t e l e o s t g i l l s . The r e s u l t s presented here and i n S e c t i o n I provide some in f o r m a t i o n on venolymphatic flow i n l i n g cod. Blood flow i s d e r i v e d from the e f f e r e n t f i l a m e n t a r t e r i e s and can proceed through a s e r i e s of narrow connecting v e s s e l s between the ECVs, CS and ACVs ( S e c t i o n I ) . Lymph1 formed at the l a m e l l a e enters the i n t e r s t i t i a l space between the e p i t h e l i u m and c a p i l l a r y endothelium. The i n t e r s t i t i a l space apparently d r a i n s i n t o the c e n t r a l s i n u s by separate channels l y i n g beneath the l a m e l l a e . These channels have not been observed before i n other t e l e o s t s . V a l v e - l i k e s t r u c t u r e s e x i s t at the 'base of the la m e l l a e i n the i n t e r s t i t i a l space i n t r o u t (Campbell et a_l. , i n p r e p a r a t i o n ) . Such v a l v e s , i f present i n l i n g cod, would prevent r e f l u x of lymph from the c e n t r a l sinus to the l a m e l l a r ' ,"' i n t e r s t i t i a l space. The CS and ACVs are r e l a t i v e l y f r e e of red blood c e l l s arid the ECVs c o n t a i n reduced rbc numbers compared to a r t e r i a l blood. A high r a t e of lymph formation would produce t h i s s i t u a t i o n i n the venolymphatics. Plasma skimming would a l s o reduce rbc numbers in the venolymphatics. The small v a s c u l a r connecting v e s s e l s are i d e a l f o r excluding red blood c e l l s but a l l o w i n g plasma through. In t r o u t Vogel et a l . (1976) noted that - 149 -the openings of venolymphatic connecting vessels are surrounded by microscopic "hairs". These hairs could serve to exclude red blood c e l l s in trout, but they have not been looked for in l i n g cod. Blood flow from the EFA to the main ECV may be controlled in l i n g cod by the smooth muscle that surrounds the connecting vessel. Based on the micropressures measured here, venolymphatic flow i s driven by low pressures. This conclusion could be predicted from the presence of narrow inter-connecting vessels of the venolymphatics. The micropressures were p u l s a t i l e , confirming that venolymphatic flow is p u l s a t i l e . Mammalian lymphatic systems are low pressure and have a p u l s a t i l e flow. Increased p u l s a t i l i t y is known to enhance lymphatic flow in mammals (Parsons and McMaster, 1938; McMaster and Parsons, 1938; Ruszyak et a l . , 1967). Some lymphatics can propel f l u i d by their own i n t r i n s i c a c t i v i t y and increased lymphatic transmural pressure w i l l increase the rate of lymph propulsion (McHale and Roddie, 1976). Arterio-lymphatic interactions also influence lymph flow since in mammalian lungs lymphatic flow is enhanced by the a r t e r i a l pulse pressure (Shephard and K i r k l i n , 1969; . Nicolaysen and Hauge, 1977). Furthermore non-pulsatile a r t e r i a l lung perfusion resulted in lymph accumulation which was associated with reduced oxygen consumption. Arterio-lymphatic interactions may also occur in the g i l l filament,.and i t is predicted that the p u l s a t i l e nature of a r t e r i a l flow could have important effects on venolymphatic- flow. This prediction is - 15 0 -examined i n S e c t i o n I I I . Venolymphatic flow i n g i l l s might a l s o be under n e u r a l or humoral c o n t r o l mechanisms, since i n t r o u t , a-adrenergic c o n s t r i c t i o n decreases venolymphatic volume ( G i r a r d and Payan, 1976; Payan and G i r a r d , 1977; Dunel and Laurent, 1977). The. importance of m a i n t a i n i n g venolymphatic flow i n the f i l a m e n t i s q u i t e simple. I f lymph formed at the l a m e l l a e were to accumulate, 0 2 t r a n s f e r would be l i m i t e d because of i n c r e a s e d d i f f u s i o n d i s t a n c e s . . . . . . . In summary I have demonstrated e x p e r i m e n t a l l y that the ALAs are the,major r e s i s t a n c e s i t e . t o g i l l blood flow and d i s t a l l a m e l l a e are not perfused under c e r t a i n i n v i v o conditions.. Changes i n v a s c u l a r dimensions through c h o l i n e r g i c and a d r e n e r g i c a c t i o n s i n i t i a t e a l t e r a t i o n s i n g i l l blood flow patterns.. G i l l r e s i s t a n c e i s a l t e r e d by the v a s c u l a r changes, but flow p a t t e r n changes are e f f e c t e d by Q r e g u l a t i o n . Increased Q _in v i v o w i l l t h e r e f o r e be a s s o c i a t e d with l a m e l l a r recruitment and i n t r a l a m e l l a r shunting, which i n c r e a s e the g i l l d i f f u s i n g c a p a c i t y . The changes i n flow p a t t e r n a s s o c i a t e d with i n c r e a s e d Q and input pressure were not v i s u a l i z e d , but are assumed to occur a u t o m a t i c a l l y based on previous f i n d i n g s . I t i s necessary to e x p e r i m e n t a l l y confirm that flow changes do occur . a u t o m a t i c a l l y with Q and pressure changes. This c o n f i r m a t i o n was c a r r i e d out i n S e c t i o n I I I by observing l a m e l l a r p e r f u s i o n i n i s o l a t e d g i l l arches. . - 150a -SECTION I I I AN EXAMINATION OF THE VASCULAR RESISTANCE AND COMPLIANCE AS THEY AFFECT GILL BLOOD FLOW USING IN VITRO PREPARATIONS The e f f e c t s of a l t e r a t i o n s i n pressure, flow and pulse rate of g i l l blood flow patterns i n the i s o l a t e d , perfused holobranch Pressure/volume curves f o r the bulbus a r t e r i o s u s and v e n t r a l a o r t a - 151 .-INTRODUCTION In 'the p r e v i o u s S e c t i o n s i t was demonstrated that p r e ^ l a m e l l a r a r t e r i o l e s c o n t r o l l a m e l l a r blood. A - d e l i c a t e balance e x i s t s between the o v e r a l l r e s i s t a n c e of each p a r a l l e l l a m e l l a r u n i t and- the d i s t a l l a m e l l a e -may not be perfused under c e r t a i n c o n d i t i o n s . Furthermore the p a t t e r n of flow d i s t r i b u t i o n w i t h i n a s i n g l e l a m e l l a i s not uniform. In these s i t u a t i o n s an i n c r e a s e i n Q and i n g i l l input pressure i s p r e d i c t e d to a l t e r g i l l blood flow p a t t e r n s i n such a way that 0 2 t r a n s f e r i s enhanced. Is t h i s t r u l y the s i t u a t i o n ? E x e r c i s i n g t r o u t do show marked changes in. Q and VA pressure which are a l s o a s s o c i a t e d with an.elevated Mo 2 (Stevens and R a n d a l l , 1967a and b; Kiceniuk and Jones, 1977). However the d i r e c t e f f e c t s of increased Q and input pressure on observed p a t t e r n s of g i l l blood flow have not been s t u d i e d . Such an i n v e s t i g a t i o n was made here using the i s o l a t e d perfused g i l l p r e p a r a t i o n , .'where l a m e l l a r ' p e r f u s i o n can be observed d i r e c t l y . A l s o , only the p a s s i v e p r o p e r t i e s of the v e s s e l s were examined when p e r f u s i o n c o n d i t i o n s were changed, s i n c e i n v i t r o g i l l p r e p a r a t i o n s have l i t t l e or no v a s c u l a r tone ( S e c t i o n I I ) , and they are denervated. Our understanding .of • • g i l l blood flow c o n t r o l has b e n e f i t e d from many s t u d i e s using an _in v i t r o i s o l a t e d perfused holobranch or head p r e p a r a t i o n (Ostlund and Fange,. 1962; Richards and Fromm, 1969; Wood, 1974b and 1975; Bergmann e t a l . , 1974 - 152 -F o r s t e r , 1976; Payan and G i r a r d , 1977). These p r e p a r a t i o n s have a l s o been used to i n v e s t i g a t e g i l l ion t r a n s f e r (Payan and Matty, 1975; P i c e t a i . , 1974 a and b; G i r a r d and Payan, 1976; Payan, 1978). If input pressure and Q can a l t e r blood flow p a t t e r n s , then care must be taken i n s e l e c t i n g in v i t r o p e r f u s i o n c o n d i t i o n s . At the very l e a s t , p e r f u s i o n pressures should be high enough to avoid c r i t i c a l c l o s u r e of l a m e l l a r blood channels ( S e c t i o n I ) . Yet i n a l l : t h e s e p revious i n v e s t i g a t i o n s i n v i v o p r e s s u r e s and flow regimes were l a r g e l y ignored. T e l e o s t f i s h , i n v i v o , have n e i t h e r constant Q nor constant g i l l p e r f u s i o n p r e s s u r e . Blood flow i s p u l s a t i l e and the DA blood pressure i s of the order of 20 to 40 cm H 20. I t i s r e a s s u r i n g that r e c e n t l y Shuttleworth (1978) d i d c o n s i d e r the i n v i v o s t a t e of blood flow and l a m e l l a r p e r f u s i o n in.the design of h i s i s o l a t e d , perfused holobranch p r e p a r a t i o n , as d i d Wood et a l . (1978). The d i r e c t e f f e c t s of p u l s a t i l e flow and pressure changes on the p a t t e r n of g i l l blood flow c l e a r l y need to be examined and the r e s u l t s of t h i s i n v e s t i g a t i o n may w e l l a f f e c t the i n t e r p r e t a t i o n of p r e v i o u s i n v i t r o experiments. I n t e r p r e t a t i o n of _in v i t r o r e s u l t s can be complicated by the nature of the p e r f u s i o n conditons ( S e c t i o n I I ) . The q u a n t i t a t i v e nature of v a s c u l a r responses i s d e f i n i t e l y a f f e c t e d by p e r f u s i o n c o n d i t i o n s . For example5, i n the perfused mammalian kidney p r e p a r a t i o n , p r e s s o r responses using constant Q p e r f u s i o n s were 2 to 5 times lower than responses with constant pressure p e r f u s i o n ( K h a j u t i n , .1964). There are a l s o d i f f i c u l t i e s - 15 3 -a p p l y i n g in v i t r o r e s u l t s to i n v i v o c o n d i t i o n s because of d i f f e r e n t p e r f u s i o n c o n d i t i o n s . I_n v i t r o a d r e n e r g i c and c h o l i n e r g i c drug i n f u s i o n s w i l l mimic p o s s i b l e n e u r a l v a s c u l a r c o n t r o l , but they only inform us whether v e s s e l s c o n s t r i c t or. d i l a t e . I_n v i v o a d m i n i s t r a t i o n of these drugs produces more complex responses than a r e s i s t a n c e change e.g. changes in p u l s a t i l i t y and Q ( S e c t i o n I I ) . I f p u l s a t i l i t y and Q can a l s o change g i l l blood flow p a t t e r n s , then c l e a r l y the e x t r a p o l a t i o n of in v i t r o f i n d i n g s to the d i s c u s s i o n of how b r a n c h i a l a d r e n e r g i c and c h o l i n e r g i c e f f e c t s a l t e r blood flow p a t t e r n s i n v i v o must be approached c a r e f u l l y . Indeed, I suggested i n . S e c t i o n II that i n a d d i t i o n to a l t e r i n g Rg, Q must a l s o be maintained or e l e v a t e d to a l t e r p e r f u s i o n p a t t e r n s . I t i s s u r p r i s i n g then, that previous i n t e r p r e t a t i o n and a p p l i c a t i o n of i n v i t r o f i n d i n g s r a r e l y considered or acknowledged the d i f f e r e n c e s i n p e r f u s i o n p r e s s u r e s and flows between the i n v i v o and the in v i t r o s i t u a t i o n s . In t e l e o s t s blood flow remains p u l s a t i l e throughout the g i l l s . Lymphatic flow i s a l s o p u l s a t i l e . I t has a l r e a d y been suggested i n S e c t i o n II that a r t e r i a l p u l s a t i l i t y c ould a f f e c t M o 2 bY i - t s e f f e c t on lymphatic flow, based on the observed a r t e r i o - l y m p h a t i c i n t e r a c t i o n s i n mammalian lungs. I t is. noteworthy that the f i l a m e n t venolymphatics are e x t e n s i v e i n l i n g cod ( S e c t i o n I ) , and that the pulse p r e s s u r e s throughout the g i l l bed, i n p a r t i c u l a r the l a m e l l a r c a p i l l a r i e s , are comparatively high ( S e c t i o n II)... Is p u l s a t i l i t y per se, t h e r e f o r e , important., i n determining b r a n c h i a l blood flow p a t t e r n s ? Furthermore, to - 154 -what extent can p u l s a t i l i t y of blood flow be c o n t r o l l e d ? A r t e r i a l p u l s a t i l i t y i s determined l a r g e l y by the a c t i o n of the' heart. Blood flow i s , however, depulsed - a Wind k e s s e l e f f e c t - by the e l a s t i c bulbus a r t e r i o s u s ( R a n d a l l , 1968; S a t c h e l l , 1971; Jones e_t a_l. , 1974). In a number of t e l e o s t s there i s sympathetic i n n e r v a t i o n of the smooth muscle of the bulbus (Gannon, 1972; Watson pers. comm.). It i s p o s s i b l e then, that sympathetic r e l e a s e could a l t e r the.tone of the bulbus a r t e r i o s u s and thus modulate the degree of blood flow d e p u l s a t i o n . The e f f e c t of p u l s a t i l i t y per se on b r a n c h i a l blood flow p a t t e r n s and whether or not the bulbus. can c o n t r i b u t e to the c o n t r o l of p u l s a t i l i t y were examined here. In t h i s S e c t i o n I conducted i n v e s t i g a t i o n s on how a l t e r a t i o n s i n p u l s a t i l e flow, pressure and pulse r a t e p a s s i v e l y a f f e c t g i l l p e r f u s i o n and p a t t e r n s of l a m e l l a r p e r f u s i o n by using the i s o l a t e d holobrarich p r e p a r a t i o n . - i i ! -MATERIALS AND METHODS In v i t r o i s o l a t e d holobranch p e r f u s i o n ; the e f f e c t of  a l t e r a t i o n s i n p r e s s u r e , flow and pulse r a t e on g i l l  blood flow p a t t e r n s The c l e a r i n g , p r e p a r a t i o n and c a n n u l a t i o n of the i s o l a t e d g i l l holobranchs has been d e s c r i b e d . i n the g e n e r a l methods.. The p e r f u s i o n p r o t o c o l Was as f o l l o w s . Each arch Was perfused s e p a r a t e l y i n a constant temperature (11°C).saline bath using a Watson-Marlow p u l s a t i l e . p e r f u s i o n pump ( F i g . 26). Inflow (Qj_) was c o n t r o l l e d with a s e r i e s of p r e c a l i b r a t e d , interchangeable tubes and the v a r i a b l e p u l s e irate of the pump. Input pressure (P^) was monitored w i t h i n 2 cm of the g i l l arch using a Statham P23Db p r e s s u r e transducer and. a Brush c h a r t recorder.. The a f f e r e n t cannula r e s i s t a n c e was measured a f t e r each experiment and deducted from mean P-^  v a l u e s . Outflow from the e f f e r e n t arch v e s s e l (QQ) was measured by drop counting and outflow pressure (P Q) was set by the height of the cannula above the s a l i n e l e v e l of the p r e p a r a t i o n ( F i g . 26). Thus Q-^ , pulse r a t e (hence s t r o k e volume) and P 0 could be manipulated, w h i l s t mean P^ and Q 0 were the measured v a r i a b l e s . E x c e s s i v e pulse p r e s s u r e s that were generated by the pump were damped by a "Wind-Kessel" which c o n s i s t e d of an a i r f i l l e d s y r inge as a s i d e branch of the main p e r f u s i o n l i n e ( F i g . 26). "Resting" values f o r pulse r a t e (3.0 m i n - 1 ) , Q-^  t o each arch (1.25 to 1.5 ml.min - 1.kg body w e i g h t - 1 ) and P Q (.30 to 40 cm H 20) were based. on - 156 -F I G U R E 26 A schematic diagram to r e p r e s e n t the in v i t r o p e r f u s i o n system used. The. p e r f u s a t e i n f l o w and p e r f u s i o n bath were both kept at constant temperature by water j a c k e t s . The Wind-kessel c o n s i s t e d of an a i r - f i l l e d s y r i n g e , the volume of which could be adjusted to vary the damping of input p u l s e p r e s s u r e . P Q = output pressure and the height of the e f f e r e n t c a t h e t e r above the s a l i n e . jackets bath - 157 -the i n v i v o measurements from l i n g cod. S i m i l a r i l y , p ulse r a t e s of 4 5 . m i n - 1 and 15 .min--'- approximate the maximum he a r t , r a t e ( a t r o p i n i s e d ) and the minimum heart r a t e (hypoxic bradycardia) recorded i n l i n g cod (see S e c t i o n s II and I V ) . T y p i c a l l y , each holobranch was i n i t i a l l y perfused at r e s t i n g and pulse r a t e , while g r a d u a l l y f e l l and QQ g r a d u a l l y rose with time towards s t a b l e v a l u e s . A f t e r e q u i l i b r a t i o n (10 to 15 min), i n d i v i d u a l manipulations were made to pulse r a t e / s t r o k e volume, Q-j. or P Q, while r e c o r d i n g P^: and Q Q . The subsequent e f f e c t s on l a m e l l a r recruitment were determined. To v i s u a l i z e the number of lam e l l a e perfused a f t e r a give n m a n i p u l a t i o n , a 1 to 2 min e q u i l i b r a t i o n p e r i o d was allowed, then a P r u s s i a n Blue dye suspension was introduced t o the i n f l o w . T h i s procedure a l s o served to demonstrate s u c c e s s f u l a f f e r e n t c a n n u l a t i o n s s i n c e dye never leaked from the a f f e r e n t s i d e of the p r e p a r a t i o n . The p a r t i c u l a t e dye lodged i n the l a m e l l a r c a p i l l a r y channels to i n d i c a t e the flow pathways at that time. P e r f u s i o n was stopped immediately s i n c e P^ was r i s i n g due to v e s s e l o c c l u s i o n . The d i s t r i b u t i o n of dye was observed by c l e a r i n g the g i l l t i s s u e i n methyl s a l i c y l a t e a f t e r alcohol, d e h y d r a t i o n . The i n t a c t holobranch was photographed and the extent of dye p e n e t r a t i o n along the f i l a m e n t l e n g t h , as w e l l as the t o t a l l e n g t h were measured i n every 10th f i l a m e n t , which were i s o l a t e d and viewed under a microscope. The dye penetrated along a p o r t i o n of the a f f e r e n t f i l a m e n t a r t e r y and entered a l l la m e l l a e connected to that segment of a r t e r y c o n t a i n i n g dye. - 158 .-There was no evidence of dye i n the remaining length of f i l a m e n t or l a m e l l a e . Since the la m e l l a e are evenly d i s t r i b u t e d along the f i l a m e n t ( F i g . 4 , S e c t i o n I ) , the percentage of la m e l l a e perfused was c a l c u l a t e d from the extent of dye p e n e t r a t i o n along the f i l a m e n t expressed as percentage of t o t a l f i l a m e n t l e n g t h . Six holobranchs were r o u t i n e l y used from each f i s h ; the f o u r t h g i l l arches, which were sma l l e r than the r e s t and had an e x c e s s i v e amount of cut t i s s u e , were not used. Holobranchs, l a c k i n g Q Q or that had e x c e s s i v e l y high P-^  v a l u e s , were not used. The experiments were conducted oyer a 2 to 3 hours p e r i o d proceding g i l l removal, thereby minimising experimental time and the a s s o c i a t e d changes i n the c h a r a c t e r i s t i c s of the g i l l s with time. S t o r i n g the holobranchs i n i c e - c o l d s a l i n e was h e l p f u l i n th i s r e s p e c t . • -159 -V The pressure/volume curves f o r the bulbus a r t e r i o s u s  and v e n t r a l a o r t a I t i s d i f f i c u l t to separate the bulbus a r t e r i o s u s from the v e n t r a l a o r t a . Therefore the pressure/volume curves were e s t a b l i s h e d f o r the i n t a c t bulbus a r t e r i o s u s and v e n t r a l , a o r t a v e s s e l from two l i n g cod and s i x rainbow t r o u t . The v e s s e l l e n g t h was measured i n s i t u i n the an a e s t h e t i s e d f i s h p r i o r to being e x c i s e d in v i v o and c l e a r e d of blood. The v e s s e l was cannulated at both ends with PE 200 tubing and placed Into a p e r f u s i o n bath c o n t a i n i n g the a p p r o p r i a t e oxygenated s a l i n e at 9 to 10°C. The v e s s e l was s t r e t c h e d t o and held at i t s i n v i v o l e n g t h . The bulbus c a t h e t e r was connected to a constant flow Harvard i n f u s i o n s y r i n g e pump and, v i a a s i d e arm, to a Statham P23Db pressure transducer ( F i g . 