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Beta-adrenergic effects of catecholamines on ventilation in fishes Aota, Sumihisa 1993

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f3-ADRENERGIC EFFECTS OF CATECHOLAMINES ON VENTILATION INFISHES.BYSUMIHISA AOTAB.Sc. (Hons.), The University of British Columbia, 1988.A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCI’OR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(The Department of Zoology)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIANovember 1993@ Sumihisa Aota, 1993.In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of cOQThe University of British ColumbiaVancouver, CanadaDate ZEc / /9DE6 (2/88)AbstractIn this thesis, the suggestion that the catecholamines, adrenaline (AD) and/ornoradrenaline (NA) may have a role in the control of ventilation in fish was tested. Thisidea was developed from the observations that catecholamines could modify ventilationin eels, and that situations that cause changes in ventilation are usually associated withraised plasma catecholamine levels. Specifically, the focus of this thesis was toinvestigate the possible role of plasma NA and AD in 6-adrenergic stimulation ofventilation in fishes.The first study looked at whether it was possible for catecholamines present in thecirculatory system to stimulate ventilation. Ventilation frequency, opercular pressureamplitude, heart rate, dorsal aortic pressure, arterial pH, arterial 02 tension, and plasmacatecholamines concentrations were recorded in rainbow trout, Oncorhynchus mykiss,during normoxia after injection of various dosages of catecholamines. This was to assessif catecholamines injected into fish could change ventilation. AD injection resulted in adepression of arterial 02 tension, hypoventilation due to a drop in ventilation frequency,and a drop in heart rate, while dorsal aortic pressure increased. NA depressedventilation frequency, but opercular amplitude increased, and dorsal aortic pressureincreased. To eliminate the possibility that the ventilatory responses observed were amixed response to both a- and f3-adrenoceptors, I repeated the test using the aantagonist phentolamine, and measured ventilation frequency, opercular amplitude,arterial pH, red blood cell pH, arterial 02 tension and content, and haematocrit. ADinjection after a-blockade had no significant effect on blood gas measurements, possibly11because they had returned to resting levels by the time the blood sample was taken. NAinjection after r-blockade led to a significant increase in opercular amplitude, but theother cardiovascular and ventilatory variables were unchanged. These results suggestthat NA can modify ventilatory output from the respiratory centre.The possible role of catecholamines in the ventilatory response to acute externalhypercapnia was studied in rainbow trout. The ventilatory response to hypercapnia offish pre-treated with the 13-adrenoceptor antagonist D,L-propranolol, was compared tofish pre-treated with D-propranolol (an isomer with minimal 13-antagonistic activity) andsaline pre-treated fish. A sustained 3.6 fold increase in gill ventilation volume wasobserved in the saline and D-propranolol treated groups during the 30 mi interval ofhypercapnia. Fish pre-treated with D,L-propranolol displayed a blunted (1.9-foldincrease) increase in gill ventilation volume. These fish could not maintain ventilationduring hypercapnia. These results indicate that the 13-component of an adrenergicresponse is involved in the usual hyperventilatory response to external hypercapnia.The possible role of catecholamines in the ventilatory response to exhaustiveexercise was investigated, with and without 13-blockade, in rainbow trout. In the controlfish, both ventilation frequency and opercular amplitude increased, PHa dropped, pHincreased slightly, haematocrit and catecholamine levels increased. Nadolol itself haseffects on blood oxygen status, which sets ventilatory effort to a different level thananimals that are not so treated. Fish pre-treated with nadolol had an impaired ability toincrease opercular amplitude, due to the higher level of oxygen present in the blood.However, there was also an inability of the nadolol treated fish to maintain ventilation to111the same extent as the sham treated fish, which was not due to differences in bloodoxygen status. These results demonstrate that catecholamines may have a role in thecontrol of ventilation during recovery from exercise in fish.The ability of catecholamines to alter the activity of respiratory neurons in themedulla was studied in dogfish. First, however, it had to be established thatcatecholamines could cross the blood-brain barrier. To study this, dogfish were injectedwith[3H]-AD or[3H]-NA into the caudal vein to see if these compounds could bedetected in the medulla and cerebellum. The medulla accumulated NA over AD, whilethe cerebellum accumulated equivalent amounts of both. This shows that catecholaminesdo cross the blood-brain barrier in dogfish, and that different areas of the brainaccumulate catecholamines differently.When NA was applied to respiratory-related neurons, there were both excitatoryand inhibitory effects on the activity of these respiratory-related neurons. The excitatoryeffects were blocked by propranolol, indicating that the excitatory effects are a 13-adrenoceptor mediated response. This demonstrates that catecholamines can affectcentrally located respiratory neurons.In conclusion, though the initial ventilatory responses to various environmentalconditions are neural in origin (i.e.02-chemoreceptor mediated), there are situationswhen catecholamines are modulating ventilation, as ventilation could not be maintainedin 13-blocked fish exposed to hypercapnia, or in 13-blocked fish during recovery fromexhaustive exercise. NA is the most likely candidate for stimulating ventilation fish as itcan cross the blood-brain barrier, and can modulate ventilation in fish by its effects onivcentrally located respiratory neurons via 13-adrenergic receptors.VTABLE OF CONTENTSPageAbstract iiTable of Contents viList of Tables viiiList of Figures xList of Abbreviations xiiiAcknowledgements xvChapter 1 General Introduction 1Chapter 2 The effect of exogenous catecholamines on the ventilationand cardiac function in normoxic rainbow trout, Oncorhynchusmykiss. 13Introduction 14Materials and Methods 16Results 23Discussion 48Chapter 3 The effects of D- and D,L-propranolol on the ventilatoryresponses of rainbow trout, Oncorhynchus mykiss, to acutehypercapnia. 52Introduction 53Materials and Methods 54Results 59Discussion 67Chapter 4 The effects of 13-adrenoceptor blockade on ventilation ofrainbow trout, Oncorhynchus mykiss, after exhaustive exercise. 70Introduction 71Materials and Methods 73Results 77Discussion 93viChapter 5 The effects of exogenous catecholamines on the respiratorycentre in dogfish, Scyliorhinus canicula. 99Introduction 100Materials and Methods 103Results 107Discussion 122Chapter 6 General Discussion 128References 140viiList of TablesPageTable 1: Mean values of arterial pH and arterial oxygen tension inpre- and post-injection blood samples. 34Table 2: Mean values of arterial pH, intracellular red blood cell pH, arterialoxygen tension, and arterial oxygen content in pre and post-injectionblood samples following a-blockade. 41Table 3: Catecholamine levels measured in untreated fish injected withcatecholamines. 44Table 4: Catecholamine levels measured in a-blocked fish injected withcatecholamines. 46Table 5: Mean values of various blood respiratory variables after 0, and 30minutes exposure to hypercapnia, and at recovery. 62Table 6: Mean values of various blood respiratory variables pre-exercise andduring 2 hours post-exhaustive exercise. 84Table 7: Mean values of various blood respiratory variables prior to and during4.5 hours following injection of nadolol or saline in resting trout. 91viiiTable 8: The activity of H] in the cerebellum and medulla of the dogfish(counts per minute) following the injection into the caudal vein of[3HJ-ADor[3H]-NA. 108ixLIST OF FIGURESPageFigure 1: Percent change in ventilation frequency before and after injection ofAD, NA and saline. 24Figure 2: Percent change in opercular pressure before and after injection of AD,NA and saline. 27Figure 3: Percent change in heart rate before and after injection of AD,NA and saline. 29Figure 4: Percent change in dorsal aortic pressure before and after injection ofAD, NA and saline. 32Figure 5: Percent change in ventilation frequency in a-blocked fish, before andafter injection of AD, NA and saline. 37Figure 6: Percent change in ventilation amplitude in a-blocked fish, before andafter injection of AD, NA and saline. 39xFigure 7: Gill ventilation volume of rainbow trout during exposure tohypercapnia following pre-treatment with saline, hypercapnia followingpre-treatment with the mock 13-adrenoceptor antagonist D-propranolol,and hypercapnia following pre-treatment with the 13-adrenoceptor antagonistD,L-propranoloL 60Figure 8: Measured plasma catecholamine levels in trout during exposure tohypercapnia following pre-treatment with saline, hypercapnia followingpre-treatment with the mock f3-adrenoceptor antagonist D-propranolol,and hypercapnia following pre-treatment with the 3-adrenoceptor antagonistD,L-propranolol. 65Figure 9a and 9b: Percent change from pre-exercise in ventilation frequency andventilation amplitude during 2 hours post-exhaustive exercise. 79Figure lOa and lOb: Whole blood pH and red blood cell pH pre-exercise andduring 2 hours post-exhaustive exercise. 81Figure 11: Arterial plasma catecholamine levels pre-exercise and during 2 hourspost-exhaustive exercise. 86Figure 12a and 12b: Percent change in ventilation frequency and ventilationamplitude prior to and during 4.5 hours following injection of saline or nadololinto resting fish. 89xiFigure 13: Recording from a curarized and force ventilated dogfish ofrespiratory-related activity from a central respiratory neuron and from thethird branchial branch of the vagus. 110Figure 14: Recording from a curarized and force ventilated dogfish from acentral respiratory neuron and the second branchial branch of the vagus,after NA injection, demonstrating an inhibitory response. 113Figure 15: Recording from a curarized and force ventilated dogfish from acentral respiratory neuron and the second branchial branch of the vagusafter saline injection. 115Figure 16a, 16b and 16c: Recording from a curarized and force ventilateddogfish from a central respiratory neuron and the third branchial branchof the vagus with no treatment, NA injection, and NAinjection following pre-treatment with D,L-propranolol. 117Figure 17: Recording from a curarized and force ventilated dogfish from acentral respiratory neuron and the third branchial branch of the vagusfollowing mechanical stimulation. 120xliLIST OF ABBREVIATIONSPo2: Oxygen tensionPao2: Arterial oxygen tensionCNS: Central nervous systemP0: Opercular pressureVentilation frequencypHa: Arterial blood pHpH1: Intracellular red blood cell pHCao2: Arterial blood oxygen contentHct: HaematocritP: Dorsal aortic pressureA0: Opercular amplitudeAD: AdrenalineNA: NoradrenalineHPLC: High performance liquid chromatographyANOVA: Analysis of varianceS.E.M.: Standard error of the meanVw: Ventilation volumePwco2: Water carbon dioxide tensionPco2: Carbon dioxide tensionPwo2. Water oxygen tensionxliiHb: HaemoglobinMCHC: Mean cell haemoglobin concentrationRN: Central respiratory neuronBV: Branchial branch of the vagusxivAcknowledgementsFirst and foremost, I would like to thank my supervisor, Dr. David J. Randall forhis support and guidance during my studies and the writing of this thesis. I am verythankful of the collaboration of Richard Kinkead and Dr. Steve Perry in Chapter 3,Cohn Brauner in Chapter 4, and Dr. Ted Taylor and Mike Young in Chapter 5. I wouldalso like to thank current and past members of the Randall lab: Nicholas Bernier, CohnBrauner, Larry Fidler, Juan Fuentes, George Iwama, Joanne Lessard, Hong Lin, DavidMcKenzie, Bernice Miller, Mark Shrimpton, and Rong Yang. For very helpful andthought provoking discussions, I appreciate and thank the assistance of Dr. Bill Milsom.For just being there to listen to me ramble on, and thereby allowing me to keep mysanity, I would especially like to thank Michelle King. I would like to thank KellyLangille for keeping me on my toes, by surprising me all of the time. I am also verygrateful for the friendship and support I received from 3 close friends, Joëlle andJonathon Harris, as well as Thunder. I am grateful to my parents, Junpachi and AtsukoAota, and my sister, Eriko Aota, for their support.xvChapter 1General Introduction1Fish breathe by pumping water over the gills at levels that maintain adequaterates of gas transfer. Situations where either oxygen supply decreases, or oxygen demandincreases, lead to an increase in ventilation. Conversely, when there is an increase inoxygen supply due to external hyperoxia, there is a drop in ventilation (Smith and Jones,1982). Stimulation of ventilation occurs very rapidly when the animal is exposed toenvironmental hypoxia or exercise, which implies that the initial increase in ventilation isneural in origin, resulting from the stimulation of oxygen sensitive chemoreceptors, mostlikely located within the gills (Dunel-Erb et al., 1982; Milsom and Brill, 1986; Burlesonand Smatresk, 1990; Burleson, 1991).Pharmacological studies have shown that circulating catecholamines can affectventilation in fish (Peyraud-Waitzenegger, 1979; Peyraud-Waitzenegger et a!., 1980;Randall and Taylor, 1989; Taylor and Wilson, 1989; McKenzie et a!., 1991a). It is welldocumented in fish that catecholamine levels in the blood are elevated during periodswhen oxygen delivery to tissues may be compromised, such as following exhaustiveexercise (Primmett et a!., 1986). Other stimuli that cause ventilatory increases, likehypoxia (Randall, 1982; Boutilier et a!., 1988), acidosis (Boutilier et a!., 1988), andhypercapnia (Perry et a!., 1989) are associated with increased levels of catecholamines.Catecholamines have many effects on the physiology of fish. For example, they affectthe distribution of red cells in the gill vasculature (Holbert et a!., 1979); oxygen transportin the blood (Nikinmaa, 1982b); red blood cell volume and intracellular pH (Nikinmaa,1982a); heart rate and cardiac output (Wood and Shelton, 1980a, b).Catecholamines can modify ventilation frequency and stroke volume/tidal volumein fish and mammals. Whelan and Young (1953) reported that intravenous2catecholamines stimulates correlation between the ventilatory response to hypoxia andthe size of the increase in plasma catecholamines in fasting men. In the same study, theyshowed that the ventilatory response to hypercapnia was also positively correlated withincreases in AD. In normal, human subjects, 13-adrenergic blockade resulted in areduction of exercise hyperventilation (Scheen et a!., 1976). As well, it has beenobserved that in the neonate, the f3-adrenergic blocker timolol in high dosages resulted inbouts of apnea (Olson et a!., 1979).In fish, there is some evidence for a link between ventilatory changes and therelease of catecholamines into the circulation. Peyraud-Waitzenegger (1979) andPeyraud-Waitzenegger et aL (1980) showed that injection of catecholamines into eels(Anguila anguila) resulted in two responses, depending on the season. During thewinter, injection of NA and AD produced a hypoventilatory response, and treatment withthe -adrenoceptor blocker phentolamine prevented the response, suggesting that thehypoventilation was due to stimulation of cr-adrenoceptors. During the summer,intravenous injection of catecholamines increased ventilation, and this response could beblocked by the 13-antagonist propranolol. Catecholamine infusion in the bowfln, Amiacalva, also resulted in a stimulation of gill ventilation (McKenzie et aL, 1991a).Release of endognous catecholamines into the circulation of fish is dependent onthe lowering of oxygen content (Perry et a!., 1989). In trout exposed to normoxichypercapnia, there was an elevation of plasma catecholamines, and a decline in bothwhole blood pH and oxygen content. If, however, oxygen content was maintained bysubjecting the fish to hyperoxia, plasma catecholamines did not increase even in the faceof severe reductions of whole blood or red blood cell pH.Infusion of acid into trout, or exposure of the animal to severe hypoxia results in3a rise in ventilation, and there is an associated increase in plasma catecholamine levels(Aota et at., 1990). Administration of propranolol partially prevented thehyperventilatoiy response in fish, but the release of catecholamines was not affected inthe normoxic or hypoxic fish. When the animal was exposed to hyperoxia, infusion ofacid did not result in a change in circulating catecholamines or gill ventilation. Onepossible explanation for these observations is that the increase in ventilation that occursis mediated by the release of catecholamines into the circulation. It appears that oxygencontent is the stimulus for both the increase in ventilation and the release ofcatecholamines into the blood. The site of action of the catecholamines could beperipheral or central, but was blocked by propranolol, and therefore involves 13-receptors.Propranolol could reduce the hyperventilation during hypoxia by hindering bothperipheral chemoreceptors and sites within the central nervous system, as propranololeasily crosses the blood-brain barrier (Hoffman and Lefkowitz, 1990).The initial modification of ventilation in fish during hypoxia is a rapidly occurringevent, which implies that the response is neurally mediated. As the stimulus for theseeffects is low levels of oxygen, the assumption made is that the neural pathway involvedis via some oxygen sensitive chemoreceptor. Milsom and Brill (1986), recording nervefibre activity from an isolated gill preparation, found fibres that increased firing withenvironmental hypoxia. They postulate that these receptors are closely associated withthe gill vasculature, because all of the fibres were sensitive to changes in the oxygen levelor flow rate of the perfusate, but not all were affected by changes in the oxygen tension(Pa2) of the bathing fluid. As well, the latency to changes in perfusate Po2 was short,while the response to a variation in the bathing fluid Po2 was slow.4Recent evidence indicates the existence of two chemoreceptor systems in fish.Externally applied NaCN stimulated bradycardia and ventilation, while administration ofNaCN internally results in a stimulation of ventilation only (Burleson and Smatresk,1990) in the channel catfish. By using the latencies for the cyanide injections, and nervesection experiments, Burleson and Smatresk (1990) demonstrated that the gills are themost likely location for 02 chemoreceptors. They propose that the location of theexternal chemoreceptors that control heart rate is superficial, or close to the surface ofthe gill, and in the primary epithelium of the gill filament facing the water flow. Theinternal chemoreceptors that regulate ventilation are probably deep in the primaryepithelium of the gill filaments (Dunel-Erb et a!., 1982) and are sensitive to efferentblood oxygen levels.In mammalian studies, there is evidence for /3-adrenoceptors mediating oxygenchemoreception. Folgering et al. (1982) investigated the question in cats and rabbits.Bolus injections of AD and NA resulted in a transient inhibition, followed by a distinctexcitation of chemoreceptor discharge, while isoproterenol (a specific 13-agonist) resultedin excitation of chemoreceptor discharge only. The excitation could be blocked bypropranolol or metoprolol, suggesting that the catecholamines excite chemoreceptors bya /3-adrenergic action. Chemoreceptor response to hypoxia could be reduced orabolished using blocking agents, implying that these adrenergic receptors may be anintegral part of oxygen chemoreceptor function.Milsom and Sadig, (1983) used both bolus injections and infusion of NA toappraise the activity of single nerve fibres from the rabbit carotid body in normoxia andhypoxia. The major effect of NA was to excite chemoreceptor fibres dose-dependently.As well, there is a strong inverse relationship between the relationship between the level5of stimulation of the chemoreceptor by any given dosage of NA, and the level of Pao2.Propranolol abolished NA effects on the chemoreceptor, and reduced the chemoreceptorresponses to hypoxia. This indicates that 13-adrenergic receptors may be an essentialcomponent of the oxygen chemoreception mechanism. Propranolol also resulted in alowering of resting activity levels, indicating that catecholamines may contribute tochemoreceptor activity during normoxic normocapnic conditions.There are also several studies that show that catecholamines are not involved inperipheral oxygen chemoreception in some mammals. A study by Hudgel et a!. (1986)demonstrated that there is no functional