AIR-BREATHING in the BOWFIN (Amia Calva L.) by MICHAEL SCOTT HEDRICK
AIR-BREATHING IN THE BOWFIN (Amia calva L.) by MICHAEL SCOTT HEDRICK
B.Sc. Lewis and Clark College, 1980 M.Sc. Portland State University, 1985
A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
in
THE FACULTY OF GRADUATE STUDIES (Department of Zoology)
We accept this thesis as conforming to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
September 1991
© Michael Scott Hedrick, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of ^ QO
The University of British Columbia Vancouver, Canada
DE-6 (2/88) ABSTRACT
The control of air-breathing in the bowfin, Amia calva, was investigated using experimental and analytical approaches.
The air-breathing pattern of conscious, undisturbed bowfin at
22+2 °C was characterized by the responses to changes in respiratory gases in the aquatic and aerial environments.
Pneumotachographic measurements of air flows during air- breathing events revealed two distinct patterns: in type I breaths exhalation was followed by inhalation; in type II air breaths, which have not been described for this species, only inhalation occurred. Under normoxic conditions both types of air breaths occurred (60% type 1:40% type II) and the mean inter-breath interval was 19.8+0.9 (95% C.I.) min. Aquatic or aerial hypoxia stimulated air-breathing, IBI decreased to about 13 min in both conditions, and there was a change in air-breathing pattern to predominantly type I air breaths
(>80% of total breaths). Maximum expired volume for type I breaths averaged 25.1+6.2 ml kg-1. Air bladder volume was 80 ml kg-1, so that about 30% of total air bladder volume was
exchanged during a type I breath. Bowfin exposed to 100% 02
in the aerial phase, regardless of aquatic P02, switched to type II air breaths almost exclusively (>99% of total breaths).
Air bladder deflation in conscious fish initially result• ed in only type II air breaths being taken. The time to initiate an air breath and the number of air breaths following deflation were both significantly dependent upon the volume removed from the air bladder. The results suggest that dynam- ic and static characteristics of air bladder mechanoreceptors are involved in the afferent limb of the type II breathing response and that type II breaths serve a buoyancy, rather than gas exchange, function.
Branchial denervation was used to test the hypothesis
that type I air breaths were stimulated by 02-chemoreceptors
located on the gills. Bowfin were either sham-operated (SH), partially-denervated (PD) or totally-denervated (TD) and exposed to aquatic normoxia and aquatic hypoxia. Air-breath•
ing frequency, measured as total breaths, increased from aguatic normoxia to hypoxia in all three groups; air-breathing frequency was significantly higher in the TD group. This was due, however, to large numbers of type II air breaths occur• ring between 0 and 1 min as a result of excessive loss of
inspired gas during inhalation. There was no significant difference in the frequency of type I breaths for any group when analyzed separately from type II breaths; thus, the afferent limb of the air-breathing response to hypoxia was not
identified, suggesting that extra-branchial sites for 02- chemorception may be involved. The results also indicate that either sensory or motor components of nerves IX and X to the gill arches are important for proper air-breathing function.
The role of central chemosensitivity was examined by perfusing a mock extra-dural fluid equilibrated with normoxic, hyperoxic, hypoxic or hypercapnic gas mixtures through the cranial space in conscious fish. Air-breathing was only stimu• lated by aquatic hypoxia, not changes in extra-dural fluid
composition, thus implicating peripheral.sites for 02-mediated
iii effects on aerial ventilation. Unfortunately, these results,
along with gill denervation data, do not yield any informa•
tion about the location of 02-chemosensitive sites or afferent pathways that modulate air-breathing in bowfin.
The temporal, intermittent pattern of air-breathing was
examined by spectral analysis. The intermittent pattern was
found to have significant, non-random frequency components. A
significant low frequency component, corresponding with a 30 min period, was found in the periodogram of 6 bowfin in nor- moxic conditions. In aquatic or aerial hypoxia, the dominant periods ranged between 5 and 10 min. The dominant periodici• ties in normoxia, or either hypoxic condition, were correlated with the mean inter-breath interval for type I breaths. Since type I breaths were affected by changes in external and/or
internal P02, the results indicate that air-breathing behavior
occurs periodically and may be driven by 02-sensitive chemore- ceptors.
A computer model was formulated to simulate the intermit• tent air-breathing pattern. The model used two independent thresholds for triggering type I and type II air breaths.
Type I air breaths were modeled as threshold responses to
reductions in intravascular P02. Type II air breaths were simulated as feedback responses to reductions in air bladder volume. Using empirical data from this study and other pub•
lished work, the model produced intermittent air-breathing simulations that closely resembled the responses of bowfin exposed to aerial normoxia, hypoxia and hyperoxia. Quantita• tive and qualitative similarities between the model and data
iv from bowfin suggest the model is realistic in its assumptions regarding mechano- and chemoreceptive inputs controlling intermittent air-breathing.
The results from this study indicate that bowfin normally use two types of respiratory behaviors that serve gas exchange and buoyancy functions. Intermittent breathing in this spe• cies is shown to be periodic, and the rhythmicity appears to
be generated by feedback from 02-sensitive chemoreceptors located in a position to monitor intra-vascular changes in
PO2.
v TABLE OF CONTENTS
ABSTRACT ii
LIST OF TABLES viii
LIST OF FIGURES ix
ACKNOWLEDGEMENTS xi
INTRODUCTION 1
The Evolution of Air-Breathing 4
Mechanisms of Aerial Ventilation
in Air-Breathing Fish 6
Physiological Control of Air-Breathing in Fish 9
Intermittent Breathing in Air-Breathing Fish 15
MATERIALS AND METHODS 2 0
Animals 2 0
Air-Breathing Behavior in Undisturbed Fish 20
Measurement of Air Flow and Expired Volume 24
Air Bladder Deflation and Inflation 29
Maximum Air Bladder Volume Measurements 3 0
Branchial Nerve Denervation 31
Intra-Cranial Perfusion 33
Spectral Analysis of the Intermittent Air- Breathing Pattern 3 8
Data Analysis and Statistics 42
RESULTS 4 3
Air-Breathing Rates and Behavior for
Undisturbed fish 4 3
Air Bladder Deflation and Inflation 62
Maximum Air Bladder Volume 72
Branchial Nerve Denervation 72
Intra-Cranial Perfusion 86
vi Spectral Analysis of the Intermittent Air- Breathing Pattern 93
DISCUSSION 101
Air-Breathing Patterns and the Responses to Changes in Aquatic and Aerial Gas Composition 101
Air Bladder Mechanoreceptors in the Control of Air-Breathing 110
Chemoreceptor Sites in the Control
of Air-Breathing 116
Peripheral Sites of Chemoreception 116
Central Sites of Chemoreception 123
Intermittent Air-Breathing in Amia 12 6
A Computer Model of Intermittent Air-Breathing in Amia 129 Results and Comparison with Data from
Undisturbed Amia 137
Evolutionary Implications 152
LITERATURE CITED 154
APPENDIX 1 165
APPENDIX 2 169
vii LIST OF TABLES
Table I. Mean inter-breath interval (min) for undisturbed Amia in various combinations of aquatic and aerial conditions 53
Table II. Mean inter-breath intervals (min) for sham-operat• ed, partial or total branchial denervate fish 83
Table III. Mean inter-breath intervals (min) for type I breaths only for branchial denervate groups 85
Table IV. Mean gill ventilation rates (fg) for branchial denervate groups 88
Table V. Mean values for gill ventilation (fg) and buccal pressure amplitude (Pb) and air-breathing during intra-cranial perfusion with mock EDF equilibrated with four different gas mixtures in aquatic normoxia or hypoxia 90
Table VI. Dorsal aortic P02, pH and PC02 and air-breathing rates during EDF perfusion in aquatic normoxia and hypoxia 9 2
Table Al. Summary of initial parameters used in the model of intermittent breathing 165
viii LIST OF FIGURES
Figure 1. Cladogram showing the relationship of Amia calva to other vertebrate groups 3
Figure 2. Schematic diagram of the set-up used to record air- breathing behavior and frequency in undisturbed Amia 23
Figure 3. Typical calibration records of manually-generated air flow measured with the pneumotachograph and pressure transducer system 27
Figure 4. Inter-breath interval as a function of time for a single fish in normoxia. Resolution of the time series of air-breathing into positive and negative delta functions for use in spectral analysis 40
Figure 5. Recordings of expiratory air flow from two fish in normoxic conditions. Type I air breaths and corresponding type II air breaths are from the same fish 45
Figure 6. Diagram of the sequence of events involved in a type I air breath 48
Figure 7. Diagram of the sequence of events involved in a type II air breath 50
Figure 8. Histogram of the distribution of inter-breath intervals (IBI; min) for eight fish in aquatic and aerial normoxia at 22+2 °C 55
Figure 9. Histogram of the distribution of IBI (min) for eight fish in aquatic normoxia and aerial hypercapnia (5% C02 in air) 57
Figure 10. Histogram of the distribution of IBI (min) for eight fish in aquatic hypoxia and aerial normoxia 59
Figure 11. Histogram of the distribution of IBI (min) for eight fish in aquatic normoxia and aerial hypoxia (8%
02) 61
Figure 12. Histogram of the distribution of IBI (min) for eight fish in aquatic normoxia and aerial hyperoxia (100%
02) 64
Figure 13. Histogram of the distribution of IBI (min) for eight fish in aquatic hypoxia and aerial hyperoxia (100%
02) 66
Figure 14. Relationship between the time taken to initiate an air breath (s) as a function of gas removed (ml kg-1) from the air bladder in aquatic normoxia, hypoxia and hyperoxia.... 69
Figure 15. Relationship between the number of air-breathing events in 10 min after air bladder deflation as a function of
ix volume removed (ml kg-1) 71
Figure 16. Relationship between air bladder volume (ml) and body mass (g) determined in vitro 74
Figure 17. Histogram of the distribution of IBI (min) for type I and type II breaths for sham-operated fish. A. Aquatic and aerial normoxia. B. Aquatic hypoxia/aerial normoxia...77
Figure 18. Histogram of the distribution of IBI (min) for partial branchial denvervate fish. A. Aquatic and aerial normoxia. B. Aquatic hypoxia/aerial normoxia 79
Figure 19. Histogram of the distribution of IBI (min) for total branchial denervate fish. A. Aquatic and aerial normox• ia. B. Aquatic hypoxia/aerial normoxia 81
Figure 20. Inter-breath intervals (IBI; min) plotted against cumulative time (min) for two fish in aquatic and aerial normoxia (A) and aquatic hypoxia/aerial normoxia (B) 95
Figure 21. A. Spectrum-averaged periodogram for 6 fish in aquatic and aerial normoxia and aquatic hypoxia/aerial nor• moxia and B. Average periodogram before spectrum averaging in normoxia 97
Figure 22. Spectrum-averaged periodogram for 6 fish in aquatic normoxia/aerial hypoxia (8% 02) 100
Figure 23. A schematic diagram of the essential features of the model used to simulate air-breathing in Amia 133
Figure 24. A. Simulated IBI (min) as a function of cumulative time (min) for the model with 0% error in the breaths. B. Simulated changes in efferent blood PO, (Torr) as a function of cumulative time (min) from model results 139
Figure 25. A. Simulated IBI (min) plotted as a function of cumulative time (min) in the model with ± 10% error in both types of breaths. B. Simulated changes in efferent blood P02 (Torr) as a function of cumulative time (min) 142
Figure 26. Periodogram generated from 10 random data sets from the model with + 12.5% error in both types of breaths 14 5
Figure 27. A. Plot of IBI (min) vs. cumulative time (min) for model data with simulated 100% 02 in inspired gas and + 15% error in both types of breaths. B. Plot of IBI vs time for a single fish exposed to 100% 02 in the aerial phase in aquatic normoxia 149
Figure 28. A. Plot of IBI (min) vs. cumulative time (min) for model data with simulated 8% 02 in inspired gas and + 10% error in both types of breaths. B. IBI vs. cumulative time for a single fish exposed to 8% 02 in the aerial phase....151
x ACKNOWLEDGEMENTS
There are several people I must acknowledge and thank for their support during my years in Vancouver. First, I thank my supervisor, David Jones, for introducing me to the "bald fish." Although the project took off in a direction that neither of us had anticipated, I think we can both be pleased with the outcome. I also thank Dave for his comments on various drafts of this thesis that were extremely helpful in improving its quality. Most importantly, however, I thank Dave for the many, many hours of great darts matches in which I believe I held the edge.
I am grateful to Dr. Chris Wood and Steve Munger, Dept. of Biology, McMaster University, for supplying most of the bowfin used in this study.
I thank the members of my auto-da-fe1, D.J. Randall, P.W. Hochachka, J.D. Steeves and W.K. Milsom, for not completely giving up on me when I gave them ample reason for doing so. I would also like to thank Bill Milsom for commenting on the penultimate version of this thesis.
I am grateful to several of my friends, including Hugo Bergen, Barry and Joanie McKeon, Heather Kirk and Bernhard Weber, for many hours of enjoyment away from the department. Also, my heartfelt thanks for the support of family and friends in the Rose City whom I did not see often enough while here in Vancouver: my parents, Gene and Judie Hedrick, Marcus "Farmer-of-the-Year" and Marilyn Simantel, the rest of the Hedrick and Simantel clans, Wayne Palioca, Debbie Duffield and Stan Hillman.
Several of my past and present lab-mates must take re• sponsibility for making life in the Jones' lab at times actu• ally enjoyable: Richard Stephenson, Molly Lutcavage, Peter Bushnell, Geoff Gabbott, Agnes Lacombe and Phil Davies. My special thanks to Claudia Kasserra for sharing office space, many laughs and for helping to improve my English on occasion- including the Introduction to this thesis.
I am greatly indebted to my good friend, Steve Katz, for literally hundreds of hours of intense discussions, bitch sessions and infotainment, all over the approximately 1000 liters of coffee we have drunk together over the past five years. I also thank Steve for helping with various aspects of my work including the spectral analysis of bowfin breathing, and for discussions and assistance that led to the formulation of the model of intermittent air-breathing presented in this thesis. Steve wrote the Turbo Pascal program for the inter• mittent air-breathing simulations that appears in Appendix 2. I also appreciate his valuable comments on parts of the Dis• cussion section of this thesis.
xi I also gratefully acknowledge the collaborative effort of Mark Burleson on the intra-cranial perfusion section of this the• sis. This work has been published in the Journal of Experi• mental Biology (Hedrick, Burleson, Jones and Milsom, 1991); however, the views expressed in this thesis are my own and do not necessarily agree with any of the co-authors.
Last - and certainly the most - I cannot thank my wife, Amy, enough for her love and support that has allowed me to complete this dissertation.
xii INTRODUCTION
The bowfin, Amia calva, is the only extant member of the subdivision Halecomorphi, the most advanced Actinopterygian
(ray-finned) fishes not included among the teleosts (Carroll
1988). The earliest known fossils of the genus Amia are from lower Cretaceous deposits, and the family Amiidae and closely related forms are known from the upper Jurassic (Boereske
1974). Anatomically, A. calva and its fossil congeners are characterized by a unique jaw articulation: the symplectic and quadrate bones both articulate with the lower jaw (Lauder and
Liem 1983). The current geographic distribution of A. calva is entirely North American and includes freshwater lakes and streams in the Great Lakes region (except Lake Superior), and most of the major river drainage systems in the Eastern United
States (Boreske 1974). Amia has long been known as a vora• cious predator and was much despised by fishermen around the turn of the century (Dean 1898), which probably accounted for its then popular name of "lake-lawyer."
Amia is in an important evolutionary position since it is considered to represent the primitive sister lineage of modern teleosts (Lauder and Liem 1983; fig. 1). Air bladder ventila• tory mechanisms in Amia are thought to represent the primitive condition of the Teleostei (Liem 1989). Thus, detailed knowl• edge of the mechanisms and control of air-breathing in this species may yield further insight into the evolution of aerial ventilation in teleosts and, perhaps, other Actinopterygian fishes.
1 Figure 1. Cladogram showing the relationship of Amia calva to selected extant vertebrate groups. Modified from Carroll
(1988) .
2 TELEOSTS Amia Gar Polvpterus
ACTINOPTER YGII SARCOPTERYGII
OSTEICHTH YES Wilder (1877) first reported that Amia used its vascular•
ized air bladder as a respiratory organ by demonstrating that
Amia had the ability to exhale and inhale atmospheric air.
There were few studies on Amia after Wilder's initial observa• tions until Reighard (1903) published the only detailed ac• count of the life history of this species. Reighard (1903) primarily described the breeding habits of Amia, but did note they often came to the surface for air. Later observations also confirmed the use of aerial respiration by bowfin in nature (Doan 1938), but it has not been demonstrated how the adaptation to air-breathing in Amia contributes to its ecolog• ical or evolutionary success (see Endler 1986) . Other studies have reported its ability to estivate during conditions of drought (Dence 1933, Neill 1950), with the gills modified to prevent collapse in air during this process (Bevelander 1938;
Daxboeck et al. 1981). Recent studies on Amia have focused primarily on the physiological aspects of gill ventilation and air-breathing (Johansen et al. 1970; Randall et al. 1981;
McKenzie 1990), and the morphological basis for aerial venti• lation (Deyst and Liem 1985; Liem 1988, 1989).
The Evolution of Air-Breathing
Air-breathing in fishes has evolved independently sever• al times (Gans 1970) . It is generally conceded that air- breathing originally evolved in a freshwater piscine ancestor
(Romer 1972), but a marine origin for air-breathing has also been proposed (Packard 1974). Current theory also suggests that lungs are more primitive structures than swim bladders,
4 and the original selective force for the evolution of lungs was probably the need for gas exchange in environments where
seasonal droughts and hypoxia were prevalent (Romer 1972). In
extant teleosts, the group from which most of the current
knowledge of air-breathing mechanisms is derived (Liem 1980,
1989), the diversity of structures used for air-breathing (see
Carter 1957; Johansen 1970) reflects secondary adaptations to hypoxic environments. The majority of teleosts, however, are
not air breathers and have retained a swim bladder, a homo-
logue of the original lung, strictly for buoyancy regulation
in the aquatic environment. In teleosts that have secondarily
evolved air bladders for respiratory gas exchange, the buoyan•
cy function of the air bladder has also remained (Liem 1989) .
Liem (1988) suggested that of the two major functions of the original piscine lung, hydrostatic and respiratory, selec• tion has operated to intensify the hydrostatic function of the
lung in the Actinopterygii, one of the two subclasses of bony
fishes (Class Osteichthyes). In this group there has been a
shift toward the need for buoyancy control in the aquatic environment, which has led to the evolution of non-respiratory
swim bladders in teleosts. However, in the extant primitive
air-breathing actinopterygians, Amia, gar {Lepisosteus spp.) and the chondrostean polypterids, air bladders or lungs have retained the dual functions of gas exchange and buoyancy.
These groups all rely to some extent upon aquatic gas exchange through gill ventilation, and the degree to which air-breath•
ing is used determines whether they are considered physiologi• cally, but not taxonomically, as faculative or obligate air-
5 breathers (Shelton et al. 1986). Amia and Lepisosteus are
generally considered faculative air-breathing fish since they
are not completely dependent upon aerial respiration for gas
exchange. Some air-breathing teleosts, such as the electric
eel, Electrophorus electricus, are highly specialized obligate
air-breathers (Johansen et al. 1968).
In the other subclass of bony fishes, the Sarcopterygii, however, the lineage from which all terrestrial vertebrates
descended, the respiratory gas exchange function of the lung has superceded the hydrostatic function (Liem 1988). There is
only one extant group of air-breathing sarcopterygians, the
lungfishes (Order Dipnoi) , with representive species found in
Africa, South America and Australia (Carroll 1988). Adult
African and South American lungfishes, Protopterus spp. and
Lepidosiren paradoxa, have reduced gills, and are thus com• pletely dependent upon aerial ventilation for oxygen ac• quisition. The Australian lungfish, Neoceratodus forsteri, however, is a faculative air-breathing fish with functional gills able to meet metabolic demands in addition to occasional air-breathing (Grigg 1965b; Johansen et al. 1967). The overall view of the evolution of air-breathing is that the primary selection pressure, regardless of phylogenetic histo• ry, was the need for oxygen acquisition in hypoxic environ• ments (Romer 1972; Randall et al. 1981).