21). The pre s s u r e transducer s i g n a l s were a m p l i f i e d and recorded on a Harvard c h a r t r e c o r d e r . I n f u s i o n r a t e s comparable to those found iri v i v o were e s t a b l i s h e d i n p r e l i m i n a r y t r i a l s , s i n c e the shape of pressure/volume curves vary with the ra t e of in c r e a s e of i n t e r n a l volume ( B e r g e l , 1961a and b; Fung, 1972). The major experiments were performed on v e s s e l s from t r o u t , f o r which the f i l l i n g time could be approximated to those a s s o c i a t e d with, r e s t i n g heart r a t e s found i n t r o u t ( R a n d a l l , 1970). F i l l i n g of the l i n g cod v e s s e l was at best 2 to 3 times slower than in v i v o because of the l i m i t e d d e l i v e r y speed of the syr i n g e pump compared to the l a r g e v e s s e l s from the specimens used. For t h i s reason only the c o n t r o l pressure/volume curve was e s t a b l i s h e d i n 2 l i n g cod so ,that i t s g e n e r a l s i m i l a r i t y with the t r o u t v e s s e l could be a s c e r t a i n e d . •it •• -160- i -F I G U R E 27 A s c h e m a t i c d i a g r a m o f t h e w a t e r - j a c k e t e d , s a l i n e - f i l l e d p e r f u s i o n b a t h used f o r e x a m i n i n g t h e p r e s s u r e / v o l u m e c u r v e s o f t h e b u l b u s a r t e r i o s u s and v e n t r a l a o r t a v e s s e l . saline syringe pump • i 1 to p>rSssure transducer water ^ jacket * inflow outflow perfusion bath o SB gas * supply clamp - 161 -Each experiment with the t r o u t v e s s e l c o n s i s t e d of a s e r i e s of p r e l i m i n a r y v e s s e l d i s t e n s i o n s , c o n t r o l s a l i n e d i s t e n s i o n s and the t e s t i n g of an a g o n i s t drug. For each v e s s e l ACH, ISOP and NAD were t e s t e d i n s e q u e n t i a l experiments and i n the f i n a l experiment the s a l i n e bath was p r o g r e s s i v e l y deoxygenated with N 2 gas while a s e r i e s of d i s t e n s i o n s were made. The p r o t o c o l adopted f o r each experiment was; i n f u s i o n s t a r t e d with the v e s s e l c o l l a p s e d to a n e g l i g i b l e volume and the outflow cannula clamped. The i n t e r n a l pressure was monitored on the c h a r t r e c o r d e r and i n f u s i o n was stopped at an i n t e r n a l pressure no g r e a t e r than 100 cm H 26. The i n t e r n a l pressure was re l e a s e d immediately. Fi v e p r e l i m i n a r y d i s t e n s i o n s ensured a p r e - s t r e t c h i n g of the ex c i s e d t i s s u e p r i o r to determining any s e r i e s of pressure/volume curves (Fung, 1972). Three c o n t r o l curves were determined f i r s t . Then an ag o n i s t drug was added to the s a l i n e bath (1 x 10~ 4 mg.I - 1 f i n a T c o n c e n t r a t i o n ) and three to f i v e d i s t e n s i o n s made afterwards. The bath and v e s s e l were washed thoroughly with three changes of f r e s h s a l i n e and the v e s s e l was f r e e l y p e r f u s e d . A f t e r 15 min a f i n a l s a l i n e change preceded the next experiment. When a l l three a g o n i s t drugs had been t e s t e d , the s a l i n e was p r o g r e s s i v e l y deoxygenated f o r the f i n a l experiment i n which four to s i x d i s t e n s i o n s were made over a 5 to 10.min p e r i o d . The r e p e t i t i o n of the c o n t r o l curves with s a l i n e before each drug experiment ensured that the p r e p a r a t i o n d i d not d e t e r i o r a t e . A l l r e s u l t s from :any.preparation that developed a leakage p a r t way through the experimental s e r i e s were - 162 -d i s c a r d e d . The p r e s s u r e / v o l u m e c u r v e s were c o n s t r u c t e d d i r e c t l y f r o m t h e p r e s s u r e r e c o r d i n g s s i n c e v o l u m e was d i r e c t l y r e l a t e d t o t i m e a t t h e c a l i b r a t e d , c o n s t a n t f l o w r a t e s o f t h e s y r i n g e pump. - 1 6 3 -R E S U L T S T h e e f f e c t o f a l t e r a t i o n s i n p r e s s u r e , f l o w a n d p u l s e r a t e o n  t h e l a m e l l a r b l o o d f l o w i n i s o l a t e d , p e r f u s e d h o l o b r a n c h s ; A t o t a l o f 5 2 s u c c e s s f u l g i l l p e r f u s i o n p r e p a r a t i o n s f r o m 1 0 l i n g c o d w e r e s t u d i e d . C a r d i a c o u t p u t i n r e s t i n g l i n g c o d a t 1 1 ° C i s a b o u t 1 0 m l . m i n - 1 . k g - 1 . T h e I s o l a t e d a r c h e s w e r e p e r f u s e d i n i t i a l l y a t . 1 / 8 - t h t h e e s t i m a t e d c a r d i a c o u t p u t g i v e n t h e w e i g h t o f t h e d o n o r f i s h . At t h i s i n i t i a l f l o w r a t e , r e f e r r e d t o a s " r e s t i n g Q±" , t h e m e a n i n p u t p r e s s u r e ( P i ) w a s b e t w e e n 7 0 t o 9 5 c m H2O. T h e g i l l r e s i s t a n c e t o f l o w , R g ( c a l c u l a t e d f r o m A P g / Q ^ , w h e r e A P g = P ^ - P 0 ) , w a s i n i t i a l l y h i g h , b u t f e l l t o a s t a b l e v a l u e 1 0 t o 1 5 m i n a f t e r t h e o n s e t o f p e r f u s i o n . A s t e p w i s e i n c r e a s e i n P 0 r e s u l t e d i n s m a l l e r c h a n g e s i n P • At l o w l e v e l s o f P Q , a n i n c r e a s e c a u s e d a f a l l i n P-^ w h e r e a s a t h i g h l e v e l s a n i n c r e a s e P Q r e s u l t e d i n a s m a l l r i s e i n P-L ( F i g . 2 8 ) . C l e a r l y , a s A P g f a l l s w i t h i n c r e a s i n g P Q a n d , a s i s c o n s t a n t , R g a l s o f a l l s w i t h i n c r e a s i n g P Q ( F i g . 2 8 b ) . T h e r a t i o o f Q Q t o w a s b e t w e e n 3 0 a n d 8 8 % a t t h e t i m e o f d y e i n j e c t i o n ^ S a l i n e w a s , t h e r e f o r e , a l s o l o s t f r o m t h e p r e p a r a t i o n v i a o t h e r o u t l e t s o r b y f i l t r a t i o n . T h e r e w e r e n e v e r a n y l e a k s i n t h e a f f e r e n t p o r t i o n o f t h e s y s t e m . a n d a l l s a l i n e m u s t / t h e r e f o r e , e n t e r t h e s e c o n d a r y l a m e l l a e . F l u i d c o u l d b e l o s t b y f i l t r a t i o n b u t t h i s i s p r o b a b l y n o t . a m a j o r p a t h w a y - 164 -because when Pj_ i n c r e a s e d the d i f f e r e n c e between Q Q and decreased ( F i g . 29b). Thus there i s more than one outflow channel i n the e f f e r e n t c i r c u l a t i o n but f l u i d was only c o l l e c t e d from the e f f e r e n t b r a n c h i a l a r t e r y . The r a t i o of Q0:Qj[ was a l s o Increased by r a i s i n g and/or i n c r e a s i n g the pulse r a t e ( F i g . 29a). E l e v a t i o n of P Q at constant i n i t i a l l y decreased Q Q which then returned to the o r i g i n a l l e v e l . I f , however, P Q was g r e a t e r than 30 cm H2O the pre-change Q Q was not always a t t a i n e d . The p r o g r e s s i v e deoxygenation of the p e r f u s a t e with N 2 gas f a i l e d to produce any change i n P^ or Q 0 i n three p r e p a r a t i o n s . Changes i n l a m e l l a r p e r f u s i o n p a t t e r n s Under " r e s t i n g " p e r f u s i o n c o n d i t i o n s 66.8% * 3.0 (n = 10) of a l l l a m e l l a e were pe r f u s e d . In any one holobranch the % p e r f u s i o n v a r i e d s l i g h t l y from f i l a m e n t to f i l a m e n t . In p a r t i c u l a r , f i l a m e n t s i n the r e g i o n d o r s a l to the bend i n the arch u s u a l l y had s l i g h t l y more lam e l l a e perfused than elsewhere on the arch ( P l a t e 17). There was no obvious c o r r e l a t i o n between the number of l a m e l l a e perfused and g i l l r e s i s t a n c e Rg ( F i g . 30). There was a s i g n i f i c a n t , change i n l a m e l l a r p e r f u s i o n with i n c r e a s e s i n Q i , P i and pulse r a t e ( F i g s . 31 and 32). The e f f e c t of i n c r e a s e d a n d P ^ : In p r e p a r a t i o n s with a 50 t o 100% i n c r e a s e (x = 76.9%) i n Qi and an e l e v a t e d P^ l a m e l l a r p e r f u s i o n was inc r e a s e d from 66.8% to 77.8 1 3.0, n = 13) ( F i g . 31). Not a l l f i l a m e n t s of one arch showed the same i n c r e a s e i n l a m e l l a r p e r f u s i o n . -Qj. was increased through e i t h e r i n c r e a s i n g s t r o k e volume or pulse r a t e . The changes i n were c o n t r o l l e d by me and r e s u l t e d i n changes i n P^. However, the change i n Q^ and P^, although in.the same d i r e c t i o n were not i n phase. T y p i c a l l y when Q^ was increased P^ i n i t i a l l y rose by a mean of 37.9%, (range 15 to 55%) and overshot i t s l a t t e r , s t a b l e i n c r e a s e 23.5% above the i n i t i a l l e v e l (range 8 to 42%) ( F i g . 29). The e f f e c t of i n c r e a s e d 'PQ and Pj_: In t h i s system, e l e v a t i o n of P Q, and the consequent l i m i t e d r i s e i n Pj., d i d not a l t e r % l a m e l l a r p e r f u s i o n but, Apg f e l l p r o p o r t i o n a t e l y ( F i g . 29). The e f f e c t of changes i n pul s e r a t e / s t r o k e volume at constant Q^ Increased r a t e / d e c r e a s e s t r o k e volume: The % la m e l l a e perfused was reduced (x =59.3% ±6.0) i n 5 out of 6 p r e p a r a t i o n s when pulse r a t e was increased to .-4 5 ..min"1, but Q^ was maintained at or c l o s e to a r e s t i n g value by dec r e a s i n g s t r o k e volume ( F i g . 32). Under these c o n d i t i o n s , pulse pressure was reduced and mean P-[ always in c r e a s e d 4 to 10 cm H2O. - 166 -Increased s t r o k e volume/decreased pulse r a t e : When, stroke volume was doubled and kept constant by de c r e a s i n g pulse r a t e to 15.min - 1, pu l s e P-^  was a l s o i n c r e a s e d as a r e s u l t . The mean P^ always decreased by 2 to 10 cm a f t e r these m a n i p u l a t i o n s . These changes r e s u l t e d , i n a mean of 68.2 ± 2.9% (n = 9) of the l a m e l l a e being perfused, a value s i m i l a r to that at r e s t i n g s t r o k e volumes. Increased stroke volume/decreased p u l s e r a t e per ^ e can i n c r e a s e l a m e l l a r p e r f u s i o n s i n c e i n 6 of the 9 p r e p a r a t i o n s there was always.an i n c r e a s e i n l a m e l l a r p e r f u s i o n ( F i g . 32). C o n s i d e r i n g o n l y these six. p r e p a r a t i o n s , l a m e l l a r p e r f u s i o n was 73.1 ± 1 .4% compared with 66.8 ± 3 . 0 % , o f the t o t a l number of la m e l l a e under " r e s t i n g " c o n d i t i o n s . In one experiment,, stroke volume was s l i g h t l y l e s s than doubled and was, t h e r e f o r e , below the r e s t i n g v a l u e . Here the % lam e l l a e perfused was again higher than r e s t i n g v a l u e s . -167 L FIGURE 28 The e f f e c t of stepwise i n c r e a s e s i n P Q on the mean and A Pg (or k.Rg where k = 1/Q^ a t r e s t i n g Qj_.) . a) absolute values of P Q vs_ mean Pj^: each p o i n t r e p r e s e n t s the mean value ± 1 s.e. of riobservations (n above p o i n t s ) , except at P Q - 60 cm H 20, where the range of the two values i s i n d i c a t e d ; b) the decrease i n APg across the holobranch with i n c r e a s i n g P Q. Each p o i n t r e p r e s e n t s the value f o r P i - P 0 taken from F i g . 28A at each P Q. Note: 1 cm H 20 =0.09 8 kPa. FIGURE. 29 A t y p i c a l recording of P^ when pulse rate was elevated from 30 to 45 p u l s e s . m i n - 1 at the arrow. was not changed from the r e s t i n g value but Q Q:Qi increased. Note the x5 increase i n chart speed. A t y p i c a l recording of the e f f e c t of e l e v a t i n g to 150% r e s t i n g flow by e l e v a t i n g pulse rate i n a stepwise fashion from 30 to 45 p u l s e s . m i n - 1 ; upon P^ and the r a t i o Q 0 to Q^ . The time trace i s i n seconds and minutes. - 168a -cm H z O 100 60 20 t sec. Q 7o 80 - I o / 6 0 j ' 40 - J 20 - I B 100. 1 i . . i . i l i i l i i i i i i u i a i i i i m i i i i i i i M i i i i i cm H 2 0 pul ra •min 50-1 $e te 40 J 1 3 0 H Q /o 80-o/ 60-' 40H 20 J mm. -163 -PLATE 17 A/ An i s o l a t e d holobranch bf Ophiodon elongatus i l l u s t r a t i n g dye d i s t r i b u t i o n i n . t h e arch f i l a m e n t s . The placement of the l i g a t u r e s can'be seen at e i t h e r end of the arc h . The dye w i t h i n the a f f e r e n t b r a n c h i a l a r t e r y (top of p l a t e ) i s a l s o v i s i b l e . B/ A magnified image of a f i l a m e n t t i p d i s p l a y i n g the f i l l i n g of the a f f e r e n t f i l a m e n t a r t e r y and some secondary l a m e l l a r channels t r a v e r s i n g the f i l a m e n t . Note the completely c l e a r v e s s e l s a t . t h e t i p . C/ A more c e n t r a l r e g i o n of a f i l a m e n t at higher m a g n i f i c a t i o n showing c l e a r l y the f i l l i n g of the base of the l a m e l l a r v e s s e l s . - 169a -- 170 -FIGURE 30 G i l l resistance,. APg/Q^ (or Rg) should be d i r e c t l y c o r r e l a t e d with the r e c i p r o c a l of the number of lamellae perfused (n) as i n d i c a t e d by the continuous l i n e , i f the g i l l s are a simple ohmic r e s i s t a n c e • 1 = n ; Rg Rlam sin c e the l a m e l l a r are a p a r a l l e l array. Each point represents the values f o r one holobranch prep a r a t i o n and o v e r a l l the po i n t s represent the complete range of pe r f u s i o n c o n d i t i o n s examined here. There i s no c o r r e l a t i o n of these p o i n t s . t - 170a -ro 01 - i n -FIGURE 31 A 3-dimensional r e p r e s e n t a t i o n of the e f f e c t of e l e v a t e d Q±, expressed as a r a t i o of r e s t i n g Q± (=1) on % l a m e l l a e perfused and on the input pressure P^. Each p o i n t r e p r e s e n t s one holobranch p r e p a r a t i o n , where ^ = r e s t i n g and pulse r a t e , • =elevated at a pulse r a t e of 45 pu l s e s .min--'- and 4 = e l e v a t e d a t a pu l s e r a t e of 30 p u l s e s . m i n - 1 . Note: The length of the s o l i d l i n e s connecting each p o i n t i n X/Z plane r e p r e s e n t s the % l a m e l l a e , as i n d i c a t e d on the Y-axis of the f i g u r e . The broken l i n e s , are p r o j e c t i o n s of the Y-coordinate onto the. X/Z plane. T h i s i s not a standard 3 - D p l o t . However i t allows simultaneous r e p r e s e n t a t i o n of 3 - v a r i a b l e s , when each p o i n t i s independent of the other p o i n t s . With r e s t i n g p e r f u s i o n c o n d i t i o n s 67% of l a m e l l a e are p e r f u s e d . A l i n e f o r 67% p e r f u s i o n i s p r o j e c t e d on the X-Y plane. - 171a -- i7^ -FIGURE 3 2 A summary of the e f f e c t s upon l a m e l l a r r e c r u i t m e n t of a l t e r i n g pulse r a t e with, stroke volume adjusted to maintain at r e s t i n g l e v e l s . Each p o i n t i n the 3-dimensional r e p r e s e n t a t i o n is. one holobranch p r e p a r a t i o n where •= 45 pulses-.min" 1,' ® - 30 p u l s e s . m i n " 1 and = 15 p u l s e s . m i n - 1 . See legend of Figure 31 f o r f u r t h e r e x p l a n a t i o n bf f i g u r e . - 172a -173 -Pressure/volume curves f o r the bulbus a r t e r i o s u s and  v e n t r a l a o r t a v e s s e l During surgery when these v e s s e l s were exposed, expansion of the v e s s e l could be observed with each v e n t r i c u l a r e j e c t i o n of blood. The bulbus a r t e r i o s u s was always distended more than the v e n t r a l a o r t a . The r e p r o d u c i b i l i t y of the v e s s e l s d i s t e n t i o n s can be seen i n f i g u r e 33. The pressure volume curves show that the v e s s e l s from t r o u t and l i n g cod have a high compliance at pressures between 40 and 60 cm H2O ( F i g . 33). The l i n g cod v e s s e l s had a high compliance up to p r e s s u r e s of 75 cm H2O, but these v e s s e l s were distended at reduced r a t e s to those found iri v i v o . The e f f e c t s of a d r e n e r g i c and c h o l i n e r g i c a g o n i s t drugs f o r v e s s e l s from t r o u t are summarised i n f i g u r e 34. A c e t y l c h o l i n e decreased the v e s s e l compliance over the 40 to 60 cm H 20 pressure range. The 0 - a g o n i s t i s o p r e n a l i n e i n c r e a s e d compliance more than n o r a d r e n a l i n e , an a -and B - a g o n i s t , over the same pressure range. Hypoxia increased v e s s e l compliance to the greatest, extent and i t s p r o g r e s s i v e e f f e c t s are shown i n more d e t a i l i n f i g u r e 34b. - 174' 'L: FIGURE 33 A) T y p i c a l pressure/volume curves f o r the bulbus a r t e r i o s u s and v e n t r a l a o r t a v e s s e l i n t r o u t . The d i s t e n s i o n s were performed at r a t e s a s s o c i a t e d with in v i v o c o n d i t o n s . '1' = 3 i n i t i a l s a l i n e c o n t r o l c urves. '2' = 3 curves a f t e r ACH treatment. '3' = 3 s a l i n e c o n t r o l curves about 1 hr a f t e r the i n i t i a l c o n t r o l s and subsequent ACH treatment. B) Three pressure/volume curves f o r the. l i n g cod v e s s e l with s a l i n e f i l l i n g r a t e s that were 2 to 3X slower than those encountered i n v i v o . - 174a -- 175-'4 F I G U R E 3 4 A) A summary of the e f f e c t s of a d r e n e r g i c and c h o l i n e r g i c treatment, and hypoxia on the pressure/volume r e l a t i o n s h i p s of the bulbus a r t e r i o s u s and v e n t r a l a o r t a of t r o u t . These curves are r e p r e s e n t a t i v e of the changes found i n a l l experiments. B.) The e f f e c t of p r o g r e s s i v e hypoxia ( i n the d i r e c t i o n of the arrow) on. the v e s s e l compliance. - 175a -volume, fj\ - Lie -DISCUSSION La m e l l a r p e r f u s i o n i s not complete i n the i s o l a t e d p e rfused holobranch when Qj/, P 0, pulse r a t e and pulse P i are comparable to i n v i v o r e s t i n g values f o r l i n g cod. The obtained value of 67% p e r f u s i o n , i s i n good agreement with the 60% l a m e l l a r p e r f u s i o n i n r e s t i n g rainbow t r o u t (Booth, 1978) . The agreement may w e l l be c l o s e r , s i n c e " r e s t i n g Q i " i n the i s o l a t e d g i l l arch was s l i g h t l y high, i f anything, due to some f i l a m e n t s at e i t h e r end of. the arch not being p e r f u s e d . Qi was based on p e r f u s i o n of complete holobranchs, as i_n v i v o . I n t e r e s t i n g l y , the mean value f o r l a m e l l a r p e r f u s i o n was 59.3% i n experiments with high pulse rate/low stroke volume p e r f u s i o n . I t i s p o s s i b l e then that r e s t i n g l i n g cod l i k e t r o u t , perfuse a minimum of 6.0% of t h e i r g i l l l a m e l l a e . Increased Qi and P i brought about l a m e l l a r r e c r u i t m e n t . So too d i d i n c r e a s e d stroke volume/decreased pulse r a t e . However, in c r e a s e d Qi and P i had the g r e a t e r e f f e c t on l a m e l l a r r e c r u i t m e n t . In f i s h , e x e r c i s e i s accompanied by e l e v a t e d Q and VA p r e s s u r e s (Stevens and R a n d a l l , 1967a and b; Stevens e_t a l . , 1972; S e c t i o n IV). F i s h i n hypoxic c o n d i t i o n s , however, ma i n t a i n Q i n the face of a b r a d y c a r d i a by i n c r e a s i n g stroke volume (Holeton and R a n d a l l , 1967a and b; T a y l o r et a l . , 1977; S e c t i o n I V ) . These i n v i v o changes i n blood flow and pressure i n the v e n t r a l a o r t a could lead to changes i n the number of g i l l l a m e l l a e perfused i n a p u r e l y p a s s i v e manner i n the above s i t u a t i o n s without the involvement of any l o c a l neural or humoral responses w i t h i n the g i l l . L a mellar recruitment i n the i s o l a t e d . g i l l of l i n g cod occurs by p e r f u s i o n of the more d i s t a l ; l a mellae on each f i l a m e n t . and could not be v a r i e d independently i n my experiments and i t cannot be determined i f recruitment i s r e l a t e d to changes i n flow alone, pressure alone or both together. The f o l l o w i n g i s an e x p l a n a t i o n of a p o s s i b l e way by which changes in pressure may lead to l a m e l l a r r e c r u i t m e n t . The s i z e of the filament, a f f e r e n t a r t e r y and the change i n flow along i t s length are such i n l i n g cod that each l a m e l l a r a r t e r i o l e along a f i l a m e n t i s s u b j e c t to almost the same input pressure ( S e c t i o n s I and I T ) . The c r i t i c a l c l o s u r e pressure f o r any l a m e l l a r a r t e r i o l