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Modulation of respiratory rhythm and pattern in rana catesbeiana the bullfrog Meier, Janice T. 2000

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M O D U L A T I O N O F R E S P I R A T O R Y R H Y T H M A N D P A T T E R N m RANA CATESBEIANA T H E B U L L F R O G by Janice T. Meier B. Sc., University of Winnipeg, 1994. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA APRIL, 1999 © Janice T. Meier in presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of L-O C7 V The University of British Columbia Vancouver, Canada • a t e AcrStL /<79 -6 (2/88) 11 ABSTRACT The present study examined the role of neurons in the optic tectum in the formation of the periodic, episodic breathing pattern in the bullfrog, Rana catesbeiana. The first objective of the present study was to search for the presence of an "episodic centre", v ia progressive transections through the optic tectum of a decerebrate, artificially-ventilated in situ preparation. The results of these experiments revealed that the rostral optic tectum provides inhibitory, and the mid-optic tectum excitatory inputs to medullary centres, with respect to breathing frequency. Furthermore, the latter region also modulates burst pattern (providing an inhibitory input), as well as respiratory pattern, in concert with peripheral feedback from the vagus nerve. More specifically, following transections through the mid-optic tectum, the episodic breathing pattern was converted to one of evenly-spaced single breaths. In animals with at least one vagus intact, lung inflation restored the episodes. The caudal optic tectum, like the mid-region, appeared to influence both breathing pattern, as well as a component of burst pattern. Following transections at this level, the average burst duration increased significantly, while the integrated activity was not significantly altered. With respect to breathing pattern, the episodic breathing pattern was converted to one of evenly-spaced single breaths, although the overall breathing frequency was not changed significantly. The episodes could not be restored by lung inflation, suggesting that the vagal input which resulted in the reappearance of episodes following transections in the mid-optic tectum, acted rostral to the site of the caudal transection of the optic tectum. The possibility that an "episodic . centre" was located in the caudal optic tectum was refuted in one preparation in which Ill episodes were observed following a transection at both the caudal optic tectum and rostral spinal cord. This suggested the presence of a neuronal input arising caudal to the site of transection in the spinal cord. Episodes re-occurred when this input was removed with only the medulla intact. These results implied that the centre(s) responsible for the formation of episodes exists not within the optic tectum, but rather within the medulla. A second objective of the present study was to examine the medulla of the bullfrog brain for the presence of multiple central rhythm generators for breathing. While recording from both the Vth and Xth cranial nerves, transections were made between the two, following which all neural activity from both nerves ceased. This suggested the presence of a centre, at the site of the transections, which is essential for the production of respiratory rhythm in the bullfrog, Rana catesbeiana. iv TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES vi ACKNOWLEDGEMENTS viii INTRODUCTION , 1 Hypotheses 25 MATERIALS AND METHODS 26 Decerebrations 26 Decerebrations 26 Experimental Set-up 26 The effects of decerebration 30 The effects of transections in the optic tectum 33 The effects of transections between the Vth and Xth cranial nerves 33 Brain tissue histology 33 Data analysis 33 RESULTS 35 The effects of transections caudal to the cerebrum (decerebration) 35 The effects of transections of the rostral optic tectum 40 The effects of transection of the mid-optic tectum 40 The effects of transections in the caudal optic tectum 58 The effects of transections between the Vth and Xth cranial nerves 58 V DISCUSSION 74 Critique of methods 74 Transections of the rostral optic tectum 75 The effects of lung inflation 76 Transections at the mid-optic tectum 77 The effects of lung inflation 77 Transections at the optic tectum-medulla border 80 Is the transection of the mid-optic tectum at the site of the nucleus isthmi? 82 The effects of transection between the Vth and Xth cranial nerves 83 CONCLUSIONS 85 Respiratory Rhythm 85 Burst Pattern 85 Breathing Pattern 86 Respiratory Frequency 86 REFERENCES 88 vi L I S T O F F I G U R E S Figure 1: Continuous versus periodic breathing 4 Figure 2: Fictive breath recordings from the trigeminal nerve of the bullfrog illustrating the 3 characteristic cycles which comprise breathing in this animal 8 Figure 3: Schematic diagram illustrating the mechanisms of breathing in the bullfrog 11 Figure 4: The effects of transections at the rostral optic tectum in the in vitro bullfrog brain 17 Figure 5: The effects of transection between the Vth and Xth cranial nerves in an in vitro bullfrog brain 21 Figure 6: Diagram illustrating the experimental apparatus employed in the present study 28 Figure 7: Schematic diagram of the bullfrog brain indicating the level of decerebration 31 Figure 8: Representative traces comparing the respiratory pattern of an intact animal breathing air with that of an in situ animal unidirectionally-ventilated with air 36 Figure 9: The effects of CO ? and lungs inflation on ficitive respiratory activity in an in situ preparation of Rana catesbeiana 38 Figure 10: Cross-sectional view of the bullfrog brain at the level of the rostral transection 41 Figure 11: A comparison of the effects of transection through the rostral optic tectum of an in situ bullfrog ventilated with 2.5% C 0 2 to an animal ventilated with air 43 Figure 12: Quantitative results following transections at the rostral optic tectum 45 Figure 13: Cross-sectional image of the bullfrog brain at the level of the mid-optic tectum 47 V l l Figure 14: Quantitative results following transections at the mid-optic tectum 49 Figure 15: Representative neural traces illustrating the effects of a transection at the level of the mid-optic tectum 51 Figure 16: Representative neural traces illustrating an alternate effect of a transection through the mid-optic tectum 54 Figure 17: Representative neural traces illustrating the effects of a transection at the mid-optic tectum, following bilateral vagotomy 56 Figure 18: Cross-sectional image illustrating the level of transection at the caudal optic tectum 59 Figure 19: Quantitative results following transections at the caudal optic tectum 61 Figure 20: Representative neural traces illustrating the effects of a transection at the caudal optic tectum 63 Figure 21: Cross-sectional image illustrating the level of transection between the Vth and Xth cranial nerves 65 Figure 22: Quantitative results following transections between the Vth and Xth cranial nerves 67 Figure 23: Representative neural traces illustrating the effect of a transection between the Vth and Xth cranial nerves 69 Figure 24: Fictive breath recording from the Vth cranial nerve following transections at the caudal optic tectum and rostral spinal cord 71 Figure 25: Schematic diagram of the bullfrog brain, mapping regions with respect to their potential roles in the central control of breathing in this animal 78 V l l l ACKNOWLEDGEMENTS I would like to offer a special note of gratitude to Dr. W. K. Milsom, for the opportunity to complete my masters under his supervision, and for complying with my request to use pencil instead of red ink throughout the 54 drafts of this thesis. I am grateful for his helpful guidance and invaluable instruction, and in awe of his uncanny ability to know that it was always my fault. I would like to thank my supervisory committee, Dr. D.R. Jones and Dr. J.D. Randall, for their advice in the writing of this thesis, and for refraining from chastising me when I begged it be done in 3 days in order to accommodate my defense date. I would like to express my deepest gratitude to my parents, for their extraordinary support and unparallelled generosity, and for the 24 pairs of shoes which inhabit my closest. Truly, their encouragement and support lifted many pressures during my time at UBC. I would like to thank Elliott, for the many late evenings he offered his company and integral advice such as "shouldn't that be plugged in?". I appreciate the many hours he spent fixing the Flinstone-model Gould recorder, and the months following which he spent removing the ink from his skin. I would also like to thank my fellow lab mates, who tolerated my annoying quirks and immature antics with transcendent patience and only minor lynching. I offer a special thanks to the following: Danielle Brochu, for so many things, the most important being her lunch. In truth, I extend my gratitude for all of her help and for sharing my morbid, dark and twisted sense of humour... .