Mechanisms of Aerial Ventilation in Air-Breathing Fish
In all air-breathing fishes using a lung or air bladder, a positive-pressure buccal force pump is used to ventilate the
6 gas exchanger (Gans 1970). A notable exception is the recent
finding that polypterid fishes ventilate their lungs with a unique aspiratory mechanism (Brainerd et al. 1989).
The aerial ventilation mechanics of the actinopterygians
Amia, Lepisosteus and a number of air-breathing teleosts have been extensively examined (Johansen et al. 1970/ Rahn et al.
1970; Kramer 1978; Ishimatsu and Itazawa 1981; Greenwood and
Liem 1984; Deyst and Liem 1985; Liem 1980, 1984, 1988, 1989).
Although details concerning mechanisms vary among species, the fundamental breathing sequence appears to be consistent: a double pulse buccal mechanism with exhalation preceding inha• lation, with the inhaled gas forced into the air bladder by the action of buccal musculature (Gans 1970; Liem 1989).
Exhalation and inhalation in these species are therefore active processes involving intra-pulmonary and buccal pressure gradients. The action of the buccal pump forcing air into the gas bladder distinguishes this mechanism as a positive-pres• sure pump, to contrast with aspiration breathing where air is drawn into an expanding lung by negative pressure (Gans 197 0;
Brainerd et al. 1989). Recent studies of the air-breathing mechanism in Amia (Randall et al. 1981; Deyst and Liem 1985) have, therefore, confirmed Wilder's (1877) original observa• tions that exhalation preceded inhalation in this species.
Liem (1989) has re-examined air-breathing in Amia and has suggested that ventilation may also occur as a passive event, without the active facilitation of air flow by the buccal cavity. Thus, an accurate picture of air-breathing mechanisms in Amia appears incomplete.
7 Aerial ventilation in lungfishes, while using a similar buccal force pump compared with actinopterygians, produces a somewhat different pattern of air transfer. McMahon (1969) reported that Protopterus first inhaled atmospheric gas by drawing air into the buccal cavity, exhaled by passive recoil of the lung through an open glottis, then forced the buccal air into the lung. This sequence of air flow may potentially mix inspired and expired gases. McMahon (1969) also reported that Protopterus occasionally inspired without expiring, and this occurred when lung pressure was held at atmospheric pressure. A similar sequence of air flow events has been found for Lepidosiren (Bishop and Foxon 1968). The mechanics of air-breathing in Neoceratodus have apparently not been examined in detail (Grigg 1965a). The major difference be• tween the ventilatory patterns of actinopterygian and sarcop- terygian fish appears to be in the sequence of air flow. In actinopterygians, exhalation nearly always precedes inhala• tion, while in sarcopterygians, inhalation may precede exhala• tion, with some mixing of inspired and expired air in the buccal cavity. The sequence of air-breathing events has been examined in detail only in Protopterus, but the variability reported for breathing mechanisms in lungfish would indicate further examination is probably necessary. The reasons for the basic differences in air flow patterns between actinopter• ygians and sarcopterygians are unknown, but probably reflect the divergent phylogenetic histories of these two osteich- thyean groups.
8 Physiological Control of Air-Breathing in Fish
Water and air, considered as respiratory media, have placed different demands and constraints on the evolution of gas exchange mechanisms and their control (Dejours 1975) .
Water has a lower capacitance for 02 than it does for C02 and fish using aquatic respiration must ventilate large volumes of
water to extract enough 02 to meet metabolic demands. The
relatively high ventilation rates for 02 extraction, and the
high solubility of C02 in water, result in low partial pres•
sures of C02 in the blood of fish; consequently, the control
of gill ventilation in fish is dominated by 02 rather than C02
(Shelton et al. 1986). Although some attention has been given
to the effects of C02 and pH on aquatic ventilation in fish
(Janssen and Randall 1975; Wood et al. 1990), a large body of
work supports the hypothesis that 02 exerts the dominant effect on control of ventilation in fishes (Shelton et al.
1986).
Almost without exception, aquatic hypoxia stimulates aerial ventilation in all air-breathing fish (see Carter 1957;
Johansen 1970; Shelton et al. 1986). This has been taken as strong evidence that the common selection pressure of environ• mental hypoxia was responsible for the independent development of air-breathing mechanisms in phylogenetically diverse groups of fishes. Fish that reportedly do not respond to aquatic oxygen levels are obligate air-breathing fish, such as the teleost, electric eel (Electrophorus; Johansen et al. 1968;
Farber and Rahn 1970) and the lungfishes, Lepidosiren (Johan• sen and Lenfant 1967) and Protopterus (Johansen and Lenfant
9 1968a), where metabolic demand is met entirely through aerial ventilation. In these species, the gills are morphologically
reduced, thereby limiting branchial 02 extraction or loss, but
C02 excretion continues through branchial routes owing to the
much higher solubility of C02 in water. In faculative air- breathers, such as Amia and Lepisosteus, that rely to varying
extents upon branchial 02 uptake, C02 elimination also takes place almost completely at gill respiratory surfaces; conse•
quently, the lung or air bladder respiratory ratio (C02 elimi• nation/02 uPtake) in air-breathing fish is usually less than
0.2, compared with a value of 1.0 in the lungs of completely terrestrial vertebrates (Shelton et al. 1986).
Although there is a vast literature documenting the physiological and behavioral (see Kramer 1987) responses of
air-breathing fish to changes in aquatic 02 concentrations, few studies have examined potential sites of control for these reflexes. Considerably more study has been devoted to the control of gill ventilation in strictly water-breathing fish.
Several lines of evidence from different species of water-
breathing fish suggests that 02-sensitive chemoreceptors are
located in or near gill vasculature, where 02 may be sensed from both aquatic and intravascular sites (Shelton et al.
1986). Aquatic hypoxia and chemical stimulants such as sodium cyanide (NaCN) applied to either or both locations stimulates gill ventilation (Saunders and Sutterlin 1971; Bamford 1974;
Eclancher and Dejours 1975; Daxboeck and Holeton 1977; Smith and Jones 1982; Burleson and Smatresk 1990a). Neurophysiolog- ical evidence also supports the hypothesis of branchial loca-
10 tions for chemoreception since afferent neural discharge,
responding to changes in P02 or pH has been recorded from cranial nerves IX (glossopharyngeal) and X (vagus) innervating the pseudobranch (Laurent and Rouzeau 1972), and the first gill arch in teleosts (Milsom and Brill 1986; Burleson and
Milsom 1990, Burleson 1991). Ablation or denervation of these sites, however, has generally failed to abolish the ventilato• ry responses to aquatic hypoxia (Hughes and Shelton 1962;
Saunders and Sutterlin 1971; Randall and Jones 1973). A recent study on catfish reports the abolition of gill ventila• tory responses to branchial denervation (Burleson and Smatresk
1990b): transection of all branchial branches of cranial nerves IX and X were required to abolish ventilatory reflex responses to aquatic hypoxia, indicating that there are sever•
al locations on the gills that convey 02-sensitive informa• tion.
The equivocal evidence in support of branchial chemosen- sitive sites for mediating hypoxic ventilatory reflexes in fish has led some authors to suggest that extra-branchial locations may also be involved. These include the venous vasculature, which may be branchial or non-branchial, for which there is limited evidence (Barrett and Taylor 1984), and the central nervous system (CNS) (Saunders and Sutterlin 1971;
Bamford 1974; Jones 1983). Chemosensitive sites in the CNS have been proposed primarily on the basis of failure to delin• eate peripheral sites, however, there is no evidence support• ing this hypothesis in water-breathing fish (Graham et al.
1990).
11 Branchial reflex responses to aquatic hypoxia vary be• tween air-breathing fish. For instance, in Lepisosteus (gar) gill ventilation increases initially with aquatic hypoxia, but
is depressed with further reductions in aquatic P02 (Smatresk and Cameron 1982a; Smatresk 1986). Johansen et al. (1970) reported a similar pattern for Amia, but a recent study sug• gests depression of branchial ventilation does not occur with
Amia in aquatic hypoxia (McKenzie 1990). In the polypterid reedfish, Erpetoichthys (=Calamoichthys) calabaricus, branchi•
al ventilation is also inhibited at low aquatic P02 (Pettit and Beitinger 1985), similar to the pattern in gar. The gill circulation in these fish is distal to the lung or air blad• der, so the inhibition of gill ventilation in hypoxia has been
interpreted as a mechanism to avoid branchial 02 loss through diffusion (Johansen et al. 1970; Smatresk and Cameron 1982a).
In air-breathing fish, there is some evidence that inter• nal and external chemosensitive sites modulate branchial and aerial ventilation. For instance, in the lungfish, Protopter• us , intra-vascular injection of sodium cyanide (NaCN) or hypoxic blood increased air-breathing rates that were attenu• ated, but not abolished, by branchial denervation (Lahiri et al. 1970). Recent evidence from spontaneously-breathing, anesthetized gar indicates that both external (aquatic) and internal (intravascular) chemosensitive sites mediate branchi• al and aerial reflexes (Smatresk et al. 1986). In gar, exter•
nal receptors respond to reductions in aquatic P02 by inhibit• ing gill ventilation and stimulating air-breathing; internal chemoreceptors, however, appear to stimulate both gill and
12 air-breathing reflexes. Smatresk (1986) also demonstrated similar responses using NaCN in conscious gar.
In several other air-breathing fish, external versus internal chemosensitive sites have been delimited non-inva- sively by independently manipulating aquatic and aerial gas concentrations. Although allowing fish to breathe hypoxic, hyperoxic or hypercapnic gases from the aerial environment has doubtful ecological significance, these manipulations can be useful in potentially separating aquatic and intravascular sites for the control of air-breathing. In general, aerial hypoxia stimulates, and aerial hyperoxia depresses, aerial ventilation in most obligate and faculative air-breathing fish
(Lahiri et al. 1970; Garey and Rahn 1970; Lomholt and Johansen
1974; Burggren 1979), suggesting that intravascular 02 chemo- receptors, at least, mediate some of the ventilatory responses to hypoxia. An exception was found with the air-breathing catfish, Brochis splendens, where air-breathing continued in
aerial hyperoxia (100% 02) at rates similar to those in aerial normoxia (Gee and Graham 1978). These results indicated that
Brochis maintained air-breathing in response to reductions in volume, and hence buoyancy, of the respiratory organ, owing to
greater losses of 02 by diffusion between breaths. Gee (1981) also found that the faculative air-breathing teleost, Umbra limi, used a number of strategies associated with air-breathing to coordinate the respiratory and hydrostatic functions of its swim bladder.
The two functions of an air bladder, respiratory and hydrostatic, introduce potential conflicts (Gee and Graham
13 1978). Any air-filled cavity used as a gas exchanger will automatically change the density and, therefore, buoyancy of the animal (Alexander 1966). To be effective as a gas exchang• er, an air bladder must permit oxygen to diffuse through its thin, vascularized membrane into the circulation. Oxygen lost through diffusion is generally not replaced by equal volumes of carbon dioxide (Johansen 1970), therefore, the volume of the organ decreases and the animal becomes negatively buoyant.
In order to remain neutrally buoyant, a fish must replace the lost volume either by secretion of gases into the air bladder or, if secretory mechanisms are too slow or not present, they may gulp air (Alexander 1966). Since efficient hydrostatic organs have low permeabilities to gas loss, they are not useful as gas exchangers. The swim bladders of teleosts are usually lined with guanine crystals that prevent significant amounts of oxygen diffusion (Fange 1976). Secretory mecha• nisms, which regulate buoyancy in physoclistous fishes (those without connections between the swim bladder and gut), are usually slow or absent in physostomous (those with connections between the air bladder and gut) fishes (Jones and Marshall
1953). In most physostomous teleosts, therefore, air-breathing is essential for buoyancy regulation (Jones and Marshall
1953) .
There is evidence in primitive fishes that air bladders or lungs have a hydrostatic, in addition to respiratory gas exchange, function. In Amia, Lepisosteus and Protopterus, lung deflation stimulates, and inflation inhibits, air-breath• ing reflexes (Johansen et al. 1970; Babiker 1979; Smatresk and
14 Cameron 1982b; Pack et al. 1984). The air bladders or lungs of these fishes contain slowly-adapting and/or rapidly adapting mechanoreceptors with afferents in the vagus nerve (DeLaney et al. 1983; Milsom and Jones 1985; Smatresk and Azizi 1987).
These mechanoreceptors respond to both dynamic and static changes in lung volume; afferent discharge increases with inflation while deflation reduces afferent discharge (Milsom
1990) . Increasing lung pressure, and presumably afferent mechanoreceptor discharge, in Protopterus has also been shown to increase the inter-breath interval (Pack et al. 1990), suggesting that pulmonary mechanoreceptors influence the breathing pattern. The mechanoreceptor-related reflexes in air-breathing fish are similar to the inspiratory-inhibiting, expiratory-facilitating Hering-Breuer reflexes in mammals
(Pack 1981).
Intermittent Breathing in Air-Breathing Fish
Air-breathing in fish requires intermittent excursions to the water's surface for aerial ventilation. The control over the timing of intermittent air breaths in fish, and other intermittently-breathing vertebrates, has received considera• ble attention in recent years (see Shelton et al. 1986; Sma• tresk 1990; Milsom 1991, for reviews). It has been suggested that intermittent air-breathing in fish represents an "on- demand" phenomenon, dependent only upon peripheral afferent feedback from receptors for its initiation (Smatresk 1990).
Afferent feedback from chemoreceptors and mechanoreceptors are therefore thought to play dominant roles in determining the
15 timing of air-breathing events in intermittently-breathing vertebrates (Shelton et al. 1986). The ability of aquatic and
aerial 02 concentrations and lung volume manipulations to markedly affect inter-breath intervals, regardless of uncer• tainties about receptor locations, clearly illustrates the role of receptors in modifying intermittent breathing pat• terns .
Intermittent breathing contrasts with the near-continuous rhythmic ventilatory patterns of fish and mammals that occur under normal conditions (Milsom 1991). In both groups, stud• ies have shown that rhythmic patterns can be generated from respiratory-related neural discharges from brainstem struc• tures in vitro, in the complete absence of afferent feedback
(Rovainen 1974; Suzue 1984). Although the precise mechanisms accounting for rhythmogenesis in these phylogenetically dispa• rate groups have not been determined, it has been suggested that a central rhythm generator driven by pacemaker cells
(Rovainen 1977; Ballintijn 1982; Feldman et al. 1990) or neural network interactions (Richter et al. 1986) in the brainstem are responsible. A key question with regard to intermittently-breathing vertebrates, then, is whether the observed patterns are initiated from the central nervous system, as in water-breathing fish or mammals, or solely from feedback by peripheral chemo- and mechanoreceptors. Recent evidence from bullfrog tadpole (Walker et al. 1990) and turtle
(Douse and Mitchell 1990) in vitro brainstem preparations has shown that intermittent, respiratory-related neural discharge can occur without sensory input, suggesting that intermittent,
16 or episodic, fictive breathing patterns can be endogenously generated by CNS structures in the brainstem of these animals.
Evidence from actinopterygian air-breathing fish, however, is completely lacking, so it is not possible at present to deter• mine whether intermittent ventilatory patterns are centrally or peripherally generated in this group.
In humans, there is considerable evidence that the con• tinuous breathing pattern contains several frequency compo• nents that are revealed by spectral analysis (see van den
Aardweg and Karemaker 1991). Goodman (19 64) first demonstrat• ed significant oscillations in the breathing pattern of humans with frequencies in the range of 2-3 min to several hours.
Hlastala et al. (1973) found significant frequencies with periods up to 28 min in the frequency spectra of resting hu• mans; the peaks in the frequency specta were correlated with variations in a number of respiratory parameters. Studies from humans illustrate that, in spite of near-continuous ventila• tion, underlying rhythmicities in the ventilatory pattern occur under normal conditions.
Although the intermittent patterns of air-breathing fish have often been termed irregular, a few studies have subjec• tively noted regular intervals between air breaths when'fish are undisturbed (see Milsom 1991). Disturbances, such as simulated predators, can markedly alter any regularities in air-breathing rates (Gee 1980; Smith and Kramer 1986). Howev• er, there is no quantitative evidence to show whether inter• mittent breathing patterns are indeed regular. Most studies have observed air-breathing events over rather short (30 min to 2 h) time periods, which is probably insufficient for regular breathing patterns, if they occur, to become estab• lished.
The purpose of this thesis was to examine various as• pects of the control of air-breathing in the bowfin. A com• bined approach of non-invasive, invasive and analytical tech• niques were used in this investigation. In the first part of this study, breathing patterns and the reflex responses to changes in respiratory gases in the aquatic and aerial envi• ronments were examined using non-invasive techniques. This was done using long-term (8 h) videotaped recordings of air- breathing in Amia. Air flow during air-breathing events was measured by pneumotachography to resolve questions concerning aerial ventilatory mechanisms. Spectral analysis, which has been used extensively in studies of human respiratory pat• terns, was used to determine whether intermittent breathing patterns in Amia are rhythmic or simply random.
An invasive approach was used to examine potential sites of control for air-breathing patterns. Both peripheral and central nervous system sites for the control of air-breathing were examined. Peripheral control of air-breathing was exam• ined in two ways. First, the cranial nerve innervation to the gills of bowfin was eliminated to test the hypothesis that ventilatory reflex reponses to aquatic hypoxia are mediated by
02-chemoreceptor sites on the gills. Secondly, the role of pulmonary mechanoreceptors in air-breathing was examined by manipulating the air bladder volume in conscious fish. Cen-
18 tral chemical sites for air-breathing were examined by chang• ing the chemical compostition of the extradural fluid sur• rounding the brain.
Finally, the empirical data obtained from bowfin in this study and from other sources were used to formulate a computer model to simulate intermittent air-breathing in Amia.
19 MATERIALS AND METHODS
Animals
Bowfin were netted by commercial fishermen in Lake Ontar• io and air-freighted to the University of British Columbia.
There was no mortality during shipment and the fish appeared to be healthy. The fish were kept in large circular fiberglass tanks in continuously running dechlorinated water at 6-15 °C on a 12:12 L:D cycle. The fish were not fed during winter months when ambient water temperature was low (4-6 °C) , but were occasionally fed live goldfish during spring and summer when water temperature was higher and the fish were more active. Bowfin were not fed for at least two weeks before any experiments began.
Air-Breathing Behavior in Undisturbed Fish
Eight bowfin, ranging in size from 246 g to 940 g (Mean
Mass = 503 g), were brought into the laboratory and placed individually into 40 L plastic bins containing aerated water
at the same temperature as the holding tank (Tw= 6-10 °C).
The fish were acclimated to room temperature (22 + 2 °C) by allowing the water to warm slowly overnight. The water was continuously aerated during acclimation. Depending on the initial temperature, equilibration with room temperature took from 12 to 24 h. After 4-5 days at room temperature, the fish were transferred to a 68 L rectangular aquarium (60 cm X 3 0 cm
X 38 cm deep) filled with water pre-equilibrated to room
20 temperature. The surface of the aquarium was covered with a perspex barrier containing several small (1 cm dia.) holes, and one large hole (either 14 cm dia. or 10 cm dia.) through which the fish could breathe gases. A diagram of the experimetal set-up is shown in figure 2. An inverted funnel (vol.= 650 ml or 300 ml) with a pneumotachograph (Fleisch) attached at the top was used to record air flow changes during air-breathing events. The pressure drop across the pneumotachograph was measured with a differential pressure transducer (Validyne, model DP103-18). A constant gas flow of 200 ml min-1, regu• lated by precision gas flow meters, was delivered through the funnel. The constant gas flow resulted in a voltage offset that was manually readjusted to zero on the carrier demodula• tor of the pressure transducer. The differential pressure signal was fed through a voltage-frequency converter (A.C.