e w i l l be set by Its o v e r a l l r e s i s t a n c e and i t s v a s c u l a r tone (Burton, 1951). The r e s i s t a n c e of d i s t a l a f f e r e n t l a m e l l a r a r t e r i o l e s i s g r e a t e r s i n c e they are s l i g h t l y narrower than proximal ones "and supply . more than one l a m e l l a , and i t i s l i k e l y then that t h e i r c r i t i c a l c l o s u r e p ressure i s a l s o g r e a t e r . '. If the c r i t i c a l c l o s u r e pressure of a r t e r i o l e s does i n c r e a s e towards the d i s t a l end Of the f i l a m e n t then a r i s e i n P^ w i l l r e s u l t i n the recruitment of d i s t a l l a m e l l a e . I n v i t r o v a s c u l a r compliance must a l s o be c o n s i d e r e d . The a r t e r i o l e s w i l l remain patent, once opened, at pressures below the' opening pressure (Busby and.Burton, 19 65) . Thus i n c r e a s e s i n pulse pressure which e l e v a t e d peak ( s y s t o l i c ) p ressure above the c r i t i c a l c l o s u r e p r e s s u r e , w i l l a l s o cause - 178 -l a m e l l a r r e c r u i t m e n t even though there may be no change . i n mean pr e s s u r e . Thus both the r i s e i n VA pressure during e x e r c i s e and the marked r i s e i n VA pulse pressure during hypoxia i n f i s h may account f o r the l a m e l l a r recruitment that has been observed i n t r o u t during hypoxia (Booth, p e r s . comm.) and hypothesized during, e x e r c i s e (Jones and R a n d a l l , 1978). My hypothesis above e x p l a i n s how mean pressure and peak s y s t o l i c pressure per se overcome c r i t i c a l c l o s i n g p r e s s u r e s . U n f o r t u n a t e l y i t does not e x p l a i n why increased pulse rate/decreased s t r o k e volume c o n d i t i o n s reduced l a m e l l a r p e r f u s i o n , when mean pressure rose s l i g h t l y and o f f s e t any r e d u c t i o n i n s y s t o l i c p r e s s u r e , and why there was inc r e a s e d l a m e l l a r p e r f u s i o n with low pulse r a t e / h i g h s t r o k e volume . c o n d i t i o n s . A f a l l i n i n t e r s t i t i a l pressure could lower the pressure f o r c r i t i c a l c l o s u r e i n the a r t e r i o l e s . In l i n g cod there i s an e x t e n s i v e venolymphatlc drainage of the g i l l s and an in c r e a s e i n lymph flow could; decrease i n t e r s t i t i a l p r e s s u r e . Many of the lymphatic v e s s e l s i n the g i l l are i n c l o s e a s s o c i a t i o n with f i l a m e n t a r t e r i e s such that there may be a mechanical i n t e r a c t i o n between the v e s s e l s , i n p a r t i c u l a r the a f f e r e n t companion v e s s e l s and f i l a m e n t a r t e r i e s and a r t e r i o l e s ( P l a t e I I , , S e c t i o n I ) . The e f f e c t of the a f t e r i o - v e n o l y m p h a t i c i n t e r a c t i o n i n t r o u t g i l l s on Rg has been b r i e f l y d i s c u s s e d by Smith (19 77). Volume changes i n the compliant f i l a m e n t v e s s e l s i n v i t r o that are a s s o c i a t e d with the pulse pressure may then promote lymphatic. d r a i n a g e . Thus i f the pressure p u l s e i n c r e a s e s -179. -then there may be more adequate venolymphatic drainage and a f a l l i n i n t e r s t i t i a l p r e ssure r e s u l t i n g i n a f a l l i n the c r i t i a l c l o s u r e pressure of. the l a m e l l a r a r t e r i o l e s . More e f f e c t i v e lymphatic drainage may then by i t s e f f e c t on i n t e r s t i t i a l p r essure lead to l a m e l l a r r e c r u i t m e n t . If there are a r t e r i o - v e n b l y m p h a t i c i n t e r a c t i o n s then venolymphatic flow would a l t e r when a r t e r i a l flow was changed. In t h i s s e r i e s of p e r f u s i o n experiments only the outflow from the EFA (Q 0) was measured. Q Q, although always l e s s than Qi, i n c r e a s e d i f e i t h e r P^, Qi or pulse r a t e was r a i s e d . The a f f e r e n t b r a n c h i a l a r t e r y was cannulated and t h i s v e s s e l s u p p l i e s o n l y the l a m e l l a r a r t e r i o l e s . A l l of Q^ passes through the secondary l a m e l l a e and l a m e l l a r f i l t r a t i o n was not a major a l t e r n a t e pathway.for f l u i d l o s s . Thus the s a l i n e , having perfused the lamellae enters the e f f e r e n t f i l a m e n t v e s s e l and flows out e i t h e r by the cannula i n s e r t e d i n the a f f e r e n t b r a n c h i a l a r t e r y or v i a some other pathway. The e f f e r e n t c i r c u l a t i o n g i v e s r i s e to a r e c u r r e n t n u t r i t i t i v e supply to the f i l a m e n t that forms a p a r t of an arch venolymphatic drainage and a l s o a hypobranchial a r t e r i a l supply to the v e n t r a l b r a n c h i a l muscles.. Both v e s s e l s pass v e n t r a l l y down the g i l l arch ( F i g . 35). The hy p o b r a n c h i a l a r t e r y was t i e d o f f s e p a r a t e l y and a l s o l i g a t u r e s were t i e d around both the a f f e r e n t and e f f e r e n t cannulae to s e a l o f f the venolymphatic drainage i n the a r c h . - 180 ~ FIGURE 3.5 Schematic r e p r e s e n t a t i o n of the c i r c u l a t i o n through the g i l l l a m e l l a and the body of the g i l l f i l a m e n t i n d i c a t i n g the s i t e s of c a n n u l a t i o n . (Based on S e c t i o n I ) . = flow d i r e c t i o n of p e r f u s a t e = l i g a t u r e cd O co OUTPUT CATHETER lamella hypobranchial artery veno-lymphatic system INPUT CATHETER - 181 -It seems probable, however, that s a l i n e leaked out v i a t h i s venolymphatic supply. Why then should an inc r e a s e In be a s s o c i a t e d with a decrease i n venolymphatic outflow? One answer i s that at hig h e r a r t e r i a l p r e s s u r e s , compliant a r t e r i a l v e s s e l s expand and compress the venolymphatic v e s s e l s , thereby r a i s i n g the r e s i s t a n c e to flow i n the venolymphatic system and thus reduce venolymphatic outflow. Such i n t e r a c t i o n would occur where the l a m e l l a r a r t e r i o l e s and venolymphatic v e s s e l s i n t e r d i g i t a t e (See P l a t e s 11 and 12, S e c t i o n I ) . Hence the o b s e r v a t i o n that high a r t e r i a l p u l s e r a t e s reduced venolymphatic flow Is c o n s i s t a n t with the concept, of the^on-c'e~px>.of a r t e r i o - ~~ venolymphatic i n t e r a c t i o n s . In v i t r o g i l l s do not act as simple ohmic r e s i s t a n c e ( F i g . 30) and i t i s c l e a r that g i l l compliance must be taken i n t o account ( F i g . 29). Under constant flow c o n d i t i o n s , a stepwise e l e v a t i o n of P Q decreased Rg without l a m e l l a e recruitment and the changes i n mean P^ were much s m a l l e r than P Q. V e s s e l dimensions must t h e r e f o r e i n c r e a s e to lower Rg. The e x t e n t . t o which compliance a f f e c t s o v e r a l l Rg can be estimated. In these g i l l p e r f u s i o n experiments, when was el e v a t e d by a mean value of 76.9%, l a m e l l a r p e r f u s i o n increased from 67.8% t o 76.5%. If. the g i l l s were a simple ohmic r e s i s t a n c e , the mean P^ should have in c r e a s e d by 55%. In f a c t , mean.P^ onl y rose an average of 37.9%, to l a t e r s t a b i l i s e a t 23..5% above the average pre-change mean P^. When Wood et. a l . (1978) e l e v a t e d P Q - 18 2 .- • in t h e i r g i l l p r e p a r a t i o n , Rg was decreased and. there was l i t t l e e f f e c t on P^. C l e a r l y , then, the range of the g i l l compliance i n v i t r o and i t s e f f e c t on Rg i s indeed c o n s i d e r a b l e . There i s , however, an important d i f f e r e n c e between the v e s s e l ' t o n e iri v i v o and i n v i t r o . In v i t r o there i s l i t t l e or none, whilst, i n v i v o the muscular l i n e d arch and f i l a m e n t a r t e r i e s and a r t e r i o l e s have r e s t i n g a d r e n e r g i c tone (Helgason and N i l s s o n , 1973; Wood, 1974b; Table I I I , S e c t i o n I ) . The lamellae l a c k v a s c u l a r tone and ,are very compliant ( S e c t i o n I ) . S w e l l i n g of the l a m e l l a e and of v e s s e l s l a c k i n g tone would account f o r the number of lamellae, perfused not being p r o p o r t i o n a l to Rg when pressures and flow regimes are changed. Decreases i n Rg cannot now, be. taken as unequivocal evidence f o r l a m e l l a r recruitment i n v i t r o . They only I n d i c a t e a change: i n the pressure p r o f i l e across c e r t a i n p o r t i o n s of the g i l l v a s c u l a t u r e .that r e s u l t from changes in v e s s e l dimensions. Of course l a m e l l a r recruitment may s t i l l occur' as a re'sult of the changes i n the pressure p r o f i l e . These f i n d i n g s c l e a r l y support the p r e d i c t i o n s made i n S e c t i o n II concerning changes i n g i l l blood flow p a t t e r n s . I t was concluded that e l e v a t e d Q a c t u a l l y e f f e c t e d the l a m e l l a r r e c r u i t m e n t , that was i n i t i a t e d by Rg or input pressure changes which represent changes in v e s s e l dimensions. In my experiments i n S e c t i o n I I I , Qi was not a l t e r e d when Rg was lowered and no recruitment was observed; E l e v a t i o n s i n Q have now been a s s o c i a t e d with l a m e l l a r recruitment i n v i v o and i n v i t r o . - 18'3 -The consequences of these f i n d i n g s are important to the i n t e r p r e t a t i o n , of r e s u l t s i n other i n v e s t i g a t i o n s using i n v i t r o p r e p a r a t i o n s . P^, Q^, Q D P Q, pulse r a t e / s t r o k e volume must a l l be c o n s i d e r e d . The pressure needed to overcome c r i t i c a l c l o s u r e of a f f e r e n t l a m e l l a r a r t e r i o l e s as w e l l as l a m e l l a r channels ( S e c t i o n I.) w i l l d i c t a t e how much of the s u r f a c e area of the g i l l s i s a v a i l a b l e f o r d i f f u s i o n . A r t e r i o - v e n o l y m p h a t i c I n t e r a c t i o n s w i t h i n the g i l l must a l s o be considered when de s i g n i n g i n v i t r o p e r f u s i o n experiments. In the past these f a c t o r s were hot u s u a l l y c o n s i d e r e d . Thus the q u a n t i t a t i v e i n t e r p r e t a t i o n of _in v i t r o p harmacological s t u d i e s w i l l , forf i n s t a n c e , be a f f e c t e d by the pressure and flow regimes s i n c e i n  v i t r o v a s c u l a r compliance i s high and v e s s e l s w i l l be more or l e s s d i l a t e d depending on the regime chosen. In ion and water t r a n s p o r t s t u d i e s the s e l e c t i o n of a p p r o p r i a t e flows .and p r e s s u r e s i s imperative, s i n c e d i f f u s i n g c a p a c i t y w i l l be a f f e c t e d by the number of l a m e l l a e perfused and ^ P i a m . The APlam d i c t a t e s c a p i l l a r y f l u i d f i l t r a t i o n r a t e s and low lymphatic c l e a r a n c e r a t e s w i l l i n c r e a s e l a m e l l a r d i f f u s i o n d i s t a n c e s . A l l these changes a f f e c t p a s s i v e exchange q u a n t i t a t i v e l y . Pulse r a t e and p u l s a t i l i t y are a l s o important i n determining b r a n c h i a l blood flow p a t t e r n s because of a r t e r i o - v e n o l y m p h a t i c i n t e r a c t i o n s . P u l s a t i l i t y w i l l be a l t e r e d by the tone of the b r a n c h i a l v e s s e l s and the bulbus a r t e r i o s u s . The bulbus was demonstrated to a c t as a Wind-kessel over a - 184 .-range of p r e s s u r e s s i m i l a r to r e s t i n g VA blood p r e s s u r e s . The degree to which blood flow i s depulsed by the bulbus. can be a l t e r e d however, by.adrenergic and c h o l i n e r g i c a g o n i s t s , and hypoxic c o n d i t i o n s . What i s the importance of these o b s e r v a t i o n s i n vivo? Changes i n VA pulse pressure i n the i n t a c t f i s h w i l l depend on a l a r g e number of f a c t o r s , of which the p r o p e r t i e s of the bulbus i s only one. However, with no other changes, ad r e n e r g i c s t i m u l a t i o n would increase d e p u l s a t i o n . Hypoxia,would have a s i m i l a r e f f e c t , but c h o l i n e r g i c s t i m u l a t i o n would in c r e a s e p u l s a t i l i t y . If blood pressures are e l e v a t e d at^the same time as bulbus s t i m u l a t i o n , then an a d r e n e r g i c action.would i n c r e a s e p u l s a t i l i t y , as would a c h o l i n e r g i c a c t i o n . A d r e n e r g i c a l l y mediated bulbus compliance changes Ln v i v o appear f e a s i b l e , s i n c e a number of t e l e o s t s have sympathetic i n n e r v a t i o n . o f the bulbus (Gannon, 1972; Watson, pers. comm.). Sympathetic pathways have not been examined i n Ophiodon. Whether c h o l i n e r g i c a l t e r a t i o n s occur in v i v o i s s p e c u l a t i v e s i n c e nothing i s .'known concerning c h o l i n e r g i c i n n e r v a t i o n ; o f the bulbus. The. sympathetic i n n e r v a t i o n of the bulbus i n t r o u t i s d e r i v e d as a branch of the l a r g e l y c h o l i n e r g i c c a r d i a c vagus nerve (Gannon, 1972) and t h e r e f o r e c h o l i n e r g i c i n n e r v a t i o n may a l s o be present. . The e f f e c t of hypoxia on the bulbus a r t e r i o s u s i s viewed as an hypoxic v a s o d i l a t i o n through reduced v a s c u l a r tone. Haswell et a l . (1978) reported a s i m i l a r phenomenon i n the b r a n c h i a l v a s c u l a t u r e of t r o u t . I found no hypoxic v a s o d i l a t i o n i n i s o l a t e d perfused g i l l arches but my p r e p a r a t i o n s had l i t t l e or - 185 -v a s c u l a r tone, which e x p l a i n s the apparent d i s c r e p a n c y . The . importance of hypoxic v a s o d i l a t i o n and whether i t even occurs i n v i v o i s unknown. In summary, I have now demonstrated that not a l l l a m e l l a e w i l l be perfused i n r e s t i n g l i n g cod. Increases i n Q and input pressure w i l l r e c r u i t l a m e l l a e a u t o m a t i c a l l y , as w i l l c o n d i t i o n of reduced heart r a t e / h i g h s t r o k e volume ( p u l s a t i l i t y ) . Mean and pulse p r e s s u r e s a l s o a f f e c t venolymphatic flow r a t e s . Since i n c r e a s e s i n Q and input pressure w i l l a l s o i n c r e a s e . A P i a m the f o l l o w i n g changes i n g i l l blood flow p a t t e r n s can t h e r e f o r e occur i n l i n g cod. Lamellar r e c r u i t m e n t , i n t r a l a m e l l a r shunting, increased l a m e l l a r volume and the a s s o c i a t e d reduced e p i t h e l i a l t h i c k n e s s , and i n c r e a s e d •venolymphatic flow and a s s o c i a t e d reduced l a m e l l a r i n t e r s i t i a l space. A l l these changes 1 can i n c r e a s e d i f f u s i n g c a p a c i t y . They were, however, a l l demonstrated i n v i t r o , i n s i t u or i n v i v o when drugs were ad m i n i s t e r e d . Do they occur n a t u r a l l y i n vivo? I t i s p r e d i c t e d that i f 0 2 demands in c r e a s e or 0 2 a v a i l a b i l i t y decreases one or more of these changes i n g i l l blood flow w i l l be brought about to maintain 0 2 t r a n s f e r at the g i l l s . T h i s p r e d i c t i o n i s t e s t e d i n S e c t i o n IV, - 185a -SECTION IV A STUDY OF GILL BLOOD FLOW AND ITS REGULATION IN OPHIODON ELONGATUS IN VIVO In v i v o monitoring of blood flow and v e n t i l a t i o n d u r i n g the s t r u g g l e responses. The e f f e c t of an hypoxic and an hyperoxic environment the c a r d i o v a s c u l a r system, g i l l v e n t i l a t i o n and oxygen uptake. on - 186 .-I N T R O D U C T I O N . ' R a n d a l l e_t a l . (1967) showed that e x e r c i s e and hypoxia i n t r o u t was a s s o c i a t e d with a change i n g i l l t r a n s f e r f a c t o r f o r oxygen, T o 2 , (see page 250 f o r d e f i n i t i o n ) . The i n c r e a s e in t r a n s f e r f a c t o r was subsequently suggested to be caused by changes in b r a n c h i a l blood flow ( R a n d a l l , 1970; Jones and R a n d a l l , 1978). In p a r t i c u l a r i t was suggested that there were changes i n both the number of l a m e l l a e perfused and the d i s t r i b u t i o n of blood w i t h i n each l a m e l l a . ' .Gaseous exchange i n f i s h g i l l s i s d i f f u s i o n l i m i t e d ( F i s h e r et a l . , 1969; R a n d a l l , 1976; Scheid and P i i p e r , 1976) and l a m e l l a r r e c r u i t m e n t and the r e d i s t r i b u t i o n of blood w i t h i n .the l a m e l l a enhance the c o n d i t i o n s f o r d i f f u s i o n such that e l e v a t e d Q and Vg ( g i l l v e n t i l a t i o n ) are a s s o c i a t e d with increased Mo 2 d u r i n g e x e r c i s e (Stevens and R a n d a l l , 1967a and b). The c a p a c i t y of the g i l l s to t r a n s f e r gases must be enhanced during .exercise otherwise the i n c r e a s e s i n 0 2 d e l i v e r y ( v e n t i l a t i o n ) and . removal (blood p e r f u s i o n ) would be p o i n t l e s s . Thus the i n c r e a s e s • • • i n Q, Vg and T o 2 are coupled during e x e r c i s e to enhance Mo 2. During hypoxia T o 2 and Vg are Increased to maintain Mo2. at reduced l e v e l s of 0 2 i n the water (Holeton and R a n d a l l , 1967a and b). Changes in l a m e l l a r recruitment .have been observed d u r i n g hypoxia i n t r o u t (Booth, p e r s . comm.) and I have demonstrated a number Of ways i n which c a r d i o v a s c u l a r changes i n c r e a s e g i l l s u r f a c e area ( l a m e l l a r recruitment), and a l t e r d i f f u s i o n d i s t a n c e s ( i n c r e a s e d sheet t h i c k n e s s , i n t r a l a m e l l a r shunting and lymphatic drainage) ( S e c t i o n s I, II and I I I ) . From the i n f o r m a t i o n we have, some of the mechanisms that bring about these changes are e v i d e n t . A l l changes i n l a m e l l a r p e r f u s i o n can now be e x p l a i n e d i n terms of automatic, changes r e l a t e d to a l t e r a t i o n s i n blood flow and input pressure to the l a m e l l a e ( S e c t i o n I I I ) . There are no o b s e r v a t i o n s of d i r e c t humoral,or neural a c t i o n s on the l a m e l l a r c a p i l l a r y sheet per se. Humoral agents do, however, produce the changes in blood flow and pressure and some of these e f f e c t s have been determined f o r l i n g cod ( S e c t i o n .II). Cardiac output can be r e g u l a t e d by humoral agents a f f e c t i n y heart r a t e and s t r o k e volume. C h o l i n e r g i c a g o n i s t drugs have negative c h r o n o t r o p i c e f f e c t s , and r e s t i n g heart r a t e s are i n f l u e n c e d by a c h o l i n e r g i c tone. P o s i t i v e i n o t r o p i c e f f e c t s are apparently mediated by 8-adrenergic r e c e p t o r s i n the heart. The p r e s s o r effe'c.ts produced by ACH and NAD i n j e c t i o n ^n v i v o were l a r g e l y a r e s u l t of d i r e c t v a s c u l a r a c t i o n s of the drugs, w h i l s t the increased Q was r e f l e x o g e n i c , as i t could not be accounted f o r by r e c i r c u l -a t i o n of the drugs to the h e a r t . The c a r d i o v a s c u l a r changes i n t h a t were mediated by NAD and.ACH i n l i n g cod were concluded to produce a l t e r a t i o n s i n g i l l blood flow p a t t e r n s , which would a l t e r T02 ( S e c t i o n I I ) . S i m i l a r c a r d i o v a s c u l a r changes have been p r e v i o u s l y demonstrated to a f f e c t T02 i n other f i s h . A d r e n a l i n e i n f u s i o n i n c r e a s e d a r t e r i a l oxygenation ^n v i t r o i n - 188 r t r o u t (Wood e_t a_l., 1978).. .Also intravenous I n j e c t i o n of a d r e n a l i n e increased a r t e r i a l P o 2 and 0 2 content i n r e s t r a i n e d e e l s (Steen and Kruysse, 1964). These changes i n T o 2 a s s o c i a t e d with a d r e n a l i n e i n f u s i o n could a l s o be explained, i n terms of a l t e r a t i o n s i n b r a n c h i a l blood flow and pressure causing automatic changes i n l a m e l l a r recruitment and p e r f u s i o n , based on my f i n d i n g s f o r l i n g cod. Thus, w h i l s t I now have reasonably d e t a i l e d i n f o r m a t i o n on how blood flow and pressure may be r e g u l a t e d i n l i n g cod, there are few data on how M6 2 i s a f f e c t e d by blood flow and pressure changes _in v i v o . The f o l l o w i n g q u e s t i o n s are t h e r e f o r e addressed i n t h i s S e c t i o n . What c a r d i o v a s c u l a r changes are made i n v i v o to ensure an 0 2 supply that meets t i s s u e demands and how are these changes r e g u l a t e d i n v i v o . Two .experimental approaches were used to answer these q u e s t i o n s . In the ocean l i n g cod are bottom d w e l l e r s , and the nearest they appear to come to e x e r c i s e i s b u r s t type swimming. An analogous response was examined here to e s t a b l i s h what c a r d i o v a s c u l a r changes accompany an increased 0 2 demand. Secondly, the c a r d i o v a s c u l a r responses to hypoxia and hyperoxic environments were e s t a b l i s h e d to determine what changes can be evoked i f environmental 0 2 . a v a i l a b i l i t y i s reduced or i n c r e a s e d . - 189 .