(compliment). "Bethie", foremost for her laugh. Secondly, for her help: fixing slides, writing my thesis, preparing my seminar, working in windaq, quattro pro, sigma plot, sigma stat, corel draw, power point, etc, etc, etc. Finally, for her encouragement and support, and for admitting aloud that the Canadians won in 1812. "Rete", for her milk money. The Reverand Michael B. Harris, for trying many times in vain to outline for me the difference between a crescent wrench and a power saw, for successfully convincing me that "old men with beer bellies" can run stellar lOK's, and of course, for his valient attempts to ordain Steve. IX The less-than-reverand Dr. Stephen G. Reid, for instructing me on the surgical techniques and experimental procedures necessary for completing this degree and for inaverdtently prompting the response, "what in the hell kind of technique is that?" from Bill; for consistently reminding me that every Calorie consumed would rapidly find its way to my thighs; and most importantly, for entrusting me, at any cost, to protect the life his dearly beloved, "mouse". On this note, I would like to acknowledge "mouse", to whom I dedicate this thesis, if not to pay homage then in hopes of divorcing myself from the ungracious alias "The Angel of Death". Finally, I would also like to thank NSERC of Canada, whose operating grant to Dr. W.K.Milsom allowed me to do research in this lab. 1 INTRODUCTION Although it is difficult to define the term "life", it is generally agreed that three of the most important properties defining a living being are replication, catalysis, and mutability. In turn, each of these is interdependent upon one another, for example in order for an organism to replicate, energy must be expended. Many eons ago, organisms were able to use ATP and polyphosphates directly from the environment around them (Voet and Voet, 1990). However, as these components were depleted, organisms were forced to develop metabolic pathways which would enable them to produce necessary energy from simpler, more abundant precursors. Eventually, a seemingly inexhaustible resource, the sun, became the source of photosynthetic processes, with H 2 O serving as the reducing agent. Yet at this time, this created another problem for organisms: the new, more advanced photosynthesis released highly reactive O2, which accumulated in the atmosphere. Over time, the reducing atmosphere that once existed was transformed into an oxidizing one consisting of 21% O2. As a result, organisms were once again forced to adapt, and eventually evolved an oxidative method of metabolism, known as respiration, in which the now abundant O2 could serve as the oxidizing agent. Today, the most urgent requirement of any living organism is the need to exchange respiratory gases. The failure to supply critical organs with oxygen, if even for minutes, can result in the animal's death. Consequently, it is essential that an animal develops efficient mechanisms of gas exchange with the media which it breathes. The two most common environments surrounding animals, air and water, differ in several aspects and thereby exert very different influences upon respiratory-related 2 functions. More specifically, gas properties such as solubility, and hence concentration, vary significantly depending on whether the gas is dissolved in air or water. Moreover, differences in density and viscosity between the two media present further consequences upon respiratory-related functions. The result of these differences is manifest in the ease with which oxygen can be extracted from the given media. As a consequence of oxygen's relatively low solubility in water, the concentration of oxygen in air far outweighs that in water. For example, at a partial pressure of 159 mm Fig and a temperature of 12 degrees Celsius, the concentration of oxygen in air is 200 mL/L and only 7.7 mL/L in fresh water (Hill and Wyse, 1989). In salt water, this value drops to 6.1 mL/L, as gas solubility in water decreases with increased salinity. Furthermore, as the temperature rises, the solubility of oxygen in water is significantly reduced, while in air it is negligibly altered. Compounding the limitations imposed by the lowered solubility of oxygen in water as compared with air are the debilitating effects resulting from water's greater density and viscosity. The density of fresh water at 17 degrees Celsius is over 800x that of air at 1 atm pressure and at the same temperature, while the viscosity of water at 0 degrees Celsius is over lOOx higher than that of air. The consequence of these differences in density and viscosity, as well as the differences in solubility, is that organisms which acquire oxygen from water have significantly lowered accessibility to this respiratory gas than land animals and therefore must commit substantially more energy to the task of obtaining an equal volume of oxygen. As a result, water-dwellers such as fish tend to breathe continuously due to low oxygen stores. 3 Mammals, too, breathe continuously however for different reasons: mammals must service a demanding metabolic rate. It is therefore understandable that animals with low metabolic rates and with unchallenged access to oxygen via air may not require continuous breathing. Instead, animals suiting this description, such as amphibians, reptiles, and air-breathing fish, tend to breathe periodically. Periodic breathing is a pattern of respiration characterized by breaths which are separated by apneic spans (Fig. 1). The breaths may occur singly or in clusters of two or more (commonly referred to as episodes), and may be rhythmic or arrhythmic. Therefore, animals can be classified as either continuous breathers (in which there are constant inspiration/expiration couplings), or periodic breathers (in which the breaths are separated by apneas). The latter group can be further subdivided into episodic breathers versus non-episodic breathers, and rhythmic versus non-rhythmic breathers. Episodic periodic breathing consists of clusters of 2 breaths or more, separated by apneic spans, while non-episodic periodic breathing refers to single breaths followed by apneic spans. Both episodic periodic as well as non-episodic periodic breathing can be further classified as either rhythmic (having a consistent cadence) or non-rhythmic (without consistent cadence). It follows then, that in the transition from water to land, vertebrates underwent profound evolutionary modifications to the respiratory system which are manifest not only in the structure of their respiratory organs, but also in the design product of their breathing. In obligate water-breathers such as most fish, gas exchange occurs at the gills, 4 Figure 1: Continuous versus periodic breathing. Continuous breathing consists of a constancy of inspiration/expiration couplings, while periodic breathing is characterized by breaths separated by non-ventilatory apneas. Periodic breathing may be further subdivided into episodic versus non-episodic, both of which may be either rhythmic or non-rhythmic. Note that "v" denotes raw nerve activity from the trigeminal nerve, while "Jv" indicates integrated activity from this nerve. 5 6 across the gill lamellae, which are situated on the gill arches housed within the opercular chamber. Briefly, 02-containing water is first drawn into the buccal cavity when the floor of the mouth is lowered, thereby decreasing the pressure to below ambient. The water is then drawn across the gills into the opercular cavity via both opercular suction-, and buccal pressure-pumps, respectively, before rejoining the ambient water. Although most fish utilize well-developed gills for gas exchange, some, which reside in sluggish waters with low O 2 concentrations, have evolved mechanisms to exploit the Cte-rich air. Often the gills of such animals will be reduced and instead most gas exchange will occur across a modified organ of the alimentary canal. The extent to which the animal uses its gills as opposed to the modified organ is dependent primarily upon the 02-availability of the surrounding water. In some cases, such as those of the African and South American lung fish, well-developed "lungs" have dominated the reduced gills to the point that these fish have become obligate air-breathers. In fact, the organization of the dipnoan lung is very similar to that of the primarily terrestrial amphibian. Ancestral amphibia represent a unique class of animals from an evolutionary viewpoint, as they served as the link in the transition from water to land. This role served particular importance with regards to the respiratory system, requiring the animal to adapt to air- rather than water-breathing. In larval bullfrogs, the skin persists as the primary organ for both oxygen uptake (60%) and carbon dioxide excretion (60%) until metamorphosis is nearly complete, with the gills serving as secondary respiratory organs (Burggren and West, 1982). Following metamorphosis, the lungs become the primary 7 organ for oxygen uptake (80%), while the skin remains the major site for carbon dioxide excretion. In fact, the lungs of adult bullfrogs expel a maximum of just 20% of the total carbon dioxide excreted, but play a larger role under conditions of increased metabolic rates (Gottlieb and Jackson, 1976) and temperatures (Mackenzie and Jackson, 1978). Most importantly during metamorphosis, the gills of the tadpole degenerate as it progresses from water- to air-breathing, while the lungs develop. Other amphibia, such as mudpuppies, retain their external gills throughout adulthood and ventilate them via muscular