f3-adrenergic activity in the carotid body of bothawake and anaesthetized goats. They observed that the ventilatory response toisoproterenol infusion was not curtailed by carotid body denervation, ventilatoryresponses to hypoxia were not quelled by 13-adrenoceptor blockade, and there was noincrease in single fibre carotid body chemoreceptor activity as a result of intravenousinfusion or bolus injection of isoproterenol. They attribute these results to speciesdifferences between all of the animals used with regard to the presence and importanceof carotid body f3-adrenergic receptor involvement in 02 chemoreception.Work by Mulligan et a!. (1986) indicates that f3-adrenergic mechanisms do notplay a role on 02 chemoreception in cat aortic bodies. They recorded the activity of theaortic body to see if 13-blockade would eliminate aortic chemoreceptor responses tohypoxia. Following propranolol application, chemoreceptor responses to hypoxia werenot diminished. This does not support the idea 13-receptors have a role in 02chemoreception. A possible explanation of this observation may be related to the use ofthe aortic body rather than the carotid body, for there is no guarantee that the twoorgans use the same mechanisms. As the authors discussed, the /3-adrenergic mechanism6in the carotid is stronger than that in the aortic body. What they have shown is that 13-receptor mediated mechanisms do not have a role in the °2 chemoreception in the aorticbody.As there is evidence both for and against involvement of catecholamines inperipheral chemoreceptor mediation in mammals, more recent work has looked for thepresence of catecholamines in the mammalian peripheral chemoreceptor system. Inadult rats, 41% of carotid body afferent neurons were found to contain tyrosinehydroxylase, which is the rate-limiting enzyme in catecholamine biosynthesis(Finley et aL, 1992). A majority of these neurons also contained DOPA decarboxylase,which is the dopamine-synthesizing enzyme. These neurons, however, did not containdopamine f3-hydroxylase, which is the NA-synthesizing enzyme. From this information, itwas inferred that these neurons are dopaminergic, and that dopamine is involved incarotid body chemoreception.The levels of dopamine and NA present in the carotid bodies was studied byHertzberg et aL (1990) in newborn rats. They found that the content of dopamine andNA in the carotid bodies both increased, with the peak in dopamine levels occurring 6-12hours after birth, and NA peaking 2-4 days after birth. These changes in catecholaminelevels in the carotid body were followed by the development of the chemoreflex, i.e.changes in ventilation following changes in ambient gases. With respect to time course,the development of the chemoreflex was correlated with the decrease in dopamine. Itwas suggested that the emergence of the chemoreflex was due to the removal of aninhibitory dopamine chemosensory mechanism. The change in NA content was thoughtto be a result of an ingrowth of new sympathetic nerve fibres.7In the rabbit, the release of dopamine and NA was studied using twochemoreceptor stimulants, hypoxia and nicotine (Gomez-Niño et a!., 1990). Stimulationwith hypoxia resulted in a predominant release of dopamine, whereas NA releasedominated after stimulation with nicotine. This selectivity was not complete, as smallamounts of NA were released during the hypoxia stimulus, and dopamine was alsoreleased in small amounts in response to nicotine. It is suggested that these stimuli(hypoxia versus nicotine) are activating different mechanisms for mobilizing thesecatecholamines from the carotid body.Wang et a!. (1992) have demonstrated that both NA and dopamine are present inthe cat carotid body, and that these catecholamines co-exist in the same cells. This doesnot conflict with the results of Gomez-Niño et a!. (1990), as Wang et a!. (1992) suggestthat there are different sub-cellular distributions and/or selective mechanisms for releaseand re-uptake of dopamine and NA in the carotid body.Tyrosine hydroxylase was localized in post-mortem human carotid bodies, but thedopamine, DOPA decarboxylase, NA, and dopamine /3-hydroxylase were not (Kummerand Habeck, 1992). This indicates that, in man, catecholamines are not involved in thechemosensory mechanisms of the carotid body.Whether or not catecholamines, specifically dopamine and NA, are involved inperipheral chemoreceptor transduction mechanisms in mammals is obviously notresolved. Again, these differing results may be due to species-specific differences.In fish, using an isolated perfused gill preparation, Burleson (1991) showed thatcatecholamines and f3-agonists in the perfusion fluid resulted in either no effect or aninhibition of activity. Propranolol also caused an inhibition of activity, which may be8related to the non-specific effects of propranolol rather than a 13-blocking effect. InAmia calva that had the gills denervated, injection of catecholamines still had an effect,that is the animal still did increase gill ventilation (McKenzie et a!., 1991b). Theventilatory increase to cyanide injection was abolished in denervated Amia, implying thatthe peripheral chemoreceptor response was abolished. These observations suggest thatalthough the peripheral chemoreceptor system is important to ventilatory responses,there appears to be another component to ventilatoiy increases, although the site ofaction is not known. Catecholamines may have a part in this component of the response.The peripheral chemoreceptors, however, are not the only sites of action for endogenouscatecholamines, indeed in fish it seems highly unlikely that the effects of catecholamineson ventilation are by peripheral chemoreceptor stimulation. The possibility exists ofcatecholamine effects within the central nervous system.The gills of fish are ventilated by buccal and opercular pumps that pass watercontinuously over the gills. These pumps are operated by the contractions of cranialmuscles innervated by motorneurons in the brainstem (Ballintijn, 1982). Transections ofthe fish brain that leave the medulla oblongata intact do not disrupt rhythmic ventilation(Shelton, 1959), although there are changes in the ventilatory pattern (Burleson andSmatresk, 1989), suggesting that the higher centres of the brain do have effects onbreathing. A longitudinal strip of nervous tissue with respiratory-related neurons hasbeen located on each side of the midline along the entire medulla (Ballintijn, 1982).This tissue consists of the trigeminal, facial, glossopharyngeal and vagal motor nuclei; thedescending trigeminal nucleus and the reticular formation next to these nuclei. This areais considered the medullary respiratory centre of fish (Ballintijn, 1982). Retrogradehorseradish peroxidase tracing of respiratory neurons has shown that these neurons are9distributed in a consecutive series in the brainstem of elasmobranchs (Withington-Wrayet aL, 1986), and recording of efferent activity from these nerves shows that the branchesof the V, VII, IX, and X cranial nerves fire in order of the anterior-posteriorarrangement of the motor nuclei in the brainstem (Barrett and Taylor, 1985). Thereticular formation is thought to be the source of the respiratory rhythm. This centralrespiratory rhythm generator in fish appears to be highly redundant and diffuselyorganized, because lesions anywhere in the reticular formation do not stop rhythmicbreathing unless the area is large (Ballantijn, 1988). Central effects of catecholamines onventilation would most likely be on one of these components, changing the drive ofeither the central rhythm generator, which sets the frequency of breathing, or the centralpattern generator, which sets the depth/pattern of breathing. In fish, not much work hasbeen done on the central effects of catecholamines, so I will now review mammalianstudies to see if there is any evidence for central effects of catecholamines on breathing.Hilaire et a!. (1989) looked at the modification of ventilation as a result of NAapplication to the medulla in newborn rats. When NA was added to the caudalventrolateral pons, there was a decrease in respiratory frequency, whereas the use of ana-adrenergic antagonist, yohimbine, resulted in an increase in respiratory frequency. Aswell, a depression of ventilatory frequency that occurred when the caudal ventrolateralpons were electrically stimulated, was blocked by yohimbine. The authors suggest that anoradrenergic inhibitory drive originating from the caudal ventrolateral pons modulatesactivity of the medullaiy respiratory generator by an a-adrenergic mechanism.Farber et a!. (1981) looked at the higher centres of the brain (rostralhypothalamus), and possible catecholamine influence on breathing in rats. The rostralhypothalamus was investigated as there is the suggestion that the increase in breathing10frequency that occurs during hypoxia is in part due to hypothalamic mechanisms (Millerand Tenny, 1975; Tenny and Ou, 1977). When NA was infused into the rostralhypothalamus, an increase in ventilation frequency was observed. NA can inhibit cellfiring in the rostral hypothalamus (Winokur and Beckman, 1978), so the authors suggestthat the increase in ventilation frequency was due to suppression of an inhibitorypathway in the rostral hypothalamus. They then infused lidocaine, which inhibitsneuronal firing in the CNS (Ritche and Green, 1980), and noted a similar stimulation,providing corroborative evidence that the stimulatory effect of NA on breathingfrequency may be due to inhibition of neural pathways.Although these studies are not in the medulla of the mammals, it does show thatboth central stimulatory and inhibitory effects of catecholamines can be observed toaffect ventilation, and so catecholamines could have an effect in the respiratory centre ofthe medulla. In fish, there are a few studies on the effects of catecholamines centrally.Studies have shown that in the eel (Peyraud-Waitzenegger, 1979) and trout (Nekvasiland Olson, 1986) catecholamines can cross the blood-brain barrier, so it is possible thatcirculating catecholamines have access to the CNS, where they may have direct effects onventilation.This thesis focuses specifically on the excitatory f3-adrenergic effects on ventilationin fish. The effects of a-adrenoeeptor stimulation, and possible inhibitory effects on fishventilation, though occasionally observed, were not studied. This is because fish aremore likely to be in conditions when the ability to increase or at the very least, maintainventilation would be important to the animals survival, so I examined the possibility thatplasma catecholamines have a role in maintaining ventilation through stimulation of f3-11adrenoceptors when the fish would most likely require it. However, it must be kept inmind that the cr-adrenoceptor effects, may be important in understanding the influenceof circulating catecholamines on ventilation in fish.Three aspects of the problem of catecholamine involvement in /3-adrenergicincrease of ventilation in fishes were investigated. First, do catecholamines that arepresent in the blood affect ventilation? This will be assessed by injecting varyingconcentrations of NA and AD into trout to see what effects this has on the ventilation oftrout. This will also be assessed by blocking cr-adrenoceptors with phentolamine, toremove any possible a-adrenergic effects on ventilation. The second focus is to look atthe potential situations when catecholamines may be having a role in mediatingbreathing. As acidosis causes a release of catecholamines, external hypercapnia will beused to both mobilize catecholamines and stimulate ventilation. The interaction betweenventilation and catecholamine will be evaluated by using a /3-blocker to see if ventilationwill be affected by the blockade. Exhaustive exercise is another situation wherecatecholamines are thought to have a role in modulating ventilation, so the ability of fishto breathe following exhaustive exercise and /3-blockade will be followed. The final focusof this thesis will be to look at the central effects of catecholamines on fish breathing.This will be done by pressure injecting catecholamines onto respiratory-related neuronsin the area of medullaiy respiratory neurons and observing their effect on ventilation.12Chapter 2The effect of exogenous catecholamines on ventilation and cardiac function in normoxicrainbow trout, Oncorhynchus mykiss.13IntroductionThe role of plasma catecholamines in the control of breathing in fishes has beenthe subject of much debate. Increases in ventilation in trout have often been associatedwith an increase in endogenous plasma catecholamines. It was postulated, therefore, thatcirculating catecholamines may stimulate the respiratoly centre, which would bemanifested as an observed increase in ventilation (Randall and Taylor, 1991). However,both stimulatory and depressant effects of circulating catecholamines have beenobserved, which might be partly the result of seasonal and species-specific differences.In the eel, Peyraud-Waitenegger (1979) demonstrated that a bolus injection ofcatecholamines stimulated ventilation in summer, but depressed ventilation in winter.These two responses appear to be the effect of Cr- (winter eels) versus 13- (summer eels)adrenoceptor activation. In rainbow trout, Kinkead and Perry (1990) observed only atransient or persistent hypoventilatory response to AD and NA injections, respectively.Catecholamines also have a marked action on the cardiovascular system and blood °2content, so the effects of catecholamines on breathing could be secondary to these effectsor due to a direct stimulation of breathing. The null hypothesis for this study was thatexogenous catecholamines injected into the circulatory system does not affect ventilationin trout. This was investigated by the injection of various dosages of catecholamines intorainbow trout.As observed in Peyraud-Waitzenegger’s (1979) studies, catecholamines can haveboth stimulatory and depressant effects on ventilation, due to the stimulation of eitherthe Cr- or 13-adrenergic receptor system. From simply injecting different dosages ofcatecholamines in trout, the observed response could be due to the stimulation of both14systems. There exists the possibility that stimulation of the c-adrenergic receptors couldbe masking or interfering with part of the 13 response. Therefore, in a second series ofexperiments, I used fish that had been pre-treated with an a-blocker, phentolamine. Thepurpose of using this blocker was to remove the effects of a-receptor stimulation. Inother words, the a-blocker would allow me to stimulate the 13-adrenoceptor system only,and so the observed responses were a pure /3-adrenergic response, and not the result ofthe stimulation of both adrenoceptor systems.15Materials and methodsExperimental animals.Rainbow trout (Oncorhynchus mykiss, average 439 ± 12 g (mean ± 1 SEM)) froma local hatchery (West Creek Trout Ponds) were held outdoors in a large fibreglass tank.They were fed commercial trout pellets once a week, and were not fed at least 48 hoursbefore surgery. Aerated, dechlorinated Vancouver tap water flowed through the tank,the temperature ranging from 10 ° to 15 ° centigrade (May through mid-September).The temperature of the water used in each experiment was the same as that in theholding tanks. Temperature varied ± 0.5 °C during each experiment. There were sixfish used in each treatment group.Surgical procedures.Series I: Ventilatoiy responses to catecholamine injections.All surgery was done under general anaesthesia. After catching the fish, it wasinitially anaesthetized in a 1:10,000 solution of tricaine methanesulfonate, MS-222(Syndel Laboratories) buffered to pH 7.5 with sodium bicarbonate. The fish was thenweighed and transferred to an operating table similar to the one used by Smith and Bell(1967). During surgery, the gills were irrigated with a less concentrated anaestheticsolution (1:20,000 MS-222). All animals had the dorsal aorta chronically cannulated withpolyethylene tubing (PE-50, Intramedic), using the technique of Soivio, Nynolm, andWestman (1975). This cannula was used for blood sampling and drug injection purposes.A second cannula (PE-190) was placed through a small hole drilled in the operculum,such that the flared end was resting against the inside the operculum. This cannula was16used for measurement of opercular pressure (P0) and ventilation frequency (fg). Aftersurgery, the animals were force ventilated with dechlorinated water until they came outof the anaesthesia. They were then placed singly in black perspex boxes (volume = 2.51)with each cannula led out of the box to a pressure transducer. Water was passedthrough an aeration tower to ensure that the water going to the boxes was normoxic.The fish were given 48 hours to recover from the surgery before any experimentalmanipulations were performed.Series II: Ventilatoiy responses to catecholamine injection following -adrenoceptorblockade.In this series, the dorsal aorta was cannulated as described above. For theventilation parameters, a different technique was employed. A flat 1 cm2 brass plate wassutured to each opercular flap. These were used to measure the displacement of theopercular flaps and the frequency of the displacement (see below, Ventilatory andcardiovascular measurements) which were used as a measure of ventilatory effort. Theleads from the plates were held in place by a single suture anterior to the dorsal fin.After surgery, the animals were treated as described in Series I, above. The reason forchanging to this monitoring system was that it was not as susceptible to swimmingmotion and water current artifacts as the opercular pressure method was. I did check tosee if the changes ventilatory effort that I was measuring with each technique gavesimilar relationships by preparing a few animals with both recording devices. Thealterations observed with both methods were identical.17Experimental protocol.Series I: Ventilatoiy responses to catecholamine injections.The animals in this series received bolus injections of either AD or NA. Fourlevels of catecholamines were chosen based on literature values for endogenouscatecholamine release in fish (Hart et a!., 1989; Kinkead and Perry, 1990, 1991). Theconcentrations I wished to achieve in the animal were 1, 10, 50, and 100 nM undernormoxia. The actual values obtained in the fish were close to the values I wanted toachieve (Table 3,4). Dosages that were injected to give final values higher than 100 nM(ie 250 nM) caused animals to struggle violently and data were not used. For the controlcondition, Cortland saline (Wolf, 1963) carrier alone was injected into the fish to be surethat the effects that I observed were not a direct result of the injection itself. Eachanimal received both catecholamines at the same dosage separately, with a three hourrecovery period between the first and second injections. NA was injected first in half ofthe treatments, AD in the other half. There was no effect of the order of catecholamineinjection on the response observed.Series II: Ventllatoiy responses to catecholamine responses following a-adrenoceptorblockade.The fish in this series received an injection of phentolamine, which is an aadrenoceptor blocker (Hoffman and Lefkowitz, 1990). The amount of phentolamineadministered was 2 mg kg1, 2 hours before the start of the experiment, that is before theinjection of the catecholamines as described above.Blood samples.18Series I: Ventilatoiy responses to catecholamine injections.Two blood samples of 1.0 ml were taken from the dorsal aortic cannula for eachcatecholamine injection, one control (cont.) prior to injection and the second six minutesafter the injection (expt.). For each 1.0 ml sample, 500 l was used to measure arterialblood pH (pHa) and arterial blood oxygen tension (Pao2). The balance of the blood wascentrifuged and the 250 tl of plasma removed and frozen in liquid nitrogen. The frozenplasma was stored in a freezer at -80 °C for later analysis of catecholamines. The redblood cell fraction remaining in the centrifuge vessel was re-suspended in the remainingplasma plus 250 jil cDortland’s saline (to replace the volume of plasma removed for thecatecholamine assay), and re-injected into the fish.Series II: Ventilatoiy responses to catecholarnine injection following ci-adrenoceptorblockade.Again, blood samples of 1.0 ml were withdrawn from the dorsal aortic cannula asdescribed above. Besides the measurements described above, I also measured red bloodcell pH (pHi), arterial blood oxygen content (Cao2), and haematocrit (Hct). The reasonfor adding these additional measurements in this series is that it is possible that the cradrenoceptor blockade may affect blood acid-base and/or oxygen status directly, so thesevalues were compared before and after the administration of the drug. As the red bloodcell fraction in this series was used to determine p1-I, 1.0 ml of Cortland saline wasinjected back into the fish to replace the volume of blood withdrawn.Ventilatory