Vetter, Inc., model 2D) and stored on the audio track of a JVC
(model TU-S2U) video cassette recorder. Each animal's breath• ing behavior was recorded with a video camera (JVC) and also stored on the video tape. Each recording session was 8 h and most sessions were recorded between 1800h and 0600h to mini• mize numerous vibrational disturbances that occurred during daytime. After a recording session, the tape was replayed and air-breathing rates were counted directly from the video tape.
Inter-breath interval was recorded to the nearest minute using a digital clock displaying real time in view of the camera.
Voltage signals from the pressure transducer were played back through the frequency-voltage converter at this time and displayed on a storage oscilloscope (Tektronix, model 5113).
21 Figure 2. Schematic diagram of the set-up used to record air- breathing behavior and frequency in undisturbed Amia. See
Methods for details. Abbreviations: PT, Pressure Transducer;
Freq.-Volt., Frequency to Voltage Converter; VCR, Video Cas• sette Recorder.
22 VCR 1 1 Freq. - Volt. The air flow changes that occurred with breathing events were examined along with the breathing behavior recorded on the video tape. The voltage signals were transferred to a chart recorder (Gould, model 220) writing on rectilinear coordi• nates .
Measurement of Air Flow and Expired Volume
The pneumotachograph was calibrated by adjusting the air flow through the funnel from 0 to 9 L min-1 and recording the voltage change. The voltage change was linearly related to air flow over the range encountered in this study. Air flow generated by the fish during breathing events was played back through the oscilloscope and the voltage converted to air flow
(ml s-1).
The volume of each breath was measured by direct integra• tion of the air flow traces, similar to the techniques of
Glass et al. (1983), Boutilier (1984) and Funk et al. (1986), but with some modifications. It has been shown that measure• ment of breath volumes by integration of pneumotachographic signals can overestimate actual volumes, owing to inertial effects, especially at high air flow rates (Ohya et al. 1988).
Tidal volumes were, therefore, calibrated in the following manner: known volumes of air were injected manually through the funnel with a 30 ml plastic syringe to mimic the expected expired tidal volumes of the fish. The syringe barrel was modified by cutting off the standard luer-lock tip and replac• ing it with a wide bore (0.5 cm) plastic connector. This arrangement minimized inertial effects resulting from inject-
24 ing air through a narrow orifice. For a given volume, air was injected at different rates and the resulting pneumotachograph signal recorded on video tape as described above; thus, the calibration was performed using the same procedures as those used during recording of the fishes' breathing behavior.
Injecting air at different rates resulted in variable time
intervals (T E) before air flow returned to baseline. A typi• cal calibration series for 2 to 10 ml injected air is shown in figure 3. It is clear from the calibration traces that manu• ally injecting air resulted in oscillations that returned to baseline after approximately 200 ms. The oscillatory nature of the calibrated volumes was a function of changes in the water level after injecting air, since these oscillations were absent when the funnel-pneumotach arrangement was tested on a solid surface. Each flow profile was integrated from the recorder chart by measuring the area under the curve (shaded areas in fig. 3) with a digitizing tablet (Jandel Scientific) and associated software (Sigma Scan). For each calibration volume, area under the flow curve (cm ) was plotted against
T'e (ms) to yield a series of calibration curves. Expired air flow traces for each fish were integrated in the same fashion
(see Results, fig. 5), and the expired volume estimated to the nearest 0.5 ml by fitting the resulting area and expiratory
i
interval (TE) to the nearest value for area and T E.
Each fish was given one day to become accustomed to the aquarium and the breathing funnel. On the second day, video taping of the air-breathing behavior began with the fish in normoxic water and air passing through the funnel. The water
25 Figure 3. Typical calibration records of manually-generated air flow measured with the pneumotachograph and pressure transducer system. Known volumes (2 ml to 10 ml) of air were
injected manually with a syringe and measured by integrating
i
the area under each flow curve (shaded areas) . T E was de•
fined as the initial time for positive flow to return to baseline. The baseline oscillates for approximately 200-300 ms after the injection. Note the change in scale for the 8 ml and 10 ml volumes.
26 VOLUME (ml)
4 6 8 10
to 100 ms was continuously aerated to maintain a partial pressure of oxygen above 140 Torr (normoxia). Water samples from the aquarium were injected onto a Radiometer oxygen electrode
(E5046), maintained at the same temperature as the aquarium, and measured with a Radiometer (model PHM 71) oxygen meter.
Water temperature and P02 were taken at the beginning and end of each recording session. Water samples taken at various
locations in the aquarium revealed no differences in P02 or
temperature. There were no detectable levels of C02 in the water. Several recordings were made for each fish in a variety of combinations of aquatic and aerial gas concentrations. The gas composition of the aquatic or aerial phases was changed by mixing combinations of gases with the flow meters, or with precision gas mixing pumps (Wosthoff) prior to being delivered through either the aquatic or aerial phase. In all, the fish were exposed to seven combinations of aquatic and aerial conditions, and are referred to in the text with the aquatic followed by the corresponding aerial condition: (1)
Normoxia/Air, (2) Normoxia/Hypercapnia, (3) Hypoxia/Air, (4)
Normoxia/Hypoxia, (5) Normoxia/Hyperoxia, (6) Hypoxia/Hyperox-
ia and (7) Hyperoxia/Air. Aquatic P02 (Pw02) in normoxia was
n 140 in hypoxia/hyperoxia was 50.0+4.0 Torr. The PC02 of aerial hypercapnia (5% C02 in air) and P02 of aerial hypoxia (8% 02 bal. N2) was confirmed with Radiometer C02 and 02 electrodes, respectively. The electrodes were maintained at the same temperature as the fish (22 + 2 °C) and calibrated with pre• cisely-mixed gases from a Wosthoff gas mixing pump (C02 elec- 28 trode) or air-saturated water and a zero P02 solution (02 electrode) or N2~equilibrated water. 100% oxygen was used for aquatic or aerial hyperoxia and the resulting P02 in either phase measured with the 02 electrode. The water was changed every 5-7 days with dechlorinated water pre-equilibrated to room temperature. After each water change, fish were returned to control conditions (normoxic water and air). The fish showed no apparent ill effects from disturbance due to this procedure and there was no correlation between air-breathing rates and water change of the aquarium. Air Bladder Deflation and Inflation Four bowfin (mean mass=348 g) were brought into the laboratory and acclimated to room temperature (22 + 2.0 C) as described above. Following acclimation, fish were anesthe• tized initially in 1:7,500 buffered tricaine methanesulfonate (MS 222; Sydell Laboratories) until gill ventilatory movements ceased, transferred to a surgical table where the gills were artificially ventilated with oxygenated, dechlorinated water (22 °C) containing a lower concentration of anesthetic (1:20,000). The glottis was located in the dorsal gut wall and held open with forceps. A 50 cm length of PE 90 tubing with a flared end containing side holes to minimize blockage was placed approximately 3-5 cm into the lumen of the air bladder. The cannula was secured to the palate with two sutures (0 silk) and passed through a PE 200 grommet that had been placed through the frontal bone into the buccal cavity. The outside diameter of PE 50 is 0.965 mm, while the diameter 29 of the glottal aperture in Amia of the size used in this study was about 5 mm (pers. obs.); therefore, cannulating the air bladder by this method would be expected to occlude the glot• tis by less than 5%. Each fish was allowed to recover for 24 h in an aquarium contining aerated water. After recovery, deflations and inflations, of various volumes were done with the fish in normoxic (Pw02=155 Torr), hypoxic (Pw02=55 Torr) and hyperoxic (Pw02=270) water over 2-3 days. Air bladder volume manipulations were done with as little disturbance as possible to the fish. Air-breathing behavior was observed for 10 min, with the experimenter out of view of the fish, follow• ing each deflation. At least 30 min was allowed between trials. During the 10 min observation period, the time taken for the fish to initiate an air-breath following deflation, the number of air-breaths taken during the 10 min observation period and types of air-breaths were recorded. Maximum Air Bladder Volume Measurements Six bowfin ranging in mass from 217 g to 1201 g were over-anaethetized in a concentrated solution (1.5 g/L) of MS- 222. After 2-3 hours, the fish were weighed to the nearest 0.1 g with a triple beam balance and then opened ventrally and the lung carefully dissected from the surrounding musculature and connective tissue. A 1 ml syringe tube, cut in half, was placed through the glottis into the lumen of the air bladder and tied in place with a short length of 0 silk. The outside end of the tube was connected to an air source and the air bladder was slowly inflated to maximum distension, taking care 30 not to rupture the lung wall. The inflated air bladder was then grasped at the bottom with long forceps and immersed completely in a 1500 ml Erlynmeyer flask filled to the top with a 0.7% saline solution. The immersed air bladder dis• placed the saline which flowed over the top of the flask and was collected in a plastic container. The container was weighed to the nearest 0.1 g before and after collecting the displaced saline. The mass of saline displaced by the forceps and deflated air bladder were subtracted from mass displaced by the inflated air bladder. The difference in mass repre• sented the volume displaced by the inflated air bladder. The procedure was repeated three times and the average mass of displaced saline taken as air bladder volume. Branchial Nerve Denervation A total of 16 bowfin (Mean Mass = 433 g) were brought into the laboratory and anesthetized. These fish were not acclimated to room temperature before surgery. Following the experiment, the fish were over-anesthetized with MS-222 for post-mortem dissection (see below). The branchial (gill) branches of cranial nerves IX (glossopharyngeal) and X (vagus) were exposed by lifting the operculum and cutting the thin membrane lying medial to the gills. The vagus nerve innervates all four branchial arches, while the glossopharyngeal nerve innervates only the first gill arch and pseudobranch. In some fish, the pseudobranch is also innervated by cranial nerve VII (facial) (Nilsson 1984); however, it is not known whether the facial nerve innervates 31 the pseudobranch in Amia. Attempts to locate a facial nerve innervation to the pseudobranch by dissection were unsuccess• ful. The nerves were identified, carefully separated from surrounding fascia, and cut with scissors. Bleeding was con• trolled with cotton applicators. Following the operation, the membrane was closed with 5-0 silk sutures. The entire opera• tion lasted between 30-60 min and the fish were allowed to recover in oxygenated dechlorinated water until spontaneous gill ventilatory movements returned, and the fish had fully righted itself. Sham-operated control animals were treated in the same manner, but the nerves were not cut. The operation and initial recovery were performed at the temperature to which the fish were acclimated (4-6 °C); the fish were then put into holding tanks at the same temperature and allowed to recover for at least 3 weeks. After the minimum 3 week recovery period, fish were brought back into the laboratory and allowed to acclimate to room temperature for five days. Fish were placed individually into aquaria with aerated, dechlorinated water at 22 + 2°C. Air flowed through the breathing funnel at 200 ml min-1. Videotaping began on day 2 with the fish in normoxic water (PwO2>140 Torr). On day 3, the aquarium water was bubbled with a mixture of nitrogen and air to lower Pw02 to 49.0+1.0 Torr. When the desired P02 was reached, after 2-3 hours, the videotape was started. On day 4, a second normoxic treatment was recorded on videotape and on day 5 the fish was over- anaesthetized with MS-222. After each recording session, air- breathing events and, whenever possible, gill ventilatory 32 rates, were counted directly from the videotape. The efficacy of each surgical operation was carefully checked by post• mortem dissection. Branchial nerves were identified and compared with published anatomical descriptions (Allis 1897). Fish were placed into one of three categories based on the results of the post-mortem dissection: Partially-denervated (PD) fish were those in which nerve re-growth was indicated or if all branchial branches of nerves IX and X were not sec• tioned (5 fish); sham-operated (SH) controls were those in which no nerves were cut (4 fish); total branchial denervates (TD) were those fish in which all branchial branches of cranial nerves IX and X were clearly sectioned (3 fish), or if all branchial branches plus the branch of cranial nerve IX inner• vating the pseudobranch (psb) was also sectioned (4 fish; for a total of 7 fish in this category). The study was blind in that the results of the videotaped breathing patterns could not be correlated with any particular category until after post-mortem dissection. Intra-Cranial Perfusion A total of eight bowfin were used to examine the effects of intra-cranial perfusion with mock extradural fluid (EDF) on branchial and aerial ventilation. Fish were initially anes• thetized as described in the preceding section. The dorsal aorta was cannulated with PE 50 tubing using a canine catheter placement unit as described by Smatresk and Cameron (1982). A PE 160 cannula was implanted in the buccal cavity through the nasal bone to monitor ventilation. 33 Each fish was prepared for cranial perfusion by drilling two small holes in the mid-saggital plane of the cranium using a dental drill. The first hole was drilled at an angle 10-15° from perpendicular, and 2-5 mm from the posterior margin of the parietal bone. A second hole was drilled, at approximate• ly the same angle as the first hole, in the frontal bone about 10 mm anterior to the first hole. Fluid samples were taken at this time with a syringe and placed into capillary tubes for later ion analysis. The fluid was extradural fluid (EDF), and not true cerebrospinal fluid (CSF), since it was sampled from the meningeal space (Davson 1967). Erythrocytes were present in some EDF samples and was subsequently removed by centrifu• gation. Perfusion cannulae made from the shanks of 18 gauge stainless-steel hypodermic needles, approximately 15 mm to 20 mm long, were implanted in the cranial holes. The steel tubing extended down into the meningeal space about 10 mm in the posterior (inflow) port and 6 mm in the anterior (outflow) port. It was found in preliminary experiments that this configuration situated the inflow tube about 2 mm above the roof of the fourth ventricle and the outflow tube above the cerebellum. Patency of the perfusion arrangement was checked by perfusing mock EDF with a syringe connected to the inflow tube. Animals in which flow could not be freely maintained, or in which there was excessive bleeding, were not used in the experiment. Once patency was ensured, the perusion tubing and meningeal space were filled with mock EDF and plugged. The fish were subsequently transferred to a darkened perspex box with continuously flowing, normoxic water. The box had a 34 forward air space to allow air-breathing. Surgery seldom required longer than 20 min to complete and all fish recovered quickly after withdrawal of the anesthetic. EDF concentrations of Na+ and K+ were determined by flame photometry (IL model 143); Cl~ concentration was determined with a Buchler digital chloridometer. Experiments began after a 24 h recovery period and were conducted at water temperatures between 14 and 16 °C. The temperature throughout a single experiment did not vary by more than 0.5 °C. Mock EDF was freshly made with the follow- -i ing composition (all concentrations in mmol 1 ) : NaCl (120) , KCI (4.0), MgS04 (1.0), CaCl2 (1.0) and NaHC03 (10.0). This fluid was placed into 500 ml bell jars which were maintained at the same temperature as the fish throughout the experiment. The mock EDF was equilibrated with one of four gas mixtures: (1) air (P02=156.4+1.0 Torr, pH=7.77+0.02); (2) 100% N2 (PO2=4.5±0.7 Torr, pH=8.03±0.04); (3) 100% 02 (PO2=705+8.4 Torr, pH=8.03+0.04); (4) 3% C02 in air [three fish; P02=145+3.8 Torr, PC02=21.1 Torr (calculated), pH=7.02+0.03] or 5% C02 in air mixture [five fish; P02=151+2.5 Torr, PC02=35.3 Torr (calculated), pH=6.74+0.01]. Oxygen and carbon dioxide partial pressures and pH of blood and mock EDF were measured with a Radiometer PHM-71 acid-base analyzer and associated electrodes maintained at the same temperature as the fish. The pH electrode was calibrated with Radiometer standard pH buffers; the oxygen electrode was calibrated with air-saturated water and a Radiometer zero P02 solution; the CO? electrode was calibrated with precise gas mixtures from 35 Wosthoff gas-mixing pumps. In order to measure PC02 of blood, the C02 electrode was adjusted to give full-scale readings for samples in the expected range (0 to 2 0 Torr). This precluded accurate measurements of PC02 in mock EDF equilibrated with 3% or 5% C02 gas mixtures. These samples were, however, injected onto the C02 electrode and, in both cases, resulted in read• ings that were off-scale, indicating that PC02 was at least 20 Torr. A blood sample was taken from the dorsal aortic (DA) cannula before an experiment began in normoxic or hypoxic water for measurement of P02, PC02 and pH. The DA and buccal cavity cannulae were then attached to Statham P23Db and Hew• lett-Packard 267BC pressure transducers, respectively, to monitor blood and buccal pressures. The outputs of the trans• ducers were recorded on a Gulton Techni-rite (model 722) two- channel chart recorder. A 2 min record of DA and buccal pressures was taken as the pre-perfusion baseline of these variables. Perfusion then began by switching a three-way stopcock which connected the perfusate to the inflow cannula attached to the fish's cranium. The mock EDF flowed through the meningeal space for 30 min while DA and buccal pressures were recorded from 0 to 6 min, then at 2 min intervals span• ning the 10, 15, 20, 25 and 30 min perfusion time periods; therefore, total recording time was about 18 min. The perfu• sate flow through the meningeal space varied between 1 and 3 ml min-1 and was maintained by adjusting the pressure head between the inflow and outflow cannuale; this pressure differ• ential ranged between 3.7 and 19 Torr and had no apparent 36 effect on resting cardiovascular or ventilatory variables. Normoxic water (PO2=156-160 Torr) flowed continuously through the perspex box at a rate of about 1000 ml min-1. Fish were allowed 30-60 min recovery time between perfusate treatments until all four treatments were completed. Each experiment began with the normoxic (air-equilibrated) perfusate, but the other three perfusates were given in random order. The fish were allowed to recover overnight and the perfusate treatments repeated the next day with the fish in hypoxic (PO2 = 30-40 Torr) water. Experiments were also performed in which sodium cyanide (NaCN) dissolved in mock EDF at initial concentrations ranging from 5 to 1000 ug ml-1, or hydrochloric acid (HCl) with pH ranging from 3.6 to 7.0, were added to the mock perfusate with fish in normoxic water. Although the final outflow pH was greater than the initial pH owing to the dilution of H+ in the cranial space, the final H+ concentrations measured were one to two orders of magnitude higher than those used to stimulate central chemoreceptors in mammals (Shams 1985) and turtles (Hitzig and Jackson 1978). NaCN is a metabolic inhibitor of the mitochondrial electron transport chain and is known to stimulate all oxygen-sensitive chemoreceptors, including carotid body oxygen chemoreceptors in mammals (Mulligan and Lahiri 1981) and oxygen-sensitive chemoreceptors in fish gills (Burleson and Milsom 1990). NaCN at the concentrations used here has also been used to elicit ventilatory responses in gar (Smatresk et al. 1986). After an experiment was completed, the fish was killed by 37 over-anesthetization with concentrated MS-222. Sudan black dye dissolved in 95% ethanol, which has been used previously as a neural stain (Filipski and Wilson 1984), was perfused through the cranial space for 3 0 min at the same pressure as during the experiment. After dye perfusion, fresh ethanol was perfused through the cranium to rinse out excess dye. The brain was then removed and examined for the presence of dye. The location of stain was taken to indicate where the mock perfusate had come in contact with CNS structures during the experiment. Spectral Analysis of the Intermittent Air-Breathing Pattern The temporal air-breathing pattern of undisturbed bowfin was more closely examined in the frequency domain using spec• tral analysis as outlined in a commercially available time- series analysis software package (ITSM; Brockwell and Davis 1991). One 8 h recording session of air-breathing events for each of 6 fish in (1) aquatic/aerial normoxia, (2) aquatic hypoxia/aerial normoxia, and (3) aquatic normoxia/aerial hypoxia (8% 02) were used in the analysis. Air-breathing in Amia occurs intermittently as single events with inter-breath intervals of varying lengths. A typical series of type I and type II air breaths, expressed as inter-breath intervals, as a function of time is shown in figure 4 (top panel; see Results for descriptions of air breaths). Air-breaths were analyzed as a series of discrete events in time (see French and Holden 1971); discrete events (or spikes) in a time series have also been called delta functions 38 Figure 4. Top panel: Plot of inter-breath interval (IBI) as a function of cumulative time (min) for a single fish in normox• ic conditions. Open symbols denote type II air breaths and closed symbols denote type I breaths (see Results for descrip• tions of both air breaths). Bottom panel: The same time series as the top panel, except the breaths have been replaced by positive and negative delta functions representing type II and type I air breaths, respectively (see text for details). The delta functions were plotted after 5-point smoothing. 