movement. Interestingly, the larvae of anurans also utilize muscular movements to ventilate their gills, enclosed in an opercular chamber, in similar fashion to fish. Following degeneration of the gills, the buccal movements persist. There is debate regarding the purpose of buccal pumping in adults. While some researchers believe the buccal pumping is merely an evolutionary remnant (lacking purpose in the now air-breathing animal), others believe these movements serve to ensure fresh air is available for the ensuing lung ventilations. Still others have suggested an olfactory role. Among amphibians, the anatomy of the lungs vary from being simple sacs with little folding in most adult species, to more elaborate, better-divided structures in anurans. The lungs, skin and oropharynx are all components of the respiratory process. (Kogo et al., 1994). Three characteristic cycles comprise frog breathing: buccal ventilations, lung ventilations, and lung inflation cycles (Fig. 2). Buccal ventilation describes the ventilation of the oropharynx. For reasons which remain unknown, they may or may not occur consistently between lung breaths. Buccal oscillations occur via vertical movements of the buccal floor, with the nares remaining 8 Figure 2. Fictive breath recordings from the trigeminal nerve of the bullfrog, Rana catesbeiana, illustrating the 3 characteristic cycles which comprise breathing in this animal. 1) Low amplitude events, known as buccal ventilations. 2) High amplitude events, known as lung ventilations. 3) Events of increasing amplitude, known as lung inflation cycles. Note that "v" indicates raw nerve activity from the trigeminal nerve. 9 B U C C A L V E N T I L A T I O N S L U N G V E N T I L A T I O N S and L U N G I N F L A T I O N C Y C L E S ) 10 open and the glottis closed (deJongh and Gans, 1969; Vitalis and Shelton, 1990). The cranial nerves and corresponding muscles responsible for these buccal movements can be classified as either levators (which raise the buccal floor) or depressors (which lower it). The primary buccal levator muscle is the intermandibularis, and is innervated by the mandibular branch of the trigeminal nerve (Vmd). Accessory levator muscles include the muscularis omohyoideus and the muscularis geniohyoideus, both of which are innervated by the main branch of the hypoglossal nerve (Hm) (deJongh and Gans, 1969; Sakakibara, 1984). The sternohyoid branch of the hypoglossal, on the other hand, is responsible for innervating the primary buccal depressor muscle, or the muscularis sternohyoideus (Sakakibara, 1984). The purpose of these buccal oscillations, as mentioned above, is not completely understood. A second characteristic pattern in the bullfrog consists of lung breaths, which occur in episodes under conditions of elevated drive (deJongh and Gans, 1969; Vitalis and Shelton, 1990). In the bullfrog, lung ventilation is seen as a larger than normal inhalation of air into the oropharynx following which the glottis opens. This allows air to flow from the lungs, through the oropharynx, and out of the open nares (Fig. 3). The nares then close, and the buccal cavity pushes air from the oropharynx into the lungs. This event immediately precedes the pulmonary, non-ventilatory interval, as the glottis closes and the lungs remain inflated (de Jongh and Gans, 1969; Vitalis and Shelton, 1990). Although the potential exists for mixing of expired and inspired air, it has been suggested that very little mixing actually takes place. DeJongh and Gans (1969) have proposed that air leaving the lungs through the oropharyngeal cavity does so in a jet-11 Figure 3. Diagrammatic representation of a lung ventilation in the bullfrog. 1 ) The buccal floor lowers and fresh air enters the buccal cavity through the nares while the glottis remains closed and the lungs remain inflated. 2) The glottis then opens and the air from the lungs is released across the buccal cavity and out through the nares. 3) The nares then close and the buccal floor rises, pushing the air from the buccal cavity into the lungs, after which the glottis closes until the next lung ventilation. [From C. Gans, Evolution 24: 723-724 (1970)] 1 2 Buccal cavity Glottis 13 stream fashion, minimizing the potential for mixing. However, this hypothesis is not conclusive, and in fact has been disputed by Vitalis and Shelton (1990). The respiratory nerves involved in the lung ventilation cycle include the laryngeal and pulmonary branches of the vagus, the former of which influences the dilation/constriction of the glottis as well as the nerves mentioned previously for buccal ventilation (Kogo et al., 1994). The final type of ventilation exhibited by bullfrogs is known as a lung inflation cycle and consists of a characteristic pattern of lung breaths. More specifically, a lung inflation cycle describes a series of several lung ventilations without significant expiratory activity, resulting in a gradual, progressive inflation of the lungs. The neural mechanisms producing lung breaths also produce the lung inflations. While the purpose of these breaths of progressively-increasing tidal volume is not clearly understood, it has been suggested that it results from the limitations imposed by pulse pump respiration. Since the volume of air that enters the lungs is limited by both the volume within the oropharyngeal cavity and the pressure differential between the atmosphere and the lungs, frogs will employ lung inflation cycles when the need for oxygen increases. As the evolutionary progression of organisms continued towards land, development of the lungs became more complex. For example unlike amphibians, most reptiles employ the lungs exclusively for gas exchange. Reptiles provide an interesting turning point in respiratory development. Although some reptiles have simple, unicameral lungs (having only one chamber), like those in amphibians, most reptiles have developed more complex, multi cameral lungs with bronchi entering the separate 14 chambers. Also innovative with this group was the mechanism of ventilation: reptiles, like birds and mammals, exhibit an aspiration- or suction-pump to fill the lungs rather than the buccal pressure mechanism utilized by amphibians. Yet similarities to amphibians persist, with both the presence of buccal ventilations as well as the pattern of respiration they exhibit. Most reptiles (excluding certain species of varanid lizard), display an episodic breathing pattern akin to amphibia and air-breathing fish, in which clusters of breaths are separated by apneic spans (Shelton et al., 1986). In birds and more so in mammals, the lungs are highly branched and subdivided to provide maximum surface area for gas exchange, in order to their high metabolic rates. Furthermore, unlike the episodic breathing pattern mentioned above, these animals tend to breath continuously, whereby there is a constant series of inspiration/expiration couplings (Milsom, 1991). Interestingly, fish also exhibit a continuous patten of respiration but as a consequence of the relative difficulty inherent in obtaining oxygen from water rather than air. Both water- and land-residing animals have been known to exhibit an episodic pattern when metabolic activity becomes depressed. For example, while most fish display only continuous ventilation, episodic breathing occurs in the carp Cyprinus carpio, the bullhead Ictalurus nebulosus, and the sucker Catostmus commersonii when their metabolic needs are low and the oxygen availability is high (Shelton et at., 1986). Many diving animals such as elephant seals have also been known to breath episodically, even on land, when their metabolism is depressed in sleep (Milsom et al., 1996). Furthermore, certain hibernating mammals (such as the golden-mantled ground squirrel) alternate between continuous and episodic patterns of breathing as their 15 metabolic needs change. These lines of evidence suggest that all vertebrates possess common mechanisms for breathing integrated by a central pattern generator, the product of which (either continuous or episodic breathing), depends on respiratory drive. In order to effectively define the role of a "central pattern generator", it is first necessary to resolve a common inconsistency when referring to "respiratory pattern". Most researchers, particularly those studying respiration in continuous rather than periodic breathers, describe the shape of the respiratory discharge associated with a single breath when referring to "respiratory pattern". More specifically, this definition describes the total motor output (a correlate of tidal volume), as well as temporal activity (such as rapid onset versus incrementing-decrementing activity) associated with an individual breath. However, researchers who study periodic breathers would refer to this as "burst pattern" and would instead define "respiratory pattern" as the arrangement of the respiratory discharge; that is, the organization of breaths to generate an overall pattern of breathing. Since the present paper focuses on respiration in a periodic breather {Rana catesbeiana), the term "respiratory/breathing pattern" will therefore refer to the arrangement of breaths, while "burst pattern" will describe their individual shape. A "central respiratory pattern generator", then, may be defined as a group of neurons in the brain which