and cardiovascuiar measurements.In the first series, the opercular cannula was connected to a pressure transducer19(Statham P23BB, AST/Servo Systems Inc.). The pressure transducer was connected toan amplifier, which in turn was connected to a six-channel chart recorder (Gould Brush260, Gould Inc.). From the trace, gill ventilatory frequency (ft, breaths mm-i) andopercular pressure (P0, kPa) could be measured. The latter were used as an indicationof ventilatory stroke volume, assuming that the resistance does not change. Theventilation was measured in 7 periods of 1 minute each: control (10 minutes prior toinjection), 0 (after completion of injection), 2, 5, 10, 15 and 20 minutes post-injection.Ventilation frequency was calculated by counting the pressure peaks for the full minutein each of the assigned time intervals. Opercular pressure was taken as the average peakpressure averaged over 30 seconds, measured from 10-second intervals at 0, 25, and 50seconds in each designated minute. The dorsal aortic cannula was attached to a secondpressure transducer (Statham P23BB), which allowed heart rate (fh, beats mm-i) anddorsal aortic blood pressure (PDA, kPa) to be recorded on the above chart recorder. Thecardiovascular measurements were taken in 6 periods of one minute each: control (10minutes prior to injection), 0 (after completion of injection), 2, 5, 15, and 20 minutespost-injection. Heart rate and dorsal aortic pressure were measured in the same way asfg and P0,. Readings were not taken at 10 minutes, as the re-injection of the blood cellsran partially into this time period.In the second series, the leads from the opercular plates were connected to adevice that measures the difference in transopercular impedance between the two plates.The variation in this impedance during ventilation was recorded as an operculogram, andallowed me to record fg and opercular amplitude (A0). A0 was used as an indication ofventilatory stroke volume. This device is based on an operculogram designed by Peyraudand Ferret-Bouin (1960); and Peyraud and Piquemal (1962). The ventilation was20measured using an AST 286 computer with a Data Translation data acquisition board,running the program Labtech Notebook (Interworid Electronics and Computers). Thecomputer then calculated fg, and A0.Although in each experimental group, water temperature was constant, there werechanges in temperature between treatments (May -September). As water temperaturechanges, there are associated changes in both resting ventilation and cardiac function(Hughes and Roberts, 1970; Randall and Cameron, 1973; Barron et a!., 1987), andtherefore significant differences between the various experimental control groups. Toremove the effects of temperature, ventilation and cardiovascular measurements areexpressed as percent change.Analytical techniques.Measurement of whole-blood oxygen tension was made using a RadiometerCopenhagen E5046 Po2 electrode (Bach Simpson) in a Radiometer D616 thermostattedcell and a Radiometer PHA 930 Po2 module together with a Radiometer PHM 71 acid-base analyser. The Po2 electrode was calibrated using nitrogen gas and air-saturatedwater. Blood oxygen content was measured according to the method of Tucker (1967),using a second E5046 Po2 electrode in a thermostatted Tucker chamber. Whole bloodpH was measured using a Radiometer G297/G2 glass capillary electrode in a RadiometerG297 water jacket with the PHM 71 acid-base analyser. Calibration of the pH electrodewas made using Radiometer Precision Buffer Solution Standards S1510 and S1500.Intracellular red cell pH was measured with the same electrode, using the fast freezethaw technique (Zeidler and Kim, 1977). Haematocrit was determined by centrifugingthe blood in a heparinized capillary tube for 5 minutes at 11,500 rpm in a Damon IEC21MB microhaematocrit centrifuge. Plasma AD and NA levels were detennined onalumina-extracted plasma samples using high pressure liquid chromatography (HPLC)based on Woodward (1982). The HPLC incorporates a Waters 460 ElectrochemicalDetector using a glassy carbon electrode (applied potential = +0.60 V), a reverse-phaseWaters Plasma Catecholamine Column, a Waters Model 510 HPLC Pump with pulsedampeners and a Waters U6K Universal Liquid Chromatograph Injector (WatersChromatography Division of Millipore Ltd.). Concentrations were calculated by anintegrator (Waters 746 Data Module) connected on-line to the electrochemical detector.Statistical analysis.Data are presented as means ± 1 Standard Error of the Mean (± 1 S.E.M.).Statistical significance of data was determined by two-way and one-way analysis ofvariance (ANOVA) and Newman-Keuls multiple comparison, and repeated-measuresANOVA with Dunnett’s multiple comparison to a control. A P value of 0.05 was takenas the statistical level of significance.22ResultsAlthough the animals are responding to the circulating levels of catecholamines,and this paper is discussed in reference to those measurements, for convenience, I referto the final concentrations I wished to obtain, not the measured catecholamine valuesthroughout the text and figure labels. However, the measured catecholamine values foreach injection are given in Tables 3 and 4.Series I: Ventilatoiy responses to catecholamine injection.Ventilatory responses.In these trout, there was a significant drop in f in the mM (-8.5%, peakresponse) and 50 nM (-8.9%, peak response) NA treatments (Fig. 1). The fg returned toresting levels within 10 minutes post-injection. There was no significant change in fgafter the 10 and 100 nM NA treatment. The highest AD concentrations (100 nM) hadno significant effect on fg, while the three lower concentrations showed significantchanges from the control, as well as different responses from each other. With 10 nMAD, there was a transient but significant drop in fg (-15.1%, peak response) whichrecovered to resting levels in 5 minutes (Fig. 1); similarly with the 50 nM concentration(-12.3%, peak response). 1 nM AD however, resulted in a biphasic response; initially asignificant drop in fg (-5.9%, peak response), followed by a significant increase in fg(+5.4%, peak response). Injection of the saline vehicle had no significant effect onventilatory frequency (Fig. 1). 1 also23Figure 1: Percent changes in ventilation frequency (± SEM) after injection of AD (a),NA (1) and saline (0). * indicates a significant difference of the post-injection valuefrom the control value in adrenaline-treated fish. x indicates a significant difference ofthe post-injection value from the control value in NA-treated fish. The arrow indicateswhen the injection was administered. N = 6 for all groups.24—>0CD :5 (H CD Cl)r’3 0.Do>zu, U-‘-‘—F’3-000C)%AVentilationFrequencyIIIIII-j.JFJ--1’31%)-.-F’3-00000000000000000Ll**Dtested for differences between NA and AD effects at each concentration. There was nosignificant difference between the catecholamines in the drop in ventilatoiy frequency;that is NA or AD injection resulted in similar depressions of ventilatory frequency.AD and NA had veiy different effects on opercular pressure. At all dosages, ADdid not have any significant effect on P0,, similarly for the 1 and 100 nM NA treatments.However, there was a significant increase in P0 in the 10 nM (43.6%, peak response)and the 50 nM (66.3%, peak response) NA condition, showing a concentrationdependant response (Fig. 2). Although P0k, had not returned to the resting level at theend of the 20 minute run (50 nM NA), the trend of P0, returning to control values canbe seen. Saline injection did not result in any significant change of P0, from restingvalues (Fig. 2). Comparisons between NA and AD P0, were not made, as AD did notsignificantly change P0, from rest.Cardiovascular responses.The heart rate and dorsal aortic pressure were measured in this series. The 1 nMNA injection caused a slight tachycardia at 15 and 20 minutes. The higherconcentrations of NA did not have any significant effect on heart rate, although there isa trend for an initial bradycardia followed by a tachycardia (Fig. 3). At all dosages, theAD treated fish exhibited a significant bradycardia. The fish treated with the two lowerconcentrations of AD exhibited a transient bradycardia. The fish in the remaining twotreatment groups displayed a significant bradycardia from pre-injection measurements atall post-injection readings (Fig. 3).26Figure 2: Percent changes in opercular pressure (± SEM) after injection AD (•), NA(L), saline (0). x indicates a significant difference of the post-injection value from thecontrol value in NA-treated fish. The arrow indicates when the injection wasadministered. N = 6 for all groups.2760 1 nM402:-20:5 —4060• x I ‘lOnM’40•20T/___0 — I-20‘50 n20- i0 0H I I...-.---.-——20 -—40-60- 100 nM20-::H: IC 0 5 10 15 20 SalDNorI Time (minutes) • Adr28Figure 3: Percent changes in heart rate (± SEM) after injection of AD (•), NA (s),saline (0). * indicates a significant difference of the post-injection value from thecontrol value in adrenaline-treated fish. x indicates a significant difference of the postinjection value from the control value in NA-treated fish. The arrow indicates when theinjection was administered. N = 6 for all groups.29xmMx*6040200-—20—40—606040200—20—40—60604020a)c30a). I I I10 nM’. I I I50 nM<0—20-40—606040200—20—40—60: I‘100 nM’. * *. *I I I I IC 0 5 10 15 20Time (minutes)o SalD Nor• Adr30Administration of the saline vehicle did not have any significant effect on heart rate (Fig.3).At all concentrations, with both catecholamines, there were significant increases inDA at all treatment levels (Fig. 4). For NA, increasing the concentration resulted inincreasing the pressure at each time interval, as well as increasing the duration of timeDA was elevated from control. For the 1 and 10 nM NA conditions, significant increasesin DA were observed at 0, 2, and 5 minutes; 50 nM had a significant effect at 0, 2, 5, and15 minutes; and 100 nM raised DA significantly at all post-injection measurements (Fig.4). The AD injected fish responded similarly, except that from the lowest dosage, DAwas significantly elevated from the resting values, that is it never returned to control DAlevels. Saline did not have any significant effect on DA (Fig. 4).Blood pH and Pao2.In all four treatment groups, there was no significant effect of either AD or NAon the arterial pH of these trout (Table 1). NA showed a trend towards a depression ofPao2, however, it was not a statistically significant effect. AD infusion did result in asignificant depression of Pao2 in the 10, 50, and 100 nM dosages, but not at 1 nM.Control Pao2 and pH values between all dosages were not significantly different. Theinjection of the saline vehicle did not have any significant effect on blood pH or Pao2(Table 1).31Figure 4: Percent changes in dorsal aortic pressure (± SEM) after injection of AD (•),NA (E), saline (0). * indicates a significant difference of the post-injection value fromthe control value in adrenaline-treated fish. x indicates a significant difference of thepost-injection value from the control value in NA-treated fish. The arrow indicates whenthe injection was administered. N = 6 for all groups.32mM***xx x -uci):3C,)U)ci)0C-)L.0C(I)0CD500400 -300200100 -050040030020010005004003002001000500400 -300 -200 -100 -0‘ I ‘lOnM** **x *z.‘5OnM’I*T 100 nM.F-- I I I - IC 0 5 10 15 20 0 Sal1’ . DNori Time (minutes) • Adr33Table 1: Mean values of arterial pH and arterial oxygen tension (± SEM) in pre- andpost- injection blood samples. * indicates a significant difference of the Post value fromthe Pre value. N = 6 for all groups.34Treatment Sample PHa Pao2 (Torr)AD Pre 7.80 ± 0.02 108.8 ± 4.21 nM Post 7.82 ± 0.02 108.3 ± 5.0AD Pre 7.83 ± 0.01 101.2 ± 1.810 nM Post 7.84 ± 0.02 88.8 ± 3.2 *AD Pre 7.82 ± 0.03 112.0 ± 5.850 nM Post 7.80 ± 0.04 82.0 ± 8.8 *AD Pre 7.89 ± 0.03 117.2 ± 3.4100 nM Post 7.84 ± 0.03 84.0 ± 12.2 *NA Pre 7.82 ± 0.01 116.2 ± 5.71 nM Post 7.78 ± 0.03 115.8 ± 3.8NA Pre 7.84 ± 0.03 101.0 ± 3.510 nM Post 7.82 ± 0.05 96.0 ± 3.9NA Pre 7.87 ± 0.03 119.3 ± 4.750 nM Post 7.80 ± 0.05 108.7 ± 6.7NA Pre 7.89 ± 0.05 110.2 ± 4.9100 nM Post 7.80 ± 0.04 98.7 ± 4.4Saline Pre 7.79 ± 0.03 123.0 ± 4.5Post 7.80 ± 0.02 124.2 ± 5.735Series II: Ventilatoiy responses to catecholamine injection following a-adrenoceptorblockade.Ventilatory responses.These fish, which were treated with the tie-blocker phentolamine, exhibited adifferent response from untreated fish to catecholamine injection. At all doses used, forboth NA and AD, there was no significant effect of the injections on the fg of these fish(Fig. 5). Again, the changes observed in A0 were dependent upon which catecholaminewas injected. There was no significant difference after AD injection, at allconcentrations used, from the pre-injection controls (Fig. 6). With NA, there was asignificant increase in A0 at the 1 nM (37%, peak response), 10 nM (56%, peakresponse), and 50 nM (63%, peak response) concentrations, again showing aconcentration dependant response (Fig. 6). Like in Series I, the 100 nM NAconcentration did not have any significant effect on P0,,. In the fish injected with the twolower concentrations, A, returned to pre-injection levels by 20 minutes. For fish treatedwith 50 nM NA, though A, has not returned to pre-injection levels, the trend forreturning to control values can be seen. Injection of the saline vehicle did not result inany significant change in fg or A from control values (Figs. 5,6).Blood pH and Pao2.There was no significant impact of NA or AD on the arterial pH of the a-blockedfish (Table 2). Similarly, there was no significant change in the intracellular red bloodcell pH at all catecholamine concentrations used. With both arterial oxygen tension andcontent, there was no significant difference between the pre and post injectionmeasurements. There was also no significant effect of the injection36Figure 5: Percent changes in ventilation frequency (± SEM) in a-blocked fish, afterinjection of AD (a), NA (1J), saline (0). The arrow indicates when the injection wasadministered. N = 6 for all groups.37%AVentHationFrequency___;I]001—N000>Z(J)DDOOD1-Figure 6: Percent changes in ventilation amplitude (± SEM) in a-blocked fish, afterinjection of AD (n), NA (E1), saline injection (0). * indicates a significant difference ofthe post-injection value from the control value in NA-treated fish. The arrow indicateswhen the injection was administered. N = 6 for all groups.39*105-01—5—101’ Time (minutes)40*1 nI—I I I I I I40 -30 -2010a)0,D10— 60C<20.o 0c 60•-‘ 4020><02015lOnM—.----—.—.I50: */lOOnMI5 10 15C 0 20 o Salo Nor• AdrTable 2: Mean values (± SEM) of arterial pH, intracellular red blood cell pH, arterialoxygen tension and arterial oxygen content in pre- and post- injection blood samplesfollowing a-blockade. N 6 for all groups.41TreatmentSamplepHpHPao2Cao2HctTorrmg02d11ADPre7.85±0.057.46±0.02120.8±5.77.67±0.5726.5±1.91nMPost7.80±0.057.45±0.03116.0±7.37.07±0.4329.7±2.0ADPre7.80±0.037.43±0.02124.7±3.87.52±0.7027.2±1.910nMPost7.72±0.037.46±0.02116.0±5.56.47±0.6527.7±1.8ADPre7.81±0.027.45±0.02120.2±5.28.06±0.7124.9±1.950nMPost7.75±0.037.47±0.01111.0±5.77.61±0.8329.3±1.5ADPre7.86±0.037.45±0.01126.2±2.28.68±0.9124.2±1.1100nMPost7.78±0.037.47±0.02118.0±3.19.06±1.0027.0±1.2NAPre7.86±0.057.49±0.02132.7±2.37.42±0.5027.1±1.51nMPost7.80±0.047.47±0.03122.3±4.88.18±0.4830.1±1.6NAPre7.89±0.057.45±0.03129.8±5.16.96±0.5125.0±1.910nMPost7.76±0.057.50±0.02115.3±5.86.11±0.2228.5±1.6NAPre7.81±0.047.46±0.02125.8±0.97.67±0.7624.8±1.950nMPost7.72±0.037.48±0.02120.5±0.97.13±0.9828.0±2.0NAPre7.86±0.037.45±0.02132.2±3.18.15±0.5628.2±1.5100nMPost7.77±0.027.47±0.02126.0±1.58.23±0.7831.0±2.5SalinePre7.88±0.057.43±0.03126.3±3.88.14±0.4026.0±1.3Post7.87±0.047.40±0.02122.3±4.98.23±0.2628.3±0.9of catecholamines on the haematocrit of these a-blocked fish. Control values of PHapH1, Pao2, and Hct were not significantly different between the concentrations used. Theinjection of the saline injection also did not have any effect on these blood variables(Table 2).Catecholamine responses of series I and II.In all treatments, the post-injection values of the injected catecholamine weresignificantly higher than the pre-injection control. The levels measured were in the sameorder of magnitude as the levels I was attempting to obtain (Table 3,4). Except for thefish in Series I injected with 1 nM concentrations of catecholamines, injection of eithercatecholamine resulted in an increase in the titre of the other catecholamine, althoughnot necessarily to the same extent as the injected catecholamine. There was nosignificant catecholamine release as a result of injecting the saline carrier into theanimals.43Table 3: Catecholamine levels measured in untreated fish injected with catecholamines.* indicates a significant increase of the post-injection catecholamine level from the preinjection control. All values are given as means ± SEM. N = 6 in all groups.44Treatment Sample NA AD(nM) (nM)AD Pre 0.68 ± 0.12 0.48 ± 0.11mM Post 0.58 ± 0.10 3.04 ± 0.49 *AD Pre 0.81 ± 0.23 0.74 ± 0.15lOnM Post 2.14 ± 0.45 * 22.0 ± 4.31 *AD Pre 0.54 ± 0.16 0.72 ± 0.115OnM Post 4.93 ± 1.13 * 56.54 ± 5.21 *AD Pre 0.65 ± 0.22 0.76 ± 0.27lOOnM Post 9.41 ± 3•77 * 278.07 ± 70.09 *NA Pre 0.66 ± 0.22 0.40 ± 0.10mM Post 3.81 ± 0.35 * 0.58 ± 0.09NA Pre 0.47 ± 0.13 0.75 ± 0.15lOnM Post 14.77 ± 2.31 * 2.50 ± 0.77 *NA Pre 0.52 ± 0.13 0.59 ± 0.205OnM Post 70.89 ± 6.09 * 4.44 ± 0.71 *NA Pre 0.29 ± 0.14 0.66 ± 0.22lOOnM Post 207.27 ± 57.81 * 21.78 ± 5.56 *Saline Pre 0.73 ± 0.18 0.63 ± 0.24Post 0.47 ± 0.13 0.49 ± 0.2145Table 4: Catecholamine levels measured in a-blocked fish injected with catecholamines.* indicates a significant increase of the post-injection catecholamine level from the preinjection control. All values are given as means ± SEM. N = 6 in all groups.46Treatment Sample NA AD(nM) (nM)AD Pre 1.01 ± 0.11 1.12 ± 0.16mM Post 2.43 ± 0.24 * 2.53 ± 0.20 *AD Pre 1.11 ± 0.18 1.15 ± 0.13lOuM Post 2.32 ± 0.37 * 24.23 ± 0.86 *AD Pre 1.29 ± 0.15 1.12 ± 0.135OnM Post 6.10 ± 0.46 * 71.59 ± 4.62 *AD Pre 1.42 ± 0.20 1.57 ± 0.06lOOnM Post 7.65 ± 0.74 * 270.90 ± 27.47 *NA Pre 0.96 ± 0.21 1.33 ± 0.30mM Post 2.51 ± 0.31 * 2.16 ± 0.41 *NA Pre 1.22 ± 0.17 1.17 ± 0.21lOnM Post 18.16 ± 1.61 * 2.16 ± 0.20 *NA Pre 1.39 ± 0.13 1.49 ± 0.135OnM Post 75.40 ± 5.07 * 5.53 ± 0.44 *NA Pre 1.39 ± 0.20 1.29 ± 0.21lOOnM Post 247.55 ± 28.24 * 9.69 ± 1.64 *Saline Pre 1.14 ± 0.10 1.31 ± 0.16Post 1.14 ± 0.09 1.33 ± 0.1647DiscussionIt is apparent that injection of catecholamines can alter ventilation in fish. Thespecific changes, however, are dependent on a variety of factors. The general ventilatoiyresponse is determined by the combined output of ventilation frequency and ventilationvolume. Catecholamine injections inhibited fg in Series I at all concentrations.Interestingly, there were no concentration dependant effects on fg; catecholaminesreduced fg at low concentrations then had no further effect as concentration wasincreased. However, in Series II, pre-treating the fish with phentolamine, an cradrenoceptor blocker, eliminated the significant effect of catecholamine injection on fg.This would indicate that the effects of catecholamines on fg seen in Figure 1 may bemediated by a-adrenergic receptors.There were also changes in P0,, but these alterations were dependent upon thetype and concentration of catecholamine used, and the presence or absence of the crblocker. In both Series I and II, AD had no effect on P0,,. The overall effect of ADinjection in Series I therefore, was hypoventilation, due to adrenaline’s effect onventilation frequency, while in cr-blocked animals, AD had no significant effect onventilation. Why a depression of ventilation should occur when AD is present is notknown, since catecholamines are released in situations when uptake of oxygen from theenvironment is important. It has been postulated that it may be an energy conservingmechanism when oxygen is limited (Perry et a!., 1992), but as ventilation accounts for lessthan 10% of a fish’s energy costs (Jones and Randall, 1978), the impact of such astrategy would likely be small. In newborn rats, electrical stimulation of the A5 nucleusin the caudal ventrolateral pons was observed to inhibit the activity of the medullary48respiratory neurons (Hilaire et al., 1989). Electrical stimulation of the same area wasineffective after the application of the cr-antagonist yohimbine or idazoxan. It may bethat a similar inhibitory drive exists in trout, and the drop in fg seen in Series I was aresult of AD stimulating this system, and this drop of fg was blocked by phentolamine inSeries II. How AD in the circulation may be activating such a system, if it exists in fish,is not known.NA’s impact, when there was an effect, was to increase P0, from control levels. InSeries I, at the mM level, as there was no effect of NA on P0k,, the overall effect again,was hypoventilation. Interestingly, when the fish were pre-treated with phentolamine,there was a highly significant increase in A,. As the a-adrenoceptor blockade