39 40 30 20 ffi M IO O O IOO 200 300 400 500 Cumulative Time (min) TYPE II TYPE I 1 r O IOO 200 300 400 500 Cumulative Time (min) 40 (DeBoer et al. 1984) . The spectrum of this type of signal is also known as the spectrum of counts (DeBoer et al. 1984). Air-breaths were treated as delta functions and given negative values if they corresponded with type I air breaths, and positive values if they were type II air breathing events. The size of the delta functions is arbitrary and does not affect the analysis. Each time series of discrete air breaths (delta functions) was smoothed using a 5-point weighted moving average (Brockwell and Davis 1991) . This removes high fre• quency components and is analgous to passing the data through a low-pass filter. The inter-breath interval series (fig. 4, top) expressed as the time series of delta functions after smoothing is shown in figure 4 (bottom panel). Each 500 min data set was subdivided into 4 equal data sets of 125 min for a total of 24 data sets (4 time series each for 6 fish). This was done to eliminate low-frequency trends in the 500 min data sets and thus maintain stationarity in the data, an assumption of Fourier analysis (Chatfield 1980) . The frequency compo• nents of the smoothed delta functions were then calculated using a discrete Fourier analysis (Brockwell and Davis 1991). The ITSM software calculates the total power of the periodo• gram and divides by the inverse square root of the number of data points in the analysis; therefore, the ordinates were expressed as normalized power. Following the calculation of the periodogram, the data were then transferred to a spread sheet (Quattro Pro) and spectrum averaged; that is, normalized power at each frequency interval was averaged for all the data sets and plotted as a function of frequency and period. 41 Frequencies in the spectrum contributing significant amounts of power in each 125 min data set were analyzed with Fisher's Exact test (Brockwell and Davis 1991). Significance deviating from the null hypothesis of random noise was accepted at the 5% level. Data Analysis and Statistics Most variables are summarized as mean + 95% confidence intervals (95% C.I.)/ unless otherwise stated. A variety of statistical tests were used to establish significance where appropriate: paired and unpaired t-tests, one-way and two-way analysis of variance, followed by the Student-Newman-Keuls (SNK) multiple range test or Tukey's multiple comparison test, and least squares linear regression analyses (Zar 1974). The probability of committing a Type I error (rejection of a true null hypothesis) was accepted at the 5% level. All statisti• cal tests were done with commercially available statistical software (Systat or Statgraphics). Departures from the above methods of statistical analyses are indicated in the text. 42 RESULTS Air-Breathing Rates and Behavior for Undisturbed Amia Intitial observations from Amia indicated that two dif• ferent breathing behaviors occurred under normoxic conditions with two different patterns of air flow that were qualitative• ly invariant; that is, every fish examined showed two, and only these two, air-breathing patterns. Since previous work on Amia has identified only one type of air-breathing behavior (Johansen et al. 1970; Deyst and Liem 1985), the breaths described here have been named type I and type II air breaths. The air flow patterns for these breath types are shown in figure 5. Type I air breaths were characterized by an exhala• tion phase, resulting in increased air flow at the pneumotach• ograph, followed by an inhalation phase resulting in a de• creased air flow (fig. 5, left column) . The inhalation phase of the type I breath was usually offset from the original baseline, owing to the characteristics of the measurement system (see Methods). The fish projecting its snout above the water's surface also contributed to the change in baseline; nevertheless, decreased air flow below the offset baseline was obviously correlated with a depression of the gular plate when viewed on videotape and, therefore, the inhalation phase of the type I breathing cycle. The small positive change in flow that occurred consistently during type I breaths approximately 200-400 ms before exhalation was due to the fish transferring lung gas to the buccal cavity upon approaching the surface and raising the water level in the funnel (T=transfer phase, fig. 43 Figure 5. Recordings of air flow (ml s-±) from two fish in normoxic conditions. Type I air breaths (left column) and the corresponding type II air breath (right column) are from the same fish. Type I air breaths are characterized by a transfer phase (T), followed by exhalation and inhalation. The expira- i tory time interval for type I breaths (TE), analogous to T E for the calibrated volumes (fig. 3), was used as the crite• rion for integrating the area under the expiratory flow curve for type I breaths (shaded area) for measurement of expired tidal volume. Type II air breaths show only an inhalation phase, corresponding with a negative flow pattern. Shaded areas for type I and type II inhalations correspond with inhaled volumes, but were not quantified owing to gas lost during inhalation and transfer (see text for details). The time scale bar equals 100 ms and applies to all traces. 44 TYPE I TYPE II INHALE i °] 10 J tn CC > tr 80 ni O INHALE INHALE CC 40-1 i I o X 0 -J HI -20 3 100 ms 5). This positive flow pulse was not due to the animal pro• jecting its snout above the water's surface since this pulse was not apparent during type II breaths when the fish also projected its snout slightly above the surface. Type II air breaths were characterized by a decreased air flow, indicating a single inhalation (fig. 5, right column). Both types of breaths were easily distinguished from each other by direct observation. In type I breathing, there was a clear depression of the gular plate twice during the breathing cycle. In type II breaths, the gular plate was depressed only once; furthermore, the amount of gular depression was notice• ably less in type II than in type I breathing. The sequence of events during both types of breathing are shown schemati• cally in figures 6 and 7. During the compression phase, there was usually, but not always, a loss of inspired gas from the opercular cavities as the fish descended below the surface (D in fig. 6, B in fig. 7) . Although the amounts lost could not be quantified, a greater amount of gas appeared to be lost during type I breathing, and in cases where there appeared to be large amounts of inspired gas lost, regardless of the aquatic or aerial conditions, type II breaths, often within one minute, occurred. Thus, owing to the loss of inspired gas, inspired volume could not be quantified. Inspired vol• umes are given only as estimates since measurement of these volumes would overestimate, by an unknown amount, the actual inhaled volume; breath volumes were, therefore, quantified only for the expiratory phase of the type I breaths. There was a considerable range of intra-individual and 46 Figure 6. Diagram of the sequence of events involved in a type I air breath. There are four distinct phases of the type I breath: (1) transfer of air bladder gas to buccal cavity, (2) exhalation of the transferred gas, (3) inhalation of atmospheric gas and (4) compression (i.e. transfer of inhaled gas to air bladder). During the compression phase there is usually a loss of gas from the opercular cavities. 47 TYPE AIR-BREATH 1. Transfer 4. Compression Figure 7. Diagram of the sequence of events involved in a type II air breath. There are only two phases: (1) inhalation of atmospheric gas and (2) compression (transfer of inhaled gas to the air bladder). 49 TYPE II AIR-BREATH 1. Inhalation 2. Compression AIR inter-individual values for expired volumes (VE) in the dif• ferent aquatic and aerial conditions. In 6 of the 8 fish -1 examined in undisturbed conditions, VE ranged from 6.3 ml kg -1 to 36.4 ml kg . The average maximum VE values for these 6 fish was 25.1 + 6.2 ml kg-1. The responses of undisturbed Amia to changes in aquatic and aerial gas concentrations are summarized in Table I and presented as frequency histograms figures 8-13. The frequency distributions under all conditions were positively skewed (i.e. the median value was less than the mean), due to breaths that occurred at time intervals greater than 90-100 min (not shown). The distributions (figs. 8-13) are presented as stacked bars, rather than overlapping, to differentiate the two breath types. In normoxic conditions, Amia took approximately 3 air- breaths h-1: inter-breath interval (IBI) was 19.8 + 0.9 min (Mean + 95% C.I.; n=1950 observations from 8 fish, fig. 8), and there were slightly more type I (60%) than type II air breaths (40%). Carbon dioxide (5% in air; fig. 9) delivered through the aerial phase had no significant effect on air- breathing rates compared with fish in normoxic conditions (Student-Newman-Keu1s (SNK) multiple range test, = 98 c[3744 2 1- )- When bowfin were exposed to aquatic or aerial hypoxia (8% 02), air-breathing increased significantly to about 5 breaths h-1 compared with normoxia: IBI decreased to 12.3+0.8 min (n=579) in aquatic hypoxia (fig. 10; SNK, q3744 4=13.4, P<0.001) and 12.9+2.0 min (n=328) in aerial hypoxia (fig. 11; SNK, ^2744 3=9-7/ P<0.001); however, there 51 Table I. Mean Inter-breath Intervals (IBI; min) for undis• turbed Amia in various combinations of aquatic and aerial conditions. Values are Mean + 95% C.I. and the number of observations (n). Type I and type II breaths are given as the percentage of total breaths for each condition. 52 CONDITION IBI BREATHS Aquatic Aerial Type I Type II Hyperoxia Air 57 .,8 + 15. 1 (30) 0 100 Normoxia 5%C02* 21.. 6 + 22 . (185) 55 45 Normoxia Air 19.. 8 + 0.9 (1950) 60 40 Normoxia ioo%o2 15.. 3 + 16. (350) 0.6 99. 4 Normoxia 8%02 12 . 9+2 .0 (328) 94 6 Hypoxia Air 12 . 3+0.8 (579) 80 20 Hypoxia ioo%o2 11.. 1+1.0 (329) 0.3 99. 7 N=7 fish, N=8 in other groups 53 Figure 8. Histogram of the distribution of inter-breath intervals (IBI; min) for eight fish in aquatic and aerial normoxia at 22 + 2 °C. Closed bars represent numbers of type I air breaths, open bars denote type II air breaths. The bars are stacked rather than overlapped. IBI greater than 90 min are not shown. The mean (X) and median (m) values for the distribution are indicated. 54 55 Figure 9. Histogram of the frequency distribution of IBI (min) for eight fish in aquatic normoxia and aerial hypercap- nia (5% C0? in air). 56 m X • Type I • Type H •JL IO 20 30 40 50 60 70 1 Inter - breath Interval (min.) Figure 10. Histogram of the frequency distribution of IBI for eight fish in aquatic hypoxia and aerial normoxia (air). 58 O H M O LO ft ft >> >> H H s I i 59 Figure 11. Histogram of the frequency distribution of IBI (min) for eight fish in aquatic normoxia and aerial hypoxia (8% 02). 60 m X Inter - breath Interval (min.) was no significant difference in IBI between the two hypoxic treatments (SNK, 2= 0'7)* There was also a clear change in the breathing pattern when Amia were placed in hypoxic conditions: type I air-breaths were used predominantly, ac• counting for about 80% of total breaths in aquatic hypoxia (Table I) ; of the remaining 2 0% of air breaths (type II) that occurred in aquatic hypoxia, the large majority of these (ca. 75%) occurred at intervals of less than 10 min (fig. 10) . In aerial hypoxia, 94% of all breaths were type I (Table I). When Amia were given 100% oxygen to breathe from the aerial phase, and aquatic conditions remained normoxic, IBI significantly declined to 15.3+1.6 min (n=350) compared with air flowing in the breathing funnel (fig. 12; SNK, =6 5 q3744 2 - ' P<0.001); furthermore, with the exception of 2, every one of 350 breaths was type II (fig. 12) . When aquatic conditions were made hypoxic, and 100% oxygen remained in the aerial phase, type II breaths were still used almost exclu• sively (fig. 13), and IBI declined even further to 11.1+1.0 (n=329) min (SNK, q3744 4=4.6, P<0.01) as compared with fish in normoxic water and breathing 100% 02 from the aerial phase (Table I). Aquatic hyperoxia resulted in a significant reduc• tion of air-breathing frequency, to about 1 breath h-1 (Table I). Of the few breaths that occurred in this condition, all were type II air breaths. Air Bladder Deflation and Inflation Air bladder deflation initiated air-breathing responses. The time taken to initiate an air-breath following deflation 62 Figure 12. Histogram of the frequency distribution of IBI (min) for eight fish in aquatic normoxia and aerial hyperoxia (100% 02). 63 m X 30n | | 25 20 • Type I 15 • Type H IO 5 11 n rm TrThrmnlKIn n n 11 O 1 1 1 'r 1 O IO 20 30 40 50 60 70 80 Inter - breath Interval (min.) Figure 13. Histogram of the frequency distribution of IBI (min) for eight fish in aquatic hypoxia and aerial hyperoxia (100% 02). 65 m X 40 30 > • Type I 0) 20 • Type H IO rrfU n rlT XL O o n IO 2III0 30I 4T0 " ' "I" " ' 5" 10 " ' I 60 Inter - breath. Interval (min.) was significantly dependent upon the volume removed from the air bladder (fig. 14). A hyperbolic function (Y=f(l/X)) between time to initiate an air breath and the volume removed was initially assumed since asymptotic values of X and Y should physically exist. An asymptote will exist at some volume (X) where volume removal from the air bladder could not be detected by the fish. An asymptote should also exist as a limit to the speed (Y) at which the fish could make an excur• sion to the water's surface for an air-breath. Thus, a linear regression model of time (s) to initiate an air breath was . — i . plotted as a function of (volume) removed to obtain a linear equation for the relationship between these variables. The equation relating these variables was : Time = 2 468.3*(l/vol.)+46.6 ; F1^64=4.66, r =0.07, P<0.05. The curve in figure 14 was calculated by converting the equation to its curvilinear form. The threshold (T, fig. 14) was approximate• ly 3 ml kg-1; deflations below this value produced no re• sponses within 10 min following deflation. The fastest time for an air breath response to deflation was 5 s. All 66 air- breaths observed following lung deflation were type II breaths, regardless of the aquatic P02 (fig. 14). Most of the air breaths (82%) were initiated within 2 min after air blad• der deflation. There was also a significant relationship between the number of breaths taken during the 10 min observation period, and the volume removed from the air bladder (fig. 15): No. 2 Breaths = 0.089*Vol. + 0.81; F± ?1=33.0; r =0.32; P<0.001. During the 10 min observation period following deflation, 67 Figure 14. Relationship between the time taken to initiate an air breath (seconds) as a function of volume of air removed (ml kg-1) from the air bladder (N=4, n=66) in aquatic normoxia (•), aquatic hypoxia (A) and aquatic hyperoxia (O)* AH breaths observed following air bladder deflation were type II air breaths. The linear equation relating these variables is: Time = 468.3*(1/Vol.) + 46.6; r2=0.07; P<0.05. The regression line in the figure was calculated from this equation and converted to a curvilinear form. -The threshold (T) for detec• tion of volume removed is approximately 3 ml kg-1. 68 Air Bladder Inflation 300H • Normoxia A Hypoxia O Hyperoxia 200 • 2 min I IOO • •H 1 O ° o -QD- min O H ACt] _ • o A a n ftp •o tr O T T T O T IO 20 30 40 50 60 Volume Removed (ml/kg) Figure 15. Relationship between the number of air-breathing events in 10 min after air bladder deflation as a function of the volume removed (ml kg ). The linear equation relating these variables is: Breaths = 0.089*Vol. + 0.81; r2=0.32; P<0.001. 70 there were occasionally type I breaths; however, they occurred only when the fish was in hypoxic water and were usually the third or fourth breath in the series. The initial breath following deflation was always a type II, regardless of the aquatic oxygenation (fig. 14) . Air bladder inflations produced a variety of responses from the fish. Small inflations (3 ml kg-1) that were deliv• ered as the fish approached the surface had no discernable effect; that is, air-breathing proceeded normally, with both type I and type II breaths evident. Larger inflations some• times caused the fish to stop its ascent to the surface. Air- breathing sometimes proceeded despite large inflations, but the breaths were nearly always of type I when this occurred. Maximum Air Bladder Volume Maximum air bladder volume (ml), measured in vitro, increased linearly with increasing body mass (g) (Vol.=0.08*Mass+12.2; r2=0.86, P<0.001; fig. 16) over the range used in this study. The slope (0.08) indicated that air bladder volume increased at a rate of 8% per 100 g of body mass over this range. The intercept (12.2 ml) was not signif• icantly different from zero (T5=1.05; P>0.05). Branchial Nerve Denervation Denervation of branchial branches of cranial nerves IX and X were done to test the hypothesis that type I air-breaths are caused by afferent stimulation of chemoreceptors located on the gills innervated by cranial nerves IX and X. All three 72 Figure 16. Relationship between maximum air bladder volume (ml) and body mass (g) determined in vitro. Values represent the mean of three determinations for each air bladder. The linear equation relating these variables is: Vol = 0.08*Mass + 12.2; r2=0.86; P<0.001. 73 74 groups of surgically treated fish exhibited both types of breaths in aquatic normoxia and increased air breathing fre• quency in aquatic hypoxia (figs. 17-19). Since air-breathing frequency for the two normoxic trials were not significantly different within a group, only the first normoxic trial was presented in figures 17-19. When both breath types were con• sidered together, there was a significant decrease in IBI for total denervate (TD) fish compared with sham-operated (SH) or partial denervate (PD) fish in normoxic conditions (Table II). Within group comparisons revealed that IBI significantly decreased in aquatic hypoxia compared with either normoxic treatment. The increased air-breathing rate for the TD group was due entirely to large numbers of type II breaths that occurred at IBI of 1 min or less in aquatic normoxia (fig. 19A) and aquat• ic hypoxia (fig. 19B), thus significantly reducing the mean IBI (Table II) . Direct observations from TD fish indicated qualitatively that larger amounts of inhaled gas were lost during the transfer phase of the air-breathing event in these fish compared with SH and PD groups. Since the hypothesis of this experiment was to test whether type I breaths were stimu• lated by chemoreceptors located on the gill arches, the re• sults were re-analyzed after removing the type II breaths from the data set. After removing type II breaths, there were no significant differences in the frequency of type I breaths between groups in normoxia and hypoxia (Table III) . One exception was a significantly lower IBI in SH during the first normoxic treatment compared with the other groups (Table III). 75 Figure 17. Histogram of the frequency distribution of IBI (min) of type I (closed bars) and type II (open bars) air breaths for sham-operated (SH) fish (N=4). A. Aquatic and aerial normoxia; B. Aquatic hypoxia/aerial normoxia. The mean (X) and median (m) values are indicated. 76 20i m X I I 16 • Type I 12 • Type H 8 4 O • lAJL. HTI rBi. XL O IO 20 30 40 50 60 Inter-Breath Interval (min) • Type I • Type H 20 30 40 50 GO Inter-Breath Interval (min) 77 Figure 18. Histogram of the frequency distribution of IBI (min) of partial branchial denervate (PD) fish (N=5). A. Aquatic and aerial normoxia; B. Aquatic hypoxia/aerial normox• ia. 78 • Type I • Type H ,nrl,n nfl , ri, W- 30 40 50 60 Inter-Breath Interval (min) 79 Figure 19. Histogram of the frequency distribution of IBI (min) for total branchial denervate (TD) fish (N=7). A. Aquatic and aerial normoxia; B. Aquatic hypoxia/aerial normox• ia. Note the distinct mode of type II breaths at 1 min com• pared with SH or PD groups in aquatic normoxia (figs. 16, 17). 80 A • Type I > • Type H fl> 0 20 30 40 50 60 Inter-Breath Interval (min) 81 Table II. Mean Inter-breath Intervals (min) + 95% C.I. and the number of observations (n) for branchial denervation groups. Groups: sham-operated (SH), partial-denervates (PD) and total-denervates (TD). Values are means for combined type I and type II air breaths. NI and N2 denote first and second aquatic normoxia treatments separated by an aquatic hypoxia (H) treatment. 