-MATERIALS AND METHODS . . . E x p o s u r e t o h y p o x i c o r h y p e r o x i c w a t e r R o u t i n e s u r g e r y was p e r f o r m e d on 6 l i n g cod and b l o o d f l o w and p r e s s u r e s were m o n i t o r e d c o n t i n u o u s l y (See g e n e r a l m e t h o d s ) . G i l l v e n t i l a t i o n . a n d the w a t e r oxygen t e n s i o n ( P 0 2 ) were m o n i t o r e d d u r i n g the e x p e r i m e n t s . F o r each e x p e r i m e n t t h e i n f l o w i n g w a t e r t o the a q u a r i u m was made p r o g r e s s i v e l y h y p o x i c o r h y p e r o x i c w i t h N 2 o r 0 2 g a s e s , r e s p e c t i v e l y . The c h a n g i n g P o 2 s t a b i l i z e d a f t e r a b o u t 10 min and was m a i n t a i n e d a t t h i s l e v e l f o r a f u r t h e r 10 t o 20 m i n . The t o t a l e x p o s u r e time was 30 min, o r l e s s , i f the f i s h s t r u g g l e d e x c e s s i v e l y . The f i s h were t h e n r e t u r n e d t o n o r m o x i c w a t e r c o n d i t i o n s i n a s i m i l a r b u t more r a p i d f a s h i o n . The n o r m o x i c w a t e r P o 2 (160 t o .170 mm Hg) was s l i g h t l y h i g h e r t h a n a m bient b e c a u s e o f a s m a l l a i r l e a k i n t h e main s a l t w a t e r pump o f the b u i l d i n g . A f t e r each e x p e r i m e n t f i s h were a l l o w e d t o r e c o v e r f o r s e v e r a l h o u r s o r o v e r n i g h t ' p r i o r . ' t o . f u r t h e r e x p e r i m e n t s . Oxygen u p t a k e (Mo 2) d e t e r m i n a t i o n s Mo 2 d e t e r m i n a t i o n s were made on f i v e o t h e r f i s h t h a t had been v e r y l i g h t l y a n a e s t h e t i s e d (MS222) when b e i n g p l a c e d i n t o t h e a q u a r i u m and had t h e n .been a l l o w e d t o r e c o v e r and a c c l i m a t e o v e r n i g h t . The s e a l e d a q u a r i u m was d a r k e n e d e x c e p t a t one end t o a l l o w v i s u a l o b s e r v a t i o n o f t h e f i s h . . The gas t e n s i o n o f i n f l o w i n g w a t e r c o u l d be a l t e r e d w i t h 0 2 o r N 2 g a s e s - 190 -( F i g . 36). Mo 2 was measured i n two ways. F i r s t , using a constant flow-through system ( u s u a l l y 2.1.min - 1), i n l e t and o u t l e t water samples were taken s i m u l t a n e o u s l y . The oxygen t e n s i o n s of the water samples were measured using the Radiometer O2 e l e c t r o d e . M02 = APo 2.ao^.flow wt where Mo 2 was measured i n ml O2•hr-1.Kg-1; P02 i s the d i f f e r e n c e i n the i n l e t and o u t l e t water oxygen t e n s i o n s measured simu l t a n e o u s l y ; a 0 2 i s the oxygen bunsen s o l u b i l i t y constant f o r 23 0/00 seawater a t 10 to 11°C. (Green, 1965). Flow ( l . h r - 1 ) was measured by r e g u l a r outflow c o l l e c t i o n during the experiment. The f i s h was weighed (kg) p r i o r to the experiment. A c l o s e d box system was a l s o used to measure Mo2» Here the water was r e c i r c u l a t e d ( F i g . 36) and the f i s h g r a d u a l l y reduced the water P02 due to i t s own consumption. Water samples were c o l l e c t e d a t 5 minute time i n t e r v a l s and the water P02 measured Mo 2 = (Po ' 2 - Po 1 ^) a o 2.V where Pd"2 and.Po'2 are water oxygen te n s i o n s at the f i r s t and second samples of a given time p e r i o d , t (hr.) and V i s the volume of water i n the r e c i r c u l a t i n g system = (19.6 - f i s h wt) i n l i t r e s . - -F I G U R E 3 6. A schematic diagram of the. s e a l e d experimental h o l d i n g aquarium used f o r measurements of oxygen uptake i n l i n g cod. The p l e x i g l a s s c o n t a i n e r was round and darkened. One end was c l e a r to allow v i s u a l o b s e r v a t i o n s . Art aerated water supply could flow d i r e c t l y to the aquarium or be d i v e r t e d through a gas s t r i p p e r . A l t e r n a t i v e l y the water i n s i d e the aquarium could be r e c i r c u l a t e d with the pump. pump - 192 -Experimental p r o t o c o l used during M02 d e t e r m i n a t i o n s : At l e a s t f i v e M02 d e t e r m i n a t i o n s were made at a normoxic P02 f o r each f i s h using the flow-through system. These determinations were made durin g a 30 min p e r i o d and preceded the experiments using the c l o s e d box system. The e f f e c t of hypoxic water c o n d i t i o n s on M02 was examined using the c l o s e d box system. A 3 min e q u i l i b r a t i o n p e r i o d was allowed whenever the r e c i r c u l a t i n g system was s t a r t e d . During t h i s time a l l a i r bubbles were removed from the system. The f i r s t water sample was taken a f t e r t h i s 3 min p e r i o d , f o l l o w e d by two o t h e r s , 5 min and 10 min l a t e r . The Mo 2 was c a l c u l a t e d f o r each 5 min p e r i o d ; T h i s f i r s t s e t of two M02 determinations was made near to normoxic water c o n d i t i o n s . The r e c i r c u l a t i n g water was then made p r o g r e s s i v e l y hypoxic and f u r t h e r M02 d e t e r m i n a t i o n s were made. To reduce the water P02 the aquarium was f l u s h e d with deoxygenated water f o r 2 minutes. A three minute e q u i l i b r a t i o n p e r i o d f o l lowed the r e s t a r t i n g of the pump, before the second set of two M02 det e r m i n a t i o n s were made. T y p i c a l l y each set of M02 determinations was separated by about 6 minutes, during which time the water P02 was reduced 5 to 10 mm Hg. Th i s p r o t o c o l was repeated and. fou r or f i v e sets of M02 dete r m i n a t i o n s were made on each f i s h as the. water became p r o g r e s i v e l y more hypoxic. P r o g r e s s i v e hypoxia was stopped at a P02 of 20 mm Hg, or.when the f i s h appeared d i s t u r b e d . F l u s h i n g of the aquarium with deoxygenated water prevented CO2 accumulation i n the - 193. -r e c i r c u l a t i n g water as w e l l as producing stepwise r e d u c t i o n s i n water P02« V e n t i l a t i o n r a t e was measured by observing and counting o p e r c u l a r movements over 1 min. p e r i o d during a l l Mo 2 d e t e r m i n a t i o n s . V e n t i l a t i o n amplitude was v i s u a l l y assessed at the same time, where 0.5 = l i t t l e d i s c e r n i b l e movement and 5 = maximum f l a r i n g of the buccal and o p e r c u l a r c a v i t i e s . For d e t a i l s of a n a l y s i s and g r a p h i c a l techniques see M a t e r i a l s and Methods, S e c t i o n I I . - 1 9 4 ..-RESULTS The s t r u g g l e response The r e s t i n g behaviour of l i n g cod i n the ocean and the r e s t i n g values f o r c a r d i o v a s c u l a r v a r i a b l e s and g i l l v e n t i l a t i o n i n l i n g cod have been presented ( S e c t i o n I I ) . Resting l i n g cod have an Mo 2 of 22.8 ± 0 . 6 ml 0 2 . h r - 1 . k g - 1 (n=26 f o r 5 f i s h ) . When f i s h were d i s t u r b e d i n the ocean, they would make 1 or 2 qu i c k l a t e r a l t a i l movements and ' g l i d e ' to a new r e s t i n g V • p o s i t i o n a few meters away. In the ho l d i n g aquaria I observed a s i m i l a r behaviour p a t t e r n . S i m i l a r movements are a l s o used f o r fe e d i n g , which I observed both i n the ocean and i n the ho l d i n g a q u a r i a . The f i s h l i e i n wait and then pounce q u i c k l y on a passing f i s h . In. the experimental aquarium movements were l i m i t e d but the l i n g cod's c h a r a c t e r i s t i c sharp l a t e r a l t a i l movements were not prevented. The t a i l movements were v i s u a l l y a s s o c i a t e d with c h a r a c t e r i s t i c r e s p i r a t o r y and c a r d i o v a s c u l a r changes were the " s t r u g g l e response" (See p.14 a l s o ) . Many, s t r u g g l e s were observed and recorded during t h i s i n v e s t i g a t i o n . The nature of the s t r u g g l e g e n e r a l l y moved the flow probes or cannulae, which caused a r t i f a c t s . Complete a r t i f a c t f r e e records of a l l ^cardiovascular and r e s p i r a t o r y v a r i a b l e s during s t r u g g l e s were t h e r e f o r e . d i f f i c u l t to o b t a i n . Four r e p r e s e n t a t i v e s t r u g g l e s from two d i f f e r e r i t ' f i s h were, however, obtained and were s u i t a b l e f o r the d e t a i l e d a n a l y s i s that was performed here. - 195. -V e n t i l a t i o n r a t e and a m p l i t u d e b o t h i n c r e a s e d a f t e r t h e t a i l b e a t o f a s t r u g g l e ( F i g . 3 7 ) . F i s h t h a t were s e e n t o s t r u g g l e f r e q u e n t l y had h i g h e r M 0 2 v a l u e s t h a n t h e more q u i e t e r f i s h ( s e e v a r i a b i l i t y i n r e s t i n g M 0 2 v a l u e s i n F i g . 41 l a t e r ) . D u r i n g a s t r u g g l e c a r d i o v a s c u l a r v a r i a b l e s d i s p l a y e d v e r y s t r i k i n g p a t t e r n s when t h e y c h a n g e d ( F i g s . 37 and. 3 8 ) . The t a i l b e a t was a s s o c i a t e d w i t h a) a r e d u c e d Q; b) a marked b r a d y c a r d i a and c) an e l e v a t e d s t r o k e v o l u m e o f up t o t w i c e t h e r e s t i n g -v a l u e . The b r a d y c a r d i a d e v e l o p e d g r a d u a l l y o v e r t h e two t o e i g h t h e a r t b e a t s p r i o r t o t h e t a i l b e a t . S t r o k e volume was e l e v a t e d * • d u r i n g t h i s p e r i o d and Q was m a i n t a i n e d . The r e d u c t i o n i n Q a t t h e t a i l b e a t was a v e r y b r i e f and s u d d e n e v e n t , w i t h Q r i s i n g e q u a l l y r a p i d l y a f t e r t h e f i r s t t a i l b e a t up t o a v a l u e 70 t o 80% a b o v e t h e r e s t i n g v a l u e . I n c r e a s e d Q was l a r g e l y a r e s u l t o f a t a c h y c a r d i a ( o f 45 b e a t .min--'- maximum), b u t s t r o k e volume was s t i l l e l e v a t e d somewhat, a l b e i t d e c l i n i n g . H e a r t , r a t e and s t r o k e v o l u m e u s u a l l y showed r e c i p r o c a l c h a n g e s d u r i n g t h i s p h a s e o f t h e s t r u g g l e . The Q r e t u r n e d t o a r e s t i n g V a l u e a b o u t 100 s e c a f t e r t h e t a i l b e a t i n t h r e e o f t h e s t r u g g l e r e s p o n s e s a n a l y s e d . I n One o f t h e s t r u g g l e r e s p o n s e s , ' h o w e v e r , Q was r e - e l e v a t e d a b o u t 1, m i n a f t e r t h e I n i t i a l t a i l b e a t . H ere t h e s t r o k e v olume and. h e a r t r a t e i n c r e a s e d i n p h a s e w i t h e a c h o t h e r and t h e r e was no t a i l b e a t . . G i l l r e s i s t a n c e (Rg) r e m a i n e d r e m a r k a b l y s t a b l e f o r m o s t o f t h e t i m e d u r i n g t h e s t r u g g l e ( F i g . 31) c o n s i d e r i n g t h e f l u c t u a t i o n s i n Q.. The Rg d i d , however., i n c r e a s e c o n s i d e r a b l y p r i o r to and a t the t i m e of.the t a i l b e a t , b u t o n l y f o r s e v e r a l s e c o n d s . The i n c r e a s e d Rg was a s s o c i a t e d w i t h t h e b r a d y c a r d i a . The Rs a l s o rose j u s t p r i o r to the t a i l beat, but f e l l markedly at the time of and a f t e r the t a i l beat ( F i g . 39). Rg increased during the second, delayed e l e v a t i o n i n Q ( F i g . 39). As a consequence of the r e l a t i v e l y s t a b l e Rg during the s t r u g g l e , the p a t t e r n f o r the APg mean pressure resembled that of Q ( F i g . 40). The damping of the pressure pulse ( APg-pulse) d i s p l a y e d a p a t t e r n s i m i l a r to that of stroke volume ( F i g . 40), and was a l s o i n c r e a s e d when Q was e l e v a t e d during a s t r u g g l e . F i s h d i d o c c a s i o n a l l y s t r u g g l e f o r sometime a f t e r a t r o p i n e had been i n j e c t e d v i a the d o r s a l aorta,'but the e l e v a t i o n of Q f o l l o w i n g the t a i l beat became much reduced compared to u n a t r o p i n i s e d f i s h . The a t r o p i n e i n j e c t i o n g r a d u a l l y blocked changes i n c a r d i a c r a t e and a f t e r 1 hr there was no . br a d y c a r d i a a s s o c i a t e d with t a i l beats ( F i g . 37b). In f a c t heart rate remained e l e v a t e d above r e s t i n g values ( S e c t i o n ' I I ) . In a t r o p i n i s e d f i s h , t a i l beats were a s s o c i a t e d with only small changes i n v e n t i l a t i o n , u n l i k e r e s t i n g f i s h ( F i g . 37)'., A t r o p i n i s e d f i s h developed another response where Q was el e v a t e d ( F i g . 37c). This response was only n o t i c e d i n a t r o p i n i s e d f i s h (.3 f i s h ) , and i t was much akin to the response produced by a NAD i n j e c t i o n i n t o the v e n t r a l a o r t a of r e s t i n g f i s h (See F i g . 19). The increased Q was not a s s o c i a t e d with any body movement, u n l i k e the s t r u g g l e response, and there were major e l e v a t i o n s i n VA and DA pressures ( F i g . 37c). The amplitude and rate of v e n t i l a t i o n was n o t i c a b l y unchanged during t h i s response, except f o r the v e n t i l a t o r y pause at the begining of the response ( F i g . 37c). -197 -FIGURE 37 . ;' ' '•' Records of cardiovascular and respiratory varibles taken simultaneously during three types of "struggle response" t y p i c a l l y seen in ling cod. In a l l cases Q •= cardiac output . (ml .min - 1. kg" 1 j , . hr. = beat to beat heart rate (beats.min" 1), VA = ventral aorta blood pressure (cm H2O), DA = dorsal aorta blood pressure (cm H2O), OP = opercular pressure (cm H2O). A l l pressures were measured from ambient, pressure (amb). The start'of t a i l movements are indicated by s o l i d v e r t i c a l bars at!the bottom of the figure. A. The f i r s t trace shows a struggle with a single t a i l beat and the second trace i l l u s t r a t e s the eff e c t of several t a i l beats. B. The effect of atropine inje c t i o n with time .(+ 15 min, + 24 min and + 48 min). The cardiovascular events associated with t a i l movements are reduced, there is a tachycardia with no beat to beat rate fluctuations. C. Two records of a response peculiar to atropinised f i s h (+ 65 and + 95 min after injection) which caused elevations in Q and blood pressures. This response was not associated with a t a i l movements and can be contrasted with Fig. 37A and compared with Fig. 19. - 138 -FIGURE 38 A summary of the a n a l y s i s of the c a r d i o v a s c u l a r v a r i a b l e s d u r i n g f o u r t y p i c a l s t r u g g l e responses i n ling' cod. A l l values were measured si m u l t a n e o u s l y . Each p o i n t r e p r e s e n t s an i n d i v i d u a l value f o r cardiac, output (Q), s t r o k e * volume (S.V.) and heart r a t e ( h . r . ) . The r e s t i n g v^lue f o r each v a r i a b l e i s a l s o known. The t a i l movements s t a r t e d when t = 0. - 198a -25:-Q ml.mirf.'Kg"1 15-*k - tail beat — = rest value ± s.e. (I) -100 100 200 300 5 0 1 • B <n o ru beats, min"1 B • • • * 100 -100 200 300 i.0 4 S.V. ml.Kg-' 05 4 4 * ' i V i • -100 100 200 300 TIME . sec - J39 ~ • F I G U R E 39 '. A summary of the a n a l y s i s of the r e s i s t a n c e to blood flow i n the b r a n c h i a l (Rg) and systemic (Rs) c i r c u l a t i o n s during four t y p i c a l s t r u g g l e responses i n l i n g cod. See the legend of f i g u r e 38 f o r f u r t h e r explanation of t h i s f i g u r e . - 199a -4 -J it = tail beat — = rest value t s.e. (I) x Q Rg 2 -I -80 • 70 170 270 a o O , ' 3 " 1 2 z g-55 RS 4 -J 2 -J -80 • 1 70 170 270 TIME , sec - 200 -F I G U R E 40 * • A summary o f the a n a l y s i s o f changes i n b i p o d p r e s s u r e s a c r o s s t h e g i l l bed d u r i n g f o u r t y p i c a l s t r u g g l e r e s p o n s e s In l i n g c o d . APg mean i s t h e . c h a n g e mean b l o o d p r e s s u r e s a c r o s s t h e g i l l s . APg p u l s e i s t h e change i n p u l s e b l o o d p r e s s u r e , w h i c h i n d i c a t e s the d e g r e e o f damping o f p u l s a t i l e f l o w . See t h e l e g e n d o f f i g u r e 38 f o r f u r t h e r e x p l a n a t i o n o f t h i s f i g u r e . 1 25 if - tail beat — = rest value ± s.e APg M E A N , c m H 20 15 H 5H . 1 " • 1— * 300 -too 100 l 200 20 APg PULSE, 1 0 cm H 20 "7" 1 -100 I 0 100 T I M E , sec 200 300 - 201 -The e f f e c t s of exposure to hypoxic water • V e n t i l a t i o n : Increases i n v e n t i l a t i o n r a t e s and amplitude accompanied p r o g r e s s i v e hypoxia, but these changes were out of phase with each other ( F i g . 41). V e n t i l a t o r y amplitude increased before v e n t i l a t i o n r a t e as the water P02 was reduced. Furthermore, maximum v e n t i l a t o r y amplitude i . e . f u l l d i s t e n s i o n of the bucca l and o p e r c u l a r c a v i t i e s , was a t t a i n e d at a water Po 2 of 70 to 80 mm Hg, w h i l s t peak v e n t i l a t i o n r a t e (30 b r e a t h s . m i n - 1 ) was reached at .50 - 60 mm Hg water Po 2• The maximum v e n t i l a t o r y a c t i v i t y was s u s t a i n e d at water P02 l e v e l s as low as 20 mm Hg. Oxygen uptake: The M02 was e l e v a t e d during p r o g r e s s i v e hypoxia and reached a peak value when the water P02 was 70 to 80 mm Hg ( P c r i t ) . The Mo 2 at P c r i t was 51.3±11.7 ml . h r - 1 . kg""1. (n-5 f i s h ) , but the range of p C r i t exceeded r e s t i n g v alues by 75% to 300%. Below a water P02 of 40 mm Hg, M02 was reduced ( F i g . . 4 1 ) . C a r d i a c output: T h i s was u s u a l l y maintained or s l i g h t l y e l e v a t e d when the water P02 was above 70 to 90 mm Hg ( F i g . 42). , F i s h would s t r u g g l e during the hypoxic exposure ( F i g . 43), but these s t r u g g l e s have been analysed s e p a r a t e l y (see above) and are not in c l u d e d here. The Q was reduced when the water P o 2 was below 6 0 to 7 0 mm Hg and i t was reduced to 69% of the r e s t i n g value at - 202 -the t e r m i n a t i o n of the p r o g r e s s i v e hypoxia (t=0, Table X I ) , when the water P02 was as low as 20 mm Hg. The Q was reduced as a r e s u l t of a marked b r a d y c a r d i a . The e f f e c t of the b r a d y c a r d i a on Q was, however, g r e a t l y o f f s e t by a concomitant i n c r e a s e i n s t r o k e volume ( F i g . 42 and Table X I ) . Upon p r o g r e s s i v e reoxygenation of the hypoxic water Q was e l e v a t e d above r e s t i n g v a l u e s ; although the water Po 2 was below normoxic l e v e l s . The Q was e l e v a t e d as a r e s u l t of a t a c h y c a r d i a and a small i n c r e a s e i n stroke volume. The s u b t l e i n c r e a s e i n stroke volume d i s p l a y e d by each f i s h was not e v i d e n t when the mean values were s t a t i s t i c a l l y compared (Table XI) due to the l a r g e range values f o r stroke volume between f i s h . The f i s h d i d not s t r u g g l e at a reduced water Pb 2 when the water was being p r o g r e s s i v e l y reoxygenated a f t e r hypoxic exposure. G i l l r e s i s t a n c e (Rg) and systemic r e s i s t a n c e (Rs): The Rg changes during p r o g r e s s i v e hypoxia were not marked u n t i l water Po 2 t e n s i o n s were below 70 mm Hg, when Rg became e l e v a t e d , with a peak value at about 40 mm Hg ( F i g . 