resultedin no changes in ventilation frequency, the overall output of these animals would havebeen hyperventilation. This indicates two things. The first is that dependant upon theconcentration used, catecholamines may have both hyper- and hypoventilatory effects.The second is that the overall output may be dependent upon which population ofadrenoceptors, a or f3, are present, or possibly on the ratio between these two receptortypes. This may also be part of the explanation for the different responses observed infish following catecholamine injection. In eels, both hypoventilatory and hyperventilatoryresponses have been observed following catecholamine injection (Peyraud-Waitzenegger,1979), and the response depended upon which adrenoceptor was stimulated. When ana-blocker (J3-adrenoceptors stimulated) was used, the eels hyperventilated, while the useof a 13-blocker (cr-adrenoceptors stimulated) resulted in hypoventilation.The increases in P0k, are the result of recruitment of more muscle groups in thebuccal/opercular area, which must therefore, be influenced by catecholamines. Incurarized, hyperoxic dogfish, AD injection resulted in respiration-related activity in the49hypobranchial nerve, which prior to the injection, showed low and intermittent activity(Levings and Taylor, unpublished observations). The hypobranchial nerve innervates thefeeding muscles, so the addition of these muscle groups would result in an increase inventilation, presumably by increasing ventilatory stroke volume.The NA results of Series I are similar to other work on dogfish (Taylor andRandall, 1990; Randall and Taylor, 1991), and bowfin (McKenzie et al., 1991a); that isan observed increase in ventilatory output following catecholamine administration. Inboth the bowfin and the intact dogfish preparations, however, an increase in ventilatoryfrequency and stroke volume is seen after injection of catecholamines (McKenzie et at.,1991a; Randall and Taylor, 1991), while trout depress ventilation frequency whileincreasing ventilatoiy stroke volume (present study). The significance of this differencein the ventilation frequency response to catecholamine administration is not known,though it may be that trout are more dependant upon changes in strokevolume/opercular pump than ventilation frequency (Smith and Jones, 1982).The cardiovascular changes observed in Series I were the typical responses toadrenergic stimuli (Wood and Shelton, 1975, 1980a). The increases in dorsal aorticpressure seen with both catecholamines are due to a direct effect on the heart bystimulating cardiac -adrenoceptors, resulting in an increase of cardiac stroke volume(Wood and Shelton, 1980a). In addition, there is an r-adrenoceptor mediatedvasoconstriction of the systemic vasculature (Wood and Shelton, 1975), which increasesperipheral resistance and is manifested as an increase in blood pressure. Heart rateshowed stereotypical responses to AD, that is a bradycardia, due to activation of arterialbaroreceptors (Wood and Shelton, 1980a). On the other hand, NA did not result in a50change in rate, unlike Wood and Shelton (1980a), but as they state, compared withchanges in cardiac stroke volume, changes in heart rate will have a small effect on theoverall cardiac output.I was surprised that I did not detect a blood acidosis after catecholamine infusion,as other studies have shown that catecholamine infusion can cause a blood acidosis(Nikinmaa, 1982a; Thomas et a!., 1991) due to activation of sodium proton exchange.Nikinmaa (1982a) demonstrated, however, that 10 minutes after catecholamine infusiontrout blood pH was not significantly different from control values. Thus, an acidosisprobably did occur in our fish but blood pH had returned to control levels by the timethe blood was sampled.In conclusion, catecholamines do have an effect on ventilation in trout. ADgenerally results in a depression of ventilation, while NA generally stimulates it. Theeffects are mediated by both a- and 13-adrenergic receptors, and the overall output fromadrenergic stimulation is likely dependent upon the ratio between these two populationsof receptors.51Chapter 3The effects of D- and D,L-Propranolol on the ventilatory responses of rainbow trout,Oncorhynchus mykiss, to acute hypercapnia.52IntroductionIn Chapter 2, I demonstrate that it is possible for catecholamines that are presentin the circulatory system to have an effect on the ventilation of fish. The second focus ofthis thesis is to look for conditions when catecholamines may be playing a role inmoderating ventilation in fish. In a previous study (Aota et a!., 1990), it has beendemonstrated that during severe hypoxia, there was a rise in endogenous plasmacatecholamines and ventilation in trout. This rise in ventilation could be partiallyblocked by the use of the $-blocker D,L-propranolol. Are there other situations whenplasma catecholamines and ventilation are seen to rise? The null hypothesis for thisstudy was that the increase in endogenous plasma catecholamines in fish exposed toenvironmental hypercapnia does not have an effect on ventilation. The advantage ofusing environmental hypercapnia to test for the effects of catecholamines on ventilationin fish is that this condition can stimulate ventilation (Janssen and Randall, 1975; Smithand Jones, 1982), and simultaneously stimulate catecholamine release into the circulation(Perry et a!., 1989). In this study, the role of catecholamines in the ventilatoiy responseto acidemia was assessed with fish injected with the /3-adrenoceptor antagonist D,Lpropranolol before exposing the animal to external hypercapnia and comparing theirresponses to untreated and sham treated fish.53Materials and MethodsExperimental animalsRainbow trout, (Oncorhynchus mykiss, average 334 ± 14g (mean ± SEM) from alocal hatcheiy (West Creek Trout Ponds) were held outdoors in a large fibreglass tank.They were fed commercial trout pellets once a week, and were not fed at least 48 hoursbefore surgery. Aerated, dechlorinated Vancouver tap water flowed through the tank,the temperature ranging from 9 to 11 centigrade. The temperature of the water usedin each experiment was the same as that in the tank. Temperature varied ± 0.5 Cduring each experiment.Surgical procedures.All surgeiy was done under general anaesthesia. For details on the anaesthetic,operating table and surgical procedure for implanting the indwelling catheter into thedorsal aorta, please see Materials and Methods, Chapter 2. This catheter was used forblood sampling and drug injection. The fish was then fitted with a latex mask suturedaround the mouth and attached to the divider of an opaque van Dam box (Cameronand Davis, 1970) supplied with flowing, aerated water. This setup allows the separationof the box into two compartments. When the water levels in the two compartments areset equally, the movement of water from the anterior to posterior chamber results in anoverflow, which can be monitored, allowing a direct measurement of ventilation volume(‘1w).All animals were allowed to recover from anaesthesia and surgery for 48 hoursbefore any experimental manipulation was performed. During the first 24 hours, a slight54pressure head in the anterior chamber (about 1 cm) was created to aid in the recoveiy ofthe fish from the surgeiy. The pressure difference between the anterior and posteriorcompartments was eliminated in the last 24 hours of recovery. The dorsal aortic cannulawas flushed at least once daily with 0.2 ml of heparinized (10 i.u. m11 heparin) Cortlandsaline (Wolf, 1963).Experimental protocol.Series I: Ventilatoiy responses to external hypercapnia in saline injected-flh.External hypercapnia was quickly achieved by gassing the anterior chamber of thevan Dam box, and by using a counter-current gas exchange column (which supplied waterto the anterior chamber of the box), with either air or a pre-analyzed commercial 1%CO2 in air gas mixture to obtain a Pwco2 of approximately 6 torr. The Pwco2 of theanterior chamber was continuously measured throughout the experiment using aRadiometer Pco2 electrode in a thermostatted cuvette. The fish in this series were acontrol group for the fish treated with the -adrenoceptor antagonists (see below).These fish were injected with 0.2 ml of saline 2 hours before the exposure tohypercapnia. The cannula was then flushed with an additional 0.2 ml of saline to ensurecomplete delivery of the carrier to the circulation.Series II: Effect of f3-adrenoceptor blockade on the ventilatoiy adjustments to hypercapnia.Fish were injected with the /3-adrenoceptor antagonist D,L-propranolol (SigmaChemical Co.) 2 hours before being exposed to external hypercapnia. The drug wasdissolved in Cortland saline just before use and injected into the dorsal aorta cannula ata dose of 2 mg kg1 body mass. The cannula was cleared with an additional 0.2 ml saline55after each injection to ensure complete delivery of D,L-propranolol to the circulation.This dose of D,L-propranolol has been shown to be more than adaquate to ensurecomplete blockade in trout to 6-adrenergic stimulation by catecholamines (Kinkead andPerry, 1990).Series III: Non-specific effects ofpropranolol on the ventilatoty adjustments to hypercapnia.The racemic mixture of propranolol (D,L-proprauolol) is often used to block the13-component of an adrenergic response. D,L-propranolol, however, is known to haveeffects that are not associated with the 13-blocking properties of the drug, and theseeffects are not stereospecific (Barrett and Cullum, 1968). The 13-adrenoceptor blockingeffects of propranolol, however, are stereospecific. The D- isomer has less than one-hundredth the potency of the L- or D,L- isomers of propranolol (Barrett and Cullum,1968). To determine if the results observed in fish injected with D,L-propranolol weredue to the blockade of the f3-adrenoceptor sites, fish were injected with D-propranolol(Sigma Chemical Co.) 2 hours before being exposed to external hypercapnia. Thepreparation and injection protocol used for this series was the same as was used in SeriesII.Blood sampling.Three blood samples of 750 d each were taken from all fish. An initial samplewas withdrawn from the dorsal aortic cannula before the start of the experiment (0 mm.),a second sample after 30 minutes exposure to hypercapnia (30 mm.) and a final sample30 minutes after the end of the experiment (recovery). After each blood sample, anequivalent volume of heparinized saline was injected into the fish to restore blood56volume. Arterial blood was analysed immediately after sampling to determine oxygentension (Pao2), arterial blood pH (PH,,), and oxygen content (Cao2). The remainingblood was centrifuged and the plasma (250 tl) was immediately frozen in liquid N2 andstored in a -80° C for later analysis of catecholamine levels. The remaining red bloodcell fraction was used to measure pH1.Ventilatory measurements.Gill ventilation volume of each fish was monitored as follows: two preexperimental measurements (Pre 1 and Pre 2), taken 10 minutes apart. Anothermeasurement was taken 10 minutes later (0 mm.), just before the first blood sample wastaken. The fish was then exposed to hypercapnia for 30 minutes. During this period,‘Clw was monitored every 10 minutes from 0 minutes. The fish was then allowed torecover under normocapnic conditions for 30 minutes, then a final gill ventilation volumemeasurement was taken, after which the experiment was terminated. Gill ventilationvolume was determined by collecting and measuring the water leaving the overflowstandpipe of the posterior chamber of the van Dam box for 1 minute. All fish survivedthe entire experimental procedure.Again, similar to the experiments of Chapter 2, water temperatures between serieswere not constant, though the temperature was constant during any experimental series.Therefore, to remove the effects of temperature, ventilation volume is expressed aschange in ‘w relative to the respective time 0 value in each series.Analytical techniques.For details on the methods used to measure PHa, pH1, Pao2, Cao2, Hct, AD and57NA levels, see Materials and Methods, Chapter 2. Water Pco2 was measured using aRadiometer E5037 electrode, and calibrated using pre-analyzed commercial gas mixtures.Statistical analysis.Data are presented as means ± 1 S.E.M. Statistical analysis was performed asdescribed in Materials and Methods, Chapter 2. In addition, the equivalent nonparametric tests, Friedman repeated measures ANOVA on ranks, and Dunnett’s orDunn’s multiple comparison to a control were used, as necessary. A P level of 0.1 wastaken as the significance level.58ResultsVentilatory responses.The control fish that were sham-injected with saline only (Series I) had anapproximate 3.6-fold increase in “w when they were exposed to hypercapnia (Fig. 7).This elevation of ‘w was maintained for the duration of the exposure to hypercapnia.The animals exposed to the /3-adrenoceptor propranolol (Series II) also displayed anincrease in At 10 minutes exposure to hypercapnia, the increase was notsignificantly different from the fish in Series I. However, unlike Series I, the animals iuSeries II could not maintain ‘w for the duration of the hypercapnia exposure. By 30minutes of hypercapnia, there was oniy a 1.9-fold increase in compared to the 3.6-fold increase in Series I. The fish that were pre-treated with D-propranolol (Series III)had a ventilatory response to hypercapnia that was similar to Series I, a sustained 3.6-fold increase in ‘w for the duration of the exposure to hypercapnia.Blood pH, Pao2 Cao2.In all of the treatments, arterial oxygen tension and arterial oxygen content didnot show any significant change throughout the experiment (Table 5). There was asignificant drop in PHa in all treatment groups. With pH1, the fish in Series II had asignificant decrease after 30 minutes exposure to hypercapnia. In the other treatments,there was either a significant increase in pH (Series I), or no change (Series III). Inboth the hypercapnia + saline, and the hypercapnia + D-propranolol groups, the ApHvalue significantly fell to similar levels, mainly due to the similar reductions in pHa inthese groups. In Series II, on the other hand, pH was unchanged at the 30 minute59Figure 7: Gill ventilation volume of rainbow trout during exposure to hypercapniafollowing pre-treatment with saline, hypercapnia following pre-treatment with the mockj3-adrenoceptor antagonist D-propranolol, and hypercapnia following pre-treatment withthe 13-adrenoceptor antagonist D,L-propranolol. The 30 minute experimental period issituated between the two vertical dotted lines. Data is shown as means ± S.E.M. *indicates that A’1w is significantly different from the 0 minute value; t indicates that thevalue is significantly different from the corresponding D,L-propranolol value. N = 6 inall groups.60-*600C 500E400ci) E :3 0 > C 0 C C ci) >•Hypercapnia+saline(sham)Hypercapnia+D—propranolol(mockfl—block)oHypercapnia+D—Lpropranolol(fl—antagonist)300200100 0PRE1PRE20102030Time(mm)RECOVERYTable 5: Mean values of various blood respiratory variables (± S.E.M.) after 0, and 30minutes exposure to hypercapnia, and at recovery. * indicates a significant differencefrom the 0 minute value. N = 6 for all groups.621%C02-i- 1%C02+1%C02+ D- DL-Sample Saline propranolol propranololCao2 0 mill. 8.4±0.9 8.1± 1.3 8.3±0.8mg 02 d11 30 mm. 5.6±0.7 5.9±0.6 6.7±0.9Recovery 7.6±0.6 5.4±1.0 7.4±1.1Pao2 0mm. 112±6 110±9 108±10Ton 30mm. 103±5 103±6 112±9Recovery 114±5 115±9 115±7PHa 0 mill. 7.96±0.05 8.04±0.06 7.96±0.0430 mm. 7.65±0.06* 7.81±0.04* 7.69±0.06*Recovery 7.93 ± 0.02 8.10±0.01 7.92±0.05pH 0 mill. 7.40±0.02 7.49±0.02 7.46±0.0230 mill. 7.46±0.02* 7.54±0.05 7.23±0.03*Recovery 7.43±0.01 7.53±0.02 7.37±0.02*ApH 0 mill. 0.57±0.04 0.55±0.06 0.50±0.04PHaPH1 30 mm. 0.19±0.07* 0.28±0.08* 0.46±0.04Recovery 0.50±0.03 0.57±0.03 0.55 ± 0.05Hct 0 mm. 25.5±1.4 28.5±2.6 25.1±2.930 mm. 32.5±1.4* 28.9±2.8 27.6±2.4Recovery 24.2±2.1 17.7±1 •4* 17.7±2.0*63reading, as in this group, both PHa and pH fell (Table 5). By the end of the experiment,gill ventilation volume, and most of the other blood respiratory variables had returned totheir pre-experimental value. The exception was haematocrit. At 60 minutes,haematocrit was at the pre-experimental value in the hypercapnia + saline treatment. Itwas, however, significantly lower than the control value in the other groups.Catecholamine Responses.All of the hypercapnic groups showed a significant elevation of both plasmacatecholamine, AD and NA. The levels measured were in the range measured in other,similar studies. Surprisingly, there was no significant difference in the NA or AD levelsbetween treatments at 0, 30 or 60 minutes.64Figure 8: Measured plasma catecholamine levels in trout during exposure to hypercapniafollowing pre-treatment with saline, hypercapnia following pre-treatment with the mockf3-adrenoceptor antagonist (hypercapnia+D-propranolol) and pre-treatment with the /3-adrenoceptor antagonist (hypercapnia+D,L-propranolol). NA (A) and AD (B) levelsbefore (0 mm.; hatched column), after 30 minutes exposure to the experimentalcondition (30 mm.; filled column), and 30 minutes after the cessation of the experimentaltreatment (Recovery; open column). The data (mean ± S.E.M.) are given as plasmaconcentration (nM). * indicates a significant difference from the 0 minute value. N = 6for all groups.6530IIci)CCci-oC0zLJ500—. 4005 300IIci)CCCa)-oLJ35A25*20V/,l 0 mm30 mmI I Recovery15*10*50IB*150100500*1% CO2 1% CO2 1% 002saline D— prop D—L— prop66DiscussionThe ventilatory responses in this study shows that the f-component of anadrenergic response is required to obtain a full hyperventilatory response to acutehypercapnia. The changes in ventilation seen in this study after exposing fish toenvironmental hypercapnia were similar to changes observed in other studies (Janssenand Randall, 1975; Smith and Jones, 1982; Graham et a!., 1990; Kinkead and Perry,1991). Though ventilation frequency was not measured in the current study, otherinvestigators, using similar protocols, have shown that hypercapnia increases ventilationvolume by increasing ventilatory stroke volume. Ventilation frequency does notsignificantly change (Kinkead and Perry, 1991). The ‘1’w values of the D,L-propranololtreated fish after 20 and 30 minutes of hypercapnia were significantly lower than thecorresponding values in the saline and D-propranolol groups. The blunted response ofthe D,L-propranolol group was not a result of a lower level of hypercapnia orhypoxemia. When the three hypercapnic groups were compared at 0 minutes (preexperimental) or at 30 minutes, none of the respiratory variables monitored in the bloodthat may modulate ventilation (PHa, Pao2, Cao2) or water (Pwco2 and Pw02) showed anysignificant differences. The observation that the changes in pH1 did not alter Cao2 in theD,L-propranolol group was surprising, as I would expect the drop in pH1 to result in adecrease in Cao2. It may be that, although there was not a statistically significantincrease in Hct, the change from 25.1% to 27.6% was sufficient enough to offset thedrop in pH in the red blood cell, so that overall, there was no change in Cao2. Thedifferences in the size of the hyperventilatory response between the f3-blocked and theother hypercapnic groups therefore reflects the direct contribution of f3-adrenoceptor67stimulation. It appears, therefore that the initial ventilatory response to acutehypercapnia is not dependent upon 13-adrenergic stimulation. However, thecatecholamines, more specifically, the 13-adrenergic component, is required to maintain orsustain a full hyperventilatory response to acute hypercapnia.As it has been observed that D,L-propranolol can inhibit the firing activity of 02-sensitive chemoreceptors located in the first gill arch of rainbow trout (Burleson andMilsom, 1990), it is possible that the impaired ventilatory response to hypercapnia (Fig.7) reflects the absence of02-related ventilatory drive. However, Burleson (1991)demonstrated that catecholamines did not stimulate 02 chemoreceptor discharge, so itseems unlikely that there is an adrenergic component to peripheral chemoreceptordischarge in trout. It was speculated that the inhibition of02-chemoreceptor activityobserved may have been due to the anaesthetic properties of D,L-propranolol (Burleson,1991). If this was the case, then the non-specific effects of D,L-propranolol was not thecause of the reduced hyperventilatory response in the current study. This is because onetreatment was to pre-treat with D-propranolol. This served as a control group for thenon-specific effects of D,L-propranolol, and these fish did not show any significantdifference in their ventilatory response to acute hypercapnia, so it is unlikely that theblunted response seen in the D,L-propranolol group was a result of the non-specificeffects of D,L-propranolol.To summarize, the hyperventilatory response to hypercapnia was blunted inanimals that had been pre-treated with the $ adrenergic blocker D,L-propranolol.Animals that were