exert an influence upon the central rhythm generator to collectively produce a breathing pattern. If it is true that a central pattern generator is the key to producing either continuous or episodic breathing, then it is possible that if removed from an episodic breather, the resultant pattern may resemble continuous breathing. To this end, Kinkead 16 et al. (1997) explored the optic tectum for a potential site responsible for clustering breaths into episodes (an episodic centre). While recording fictive breathing from both the Vth and Xth cranial nerves, a region of the optic tectum known as the "nucleus isthmi" (located between the roof of the midbrain and the cerebellum) was lesioned by microinjections of kainic acid. As a result of the ablations, the respiratory pattern was converted from episodic breathing to periodic single breaths, and an overall decline in breathing frequency was noted. Initially, this indicated the potential discovery of an "episodic centre", responsible for clustering breaths. However, the episodes (and a return to the original breathing frequency), could be restored by hypercarbia, indicating that an excitatory input, rather than an "episodic centre", had been lesioned by the kainic acid. The results of this study, nonetheless, indicated that the respiratory pattern was modulated by sites located in the optic tectum of the bullfrog. The only results to date which may indicate the presence of an episodic centre in the bullfrog brain were obtained from an in vitro study, in which a transection through the rostral optic tectum transformed the breathing pattern from an episodic one to one of high frequency single breaths (Fig. 4). However, in vitro studies have often produced inconsistent results. For example, an episodic respiratory breathing pattern was exhibited during in vitro experiments by Kinkead et al. (1994), however not in the two studies by McLean et al. (1995a and b). A fourth study detailed the occurrence of episodic breathing in 35% of the preparations, and non-episodic breathing in the other 65% (Reid and Milsom, unpublished data). Moreover, while both McLean et al. (1995a) and Kinkead et al. (1994) described a significant pH sensitivity associated with the in vitro 17 Figure 4. The effects of transections at the rostral optic tectum in the in vitro bullfrog brain. A) Episdoic breathing recorded from the trigeminal nerve prior to the transeciton. B) Conversion from an episodic breathing pattern to a continous one following the transection. The arrow denotes the transection event. C) High-frequency single breaths persisting after the transection. (Reid and Milsom, unpublished data). 18 1 _ 30 s B 30 s C 30 s 19 preparation, Reid and Milsom (unpublished data) noted a blunted pH response. Finally, inconsistencies have also been revealed with respect to central rhythm generation. While Perry et al. (1995) noted that a transection caudal to the trigeminal nerve abolished discharge from the vagus, an in vitro study by Reid et al. (unpublished data) recorded fictive breathing from both the trigeminal and vagus nerves even after a transection was made between the two. The role of central rhythm generators (CRGs) in breathing has been investigated since 1923, when Lumsden was able to demonstrate the progression from eupnea to apneusis following transections through the pons of the cat. Since then, various studies have explored this issue over several years. A respiratory central rhythm generator may be defined as a group of neurons located in the brain whose cadent depolarizations induce respiratory-related movements which enable the animal to breathe. Whether the rhythm is generated from pacemaker cells or a more complex neural network remains unknown. In vertebrates, CRGs have been demonstrated to exist in the medulla, as transections eliminating the brain both rostral and caudal to this region have not eliminated respiratory bursts (Smith and Feldman, 1987). A more specific location is currently being sought after, with the prime candidate being a region in the ventral medulla known as the pre-Botzinger complex (Smith et al , 1991). To date, the mechanisms underlying rhythrnogenesis remain unknown, however two hypotheses dominate the literature: one of these attributes the generation of rhythm to a set of pacemaker cells, while the other involves a more complex neural network. Neither have been proven. In the frog, numerous studies have examined the endogenous respiratory-related 20 rhythm generated by the medulla (Langendorff, 1887; McLean et al., 1995b; Reid et al., unpublished data; Schmidt, 1973). Chemical lesion studies using microinjections of lidocaine, GABA, and glutamate into the medulla have suggested two sites within the ventral medullary reticular formation which appear to influence endogenous respiratory activity in the bullfrog: one is believed to be located between the Vllth and IXth cranial nerves, and the other is suggested to exist at the level of the vagus nerve root (McLean et al., 1995b). This supports earlier work by both Langendorff (1887) and Schmidt (1973), who documented respiratory-related activity from the medulla of the frog following transections between the Vth and Xth, and VHIth and Xllth cranial nerves, respectively. Perry et al. (1995) performed transections caudal to the trigeminal nerve following which activity from the vagus ceased. More recently, in vitro transection studies have suggested not just one, but potentially several central respiratory rhythm generators in the medulla of the bullfrog (Reid et al., unpublished data). In this set of experiments, suction electrodes were used to record fictive breathing from the Vth and Xth cranial nerves of the in vitro brainstem. Following a transection between the two nerves, activity persisted in both. However, while the bursts in both nerves occurred simultaneously prior to the transection, the bursts now occurred asynchronously (Fig. 5). Interestingly, multiple CRGs which operate as a unit when the brain is intact have been described in both chick and lamprey studies (Fortin et al., 1995; Rovainen, 1983; Thompson, 1985; Russell, 1986). In the chick embryo, transverse slices of the hindbrain are able to generate rhythmic respiratory activity in isolation (Fortin et al., 1995). In larval lamprey, a trigeminal pacemaker has been proposed as a transection at the level of 21-Figure 5. The effects of transection between the Vth and Xth cranial nerves in an in vitro bullfrog brain. A) Prior to the transection, bursts from both nerves occur simultaneously. B) Following transection between the two nerves, however, the bursts occur asynchronously. Asterisks denote neural activity from the trigeminal nerve, while arrows indicate activity from the vagus nerve. Note that the burst pattern from the vagus nerve, and in some cases the trigeminal is rapid-onset, decrementing rather than a "normal" incrementing-decrementing pattern. (Reid et al., unpublished data). 22 A Isolated Medulla 4 ' p i 1 h M ' " Mi I B Transection between nerves V and X V i 1 i fr» ' m 'lull i . i ' i i j^MMIl'I'lll^ in «H»lUi> v W « I I I II [ ni j >iin»iiimi — ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^  T V .1 UN,,,, • .j! ^ . Mil i | »» ill I I I i l 30 s 23 the V nucleus eliminated respiratory bursts caudal to the cut, while sparing activity located rostrally (Homma, 1975). Initially, the V nucleus was also believed to be the site of the pacemaker in adult lamprey (Rovainen, 1983), however other studies suggested a more caudal component of the respiratory rhythm generator (Kawasaki, 1979, 1984). More recently, central rhythm generation in adult lamprey has been attributed to a medullary system comprised of 2 pairs of CRGs and the motoneurons they synapse with (Thompson, 1985; Russell, 1986). One pair of CRGs is believed to be located in the trigeminal region while the other has been suggested to exist caudally, in the region of the Vllth, IXth, and Xth motor nuclei. In fact, it has been suggested that respiratory pacemakers in the lamprey may be arranged segmentally much like the respiratory motoneurons. Indeed, the central rhythm generators associated with swimming movements are known to be segmentally arranged along the spinal cord, as are the motoneurons (Cohen and Wallen, 1980). In addition to the chick embryo and the lamprey, multiple CRGs have been suggested to exist in the bullfrog, Rana catesbeiana (Reid et al., unpublished data). Unfortunately in the bullfrog, the evidence which supports the presence of multiple CRGs for breathing remains debatable as the burst pattern following the transection does not appear to be "normal" respiratory activity. That is, the bursts of neural activity from both the trigeminal and vagus nerves are rapid in onset and their activity is decrementing over time, rather than the incrementing-decrementing pattern of activity exhibited by conventional "normal" breaths (Fig. 5). As a consequence of these results, as well as other inconsistencies associated with the in vitro preparations (aforementioned), further data would be required to confidently support 24 the presence of multiple CRGs in the bullfrog. Utilizing an in situ, rather than the in vitro preparation previously used, the presence of multiple CRGs can be explored in a more intact animal. Based on the information