82 NI H N2 SH 16.4±5.1 (103) 6.3±0.4a (306) 17.3+3.0 (105) PD 20.5±5.5 (132) 8.2+0.8a (292) 16.7+3.8 (124) TD 7.5+1.0b (451) 5.1+0.4a'c(680) 9.0+1.2b (339) aSignificantly different NI and N2 within same group. bSignificantly different from SH and PD for same treatment. cSignificantly different from PD-H for same treatment. 83 Table III. Mean + 95% C.I. of Inter-breath Intervals (min) for branchial denervation groups. Values are for type I air breaths only. Same abbreviations as Table II. 84 NI H N2 SH 23.7+4.0 (57) 8.6+0.73 (224) 22.6+5.9 (59) PD 35.5+12.5b (58) 12.9+0.9a (178) 25.2±9.1 (70) TD 31.3+5.5 (92) 12.6+0.8a (262) 23.7+4.3 (117) a Significantly different from NI and N2 within same group, k Significantly different from SH-N1. 85 All three groups increased the frequency of type I air breaths to about 5 breaths h-1 when exposed to aquatic hypoxia compared with 2-3 breaths h-1 in normoxic conditions (Table III) . Mean gill ventilation rates ranged between 4.5 to 10.5 breaths min-1 and were significantly lower in the TD group in normoxic and hypoxic conditions (Table IV). Within groups, fg did not change significantly in aquatic hypoxia (Table IV). Intra-Cranial Perfusion Gill ventilation rates increased significantly from about 10 cycles min in aquatic normoxia to about 17 cycles mm in aquatic hypoxia before the start of perfusion (Table V). Air-breathing rates in aquatic hypoxia were calculated by dividing the total number of air breaths during a perfusion treatment by the total minutes recorded for the treatment; — i these values were converted to breaths h . Air-breathing was =0,15 not affected by any perfusate treatment (ANOVA, F3 26 ) and the rate of air-breathing during aquatic hypoxia was 3.0+0.6 breaths h-1 (n=8), and was significantly different from zero (T7=5.0, P<0.001; Table VI). The ventilatory responses to intracranial perfusion with NaCN were variable. NaCN at a concentration of 500 ug ml-1 increased ventilation in one fish, 1000 ug ml-1 increased ventilation in another fish, but there was no effect at lower doses; however, at concentrations ranging from 5 to 500 ug ml" 1, NaCN depressed ventilation in three other fish. Ventila• tion was unaffected in five fish perfused intracranially for 86 Table IV. Mean + 95% C.I. values for gill ventilation rate (breaths min-1) for branchial denervation groups. Abbrevia• tions same as Tables II and III. 87 NI H N2 SH 9.6+1.8 (14) 10.5+2.0 (18) 7.0+1.5 (8) PD 9.5+2.5 (10) 8.9+1.6 (16) 6.6+0.8 (21) TD 6.1±2.4a(14) 5.7±0.7b (40) 4.5±0.7 (15) aSignificantly different from SH-N1 for same treatment. bSignificantly different from SH and PD for same treatment. 88 Table V. Mean + 95% C.I. for gill ventilation rate (fg; breaths min-1), buccal pressure amplitude (Pb; mmHg) and air- breathing (AB; breaths h_1) during intra-cranial perfusion with mock EDF equilibrated with four different gas mixtures under aquatic normoxic or hypoxic conditions. Values are provided for pre-perfusion and 15 min after the start of perfusion. 89 Aquatic Normoxia 15 min Perfusate Variable Pre-perfusion perfusion AB Normoxia fg 9.8+3.0 9.8+2.3 (N=8) Pb 0.44±0.2 0.51±0.2 0 Hypoxia fg 11.8+2.5 11.1+2.1 (N=8) Pb 0.51±0.2 0.51+0.2 0 Hypercapnia fg 11.2+2.1 11.6+2.5 (N=8) Pb 0.44+0.2 0.51±0.2 0 Hyperoxia fg 10.3+2.8 9.7±2.5 (N=7) Pb 0.44+0.2 0.44+0.2 0 Aquatic Hypoxia 15 min Perfusate Variable Pre-perfusion perfusion AB Normoxia fg 16.4+3.5 16.1+4.1 (N=7) Pb 0.74±0.3 0.59+0.3 3.2+2.1 Hypoxia fg 17.6±4.1 16.7+3.7 (N=7) Pb 0.74+0.3 0.74+0.3 3.2+2.1 Hypercapnia fg 18.1±3.9 18.5+3.7 (N=8) Pb 0.81+0.3 0.74+0.3 2.4+2.1 Hyperoxia fg 18.8+3.7 18.3+3.9 (N=8) Pb 0.81±0.3 0.81+0.3 3.1+2.3 Three fish perfused with 3% C02 perfusate and five fish perfused with 5% C02 perfusate. 90 Table VI. Summary of dorsal aorta P02, pH and PC02 and air- breathing rates (AB) during aquatic normoxia and hypoxia. Values are mean ± 95% C.I. and number of animals used in each treatment (N). 91 Condition PO2 PH PCO2 AB (mmHg) (mmHg) (h"1) Aquatic Normoxia 68.6±27.6 7 . 74 + 0.05 3.0+0.5 0 (8) (8) (8) (8) Aquatic Hypoxia 19.3+4.4 7.81+0.07 1.4+0.2 0 + 1.4 (8) (6) (8) (8) Signif. (P) <0.005 <0. 02 <0.001 <0.001 92 30 min with HCl solutions ranging in pH from 3.8 to 6.8. Sudan Black dye stained extensive areas of the brain and associated structures. Dye was found occasionally within the third ventricle and usually in the fourth ventricle. Dye was always present on structures surrounding the brain, including the ventral surface of the medulla, cerebellum, optic tectum and telencephalon. Spectral Analysis of the Intermittent Air-Breathing Pattern The temporal air-breathing pattern in Amia was intermit• tent, and appeared irregular. The contrasting patterns of air-breathing in normoxia and aquatic hypoxia are shown in figure 20. In normoxia, there was usually an alternation of type I and type II air breaths (fig. 20A), while in hypoxia, air-breathing frequency was higher, and type I breaths were used predominantly (fig. 20B). Data from these fish, and from 4 others, were used in the spectral analysis. Plots of inter-breath interval over time for fish in aerial hypoxia were very similar to those in aquatic hypoxia. The averaged periodogram for the 6 fish in normoxia revealed a significant low frequency peak, corresponding to a period of about 25 min (fig. 21A). Two other peaks, with less power, occurred at about 12 min and 7 min in normoxia. The dominant peak was halfway between the mean inter-breath inter• vals for type I breaths (29.8+2.5 min) and type II air breaths (20.5+2.1 min) for these 6 fish. Before spectrum averaging, however, the dominant peak for the six fish was centered at 29.6 min in normoxia (fig. 21B), indicating that the periodic- 93 Figure 20. Inter-breath Interval (IBI; min) plotted as a function of cumulative time (min) for two fish, one in aquatic and aerial normoxia (A) , and the other in aquatic hypoxia (PwO2=50 Torr) and aerial normoxia (B). Open symbols denote type II air breaths; closed symbols denote type I air breaths. 94 Figure 21. A. Spectrum-averaged periodogram for 6 fish (n=24 data sets) in aquatic normoxia (solid line) and aquatic hypox- la (dashed line). Normalized power (mm) is plotted as a function of frequency (cycles/min) and as an equivalent time period (min). A significant peak occurred in normoxia at about 25 min; two smaller peaks are evident at 12 and 7 min. In aquatic hypoxia, the significant peak has shifted to about 10 min, with a smaller peak at about 6 min. B. Periodogram for the same 6 fish in 21A before spectrum averaging. The dominant peak is centered at 29.6 min, indicating this peak contributes most of the power to the largest peak in A (see text). 96 B Period (min) Frequency (cycles/min) 97 ity in figure 21 was associated with type I, rather than type II, breaths. Spectrum averaging smooths the periodogram even further and thus has the effect reducing and spreading out power among closely related frequencies. At the low frequency end of the scale, resolution becomes further reduced, thus differentiation between the 30 min and 20 min peaks was not possible after spectrum averaging. In aquatic hypoxia, the 30 min low frequency peak disap• peared and a significant, higher frequency peak at 10 min was revealed (fig. 21A). A second peak, occurring at approximate• ly 7 min was also evident in aquatic hypoxia. This 10 min peak was also closely associated with the mean inter-breath interval for type I air breaths. The results from fish in aerial hypoxia were similar to those in aquatic hypoxia, but the fish fell into two characteristic groups (fig. 22). Half of the fish showed a significant high frequency peak at ap• proximately 6 min, and a second broader peak with a period of about 9 min (solid line, fig. 22) . There was also a 9-10 min peak in the 3 remaining fish; at higher frequencies, however, several peaks with lower amounts of power were evident (dotted line, fig. 22). Overall, bowfin in normoxic or hypoxic conditions exhib• ited some periodicity around 10 min; in normoxia there was a significant low frequency peak occurring at about 30 min. In aquatic or aerial hypoxia, dominant periods occurred in the range of periods between 5 and 10 min. The dominant periods in normoxic or hypoxic conditions were most closely associated with the mean intervals between type I air breaths. 98 Figure 22. Spectrum-averaged periodogram for 6 fish (n=24 data sets) in aquatic normoxia and aerial hypoxia (8% 02). The fish were divided into two groups according to their periodograms. The solid line represents the average of 3 fish with a significant peak centered at 6 min. A smaller peak occurs at about 9 min. Three other fish had a significant peak around 10 min, and a slightly smaller peak at 7 min. Note there was a common period of about 9-10 min for all 6 fish. 99 Period (min) 20 IO 5 2 . , , r- 1 o 0.1 0.2 0.3 0.4 0.5 Frequency (cycles/min) DISCUSSION Although the natural history of Amia is not well known, the few observations made to date indicate that it does use aerial respiration under natural conditions (Reighard 1903; Doan 1938). Air-breathing has been observed during breeding, and occurs at the same water temperatures used in this study (Reighard 1903). Thus, the physiological results presented here may have direct relevance to its natural behavior and ecology, but these issues are not directly assessed in this study. Air-Breathing Patterns and the Responses to Changes in Aquatic and Aerial Gas Composition Since the seminal study establishing that Amia exhales and inhales atmospheric gas (Wilder 1877), subsequent studies have confirmed the double pulse breathing mechanism of aerial ventilation in Amia (Johansen et al. 1970; Randall et al. 1981; Deyst and Liem 1985; Liem 1988, 1989): exhalation fol• lowed by inhalation, which is the breathing pattern described here as type I. This study used the technique of pneumota• chograph^ to directly measure air flow generated by the fish during air breathing events. The application of this tech• nique has independently established the exhalation/inhalation breathing sequence in Amia, in addition to finding a previous• ly undescribed breathing pattern. The type II breathing pattern has not been described in Amia prior to this study, but makes up a large proportion (ca. 40%) of the total air 101 breaths under normoxic conditions (Table I, fig. 8). The most comprehensive study of the mechanism of aerial ventilation in Amia, using X-ray cine film, pressure record• ings and electromyographic (EMG) analysis, was conducted by Deyst and Liem (1985). In addition to confirming previous studies showing that exhalation occurred before inhalation, X-ray cinematography revealed that a large residual volume in its air bladder remained following exhalation. It is not clear why their study failed to observe type II breaths. One reason may be that the experiments described by Deyst and Liem (1985) were done with fish in hypoxic water, a condition that resulted in a predominance of type I breaths in this study. The breathing pattern of Amia (Type I in this study) has been described as a four-phase process (Liem 1989): (1) Trans• fer phase; depression of the buccal floor which creates a negative pressure in the buccal cavity, thus transferring gas from the air bladder, (2) Expulsion phase; active exhalation of buccal gas by elevation of the buccal floor, (3) Intake phase; a second depression of the buccal floor draws air from the atmosphere into the buccal cavity, and (4) Compression phase; transfer of inhaled gas from the buccal cavity to the air bladder by a second elevation of the buccal floor. In this study, phases 1, 2 and 3 were detectable by pneumotachog• raph^. The initial transfer phase (phase 1) appeared as a brief positive flow (T in fig. 5), owing to the local change in water level created as the fish expanded its buccal cavity when approaching the surface during gas transfer. This flow was useful in determining the start of the transfer phase 102 during the type I breathing cycle. Exhalation (phase 2), as expected, produced a positive change in air flow at the pneu- motach as the fish actively expelled buccal gas (fig. 5). Inhalation (phase 3), produced a negative flow immediately after exhalation, but the baseline was raised as a function of the recording system (see Methods), and probably also due to the animal protruding its snout above the water surface. The compression phase (phase 4), although not apparent from the pneumotach recordings, was clearly visible on videotape, and was usually characterized by gas bubbles escaping from-the opercular cavities as the fish descended below the surface. Deyst and Liem (1985) also reported inhaled gas loss during the compression phase in Amia. Thus, observations from the video recordings and pneumotachographic analyses of type I air breaths are entirely consistent with the mechanisms described by Deyst and Liem (1985). The air breath sequence including transfer, exhalation and inhalation for the fish in this study occurred in about 0.5 s, which is similar to reported values for Amia (Deyst and Liem 1985) , electric eel (Farber and Rahn 1970) and gar (Rahn et al. 1971). Measurements of expired tidal volume and air bladder volume were similar to measurements obtained for Lepisosteus. Maximal expired tidal volume for undisturbed Amia averaged 25.1 ml kg-1, which is similar to values of 24.9 and 31.7 ml kg-1 measured for gar (Rahn et al. 1971; Smatresk and Cameron 1982b). The expired tidal volumes for gar were obtained by direct collection of air bladder gas, therefore, the indirect measurement technique used in this study yields comparable re- 103 suits. Wilder (1877) measured expired tidal volumes by direct collection ranging from 18 to 105 ml kg-1, with a mean of 44 ml kg-1, for a single Amia. Although the absolute volumes were lower in this study, compared with the single fish used in Wilder's original observations, comparisons indicate that expired tidal volume varies considerably within individual animals. Air bladder volumes for Amia (fig. 16) and Lepi- sosteus (Rahn et al. 1971) are approximately 8% of body mass, which is well within the range reported for freshwater fish (Jones 1957), and in the expected theoretical range for fresh• water fish (Alexander 1966) . When expressed as a fraction of air bladder volume, expired tidal volume for Amia is about 31% of total air bladder volume, and about 40% in gar (Rahn et al. 1971). These results support the direct observations of Deyst and Liem (1985) indicating that a substantial residual volume remains following expiration. In this study, type II breaths were identified by nega• tive air flow at the pneumotachograph (fig. 5), with no ini• tial transfer phase that was evident in type I breaths, and no positive air flow preceding the characteristic negative flow pattern. Observations from the video recordings showed that the buccal floor was depressed only once when the fish reached the surface (shown schematically in fig. 7) . There was no evidence of exhalation since air was never seen to escape as the fish approached the surface. During the compression phase of type II breaths, however, there were also occasionally small losses of inhaled gas through the opercular cavities, as in the type I breaths, but this did not always occur. Since 104 type II breaths involved a brief inhalation at the surface, the time sequence was faster (ca. 0.1 s) than type I breaths. It is not clear why previous studies have failed to observe single inhalation breaths (type II) in Amia. A likely reason is that pneumotachography has not been previously used to examine aerial ventilation in this species. Inhalation without exhalation would be difficult to detect without a direct measure of air flow. A recent re-examination of the air-breathing mechanism in Amia (Liem 1988, 1989) suggested that two types of breaths were used, but one type of breathing involved a passive, rather than active, transfer of gas from the swim bladder to the buccal cavity. The analyses are based on EMG data showing that the sternohyoideus muscle, used primarily for lowering the buccal floor during active transfer, did not fire during "passive transfer'; therefore, Liem (1989) concluded that air bladder gas was transferred to the buccal cavity passively, facilitated by the hydrostatic pressure gradient. His obser• vations suggest, however, that exhalation was always active. The results from this study do not support a passive transfer hypothesis. There was no evidence from this study to suggest that "passive transfer' occurred. In this study, transfer (phase 1) and exhalation (phase 2) were either active (type I), in which the buccal floor was raised and lowered twice during a cycle and is consistent with Liems's analysis, or not present (type II). There was no indication of a third breath type that could be interpreted as using "passive trans• fer' coupled with active exhalation. Upon closer examination 105 of Liem's (1989) results, the EMG pattern and buccal pressure recordings corresponding with "passive transfer' could be re• interpreted as a type II breathing pattern. The activity of the sternohyoidues muscle once only in his description of "passive transfer' is consistent with the observations in this study indicating that the buccal floor is depressed once during the type II breathing pattern. Since Liem (1989) did not measure expiratory or inspiratory air flow, it would have been difficult to distinguish, on the basis of EMG measure• ments, whether gas transfer had indeed occurred. The evidence from this study, therefore, indicates that two separate mecha• nisms for generating air flow occur in Amia. One mechanism, which has been previously described in several other studies, involves active exhalation of air bladder gas, and active inhalation of atmospheric air; the use of the buccal force pump during these events is evident. The second mechanism, not previously described in Amia, involves a single aspiratory inhalation by the action of the buccal cavity, with subsequent transfer of inhaled gas to the air bladder. There is no evidence of exhalation during this type of breathing sequence. Although the precise neuromuscular motor patterns responsible for generating these two air-breathing events are not known, type II breaths may be the latter half of the type I air breath cycle. The responses of Amia to alterations in aquatic or aerial gas concentrations showed obvious changes in both the frequen• cy and pattern of air-breathing. In light of the finding that Amia use two breathing mechanisms, closer attention must be 106 paid to the pattern of breathing in order to more accurately interpret the results. Breathing frequency increased signifi• cantly in aquatic or aerial hypoxia, a response typical of Amia (Johansen et al. 1970; Randall et al. 1981; McKenzie 1990) and many other species of air-breathing fishes (see Shelton et al. 1986). In aquatic or aerial hypoxia, Amia took about 5 breaths h-1, which is considerably less than rates of 15-20 breaths h-1 observed by Johansen et al. (1970). There is no apparent reason for this large discrepancy, but the breathing frequencies in this study are closer to those found in other studies (Horn and Riggs 1973; Randall et al. 1981; McKenzie 1990). In addition to increased breathing frequency in hypoxia, there was also a clear shift to a predominance of type I air-breaths (figs. 10,11) compared with fish in normox• ia, suggesting that type I breaths are stimulated by 02-sensi- tive chemoreceptors. There is also an indication that 02~ sensitive chemoreceptors monitor intravascular 02 tension. Amia exposed to aerial hypoxia, with aquatic P02 held constant above 140 Torr, increased the overall frequency of breathing and used predominantly type I air breaths. This indicates that internal chemoreceptor sites, at least, mediate the hypoxic ventilatory reflexes. The results with Amia exposed to either aquatic or aerial hypoxia are, therefore, consistent with studies on gar (Smatresk et al. 1986) and lungfish (Johansen and Lenfant 1968) showing that internal hypoxia stimulates air breathing. Aerial hyperoxia caused dramatic changes in the air- breathing pattern. Amia exposed to 100% 0P in the gas phase 107 maintained, or increased, air-breathing rates (Table I, figs. 12,13), in contrast with several other species of air-breath• ing fish where aerial hyperoxia reduced air-breathing frequen• cy (Garey and Rahn 1970; Lomholt and Johansen 1974; Burggren 1979; Pettit and Beitinger 1985; Smatresk et al. 1986). The high rate of type II breathing, especially in aquatic hypoxia, suggests that type I breaths are inhibited by elevated inter• nal PC>2, since type I breaths normally occur in aquatic nor• moxia and are predominant in aquatic hypoxia (fig 10). Gee and Graham (1978) reported that in the air-breathing catfish, Brochis splendens, air-breathing frequency was maintained in aerial hyperoxia at rates similar to when exposed to air. Their study indicated that buoyancy regulation, rather than blood PC>2 per se, was responsible for the maintenance of air- breathing frequency. This occurred since the increased 02 diffusion gradient in aerial hyperoxia, compared with air in the air-breathing organ (ABO), resulted in a faster decline in volume. Thus, a clear hydrostatic function, in addition to the respiratory function, was indicated for the ABO in B. splendens. This mechanism is probably responsible for the maintained frequency of type II air-breathing during aerial hyperoxia in Amia. The overall view of the control of air-breathing in Amia, in response to various combinations of gases in the aquatic and aerial environment, suggests that decreased intravascular P02 stimulates, and increased P02 abolishes, type I air breaths, indicating an oxygen-related role for type I breaths. Aquatic hyperoxia uniformly depressed air-breathing but, when 108 it did occur, type II breaths were used exclusively. Type I air breaths, therefore, appear to be modulated primarily by changes of internal P02 levels: low blood P02 stimulates intravascular chemoreceptors which increase type I air- breathing, while high blood P02 inhibits type I air breaths. Previous work on other water-breathing and air-breathing fishes indicates the most likely sites for the reflex stimula• tion of ventilation through 02-sensitive chemoreceptors are the central nervous system (Bamford 1974; Jones 1983) and the peripheral branchial vasculature (Powers and Clark 1942; Saunders and Sutterlin 1971; Milsom and Brill 1986; Burleson and Smatresk 1990; Burleson 1991). Since type I breaths were stimulated by aquatic and aerial hypoxia, potential central and peripheral sites controlling type I breaths were investi• gated. Increased rates of type II air breathing in aerial hyperoxia suggested one of two hypotheses: (1) elevated blood P02 simultaneously inhibits type I breathing and causes a behavioral switch to type II air-breathing, or (2) elevated air bladder P02 results in a faster reduction in lung volume, owing to an increased 02 diffusion gradient as suggested by Gee and Graham (1978), indicating type II breaths are mediated by stretch receptors located in the air bladder wall. The second hypothesis does not exclude the possibility of high blood P02 inhibiting type I breaths, but the role of stretch receptors in type II breathing could be tested independently of hypothesis 1. 