pre-treated with D-propranolol, which has minimal 13-blocking ability,did not impair the ventilatory response to hypercapnia. This indicates that the68impairment observed following pre-treatment with D,L-propranolol was not due to thenon-specific side effects of D,L-propranolol. The initial response was not dependentupon the presence of catecholamines. These results indicate that the I3-component of anadrenergic response is involved in maintaining the hyperventilatoiy response to externalhypercapnia.69Chapter 4The effects of -adrenoceptor blockade on ventilation of rainbow trout, Oncorhynchusmykiss after exhaustive exercise.70IntroductionChapter 2 has shown that catecholamines could have a role in the regulation ofbreathing in fish, and Chapter 3 has shown that during a respiratoiy acidosis, whencatecholamines are released, 13-adrenoceptor blockade can impair or blunt the ventilatoiyresponses of trout to this ventilatory stimulus. There are other situations whencatecholamines are released into the bloodstream in fish. One of these is during andafter exhaustive exercise. After exhaustive exercise in fish, there is a build up ofmetabolites, especially lactate (Moyes et aL, 1992). During recovery from exercise, 02consumption is elevated, in part to remove lactate and other metabolites (Scarbello eta!., 1991). Part of this increased oxygen consumption would be maintained by in the fishby elevating ventilation after exhaustive exercise. Blood oxygen content is notsignificantly depressed (Primmett et al., 1986) after exhaustive exercise, so Cao2 wouldnot be functioning as a ventilatory stimulus. A metabolic acidosis is also observedfollowing exhaustive exercise (Thomas et at., 1987), so this is another situation where onehas circulating catecholamines, a drop in blood pH, and a requirement for increasedventilation. It may be then, that catecholamines are acting to allow the fish to maintainbreathing after exhaustive exercise. The null hypothesis for this investigation then wasthat catecholamines are not influencing the ventilatory response in free swimmingrainbow trout following exhaustion via /3-adrenoceptors. To test for this, I injected a 1-adrenoceptor blocker prior to swimming the fish to exhaustion, and followed ventilationduring recovery. This group of fish were compared to a sham-injected group. Becausethe use of D,L-propranolol infusion has many non-specific side effects associated with it(Barrett and Cullum, 1968), a different 13-adrenoceptor blocker was used. I have71demonstrated in the previous chapter that, at the concentration I use, the non-specificeffects of D,L-propranolol do not interfere with the ventilatory response of trout.However, the non-specific effects of D,L-propranolol may affect the fish’s ability to swim.Indeed, van Dijk and Wood (1988) have shown that trout treated with D,L-propranololdo not swim well. For this reason, I have used nadolol, which is a general f3-adrenoceptor blocker that does not have these effects (Hoffman and Lefkowitz, 1990), totest the effect of a a-blockade on the breathing response of trout to exhaustive exercise.72Materials and MethodsExperimental animals.Rainbow trout (Oncorhynchus mykics, average 762 ± 48 g (mean ± SEM) from alocal hatcheiy (West Creek Trout Ponds) were held outdoors in a large fibreglass tank.They were fed commercial trout pellets once a week, and were not fed at least 48 hoursbefore surgeiy. Aerated, dechlorinated Vancouver tap water flowed through the tank,the temperature ranging from 11 to 16 0 C. The temperature of the water used ineach experiment was the same as that in the holding tank. Temperature varied ± 0.50during each experiment.Surgical Procedures.All surgery was performed under general anaesthesia. Fish were initiallyanaesthetized in a 1:10,000 solution of tricaine methanesulfonate (MS-222, SyndelLaboratories) buffered to pH 7.5 with NaHCO3before being weighed and transferred toan operating table similar to the one used by Smith and Bell (1967). During surgery thegills were irrigated with a less concentrated MS-222 solution (1:20,000). In all fish, thedorsal aorta was chronically cannulated with polyethylene tubing (Clay-Adams PE-50,Intramedic), using the technique of Soivio et al., (1975). This cannula was used for bloodsampling and injections. Flat 1 cm2 brass plates were sutured to the opercular flaps asdescribed in Chapter 2. After surgery, the fish were force ventilated with dechlorinatedwater until they recovered from anaesthesia. They were then placed individually in blackperspex boxes (volume 2.51), and allowed to recover for 48 hours from the surgicalprocedure before any experimental manipulation was performed.73Experimental protocol.Series I: Effect of nadolol on ventilation and blood gases during recoveiy from exhaustiveexercise in trout.Fish were placed in a modified Brett-type respirometer (Gehrke et a!., 1990) andforced to swim for 30 minutes at increasing velocities until they fatigued. After theyfatigued, the velocity of the swim tunnel was reduced to the initial, resting velocity whenpre-swim measurements were taken, and the recoveiy of the animals followed. In allconditions fish were swum to exhaustion, and the measurements of interest wererecorded during recovery. Two hours prior to the forced swim to exhaustion, one groupof fish were injected with 1 ml of 2 mg . kg’ body weight of nadolol (Sigma ChemicalCo.) in saline (13-blocked fish, N = 7). Nadolol is a /3-adrenoceptor blocker that doesnot have the local anaesthetic properties of DL-propranolol (Barrett and Cullum, 1968;Hoffman and Lefkowitz, 1990). Cortland saline (Wolf, 1963) alone was injected intoanother group of fish so that any effects due to the injection itself could be observed(control, N = 6).Series II: Effect of nadolol on ventilation and blood gases in resting trout.From the results of Series I, it was obvious that nadolol does have significant nonspecific effects in rainbow trout, especially on blood values, which in turn would have animpact upon ventilation. I therefore decided to look at the effects of nadolol onventilation and blood gases in resting trout. After surgery, as described above, the fishwas placed in black perspex boxes as in Chapter 2. One group was injected with nadololas in Series I, and the other group was injected with saline.74Blood Samples.Series I: Effect of nadolol on ventilation and blood gases during recoveiy from exhaustiveexercise in trout.One pre-swim blood sample of 1.0 ml was taken 2 hours after the injection of thesaline or a-blocker, then 1.0 ml samples were taken at 0, 15, 30, 45, 60, and 120 minutesafter exhaustion, for a total of 7 samples from a single fish. For each 1.0 ml sample, thefollowing measurements were taken: 100 tl of blood for haematocrit (Hct); 40 l ofblood for haemoglobin (Hb); 50 l of blood for whole blood pH (pHa); 25 .d of bloodfor oxygen content (Cao2). 500 tl of blood was centrifuged and 250 l of plasmaremoved and frozen in liquid nitrogen. The frozen plasma was stored in a -80 C freezerfor later analysis of catecholamines. The red blood cell fraction remaining was used tomeasure pHi. The balance of the blood was used to measure blood oxygen tension(Pao2).Series II: Effect of nadolol on ventilation and blood gases in resting trout.A pre-injection blood sample of 600 l was taken, then 600 tl samples were takenat 2, 2.5, 3.5, and 4.5 hours after the injection, for a total of 5 samples from a single fish.From each 600 l sample, Hct, Hb, PHa, pH1, Cao2, and Pao2 were determined. Plasmacatecholamine levels were not analysed in this series as Series I showed that nadololalone does not cause a release of catecholamines into the circulatory system.Ventilation measurements.Opercular frequency (fg) and amplitude (Ac,i,) were measured with the brass platesas described in Chapter 2. Ventilation was measured just before taking each blood75sample.Although in each experimental group, water temperature remained constant, therewere changes in temperature between treatments (11 C to 16 C). As watertemperature changes, there are associated changes in the resting ventilation (Hughesand Roberts, 1970; Randall and Cameron, 1973), and therefore significant differencesbetween the various experimental control groups. The ventilatoiy parameters areexpressed as percent changes to remove the effects of temperature on ventilation.Analytical techniques.For details regarding the methods used to measure Pao2, Cao2, PHa, pHi, Hct, AD,and NA levels, see Materials and Methods, Chapter 2. Total haemoglobin concentrationwas determined using a Sigma Total Haemoglobin (525-A) assay kit and the relativeabsorbency measured at 540 nm in a Shimadzu UV-160 visible recordingspectrophotometer. Mean cell haemoglobin concentration (MCHC) was calculated bythe formula: total [Hb]*100/Hct.Statistical analysis.Data are presented as means ± 1 S.E.M. Statistical analysis was performed asdescribed in Chapters 2 and 3. A P value of 0.05 was taken as the statistical level ofsignificance.76ResultsSeries I: Effect of nadolol on ventilation and blood gases during recoveiy from exhaustiveexercise in trout.Ventilatory responses.In both treatment groups, there was a significant elevation of fg after the fish wereforced to swim to exhaustion compared to the pre-swim controls. Both groups exhibitedsimilar changes. The increased fg was maintained throughout the 2 hour recoveiy period,although f does decline from the peak response during this period (Fig. 9a). There wereno significant differences in fg between the saline injected or /3-blocked fish.There was a significant increase in at fatigue relative to the pre-swim controlsin both conditions. There were also significant differences between the two groups. Inthe /3-blocked fish, there was about a 35% increase in A9, over control levels, but by 60minutes, it was back to resting levels. In the saline injected animals, the increase in A9,was approximately 75%, and although it does decline throughout the recovery period, at120 minutes, A0 is still significantly elevated over control values (Fig. 9b). The otherdifference between the two treatment groups is that in the saline injected group,ventilation remained elevated at the same level from 0 minutes to 45 minutes, that isthere was no significant decline in A0 between 0 and 45 minutes post-exercise.However, in the nadolol treated group of fish, A, declined significantly from 15 minutespost-exercise (Fig. 10 b).Blood parameters.Whole blood pH in both groups showed a significant decrease following77exhaustive exercise of about 0.3 pH units when compared to control values (Fig. lOa).There was no significant difference in the PHa values between the two treatments, butthere was a trend in the saline injected animals for PHa to rise back towards the restingvalue at 120 minutes post-exhaustion. On the other hand, the intracellular red blood cellpH did show different responses between the two conditions (Fig. 9b). The animals thatwere injected with nadolol had a significant drop in pH1 by almost 0.3 pH units, aboutthe same drop seen in PHa. The pH1 of the nadolol group did not return to controlvalues at the end of the recoveiy period. However, the pH1 of the saline injected groupdid not show any significant difference from the pre-swim values, indeed there was aslight trend for an increase in pH1. There were significant pH differences between thetwo treatments at all post-swim sampling times. Blood haematocrit had two differentresponses between the nadolol and saline treatment groups. The saline conditionshowed a significant increase in Hct post-swim from resting values. This elevated Hctwas maintained for the duration of the recovery period. The 13-blocked group did notshow any significant changes in Hct between the pre- and post-swim values. In addition,there was a significant difference between the two pre-swim values (Table 6).Haemoglobin values in the nadolol condition were significantly greater than salineconditions (Table 6). There were no significant difference in the pre- and post-swimvalues for MCHC (Table 6). Similar to haemoglobin, MCHC was significantly greater innadolol injected fish than saline injected fish. Both blood oxygen tension and bloodoxygen content did not show any significant differences between the pre-swim andrecovery periods. There was, however, a significant difference between the Cao2 valuesbetween the nadolol and saline injected groups. There was no significant difference inthe Pao2 values between the two treatments (Table 6).78Figure 9a and 9b: Percent change from pre-exercise in ventilation frequency (9a) andventilation amplitude (9b) followed for 2 hours post-exhaustive exercise. Open circlesrepresent fish that were saline injected before being swum to exhaustion. Closed circlesrepresent fish that were injected with the $-blocker nadolol before being swum toexhaustion. * indicates a significant difference from the Pre value in each group. tindicates a significant difference between the saline and nadolol injected conditions. +indicates a significant difference from the 0 mm. value in each group.7920 azo 18 * 0 SALINE• —OL0CKED16 J * * *= >- 14F-0Z12> D 10wc7(LJJ 8Z<U 6I(_) 42*0 /,— I IPre 0 15 30 45 60 90 120Time (minutes)90z * * bo 80 * 0 SALINE70 *• —BL0CKED-J— LiJ 60Li D 50*J 40 +LiD-o 30z<20 ?C) 10 +Tht0-104/-I+ +71 I I IPre 0 15 30 45 60 90 120Time (minutes)80Figure lOa and lOb: Whole blood pH (P11a) and red blood cell pH (pH1) pre-exerciseand during 2 hours post-exhaustive exercise. Open circles symbolize fish that were pretreated with an injection of the saline carrier only before swum to exhaustion. Closedcircles symbolize fish that were pre-treated with an injection of the 3-adrenoceptorblocker nadolol before swum to exhaustion. * indicates a significant difference from thePre value in each group. f indicates a significant difference between the saline andnadolol injected conditions.81a8.0-io SALINE7 8f3—BLOCKED**7.2I I I I I I IPre 0 15 30 45 60 90 120Time (minutes)8.0 b7.8 0 SALINE• 3—BLOCKED7.6*EL7.472 t t 1• t t1-7.0 *****I I IPre 0 15 30 45 60 90 120Time (minutes)82Catecholamine responses.There were significant increases in circulating plasma catecholamine levels in allanimals post-swim (Fig. 11). This increase in plasma catecholamines was maintainedthroughout the entire 2 hour recovery period in all groups. There were significantdifferences in the catecholamine responses between groups as well. With NA, therewere significant differences in the peak levels attained, the /3-blocked group havinghigher levels of NA compared to the saline group. This difference was maintainedthroughout the entire 2 hour recovery period. AD measurements, however, did not showany significant differences between saline and nadolol treatment groups. There were nosignificant differences between the NA and AD values obtained from the 13-blockedgroup, but in the saline treatment, AD concentrations were significantly higher than NAconcentrations.Series II: Effect of nadolol on ventilation and blood gases in resting trout.Ventilatory responses.In both groups of fish, fg did not show any significant difference throughout theentire 4.5 hour post-injection period from the pre-injection value (Fig. 12a). Withventilation amplitude, there were significant differences between the responses of the twotreatment groups. The nadolol treated group showed a significant (25%) reduction inventilation amplitude two hours after the injection of nadolol (Fig. 12b). This reductionwas maintained for the remaining 2.5 hours that the fish were monitored. The salinetreated group did not show any significant difference in ventilatory amplitude postinjection from the pre-injection value.83Table 6: Mean values of various blood respiratory variables (± 1 S.E.M.) pre-exerciseand during 2 hours post-exhaustive exercise. Pao2 - arterial blood oxygen tension; Cao2 -arterial blood oxygen content; Hct - Haematocrit; Tot Hb - total haemoglobin; MCHC -mean cellular haemoglobin content; S - saline injected 2 hours prior to swim (N = 6); N- nadolol injected 2 hours prior to swim (N = 7). * indicates a significant differencefrom the Pre value in each group. t indicates a significant difference between the salineand nadolol injected conditions.84Pre0Mm.15Mm.30Mm.45Mm.60Mm.120Mm.Pao2,S137.5±6.7106.4±8.5104.7±10.4115.2±11.099±8.8119.4±9.6121.1±6.3mmHgPao2,N125.9±6.9111.9±6.2103.0±7.9104.7±8.7101.7±7.0107.6±7.3125.1±5.8mmHgCao2,S10.33±2.2011.57±2.7311.81±2.7311.93±2.7411.18±2.5010.42±2.2210.33±2.50ml02d1bloodCao2,N17.42±1.5216.86±2.0619.33±2.3719.20±2.0118.25±1.5815.55±1.1915.17±1.09m102d11tttttttbloodCo UiHct,S20.2±1.828.0±0.227.6±0.728.0±1.427.8±1.327.5±1.026.4±1.1%******Hct,N29.7±1.729.0±1.529.3±1.629.6±2.631.8±0.829.3±1.428.7±1.1I-TotalHb5.47±1.105.31±1.045.08±1.064.53±0.694.23±0.864.44±0.923.77±0.865,g/mlTotalHb9.44±0.909.09±0.799.39±1.038.39±0.938.19±0.728.01±0.826.98±0.39N,g/ml1ttttttMCHC,S28.4±6.418.9±3.718.3±3.615.9±2.114.7±2.316.0±3.215.1±3.9MCHC,N32.2±3.332.0±3.231.6±2.529.1±3.325.9±2.227.6±2.724.5±1.6tttttttFigure 11: Arterial plasma catecholamine levels pre-exercise and during 2 hours postexhaustive exercise. Open symbols signify saline injected fish, filled symbols signifynadolol injected fish. Circles represent NA levels, inverted triangles represent AD levels.* indicates a significant increase from the Pre value in each treatment. t indicates asignificant difference between the saline and nadolol injected treatments for eachcatecholamine.86C100Z 80 -0FHzu-i60 -z00u-iz40 --j00LiJ20-015 30 45Time60 90(minutes)871 20o NA SALINE• NA NADOLOLv AD SALINEv AD NADOLOL****1-**1•t•1•1***Blood measurements.The values for PHa, pH1, and Pao2 did not show any significant differencesbetween the pre- and post-injection measurements in either group. Nadolol, however,had a significant effect on Hct, Hb, Cao2 and MCHC, all of which were significantlyelevated 2 hours post-injection. This increase was maintained for the remaining 2.5hours (Table 7). Injection of the saline carrier resulted in no significant changes in thesesame values.88Figure 12a and 12b: Percent change in ventilation frequency (12a) and ventilationamplitude (12b) prior to and during 4.5 hours following injection of saline (open circles)or nadolol (closed circles) into resting fish. * indicates a significant difference from thePre value. t indicates a significant difference between the saline and nadolol injectedconditions.89-H0 °CD 0 C - C’)IIII1\)_010010(31%ChangeVentilationFrequencyIIIIIIc,J(14FJIJ--I0Ci’0Ci’0(ii0C)’%ChangeVentilationAmplitude-o -‘ Ct,0(3’0*_-+*—+•o*—I.ZCI)000U01Table 7: Mean values of various blood respiratory variables (± 1 S.E.M.) prior to andduring 4.5 hours following injection of nadolol or saline in resting trout. See Table 6 forfurther explanations of symbols and abbreviations.91Pre 2 Hours 2.5 Hours 3.5 Hours 4.5 HoursPao2, S 127.2 ± 7.3 127.0 ± 7.8 129.7 ± 6.6 129.8 ± 6.8 128.0 ± 3.5mmHgPao2, N 128.0 ± 7.1 132.0 ± 6.2 127.3 ± 7.1 130.2 ± 9.1 128.3 ± 8.0mmHgCa02, S 11.25 ± 0.40 10.41 ± 0.36 11.37 ± 0.50 11.84 ± 0.30 11.00 ± 0.46ml 02 d11bloodCao2, N 10.23 ± 0.46 17.99 ± 0.99 17.22 ± 0.93 14.89 ± 1.51 15.49 ± 1.74ml °2 d11 *_JbloodHct, S 21.2 ± 0.46 22.4 ± 0.21 22.7 ± 0.33 22.3 ± 0.24 22.4 ± 0.28%Hct, N 22.7 ± 0.28 29.0 ± 0.58 28.0 ± 0.46 28.2 ± .46 27.7 ± 0.31% *.,. *j. *j.Total Hb 5.97 ± .041 5.87 ± 0.40 5.72 ± 0.41 5.61 ± 0.41 5.62 ± 0.39S, g/mlTotal Hb 5.72 ± 0.44 9.99 ± 0.46 9.73 ± 0.44 9.39 ± 0.35 9.03 ± 0.33N, g/ml *.j- *f *j.MCHC, S 27.3 ± 1.5 26.2 ± 1.7 25.2 ± 1.8 25.2 ± 1.8 25.1 ± 1.8MCHC, N 25.3 ± 2.2 34.6 ± 1.8 34.9 ± 2.0 33.2 ± 1.0 32.6 ± 1.2*t *t *tPHa, S 7.81 ± 0.31 7.81 ± 0.03 7.80 ± 0.03 7.82 ± 0.02 7.83 ± 0.02PHa, N 7.81 ± 0.02 7.82 ± 0.02 7.84 ± 0.02 7.82 ± 0.03 7.82 ± 0.02pH1, S 7.44 ± 0.02 7.45 ± 0.02 7.42 ± 0.01 7.44 ± 0.01 7.44 ± 0.01pH1, N 7.43 ± 0.02 7.44 ± 0.02 7.45 ± 0.01 7.45 ± 0.02 7.43 ± 0.0292DiscussionThere was an effect of the 13-adrenoceptor blockade on the ventilatoiy response inrainbow trout following exhaustive exercise. Specifically, treatment with a J3-adrenoceptor blocker impairs the increase in ventilation amplitude (Fig. 9b), thoughplasma catecholamine levels increased. On the other hand, the lack of an effect ofnadolol on ventilation frequency indicates that f3-adrenoceptors are not likely involved inmediating the ventilatoiy frequency response following exhaustive exercise. Whether ornot catecholamines are involved in the frequency response cannot be determined fromthe current study, however it appears unlikely. This is an interesting result, for it meansthat ventilation frequency post-exercise is responding to another stimulus. It is notresponding to Cao2, since Cao2 did not change, and a /3-adrenergic effect also is notinvolved.The changes in plasma catecholamine levels were as expected, exhaustive exerciseleads to a significant increase of circulating catecholamines (Primmett et a!., 1986). It isinteresting, however, that the catecholamine levels obtained between the groups were notthe same. Nadolol injection resulted in a much