presented above, from both in vitro and in situ studies, the optic tectum appears influential in the modulation of respiratory pattern in the bullfrog. Yet to date, no location has been deduced as a definitive "episodic centre". That is, neither chemical lesions nor transections in this region have eliminated the episodes consistently and unconditionally. Therefore, the first objective of the present study was to explore the optic tectum for the presence of a suspected "episodic centre", via progressive rostral to caudal transections and concurrent fictive respiratory recordings of an in situ preparation. The first transection will be performed in the rostral optic tectum; more specifically, slightly caudal to the optic chiasma. This location mimicks the site of the transection previously performed in the in vitro preparation which converted episodic breathing to high frequency single breaths (Reid et al., unpublished data). If the results of this study concur with the in vitro experiments, further support will be granted to this site as the potential "episodic centre". If, however, the in situ results oppose the preliminary in vitro findings, and an episodic breathing pattern persists following this rostral transection, a cut will be made further caudally, approaching the mid-optic tectum. Once again, ficitive breathing will be monitered to determine if this transection indeed serves to eliminate episodic breathing unconditionally. Following the above protocal, a third transection will be performed at the caudal optic tectum. A second objective of the present study was to confirm or refute the presence of 25 multiple CRGs for breathing in the bullfrog, again using an in situ preparation and appropriate transections while recording fictive breathing. The methodology applied in this case is again easily comprehendable: while recording from both the trigeminal and vagus nerves, a transection will be made between the two while fictive breathing continues to be monitored. If activity persists in both nerves following the transection, the hypothesis predicting the presence of multiple central rhythm generators for breathing will be supported. If only one nerve continues to fire, it is suggestive of one central respiratory rhythm generator, located in the portion of the brain from which acitivity persists. Finally, if activity ceases from both nerves, it is likely that only one central rhythm generator exists, which has been ablated by transection. Hyphotheses 1. There is a region within the optic tectum of the bullfrog brain, which is responsible for clustering breaths into episodes. The removal of such a centre would result in the unconditional abolishment of the animal's episodic breathing pattern. 2. There are multiple segemental respiratory rhythm generators in the medulla of the bullfrog brain. If a transection is made between them, respiratory activity arising from neurons located in opposing segmented regions will continue to fire independently of one another. 26 MATERIALS AND METHODS Adult bullfrogs, Rana catesbeiana, of either sex and weighing between 215 and 510g (average mass = 386g), were obtained from a commercial supplier. The animals were housed indoors, in fibreglass basins containing 15 cm of dechlorinated water at room temperature, which was flushed weekly. They were provided with platforms to hide beneath, or bask on. Live locusts were fed to the animals once/week, and photoperiod consisted of 12 hours of light and 12 hours of dark per day (12h:12h L:D). Decerebrations Frogs were anaesthetized with MS-222 until the toe-pinch response was abolished, (approx. for 1 hour). A small hole was then drilled into the skull rostral to the forebrain and the bone was chipped away back to the medulla-cerebellum border. This allowed easy access to the optic tectum on the day of transection. Decerebration was achieved with a cauterizing device and cotton balls were placed where the forebrain was removed to sustain pressure over the blood vessels, promoting clotting and thus preventing bleeding. Vaseline and saline-saturated cotton balls were then carefully positioned over the exposed brain to prevent desiccation, a small piece of dental dam was glued over the open braincase to secure the arrangement of the cotton and to prevent the entry of water, and the skin was sewn closed. A recovery time of at least 24 hours was allowed before any experiments began. Experimental Set-up Following recovery from decerebration (determined by resumption of activity by the animal), the animal was restrained without paralysation. The apex of each lung was 27 then cannulated with polyethylene tubing (PE 240), to allow unidirectional ventilation with humidified air containing varying levels of CO2. This was achieved via a small incision on each side of the body wall. An extension was sometimes added to the outflow cannula to increase resistance and thereby inflate the lungs. Further resistance was achieved by immersing this extension to different depths in water. The glottis was subsequently occluded with tissue cement to prevent the escape of gas through the mouth. In preparation for brain transection, the brain case was re-opened and the brain exposed before the animal was placed in a stereotaxic apparatus which ensured immobility. The animal was kept moist at all times to prevent desiccation. In order to monitor Active breathing, the trigeminal nerve was extracted from the lower jaw and positioned across bipolar platinum hook electrodes. In cases where Active breathing was also recorded from the vagus nreve (that is, in experiments investigating the effects of transection between the Vth and Xth cranial nerves), the vagus was extracted from a dorsal approach, just caudal to the tympanic membrane and also positioned across bipolar platinum hook electrodes. Both nerves were kept moist with a 1:1 mixture of mineral oil and Vaseline. A diagram of the experimental apparatus is provided in figure 6. Raw nerve activity, once amplified, was viewed on an oscilloscope and heard via a Grass Audio monitor. Furthermore, the signal was integrated via an integrating amplifier connected to a polygraph recorder (Gould), and both raw and integrated nerve activity were recorded on computer with a data acquisition system (Windaq DI-200 Acquisition) in addition to the polygraph. The sampling rate of analogue to digital conversion was 2500Hz. Varying levels of CO2 in air were delivered to the lungs at 28 Figure 6. Diagram illustrating the experimental apparatus: Frogs were unidirectionally ventilated with varying levels of C02 in air via cannula inserted into the apex of each lung. Fictive nerve acitivity was recorded from the trigeminal nerve (extracted from the lower jaw), and the vagus nerve (extracted just caudal to the tympanic membrane). The outflow cannulae could be immersed in a beaker of water to increase resistance and thereby inflate the lungs. Finally, the braincase was opened to allow access to the optic tectum and medulla in order to perform the transections. 29 30 different degrees of lung inflation. In order to produce episodic breathing, 2.5% CO2 was usually administered to the animal, with the lungs deflated. This is consistent with previous findings which indicate that bullfrogs under resting conditions tend to breathe in sparsely-spaced single breaths, with breathing becoming episodic only when respiratory drive is increased (Milsom, 1991). For the transection experiments, once episodic breathing was attained, the brain was then transected at various rostral-caudal locations using either a blade fitted into a stereotaxic device which held the animal in place, or a pair of fine opthalmic scissors. The effects of these ablations on fictive breathing were concurrently recorded. By increasing/decreasing outflow resistance (see above), the effects of inflating and deflating the lungs could also be monitored and compared pre-and post-transection. Furthermore, in experiments investigating the effects of vagotomy, the vagus was cut where it exited the brain. (In experiments where the vagus was being recored from, one vagus was already transected more peripherally). At the end of the experiment, the brains were removed and fixed in a 4% paraformaldehyde solution for at least 24 hours at 4 degrees Celsius and examined histologically to provide a cross-sectional view of the transection sites. The effects of decerebration This set of experiments was designed to determine the effects of decerebrations at the level indicated in figure 7. Fictive breathing was recorded from the trigeminal nerve while the animal was unidirectionally ventilated with air, 2.5% C0 2 , and 5.0%) C0 2 , with the lungs alternately inflated and deflated. In this way, it was possible to determine the effects of both lung inflation and carbon dioxide on respiration in this animal. 31 Figure 7. Schematic diagram of the bullfrog brain indicating the levels of transections. 1) Level of decerebratipn. 2) Level of transection at the rostral optic tectum. 3) Level of transection at the mid-optic tectum. 4) Level of transection at the caudal optic tectum. 5) Level of transection between the Vth and Xth cranial nerves. 