109 Air Bladder Mechanoreceptors in the Control of Air-Breathing It has been shown that Amia have slowly-adapting pulmo• nary stretch receptors (PSR), carried in the ramus intestina- lis branch of the vagus nerve, that respond to both dynamic and static changes in air bladder volume (Milsom and Jones 1985) . A rapid off-response in the discharge frequency of vagal afferents was caused by rapid air bladder deflations, illustrating the dynamic aspects of these receptors. In addition, PSR showed a discharge frequency proportional to lung volume typical of tonic stretch receptors in the air bladders of gar (Smatresk and Azizi 1987) and lungfish (DeLa- ney et al. 1983). The initial response to air bladder defla• tion elicited only type II air breaths, suggesting that a physiological and behavioral correlate for the neural observa• tions exists: the rapid off-response of afferent nerve dis• charge recorded from PSR afferents (Milsom and Jones 1985) probably elicits the initial type II breath reflex since most deflations in this experiment were done rapidly (<5 s). Also, about 60% of the responses to deflation occurred in less than 1 min, suggesting a rate-sensitive component of the reflex. When Amia use type I breaths under normal conditions, expired volume is occasionally low, or gas is lost during the inhala• tion and transfer phase; in these instances, type II breaths often occur in less than 1 min, suggesting the rate-sensitive properties of PSR may be involved. This off-response behavior of PSR may be important in maintaining air bladder volume by evoking a type II breath when the tidal volume of type I breaths alone are not sufficient to increase stretch receptor 110 firing above threshold. Since the off-response of PSR shown by Milsom and Jones (1985) was rate-sensitive, rather than volume-sensitive, it may account for the weak, although sig• nificant, correlation between the response time of initial air breath and the volume removed from the air bladder (fig. 14) . Volume removed from the air bladder only accounted for 7% of the variation in the initial response time after deflation; the initial air breath response was largely independent of the volume removed. Since tonic discharge of PSR are directly proportional to air bladder volume (Milsom and Jones 1985), increased frequency of type II breaths would be expected to be related to the degree of air bladder deflation. Indeed, a significant correlation was found between air-breathing fre• quency and volume removed (fig. 15). Since type II breaths involve inhalation only, increased frequency of these breaths would have the effect of increasing air bladder volume over time. Type II breathing after deflation probably continues until PSR afferents cause feedback inhibition of breathing. The consistent type II breathing behavior upon air bladder deflation, that is also significantly related to the volume of deflation, supports the hypothesis that type II breaths are stimulated by mechanoreceptor afferents from the air bladder. Type II breaths appear to play a role in maintaining lung volume which declines during inter-breath intervals by the diffusion of 02 from the air bladder. The most likely reason for the maintenance of air bladder volume is that type II breaths have a buoyancy, rather than gas exchange, function. The results from air bladder deflations, therefore, favor the 111 second hypothesis proposed in the previous section: Amia breathing 100% 02 continue to take type II breaths in response to a constant reduction in PSR tonic discharge as 02 diffuses from air bladder to blood. It is also likely that type I breaths are inhibited by high blood P02 in aerial hyperoxia, since aquatic hypoxia normally stimulates type I air breaths. Given that type II air breaths involve inhalation, with no exhalation, this type of breath would appear ideally suited for maintaining constant air bladder volume and neutral buoy• ancy in Amia. During inter-breath intervals oxygen diffuses from the air bladder, without being replaced by equal amounts of C02 (Johansen 1970) ; the result is a decline in air blad• der volume and a reduction in buoyancy (Alexander 1966) . Unless the gas is replaced, or other mechanisms are used, the fish will become negatively buoyant and sink. Intermittent excursions to the surface to replace reduced air bladder volume is a necessary function of aerial respiration in fish where active secretion of gas into the lung is not an option (Jones and Marshall 1953). Bishai (1961) demonstrated that the response of a number of physostomous teleosts to sudden compression, causing a reduction in air bladder volume, was to swim to the surface and gulp air. There is evidence that some physostomous teleosts can secrete gas into the swim bladder (Alexander 1966), however, the rates of secretion are very low compared with physoclists; there is no evidence that Amia can actively secrete gases into the swim bladder. It has been argued that physostomous fish are dependent upon air gulping for buoyancy regulation (Jones and Marshall 1953; Jones 1957). 'Amia, therefore, appears to fit the general pattern for a physostomous fish: air-breathing is required for buoyancy regulation; Type II breaths probably provide this function. Previous studies on Amia (Johansen et al. 1970) and Lepisosteus (Smatresk and Cameron 1982b) have shown that air bladder deflations result in air breathing events. Although the authors suggested that pulmonary stretch receptors may have been involved in mediating the responses, they suggested that air-breathing was an indirect 02-chemoreceptor response, due to changes in P02 in the lung and blood resulting from the change in lung volume. This explanation is unlikely in Amia for two reasons. First, the rapid response to air blad• der deflation indicates that stretch receptor pathways, not intravascular 02-sensitive stimuli, mediate the response. Although lung deflation would have the effect of decreasing total lung 02 stores, air bladder P02 would not change immedi• ately after deflation, therefore, it could not bring about a chemoreceptor-driven ventilatory response. Secondly, if the change in 02 stores were important in determining the defla• tion reflex, it would be expected to produce a greater number of type I breaths, as in aerial hypoxia in this study (fig. 9), rather than the 100% of type II breaths that were ob• served. Further evidence against a chemoreceptor hypothesis for lung deflation responses is shown by calculating the gain in lung 02 that would occur in a typical type II breath. Assum• ing that a 500 g Amia has an air bladder volume of about 8% of body mass, or 4 0 ml, and the average P02 in the lung is about 113 60-80 Torr (Johansen et al. 1970; pers. obs.), an air bladder with a P02 of 80 Torr has an oxygen store of 4.32 ml 02. If the apparent threshold for detection (3 ml kg-1, fig. 13) of gas removal from the air bladder is used, then 1.5 ml 02 diffuses from the lung to the blood, without being replaced by C02. The new air bladder 02 content and P02 become 2.82 ml 02 and 54.2 Torr, respectively. The fish replaces the lost volume with a 1.5 ml type II breath, which contains about 0.32 ml 02. After the type II breath, the resulting P02 is 58.1 Torr, a change of only 3.9 Torr. Thus, simple calculations suggest it is unlikely that type II breaths contribute signif• icantly to gas exchange. Furthermore, a small change in P02 of 3-4 Torr in the blood after diffusion from the air bladder is unlikely to be undetected by intravascular chemoreceptors (Burleson 1991). Type I breaths, alternatively, which are of larger volume and occur predominantly in hypoxic situations, would appear to serve mainly a gas exchange function by exhal• ing a lower P02 lung gas and replacing it with ambient air containing a higher P02. Additional support for this view of Amia air bladder mechanoreceptors being involved in buoyancy regulation comes from experiments in which these nerves were bilaterally sec• tioned. In this condition, Amia continued type II air-breaths but the air bladder eventually became very distended until the fish floated at the surface, indicating that buoyancy control was severely compromised. In the aquatic anuran amphibian, Xenopus laevis, the lung also becomes distended and the animal loses buoyancy control with lung denervation (Evans and Shel- ton 1984), suggesting that ventilation may also play an impor• tant role in buoyancy control in this species. Bowfin were occasionally subjected to air bladder infla• tions upon approaching the surface. Although the responses are more difficult to interpret, air bladder inflation some• times caused Amia to abandon the approach to the surface but,, more importantly, air bladder inflations failed to inhibit type I air breaths when they did occur. Although this is not conclusive evidence, it does further support the contention that afferent pathways controlling type I and type II air breaths are separate. Pulmonary mechanoreceptor inputs in ectothermic verte• brates appear to play a significant role in modulating the pattern of breathing in those animals that have been studied (see Milsom 1990, for review). For instance, in the African lungfish, Protopterus annectens, lung inflation significantly increased inter-breath interval, and the effect was potentiat• ed by inflating the lung with high 02 concentrations (Pack et al. 1990). It is not known whether equivalent ventilatory responses to chemoreceptor and mechanoreceptor stimulation are present in lungfish; the data from Amia suggest they are different. In the turtle, Chrysemys picta, reductions in lung volume reduced the length of the non-ventilatory period (NVP), but did not affect overall minute ventilation (Milsom and Chan 1986). The change in NVP in the turtle could not be accounted for on the basis of changes in lung 02 stores, as in this study. The general effect of increased pulmonary afferent discharge in Amia, and other ectothermic vertebrates, appears to increase the inter-breath interval (IBI), or NVP, with inflation, and reduce IBI or NVP with deflation. The remain• ing question, for which there are no data, is how these pulmo• nary afferents are integrated in the central nervous system to produce the various breathing patterns in ectothermic verte• brates . The general features of mechanoreceptor involvement in controlling ventilatory patterns in ectotherms are similar to those seen in mammals. Lung inflations in mammals character• istically shorten inspiration and prolong expiration, while deflations shorten expiration and can increase respiratory frequency (Knox 1973) . These reflexes in mammals are collec• tively known as the Breuer-Hering reflex, which are mediated through slowly-adapting pulmonary stretch receptors with afferent fibers contained in the vagus nerve (see Pack 1981). The similarities of ventilatory responses to lung or air bladder inflation and deflations across a broad phylogenetic spectrum indicates that Breuer-Hering-1ike reflexes were selected early in vertebrate evolution. Chemoreceptor Sites in the Control of Air-Breathing Peripheral Sites of Chemoreception Denervation of branchial branches IX and X to the gill arches and, in a few fish, the pseudobranch, failed to abolish the ventilatory responses to aquatic hypoxia. The hypothesis tested was that peripheral 02-chemosensitive sites on the gill arches, innervated by branches of the glossopharyngeal (crani- 116 al n. IX) and vagus (cranial n. X), were responsible for stimulating the type I air breaths during aquatic hypoxia. Mechanoreceptors innervating the air bladder are also inner• vated by a branch of the vagus nerve, but this branch was left intact; only those branches of IX and X innervating the four branchial arches were sectioned. Since type II breaths appear to be influenced mainly by air bladder mechanoreceptors, there was no a priori assumption that all air-breathing responses could be abolished; thus, any involvement of air bladder mechanoreceptors in the air-breathing responses would have remained. It was apparent that eliminating the innervation to the four branchial arches and the pseudobranch did not abolish type I or type II air-breathing, nor did it prevent or attenu• ate the increase in air-breathing during aquatic hypoxia. The failure to reduce type I air-breathing frequency with total branchial nerve section indicates there are probably extra- branchial sites for detecting low P02 - Most experiments on water-breathing fish have also failed to abolish gill ventila• tory responses to hypoxia after branchial and/or pseudobranch denervation (Hughes and Shelton 1962; Saunders and Sutterlin 1971; Randall and Jones 1973; Bamford 1974); however, a recent study indicated that hypoxic ventilatory reflexes are abol• ished by complete branchial nerve section in channel catfish, Ictalurus punctatus (Burleson and Smatresk 1990). In lung• fish, branchial nerve section attenuated, but did not abolish, the ventilatory responses to hypoxemia and cyanide injection (Lahiri et al. 1970). More recently, McKenzie (1990) reported 117 that air-breathing was completely abolished by total branchial nerve section combined with pseudobranch ablation in Amia. In this study, it is possible that failure to abolish the hypoxic ventilatory reflexes were due to intact innervation of the facial nerve (n. VII) to the pseudobranch. The pseudobranch in Amia is a glandular structure lying beneath the epithelium of the palate (Allis 1897). It receives an arterial blood supply via the internal carotid artery, but probably does not sense immediate changes in external water P02 due to its subepithelial location. If the pseudobranch in Amia func• tioned in chemoreception, it would be in position to detect changes in arterial P02. It is not yet known, however, wheth• er the pseudobranch in Amia has a chemoreceptive function or whether it also receives neural innervation from the facial nerve. Cutting its glossopharyngeal innervation had no effect on the responses to aquatic hypoxia. The approach of McKenzie (1990), however, examined the ventilatory resposes to aquatic hypoxia applied for only 15 min, which is probably too short a time period to determine whether hypoxic reflexes were still intact. Amia in this study were exposed to aquatic hypoxia for several hours, which may have caused release of humoral factors, such as catecholamines, that could influence ventila• tion. Adrenaline and noradrenaline have been implicated in the ventilatory responses to hypoxia in water-breathing fish (Peyraud-Waitzenegger 19,79; Aota et al. 1990). However, intra-vascular injections of catecholamines in Amia do not stimulate air-breathing (McKenzie 1990). Despite the demon• stration of 02-sensitive chemoreceptors with afferents con- 118 tained in the glossopharyngeal (Burleson and Milsom 1990; Burleson 1991) and vagal (Milsom and Brill 1986) branches of the gill arches, as well as the pseudobranch (n. IX) nerves (Laurent and Rouzeau 1972), the failure to abolish hypoxic ventilatory responses by denervating these nerves suggests that extra-branchial chemoreceptive sites are probably present in fish. Clearly-defined oxygen receptor sites in air-breathing fish have not been demonstrated, but indirect evidence indi• cates that vascular chemoreceptors stimulate air-breathing in lungfish (Lahiri et al. 1970) and gar (Smatresk 1986; Smatresk et al. 1986). Injections of hypoxic blood or NaCN into the anterior arches of lungfish produces a greater ventilatory stimulus than injections into more posterior arches (Lahiri et al. 1970). In gar, denervation of vagal branchial nerves abolished the ventilatory depression of aquatic hypoxia and NaCN on gill ventilation, but air-breathing reflexes were only slightly attenuated (Smatresk 1987). Although the evidence is limited, the air-breathing fish that have been studied to date may have extra-branchial sites for 0,2 chemoreception that modulate air-breathing reflexes. The walls of the posterior cardinal vein in Amia have been shown to contain chromaffin cells, associated with nerve terminals, which resemble cells of the adrenal medulla in higher vertebrates (Youson 1976). The ultrastructural charac• teristics of these cells indicate they may release catechola• mines since they closely resemble cells derived from neural crest tissue belonging to the APUD (Amine Precursor Uptake and 119 Decarboxylation) series (Youson 1976). Glomus cells in mamma• lian and avian carotid bodies, with a known 02-chemoreceptive function, are also considered part of the APUD series (Pearse 1969). Thus, if the granulated cells in the venous vascula• ture in Amia are indeed similar to the APUD cells of other vertebrates (see Jones and Milsom 1982), they may be involved in chemoreception. Their location in the walls of the poste• rior cardinal vein would place them in an ideal position to monitor blood P02 distal to the air bladder, before the blood enters the gill vasculature. This would suggest a venous location for 02-sensitive chemoreception. There is some experimental evidence for a venous location for chemoreception in water-breathing fish (Barrett and Taylor 1984). It is still not known, however, if such cells are involved in 02 chemoreception in fish. Amia normally increase gill ventilation in aquatic hypox• ia in addition to increasing aerial ventilation (Johansen et al. 1970; McKenzie 1990). In this study, gill ventilation in normoxic and hypoxic conditions did not appear to be affected to as great an extent as air-breathing (Tables III, IV). In contrast with other studies, Amia did not increase gill venti• lation in aquatic hypoxia in this experiment, although gill ventilatory increases in aquatic hypoxia were noted with Amia using a different experimental approach (next section). In previous studies, gill ventilation rates, at similar tempera• tures and levels of oxygenation, were 10 to 100% greater than reported here (Johansen et al. 1970; McKenzie 1990). In those studies, gill ventilation rates in normoxia were higher and 120 increased significantly in aquatic hypoxia. The differences in gill ventilation rates between studies suggests the type of experimental approach used to monitor ventilation may have an effect on the normal gill ventilatory pattern and the re• sponses to hypoxia. Gill ventilation rates have been usually determined using invasive techniques that involve implanting cannulae into the buccal or opercular cavities to monitor ventilation. Gill ventilation rates reported here were deter• mined by direct observation from videotape. The greater lability of air-breathing over branchial responses to aquatic hypoxia was also noted by direct observation in reedfish (Pettit and Beitinger 1985). The large number of type II breaths that occurred follow• ing total branchial denervation in Amia was an unexpected result (fig. 19). It was apparent from the videotape record• ings these fish had considerable difficulty with gas capture and transfer (phases 3 and 4 of the type I cycle), particular• ly during type I breaths; it appeared that abnormally large amounts of gas escaped from the opercular cavities. In these cases, large numbers of type II breaths occurred, probably owing to an inability of the fish to maintain a constant volume in the swim bladder. This is indirect evidence to support the hypothesis that type II breaths are mediated by air bladder mechanoreceptors. The observation that total branchial denervation adverse• ly affects gas transfer during air-breathing in Amia raises questions concerning the efficacy of this experimental ap• proach in delimiting chemoreceptive sites in air-breathing 121 fish. The underlying assumption of branchial denervation experiments is that efferent and afferent information, other than chemoreceptor afferents, carried in cranial nerves IX and X is unimportant in determining the motor output for the branchial pumps, which are mainly innervated by the trigeminal (cranial n. V) and facial (cranial n. VII) nerves (see Nilsson 1984) . Powers and Clark (1942) did observe that cutting