larger release of NA than did the salineinjection alone. Why this difference in NA levels occurred is not known. It may be aneffect attributable to a-blockade. Similar observations have been made with other 13-adrenoceptor blockers in the rat, which seems to involve a direct action of the 13-blockerson the rat adrenal medulla, as well as centrally mediated effects (Sugawara et a!., 1980).Also, during exercise in humans, 13-blockade enhanced normal increases in plasmacatecholamine levels (Irving et a!., 1974; Galbo et aL, 1976). AD, on the other hand,did not show this difference and appears to have been equally released in both situations.93This implies that trout can release catecholamines differentially depending upon thesituation. This ability has been demonstrated in the Atlantic cod (Gadus morhua)(Perry et a!., 1991).The main point, however, is that fish that were treated with j3-blockers did notmaintain ventilation throughout the 2 hour recoveiy period, as A, declined from 0minutes to the end of the measurement period. The saline group, on the other hand,maintained A at the same level from 0 minutes to 60 minutes post-exercise. Thisindicates that, similar to the results seen in Chapter 2 with fish exposed to hypercapnia,13-adrenergic stimulation is required to maintain or sustain a complete ventilatoryresponse during recovery from exhaustive exercise. The site of action of thecatecholamines on ventilation is not known. However, it has been demonstrated thatinfusion of catecholamines into the 4th ventricle of the brain, or in the area of the vagalnucleus in the medulla of the dogfish stimulates respiratory activity in dogfish (Randalland Taylor, 1991). NA can cross the blood-brain barrier in trout (Nekvasil and Olson,1986), so elevated NA levels could act on the medullary respiratory centre by a directaction on neurons in the region of the vagal motor nucleus.There was a definite effect of nadolol alone on both the blood gases andventilation in rainbow trout (Series II). With respect to ventilation, there was a decreasein operculum motion of about 25 %, with no significant effect on ventilation frequency,so the fish were hypoventilating 2 hours post-injection of nadolol. This reduction wasmaintained at a constant level from 2 to 4.5 hours post-injection. The implication is thatthe nadolol treated fish in Series I were not ventilating at the same level as the salinecontrols, but hypoventilating in a similar manner before the start of the exercise regime.The probable cause of the hypoventilation are the effects of nadolol on Cao2, Hb, and94Hct, all of which were significantly elevated over pre-nadolol injection measurements(Series II), as well as potential effects of j3-adrenoceptor blockade on the adrenergicnervous system that may be involved in the cardiorespiratory system. Does this meanthat the changes in ventilation observed in Series I were due to these effects of nadolol,rather than the effects of a /3-blockade? Not necessarily, for though the initial ventilatoryeffort between the saline and nadolol treated groups are different (Series I) due todifferences in Cao2, there was no significant difference over time of the Cao2 valueswithin the nadolol or saline groups (Series I). In other words, the lower ventilatoryeffort seen in the nadolol treated group in Series I was most likely due to an attenuationof the ventilatory response, (lower starting point, lower increase in ventilator)’ effort) dueto a higher Cao2 in the nadolol treated group. However, the patterns of the maintainedventilatory response between the saline and nadolol treated groups were quite different(Series I). It could also be argued that the nadolol-treated fish may not have beenexercising as hard as the saline-treated group, because their blood oxygen levels werehigher, and perhaps this is why there was a significant decline in ventilatory effort in thenadolol treated fish. This was not so, as there was no significant difference in thevelocities attained between the saline and nadolol fish before exhaustion (data notshown), so they were exercising to similar levels. This is interesting, for van Dijk andWood (1988) state that fish that were pre-treated with the /3-blocker propranolol wouldnot exercise. A difference in the protocol between that study and the present, however,is that the fish in van Dijk and Wood (1988) were exhausted by chasing them with a stickin a tank, while in the current study, the fish were swimming against increasing watervelocities until they fatigued. This may be why the fish in this study did exercise thoughthey were pre-treated with a /3-blocker, as the fish were swimming at a lower velocity95initially, and gradually being forced to swim harder, rather than being startled intoswimming.There is the possibility that what we observed was an effect of nadolol on theperipheral chemoreceptor system in trout. Indeed, it has been shown that propranololcan cause a reduction of the chemoreceptor discharge in putative fish gillchemoreceptors, by blocking some adrenergic component of the peripheralchemoreceptor function (Burleson and Milsom, 1990). If nadolol works in a similarmanner, then one could expect that the reduction in A, observed in this study may be adirect inhibitory effect of the nadolol on the peripheral chemoreceptors. I think that thisis unlikely. It had been demonstrated that catecholamines do not affect the firingdischarge of peripheral chemoreceptors in fish (Burleson, 1991), so it seems unlikely thatthere is an adrenergic component to the function of these chemoreceptors. It is alsoknown that propranolol has local anaesthetic properties, and it is possible that theseperipheral chemoreceptors had their discharge diminished by these non-specific effects ofpropranolol. Nadolol, on the other hand, does not have a local anaesthetic effect(Hoffman and Lefkowitz, 1990) so nadolol is unlikely to have been inhibiting peripheralchemoreceptor function in this manner.In Series I, the oxygen tension and oxygen content of the blood is not a goodsource of information for the fish to determine its ventilatory effort during recovery. Inboth cases, there was no significant difference between the pre- and post-swim values(Table 6). Therefore, it is unlikely that the fish were using this information. Indeed,with the Cao2 data, there is a trend for increased oxygen content following exhaustiveexercise in both groups, so one would expect no difference in ventilator)’ effort, whichwas not what we observed under control conditions. This is not to say that the fish are96not using Cao2 to set ventilation, as the hypoventilation in Series II was set by the higherCao2 of the post-nadolol injected group.Blood haematocrit (Series I) showed different responses between the saline andthe 13-blocked condition, however, there was no correlation of this parameter with theventilatory changes observed in this study. Within the control group, the increase inhaematocrit observed post-swim is indicative of either cellular swelling (Primmett et aL,1986), splenic release of red blood cells (Kita and Itazawa, 1989), or more likely, both.Total haemoglobin did not show any significant change in both groups. Presumablysplenic release of red blood cells offset the effects of blood sampling, and haemoglobinlevels did not change. There is, however, a trend towards a decreasing MCHC, whichimplies that there was cell swelling. The nadolol treated animals had a differentresponse, the haematocrit did not show any significant differences before and after theswim.The pre-swim haematocrits and haemoglobin levels in nadolol treated fish weresignificantly higher than the pre-swim values for the saline injected group. Theseobservations were confirmed in Series II, as injection of nadolol resulted in a significantincrease in Hb and Hct values. This increase in haematocrit and haemoglobin levelscould be due to either a reduction in plasma volume, an increase in circulatingerythrocytes, or both, some time between the injection of the 13-blocker and taking thepre-swim/2 hour post-injection measurement. Haemoglobin levels increased more thanhaematocrit in the nadolol treated groups, so as a result, MCHC was higher in thenadolol treated fish, that is cell volume was probably less. This could be due to 13-blockade on the red blood cell membrane preventing any catecholamine inducederythrocyte swelling, though only low levels of catecholamines were present in these fish.97The effectiveness of nadolol as a 13-adrenoceptor blocker can be seen by lookingat the blood PHa and pH data. After exhaustive exercise, the PHa in both groupsdropped (Fig. 2a) as has been previously described (Primmett et al., 1986). However, thepH1 of the nadolol group also showed a drop, while the saline treated group was able tomaintain pH1 (Fig. 2b). Catecholamines induce a rise in pH1 in trout red blood cells thatis mediated by a /3-adrenoceptor mechanism (Nikinmaa, 1982a). Addition of a 13-blockerprevents this rise in pH1, which is what we observed, indicating that nadolol was stilleffectively blocking /3 adrenergic receptor sites, and the blockade was effective for theduration of the 2 hour period that recovery was followed.To summarize, in the control (sham-injected) fish, ventilation frequency andopercular amplitude increased, PHa dropped, pH1 remained the same, Hct increased, andlevels of circulating catecholamines increased, with more AD being released than NA. Inthe fish that were pre-treated with a 13-blocker, again both ventilation frequency andopercular amplitude increased, but the fish were not able to maintain the initial level ofventilation. Both pHa and pH1 dropped, indicating that the 3-adrenoceptor blockade waseffective. Hct did not change between pre- and post-swim conditions. Catecholamineswere released, but there was no significant difference between the NA and AD levelsmeasured. Nadolol itself has effects on blood oxygen status, which apparently setsventilatoiy effort to a different level than animals that are not so treated, but there stillare changes in ventilation that are not due to this difference in blood oxygen. Theseresults imply that catecholamines, specifically the /3-adrenergic component, may beinvolved in mediating ventilatory responses in fish following exhaustive exercise.98Chapter 5The effects of exogenous catecholamines on the respiratory centre in dogfish,Scyliorhinus Canicula.99IntroductionIn the previous chapters, I have shown that catecholamines have an effect onventilation in fish. Chapter 2 demonstrated that catecholamines that are present in thecirculation of the fish do affect ventilatory rhythm and pattern. In Chapters 3 and 4,blockade of /3-adrenoceptors blunts the ventilatory response of acidotic and fatigued fish,respectively. In unrestrained, undisturbed dogfish, catecholamine infusion, as well ashypoxia, results in an increase in ventilation (Metcalfe and Butler, 1984; Taylor andWilson, 1989). The possibility, therefore, exists that dogfish have a ventilatoiy responseto circulating catecholamines in a manner that was similar to teleosts. The question thatremains, however, is where the possible site of action of the catecholamines onventilation in fish is.Catecholamines have an effect on ventilation, but the mode of action is not clear.It is possible that ventilation may have been modulated by catecholamines via theperipheral chemoreceptor system. In some mammals, application of /3-adrenergicagonists have been shown to excite peripheral chemoreceptors (Milsom and Sadig, 1983;Mulligan et a!., 1986). However, in the trout, this is unlikely to be the cause ofventilatory changes, because Burleson (1991) has demonstrated that the afferent inputfrom peripheral chemoreceptors in trout is not altered by the application ofcatecholamines.It may be that the changes in ventilation were a secondary effect of thecardiovascular changes that were occurring as a result of the catecholamine injection.This is also unlikely. First, the time course for the changes in the ventilatory andcardiovascular parameters does not co-vary (Chapter 2); the cardiovascular effects last100longer. Secondly, there are strong cardiovascular effects at both the low and highcatecholamine concentrations (Chapter 2), while there is virtually no effect onventilation. This indicates to that there are separate pathways for the cardiovascular andventilatory responses. Finally, it has been demonstrated in the Atlantic cod (Gadusmorhua) that hypertension does not influence ventilatoiy responses during hypoxia(Kinkead et a!., 1991).Blood pH is a potential modulator of ventilation. However, there were nosignificant changes in blood pH when ventilation was either elevated or depressedfollowing catecholamine infusion, that is blood pH and ventilation did not co-valy(Chapter 2). Therefore, I consider it unlikely that changes in the pH status of the bloodresulted in the ventilatory changes observed.Taylor and Randall (1990) demonstrated in the Pacific Spiny Dogfish, Squalusacanthias, that injection of catecholamines into the fourth ventricle resulted in a changein the rhythm and pattern of central respiratory drive. These responses were blocked bypre-treatment with the 13-blocker propranolol.The implication of these last observations is that there are sites in the centralnervous system that responds to increasing catecholamine concentrations with a changein central respiratory drive. To investigate this possibility, I looked at the activity ofrespiratory neurons located in the medulla of fish. Dogfish were used for this study, asthey are larger than trout, and their brainstem is more easily accessible than a trout.Elasmobranchs and teleosts are different fish that have been separated evolutionanly fora long time. However, they live in very similar environments, so it is not unreasonable tothink that the control systems for ventilation between these two groups might be similar.Indeed, both the work of Taylor and Wilson (1989), and Taylor and Randall (1990)101indicates that both dogfish and trout have similar ventilatory responses to exogenouseatecholamines.The purpose of this study was two-fold. First, though there is evidence thatcatecholamines in the bloodstream can affect ventilation in dogfish, it is not known if theblood-brain barrier would be effective in preventing catecholamines from crossing intothe brain from the blood. Therefore, the ability of the brain to take up radioactive NAand AD was assessed. The null hypothesis was that catecholamines present in thecirculatory system of dogfish do not appear in the brain of dogfish. To see if differentareas of the brain accumulate catecholamines differently, the ability of the cerebellumand the medulla to amass catecholamines was compared. The second objective of thisstudy was to locate respiratory motor neurons in the medulla. The null hypothesis in thesecond part of this study was that catecholamines applied to respiratory neurons in thebrainstem do not alter the firing activity of these neurons. Microelectrodes were used tofind the bursting activity of respiratory neurons in the dorsal vagal motor column of thedogfish medulla. Once found, small volumes of NA were injected onto these neurons.102Materials and MethodsLesser Spotted Dogfish (Scyliorhinus canicula, N = 30) of either sex were used forthese experiments. They were held outdoors in a concrete tank at the Laboratories ofthe Marine Biological Association (Plymouth, U.K). Aerated seawater flowed throughthe tank, the temperature ranging from 13 to 15 C for a minimum 2 weeks. Thedogfish were not fed for a week prior to being used in the experiments. Temperaturevaried ± 0.5 C during any experiment.Surgical procedures.Series I: Accumulation of catecholamines by the dogfish brain.All surgery was performed under general anaesthesia. After catching the fish,they were deeply anaesthetized in a 1:10000 solution of tricaine methanesulfonate (MS222, Sandoz) before being weighed. They were then transferred to an operating tray,packed in ice and left for a further 5 minutes. The skin between the eyes was removedand a flap was cut from the chondrocranium to expose the forebrain. A completetransection was made through the diencephalon rostral to the optic tectum and theforebrain removed after cauterizing the major blood vessels. The caudal artery and veinwere cannulated with polyethylene tubing (PE-120, Intramedic). The caudal arterycannula was used for blood sampling, while the caudal vein cannula was used for druginjection. The fish was then transferred to the experimental tank.Series II: Effect of applying catecholamines onto respiratory neurons in the dogfish medulla.The dogfish were anaesthetized and cannulated as described above. The cartilage above103the cerebellum was removed to allow a pair of retractors to be inserted into the dorsalwalls of the chondrocranium, lateral to the cerebellum. Opening the retractors allowedenough pressure to be applied to the cartilage above the hindbrain, and allow theremoval of the cartilage. Care was taken not to damage any of the blood vessels in thechoroid plexus.The branchial branches of the Xth (vagus) cranial nerve were exposed on the leftside of the animal by a lateral incision, starting about 2 cm behind the spiracle andextending posteriorly just below and parallel to the lateral line to a point dorsal to thefifth gill slit (Taylor and Butler, 1982). This incision opened into the dorsal wall of theanterior cardinal sinus, on the floor of which runs the vagus nerve and the origins of itsmajor branches. A branchial branch of the vagus nerve (II or III) was identified, clearedof connective tissue, cut distally and replaced on the floor of the sinus. The incision wastightly sutured while the fish was being transferred to the experimental tank, which wasfilled with oxygenated, filtered and recirculated seawater. The fish was prepared for theexperiment by clamping it into a stereotaxic frame (Narishige Instruments) with a plateinserted in the mouth and clamped dorsally, a lateral clamp holding the body of the fishcaudal to the pectoral fins and dorsal to the midline to avoid constriction of posteriorcardinal sinuses, and a clamp around the posterior part of the tail.Experimental Procedures.Series I: Accumulation of catecholamines by the dogfish brain.After being placed into the experimental tank, still deeply anaesthetized, the fishwas injected with 40 tCi of either[3H]-adrenaline or[3H]-NA (New England Nuclear).The isotope injection was cleared from the cannula with 1.5 ml of dogfish saline. Two104minutes after the injection of the radioisotope, a blood sample was taken from thecaudal artely. The fish was decapitated and saline was infused into the cut ventral aorta,and saline appeared from the dorsal aorta, and the blood was cleared from the head.The brain was exposed and the medulla and cerebellum removed. The medulla andcerebellum were then weighed, and each placed individually in 300 p1 of trichloroaceticacid (6 % w/v). The blood and brain samples were then counted by liquid scintillationspectrophotometry (Beckman IS 1701).Series II: Effect of applying catecholamines onto respiratoly neurons in the dogfish medulla.After the transfer to the experimental tank, the decerebrate fish recovered from theanaesthetic and started to ventilate. A small volume (<1 ml) of curare [(+)-tubocurarine chloride, Wellcome, 7 mg kg9 was injected into the dogfish via the caudalvein. In the curanzed fish, spontaneous ventilatoiy and locomotor movements stoppedalmost immediately. The gills were then force ventilated with oxygenated seawater via atube inserted into the mouth below the clamp.The incision into the anterior cardinal sinus was reopened and held in positionwith a pair of retractors (McCarthy’s Surgical). By careful positioning of the retractors,the incision was orientated so that the blood continued to flow across the floor of thecardinal sinus and drain into Cuvier’s duct, thereby maintaining venous return to theheart. The branchial branch of the vagus was placed over a pair of platinum recordingelectrodes. The respiratory related activity in the nerve was relayed via a pre-amplifier(Isleworth, type AlOl) with suitable filters and an additional amplifier (NeurologSystems, Digitimer) to a dual beam storage oscilloscope (Tektronix R5031) and recordedon magnetic tape (4-channel FM tape recorder, SE Laboratories).105Double-barrelled micropipettes, one a recording electrode filled with 4 mM NaC1having resistances of 10-20 Mfl, the other an injection electrode filled with either 106 MNA or carrier only, were driven into the hindbrain with a calibrated micromanipulator(Narishige Instruments) in the area of the vagal motorcolumn. When respiratory relatedactivity similar to the activity in the branchial nerve was picked up in the centralrecording electrode, a small amount of the fluid in the injection electrode was injected byhydrostatic pressure applied by a Picospritzer II (General Valve Corporation). Theactivity from the centrally placed electrode was amplified and filtered (Neurolog Systems,Digitimer) before being sent through the oscilloscope and FM tape recorder as above.Before and after the recording of the central respiratory-related activity, the recordingmicroelectrode was calibrated to ensure that any of the changes in activity observed werenot due to a change in the resistance of the microelectrode. A few of the animals werepre-treated by an injection of D,L-propranolol (2 mg * kg1) via the caudal vein 45minutes prior to the administration of NA. The dose-response of D,L-propranolol indogfish brains by central and peripheral injections has been investigated. The dose usedin this study was found to be more than adequate to give complete blockade tocatecholamine injections (Taylor, pers comm.) Also, 3 animals were mechanicallystimulated by moving the contralateral gill septa to compare this type of nerve activity torespiratory-related activity.Statistical analysis.Data are presented as means ± 1 S.E.M. Statistical significance was performed asdescribed in Chapter 2. A P value of 0.05 was taken as the statistical level ofsignificance.106ResultsSeries I: Accumulation of catecholamines by the dogfish brain.The radioactivity of 3H in the tissues after the injection of[3H]AD or[3HJNA isshown in Table 8. On a per gram basis, there is a difference in the accumulation ofcatecholamines in the cerebellum and medulla. The cerebellum did not show anysignificant difference in the activity of 3H after injection of either[3H]AD or[3HINA.On the other hand, in the medulla, there was significantly more [3H] activity after NAinjection than AD injection. The activity in the cerebellum and medulla was divided bythe activity in the blood sample, to give a ratio between the two. This gives an idea ofthe accumulation of [3H] in the cerebellum and medulla after injection of eithercatecholamine. In the medulla, there was a significantly larger ratio of [3HJ in themedulla after[3H]NA injection than after[3H]AD injection. There was no significantdifference in this ratio in the cerebellum after the catecholamines were injected.Series II: Effect of applying catecholamines onto respiratoly neurons in the dogfish medulla.After locating respiratory-related units in the hindbrain of the dogfish, application of NAresulted in two responses. The first was an increase in the neuronal activity of bothcentral and peripheral nerves (Fig. 13) After a latency of approximately 2.62 ± 0.32 s.