6) Level of transection at the rostral spinal cord. 32 33 The effects of transections in the optic tectum This set of experiments was designed to determine the effects of brain transections on both the respiratory and burst patterns in the bullfrog. Following decerebration, the brain was further transected at one of three locations: the rostral, mid-, or caudal optic tectum, while fictive breathing was monitored from the trigeminal nerve. In some cases, respiratory drive was increased or decreased following the transections via lung inflation or C 0 2 challenge. This was achieved by either increasing the resistance of the outflow cannulae as described above, or by decreasing the C0 2 in the ventilation gas, respectively. The effects of transections between the Vth and Xth cranial nerves This set of experiments was designed to support or refute the notion of multiple central rhythm generators for breathing in the bullfrog. In these experiments, following recovery from decerbration, the brain was transected in the medulla, between the Vth and Xth cranial nerves. Furthermore, in addition to monitoring fictive breathing from the trigeminal nerve, neural activity was also recorded from the vagus nerve. Brain tissue histology Following the experiment, the brain was removed from the animal and fixed in a 10% neutral buffered formalin solution for at least 24 hours. The tissue was subsequently embedded in paraffin, sectioned, and then stained with eosin-hematoxylin to determine the precise location of the transections. Data analysis The data comprising the quantitative analysis for fictive breathing variables were calculated as means +/- SE. In experiments involving 3 conditions, (pre-transection, 34 post-transection, and post-transection, increased drive), the results were statistically analysed using two-way repeated ANOVA followed by a Student-Newman-Keuls test. When only 2 conditions were compared (pre-transection versus post-transection), one-way ANOVA followed by a Student-Newman-Keuls test was utilised. 35 RESULTS The effects of transections caudal to the cerebrum (decerebration) Decerebrations were performed at the caudal cerebrum, at the level indicated as #1 in Fig. 7. Fig. 8 compares the fictive respiratory activity from the trigeminal nerve of a decerebrated, in situ bullfrog ventilated with air and the buccal pressure recording of an intact animal breathing air. In both preparations, the breathing was intermittent, consisting of sparsely occurring single and double breaths. These results suggest that decerebration had minimal effects on the breathing pattern. Fig. 9 compares the fictive respiratory activity from the trigeminal nerve of a decerebrated bullfrog ventilated with air, 2.5% C 0 2 and 5.0% C0 2 , both prior to and following inflation of the lungs. As described above, animals ventilated with air tended to exhibit a breathing pattern consisting of sparse single and double breaths. In instances where the animal was artificially-ventilated with 2.5% C0 2 , the pattern remained intermittent, however the number of breaths per episode increased, presumably due to the elevated respiratory drive resulting from the increased levels of C0 2 . At 5.0% C0 2 , the breathing became continuous. In each case, inflating the lungs appeared to have an excitatory effect on breathing frequency and an inhibitory effect on the tidal volume of the individual breaths. In animals ventilated with air, as well as those ventilated with 2.5% C0 2 , the respiratory pattern was altered from episodic breathing while deflated, to continuous breathing following inflation. At 5.0% C0 2 , continuous breathing was maintained following lung inflation, however the amplitude of the breaths were again decreased. In all cases, the vagi remained intact. 36 Figure 8. Representative traces comparing A) the buccal pressure of an intact bullfrog breathing air (Kinkead and Milsom, 1996), with B) the fictive breathing pattern, recorded from the trigeminal nerve, of an in situ bullfrog unidirectionally-ventilated with air. Note the similar respiratory patterns exhibited. 37 Buccal pressure recording of animal on air cm/HjO 5 ; 4 -3: 2 -1 Fictive breathing trace of animal on air 5 lire 38 Figure 9. The effects of C0 2 and lung inflation on fictive respiratory activity in an in situ preparation ofRana catesbeiana. Traces illustrate the neural activity from the trigeminal nerve: A) on air, deflated; B) on air, inflated; C) on 2.5% C0 2 , deflated; D) on 2.5% C0 2 , inflated; E) on 5.0% C0 2 , deflated; and F) 5.0% C0 2 , inflated. 39 < < 40 2. The effects of transections of the rostral optic tectum Fig. 7 (transection #2) illustrates the level of the transections performed in the rostral optic tectum, via a schematic image. A cross-sectional view can be seen in figure 10. Transections at this level'affected both the breathing frequency as well as respiratory pattern. In 4 of 4 trials, the breathing frequency increased following the transections, and the episodes were converted to continuous breathing (Fig. 11, A and B; Fig. 12). Conversely, neither the burst duration nor integrated activity were significantly altered. In 2 of these animals, one vagus remained intact, while the remaining two animals were bilaterally vagotomized. Lung inflation did not affect a change in respiratory pattern nor frequency in any case. Interestingly, however, in 2 animals, the episodes returned over time (approximately 10 minutes following the transections), and in one animal the episodic breathing pattern returned when respiratory drive was reduced by removing the C 0 2 from the gas mixture ventilating the lungs (Fig. 11C). < 3. The effects of transection of the mid-optic tectum Schematic and cross-sectional images of the transection at the mid-optic tectum are illustrated in Figs. 7 (transection #3) and 13, respectively. Transections at this level produced 5 significant results which are outlined in the graphs presented in Fig. 14 (n=10). A representative trace is provided in Fig. 15. The pre-transection state represents the fictive breathing activity following a decerebration, but prior to any further transections. With respect to individual breaths, that is burst pattern, both the average bust duration (p=0.00855), as well as the average integrated activity (p=.0217), increased following a transection at the mid-optic tectum. Following lung inflation, however, both 41 Figure 10. Cross-sectional view of the bullfrog brain, at the level of the rostral transection (mag.: 6.25X). Third: third ventricle. 43 Figure 12. A comparison of the effects of transection through the rostral optic tectum of an in situ bullfrog artificially-ventilated with 2.5% C 0 2 to an animal ventilated with air. A ) Episodic breathing pattern exhibited by an in situ bullfrog ventilated with 2.5% C 0 2 , prior to transection. B) Continuous breathing pattern exhibited following a transection through the rostral optic tectum, still ventilated with 2.5% C 0 2 . C) Episodic breathing pattern displayed following alteration of ventilatory gas to air. A Pre-transection, 2.5% C02 ^ i i 'mm in ni liMiimii i MIL j v J l lL^J^ 75 sec B Post-transection, 2.5% C02 ^ IHHIIIIllll llll'dllllUnillllllll lll'ill 15 sec C Post-transection, air v II il Ml l | H ll^l llliH H. 15 sec 45 Figure 12. Quantitative results following transections at the rostral optic tectum. o UJ I-o I-Q. O > 3^ o < •a 3 CB 4-1 Qi Ui C co O CD 1— Q. CO O r_: i -< CO o UJ CO < • • CO V-_J CO UJ oo o o CO o o o o CM o o o o o ( s SI|OA) AijAjpe paiejBaiui c .2 +3 CD i _ 3 T3 _ 3 m H H r~ CM co o Q. CD i— Q. u c Qi 3 O" tl) c m H CO o a . a) CL to CM O CM to ainuiw/sineajg (spuooas) uoriejnp isjng 47 Figure 13. Upper panel illustrates a sagittal section of the optic tectum and rostral medulla of the bullfrog brain (mag.: 25X) (Kinkead and Milsom, 1997). The broken line indicates the level of transection performed in the mid-optic tectum in the present experiment. The lower panel illustrates a cross-sectional image of the bullfrog brain at the level of the mid-optic tectum (mag.: 6.25X). Aqu S: Sylvian aqueduct; cer: cerebrellum; chor. plex.: choroid plexus; fourth: fourth ventricle; nu. ist.: nucleus isthmi; tec: optic tectum; tec ven: ventricle of the optic tectum. 49 Figure 14. Quantitative results following transections at the mid-optic tectum. Along the x-axis, "pre" indicates measurements made prior to the transection, while "post" denotes results following the transection. In both cases, the lungs remained deflated. "Post, increased drive" represents measurements made following the transection with the lungs inflated, thereby increasing the respiratory drive. 50 O UJ h-o i-Q. o I Q < CO z o o UJ CO z 2 CO h-_J 3 CO UJ on u < T3 CD «-> 2 O) © •o o 13 5> • E - c o -a c o Q. a c a> 3. O" a> k. u. o !E CO 2 m a. o o CM —• o o o o (zs SJIOA) AijAjpe p9)ej6aiu| o CO (2 3 m H r CO CM "8 13 aj o -a c o Q. 2 a. (OBS) u o r j e j n p l s j n g r CM o 00 CD CM •a v 8 5> O T3 C o Q. 2 a. ainujUJ/siJiBSjg >» o c CD 3 CT £ u. <D TJ O w a UJ r T -a v 13 2> o -a c o a. 2 a. CO CM ainujuj/saposjdg —• o CD •o O W a UJ w CO CQ i i i i r to IO i f CO CM T J w 13 £ a -c a TJ c o a. 2 sposjda/sqieajg 51 Figure 15. The effects of a transection at the level of the mid-optic tectum. Note that the large bursts of activity represent a fictive lung breath, while the small events denote buccal oscillations. A) Episodic breathing pattern prior to the transection. B) Sparsely-occurring single breaths following the transection. C) Episodic breathing pattern reclaimed following the lung inflation. 