44). Hypoxic exposure was terminated at a water Po 2 below 40 mm Hg, and Rg had g e n e r a l l y d e c l i n e d from the peak value but was s t i l l e l e v a t e d above the r e s t value (Table XI, F i g . 44). The Rs increased more markedly than Rg during hypoxia and a l s o d i s p l a y e d at peak value at about a water Po 2 of 40 mm Hg ( F i g . 44, Table XI).. Rs a l s o d e c l i n e d i n some f i s h at Po 2 v a l u e s lower than 40 mm Hg.. ~ 20 3 ~ During the f i r s t f i v e minutes of the reoxygenation.of the hypoxic water Rs was reduced s i g n i f i c a n t l y , d e s p i t e below normoxic Po 2 l e v e l s (Table X I ) . The Rg, however, was at a value i n between the e l e v a t e d value at t = 0 and the r e s t i n g value (Table X I ) . During p r o g r e s s i v e hypoxia the APg p u l s e was i n c r e a s e d compared to that i n r e s t i n g f i s h . The APg was a l s o e l e v a t e d d u r i n g the reoxygenation of hypoxic water when Q was e l e v a t e d (Table XI) . - 204 -FIGURE 41 The e f f e c t of environmental hypoxia on oxygen uptake (Mo 2) and v e n t i l a t i o n r a t e and amplitude ( a r b i t r a r y u n i t s ) i n 5 l i n g cod. The oxygen uptake during p r o g r e s s i v e hypoxia f o r each f i s h i s presented as a continuous l i n e . These determinations were made i n the " c l o s e d box" system. The Mo 2 v a l u e s j o i n e d by a v e r t i c a l l i n e at the normoxic Po 2 are r e s t i n g values determined using the "flow-through" system. Each p o i n t f o r the v e n t i l a t i o n v a r i a b l e s i s an i n d i v i d u a l value, but many values o v e r l a p . ro o I OXYGEN UPTAKE, (ml02.hr-1.Kg-1) o I VENTILATION RATE, (per min) ro w O o r o . O o O " o o a ~ r, °° • D o o a • • • a a o o o • • • • • • o • a • •a o a • • o P3 • • r b T — r VENTILATION AMPLITUDE - : 2 0 5 -FIGURE 42 A summary of the a n a l y s i s of c a r d i a c output (Q), heart r a t e (h.r.) and stroke volume (S.V.) during the p r o g r e s s i v e hypoxic exposure i n 6 l i n g cod. I n d i v i d u a l values are p l o t t e d and the r e s t i n g value i s i n d i c a t e d as a r e f e r e n c e . - 205a 15 -I Q 1 ° -ml. min".'Kg"' 5 H 45 HR beats.min' :-, 25 H 5 A 1-5 1 vo H S.V. ml. Kg"' Q.5 J rest value £ s.e. i L* *• «t 5 • . •» , • e • • • ••• 35 75 112 150 - I • • • • B a • e _ a 1 35 75 l 112 150 i 35 i 75 112 150 WATER OXYGEN TENSION, P Q ^ , mm. Hg - 206 -F I G U R E 4 3 T h e c a r d i o v a s c u l a r r e s p o n s e s o f a l i n g c o d t o e x p o s u r e t o p r o g r e s s i v e e n v i r o n m e n t a l h y p o x i a a n d t h e n r e o x y g e n a t i o n o f t h e w a t e r . T h e f o u r s e l e c t e d a r e a s a r e t a k e n f r o m a c o n t i n u o u s r e c o r d . " R e s t " i s p r i o r t o t h e e x p e r i m e n t "N2 o n " r e p r e s e n t s t h e s t a r t o f w a t e r d e o x y g e n t i o n . T h e " 9 t h rain" i s a s e l e c t e d t r a c e c o n t i n u i n g f r o m t h e 9 t h m i n o f e x p o s u r e t o p r o g r e s s i v e h y p o x i a . T h e " 1 5 t h m i n " s e c t i o n d i s p l a y s t h e t e r m i n a t i o n o f t h e h y p o x i c e x p o s u r e i n t h e 1 8 t h m i n ( t = 0 ) w h e n N 2 w a s t u r n e d o f f a n d " a i r t u r n e d o n " . I t c a n b e s e e n t h a t r e o x y g e n a t i o n w a s m u c h f a s t e r t h a n d e o x y g e n a t i o n . N o t e t h e s y n c h r o n y b e t w e e n t h e h e a r t b e a t a n d b r e a t h i n g d u r i n g t h e 9 t h m i n o f p r o g r e s s i v e h y p o x i a . S e e F i g . 3 7 f o r e x p l a n a t i o n o f a b b r e v i a t i o n s u s e d . - 207 -TABLE XI ; •• • . .. A/ The e f f e c t of hypoxia on. mean values s.e. (n values) f o r measured c a r d i o v a s c u l a r v a r i a b l e s i n 6 l i n g cod. Resting values were obtained p r i o r to the exposure, and are s t a t i s t i c a l l y compared with t - 0 values ( r e p r e s e n t i n g the t e r m i n a t i o n : o f p r o g r e s s i v e hypoxia) and t = +1 to +5min values ( r e p r e s e n t i n g the f i r s t 5 min of reoxygenation of hypoxic water). S t a t i s t i c a l d i f f e r e n c e s are i n d i c a t e d by *. VA mean, VA p u l s e , DA mean and DA pulse are the mean^and pulse pressures (em H^ 'O) i n the v e n t r a l a o r t a and d o r s a l . a o r t a . B/ The a f f e c t of hypoxia on c a r d i o v a s c u l a r V a r i a b l e s c a l c u l a t e d from those v a r i a b l e s measured i n Table XI A above. Q = SV x HR, Apg = VA mean - DA mean, APg pulse '.' = ' VA pulse '- DA p u l s e . Rg = Apg/g, Rs = DA mean/Q. A b b r e v i a t i o n s are as exp l a i n e d above. 207a TABLE XI Heart Rate beats .min - 1 Rest(n=18) 29.4 ± 0.9 t=0(n=6) 12.1 ± 1.2' t=lto+5(n=14) 32.8 ± 1.9* Stroke Volume ml. kg - 1 0.384 ± 0.015 0.696 ± 0.108* 0.421 ± 0.028 VA mean 51.7 ± 0.5 46.0 ± 1.6' 57.7 ± 1.5* VA p u l s e 12.2 ± 0.5 22.3 ± 1.8' 15.5 ± 0.9* DA mean 38.5 ± 0 . 6 32.5 ± 1.3* 38.4 ± 1 . 1 DA p u l s e 6.1 ± 0.3 7.4 ± 0.8' 6.0 ± 0.2 C a r d i a c output,Q ml .min - 1. kg"1 Rest 11.2 ± 0 . 4 t=0 7.7 ±0.5* t = l t o 5 min-' 13.0 ± 0.7' Rg: 1.20 ± 0.09 1.88 ± 0.23* 1.49 ± 0 . 6 1 Rs 3.58 ± 0.20 4.67 ± 0.19* 3.23 ± 0 . 1 9 APg APg p u l s e •13.3 ± 0.8 6.1 ± 0.4 13.6 ± 1 . 2 8.5 ± 1 . 5 ' 18.9 ± 1.3* 9.7 ± 0.7' - 208 -FIGURE 4 4 A summary of the a n a l y s i s of the r e s i s t a n c e to blood flow through the g i l l (Rg) and systemic (Rs) c i r c u l a t i o n s during the progressive hypoxic exposure i n 6 l i n g cod. I n d i v i d u a l values are presented and the r e s t i n g value i s i n d i c a t e d . RESISTANCE TO BLOOD FLOW, cm H20.min.Kg.ml R S , • (SYSTEMIC) Rg, (GILL) 4 2 H 8 t • 9 * B B B 13 B B B B „ H B _ B B B \ " B B B B * 0 3 5 7 5 1 112 rest values ± s.e. B B I 1 5 0 1\> o CO SB WATER OXYGEN TENSION , PQ , mm Hg - 209 -The e f f e c t s of exposure to hyperoxic water P r o g r e s s i v e hyperoxla up to 9 0 % 0 2 s a t u r a t e d produced few c a r d i o v a s c u l a r changes. There were small changes i n the mean values f o r VA mean and DA mean pressures at t = 0. (Table X I I ) . Only i n one of the four experiments were Q and stroke volume reduced markedly. -1 TABLE XII A/ The. a f f e c t of hyperoxia on measured c a r d i o v a s c u l a r v a r i a b l e s i n 4 l i n g cod. For an e x p l a n a t i o n of the a b b r e v i a t i o n s see Table XI. B/ The e f f e c t of hyperoxia on c a r d i o v a s c u l a r v a r i a b l e s c a l c u l a t e d from those v a r i a b l e s measured i n Table XII A . above. See Table XI f o r an e x p l a n a t i o n of the a b b r e v i a t i o n s used. . -210a- TABLE XII A/ Heart Rate beats .min"1 ,Rest(n=12) t=0 (n=4) t=lto+5min(n=8) 29.5 ± 0.9 27.3 ± 1 . 7 28.9 ± 0 . 8 Stroke Volume ml .kg - 1 VA mean VA pul s e DA mean DA pulse 0.348 ± 0.026 0.303 ± 0.041 0.309 ± 0 . 0 2 5 53.2 ± 0.5 40.8 ± 1.0 5.4 ± 0.4 49.4 ± 0 . 7 ' 4.6 ± 0.7 50.4 ± 1.1' 11.9 ± 0.3 11.4 ± 0.4 11.1 ± 0.4* 38.1 ± 2 . 1 38.1 ± 1.2' 5.0 ± 0.5* B/ C a r d i a c output, Q ml .min"1 .kg"J Rg Rs APg APg p u l s e Rest 10.2 ± 0.8 t=0 8.5 ± 1 . 6 t=lto+5min 9.0 ± 0.9 1.27 ± 0.10 1.61 ± 0 . 5 4 1.29 ± 0.18 4.00 ± 0 . 2 1 4.48 ± 0 . 2 5 4.23 ± 0 . 2 3 12.4 ± 0.9 11.3 ± 1.6 11.9 ± 0.7 6.4 ± 0.1 6.9 ± 0 . 3 6.1 ± 0 . 3 - X I I -DISCUSSION The s t r u g g l e response i n l i n g cod My v i s u a l observations confirm that the s t r u g g l e r e -sponse analysed here i s one form of e x e r c i s e normally found i n l i n g cod. The s t r u g g l e response i s c h a r a c t e r i s e d by a . t a i l move-ment. A bradycardia. precedes /and an increase i n Q f o l l o w s the t a i l movement. Stevens et a l . , (1972) made observations on e x e r c i s i n g and d i s t u r b e d l i n g cod, but t h e i r a n a l y s i s was l e s s d e t a i l e d and they .only reported the marked bradycardia and cori-c u r r e n t r e d u c t i o n i n Q. Increased Q duri n g swimming has a l s o been observed i n t r o u t (Stevens and R a n d a l l , 1967b; Kiceniuk and Jones, 1977) and d o g f i s h ( P i i p e r e t a_l. , 1977) . The a n a l y s i s presented here revealed ..two s t r i k i n g p o i n t s . F i r s t , c a r d i o v a s c u l a r changes during the s t r u g g l e show patterns which r e v e a l c l e a r i n t e r r e l a t i o n s h i p s between the v a r i -a bles. Heart r a t e and Q shared a s i m i l a r p a t t e r n . Stroke volume had a d i f f e r e n t p a t t e r n t o heart r a t e , but i t was s i m i l a r to the pa t t e r n of APg pulse. In l i n g cod, t h e r e f o r e , heart r a t e has the major i n f l u e n c e on Q. This c o n c l u s i o n d i f f e r s from those f o r t r o u t and.dogfish, where increases i n Q during e x e r c i s e are l a r g e l y brought about through increased stroke volume (Stevens and R a n d a l l , 1967b; Kiceniuk and. Jones, 1977; P i i p e r et a l . , 1977) Secondly, during the. s t r u g g l e response, Rg changes were small compared, w i t h flow changes except f o r the b r i e f , but dramatic r i s e i n Rg p r i o r to and at the t a i l beat. S i m i l a r l y . i n e x e r c i s i n g t r o u t Rg remains r e l a t i v e l y unchanged (Stevens - a.U -and R a n d a l l , 1967) or i n c r e a s e s (Kiceniuk and Jones, 1977). The e l e v a t e d Q d u r i n g s t r u g g l i n g w i l l r e c r u i t l a m e l l a e , which i s con-s i s t e n t w i t h the observed i n c r e a s e i n t h e damping of the pre s s u r e pulse.: L a m e l l a r r e c r u i t m e n t and no other changes should, however, reduce Rg. Why does l a m e l l a r r e c r u i t m e n t . n o t a l t e r Rg d u r i n g the s t r u g g l e response i f the major g i l l r e s i s t a n c e r e s i d e s i n the l a m e l l a r u n i t ? Q must be e l e v a t e d t o e f f e c t l a m e l l a r r e c r u i t m e n t . One e x p l a n a t i o n i s t h a t the i n c r e a s e d flow i s the same'or n e a r l y \ • • the same as the i n c r e a s e d blood volume of the g i l l s , as a r e s u l t of l a m e l l a r r e c r u i t m e n t and i n c r e a s e d v a s c u l a r sheet t h i c k n e s s ( S e c t i o n I ) , and thus any p o t e n t i a l change i n Rg i s o f f s e t . T h i s e x p l a n a t i o n i s supported by c a l c u l a t i o n s of increased, flow and p o t e n t i a l changes i n Rg due to l a m e l l a r r e c r u i t m e n t i n my e x p e r i -ments. R e s t i n g l i n g cod probably p e r f u s e about 60% of t h e i r l a m e l l a e a t r e s t . ( S e c t i o n I I I ) and i f d u r i n g a s t r u g g l e a l l l a m e l l a e were p e r f u s e d , Rg c o u l d decrease by 66% wit h no.other changes. During the s t r u g g l e response Q i s i n c r e a s e d by 70 to 80%, which would c l e a r l y o f f s e t the p o t e n t i a l e f f e c t of l a m e l l a r r e c r u i t m e n t on Rg. Any d i s c u s s i o n of the important changes i n v a s c u l a r dimensions d u r i n g the s t r u g g l e response would be specu-l a t i v e s i n c e l i t t l e i s known concerning v a s c u l a r compliance i n  v i v o , except t h a t the lamellae, are the major g i l l compliance s i t e . The t a i l , beat does not i n i t i a t e the s t r u g g l e response. There i s a pr e c e d i n g b r a d y c a r d i a which i n d i c a t e s some a n t i c i p a t i o n of the t a i l beat. The b r a d y c a r d i a reduces Q even though s t r o k e volume i s i n c r e a s e d due to i n c r e a s e d f i l l i n g times and assuming unimpeded venous r e t u r n . The bradycardia can r e s u l t from an increase i n c h o l i n e r g i c tone to the heart. The Rg a l s o increased c o n c u r r e n t l y . :Increased Rg can be produced by a c h o l i n e r g i c a l l y mediated b r a n c h i a l v a s o c o n s t r i c t i o n (Section I I ) . I t i s proposed that the observed bradycardia and increased Rg are both produced bygia- s h o r t - l i v e d i n crease i n vagal c h o l i n e r g i c a c t i v i t y . A t r o -pine, which blocks c h o l i n e r g i c r e c e p t o r s , prevented the brady-c a r d i a and the increase i n Rg during a s t r u g g l e response. The elevated Q and ta c h y c a r d i a d i s p l a y e d by e x e r c i s i n g 1 t r o u t or d o g f i s h may be r e l a t e d e x c i t a t o r y c a r d i a c e f f e c t s of c i r c u l a t i n g catecholamine's during e x e r c i s e . These hormones are known to increase during exercise- i n t r o u t (Randall et al_. , pers. comm.) and a l s o during s t r e s s i n salmon (Mazeaud et al_. , 1977), Gadus (N i l s s o n et al_. , 1976) and d o g f i s h ( B u t l e r et a l . , 1978) . The importance, i f any, of adrenergic mechanisms during the st r u g g l e response i n Ophigdon i s d i f f i c u l t to determine. However, the second^ delayed e l e v a t i o n of Q has many . s i m i l a r i t i e s ' to c a r d i o -v a s c u l a r e x c i t a t i o n through c i r c u l a t i n g catecholamines (Section I I ) , i n which there i s elevated Q , elevated VA mean pressure, no change i n APg mean and an increased damping of the pressure • pulse 1 The simultaneous e l e v a t i o n s of stroke volume and heart r a t e , without t a i l beats, i n d i c a t e s an a d r e n e r g i c a l l y mediated • i n o t r o p i c e f f e c t i n a d d i t i o n t o .a l o s s of c h o l i n e r g i c tone to the heart (Section I I ) . Ling cod are a l s o apparently capable of a d r e n e r g i c a l l y mediated c a r d i o v a s c u l a r responses. In a t r o p i n i s e d f i s h spontaneous c a r d i o v a s c u l a r changes occur, without a t a i l beat. There i s an elevated Q and pressor response without any heart - A i 4 -r a t e change ( F i g . 37), which are very s i m i l a r to the car d i o v a s -c u l a r changes brought about by a NAD i n j e c t i o n i n t o the v e n t r a l aorta of r e s t i n g l i n g cod. There i s even the c h a r a c t e r i s t i c r e s p i r a t o r y pause a s s o c i a t e d / w i t h the i n i t i a t i o n of the pressor response ( F i g . 37). I t i s i n t e r e s t i n g t h a t Stevens et al.',' (1972) noted that e x e r c i s e i n a t r o p i n i s e d l i n g cod was accompan-i e d by an elevated Q and no heart r a t e changes. Li n g cod, th e r e -f o r e , probably can and do r e l e a s e catecholamines during s t r e s s f u l s i t u a t i o n s l i k e other f i s h . I propose that the s t r u g g l e response i n •'ling cod i s l a r g e l y c h o l i n e r g i c a l l y mediated and r e s u l t s i n l a m e l l a r r e c r u i t -ment wi t h a matched e l e v a t i o n i n blood flow. Increased Q a l s o a l t e r s i n t r a l a m e l l a r flow to f u r t h e r increase d i f f u s i n g c a p a c i t y . The increased d i f f u s i n g c a p a c i t y , Q, and Vg permit an e l e v a t i o n i n Mo,, t o meet the increased t i s s u e 0 2 demands during the s t r u g g l e . Exposure to hypoxic water .' ' During hypoxia l i n g cod elevated t h e i r Mo 2 as much as t h r e e f o l d arid maintained Mo 2 l e v e l s even at water Po 2 as low as 40 mm Hg. My observations show a peak M© 2 a s s o c i a t e d w i t h max-imum v e n t i l a t o r y a c t i v i t y at the water p c r £ ^ . °f 70 mm Hg. Q u a l i t a t i v e l y s i m i l a r observations have been made f o r t r o u t (Holeton and R a n d a l l , 1967 a and b ) . Ling cod can eleva t e or maintain t h e i r Q at Po 2 l e v e l s above the p c r i t - T h e e f f e c t of p e r i o d i c s t r u g g l e s i s to f u r t h e r increase Q by 70 to 80% f o r . short periods (see above). S t r u g g l i n g , which occurred to a — e U 3 v a r i a b l e extent, i s t h e r e f o r e i n i t s e l f an important response to hypoxia. Oxygen: d e l i v e r y to and 0^ removal from the g i l l s are c l e a r l y increased during hypoxia. '; • • . I t i s c l e a r t h a t the increased p e r f u s i o n during hypoxia cannot, per se account f o r the observed t h r e e f o l d e l e v a t i o n i n . Mo,,. The r a t e of d i f f u s i o n must a l s o change, which would i n v o l v e changes i n surface area, d i f f u s i o n d i s t a n c e s and Po 2 g r a d i e n t s . Thus d i f f u s i o n l i m i t a t i o n s must be o p e r a t i v e during hypoxia i n l i n g cod,, a conclusion f i r s t made f o r t r o u t (Randall et a l . , • 1967). This c o n c l u s i o n i s c o n s i s t e n t w i t h gas t r a n s f e r i n , f i s h being d i f f u s i o n l i m i t e d (Fisher et a l . , 1 9 6 9 ; R a n d a l l , 1976; P i i p e r and Scheid, 1976). How then do observed c a r d i o v a s c u l a r changes i n l i n g cod r e l a t e to the a l t e r e d To,, during hypoxia? The noted c a r d i o -v a s c u l a r events are those a s s o c i a t e d w i t h the bradycardia and those w i t h s t r u g g l i n g . These observed c a r d i o v a s c u l a r events gan promote l a m e l l a r recruitment, i n t r a l a m e l l a r shunting, l a m e l l a r volume changes and elevated venolymphatic flow, as w i l l be deduced below. A l l these changes increase the g i l l d i f f u s i n g c a p a c i t y (Section I I I ) . The e f f e c t s of s t r u g g l i n g on Td 2 have already been discussed i n d e t a i l . Lamellar recruitment occurs during hypoxia when t h e - f i s h s t r u g g l e s . Bradycardia per se w i l l a l s o promote l a m e l l a r recruitment based on the i n v i t r o f i n d i n g s . (Section I I I ) since t h i s i s a c o n d i t i o n of high stroke volume/ low heart r a t e . The bradycardia probably maintains a higher - 216 -percentage of lamellae perfused:in between, s t r u g g l e s than are perfused d u r i n g . r e s t i n g s t a t e s . I f t h i s i s , t h e case then a con-d i t i o n of high l a m e l l a r p e r f u s i o n w i l l e x i s t during hypoxia i n ' l i n g cod, which i s s i m i l a r to the observed l a m e l l a r recruitment-during hypoxia i n t r o u t (Booth, pers. comm.). A high pulse pressure/low heart r a t e c o n d i t i o n a l s o enhances venolymphatic flow (Section I I I ) . Improved venolymphatic flow may r e s u l t i n b e t t e r drainage of the l a m e l l a r i n t e r s t i t i a l space. I f t h i s i s . t r u e , then the blood-water d i f f u s i o n b a r r i e r w i l l be reduced during hypoxia. ; ' • The- increase i n Rg during hypoxia i s due to a c h o l i -n e r g i c a l l y mediated v a s o c o n s t r i c t i o n of the b r a n c h i a l outflow a r t e r i e s (Section I I ) . As discussed above t h i s a l t e r s g i l l blood flow patterns through an elevated AP-^am, provided Q i s ele v a t e d . During hypoxia Q i s elevated a t P O 2 l e v e l s above the ^ c r ^ t but at P O 2 l e v e l s below the p- c ri t» Q i s n o longer maintained and Mo 2 i s no longer maximal ( F i g . 41). 0 2 delivery,however, remains maximal. As Q f a l l s , so w i l l AP, and l a m e l l a r recruitment. . lam I t i s suggested that because l i n g cod are unable t o maintain Q* below the Pcr£^.f R<? i s increased to compensate and thus maintain * the AP, to some degree. This s t r a t e g y i s s u c c e s s f u l to an lam . extent since below the p c r ; j _ t / oxygen uptake can s t i l l be elevated ( F i g . 