(N = 5), there was an increase in the firing rate. The response was of a short duration,lasting 2.8 ± 0.22 s. The intervals between bursts were significantly reduced (0.98 ± 0.03s to 0.56 ± 0.3 s), and there was also a significant increase in the burst duration (0.19 ±0.02 s to 0.32 ± 0.01 s). It can also been seen that there is some change in the activitywithin the central burst. Before the application of NA107Table 8: The activity of [3H] in the cerebellum and medulla of the dogfish (counts perminute, CPM) following the injection into the caudal vein of[3H]-adrenaline (8a) or[3H]-NA (8b). The * indicates a significant increase in [3H] activity following theinjection of[3H]-NA than following the injection of[3H]-AD. N = 5.108Table 8a:Cerebellum Cerebellum Medulla MedullaCPM * g tissue’ CPM * g tissue’/ CPM * g tissue’ CPM * g tissue’/CPM * m1’ blood CPM * ml’ bloodAvg. 20926.70 3.736 18159.40 2.736S.E. 4158.26 2.116 2077.13 0.292Table 8b:Cerebellum Cerebellum Medulla MedullaCPM * g tissue’ CPM * g tissue-’! CPM * g tissue-’! CPM * g tissue1!CPM * m1’ blood CPM * m11 blood CPM * m[’ bloodAvg. 25187.39 6.720 26730.92 * 8.627 *S.E. 7172.93 1.317 2571.52 2.175109Figure 13: Recording from a curarized and force ventilated dogfish of respiratoiyrelated activity from a central respiratory neuron (RN) and the third branchial branch ofthe vagus (BV). The bursting activity accelerated briefly after the pressure injection ofNA into the central recording site (arrow). The response (R) was of short duration (2.6s), starting 3.5 s after injection, and consisted of an acceleration in bursting rate, and anincrease in the number of spikes per burst. The “A” indicates an artifact in therecording.110RN“IR }-43sto the hindbrain, there were between 2 to 3 spikes in a burst (Fig. 13). In the response,there is an increase in the number of spikes observed within a burst to between 3 and 5spikes. After the response, the number of spikes in the burst return to the pre-injectionlevel.These responses to the application of NA contrasts from the other response thatwas observed, which was an inhibition of the activity recorded from the area of the vagalmotor column in the brainstem (Fig. 14). The latency between injection of NA, and theinhibition of respiratory-related activity (6.30 ± 0.26 s, N = 3) was significantly longerthan the latency that occurs when an increase in respiratory related activity is observed(Figs. 13, 14). The period when respiratory-related activity was not observed lasted for3.06 ± 0.19 s. There were significant changes in the burst intervals and burst durations,as they went from 0.37 ± 0.02 s. to 0.58 ± 0.02 s. (burst interval), and 1.05 ± 0.05 s. to0.76 ± 0.04 s. (burst duration). The animals injected with the carrier solution alone didnot show any changes in the activity of the neurons centrally or peripherally (Fig. 15).In the animals that were pre-treated with an injection of D,L-propranolol prior tothe injection of NA centrally, the increase in bursting activity seen before propranololtreatment (Fig. 16b, burst interval from 1.03 ± 0.44 s. to 0.63 ± 0.4 s, burst durationfrom 0.33 ± 0.02 to 0.57 ± 0.02 s. N = 5) did not occur (Fig. 16c, burst interval from1.03 ± 0.03 s. to 1.04 ± 0.04 s., burst duration from 0.33 ± 0.03 s. to 0.37 ± 0.03 s. N =5). Comparing the activity before and after the administration of D,L-propranolol didnot reveal any significant difference due to the /3-adrenoceptor antagonist itself.112Figure 14: Recording from a curarized and force ventilated dogfish from a centralrespiratory neuron and the second branchial branch of the vagus, after NA injection,demonstrating an inhibitory response. The bursting activity centrally was diminishedafter the pressure injection of NA into the central recording site (arrow). The responsewas of short duration (2.5 s), starting 6.2 s after injection, and consisted of a depressionof bursting activity centrally. The symbols in this figure are as for Figure 13.113FAARwIIi w .hi.Lii.IL1TTIII•Ir’TJ+II,rjir,,119JIsF,Figure 15: Recording from a curarized and force ventilated dogfish from a centralrespiratory neuron and the second branchial branch of the vagus after saline injection.The bursting activity was unchanged after the pressure injection of saline (arrow). Thesymbols in the figure are as for Figure 13.1159Uw z-••-.••••••••h’i”—23•aa-•.—•-..•.-—..-23••a63•__10WFigure 16a, 16b and 16c: Recording from a curarized and force ventilated dogfish froma central respiratory neuron and the third branchial branch of the vagus with notreatment (16a), NA injection (16b), and NA injection following pre-treatment with D,Lpropranolol (16c). The increase in bursting seen upon application of NA (16b, arrow) isnot present in the fish following pressure injection of NA into the central recording site(16c, arrow) after pre-treatment of the dogfish with the 13-blocker D,L-propranolol. Pretreatment of the dogfish with D,L-propranolol does not appear to have any effect on thebursting pattern, as the pattern in 16b is unchanged from 16a, before the administrationof D,L-propranolol. The symbols in the figure are as for Figure 13.117• .....:....• .• -_ei. :ee.:-._-.•. .IC’),—•.•. INz . ...• .• .• . .> zI- .. . •CI)118For comparison purposes, the activity from both the central and peripheral nervesafter mechanical stimulation of the gill septa was also recorded. This allowed me to seeif the activity observed after mechanical stimulation was similar to the activity observedafter injection of NA centrally. The activity observed from the mechanical stimulationwas quite different (Fig. 17). The burst duration following the stimulation of the gill was1.3 s, which was longer than the burst duration after NA injection. There was, however,no significant change in the interburst interval or burst duration before or after themechanical stimulation. Other differences were that the activity observed after the gillwas touched was that the large burst of activity was reflected in both the central andperipheral sites, and that there were only single bursts to any single stimulus.119Figure 17: Recording from a curarized and force ventilated dogfish from a centralrespiratory neuron and the third branchial branch of the vagus following mechanicalstimulation. When the gill on the contralateral side (right) was mechanically stimulated(arrows), large bursts of activity could be seen on both the central and peripheralrecordings. The bursting pattern after the mechanical stimulation, however, wasunaffected. The symbols in the figure are as for Figure 13.120BVRNmIDID 44isDiscussionThe results of the first series of experiments demonstrated that catecholaminesthat are in the circulation of dogfish can pass through the blood-brain barrier, and befound in the brain of dogfish. This is similar to findings in both rainbow trout,Oncorhynchus mykiss, (Nekvasil and Olson, 1986) and eels, Anguilla anguilla. It is alsointeresting that different areas of the brain take up circulating catecholaminesdifferently. While the cerebellum does not differentiate between the two catecholamines,and accumulates them equally, the medulla takes up NA preferentially to AD. This is animportant finding as it shows that it would be possible for circulating catecholamines tohave access to the brain and affect the medullary respiratoiy centre. Whether otherareas of the brain are like the medulla, and differentiate between the catecholamines, orare more like the cerebellum and don’t differentiate is not known.It is possible that the radioactivity that is being picked up is from the metabolitesof AD or NA. However, as only 2 minutes were allowed before the fish was sacrificed tocount the activity present, it seems unlikely that a large portion of metabolites would bepresent. Indeed, Nekvasil and Olson (1986) demonstrated that after 2 minutes, therewas approximately 80 % of[3H]-AD and[3H]-NA present in the blood from the originalinjection in rainbow trout.The results of the second series of experiments shows that NA administered tothe respiratory motorneurons can affect the bursting activity of these neurons. Bothexcitatory and inhibitory effects can be observed following the central injection of NA.The short latency and duration of both the increased and decreased neuronal activity to122NA stimulation indicates that these neurons can be directly affected by localconcentrations of catecholamines. The fact that both excitatory and inhibitory effects ofcentral injections of NA can be observed is similar to what was observed in Chapter 2,where both excitatory and inhibitory effects of NA on ventilation were also observed.The neuronal excitation following NA injection, increases in the bursting rate (or adecrease in the interburst interval) and the number of spikes in a burst/burst duration,indicate that these effects of NA were affecting both the rhythm (frequency) and pattern(force of ventilation) for respiration. If the activity of enough central respiratory-relatedneurons changes, I would expect in the intact animal to see a change in the activity(frequency and/or force) of the respiratory muscles of the buccal/opercular pumps. Incurarized and force-ventilated dogfish, recorded from the branchial branch of the vagusonly, intravenous bolus injection of 10.6 M NA resulted in an increase in bursting rateand burst duration in the vagus (Randall and Taylor, 1991).There have been other observations of excitatory effects of NA on respiratoryneurons. Working on rabbits, Fallert (1979) recorded the activity of respiratory neuronsin the hindbrain after microiontophoresis of various neurotransmitters. With NA,although clear effects were not established as both excitation and inhibition of respiratoryneurons was observed, excitation was the predominant effect of the application of NA.When isoproterenol, which is a 13-adrenoceptor specific agonist (Hoffman & Lefkowitz,1990) was applied, stimulation of inspiratory neurons was observed, indicating that this islikely a 13-adrenoceptor mediated effect.Folgering (1980) investigated the effects of 13-adrenergic mimetics and blockersinjected into the vertebral artery of paralysed, artificially ventilated cats. The 13-mimeticsisoprenaline and prenalterol stimulated the firing of the phrenic nerve, while the /3-123blockers propranolol, metoprolol and oxprenolol resulted in a depression of activity.Intravenous injection of these same drugs at the same dose did not have any effect onphrenic nerve activity, so he assumed that the effects observed were central effects. Thephrenic nerve activity was quite different when an anaesthetic (xylocaine) was used, andD-propranolol gave a varied response. L-propranolol, however, had depressant effects,indicating that the action of the $-blockers were not via a local anaesthetic effect.The other response observed after application of NA was inhibition of centralactivity. That administration of NA can lead to an inhibition of respiratoiy activity is notsurprising, as similar observations have been made previously. For example, in rainbowtrout, injection of NA during moderate hypoxia resulted in a hypoventilatory response(Kinkead and Perry, 1990). The latency of the response was longer when an inhibitionof central neuronal activity was observed. Why the latency was longer in these responsesis not known. It is not likely that the inhibitory effect observed was an artifact of thepressure injection itself due to the latency of the response. If the decrease in theresponse was due to the physical removal of the recording site from the electrode due tothe pressure of the injection, then this inhibition should have been immediate. Instead,it took about 6 seconds to develop, which indicates that this is a true inhibition of activityrather than the removal of the electrode from the bursting activity. It was not due to achange in the resistance of the electrode, as the calibration check of the microelectrodeafter recording was not different from before the recording. The latencies observed inthis study are long. This implies that the site of action of the catecholamines is not onthe neurons that I was recording from. Where may the site of action by NA be? Itcould be that the neurons that are being activated are interneurons located some short124distance away from the neurons that I was recording from. With the picospritzer, basedon the pressure used, the tip diameter of the electrode, and the distribution/spacing ofthe cell bodies in the vagal motorcolumn, there would be approximately 30 to 50 cellbodies (E.W. Taylor, pers. corn.) that could be affected by the injection. The observedlatency could then be due to time for NA to diffuse, and to stimulate the neurons tochange their activity. In other words, I am not dumping NA directly onto the site ofaction.Other investigations have also revealed inhibitory effects of catecholamineadministration to respiratory neurons in the medulla. Microiontophoresis of NA ontoareas of the brainstem where respiratory-related neurons in the medulla of decerebratecats were previously noted resulted predominantly in a depression of phrenic nerveactivity (Champagnat et a!., 1979). This study also looked at the effects of AD,isoprenaline (isoproterenol), and clonidine (u-adrenoceptor agonist) by iontophoresisonto respiratory related neurons. Again, depression was the dominant effect. Since theeffect was consistent across the catecholamines used, it is likely that the effect is acatecholaminergic mechanism. Clonidine led to a depression of phrenic nerve activity,even in neurons that were excited by NA, suggesting that the effect is an a-receptormediated effect. However, isoprenaline also produced a depression of phrenic activity 83percent of the time, so Champagnat et aL, (1979) speculated that -adrenergicmechanisms may be involved as well.Murakoshi et al., (1985), using an isolated brainstem-spinal cord preparation ofnewborn rats, studied the pharmacological effects of various neurotransmitters onrespiratory frequency. Both NA and AD resulted in a reduction of respiratoryfrequency. The use of an cr-adrenoceptor blocker (phentolamine) prevented the125depressant effects of NA. Isoprenaline did not have an effect on the respiratolyfrequency, and propranolol did not prevent the depressant effects of NA. This impliedthat the frequency effects of NA are mediated by an a-adrenoceptor mechanism.The lack of an effect of catecholamines on the activity of the respiratory neuronsfollowing pre-treatment of propranolol implies that the excitatory effects of NA aremediated by j3-adrenoceptors. The mammalian studies also demonstrate that thestimulatory effects of NA on respiration are a /3-adrenoceptor mediated effect, while thedepressant effects are a-adrenoceptor mediated. Similar conclusions were reached byPeyraud-Waitzenegger (1979), who observed a /3-receptor mediated hyperventilation, andan a-receptor mediated hypoventilation in eels. The other interesting observation is thatcentral excitation resulting from NA injection can be observed in the peripheral nerves,while with inhibition, the effect is much more localized, and not seen peripherally.When NA is injected into the medulla, the injection site encompasses a small area,probably stimulating 30 to 50 cell bodies. With an excitatory response, this stimulus ispassed on to the branchial branches of the vagus. Why are similar effects not observedduring inhibition? The reason for this is not known, but it is possible that the stimulusused was simply not large enough to evoke a full inhibitory response that could berecorded from the branchial branches of the vagus.The basis for the inhibitory response is also not known, but it may be that theinhibitory effects of catecholamines are not mediated within the medulla, but is due toinhibition from another site, possibly another area of the brain, sending inhibitoryneurons to the respiratory centre in the medulla. As the first series showed, differentareas of the brain in fish have different permeability to catecholamines, so the actions ofcatecholamines may occur in different areas of the brain. In newborn rats, Hilaire et a!.,126(1989) looked at the modification of ventilation as a result of NA application to abrainstem-cervical cord preparation. When NA was added, there was a drop in fictiveventilation frequency, whereas the use of an cr-adrenergic antagonist, yohimbine, resultedin an increase in fictive respiratory frequency. Electrical stimulation of the ventrolateralpons inhibited fictive respiratory rhythm. After yohimbine was applied to the perfusionmedium, electrical stimulation of the ventrolateral pons became ineffective at inhibitingrespiratory rhythm. It was therefore suggested that a noradrenergic inhibitory driveoriginating from the caudal ventrolateral pons modulates the activity of the medullaryrespiratory generator by an u-adrenergic mechanism, and something similar may beoccurring in fish. If this in fact occurs in fish, it would also explain the longer latency inthe inhibitory response to NA.To summarize, catecholamines do have a central effect on ventilation in fish.Stimulatoiy and depressant effects have been observed. Propranolol blocks the effect ofNA on respiratory neurons. There may be a separation of the stimulatory and inhibitoryeffects of catecholamines in the brain and between the a- and 13-receptors, which lead toinhibitory and stimulatory effects. Noradraline can cross the blood-brain barrier indogfish, similar to eels and trout, and different parts of the brain can take upcatecholamines differently. This suggests that catecholamines can get to the brain fromthe circulatory system, and exert their effects on the ventilation of fish.127Chapter 6General Discussion128The question asked in this thesis was, can circulating catecholamines have aneffect on the gill ventilation in fish? More specifically, is it possible that f3-adrenoceptorstimulation mediates ventilatory responses to catecholamines in fishes? The observationsof Chapter 2 indicate that catecholamines can affect gill ventilation in fish. The resultswere complicated, for whether the fish hypo- or hyperventilated was dependent upon thecatecholamine used. Other studies have also shown an effect of catecholamines in thecirculatory system affecting ventilation in fishes.In Amia calva, injection of catecholamines results in a significant increase inopercular amplitude (McKenzie et a!., 1991a). When the bowfins had their gillchemoreceptors denervated, the response of the animal to aquatic hypoxia was differentfrom sham operated animals (McKenzie et a!., 1991b). In the denervates, the