52 A Pre-transection, deflated JIMAAM 15 sec B Post-transection, deflated Jv J U J AAAAJUJIMAJIUAAJ LUlA C Post-transection, inflated JVAMJ WliLUUi IS sec 53 variables retimed to states similar to those which occurred prior to the transection (N=5). Furthermore, in 2 animals, the burst shape was altered from an incrementing-decrementing pattern pre-transection, to a rapid onset-decrementing pattern following the transection (Fig. 16). In these cases, when the lungs were inflated, the episodes again returned, however interestingly, so too did the "normal" incrementing-decrementing burst pattern. With respect to breathing pattern, 2 significant ventilatory reponses were noted following the transection. The average number of breaths within an episode as well as the average overall breathing frequency decreased significantly post-transection (p=0.00144; p=.00694), while the average episode frequency was not significantly altered. That is, the breathing pattern was converted from episodes averaging 4 - 5 breaths to a pattern of consisting primarily of sparsely-occurring single breaths (periodic, non-episodic), as exemplified in Fig. 13. Consequent to lung inflation, the breathing frequency, as well as the number of breaths per episode, significantly increased, rendering these measurements insignificantly different from the pre-transection values (p>0.05). In 2 animals, bilateral vagotomy was perfomed prior to or following the transection at the mid-optic tectum. In these cases, breathing was altered in a similar fashion to those animals with one vagus intact. That is, the respiratory pattern was altered from episodic breathing to evenly-spaced single breaths (periodic, non-episodic). However, unlike in the animals possessing vagal feedback, lung inflation did not return the pattern to a pattern similar to that seen in the pre-transection state in the bilaterally vagotomized animals (Fig. 17). Therefore, in these cases, the episodes were not reclaimed following lung inflation. 54 Figure 16. Alternate effect of transection at the mid-optic tectum (N=2). A) Episodic breathing pattern exhibited prior to the transection. Note the "normal" incrementing-decrementing burst patterns prior to the transection. B) Sparsely-occurring single breaths following the tranestion. Note the "gasp-like" rapid-onset, decrementing burst pattern following the transection. C) Reappearance of episodic pattern following lung inflation. Note the re-emergence of a "normal" incrementing-decrementing burst pattern. 55 A Pre-transection, deflated IS sec B Post-transection, deflated tV ***** 15 sec C Post-transection, inflated 15 sec 56 Figure 17. The effects of transection at the mid-optic tectum, following bilateral vagotomy (N=3). A) Episodic breathing pattern exfiibited prior to the transection. B) Sparsely-occurring single breaths following the transection. C) Reappearance of episodic breathing pattern following lung inflation, before bilateral vagotomy. D) Sparsely-occurring single breaths persist following lung inflation in bilaterally-vagotomized animals. 57 58 4. The effects of transections in the caudal optic tectum Figs. 7 (transection #4) and 18 indicate the transection level at the caudal optic tectum. Quantitative results following this transection are presented in the graphs in Fig. 19, and a representative trace is provided in Fig. 20. It should be noted that pre-transection values are those recorded following decerebration and prior to the transection at the caudal optic tectum. No other transections were made in these cases. As was the case following the transection through the mid-optic tectum, 5 respiratory alterations were noted. The average burst duration significantly increased following the transection (p=0.000378), while the average integrated activity was not significantly modified (p=0.252). With respect to breathing pattern, the average number of breaths per episode decreased significantly (p=0.00810), while neither the average episode frequency nor the overall breathing frequency were significantly altered (p=0.0543; p=0.141). Interestingly, in no case did lung inflation have an effect on breathing frequency nor pattern. Al l experiments were performed with one vagus intact. The effects of transections between the Vth and Xth cranial nerves Figs. 7 (transection # 5) and 21 illustrate the level of the transections performed between the Vth and Xth cranial nerves. In 7of 7 animals, a transection at this level terminated all activity from both the trigeminal and vagus nerves (Fig.22). A representative trace depicting activity from both nerves before, and subsequent to the transection, is presented in Fig. 23. In 1 animal, transections were made at the rostral level of the optic tectum and caudal to the Xllth cranial nerve before the transection between the Vth and Xth cranial nerves (Fig. 24). In this case, an episodic fictive 59 Figure 18. Cross-sectional image illustrating the level of transection at the caudal optic tectum. cerebellum 61 Figure 19. Quantitative results following transections at the caudal optic tectum. "Pre" and "post" values were recorded with the lungs deflated, prior to and following the transection, respectively. "Post, increased drive" denotes post-transection measurements, recorded with the lungs inflated, thereby increasing the respiratory drive. 62 O UJ h-o r- c Q. O _J < a < O < CO z g o UJ (0 I I • <«-> 2 3 Q •*-> 2 3 CD CO 3 CO UJ or > o < 2 2 O) O 1 H I CM —-O O O O O "8 1 ? a-c 3 —i o Q. a o. CO o o o ( S S)|0A) AJjAflOB p3)ej63)U| H r CM T •a u a-c o Q. a a. CO  —• O (oas) uojiBjnp lsjng >» o c CD 3 O" CD Ui c CO <D ffl CM - i — i 1—i r O 00 CD CM ainujiu/sine&ig •s 0> w > a-c O T3 C o a. a O C 0> 3 or 2 LL <D T3 O CO a LU oo ~T co —r CM "8 o ex a o. e i n u i u j / s s p o s . i d g CD TJ O (0 a LU co 2 ffl T co CM X I u a-c U T3 B tt O a o. apos|da/sin.eajg 63 Figure 20. The effects of transection at the caudal optic tectum. A) Episodic breathing pattern exfiibited prior to the transection. B) Evenly-spaced single breaths following the transection. C) Single-breath pattern persists following lung inflation. 64 A Pre-transection, deflated IS sec B Post-transection, deflated 15 sec C Post-transection, inflated 15 sec 65 Figure 2 1 . Cross-sectional image illustrating the level of transection between the Vth and Xth cranial nerves. 67 Figure 22. Figure 26. Quantitative results following transections between the Vth and Xth cranial nerves. "Pre" refers to data collected prior to the transection, while "post" denotes values recorded following the transections. 68 X > z UJ UJ UJ m co z g »-o UJ CO z < or h-co I -_ J CO UJ T3 O (0 '5. U J (0 CD CO CN T— CO O Q . Q . aposida/sineajg o c 0 3 O" Q) CO C !E •+-> (TJ O u. CQ CM o n I r -CO CD M " CM amujw/sineajg >» o c o 3 cr o i _ LL O •o O V) "EL UJ r - 1 — i — i — i — i — i — r amujLu/saposjdHi 69 Figure 23. The effects of transection between the Vth and Xth cranial nerves. A) Episodic breathing pattern recorded from both the trigeminal and vagus nerves, prior to any transections. B) Lack of neural activity from either nerve following the transection. 70 A Pre-transection •Xl IS sec B Post-transection 15 sec X J IP 75 sec 71 Figure 24. Fictive breathing recorded from the trigeminal nerve following transections at the caudal optic tectum and rostral spinal cord. A) Episodic breathing pattern exhibited prior to any transections. B) Single-breath pattern observed following a transection at the optic tectum-medulla border. C) Episodic pattern re-emerges following a transection slightly caudal to the 12th cranial nerve. 72 A Pre-transection v Jv 15 sec B Post-transection at caudal optic tectum IS sec C Post-transection at rostral spinal cord llli.lilllpilllllllllllll!!!!!!!!,! JV ^ ' V ^ * ^ ^ IS sec 73 breathing pattern was also recorded prior to the transection, after which activity from both nerves ceased. 