41) . Ling cod l a c k a coronary blood supply and thus myocar-. d i a l 0? supply i s deriv e d from the venous blood. Hypoxia i n - 217 -t r o u t i s a s s o c i a t e d w i t h reduced venous P02 l e v e l s (Holeton and R a n d a l l , 1967b), which i s l i k e l y to be true f o r l i n g cod as w e l l . I f i t does occur and there are no other changes,. myocardial hypoxia w i l l develop. Myocardial hypoxia w i l l reduce the f o r c e ' of c a r d i a c c o n t r a c t i o n i n f i s h (Cesser/ 1978). One s t r a t e g y which may maintain myocardial 0 2 supply i s to increase blood residence time i n the heart through a bradycardia. This s t r a t e g y however i n v o l v e s a compromise between maintaining myocardial a c t i v i t y and reducing Q since the heart r a t e has the major i n -fluence on Q.. L i n g cod, however, can maintain- a c e r t a i n degree of s t a t u s quo i n the face of b r a d y c a r d i a , because Q can be main-ta i n e d w i t h increased stroke volumes ( S t a r l i n g ' s law). With p r o g r e s s i v e hypoxia venous Po 2 l e v e l s must u l t i m a t e l y f a l l so low t h a t prolonged residence times can no longer maintain myo- ' c a r d i a l 0 2 supply, and Q i s reduced. ' The c h o l i n e r g i c a l l y mediated c a r d i o v a s c u l a r changes ass o c i a t e d w i t h hypoxia.are s i m i l a r to those observed to precede the t a i l movements•during the s t r u g g l e response i n l i n g cod. In other f i s h (tench; Randall and Shelton, 1962^and d o g f i s h ; Kent and P i e r c e , 1975) the hypoxic bradycardia i s prevented by atropine i n j e c t i o n . Thus these changes appear widespread amongst f i s h . In the preceding d i s c u s s i o n i t was suggested (not demon-strated) t h a t these c h o l i n e r g i c a l l y mediated changes enhanced l a m e l l a r recruitment, reduced d i f f u s i o n d i s t a n c e s and helped maintain cardiac, performance, a l l of which a f f e c t To,,. The '. - 218 -f i n d i n g s of Taylor et al_. , (1977) add credence to these sugges-t i o n s since they have e s t a b l i s h e d the importance of c h o l i n e r g i c mechanisms i n maintaining To 2 during hypoxia i n d o g f i s h . Ih •their work, at any given l e v e l of environmental hypoxia, the a r t e r i a l Po 2 was always lower i n d o g f i s h i n which c h o l i n e r g i c mechanisms had been prevented by atropine i n j e c t i o n or v a g i s e c t i o n s , compared wi t h i n t a c t f i s h . Since the q u a l i t a t i v e and q u a n t i t a t i v e cardiovascular.responses to hypoxia are s i m i l a r i h both l i n g cod and d o g f i s h , i t i s l i k e l y that' the c h o l i n e r g i c a l l y mediated changes a l s o have important e f f e c t s 'On To 2 i n l i n g cod.' ! The-analysis of c a r d i o v a s c u l a r responses of l i n g cod to a e r a t i o n of hypoxic water i s d i f f i c u l t to i n t e r p r e t since the water Po 2 increased r a p i d l y . There were, however, s i g n i f i c a n t * c a r d i o v a s c u l a r changes (Table XI, F i g . 43), which resembled those observed a f t e r NAD had been i n j e c t e d Into the v e n t r a l aorta of r e s t i n g l i n g cod (Section I I ) . Heart r a t e reached 45 beats.min , Q increased up to 50%, stroke volume was elevated * there was a» pressor response and APg pulse increased. Such changes w i l l change the p a t t e r n of g i l l blood flow, increase the g i l l d i f f u s i n g c a p a c i t y and, t h e r e f o r e , the g i l l To 2 would-be increased. These changes are c o n s i s t e n t w i t h the observed " increase i n Mo 2 i n l i n g cod during the a e r a t i o n of hypoxic water. A p o s s i b l e involvement of adrenergic mechanisms i n the flow and pressure changes a s s o c i a t e d w i t h the increase In t r a n s f e r f a c t o r cannot be discounted. . - 219 -. Hyperoxia was not a s s o c i a t e d w i t h marked c a r d i o v a s c u l a r changes. Although c o n d i t i o n s f o r oxygen d i f f u s i o n are g r e a t l y enhanced i n t h i s experimental s i t u a t i o n , no changes i n g i l l per- • f u s i o n were apparent. I t is-suggested, t h e r e f o r e , t h a t r e s t i n g l i n g cod perfuse a minimum number of l a m e l l a e , which would be about 60% based on the observations made i n Section I I I . In c o n c l u s i o n the hypoxic response and the s t r u g g l e response of l i n g cod have remarkable s i m i l a r i t i e s w i t h respect to t h e i r a s s o c i a t e d c a r d i o v a s c u l a r and v e n t i l a t o r y changes. Both.responses w i l l increase the number of lamellae perfused, a l t e r the p e r f u s i o n w i t h i n each l a m e l l a and reduce d i f f u s i o n d i s t a n c e s . These changes increase the d i f f u s i n g c a p a c i t y of g i l l s . Vg and Q are a l s o elevated which accounts f o r Mo 2 being increased. The s t r u g g l e response i n v o l v e s a t a i l beat, which increases venous r e t u r n , and c h o l i n e r g i c mechanisms t h a t a l t e r Q and Rg p r i o r to and at the t a i l beat, thus i n i t i a t i n g the changes' i n blood flow through the g i l l s . The subsequent in c r e a s e i n Q w i t h reduced c h o l i n e r g i c a c t i v i t y , e f f e c t s the changes i n - g i l l blood flow p a t t e r n s . There may a l s o be a catecholamine release a f t e r the t a i l beat a s s o c i a t e d w i t h s t r u g g l i n g . The hypoxic response i n v o l v e s a c h o l i n e r g i c a l l y medicated c a r d i o -v a s c u l a r responses during p r o g r e s s i v e hypoxia, which are i n t e r r u p t e d by numerous s t r u g g l e s . The a e r a t i o n of hypoxic - 220 water may be accompanied by catecholamine r e l e a s e and no. c h o l i n e r g i c e f f e c t s . i - 220a -G E N E R A L D I S C U S S I O N AND ' SUMMARY ' 221 -Lamellar recruitment and i n t r a l a m e l l a r shunting were proposed i n the past as p o s s i b l e ways i n which blood flow might be a l t e r e d i n t e l e o s t g i l l s . This study has confirmed these two hypotheses. During the course of my work, Booth (pers. comm.) a l s o experimentally e s t a b l i s h e d that l a m e l l a r recruitment occurs i n t r o u t . R espiratory ( l a m e l l a r ) bypass as found in A n g u i l l a , does not occur i n Ophiodon. In a d d i t i o n to demonstrating two patterns pf blood flow, my study has presented observations which permit an explanation of how these a l t e r a t i o n s i n g i l l blood flow patterns might be regulated in v i v o . The t h e s i s has emphasised passive p r o p e r t i e s of blood v e s s e l s , p a r i c u l a r l y the l a m e l l a r c a p i l l a r y ' sheet which allow passive changes i n blood flow p a t t e r n s , and the importance of A p l a m . O v e r a l l , I have revealed a number of the i n t r i c a c i e s of b r a n c h i a l c i r c u l a t i o n and i t s r e g u l a t i o n i n Ophiodon, which can be applied to other t e l e o s t s , provided the often very important i n t e r s p e c i f i c d i f f e r e n c e s are a l s o considered. Resting l i n g cod perfuse about two t h i r d s of t h e i r l a m e l l a e , and these are the proximal lamellae. C r i t i c a l c l o s u r e of small v e s s e l s , a f f e r e n t l a m e l l a r a r t e r i o l e s , l a m e l l a r channels or both, i n d i s t a l regions of the filament, i s apparently c e n t r a l to t h i s c o n d i t i o n . Blood flow through the lamellae i s best described by sheet flow. The c o n d i t i o n s of l a m e l l a r blood flow are, t h e r e f o r e , analagous to pulmonary blood flow i n mammals and p o s s i b l y amphibians, thus patterns of c a p i l a r y . blood flow i n r e s p i r a t o r y exchange s i t e s show e v o l u t i o n a r y convergence. . - 222 -Lamellar recruitment Lamellae can be r e c r u i t e d a u t o m a t i c a l l y when input pressure and/or flow are elevated. Lamellar recruitment does not cause marked changes in. Rg, "which can be r e l a t e d to changes i n r e s i s t a n c e to flow i n the input and output b r a n c h i a l vessels , instead. The lamellae are very compliant vascular sheets with a low r e s i s t a n c e to flow. Consequently o v e r a l l g i l l compliance w i l l increase with l a m e l l a r recruitment, and blood flow w i l l be damped. I_n v i v o e l e v a t i o n s i n Q are accompanied by increased damping of the pressure pulse, APg pulse. I t i s suggested, th e r e f o r e , that an increase APg pulse i s associated with l a m e l l a r recruitment. Thus APg pulse measurements can be used to • q u a l i t a t i v e l y describe changes i n l a m e l l a r p e r f u s i o n , given the compliance c h a r a c t e r i s t i c s of the l a m e l l a r sheet. More accurate p r e d i c t i o n s of l a m e l l a r recruitment could be. obtained i f VA and DA blood flow patterns were compared fo r phasic -di f f e r e n c e s . The a f f e r e n t filament a r t e r y does not have a major r o l e i n c o n t r o l l i n g l a m e l l a r recruitment i n Qphiodon. . I t s r e s i s t a n c e to flow i s s i g n i f i c a n t . b u t I disagree with the proposal of Morgan and T o v e l l (1973) that the v e s s e l taper has a major e f f e c t on r e s i s t a n c e , because flow i s reduced along the v e s s e l length. Morgan and T o v e l l (1973) apparently did not consider these flow changes. In r e s t i n g l i n g cod the a f f e r e n t l a m e l l a r a t e r i o l e s have the c o n t r o l l i n g i n f l u e n c e i n p e r f u s i o n - 223 -of i n d i v i d u a l l a m e l l a e . These vessels w i l l a l s o , set A P ] _ a m which i s extremely important to i n t r a l a m e l l a r flow (see below). F i s h r e c r u i t more lamellae when oxygen demands increase.; .We now have a c l e a r e r idea of what mechanisms may bring t h i s about i n ' v i v o . E l e v a t i o n s of Q and/or a r t e r i a l blood pressures can bring about l a m e l l a r recruitment a u t o m a t i c a l l y . So too does a reduced heart rate/increased stroke volume c o n d i t i o n , but to a l e s s e r extent. The reason for these c a r d i o v a s c u l a r changes producing l a m e l l a r recruitment has been explained based on my i n v i t r o observations. The explanations are that a) the higher c r i t i c a l c losure pressures associated With d i s t a l l a m e l l a r u n i t s are overcome by elevated s y s t o l i c blood pressures (changes i n pulse pressure) or by elevated mean pressure per se. In the compliant l a m e l l a r u n i t s p o s i t i v e feedback occurs when plam l s r a i s e d since the vascular sheet thickness Increases and r e s i s t a n c e f a l l s . Hence blood pressures need not be c o n t i n u a l l y elevated f o r l a m e l l a r recruitment. b.) Because of arterio-venolymphatic i n t e r a c t i o n s in g i l l f i l a m e n t s , increased venolymphatic flow rates during high pulse pressure/low heart rate p e r f u s i o n c o n d i t i o n s may reduce c r i t i c a l c losure pressures. The nature of the c a r d i o v a s c u l a r changes that I observed during the i n vivo responses to s t r e s s i n l i n g cod, hypoxia and s t r u g g l i n g , i s such that both the above mechanisms could operate i n v i v o . The r e s u l t i n g l a m e l l a r recruitment i s Important i n accounting f o r the elevated M02 observed i n these s i t u a t i o n s . - 224 -In l i n g cod, the changes i n Q and/or blood pressures that produce l a m e l l a r recruitment are a r e s u l t of adrenergic and c h o l i n e r g i c a c t i o n s . Heart, rate has the major in f l u e n c e on Q i n l i n g cod. Reduced c h o l i n e r g i c tone to the heart or adrenergic c a r d i a c e x c i t a t i o n increase .Q. Increased c h o l i n e r g i c tone reduces heart r a t e , but stroke volume and pulse pressures are elevated and Q i s maintained provided venous return i s maintained. T a i l beats may be important i n i n c r e a s i n g venous re t u r n to elevate stroke volume. The b r a n c h i a l v a s c u l a t u r e , excluding the l a m e l l a r vascular sheet, i s a l s o d i r e c t l y a f f e c t e d by. c h o l i n e r g i c arid.adrenergic act ions. In v i v o g i l l outflow a r t e r i e s v a s o c o n s t r i c t through l o c a l i s e d c h o l i n e r g i c a c t i o n s , which r a i s e Rg. In s t r u g g l e s the b r i e f period of elevated Rg w i l l r a i s e the filament blood pressure (a back pr e s s u r e ) , and overcome the higher c r i t i c a l closure pressures associated with the d i s t a l l a m e l l ae. ' This leads, to l a m e l l a r recruitment i f Q i s subsequently elevated. The importance of e l e v a t i n g Q at the same time ae changing Rg was demonstrated i n v i t r o where decreasing Rg 'i alone did not bring about l a m e l l a r recruitment. B r a n c h i a l v a s o d i l a t i o n occurs through adrenergic actions and reduces Rg. V a s o d i l a t i o n of input vessels reduces c r i t i c a l c l o sure pressures and may be n e u r a l l y mediated adrenergic v a s o d i l a t i o n of a f f e r e n t l a m e l l a r a r t e r i o l e s . Release of catecholamine stores also occurs i n l i n g cod and the humoral adrenergic actions cause a systemic v a s o c o n s t r i c t i o n with a r e s u l t i n g pressor response, i n a d d i t i o n - 225 -to b r a n c h i a l v a s o d i l a t i o n . The pressor response and the b r a n c h i a l v a s o d i l a t i o n led to l a m e l l a r recruitment with the accompanying increase i n Q. When c r i t i c a l c losure pressures are lowered or elevated blood pressures exceed them, l a m e l l a r recruitment can occur. Lamellar recruitment should p o t e n t i a l l y reduce Rg, unless the increase i n Q c l o s e l y matches these p o t e n t i a l Rg changes. This appears to be the s i t u a t i o n w i t h humoral adrenergic actions i n l i n g cod. Catecholamine i n j e c t i o n causes l i t t l e change i n APg, even though l a m e l l a r recruitment and v a s o d i l a t i o n w i l l occur (Sections I I and I I I ) . Since lamellae contain the greater blood volume of the filament (Section I ) , 6" increases must therefore c l o s e l y match, the volume of lamellae r e c r u i t e d . This c o r r e l a t i o n c l e a r l y emphasises the reason for no Rg changes accompanying l a m e l l a r recruitment and the importance of e l e v a t i n g Q i_n v i v o and in v i t r o to e f f e c t l a m e l l a r recruitment. Such a s i t u a t i o n may w e l l be best regulated i f the l a m e l l a r recruitment occured a u t o m a t i c a l l y with: Q and pressure e l e v a t i o n s . ' The matching of. Q with l a m e l l a r recruitment has an important consequence i n gaseous exchange: red blood c e l l t r a n s i t time through the lamellae i s unaltered by these " . c a r d i o v a s c u l a r changes. Such a . s i t u a t i o n might be expected since blood oxygenation i s dependent on residence time of red blood c e l l s i n the lamellae when exchange i s d i f f u s i o n l i m i t e d . Lamellar t r a n s i t time i n r e s t i n g l i n g cod i s about 2 seconds (Section I) which approximates to the r e s t i n g frequency of the - 226 -heart beat. C l e a r l y i f a l t e r a t i o n s i n Q occur through stroke volume changes that are matched to l a m e l l a r recruitment, then t r a n s i t time remains p r o p o r t i o n a l to heart r a t e . During hypoxia, t h e r e f o r e , when there i s l a m e l l a r recruitment, increased stroke volume and a bradycardia, the l a m e l l a r t r a n s i t time i s increased. This s i t u a t i o n f a c i l i t a t e s 0 2 t r a n s f e r i n the face of reduced p a r t i a l pressure g r a d i e n t s . Such a s i t u a t i o n was pr e d i c t e d by Randall (1970).. During s t r u g g l e s when Q i s elevated through heart rate and stroke volume incr e a s e s , the r e l a t i o n s h i p between l a m e l l a r t r a n s i t time and heart rate i s a l t e r e d . Lamellar t r a n s i t time per se i s not a l t e r e d though. I n t r a l a m e l l a r shunting M6 2 i n l i n g cod i s increased up to three f o l d during h hypoxia. Resting l i n g cod perfuse about 60% of t h e i r lamellaj. I f l a m e l l a r p e r f u s i o n became maximal, then the surface area f o r gaseous exchange would be increase by about two-thirds. Thus maximal l a m e l l a r p e r f u s i o n w i l l only increase To 2 by two-thirds i f there are no other changes, and t h i s does not account f o r observed increases i n Mo2. I n t r a l a m e l l a r shunting and the associated.changes i n the lamellae with increased APlam. (Section I) must, t h e r e f o r e , c o n t r i b u t e s i g n i f i c a n t l y to in c r e a s i n g Mo2. I n t r a l a m e l l a r shunting i s a r e d i s t r i b u t i o n of blood flow w i t h i n a la m e l l a and occurs because i n l i n g cod the compliance, e p i t h e l i u m and vascular sheet thickness of the lamellae are non-uniform. I n t r a l a m e l l a r shunting can e a s i l y be - 227 -envisaged as an automatic phenomenon, as A P ^ a m r i s e s now that I have described l a m e l l a r blood i n terms of sheet flow. Simply, flow i s p r o p o r t i o n a l to r e s i s t a n c e , which i s p r o p o r t i o n a l t o / l a m e l l a r v a s c u l a r sheet thickness to the fo u r t h power. Sheet t h i c k n e s s , and thus flow, are always greater i n d i s t a l regions of the l a m e l l a . This r e l a t i o n s h i p i s true at a l l A P ^ a m . The d i s t a l regions are associated with higher d i f f u s i o n c a p a c i t i e s ; since i n e p i t h e l i a l d i f f u s i o n distances are greater i n basal regions. The d i s t a l regions of the sheet are more compliant at low A Piam than basal regions. Thus as blood pressure r i s e s the l a m e l l a r v a s c u l a r sheet thickens more i n the d i s t a l region and a l t e r s the r a t i o of resistance's and flow between the d i s t a l and basal regions. Therefore, i r i t r a l a m e l l a r shunting to d i s t a l l a m e l l a r regions occurs a u t o m a t i c a l l y when A P i a m r i s e s and, as a consequence, the g i l l d i f f u s i n g capacity i n c r e a s e s . Adrenergic and c h o l i n e r g i c c a r d i o v a s c u l a r a c t i o n s cause pressor responses, as described above, which r a i s e A P i a m and increase g i l l d i f f u s i n g c a p a c i t y i n t h i s manner. Other e f f e c t s of r a i s i n g A P i a m are that l a m e l l a r volume i s increased (a 12% r i s e with a 20 cm increase i n AP]_ a m) and the epit h e l i u m becomes thinner as the l a m e l l a r blood sheet t h i c k e n s . Both changes increase d i f f u s i n g c a p a c i t y . The r e l a t i v e c o n t r i b u t i o n s of i n t r a l a m e l l a r shunting, increased l a m e l l a r volume and reduced e p i t h e l i a l thickness on i n c r e a s i n g d i f f u s i n g c a p a c i t y are not known. As A P i a m increases so w i l l lymph formation, however. Fi s h are noted f o r t h e i r high c a p i l l a r y p r e m e a b i l i t y (Hargens e_t a l . , 1974) and furthermore, l a m e l l a r c a p i l l a r y blood pressures are high by mammalian c a p i l l a r y standards. Lymph so formed w i l l enter the l a m e l l a r i n t e r s t i t i a l space and, i f i t i s . not removed, w i l l increase d i f f u s i o n distances and decrease d i f f u s i n g c a p a c i t y . To fish> i n c r e a s i n g A P l a m i s so important i n i n c r e a s i n g To 2 that there are c e r t a i n safeguards that appear to p r e v e n t . l a m e l l a r i n t e r s t i t i a l f l u i d accumulation associated with high A P i a m . The l a m e l l a r blood flow i s h i g h l y p u l s a t i l e compared to other c a p i l l a r y beds, and because the.lamella blood sheet i s compliant, t h i s p u l s a t i l i t y d r i v e s lymph flow from the i n t e r s t i t i u m i n t o the c e n t r a l s i n u s . In a d d i t i o n