the gill innervation of cranial nerve IX (glossopharyngeal) pro• duced gasping movements in trout that were not present when nerve X (vagus) alone was eliminated. Nerve sections in Amia, although affecting the fish's ability to breathe air normally, did not produce any effects that resembled gasping. Nerve fibers eliminated by cutting cranial nerves IX and X to the gills provide motor control for positioning gill arch rakers and vasomotor control for branchial vasculature (Nilsson 1984). Afferent information, other than 02 chemosensory, from baroreceptors (Mott 1951), nociceptors (Poole and Satchell 1979) and gill mechanoreceptors (De Graaf.et al. 1987), would also be eliminated by denervation. The inability of Amia to effectively capture and transfer inhaled air after total branchial denervation suggests that intact branchial motor and/or sensory information is required for normal air-breath• ing function. Gill ventilation, although depressed with re• spect to rate, appeared normal. It is difficult to determine, at present, which nervous pathways and structures innervated by nerves IX and X are important in facilitating normal air- breathing in Amia. The most likely candidates are gill arch motor nerves and mechanoreceptors that control and transduce 122 information about gill arch postion. These functions are probably important for positioning and sensing the changes that occur during air-breathing when the buccal cavity is briefly filled with air. These results would appear to vio• late the assumptions of this experimental approach and raise more questions about the role of branchial innervation in determining ventilatory neuromuscular motor output, particu• larly in air-breathing fish. Central Sites of Chemoreception The hypothesis that central chemoreceptors may be in• volved in the ventilatory responses to hypoxia and/or hyper• capnia was not supported by the EDF perfusion experiments (Table V). The only significant effect on branchial or aerial ventilation occurred with aquatic hypoxia; changing EDF gas concentrations had no effect on ventilation. The manipulation of EDF for the experimental protocol necessitated the use of a different type of arrangement than other experiments in this study. Fish had to be confined to a small box in order to change EDF concentrations. The type of holding box described here, which has been used previously for ventilation studies in Amia (Randall et al. 1981; McKenzie 1990) made differentiating between type I and type II breaths impossible, since air-breathing was distinguished on the basis of changes in opercular pressure during these events. The use of this type of apparatus, which is more confining to the animal, might be expected to produce different branchial or aerial ventilation responses from fish than freely-swimming 123 animals in larger aquaria. Control gill ventilation rates in this experiment were considerably higher than values obtained by direct observation from fish in aquaria (Table IV), despite the lower temperature (15 °C) in the EDF experiment. Air-breathing in Amia is highly temperature dependent (Johansen et al. 1970; Horn and Riggs 1973; pers. obs.), which probably accounts for the lack of air-breathing in aquatic normoxia (Table IV). In the same type of experimental apparatus used here, Amia have been shown to use aerial respiration in normoxia, and increase the fre• quency of air-breathing in aquatic hypoxia (Randall et al. 1981; McKenzie 1990), although the reported frequencies were slightly lower than fish in aquaria in this study. Despite the caveats regarding experimental situations, the relative responses to aquatic hypoxia remain intact regardless of the approach; therefore, the conclusion that EDF manipulations do not affect aerial ventilation would appear to remain valid. In selected species from all classes of terrestrial vertebrates studied thus far, central chemoreceptors have been shown to stimulate ventilation (amphibians: Smatresk and Smits 1991; reptiles: Hitzig and Jackson 1978; birds:- Milsom et al. 1981; mammals: Mitchell et al. 1963; Millhorn and Eldridge 1986). In these terrestrial groups, areas near the ventrolat• eral medulla oblongata, and accessible from the ventricular system (Pappenheimer et al. 1965; Hitzig and Jackson 1978), affect breathing in response to changes in CC>2 and/or pH. In this experiment, cerebrospinal fluid (CSF) was not manipulated directly but, instead, mock EDF was perfused throughout the 124 meningeal space. In fishes, the meningeal space lies between the periosteum of the cranium and the meninx overlying the brain surface; this communicates through blood vessels with the ventricular system and CSF. These pathways have been confirmed with radioactive tracers and vital dyes (see Davson 1967). The dye perfusion results in this experiment indicated that the cranial perfusion method allowed mock EDF to come into contact with areas of the brain corresponding with cen• tral reflexogenic areas of terrestrial vertebrates. The lack of significant ventilatory effects in response to hypercapnia and low pH solutions indicates that Amia do not possess cen• tral chemoreceptive sites analogous to those of terrestrial vertebrates. A recent study in the skate (Raja ocellata) also showed that EDF pH was not correlated with changes in ventila• tion (Graham et al. 1990). The lack of ventilatory responses to changes in 02 in EDF also argues against the hypothesis of a central oxygen chemo- receptor in fish. Experiments in which isolated, spontaneous• ly breathing carp heads are made hypoxemic, show that ventila• tory movements eventually stop, indicating that central hypox• ia has a depressant effect on ventilation (Kawasaki 1980); however, there was no evidence for a depressant effect of central hypoxia on ventilation in Amia. The limited evidence from Amia and Raja would indicate that chondrichthyean and actinopterygian fishes probably do not have central chemoreceptors; it is likely they have evolved with the transition tb terrestriality in Sarcoptery- gian vertebrates. 125 Intermittent Air-Breathing in Amia The intermittent breathing pattern of Amia usually showed an alternation between type I and type II air breaths in normoxic conditions (fig. 2OA). Although there did not appear to be an obvious pattern, spectral analysis of long-term recordings clearly indicated, however, that air-breathing occurred with a period of 30 min (fig. 21B) in normoxic condi• tions, and 10 min in aquatic hypoxia (fig. 21A). If the breathing pattern of Amia were irregular or random, there would be no significant peaks from spectral analysis; the spectrum would resemble, instead, broad-band noise. The results here indicate the pattern is not irregular or random. Although it has been acknowledged that intermittently- breathing fish, when undisturbed, air-breathe somewhat regu• larly (see Milsom 1991), the use of spectral analysis clearly demonstrates the underlying rhythmicity in the breathing pattern of Amia. In previous studies, the rhythmicity of the breathing patterns has been assessed subjectively, or has been examined using measurements of variance, which cannot yield much information about rhythmicity in the data (see van den Aardweg and Karemaker 1991). The finding that Amia breathe rhythmically has important implications for the control of air-breathing in Amia and perhaps other intermittently-breath• ing species. The reasons that rhythmic air-breathing have not been previously demonstrated in air-breathing fish are at least two• fold. First, long-term observations of breathing patterns are usually not recorded in the laboratory. Most of the recording 126 sessions in this study were 8 h in length, with a minimum of outside disturbance to the fish, which allowed time enough for undisturbed breathing patterns to become established. It is well known that behavioral effects, such as the simulation of a predator, increases the inter-breath interval in air- breathing fish (Gee 1980; Smith and Kramer 1986). Second, although spectral analysis has been extensively applied in analyzing the frequency components of ventilation in humans (Goodman 1964; Hlastala et al. 1973; Waggener et al. 1982; Pack et al. 1988), this analysis does not appear to have been used to examine breathing patterns in ectotherms. There is ample evidence that ventilatory patterns of humans exhibit a wide range of periodicites (see van den Aardweg and Karemaker 1991, for a recent review). For in• stance, Goodman (1964) first demonstrated significant ventila• tory oscillations occurring at approximately 50-80 s, 2-3 min, 6-8 min, and 2.5-3.5 h. Hlastala et al. (1973) identified several frequencies in humans, with periods up to 28 min, that were associated with end tidal P02 and PC02, tidal volume and functional residual capacity. Hlastala et al. (1973) suggest• ed further that any system with feedback loops, such as the respiratory system, would be expected to oscillate. Thus, spectral analysis has been shown to be a powerful tool for revealing periodicity in time series data in humans that would otherwise be overlooked. This type of analysis has wide application, and should be applied in studies in intermittent breathers before deciding whether rhythmic oscillations are present. 127 The major question that arises from this analysis is: what is responsible for the underlying periodicity in the breathing pattern in Amia? One possible explanation is that an intrinsic "clock" located in the CNS generates activity every 30 min in aquatic normoxia, which decreases to 10 min in aquatic hypoxia. This concept would be antithetical to the view that air-breathing in fish is an on-demand phenomenon (see Smatresk 1990), generated by peripheral feedback. If air-breathing is indeed on-demand, a rhythmic breathing pat• tern would imply periodic stimulation of peripheral chemo- or mechanoreceptors. Since type I and type II breaths appear related to oxygen availability and buoyancy, respectively, it seems reasonable that either of these factors could contribute significantly to the rhythmic breathing pattern. The evidence from spectral analysis that shows a dominant periodicity identical to the average inter-breath interval for type I breaths is strong evidence that periodic breathing in _ Amia is dependent on feedback from 02 sensitive chemorecep• tors; however, their locations could not be determined from this study. Although centrally-generated periodic rhythmogen- esis cannot be ruled out as a mechanism, a more attractive hypothesis for the generation of rhythmic air-breathing in Amia is that periodic stimulation of peripheral 02-sensitive chemoreceptors are responsible. In aquatic hypoxia, the dominant period is reduced to about 10 min, which is also associated with the mean of inter-breath interval for type I air breaths. Since type I air breaths are sensitive to exter• nal and/or internal P0?, this suggests the rhythmic air- 128 breathing behavior is driven by periodic stimulation of 02~ sensitive chemoreceptors. The periodicities occur in condi• tions of constant aquatic P02, which suggests that internally fluctuating oxygen levels are probably responsible for estab• lishing the ventilatory rhythmicity. Whatever the precise mechanism for generating the breathing pattern, it is apparent that Amia breathe rhythmically in undisturbed conditions, and the dominant frequency of the rhythm changes from normoxic to hypoxic conditions. The next section tests these possibilites using a computer model to simulate the intermittent breathing pattern in Amia. A Computer Model of Intermittent Air-Breathing in Amia A computer model, using empirically-derived observations from this study and elsewhere, was created to simulate the breathing pattern of Amia. The main purposes of this model were to explain the ventilatory responses of Amia to changes in respiratory gases, and test some of the underlying hypothe• ses generated from the empirical data. The basis for the model is the assumption that air blad• der oxygen mixes with systemic venous return before entering the gill vasculature. The vascular anatomy of Amia indicates this is a valid assumption (Randall et al. 1981). Gill venti• lation and its contribution to the oxygenation of the air bladder vasculature was not considered in this model. In reality, arterial blood from a branch of the fourth gill arch in Amia serves as the afferent blood supply to the air blad• der. Oxygen obtained by air-breathing, however, ultimately 129 diffues into the venous, rather than arterial, vasculature; it is this step in aerial ventilation that has been modeled. At present, there appears to be only one comparable model that has simulated the ventilatory pattern of intermittently breathing vertebrates (Shelton and Croghan 1988). Their model used data from an air-breathing teleost, Electrophorus elec- tricus, and an aquatic anuran amphibian, Xenopus laevis, to simulate intermittent breathing patterns in reponse to changes in lung, blood and tissue oxygen stores. Both models use the concept of a blood P02 threshold to trigger air-breathing events, but there are some differences between them. Simulat• ed type I breaths in the Amia model are triggered in response to reductions in blood P02 to a set-point threshold, which is similar to air-gulping simulations in the Shelton and Croghan (1988) model. In both models ventilation is dependent on a feedback response to a decline in blood P02 rather than any explicit change in chemoreceptor discharge. This is more convenient than hypothesizing chemoreceptor discharge per se since the locations and discharge characteristics of the chemoreceptors are not known. A major distinction of the Amia model is the addition of air bladder volume-related air breaths suggested by the empirical data; consequently, there are two independent variables for triggering air breaths. The Amia model also incorporates a method to simulate the pattern of breathing that is characteristic of intermittent breathers. The Shelton and Croghan (1988) model predicts that ventilatory bouts are always evenly spaced. Both models, which use activ• ity of variables decreasing (or increasing) to threshold 130 values and then resetting, are of a general type known as integrate and fire (Glass and Mackey 1988). A schematic diagram of the essential features of the model is in figure 23. A more precise description of the model, including the parameters used, their assigned values and the equations for numerical calculations are given in Appendix 1. The source code, written in Turbo Pascal, used to solve the equations iteratively is listed in Appendix 2. The values used in the model (Appendix 1) are based on empirical data from this study, or estimates from other studies on Amia or Lepisosteus, for a 500 g animal. Air breaths, whether type I or type II, were modeled as discrete events triggered by independent, thresholds (fig. 23). It was assumed that following either a type I or type II air- breath, 02 diffused into the blood flowing past the air blad• der. Oxygen flux after an air breath was modeled in two discrete steps: (1) 02 diffusion from air bladder to blood, and (2) convective flux of oxygen in blood flowing past the air bladder. Oxygen diffusing from the air bladder was assumed not to be replaced by C02, so any decline in air bladder volume was entirely dependent on 02 removal. Respira• tory exchange ratio (R) values in the air breathing organs of air-breathing fish are low owing to the preferential loss of C02 to the environment through diffusion across gill surfaces (see Shelton et al. 1986), and so this seems a reasonable assumption. Oxygen diffusion from the air bladder to blood, according to Fick's Law, is directly proportional to the diffusion 131 Figure 23. A schematic diagram of the essential features of the model used to simulate air-breathing in Amia. See text for details. 132 Air - Breathing Model Volume SenSOr (Stretch Receptor Feedback) r I TYPE II —• AIR AIR BLADDER BREATH Oz DIFFUSION TYPE I EFFERENT AFFERENT Q _ PO2 _ AB Sensor 1 (O2 Chemoreceptor) coeffiencent for 02 in tissue (D02), surface area (SA) for gas exchange and the P02 gradient (AP02) between air bladder and blood, and inversely proportional to diffusion distance (Ax) . D02, SA and Ax are often difficult to determine empirically, so it is convenient to combine them into a single parameter, pulmonary diffusion capacity (DL02). Oxygen flux from air bladder to blood is then the product of DL02 and the P02 gradient. There are no direct measurements of DL02 for Amia, so a value was estimated based on empirical data and estimates from amphibians and gar (Lepisosteus). Measured and estimated - values of DL02 for amphibians are in the range of 0.03 ml min 1 Torr-1 at 25 °C (Glass et al. 1981; Withers and Hillman 1988). Values for air-breathing fish should be less than this, owing to lower alveoarization of the air bladder. Indeed, empirical measurements of air bladder surface area in Lepi• sosteus are approximately 3 to 4 times lower than amphibians of comparable body mass (Rahn et al. 1971), so DL02 was scaled proportionately lower for Amia. A value of 0.0085 ml -1 -1 min Torr for DL02 was used in the model calculations. DL02 was called air bladder diffusion capacity (DAB02) in this model. The convective step was simplified and modeled as a single vessel containing blood flowing past the air bladder (fig. 23) . 02 flux from the air bladder was added to blood flow iteratively in discrete steps in the program. The itera• tion process resulted in the integration of the 02 flux or blood flow rates over each discrete time interval. Since volume (ml 02 or ml blood) is the integral of 0, flux or blood 134 flow rate, volumes of 02 or blood were calculated at each iteration. Therefore, the convective step was modeled as the summation of blood 02 content until the threshold was reached. Blood was thus treated as a pool, rather than a flow rate in the strict sense. Changing blood flow would have the effect of changing the size of the blood pool. Blood 02 content at any level of blood P02 was calculated by interpolating previously published oxygen dissociation curves of Amia (Johansen et al. 1970). Blood flowing toward the air bladder containing low oxygen was designated as affer• ent (aff) blood. Blood distal to the air bladder, after the accumulation of 02 from the air bladder, was termed efferent (eff) blood. Afferent blood in the model is analogous to systemic venous blood returning from the body. This blood is mixed with 02 diffusing from the air bladder. The P02 and 02 content of afferent blood was kept constant in the model. The amount of oxygenation of systemic venous blood in the animal would reflect the degree of 02 extraction from respiring tissues. In other words, the constant afferent input term is analogous to a constant tissue metabolic rate. The issue of metabolism cannot be addressed here, since only the air blad• der and its immediate vascular environment is modeled. Effer• ent blood in the model is analogous to blood that is distal to the air bladder and is a mixture of systemic venous blood and 02~saturated blood from the air bladder. These two blood pools in Amia are mixed before entering the heart, prior to entering the ventral aorta and gill vasculature (Randall et al. 1981). The PCU sensor placed in this location in the 135 model is thus analogous to a chemoreceptor sensing venous P02 between the air bladder and the gill vasculature in the ani• mal. There is experimental evidence for a venous oxygen receptor in water-breathing fish (Barrett and Taylor 1984), and a venous receptor has also been used in a model of venti• latory control in water-breathing fish (Taylor et al. 1968). A threshold value of 20 Torr was used for the efferent P02 sensor, which represents an 02 saturation of 60% (Johansen et al. 1970) measured for ventral aortic blood in Amia (Randall et al. 1981). Air bladder volume was set initially at 42 ml, which is approximately 8% of body mass for a 500 g fish, based upon measurements from this study. A threshold of 1.5 ml (i.e. 3 ml kg-1; cf. fig. 14) less than initial air bladder volume triggered a simulated 1.5 ml type II inhalation of air to replace the lost volume. A key feature of the model is the incorporation of a variable error term, associated with both breath types. The error term was added to take into account the observation that Amia usually lose some proportion of the inhaled volume during the transfer of inhaled gas from the buccal cavity to the air bladder in both type I and type II breaths (Deyst and Liem 1985; this study); therefore, gas capture and transfer is not 100% efficient. The error term was varied over a range from 0 to a maximum of +15% of the mean values for both breath types to examine the effects of inefficient gas exchange on the simulated pattern of breathing. A random walk simulation (Treloar 1975) was used to approximate a Gaussian distribution 136 for breath volumes over the desired error range. For a par• ticular percentage error, a pseudo-random number generator picked a value within the range of the approximated distribu• tion. For instance, if an error of + 10% of the mean value for a type II breath (1.5 ml) is considered, then the computer picked a value at random from the approximated Gaussian dis• tribution ranging from 1.5 ml + (1.5*0.1), or 1.35 ml to 1.65 ml. Results and Comparison with Data from Undisturbed Amia In the absence of any error associated with the simulated breaths (i.e. 100% efficiency), there was a regular alterna• tion of type I and type II air breaths (fig. 24A) . Type I breaths occurred every 22 min after a type II breath, and type II breaths followed 8 min after each type I breath. Efferent p blood P02 ( eff°2^ increased to a maximum of 32 Torr after each type I breath, then declined to the threshold value of 20 Torr every 3 0 min which triggered another type I breath (fig. 24B). It is apparent that type II breaths in the model had over very little affect on PeffC>2 time except slightly length• ening the time to reach the Peff02 threshold. This is not surprising since each simulated type II breath, 1.5 ml of air, contains only about 0.3 ml 02 and, therefore, did not appre• ciably affect the air bladder to blood P02 gradient. With fixed breath volumes, the model produced a constant inter- breath interval for both types of breaths. In this respect, the Amia model produces a constant simulated breath interval similar to the Shelton and Croghan (1988) model for simulated 137 Figure 24. A. Simulated IBI (min) as a function of Cumulative Time (min) for the model with 0 error in both breaths (see text). Open symbols indicate type II breaths in the model output, closed symbols denote type I breaths. B. Simulated changes in efferent blood P02 (Torr) associated with IBI (A, above) as a function of Cumulative Time (min). 