rapidincrease in gill ventilation was abolished, but a slow ventilatory increase was observed.This response was similar in time course to the ventilatory response of intact bowfin tocatecholamine injection, and plasma NA levels were elevated during hypoxia in thedenervates. The response was not due to catecholaminergic stimulation of anextrabranchial 02 chemoreceptor, as cyanide did not stimulate any ventilatory responsesin the denervates, indicating that bowfin do not retain02-sensitive chemoreceptormediated ventilatory responses following denervation. McKenzie et a!., (1991b)speculated that the circulating catecholamine levels may have been mediating theventilatory response of the denervated bowfin during hypoxia. If this was so, then theventilatoiy response to catecholamines could not involve peripheral chemoreceptors. Asadrenergic blockers were not used in this study, it is not known whether the ventilatoryresponse to catecholamine injection in the denervated fish was due to the stimulation ofa or 13 adrenergic receptors.129In eels, ventilatory responses to catecholamine injection were dependent upon thetime of year. In summer fish, catecholamine injection resulted in an increase inventilation (Peyraud-Waitzenegger et aL, 1980). NA and AD increased ventilationfrequency and amplitude, with increases in opercular amplitude being more importantthan increases in ventilatory frequency. Isoprenaline (isoproterenol) which is a pure 13-adrenergic agonist, produced a slightly larger increase in ventilation than NA or AD.When propranolol was utilized to block 13-receptors, catecholamine injection resulted in ahypoventilatory response. This hypoventilatory response was similar to thehypoventilation seen when catecholamines were injected into winter eels, and could beblocked with the use of an a-adrenoceptor blocker, phentolamine. As PeyraudWaitzenegger et a!., (1980) discussed, the hyperventilatory response to catecholamineinjection appears to be mediated by /3-adrenoreceptors, while the hypoventilatoryresponse is due to a-adrenoceptor stimulation.Studies in the carp, Cyprinus caipio, have also demonstrated similar ventilatoryresponses to catecholamine injection as the eels. The ventilatory responses were againseasonally dependent (Peyraud-Waitzenegger, 1972). AD and NA injection resulted in ahyperventilatory response in summer carp, but a hypoventilatory response in winter carp.Use of adrenergic blockers showed that the hyperventilatory response was due tostimulation of 13-receptors, and hypoventilation from the stimulation of ar-receptors.The seasonality of the ventilatory responses to catecholamine injection is anothercomplicating factor that could make the interpretation of my data difficult. I do notthink, however, that this is a complicating factor in my thesis, as all of the experimentswere perfonned on summer fish. The temperature range for all of the experiments werefrom 9° to 16° centigrade. This temperature range is considered within the temperature130range for summer fish (R. Kinkead, pers. corn.), so it is unlikely that the ventilatoryresponses observed were due to differences between the seasonal states of the fish.Playle et a!., (1990) demonstrated changes in ventilation following catecholamineinjection. They noted that injection of AD resulted in an immediate hypoventilation,while NA injection resulted in a biphasic response, an initial hyperventilation, followedby hypoventilation. They suggest that these responses to catecholamine injection may bedue to differential effects of the injected catecholamines on central adrenoceptors, but asadrenoceptor blockers were not used, whether the effects were due to a- or f3-adrenoceptor stimulation is not known.In Chapter 2, generally speaking, injection of NA provoked an increase in P0, anda decrease in fg, while AD injection resulted in a drop in fg, with no effect on P0k,. Withthe pre-treatment of trout with the a-blocker, phentolamine, changes in fg followingcatecholamine injection were eliminated, while an increase in was unmasked at thelower concentrations used. There are three implications from these results. The first isthat it appears that changes in POJA, are due to stimulation of f3-adrenoceptors in trout,as the a-receptors were blocked, /3-receptors are the most likely site of action for theinjected catecholamines. The second is that changes in fg due to catecholamine injectionare mediated by a-receptors. Finally, it also appears that there is some interactionbetween a and /3 receptors, as blockade of the a receptors unmasked a hyperventilatoryresponse.These studies indicate that catecholamines present in the blood system can affectventilation in fish. However, not all the investigations looking at the effect ofcatecholamines on ventilation have come to the same conclusion. Trout that wereexposed to a moderate level of hypoxia did not release catecholamines into the131bloodstream (Kinkead and Perry, 1990), but did exhibit a hyperventilatory response tohypoxia. In addition, the use of both a- (phentolamine) and 13- (propranolol) blockersdid not modify the hyperventilatory response during hypoxia. This was to ensure thattransient catecholamine changes that were not detected during sampling were notstimulating ventilation. They also observed that an inter-arterial infusion ofcatecholamine in normoxic trout resulted in a transient (adrenaline) or persistent (NA)hypoventilation. In similar study, catecholamines were again administered to rainbowtrout during moderate hypoxia or hypercapnia (Kinkead and Perry, 1991). A slightlydifferent method was used, where catecholamines were infused into trout instead ofbeing given a bolus injection. In hypoxic fish, infusion of catecholamines resulted in atransient hypoventilatory response, while catecholamine infusion during hypercapniaresulted in a more prolonged hypoventilatory response. The catecholamine infusionswere repeated under conditions of normocapnia, hyperoxia, and hyperoxic hypercapnia toassess whether or not the initial ventilation or blood respiratory status could beimportant in catecholamine-mediated ventilation. All of these initial conditions alsoresulted in no effect, or a depression of ventilation. In studies in Atlantic Cod, theventilatory responses to hypoxia were not altered by pre-treatment of the fish with eitherof the a- or 13-adrenoceptor blockers phentolamine or sotalol (Kinkead et a!., 1991).Bretylium, which is an inhibitor of peripheral catecholamine release also did not alterventilatory responses to hypoxia. From these studies, it was concluded thatcatecholamines do not have a stimulatory role in the control of ventilation in teleosts.With respect to the studies of Kinkead et a!., (1990, 1991), the hypoxic stimulusall used a moderate level of hypoxia. For the level of hypoxia utilized, it is not surprisingthat ventilatory modifications were made without the presence of catecholamines. It is132more than likely that these ventilatory responses were moderated by02-chemoreceptoractivity. It has been observed that gill oxygen chemoreceptors in trout increase theiractivity with decreasing oxygen levels, and that these receptors are not sensitive tochanges on the level of plasma catecholamines (Burleson, 1991). It would seem then,that catecholamines do not have an effect on gill ventilation in fish by stimulating gillchemoreceptors, nor do they have a stimulatory role on gill ventilation during moderatehypoxia or hypercapnia. This is not unexpected, as the ventilatory response to hypoxia inteleosts is a rapid response, and is strong evidence for an02-chemoreceptor basedventilatory reflex. However, below a Po2 of approximately 30 torr, fish chemoreceptorsshow a very strong inhibition of activity (Burleson, 1991). It is also at this Pao2 thatcatecholamines are released into the bloodstream of trout (Thomas et a!., 1988), and itmay be possible that in trout, increased ventilation during severe hypoxia is maintainedby elevated catecholamine levels, counteracting the inhibition of peripheral 02-chemoreceptors. I am not suggesting that all of the ventilatory responses of fish aremediated by catecholamines. Changes in ventilation occur very rapidly when an animalis exposed to environmental hypoxia, indicating that the initial changes in ventilation areneural in origin, resulting from the stimulation of chemoreceptors.Another focus of this thesis was, under what conditions could plasmacatecholamines mediate ventilatory responses? In a previous study, Aota et aL (1990)demonstrated that catecholamines may be mediating ventilation in trout during severehypoxia. In that study, severe hypoxia resulted in an increase in both ventilation andcirculating catecholamines. When propranolol was injected before the exposure tohypoxia, the ventilatory response to hypoxia was significantly impaired. It is also known133that acid infusion results in an increase in circulating catecholamines (Boutilier et a!.,1986), so acid infusion has been used to see what effect this would have on ventilation(Aota et a!., 1990). As expected, when acid was infused into the fish, ventilationincreased, as did circulating catecholamines. When propranolol was used to pre-treat theanimals before the injection of the acid, the hyperventilatory response was againimpaired. The fish were also injected with acid during exposure to hyperoxia, withoutpropranolol. This blocked the release of catecholamines into the bloodstream, and therewas no increase in ventilation. This indicated that the acidosis, by itself, was notsufficient enough to cause a ventilatoiy response. The simplest interpretation of theseresults is that the acidosis resulted in catecholamine release, and this, in turn, stimulatedventilation. Chapter 3 looked at an acidosis caused by exposing the fish to hypercapnicwater. As expected, this procedure resulted in an acidosis in the fish, a hyperventilatoryresponse, and a rise in plasma catecholamines. Animals that were pre-treated with D,Lpropranolol showed an impaired ventilatory response to hypercapnia, while the fish pretreated with D-propranolol had a full ventilatory response, indicating that theimpainnent of the ventilation was not a result of some non-specific side effect of D,Lpropranolol. The blood and water parameters measured (PHa, Pao2, Cao2, Pwco2, Pwo2),which are all potential modulators of ventilation were not different between treatments,indicating that the respiratory drive between treatments was the same, so the differencesin the size of the ventilatory responses reveals the direct contribution of 13-adrenoceptorstimulation.The acidosis following burst swimming is also associated with high levels ofcirculating catecholamines (Primmett et at., 1986), and so this may stimulate gillventilation in the post-exercise fish. Chapter 4 studied this possibility, utilizing the 13-134blocker nadolol, which is known not to have the anaesthetic side-effects of propranolol(Hoffman and Lefkowitz, 1990). Unfortunately, nadolol does have other side effects,specifically altering Cao2 by effects on Hct and total Hb. This meant that the overalldifferences in A013, and the smaller increase in A post-exercise in the nadolol treatedfish observed in Chapter 4 was due to the differences in Cao2. However, afterexhaustion, the group pre-treated with nadolol had high levels of catecholamines, and animpaired ability to maintain ventilation. An acidosis was present, and the effectivenessof the f3-blockade was demonstrated by the observation that the nadolol group could notregulate intracellular red cell pH. Though there were differences in blood respiratorystatus between groups due to nadolol itself, within a group, oxygen status was constant,but there were still differences in the pattern of respiratory change between the twogroups.The other difference between the studies of Aota et at., (1990), Chapters 3 and 4and those of Kinkead et aL, (1990, 1991) is that there was no acidosis in the latterstudies. It may be that there is some interaction between catecholamines and blood pHthat modulate ventilation in fish. This, however, seems unlikely. Ventilation and PHa,however, do not always co-vary. An acidosis without catecholamine release does notstimulate ventilation in fish (Aota et at., 1990). In addition, hyperoxic hypercapnia,although causing an acidosis, can result in hypoventilation (Smith and Jones, 1982), orhyperventilation (Thomas et a!., 1983). Correlations between PHa and ventilation havebeen reported (Janssen and Randall, 1975; Graham et a!., 1990; Wood et a!., 1990) butno direct effects of changes in PHa on ventilation have been shown. When mockextradural fluid was perfused into Amia calva, alterations of pI-IIPco2had no effect on gillor air-breathing (Hedrick et a!., 1991). Finally, cerebral spinal fluid pH and brain pH1 in135skates during hypercapnia does not correlate well with ventilation (Perry and Wood,1989). So, though a central H receptor is thought to exist, there is little evidence tosupport it.It is also interesting that the catecholamines have different effects on ventilationfrequency and amplitude. The effect on frequency is a depression probably via radrenoceptors, by 25% or less, while amplitude can increase, via /3-adrenoceptors by 60-90%. Why this occurs is not known, but it could be due to mechanical limitations of thesystem. It may be that as ventilation amplitude increases, it must take more time, so thatthe number of breaths that can be taken are reduced, but each breath is a deeper breath,for the same time period measured. What the trade off is between increasing amplitudeversus decreasing frequency is also not known.For water-breathing animals, because carbon dioxide is more soluble in water thanoxygen, ventilation is regulated with respect to oxygen supply rather than carbon dioxideexcretion, so a central H receptor would not be required (Randall, 1990). It is possiblethat the regulatory system for ventilation consists of peripheral oxygen chemoreceptorsand a central catecholamine-sensitive system. The peripheral chemoreceptor systemwould adjust ventilation to oxygen availability during normoxia and moderate hypoxia,while the catecholamine system would adjust ventilation to metabolism when oxygen islimiting. This is a different situation from what would be required on land, as ventilationis coupled to carbon dioxide elimination and regulation of PHa. For terrestrial animals,the central W chemoreceptor system will dominate over any actions of catecholamines.It would also be unlikely that there could be any action of circulating catecholaminescentrally in mammals, as the blood-brain barrier in mammals is not permeable tocatecholamines. It appears, then, that there must be a change in the control systems136between water- and air-breathing animals, as the criteria for water-breathing, mainlyregulation of oxygen exchange, and air-breathing, regulation of carbon dioxide and PHaare different requirements. Indeed there may be evidence for this, for in Amia calva,which is a bimodal breather, catecholamine injection results in a stimulation of gillventilation, but had no effect on air-breathing (McKenzie et at., 1991a).All of the previous studies have looked at the effect of catecholamines onventilation, but nothing specific could be said about the site of action of thecatecholamines on ventilation. The last focus of this thesis is, is there any evidence of acentral effect of catecholamines on ventilation in fish? In dogfish, the injection ofcatecholamines results in an increase in ventilation if it is an undisturbed animal (Taylorand Wilson, 1989). In a different study (Randall and Taylor, 1989), the centralrespiratory drive of dogfish was monitored from the branchial branches of the vagus thatinnervate the respiratory muscles. A bolus injection of catecholamines into the caudalvein of curarized and force-ventilated dogfish resulted in a stimulation of bursting activityin the branchial vagus nerve about 40 to 120 s after injection, which would be about thetime it would take for the bolus to reach the brain. In another study, the activity of thehypobranchial nerve, which innervates feeding muscles, and the branchial branch of thevagus, which innervates respiratory muscles were recorded simultaneously (Levings andTaylor, unpublished). The branchial branch had regular respiratory-related burstingactivity, while the hypobranchial fired occasionally, though with respiratory-relatedactivity. When catecholamines were injected, there was high levels of respiratory-relatedbursting activity in the hypobranchial nerve. This implies a potential role forcatecholamines centrally in the mediation of ventilation.Taylor and Randall (1990) have examined the possible effects of catecholamines137on the central respiratoiy neurons of dogfish by injection of catecholamines into thecentral nervous system. Small amounts of AD were injected into the fourth ventricle,while recording from a branchial branch of the vagus. The application of AD caused achange in the bursting pattern from the vagus. After about 100 s of the injection, therewas a slowing of the bursting rate, with an increase in the activity of a burst. Thisimplies an increase in the stroke volume rather than the ventilation frequency, which wasalso observed in trout (Chapter 4; Aota et a!., 1990). As the response developed, boththe rate and activity in a burst increased. These responses were blocked by injection ofpropranolol.These observations imply that there are sites within the CNS, accessible from thefourth ventricle, which can be stimulated by an increase in catecholamine concentration,and increase or decrease respiratory drive. The vagal respiratory neurons are located inthe dorsal vagal motor nucleus, positioned medially in the medulla, close to the wall ofthe fourth ventricle (Withington-Wray et aL, 1986). These neurons are rhythmicallyactive, and supply the innervation to respiratory muscles in the gills. Their location closeto the fourth ventricle may have caused them to be directly affected by the injection ofcatecholamines into the fourth ventricle. It was based on these observations that thestudy in Chapter 5 was undertaken. However, though catecholamines are known to crossthe blood-brain barrier in trout and eels, (Peyraud-Waitzenegger et a!., 1979; Nekvasiland Olson, 1986), it was not known if the same was true for elasmobrauchs, specificallydogfish. The results of Chapter 5 show that dogfish are similar to both eels and trout,for catecholamines do enter the brain. What is interesting about this, however, is thatthere appears to be differential accumulation of circulating catecholamines into differentareas of the brain. Whether this is due to different permeability of the blood-brain138barrier in different areas of the brain, or due to selective uptake in different areas is notknown. The effect of catecholamines centrally was complex, as both inhibition andstimulation were both observed. This is similar to the previous observations in trout(Chapter 2) for catecholamine injection resulted in both hyper- and hypo-ventilatoryresponses. In mammals as well, centrally applied catecholamines resulted in bothstimulatory and inhibitory respiratory activity. It appears from the mammalian studiesthat the inhibitory effects are a-receptor mediated, while the stimulatory effects are /3-receptor mediated. In the dogfish as well, administration of a /3-blocker blocked thestimulatory effect of catecholamine injection into the respiratory neurons of the medulla.To reiterate, this thesis has demonstrated that catecholamines, specifically NA inthe circulatory system, can affect ventilation by stimulation of /3-adrenoceptors.Conditions when catecholamines are released, like during an acidosis caused by anexternal factor like hypercapnia exposure, or an internal one, exhaustive exercise, areconditions when catecholamines could be playing a role in mediating ventilation, as /3-blockers impaired the ability of the fish to maintain a full ventilatoiy response to theseconditions. The mechanism, or site of action of the circulating NA is likely central. NAcan cross the blood brain barrier in fishes, and NA administered to the medullaiyrespiratory neurons alters the firing activity of these neurons, and the central stimulatoiyactions of NA can be blocked by /3-adrenoceptor antagonists.139ReferencesAota, S., Holmgren, K.D., Gallaugher, P., and Randall, D.J. 1990. A possible role forcatecholamines in the ventilatory responses associated with internal acidosis or externalhypoxia in rainbow trout Oncorhynchus mykiss. Journal of Experimental Biology. 151:57-70.Ballintijn, C. 1982. Neural control of respiration in fishes and mammals. In: Exogenousand Endogenous Influences on Metabolic and Neural Control oft Respiration, FeedingActivity and Energy Supplies in Muscles, ion- and Osmoregulation, Reproduction, Perceptionand Orientation. Eds. Addink, A.D.F. and Spronk, N. Pergamon Press, Oxford. pp 127-140.Barrett, A.M. and Cullum, V.A. 1968. 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