an increased a r t e r i a l p u l s a t i l i t y / r e d u c e d heart rate c o n d i t i o n can promote venolymphatic flow. The e f f e c t of i n t r a l a m e l l a r shunting on gas t r a n s f e r can be analysed i n more d e t a i l i n the future as an extension of my f i n d i n g s . . Accurate r e g i o n a l measurements of e p i t h e l i u m thickness and blood sheet t h i c k n e s s , and how they change with A p ^ a m are needed. P r e d i c t i o n s can then be made, as I d i d , based on the r e g i o n a l v a r i a t i o n s i n l a m e l l a r compliance. . Important questions to be answered i n future i n v e s t i g a t i o n s are how and why does t h i s r e g i o n a l v a r i a t i o n occur? I can speculate on both these p o i n t s . F i r s t l y the reasons f o r g i l l lamellae d i s p l a y i n g r e g i o n a l non-uniformity. Is i t f o r t u i t o u s that both a and'h are reduced i n the base of the lamellae where the ep i t h e l i u m i s t h i c k e r , or are they r e l a t e d ? A l v e o l i i n the b i t - 229 -lung are noted f o r a high a value and a very t h i n e p i t h e l i u m . An a l t e r n a t i v e i s that the p r o p e r t i e s of the p i l l a r c e l l s are important since they hold the l a m e l l a r vascular sheet together under blood pressure and undoubtedly a f f e c t the compliance. P i l l a r c e l l s i z e does vary r e g i o n a l l y (Table I I I ) , but whether t h i s corresponds to the r e g i o n a l v a r i a t i o n s i n ct would re q u i r e a more rigorous a n a l y s i s of p i l l a r c e l l s i z e than performed here. Collagen f i b r e content of p i l l a r c e l l s may a l s o be a f a c t o r . In some t e l e o s t s the p i l l a r c e l l s i n basal regions have a higher c o l l a g e n content than those i n d i s t a l regions (Newstead, 1967), which i s i n t e r e s t i n g since the lung and blood- v e s s e l compliance are'both reduced i f c o l l a g e n f i b r e content i s increased (Sobin, pers; comm.; GOsline and Harman, pers. comm.). Newstead's f i n d i n g appears to c o r r e l a t e w e l l with the l e s s compliant basal l a m e l l a r regions i n l i n g cod and regional, v a r i a t i o n s i n l a m e l l a r compliance may, then,' be r e l a t e d to c o l l a g e n content of p i l l a r c e l l s . Why does r e g i o n a l v a r i a t i o n i n compliance and, thus " I ' i n t r a l a m e l l a r shunting e x i s t i n f i s h ? Lamellae act as the gaseous and i o n i c exchange area. I n t r a l a m e l l a r shunting may be necessary f o r t h i s dual f u n c t i o n . Resting f i s h apparently perfuse the minimum number of lamellae to meet t i s s u e Q 2 demands. Thereby they minimise d i f f u s i o n a l losses of water o f ions , which have to be replaced by a c t i v e processes to maintain an i n t e r n a l homeostasis. .Lamellar p e r f u s i o n i s a compromise between M02 and water or i o n i c loss., and should a l t e r i n r e l a t i o n .to the demands of a given s i t u a t i o n . During swimming -2 30 Mc-2 should d i c t a t e the nature of l a m e l l a r p e r f u s i o n and. t h i s has indeed been demonstrated. Trout during swimming a c t i v i t y t o l e r a t e an increased s a l t e f f l u x and water i n f l u x w h i l s t M02 i s elevated (Wood and R a n d a l l , 1973a and b). C l e a r l y r e g i o n a l v a r i a t i o n s i n l a m e l l a r d i f f u s i n g c a p a c i t y and the a b i l i t y to shunt flow between these areas provides the f l e x i b i l i t y needed fo r a d j u s t i n g l a m e l l a r p e r f u s i o n . Some s p e c i a l i z e d f i s h e s i n h a b i t hypoxic aquatic environments and ob t a i n t h e i r Oxygen l a r g e l y by breathing a i r using another gas exchange s i t e i n a d d i t i o n to that of the g i l l s . Blood flowing through the g i l l s i s , t h e r e f o r e , oxygenated and, i n hypoxic water, 0 2 l o s s from the blood to the water can occur. In t h i s s i t u a t i o n p e r f u s i o n of regions of the l a m e l l a with a high d i f f u s i n g c a p a c i t y i s a l i a b i l i t y . A i r breathing f i s h are. noted f o r large d i f f u s i o n d i s t a n c e s over extensive regions of the l a m e l l a and p e r f u s i o n of these regions would be h i g h l y advantageous i n a hypoxic aquatic environment (Randall et a l . , 1979). Some bimodal a i r - b r e a t h i n g f i s h when i n normoxic aquatic environments, do not a i r breath and obt a i n oxygen across the g i l l s . Here p e r f u s i o n of regions of the . l a m e l l a with high d i f f u s i o n c a p a c i t y i s necessary. To a i r breathing f i s h , t h e r e f o r e , i n t r a l a m e l l a r shunting of blood flow may w e l l be a way of l i f e . There i s no i n t r a - a l v e o l a r shunting of blood flow. In c o n c l u s i o n , i t i s expected that as f i s h r a d i a t e d i n t o d i f f e r e n t niches and as the r e l a t i v e r o l e s of the g i l l s were a l t e r e d , the nature of i n t r a l a m e l l a r shunting a l t e r e d a c c o r d i n g l y by r e g i o n a l l y modifying the l a m e l l a r v a s c u l a r sheet compliance. -231 -BIBLIOGRAPHY ATTINGER, E.O..and F.M. ATTINGER (1973). Frequency dynamics of p e r i p h e r a l vascular blood flow. Am. Rev. Biophys. Bioeng. '_2: 7-36. . • BALLINTIJN, CM. & G. M. HUGHES (1965). The muscular basis of the r e s p i r a t o r y pump i n the t r o u t . J . Exp. B i o l . 43: 349-362. BARER, G., F. MOHAMMED, A. SUGGETT, C. TWELVES (1978). Hypoxic pulmonary v a s o c o n s t r i c t i o n i n the f e r r e t . J . P h y s i o l . 281: 40P-41P. BERGEL, D.H. (1961a). The s t a t i c . e l a s t i c p r o p e r t i e s of the a r t e r i a l w a l l . J . P h y s i o l . (Lond.) 156: 445-457. BERGEL, D.H. (1961b). The dynamic e l a s t i c p r o p e r t i e s of the a r t e r i a l w a l l . J . P h y s i o l . (Lohd.) 156: 458-469. BERGMAN, H.L., K.R. OLSON and P.O. FROMM (1974). The e f f e c t s of vasoactive agents on the f u n c t i o n a l surface area of is o l a t e d - p e r f u s e d g i l l s of rainbow t r o u t . J . comp. P h y s i o l . 94; 267-286. BETTEX-GALLAND, M. and G.M. HUGHES (1973). C o n t r a c t i l e filamentous m a t e r i a l i n the p i l l a r c e l l s of f i s h g i l l s . J . C e l l . S c i . 13_: 359-370. BOOTH, J.H. (1978) . The d i s t r i b u t i o n of blood flow i n the g i l l s of f i s h : a p p l i c a t i o n of a new technique to rainbow t r o u t (Salmo g a i r d n e r i ) . J . Exp. B i o l . 73.: 119-129.. BURTON, A.C. (1951). On the p h y s i c a l e q u i l i b r i u m of small blood v e s s e l s . Am. J . P h y s i o l . 164: 319-329. BUSBY, D.E.and A.C. BURTON (1965). The e f f e c t of age on the e l a s t i c i t y of the major b r a i n a r t e r i e s . Can. J . P h y s i o l . Pharmacol. 43_: 185-202. » BUTLER, P.J. and E.W. TAYLOR (197.5) . The e f f e c t of progressive hypoxia on r e s p i r a t i o n i n the do g f i s h ( S c y l i o r h i n u s canicula) at d i f f e r e n t seasonal temperatures. J . Exp. B i o l . 63: 117-130. BUTLER, P.J., E.W. TAYLOR, M.F. CAPRA and W. DAVIDSON (1978). The e f f e c t of hypoxia on the l e v e l s of c i r c u l a t i n g catecholamines i n the dogfish S c y l i o r h i n u s c a n i c u l a . J . comp. P h y s i o l . 127: 325-330 CAMERON, J.N. (1974). Evidence f o r lack of by-pass shunting i n t e l e o s t g i l l s . J . F i s h . Res. Bd. Can. 31(2): 211-213. - 23 2 " CAMPBELL, G., B.J. GANNON and D.J. RANDALL. The vasculat u r e ' o f the g i l l of the rainbow t r o u t , Salmo g a i r d n e r i i In prep. CHAN, D.K.O. and P.H. CHOW (1976). The e f f e c t s of a c e t y l c h o l i n e , biogenic amines and other vasoactive agents on the c a r d i o v a s c u l a r functions of the e e l , A n g u i l l a a n g u i l l a . J . Exp. Zool. 196: 13-26. COBB, J.L.S. and R.M. SANTER (1973).. E l e c t o p h y s i o l o g y and cardiac f u n c t i o n i n t e l e o s t s : c h o l i n e r g i c a l l y mediated i n h i b i t i o n and rebound, e x c i t a t i o n . J . P h y s i o l . 230: 561-573. -DANZER, L.A., J.E. COHN and F.W. ZECHMAN (1968). R e l a t i o n s h i p of Dm and Ve to pulmonary d i f f u s i n g capacity during e x e r c i s e . R espir. P h y s i o l . 5: 250-258. DAVIS, J . C (1970). Estimation of c i r c u l a t i o n time i n rainbow t r o u t , Salmo g a i r d n e r i . J.- F i s h . Res. Bd. Can. 27 (10) : 1860-1863. DAVIS, J.C. (1972). An i n f r a r e d photographic technique u s e f u l f o r studying v a s c u l a r i s a t i o n of f i s h g i l l s . J . F i s h . Res. Bd. Can. 29: 109-111. DAXBOECK, C. and G.F. HOLETON (1978). Oxygen receptors i n the rainbow t r o u t , Salmo g a i r d n e r i . Can. J . Zool. 56(6): 1254-1259. DOELLINGER, Dr. (1837). Ueber die Ver t h e i l u n g des Blutes i n den Kiemen der Fishe. Abhandl. der mathem. physie Klasse. Bd I I : 784-794. DUNEL, S. and P. LAURENT (1977). La v a s c u l a r i s a t i o n branchiale chez l ' A n g u i l l e : a c t i o n r e s p a r i t i o n d'une resine polymerisable dans l e s d i f f e r e n t compartiments v a s c u l a i r e s . C R. Acad. Sc. P a r i s 284: 2011-2014. FISHER, T.R., R.F. COBURN and R.E. FORSTER (1969). Carbon monoxide d i f f u s i n g c a p a c i t y i n the bullhead c a t f i s h . J . Appl. P h y s i o l . 26(6): 161-169. FORSTER, M. E. (1976). E f f e c t s of adrenergic*blocking drugs on-the c a r d i o v a s c u l a r system of the e e l , A n g u i l l a a n g u i l l a ( L ) . Comp. Biochem. P h y s i o l . 55(IC): 33-36. FRONEK, K. and B.W. ZWEIFACH (1975). Mircovascular pressure d i s t r i b u t i o n i n s k e l e t a l muscle and the e f f e c t of v a s o d i l a t i o n . Am. J . P h y s i o l . 228(3): 791-796. FUNG, Y.C (1972). Biomechanics - i t s foundations and o b j e c t i v e s . p. 182 Pub. P r e n t i c e - H a l l Inc., New Jersey. FUNG, Y.C and S.S. SOBIN (1969). Theory of sheet flow i n lung a l v e o l i . J . Appl. P h y s i o l . 2_6: 472-488. FUNG, Y.C. and S.S. SOBIN (1972). E l a s t i c i t y of the pulmonary a l v e o l a r sheet. C i r c u l a t i o n Res. 3_0: 451-469. - 233 -FUNG, Y.C. and S.S. SOBIN (1977). Mechanics of pulmonary c i r c u l a t i o n i n "Cardiovascular Flow Dynamics and Measurements^: N Ed. N.B.C. Hwang and N.A. Normann. Pub. Uni. Park Press, Baltimore. p665-730. GANNON, B . J . ( 1 9 7 2 ) . Comparative and developmental, studies of autonomic nerves i n v i s c e r a l and c a r d i o v a s c u l a r systems. PhD d i s s e r t a t i o n , Univ. of Melbourne, A u s t r a l i a . GANNON, B.J. (1978). Methods of vascular c a s t i n g f o r scanning, e l e c t r o n microscopy. In: Advances .in o p t i c a l and e l e c t r o n microscopy V o l . 7. In Press. GANNON, B.J. and G. BURNSTOCK (1969). E x c i t a t o r y adrenergic i n n e r v a t i o n of the f i s h heart. Comp. Biochem. P h y s i o l . 29; 765-774. ; ' ' • GANNON, B.J. and G. CAMPBELL and D.J. RANDALL (1973). Scanning e l e c t r o n microscopy of vascular casts f o r the. study of v e s s e l connections i n a complex vascular bed - the t r o u t g i l l . Proc. 31st.E.M. Soc. Am.: 442-443. GESSER, H. ( 1977.). The e f f e c t s of.hypoxia arid reoxygenation on ;force of- development i n myocardia of carp and rainbow t r o u t : • p r o t e c t i v e e f f e c t s of CO2/HCO3. J . Exp. B i o l . 69: 199-206. GIRARD, J.P. and P. PAYAN (19 7 6). E f f e c t of epinepherine on • vascular space of g i l l s and head of rainbow t r o u t . Am. J . P h y s i o l . ' 230: 1555-1560. GLAZIER, J.B., J.M.B. HUGHES, J.E. MALONEY and J.B. WEST. (1969). Measurements of c a p i l l a r y dimensions and blood volume i n r a p i d l y frozen lungs. J . Appl. P h y s i o l . 26_: 65-76. GOW, B.S. (1972). The inf l u e n c e of vascular muscle on the v i s c o e l a s t i c p r o p e r t i e s of blood v e s s e l s . In:.Cardiovascular FluidiDynamics, V o l . 2. Ed. D.H. Bergel. Pub. Academic Press, New York.. GUYTON, A.C. and A.W. LINDSEY (1959). E f f e c t of ele v a t e d l e f t a t r i a l pressure and decreased plasma p r o t e i n concentration on the development of pulmonary edema. C i r c u l a t i o n Res. 7_: 649-657. ... HARGENS, A.R., R.W. MILLARD and K. JOHANSEN (1974) . High c a p i l l a r y p e r m e a b i l i t y i n fishes-. Comp. Biochem. P h y s i o l . 4_8: 675-680. HASWELL, M.S., S.F. PERRY and D.J. RANDALL (1978). The e f f e c t of perfusate oxygen l e v e l s on CO2 e x c r e t i o n i n the perfused " g i l l . J . Exp. Zool. 205: 309-314. HELGASON, S.S. and S. NILSSON (1973). Drug e f f e c t s on pre- and po s t - b r a n c h i a l blood pressure and heart rate i n a free swimming marine t e l e o s t , Gadus morhua.. Acta p h y s i o l . Scand. 88: 533-540. " 234 -HOLE TON, G.F. and D.J. RANDALL (1967a). Changes .in blood pressure i n the rainbow t r o u t during hypoxia. J . Exp. B i o l . 46(2): 297-306. HOLETON, G.F. and D.J. RANDALL (1976b). The e f f e c t of hypoxia upon the p a r t i a l presure of gases i n the blood and water a f f e r e n t and e f f e r e n t to the g i l l s of rainbow t r o u t . J . Exp. B i o l . 46(2) : 317-328. HOLMGREN, S. (1977). Regulation of the heart of a t e l e o s t , Gadus  morhua, by autonomic nerves arid c i r c u l a t i n g catecholamines. Acta p h y s i o l . Scand . 99; 62-74. HUGHES, G.M. (1972). The r e l a t i o n s h i p between cardiac and r e s p i r a t o r y rhythms i n the dog f i s h S c y l i o r h i n u s c a n i c u l a . J . Exp. B i o l . 5_7: 415-434. HUGHES, G.M. and G. SHELTON (1962). R e s p i r a t o r y mechanisms and t h e i r nervous c o n t r o l i n f i s h . Advan. Comp. P h y s i o l . Biochem. lz 13-29. Ed: 0. Lowenstein. Pub. Academic Press Inc., New York. HUGHES, G.M. and A.V. GRIMSTONE (1965). The f i n e s t r u c t u r e of the secondary lamellae of the g i l l s of Gadus p o l l a c h i u s . Q. J l . micros. S c i . 106: 343-353. HUGHES, G.M. and T. KOYAMA (1975). Gas Exchange of s i n g l e red blood c e l l s w i t h i n secondary lamellae of f i s h g i l l s . J . P h y s i o l . 24 6: 82P-83P. HUGHES, G.M* and S.F PERRY (1976). Morphometric study of tr o u t g i l l s : a l i g h t microscopic method s u i t a b l e f or the ev a l u a t i o n of p o l l u t a n t a c t i o n . J . Exp. B i o l . 64(2): 447-460. HYRTL, J . Beobachlungen aus den Gebiethe der vergleichenden G e f a f s l e h r e . Medizinische Jahrbucher Osterr Staates 24: 23 2-248. INTAGLIETTA, M. (1973). Pressure measurements i n the m i c r o c i r c u l a t i o n with a c t i v e and passive transducers. Microvas. Res. j>: 317-323. INTAGLIETTA, M., R.F. PAWULA and W.R. TOMPKINS (1970). Pressure measurements i n the mammalian microvasculature. Microvasc. Res. 2: 212-220. JONES, D.R. and D.J. RANDALL (1978) . The r e s p i r a t o r y and c i r c u l a t o r y systems during e x e r c i s e . In: Fish physiology Ed: W.S. Hoar and D.J. Ran d a l l , V o l . 7. Pub. Academic Press, New York p. 425-501. JONES, D.R., B.W. LANGILLE, D.J. RANDALL and G. SHELTQN (1974). Blood flow i n d o r s a l and v e n t r a l aortae of the cod, Gadus  morhua. Am. J . P h y s i o l . 226 (1) : 90-95. \ - 2 3 5 -KALEY, G. and BiM. ALTURA (1977). M i c r o c i r c u l a t i o n V o l . I. Pub. U n i v e r s i t y Park Press. KEMPTON, R.T. (1969). Morphological features of f u n c t i o n a l s i g n i f i c a n c e i n the g i l l s of the spiny d o g f i s h , Squalus acanthias. B i o l . B u l l , 136: 226-240. KENT, B. and E.C. PEIRCE I I (1975). Reflex c o n t r o l of g i l l blood flow i n Squalus ancanthias. In: " R e s p i r a t i o n of Marine Organisms". Ed: CECH, J . J . , D.W. BRIDGES, and D.B. HORTON. p. 183 -184. KENT, B and E.C. PEIRCE IT (1978). Cardiovascular responses to changes i n blood gases i n dogfish shark, Squalus ancanthias. Comp. Biochem. P h y s i o l , 60C: 37-44. KHAJUTIN, V.M. (1964). P h y s i c a l p r o p e r t i e s of v e s s e l s and vasomotor r e g u l a t i o n s . Ed: E.O. ATTINGER. Pub. McGraw-Hill, New York. p. 331-342. KICENIUK, J.W. and D.R.,JONES (1977). The oxygen tr a n s p o r t system i n t r o u t (Salmo g a i r d n e r i ) during sustained e x e r c i s e . J . Exp. B i o l . 69_: 24 7-2 60. KLAVERKAMP, J .V.-and D.C. DYER (1974). Autonomic receptors In rainbow t r o u t v a s c u l a t u r e . European J . Pharmacol. 28: 25-34. LAMPORT, H. and S. BAEZ (1962). P h y s i c a l p r o p e r t i e s of small a r t e r i a l v e s s e l s . P h y s i o l . Rev. Supp. 5_: 328-345. . LANDIS, E.M. (1934). C a p i l l a r y pressure and c a p i l l a r y p e r m e a b i l i t y . P h y s i o l . Rev. .14: 404-481. LAURENT, P. and S. DUNEL (1976). F u n c t i o n a l o r g a n i s a t i o n of the t e l e o s t g i l l . I. Blood pathways. Acta. Zool. 57: 189-209. LUNDVALL, J . and J . J . ARHULT (1974). Beta-adrenergic microvascular d i l a t i o n evoked by sympathetic s t i m u l a t i o n . Acta p h y s i o l . Scand. 92^ 572-574. LUTZ, B.R. and L.C. WYMAN (1932). Reflex cardiac i n h i b i t i o n of branchiovascular o r i g i n i n the elasmobranch Squalus  acanthias. B i o l . B u l l . 6_2: 10-16. McDONALD, D.A. (I960). Blood flow i n a r t e r i e s . Pub. W. and J . Mackay and Co. L t d . , Chatham. p. 255. v ' , <McHALE, N.G. and I.C. RODDIE (1976). The e f f e c t of transmural • pressure on pumping a c t i v i t y i n i s o l a t e d bovine lymphatic v e s s e l s . J . P h y s i o l . 261: 255-269. - 236 -McMASTER, P.O. and R.J. PARSONS (1938). The e f f e c t of the pulse on the spread of substances through t i s s u e s . J . Exp. Med. 68(1) : 377-40 0. MALONEY, J.E. and B.L. CASTLE (1969). Pressure-diameter r e l a t i o n s h i p of c a p i l l a r i e s and small blood vessels i n frog lung. Resp. P h y s i o l . ]_: 150-162. MAZEAUD, M.M., F. MAZEAUD, and E.M. DONALDSON. Primary and secondary e f f e c t s , of. s t r e s s i n f i s h : Some new data with a general review. Trans. Am. F i s h . Soc. 106(3): 201-212. MONRO, A. (1785). The s t r u c t u r e and physiology of f i s h e s explained and compared with those of man and other animals. Univ. of B r i t i s h Columbia s p e c i a l c o l l e c t i o n s . MORGAN, M. and P.W.A. TOVELL (1973). The s t r u c t u r e of the g i l l of the t r o u t , Salmo g a i r d n e r i . A. Z e l l f o r s c h 142: 147-162. MOTT, J.C. (1951). Some f a c t o r s a f f e c t i n g the blood c i r c u l a t i o n i n the common e e l (Angui11a a n g u i l l a ) . J . P h y s i o l . . 114: 387-398. MULLER, J . (1839). Vergleichende Anatomie der Myxinoiden I I I Uber das Gefassystem. Deutsche Academie der Wissenschaften zu B e r l i n . Abhandlumger.: 175-304. . " ' MURAKAMI, T. (19 71). A p p l i c a t i o n of the. scanning e l e c t r o n microscope to the study of the f i n e d i s t r i b u t i o n of blood v e s s e l s . Arch, h i s t o l . Jap. 3_2_: . 445-454. NAKANO, T. and N. TOMLINSON (1967). Catecholamine and carbohydrate metabolism i n rainbow t r o u t (Salmo g a i r d n e r i ) i r e l a t i o n to p h y s i c a l disturbance. J . F i s h . Res. Bd. Can. 24 1701-1715. NEWSTEAD, J.O. (1965). Fine s t r u c t u r e of the r e s p i r a t o r y lamellae of t e l e o s t g i l l s . Anat. Rec. 153: 393. NEWSTEAD, J.O. ( 1967). Fine s t r u c t u r e of the r e s p i r a t o r y lamellae of Teleostean g i l l s . Z. Zel l f o r s c h u n g 79: 396-428. NICHOL, J . , F. GIRLING, W. JERRARD, E.B. CLAXTON and A.C. BURTON (1951). Fundamental i n s t a b i l i t y of the small blood v e s s e l s and c r i t i c a l c l o s i n g pressures i n ' v a s c u l a r beds. Am. J . P h y s i o l . 