138 25n 139 air-gulping events in Electrophorus. The addition of small amounts of random error (+ 10% of the mean breath volume) in both breath types had a marked effect on the simulated breathing pattern (fig. 25A). The timing between type I and type II breaths with variable vol• umes was no longer fixed, which gave the overall breathing pattern an irregular appearance. The introduction of error into breath volumes produced a simulated breathing pattern that qualitatively resembled the breathing pattern of Amia in normoxic conditions (fig. 20A). In comparison, the model and data from bowfin, showed an alternation between type I and type II air breaths, with type II breaths often occurring at short time intervals after type I breaths. In the model, the short interval type II breaths can be explained by their occurrance after type I breaths that were much less than 100% efficient (<12 ml). In these cases, air bladder volume thresh• old was not met and, therefore, a type II breath was trig• gered to raise air bladder volume above its threshold. If type I breaths were at the high end of the distribution, then air bladder volume was also high and the P02 threshold was reached before the volume threshold. When this occurred, there were no type II breaths between successive type I breaths. The simulated pattern produced by the model that resem• bles data from bowfin can be partially explained by examining the simulated Peff02 values over time (fig. 25B). Variable amounts of inhaled air contained different amounts of 02, which changed the air bladder to blood P0? gradient with each 140 Figure 25. A. Simulated IBI (min) plotted as a function of Cumulative Time (min) in the model with +10% error in both breaths. Symbols same as fig. 26. B. Simulated changes in efferent blood P02 (Torr) associated with IBI (A, above) as a function of Cumulative Time (min). 141 Cumulative Time (min) 36-i 18 1 1 1 1 1 1 1 1 O IOO 200 300 400 Cumulative Time (min) n wn n breath. This created variable decay times for Peff02 -*- i° one threshold or the other was reached at different times depending on the value of blood P02 immediately following an air breath. Even small variations in breath volume, there• fore, had profound effects on the qualitative pattern of breathing in the model. The error in breath volumes that changed the simulated ventilatory pattern from constant to variable breath intervals is analogous to incorporating feed• back delays in models of human respiratory control (Glass and Mackey 1988). Despite the irregular appearance of simulated breathing from model simulations with the addition of an error term, spectral analysis showed that the underlying rhythmicity is maintained (fig. 26) . Ten data sets taken at random from the model, all with + 12.5% error in both breaths, were analyzed by spectral analysis as previously described. The largest peak from the spectral analysis of model data was associated with the mean interval of 30 min for between successive type I tnat breaths. This reflected the decay in Peff02 occurred after each type I breath which reached the PeffC>2 threshold, on average, at the same interval in which there was a fixed breath size (fig. 24A) . Thus, the deterministic, regular breathing pattern evident with fixed breath volumes was quan• titatively revealed by spectral analysis, despite the incorpo• ration of an error term that produced irregular-appearing ventilatory patterns. In undisturbed Amia, a periodic breath• ing pattern was also associated with type I breaths that occurred at 30 min intervals (see Results). The occurrance of 143 Figure 26. Averaged periodogram generated from 10 random data sets from the model with +12.5% error in both breaths. 144 145 significant periodicities in the natural breathing pattern in undisturbed Amia is good evidence of agreement between model assumptions and real data. Since breathing frequencies in the model and from Amia can be attributed to the interval between type I breaths, there is the strong suggestion that the under• lying periodicity during air-breathing behavior is dependent on periodic feedback from 02 chemoreceptors in contact with the blood. A sensor placed in the gill arterial vasculature would probably face a nearly constant environmental P02 rather than larger fluctuations in 02 tension. Therefore, a gill arterial receptor would not be expected to produce periodic large fluctuations in output since the receptor in this loca• tion probably equilibrates with the environmental P02. The periodic stimulation of type I breathing in fish with a near- constant aquatic P02 would predict, from model simulations, that a chemoreceptor sensor in the blood should be located between the air bladder and gill vasculature before equilibra• tion with aquatic P02 occurs. This does not rule out a bran• chial receptor located in the afferent gill vasculature. Air bladder volume-related breaths are an important element of the breathing pattern in Amia, since they contribute to the appearance of irregular breathing and tend to mask the normal periodicity of type I breaths. Type II breaths appear to be responsible for making moment by moment adjustments of lung volume on a finer temporal scale than 02~related breaths. The model also accurately predicted the qualitative changes in breathing pattern when aerial hyperoxia (100% 02) or hypoxia (8% 02) was used in place of air (21% 02) in simu- 146 lated breaths. In aerial hyperoxia, PeffC>2 remained above threshold because type II breaths replaced lost volume with 100% 02, not air (21% 02), so type I breaths were not stimu• lated. The larger P02 gradient resulted in a greater 02 flux from the air bladder, causing continuous reductions in air bladder volume that were corrected by type II breaths. The result was a simulated breathing pattern dominated exclusively by type II breaths (fig. 27A). This is the pattern displayed by Amia breathing pure 02 from the aerial phase (fig. 27B) . In simulated aerial hypoxia, the reverse occurred: the blood P02 threshold was reached more often than the air bladder volume threshold, and the model predicted a greater frequency of type I breaths (fig. 28A), as occurs when Amia are made to breathe hypoxic gas (fig. 28B). The similarity between model results and data from undis• turbed Amia, suggest the basic assumptions incorporated in the model are probably realistic. Model predictions with simulat• ed aerial normoxia, hypoxia and hyperoxia, which closely resemble, both qualitatively and quantitatively, data from Amia indicate the breathing pattern is largely governed by two distinct inputs. One input monitors intravascular P02, while the other monitors changes in air bladder volume, which sug• gests that buoyancy regulation is probably a major determinant of the ventilatory pattern. Thus, a full understanding of the ventilatory pattern in Amia should incorporate air bladder volume and blood oxygen information. 147 Figure 27. A. Plot of IBI (min) vs. Cumulative Time (min) for model data with simulated 100% 02 in inspired gas and + 15% error in both breaths. All simulated breaths were type II (open symbols). In this example, QAB was lowered to 10 ml . — i — i — i x x min and DAB02 was reduced to 0.002 ml mm Torr B. Plot of IBI vs. Cumulative Time for a single data set from one Amia breathing 100% 02 from aerial phase in aquatic normoxia shows only type II breaths. 148 251 20- e 10 5-1 100 200 300 400 Cumulative Time (min) 149 Figure 28. A. Plot of IBI (min) vs. Cumulative Time (min) for model data with simulated 8% 02 in inspired gas and + 10% error in both types of breaths. Closed symbols denote simu• lated type I breaths, open symbols indicate type II breaths. B. IBI vs Cumulative Time for a single data set from one Amia breathing hypoxic gas (8% 02) from the aerial phase. Closed symbols indicate type I breaths, open symbols denote type II breaths. 150 25i 20 15 a IO O O IOO 200 300 400 Cumulative Time (min) 25 20 S 15 e IO 5 OO IOO 200 300 400 500 Cumulative Time (min) 151 Evolutionary Implications The current view of the evolution of air-breathing is that lungs originally evolved as gas exchange organs and were retained for that function in extant air-breathing fish such as Amia (see Romer 1972; Randall et al. 1981). A primitive lung in fish, or any gas-filled structure, however, would have automatically assumed a buoyancy function. It is likely, then, that strategies to overcome the problems of buoyancy would have evolved with the development of primitive lungs. The results from this study are important in suggesting that Amia have evolved two different respiratory strategies for coping with the problems imposed by having a single organ for gas exchange and buoyancy regulation. This raises a question concerning the primary selection pressure for the development of air-breathing in Amia: did air-breathing evolve first for gas exchange or to control buoyancy in the aquatic environ• ment? It has been suggested that air-breathing mechanisms evolved from re-organization of pre-existing neuromuscular patterns for aquatic ventilation, feeding or coughing (McMahon 1969; Smatresk 1990). ' Presumably, primitive fishes evolved feeding mechanisms much earlier than air-breathing, so the neuromuscular motor patterns would have been in place before the evolution of air-breathing. Lauder (1980) has suggested that the neuromuscular motor pattern for the feeding mechanism in Amia represents the primitive condition for the Teleostomi. The feeding pattern in Amia involves a suction mechanism in which negative pressures are generated by lowering the buccal 152 floor (Lauder 1980). 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Wilder, B.G. (1877). On the respiration of Amia. Proc. Am. Acad. Adv. Sci. 26: 306-313. Withers, P.C. and S.S. Hillman (1988). A steady-state model of maximal oxygen and carbon dioxide transport in anuran amphib• ians. J. Appl. Physiol. 64:860-868. Wood, CM., J.D. Turner, R.S. Munger and M.S. Graham (1990). Control of ventilation in the hypercapnic skate Raja ocellata: II. Cerebrospinal fluid and intracellular pH in the brain and other tissues. Respir. Physiol. 80:271-290. Youson, J.H. (1976). Fine structure of granulated cells in the posterior cardinal and renal veins of Amia calva L. Can. J. Zool. 54:843-851. Zar, J.H. (1974). Biostatistical Analysis. Englewood Cliffs, NJ:Prentice-Hall, Inc. 164 APPENDIX 1 Table Al. Summary of initial parameters used in the model of intermittent breathing. Values based on a 500g bowfin. Parameter Abbreviation Value Air Bladder Volume VAB 42 ml Air Bladder 02 Content VAB°2 4.32 ml 0- Air Bladder P0o PAB°2 80 Torr Air Bladder -1 Blood Flow ^AB 25 ml min Air Bladder 1 1 Diffusion Capacity DAB°2 0.0085 ml min" Torr" Afferent 02 Content Caff°2 0.0443 ml 02/ml blood c Efferent 02 Content C eff°2 Variable (= aff°2' at threshold) Afferent P02 Paff°2 20 Torr Efferent P02 P eff°2 Variable (= Paff02, at threshold) Hill n Coefficient 1.58 Hill K Coefficient 0.0116 Outline of the Model The model started with initial values for air bladder and blood parameters given in Table Al. Oxygen flux (A02/At) was calculated in two steps, one diffusive and the other convec- tive (see Discussion). The diffusive step, from air bladder to blood, required a P02 gradient to support an 0? flux. In 165 order to establish the air bladder to blood P02 gradient, a p was value for mean blood P02 ( b°2^ calculated. This was accomplished in Equations 1 to 3. An efferent- c : afferent 02 content gradient ( eff_aff°2) ceff-aff°2 = ceff°2 " caff°2 c ec ua s It is apparent that at initial values eff-aff°2 3 l zero. An average blood 02 content (Cb02) is then calculated from ceff-aff°2 as' C b°2 " nCeff„aff02)/2] + Caff02 [2], c iS zero c aS usec to which equals Ca^^02 when eff-aff°2 * b°2 * calculate an average blood P02 (Pb02). The relationship between blood oxygen saturation (S) and blood P02 is not linear, but can be approximated as a sigmoidal function de• scribed by the Hill equation: KPn S/100 = [3] . 1 + KPn Values for K and n (Table Al) were calculated from published oxygen dissociation curves for Amia (Johansen et al. 1970). Equation 3 was linearized by log-transformation and rearranged to calculate P^C^ from Cb02• Equation 3 was used for Cb02 values ranging from 0 to 0.0702 ml 02/ml blood, which is 90% saturated with oxygen at 0.0702 ml Op/ml blood, and corre- 16 6 sponds to a blood P02 of 67 Torr. A simple linear regression was used to calulate content and P02 between 0.0702 and 0.07988 ml 02/ml blood (100% saturation). An upper limit of 110 Torr was set for the blood P02 corresponding with 100% 02 saturation. The determination of P^C^ thus allowed calculation of oxygen flux (A02/At), which was dependent upon air blad• der diffusion capacity (DAB02) and the air bladder to blood P02 gradient, ]]>[ A02/At = DAB02 AP02/At [4], where AP02/At was the discrete change in the air bladder to blood P02 gradient. At each step, AP02/At was the difference between PAB02 and Pj~,02 . Since the change in air bladder av were volume (AVAB) and air bladder 02 content ( AB^2^ depend• ent only upon the oxygen flux, AVAB/At = VAB -^A02/At [5a], t and AVab02 = VAB02 -^A02/At [5b]. t After 02 diffused from the air bladder, the new PAB02 was calculated from the changes in VAB and vAB02 as: PAB°2 = (VAB°2/VAB> * (PB " WVP) ^' where PB is barometric pressure at sea level (760 Torr) and wvp is the water vapor pressure at 22 °C (20 Torr). 167 The oxygen flux calculated from Eq. 4 was used to deter• mine the amount of 02 added to efferent blood at each itera- tion (see Discussion). The calculation of A02/At and QAB at each step by the iteration procedure integrated each variable so volumes of 02 and blood were produced, from which Ceff02 was calculated, £ A02/At Ceff°2 =™ + Caff°2 ^'3 • t The new Ce^^02 value was used as the input variable for equa• tion 1. Equations 1-7 were solved numerically in discrete steps until either air bladder volume threshold (40.5 ml) or arterial blood P02 threshold (20 Torr) was reached. If the volume threshold was met, the program added 1.5 ml of air to the air bladder and new parameters were calculated and used in the next iteration. If blood P02 threshold was reached, the program decreased air bladder volume to an end exhalation volume of 30 ml, and then added 12 ml of air. After each simulated breath, Equations 1-7 were again iteratively solved to calculate new blood and air bladder parameters. 168 APPENDIX 2 PROGRAM AmexII3; { April 27, 1991 } { This program was written in Pascal using Borland's Turbo Pascal compiler (Version 5.0) with Borland's Turbo Pascal Graphix Toolbox (Version 4.0). ) USES Crt, Dos, Gdriver, Printer, Gkernel, GWindow, Gshell; CONST { Set initial constants. ) VenousPo2 =20; { Torr } VenousContent = 0.0443; { Volumes % } BloodFlow =25; { Mis/ Minute } VAR ( Set global variables. ) DataFileName : STRING; TypeOne, TypeTwo : REAL; One, Two : REAL; PROCEDURE GetFileName; { Get file name to store generated values of blood Po2 and lung volume. } VAR Name : STRING; BEGIN WRITELN('This is the Normoxic Version.'); WRITELNJ'What is the File Name?'); READLN(Name); DataFileName := 'B:'+ Name + '.DAT'; WRITE('What is the Noise Level in the Type One Breaths? (%)'); READLN(One); WRITE('What is the Noise Level in the Type Two Breaths? (%)'); READLN(Two) ; TypeOne := (One/100); TypeTwo := (Two/100); END; {GetFileName} PROCEDURE GetArterialPressure(Var A, B, M, N, S, T : PlotArray); { Generates the values for blood Po2 and lung volume for ) { plotting. ) 169 VAR { Variables local to procedure "GetArterialPressure". } ArterialContent, ArterialPressure : REAL; LungPo2, LungVolume, Lung02Volume : REAL; Lung02Content, LungBloodGradient : REAL; I : INTEGER; DataFile : TEXT; FUNCTION Log(X : REAL) : REAL; BEGIN Log := Ln(X)/Ln(10); END; FUNCTION PressureFromContent(ArterialContent : REAL) : REAL; { Calculates blood Po2 from blood content using data from Johansen et al. (1970). The sigmoid curve is solved from a content of 0.00 to 0.0702 ml 02/ ml blood. It is linearly approximated from 0.0702 to 0.07988 ml 02/ ml blood. } VAR b, c, Saturation : REAL; BEGIN IF ArterialContent > 0.07988 THEN PressureFromContent := 110.0 ELSE IF ArterialContent > 0.0702 THEN PressureFromContent := (ArterialContent * 4438.016529) - 244.5087 ELSE BEGIN Saturation := (ArterialContent/7.7988) * 100; b := Log(Saturation/(1 - Saturation)); c := (b + 1.9355)/1.582264; PressureFromContent := EXP(c * Ln(10)); END END; PROCEDURE Iterate; { Calculate new values for all physiological variables. ) VAR MeanArterioVenousContent, LungBloodGradient : REAL; Hold, 02Flux, ArterioVenousGradient : REAL; 170 BEGIN ArterioVenousGradient := (ArterialContent - VenousContent); MeanArterioVenousContent := (ArterioVenousGradient/2) + VenousContent; Hold := PressureFromContent(MeanArterioVenousContent); LungBloodGradient := LungPo2 - Hold; 02Flux := 0.0085 * LungBloodGradient; LungVolume := LungVolume - 02Flux; Lung02Volume := Lung02Volume - 02Flux; LungPo2 := (Lung02Volume/LungVolume) * 740; { PB(760) - wvp(20) = 740 } ArterialContent := VenousContent + (02Flux / BloodFlow); ArterialPressure := (PressureFromContent(ArterialContent)); END; PROCEDURE TypelE; { Exhale for Type I breath. } BEGIN LungVolume := 30; { ml } Lung02Volume := (LungPo2/740)*LungVolume; { ml } END; PROCEDURE Typell; { Inhale for Type I breath. ) VAR DeltaLungVolume, Test : REAL; Al, A2, SinAl, SinA2 : REAL; DeltaA : REAL; BEGIN Al := Random * 360; { Two-step random walk simulation. ) SinAl := Sin(Al * (PI/180)); A2 := Random * 360; SinA2 := Sin(A2 * (PI/180)); DeltaA := (SinAl + SinA2)/2; Test := (12 * TypeOne) * DeltaA; DeltaLungVolume := 12 - Test; LungVolume := LungVolume + DeltaLungVolume; Lung02Volume := Lung02Volume + (DeltaLungVolume * 0.21); LungPo2 := (Lung02Volume/LungVolume) * 740; END; PROCEDURE Type2; £ Inhale for Type II breath. } VAR DeltaLungVolume, Hold : REAL; Bl, B2, SinBl, SinB2, DeltaB : REAL; 171 BEGIN Bl := Random * 360; SinBl := Sin(Bl*(PI/180)); B2 := Random * 360; SinB2 := Sin(B2*(PI/180)); DeltaB := (SinBl + SinB2)/2; Hold := (1.5 * TypeTwo) * DeltaB; DeltaLungVolume := 1.5 - Hold; LungVolume := LungVolume + DeltaLungVolume; { ml ) Lung02Volume := Lung02Volume + (DeltaLungVolume * 0.21); LungPo2 := (Lung02Volume/LungVolume) * 740; END; BEGIN { GetArterialPressure } Assign (DataFile, DataFileName); Rewrite (DataFile); RANDOMIZE; I := 1; { Set initial conditions. } ArterialPressure := 20; { Torr } ArterialContent := 0.044 3; { mis } LungVolume := 40; { ml } Lung02Volume := 5.945911; { ml ) LungPo2 := 80; { Torr } LungBloodGradient := 60.0; A[l,l] = I; A[l,2] = ArterialPressure; M[l,l] = I; M[l,2] = LungVolume; FOR I := 1 to 400 DO BEGIN A[I,1] = I, A[I,2] ArterialPressure; M[I,1] I; M[I,2] LungVolume; S[I,1] I; S[I,2] = 2; Iterate; IF LungVolume < 40.5 THEN BEGIN Type2; S[I,1] I; S[I,2] 4 END ELSE IF ArterialPressure < 20.1 THEN 172 BEGIN TypeIE; S[I,1] := I; S[I,2] := 0; Typell; END; WRITELN(DataFile, A[I,1], A[I,2], M[I,2], S[I, END; Close (dataFile); END; { GetArterialPressure } PROCEDURE ComputeAndDisplay; VAR A, B : PlotArray; M, N : PlotArray; S, T : PlotArray; Ch : Char; XI, X2 : Integer; OneStr, TwoStr : STRING; BEGIN ClearScreen; GetArterialPressure(A, B, M, N, S, T); DefineWindow(l, 0, 5,' XMaxGlb, YMaxGlb-220); DefineHeader(l, 'Blood Po2 Through Time. '); Defineworld(1, 0, 15, 405, 50); SelectWorld(1); SelectWindow(1); SetBackground(0); SetForeGroundColor(12); SetHeaderOn; DrawBorder; DrawAxis(7, -8, 0, 0, 0, 0, 0, 0, False); DrawPolygon(A, 10, 400, 0, 0, 0); DrawLine(20, 41.6, 200, 41.6); DefineWindow(2, 0, YMaxGlb-215, XmaxGlb, YMaxGlb-80); DefineHeader(2, 'Lung Volume Through Time.'); DefineWorld(2, 0, 34.5, 405, 45); SelectWorld(2); SelectWindow(2); SetForeGroundColor(12); SetHeaderOn; DrawBorder; DrawAxis(8, -5, 0, 0, 0, 0, 0, 0, False); 173 DrawPolygon(M, 10, 400, 0, 0, 0); DrawLine(20, 41.7, 200, 41.7); STR(TRUNC(One), OneStr); STR(TRUNC(Two), TwoStr); DrawTextW(25, 38, 1, 'Noise Level: Type I:' + OneStr + '%,'); DrawTextW(130, 38, 1, 'Type II:' + TwoStr + '%'); DefineWindow(3, 0, YMaxGlb-75, XMaxGlb, YMaxGlb); DefineHeader(3, 'Breath Occurance'); DefineWorld(3, 0, -1, 405, 5); SelectWorld(3); SelectWindow(3); SetForeGroundColor(12); SetHeaderOn; DrawBorder; DrawAxis(8, 1, 0, 0, 0, 0, 0, 0, False); DrawPolygon(S, 10, 400, 0, 0, 0); DrawTextW(3, 2.2, 1, 'Type I'); DrawTextW(3, 0.5, 1, 'Type II'); Repeat Until Keypressed; END; { ComputeAndDisplay } BEGIN { main program. ) GetFileName; InitGraphic; ComputeAndDisplay; Repeat Until KeyPressed; LeaveGraphic; END. { main program. } 174