164: 330-344. NICHOLAYSEN, G. and A. HAUGE (1977). Comparison of lung c a p i l l a r y f u n c t i o n at p u l s a t i l e and non p u l s a t i l e p e r f u s i o n . Proc. 27th I n t e r . Congr. P h y s i o l . Set., P a r i s . ' - 237 -NICHOLL, P.A. (1971). M.C.S. symposium on "The P r e c a p i l l a r y Sphincter". Microvasc. Res. 3: 426-427. NILSSON, S, T. ABRAHAMSON and D.J. GRQVE (1976). Sympathetic nervous c o n t r o l of adrenaline release from the head kidney of the cod Gadus morhua. Comp. Biochem. P h y s i o l . _5_5: 123-127. OSTLUND, E. and R. FANGE (1962). V a s o d i l a t i o n by adrenaline and noradrenaline and the e f f e c t s of some other substances on perfused f i s h g i l l s . Comp. Biochem. P h y s i o l . 5^ : 307-309. PARSONS, R.J. and P.D. McMASTER (1938). The e f f e c t of the pulse upon the formation and flow of lymph. J . Exp. Med. 68(1); 353-376. PAYAN, P. and J.P. GIRARD (1977). Adrenergic receptors r e g u l a t i n g patterns of blood flow through the g i l l s of t r o u t . Am. J . P h y s i o l . 232(1): H18-H23. PERMUTT, S. (1965). E f f e c t of i n t e r s t i t i a l pressure of the lung on pulmonary c i r c u l a t i o n . Med. thorac. 2j2: 118-131. PETERSON, L.H. (1962). P r o p e r t i e s and behaviour of the l i v i n g v a s c u l a r w a l l . P h y s i o l . Rev. Suppl. _5: 309-324. PIIPER, J . , D. BAUMGARTEN and M. MEYER (1970). E f f e c t s of hypoxia upon r e s p i r a t i o n and c i r c u l a t i o n i n the d o g f i s h , S c y l i o r h i n u s s t e l l a r i s . Respir. P h y s i o l . 3_0: 221-239. RANDALL, D.J. (1967). Sinus arhythmia and bradycardia i n f i s h r e s u l t i n g from deoxygenated wter passing over t h e . g i l l s . XXII I n t e r n a t . Cong. P h y s i o l . S c i . RANDALL, D.J. (1968). F u n c t i o n a l morphology of the heart i n f i s h e s . Am. Zool. 8: 179-189. RANDALL, D.J. (1976). G i l l s t r u c t u r e and f u n c t i o n : e f f e c t s of gas on ion exchanges. P h y s i o l o g i s t 19(3) . RANDALL, D.J. (1970). The C i r c u l a t o r y System. In: F i s h Physiology V o l . 4 Ed: "E.S. Hoar and D.J. Randall. Pub. Academic Press, New York' p. 132-172. RANDALL, D.J. and E.D. STEVENS (1967). The r o l e of adrenergic receptors i n c a r d i o v a s c u l a r changes associated with e x e r c i s e in. salmon. Comp. Biochem. P h y s i o l . 2\y 415-424. RANDALL, D.J. and G.F. HOLETON and E.D. STEVENS (1967). The exchange of oxygen and carbon d i o x i d e across the g i l l s Of 1 rainbow t r o u t . J . Exp. B i o l . 46(2): 339-348. - 238 -RANDALL, D.J., D. BAUMGARTEN and M. MALYUSZ (1972) . The r e l a t i o n s h i p between gas and ion t r a n s f e r across the g i l l s of f i s h e s . Comp. Biochem, P h y s i o l . 4_1: 629-637. RICHARDS, B.D. and P.O. FROMM (1969). Patterns of blood flow through f i l a m e n t s and lamellae of i s o l a t e d - p e r f u s e d rainbow t r o u t (Salmo g a i r d n e r i ) g i l l s . Comp. Biochem. P h y s i o l . 29: 1063-1070. RIESS, J.A. (1881). Der Bau der Kiemenblatter b e i den Knocherf ischen. Acrh.lv f u r Naturgeschichte ( U n i v e r s i t a t s Buchdruckenei). _47: 518-550. ROBERTS, J.L. (1975). C a r d i o - v e n t i l a t o r y i n t e r a c t i o n s during swimming, and during thermal and hypoxic s t r e s s . In: R e s p i r a t i o n of Marine Organisms. Eds: J.J> Cech, D.W. Bridges and D.B. Horton. p. 139-151 RUSZYAK, I . , M. FOLDI and G. SZABO (1967). Lymphatics and lymph c i r c u l a t i o n . Pub. Permagon Press, New York. SATCHELL, G.H. (1962). I n t r i n s i c vasomotlon i n the d o g f i s h g i l l . J . Exp. B i o l . 39: 503-512. SATCHELL, G.H. (1971). C i r c u l a t i o n i n f i s h e s . Pub. Cambridge U n i v e r s i t y Press, London. SCHEID, P. and J . PIIPER (1976). Q u a n t i t a t i v e f u n c t i o n a l a n a l y s i s of b r a n c h i a l gas t r a n s f e r : Theory and a p p l i c a t i o n to S c y l i o r h i n u s s t e l l a r i s (Elasmobranchii). In: R e s p i r a t i o n of Amphibious Vertebrates. Ed: G.M. Hughes. Pub. Academic Press, London. p. 17-38. SHELTON, G. (1970). Regulation of breathing. In: F i s h Physiology, V o l . 4 Ed: W.S. Hoar and D.J. R a n d a l l . Academic Press, New York. p. 293-359". SHEPHARD, R.B. and JiW. KIRKLIN (1969). R e l a t i o n of p u l s a t i l e flow to oxygen consumption and other v a r i a b l e s during cardiopulmonary bypass. J . Thorac. Cardiovasc. Surg. 58: 694-702. SHORT, S., P.J. BUTLER and E.W. TAYLOR (1977). The r e l a t i v e importance of nervous, humoral and . i n t r i n s i c mechanisms i n the r e g u l a t i o n of heart rate and stroke volume i n the dogfish S c y l i o r h i n u s c a n i c u l a . J . Exp. B i o l . 7_0: 77-92. SHOUKAS, A.A. (1977). C o n s t r i c t i o n of h y d r a u l i c cuff occluders fo r blood v e s s e l s . Am. J . P h y s i o l . 232(1) : H99-H100. SHUTTLEWORTH, T.J.(1972), A new Isolated-perfused g i l l p r e p a r a t i o n f o r the study of the mechanisms of i o n i c r e g u l a t i o n i n t e l e o s t s . Comp. Biochem. P h y s i o l . 43_: 59-64. - 239 -SHUTTLEWORTH, T.J. (1978). The e f f e c t of adrenaline on p o t e n t i a l s i n the i s o l a t e d g i l l s of the flounder P l a t i c h t h y s f l e s u s j . J . Comp. P h y s i o l . B. 124: 129-136. SKIDMORE, J.F. (1970). R e s p i r a t i o n and osmoregulation i n rainbow t r o u t with g i l l s damaged by z i n c sulphate. J . E x p . B i o l . 52: 481-494. SMITH,. D.G. (1976). PhD D i s s e r t a t i o n . Melbourne, A u s t r a l i a . SMITH, D.G. (1977). S i t e s of c h o l i n e r g i c v a s o c o n s t r i c t i o n i n t r o u t g i l l s . Am. J . P h y s i o l . 233: R222-R229. SMITH, F.M. and D.R. JONES (1978). L o c a l i z a t i o n of receptors causing hypoxic bradycardia i n t r o u t (Salmo g a i r d n e r i ) . Can. J . Zool. 56(6): 1260-1265. SOBIN, S.S.', H.M. TREMER and Y.C. FUNG (1970). Morphometric basis of the sheet-flow concept of the pulmonary a l v e o l a r m i c r o c i r c u l a t i o n i n the cat. C i r c u l a t i o n Res. 2j5: 397-414 . SOBIN, S.S., R.G. LINDAL and S. BERNICK. The pulmonary a r t e r i o l e . Microvas. Res. 1_4: 227-239. SOBIN, S.S., Y.C. FUNG, H.M. TREMER and T.H.. ROSENQUIST (1972). E l a s t i c i t y of the pulmonary a l v e o l a r microvascular sheet i n the cat. C i r c u l a t i o n Res.' _30: 440-450. STARLING, E.H. (1896). On the absorption of f l u i d s from the connective t i s s u e . J . P h y s i o l . 1_9: 312-326. STEEN, J.B. and A. KRUYSSE (1964). The r e s p i r a t o r y f u n c t i o n of t e l e o s t g i l l s . Comp. Biochem. P h y s i o l . 12^ : 127-142. STEVENS, E.D. and D.J. RANDALL (1967a). Changes i n blood pressure, heart rate and breathing rate during moderate . swimming a c t i v i t y i n rainbow t r o u t . J. Exp. B i o l . 46: 307-316. STEVENS, E.D. and D.J. RANDALL (1967b). Changes of gas concentrations i n blood and water during moderate swimming a c t i v i t y i n rainbow t r o u t . J . Exp. B i o l . _4_6: 329-338. STEVENS, E.D., G.R. BENNION, D.J. RANDALL and G. SHELTON (1972). Factors a f f e c t i n g a r t e r i a l blood pressures and blood flow from the heart i n i n t a c t , unrestrained l i n g cod, Ophiodon  elongatus. Comp. Biochem. P h y s i o l . B. j4_3: 681-^695. TAYLOR, E.W., S. SHORT and P.J. TAYLOR. The r o l e of the c a r d i a c vagus i n the response of the d o g f i s h , ( S c y l i o r h i n u s c a n i c u l a to hypoxia. J . Exp. B i o l . 7 J ) : 57-75. - 240 -VOGEL, W.O.P. (1978). Arteriovenous anastomoses i n the a f f e r e n t region of t r o u t g i l l f i l a m e n t s (Salmo g a i r d n e r i ) . Zoomorphologie 90_: 205-212. VOGEL, W., V. VOGEL, and H. KREMERS (1973). New aspects of the i n t r a f i l a m e n t a l vascular system i n g i l l s of a euryhaline t e l e o s t , T i l a p i a mossambica. Z. Z e l l f o r s c h 144: 573-583. VOGEL, W., V. VOGEL and W. SCHLOTE (1974). U l t r a s t r u c t u r a l study of arteriovenous anastomoses i n g i l l f i l a m e n t s of T i l a p i a mossambia. C e l l T i s s . Res. 155: 491-512. VOGEL, W., V. VOGEL and M. PFAUTSCH (1976). Arterio-venous anastomoses i n rainbow t r o u t g i l l f i l a m e n t s . C e l l T i s s . Res 167: 373-385. WAHLQVIST, I. and S. NILSSON (1977). The r o l e of sympathetic f i b r e s and c i r c u l a t i n g catecholamines i n c o n t r o l l i n g the blood pressure and heart rate i n the cod, Gadus morhua. Comp. Biochem. P h y s i o l . 5_7: 65-67. WEIBEL, E.R. (1963). Morphometry of the human lung. Pub. Academic Press, New York. WEIDERHIELM, C A . (1968). Dynamics of t r a n s c a p i l l a r y f l u i d exchange. J . Exp. P h y s i o l . 5_2: 29-63. WEIDERHIELM, C.A. and B.V. WESTON (1973). M i c r o v a s c u l a r , lymphatic and t i s s u e pressures i n the unanaesthetised mammal. Am. J . P h y s i o l . 224(4): 992-996. WEIDERHIELM, CA., J.W. WOODBURY, S. KIRK and R.F. RUSHMER (1964). P u l s a t i l e pressures i n the microvasculature of frog mesentery. Am. J . P h y s i o l . 207(1): 173-176. WEST, J.B. (1977). V e n t i l a t i o n / b l o o d flow and gas exchange. B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s . 3rd e d i t i o n . WOLF, K. (1963). P h y s i o l o g i c a l s a l i n e s f o r freshwater t e l e o s t s . Progr. F i s h . C u l t . 25: 135-140. WOOD, CM. (1974a). A c r i t i c a l examination of the p h y s i c a l and adrenergic f a c t o r s a f f e c t i n g blood flow through the g i l l s of the rainbow t r o u t . J . Exp. B i o l . 60: 241-265. WOOD, CM. (1974b). Mayer waves i n the c i r c u l a t i o n of t e l e o s t f i s h . J . Exp. Zool. 189: 267-273. WOOD, CM. (1975). A pharamacological a n a l y s i s of the adrenergi and c h o l i n e r g i c mechanisms of r e g u l a t i n g b r a n c h i a l vascular r e s i s t a n c e i n the rainbow t r o u t , Salmo g a i r d n e r i . WOOD, CM. (1977). C h o l i n e r g i c mechanisms and the response to ATP i n the systemic vasculature of the rainbow t r o u t . J . comp. P h y s i o l . ( B ) 122(3) : 325-345. _ 241 -WOOD, CM. and D.J. RANDALL (1973). The inf l u e n c e of swimming a c t i v i t y on sodium balance i n rainbow t r o u t (Salmo  g a i r d n e r i ) . J . comp. P h y s i o l . 8_2: 207-233. WOOD, CM. and J . J . RANDALL (1973b). The inf l u e n c e of swimming a c t i v i t y on water balance i n rainbow t r o u t (Salmo g a i r d n e r i ) . J . comp. P h y s i o l . 8j2: 257-276. WOOD, CM., B.R. McMAHON and D.G. MCDONALD (1978). Oxygen exchange and vasc u l a r r e s i s t a n c e i n the t o t a l l y perfused rainbow t r o u t . Am. J . P h y s i o l . 234(5): R201-R208. YAMAUCHI, A. and G. BURNSTOCK (1968). E l e c t r o n microscopic study on the i n n e r v a t i o n of the t r o u t heart. J . Comp. Neur. 132: 567-588.. ZWEIFACH, B.W. and H.H. LIPOWSKY (1977). Q u a n t i t a t i v e studies of m i c r o c i r c u l a t o r y s t r u c t u r e and f u n c t i o n . I I I . M i c r o v a s c u l a r hemodynamics of the cut mesentery and r a b b i t omentum. C i r c u l a t i o n Res. 41(3 ) : 380-390. - 2 4 2 -t APPENDIX I In Section I , I presented a number of r e s u l t s f o r c a l c u l a t i o n s of the r e s i s t a n c e to flow and the pressure drop i n g i l l f i l a m ent v e s s e l s . The values used i n those c a l u l a t i o n s are presented here. In equation (1), used f o r determining APlam' the' f o l l o w i n g values were used. n = 4.17 x 10 -^ cm H^O.min; k = 13.8; f =1.8; L =0.05 cm; S = 0.881; a\ = 7 x I O - 6 cm. H20 - 1; h a = 1.146 x 10~ 3 cm. The t o t a l l a m e l l a r area i s 6.9 x 10^ cm 2, but i t was assumed that at r e s t only 60% of the lamellae were perfused. Thus A = 4.14 x 10^ cm 2. In P o i s e u i l l e ' s equation (5) n =0.833 x 1 0 - 6 cm H^O.min, i . e . a blood v i s c o s i t y of 5 cp. For the c a l c u l a t i o n s on the a f f e r e n t filament a r t e r y c o r r e c t i o n s were made f o r v e s s e l taper and the l o s s of flow to branches. Vessel taper increases r e s i s t a n c e to flow and r e s i s t a n c e was c a l c u l a t e d from i t s inverse r e l a t i o n s h i p to radius to the fo u r t h power. The c a l c u l a t i o n s are presented i n Table X I I I and they i n d i c a t e that a taper of 10 to 30% approximately doubles r e s i s t a n c e . The AFA tapers to 10 to 30% of i t s basal diameter by a p o s i t i o n 60% of the way along i t s length ( F i g . 14). Equation (6) was developed from a simple e l e c t r i c a l analogue to p r e d i c t the percentage decrease i n v e s s e l r e s i s t a n c e due to a progressive l o s s of flow to branches. where n = the t o t a l number of l a m e l l a r u n i t s on a filament and m = the. number of l a m e l l a r u n i t s at any l o c a t i o n along the filament length. Using equation (6), a v e s s e l r e s i s t a n c e would be 50% lower than i f there were no flow l o s s to branches. The re p r e s e n t a t i v e geometry used f o r a f f e r e n t l a m e l l a r a r t e r i o l e s i n proximal, c e n t r a l and d i s t a l l o c a t i o n s was 1 •=• 600 u with r = lOu , 1 = 300 u with r = 8.5 u and 1 = 150 y and r = 7 y , r e s p e c t i v e l y . - 2 4 4 -TABLE X I I I The e f f e c t of taper on the r e s i s t a n c e of flow i n a v e s s e l , where r e s i s t a n c e i s i n v e r s e l y r e l a t e d to radius to the f o u r t h power. - 244a -TABLE X I I I % decrease i n r a d i u s (r) 0 : io 20 15 30 40 50 R e s i s t a n c e x l O 9 (= r1* ) 1 1.5 2.4 3.16 4.2 7.7 16 - 245 -APPENDIX I I Discussion of the use of the micropressure system Many accurate i n t r a v a s c u l a r and i n t r a l y m p h a t i c pressure measurements have been made with the micropressure system (See Sec t i o n I I f o r r e f e r e n c e s ) . The f i d e l i t y of the micropressure 4 system ( F i g . 45) used here was measured using c a l i b r a t i o n s against s t a t i c pressures developed by a column of water and a dynamic t e s t of frequency response using a "pop-test" (McDonald, 1960). I n i t i a l l y each i n d i v i d u a l p i p e t was c a l i b r a t e d and the frequency response t e s t e d . A f t e r many c a l i b r a t i o n s i t was e s t a b l i s h e d t h a t , provided the p i p e t t i p was not p a r t i a l l y or completely o b s t r u c t e d , a batch of micropipets with s i m i l a r t i p diameters had the same c a l i b r a t i o n and t h e i r frequency response was between 10 to 20 Hz. Therefore, i n l a t e r experiments only a re p r e s e n t a t i v e micropipet was c a l i b r a t e d from each batch of pi p e t s and the r e s t were v i s u a l l y examined f o r tip . s i z e and i n t e r n a l p a r t i c l e s . T y p i c a l l y i f a micropipet t i p was obstructed the system could not be n u l l e d . Such p i p e t s were discarded. A high y i e l d of unobstructed p i p e t s was obtained only i f a r i g i d p r o t o c o l of c l e a n l i n e s s was adhered to: scrupulous cleaning of the c a p i l l a r y tubing with ,a s e r i e s of concentrated a c i d s , methanol and d i s t i l l e d water; t r i p l e f i l t e r i n g of a l l s o l u t i o n s where p o s s i b l e and using dust covers, with a r e l a t i v e l y dust free room. - 2 4 6 -Source of measurement e r r o r The micropipet w i l l only record pressures i f the t i p i s unoccluded and on many occasions p i p e t s were discarded because they became blocked or p a r t i a l l y blocked with mucus or t i s s u e during the micropuneture. Only two types of pressures could, t h e r e f o r e , be recorded from the micropipet. Those due to a) blood pressures with the t i p i n a v e s s e l lumen and b) i n t e r n a l volume changes as a r e s u l t of bending of the p i p e t t i p . . Despite a l l my e f f o r t s to s t a b i l i s e the p r e p a r a t i o n , movements at the micro l e v e l were problematic. Several s t r i c t c r i t e r i a were, t h e r e f o r e , used to d i s t i n g u i s h between pressure a r t i f a c t s from bending and true blood pressures. Of the s e v e r a l hundred micropressure recordings made l e s s than 40 passed t h i s s c r u t i n y . I f there was the l e a s t doubt concerning a recording i t was r e j e c t e d . Some of the c h a r a c t e r i s t i c s of bending a r t i f a c t s are o u t l i n e d below. Mean pressure: The gre a t e s t blood pressure i n the g i l l f i lament could be no greater than the v e n t r a l a o r t i c (in vivo) or input ( i n v i t r o ) pressures. Bending could generate even higher pressures. Wave form: P u l s a t i l i t y i s l o s t as blood passes through the compliant g i l l bed. There i s al s o a c h a r a c t e r i s t i c shape to the v e n t r a l a o r t i c or input pressure pulse. Bending generated pulse pressures which were i n excess of a f f e r e n t pulse pressures and - 247 -which possessed a d i f f e r e n t wave shape. Often reversed saw-tooth shapes and very sharp t r i a n g u l a r shapes were seen as a r t i f a c t s . P a r t i c u l a r e f f o r t was made to s e l e c t micropressures with s i m i l a r wave forms as the input and output pressures. Note F i g . 17, p.116. Phasic r e l a t i o n s h i p s : Bending a r t i f a c t s were out of phase with true micropressures, e i t h e r leading a f f e r e n t or lagging behind e f f e r e n t pressures. The l o c a t i o n of the micropipet t i p was never v i s u a l i z e d during pressure measurements since the t i p s i z e approaches the r e s o l u t i o n of l i g h t microscopy.and a l l g i l l a r t e r i e s and a r t e r i o l e s are located deep i n the t i s s u e s . Post-experimental h i s t o l o g i c a l examination of filament micropuneture s uninformative due to the small, s i z e of the puncture, of the t i s s u e and l i m i t e d q u a n t i t i e s of dye that cou i n j e c t e d i n t o the t i s s u e from the micropipet. I t wa assumed that the micropipet was.in a lumen of a vess p u l s a t i l e pressure was being recorded and the a r t i f a p o s s i b i l i t i e s had been e l i m i n a t e d . But what v e s s e l in? F o r t u n a t e l y , the regu l a r arrangement of the f i l v a s culature allowed an answer. On e i t h e r the e f f e r e a f f e r e n t side of the filament there are' only two typ ve s s e l s : venolymphatic and a r t e r i a l . I made no a t t d i s t i n g u i s h between recordings made i n filament a r t e l a m e l l a r a r t e r i o l e s on e i t h e r the a f f e r e n t or e f f e r e the f i l a m e n t . I t was a l s o assumed that a r t e r i a l pre not be l e s s than output pressures. ite.s was the vastness Id be s, t h e r e f o r e , e l i f a ct was the pip e t ament nt or es of empt to r i e s and nt side of ssures could - 248 -Micropressure measurements were a l s o attempted on t r o u t g i l l s i n v i t r o and C-0 sole (Pleuronichthys coenosus), a f l a t f i s h , i n v i v o , but these experiments had no b e t t e r success than the experiments with l i n g cod. _ 249 _ FIGURE 45 A schematic r e p r e s e n t a t i o n of A. the fundamental p r i n c i p l e of the servo n u l l i n g micropressure measurements and B. the experimental setup (as taken from handbook, I.P.M., San Diego, Ca.). - 250. -APPENDIX I I I The t r a n s f e r f a c t o r i s a measure of the r e l a t i v e a b i l i t y of the r e s p i r a t o r y s u r f a c e to exchange gases, and i s a f f e c t e d by changes i n s u r f a c e area a v a i l a b l e f o r exchange, as w e l l as d i f f u s i o n d i s t a n c e between blood and water (Randall e t al'. , 1967). The oxygen t r a n s f e r f a c t o r T o 2 , i s d e f i n e d as M ° 2  T o 2 ~ Pc>2 d i f f e r e n c e between water and b l o o d D i f f u s i n g c a p a c i t y of the g i l l s i s estimated from morphometric. data on the secondary l a m e l l a e . D i f f u s i n g c a p a c i t y = K.A *h where A i s the t o t a l s u r f a c e area of a l l lamellae and X^ i s the harmonic mean t h i c k n e s s of the blood to water b a r r i e r . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094751/manifest

Comment

Related Items