74 DISCUSSION Critique of methods Decerebrations, which were performed on anesthetized animals at least 24 hours prior to the experiment, appear to have minimal effect on breathing in the bullfrog. Figure 8, which compares fictive respiratory activity from the trigeminal nerve of an in situ bullfrog preparation ventilated with air, with the buccal pressure recording of an intact animal breathing air, is testimony to this claim. Following recovery from the anesthetic, and prior to any further transections, the animals were artificially-ventilated with 2.5% C0 2 . This level of carbon dioxide was chosen as it most consistently produced episodic breathing (Fig. 9). When ventilated with air, bullfrogs tend to breathe with sparsely-occurring single and double breaths rather than consistent episodes. By contrast, when the respiratory drive is increased further (5.0% C 0 2 for example), the breathing becomes continuous. These results are consistent with previous data matching arterial blood gas levels of spontaneously-breathing bullfrogs with those of bullfrogs unidirectionally-ventilated with 2.0% C 0 2 (Kinkead and Milsom, 1994). In the present paper, lung inflation was consistently used as a method of increasing the respiratory drive. This method is based on data acquired as a component of the present study (Fig. 9), in concert with results from previous studies (Kinkead et al., 1997 and Kogo et. al, 1994) demonstrating an increase in breathing frequency associated with lung inflation. Inflating the animals' lungs, rather than increasing the levels of C0 2 , was the chosen procedure for increasing the respiratory drive as the effects were seen immediately. In this way, any resulting changes in breathing pattern/frequency could 75 confidently be attributed to the imposed manipulations. Transections of the rostral optic tectum This set of experiments was designed based upon the results of previous in vitro experiments suggesting that a transection of the rostral optic tectum (slightly caudal to the optic chiasma), results in the conversion of the episodic breathing pattern to a continuous one (Reid et al., unpublished data) (Fig. 4). The present data, utilizing an in situ preparation of the bullfrog brain, also reveals that transection of the rostral optic tectum converts the episodic breathing pattern into a continous one (Fig. 11). In contrast to the in vitro work, however, 3 of 4 animals reverted to an episodic respiratory pattern with either the passing of time (in 2 of 3 animals), or following a switch to ventilation with air (in 1 of 3 animals, Fig 12). These results suggest that the region rostral to the optic chiasma provides inhibitory input to more caudal regions of the bullfrog brain. When removed, an increase in breathing frequency is observed. The effect on breathing pattern appears to be transient and can be reversed if the respiratory drive is decreased. The reasons for the difference between the in vitro results (persistent continuous breathing) and in situ results (return of episodic breathing) remain unclear. It is possible that input from an uncut cranial nerve in the in situ preparation plays a role in compensating for the inhibitory region removed following the transection. Interestingly, though, the vagus is excluded from this speculation as bilaterally-vagotomized animals exhibited a similar post-transection response to those with one vagus intact. Alternately, it is possible that a central site plays a compenentory role, and that the transient excitatory effect observed in situ is prolonged in the in vitro preparation due to the vulnerability of 76 the in vitro preparation to hypoxia/anoxia and therefore an elevated drive to breathe. A third explanation denies the presence of a compensatory site. Instead, it is possible that in the 2 animals which exhibited a spontaneous return to episodes, the transection was made through a group of neurons providing an inhibitory input and therefore this site (and consequently its inhibitory effect) was only partially removed. The effects of lung inflation In the bilaterally-vagotomized preparations, the lack of response to lung inflation is not surprising considering the vagi are the neural pathways via which pulmonary mechanoreceptors transmit their impulses to the brain. When severed, the effects of lung inflation are eliminated. More curious, however, is the lack of response following lung inflation in the non-vagotomized preparations. This probably reflects the inability to increase the drive of a nearly continuously-breathing animal. That is, if lung inflation is expected to provide an excitatory input, the response may be negated in an already continuously-breathing animal. Interestingly, however, the subsequent lung deflation (and therefore phasic reduction in the respiratory drive), did not decrease the breathing frequency or affect a return to episodes in these cases. It should be noted that while lung inflation occurred immediately following manipulations to the outflow resistance, subsequent deflation was achieved over the course of several seconds. It is possible, therefore, that the effects following lung inflation were more readily observed as more mechanoreceptors were stimulated simultaneously and subsequently the threshhold required to evoke a response (increase in breathing frequency) was more consistently met than in the case of lung deflations. 77 Transections at the mid-optic tectum This series of experiments produced several interesting results. Most formatively, transections in this region converted the respiratory pattern from episodic to one of evenly-spaced single breaths of increased amplitude and duration (Fig. 15). These results suggest that the region between the rostral and mid-optic tectum (Fig. 25a), provides both excitatory and inhibitory inputs. With respect to breathing frequency, an excitatory input appears to be eliminated, as both the breaths/episode and the overall breathing frequency are significantly reduced following the transection (Fig. 14). However, with respect to burst pattern, the opposite is true, as both the burst duration and the integrated activity (tidal volume) increase significantly post-transection. Interestingly, in 2 of 10 animals, another trend was noted. In addition to the aforementioned changes to burst pattern (involving duration and amplitude), the burst shape was altered from a "normal" incrementing-decrementing pattern pre-transection to a "gasp-like" rapid-onset, decrementing pattern following the transection (Fig. 16). It is not clear why this result occurred in only 2 of 10 preparations, however it is possible that the transection in these 2 cases rendered the brain hypoxic, thus eliciting a gasp-like respiratory response, as is known to happen in hypoxic mammals. Effects of lung inflation In animals with at least one vagus intact, increasing the respiratory drive via lung inflation reclaimed the episodic breathing pattern (Fig. 15). These results suggest that the region between the 1st and 2 n d transections (Fig. 25b) only serves to modulate the respiratory pattern, and is not solely resopnsible for clustering the breaths into episodes. In bilaterally-vagotomized animals (N=2), post-transection lung inflation did not affect a 78 Figure 25. Schematic diagram of the bullfrog brain, mapping regions with respect to their potential roles in the central control of breathing in this animal. 1) Level of decerebration. 2) Level of transection at the rostral optic tectum. 3) Level of transection at the mid-optic tectum. 4) Level of transection at the caudal optic tectum. 5) Level of transection between the Vth and Xth cranial nerves. 6) Level of transection at the rostral spinal cord. 79 80 return to the episodic pattern (Fig. 17). This is understandable presuming the vagus serves as the conduit for the mechanoreceptor feedback provoking the excitatory respiratory response which reclaims the episodes in animals with at least one vagus intact. In the 2 animals in which the burst pattern was altered from incrementing-decrementing to rapid-onset, decrementing, lung inflation again affected a return to the episodic breathing pattern. Interestingly, however, it also reverted the burst pattern to the "normal" incrementing-decrementing pattern (Fig. 16). If the gasp-like response observed in these cases was a product of a hypoxic brain, it is possible that inflating the lungs may have served to increase blood flow thus returning the animal to normoxic conditons and reinstating a "normal" incrementing-decrementing respiratory burst pattern. Transections at the optic tectum-medulla border This set of experiments suggests that the caudal optic tectum influences both respiratory pattern as well as a component of burst pattern (Fig. 19). More specifically, following transections at this level, the average burst duration increased, while the integrated activity remained unchanged. These results imply the presence of an inhibitory input in this region, with respect to burst duration. Yet more strikingly, the results of these transections suggest the presence of a site important in the production of episodes. That is, transections at this level transformed the episodic breathing pattern to one of evenly-spaced single breaths which could not be reconverted to episodes following lung inflations (Fig. 20). This may have suggested that the region between the mid- and caudal optic tectum is essential for the production of episodes, as neither lung inflation, 81 nor a return to the deflated state stimulated their reappearance. However, this is not the case, as episodes were observed in one preparation following a transection at both the caudal optic tectum and the rostral spinal cord (Fig. 24). This result, in accordance with the lack of response to lung inflations/deflations unveils 2 possible conclusions: 1) the transection at the optic tectum-medulla border did not remove the episodes unconditionally and therefore this region seems only to modulate the respiratory pattern rather than being responsible for the production of episodes, and 2) vagal inputs alter breathing pattern via projections to sites rostral to the caudal optic tectum trasections. The implication of the results following transections at both the caudal optic tectum and rostral spinal cord is the presence of neural components within the rostral spinal cord which when removed, allow the episodic breathing pattern to reappear. The nature of this input remains questionnable, however, and 2 hypotheses may be presented. The first suggests that within the spinal cord are neural components which influence the arrangement of breaths into episodes. Yet such pattern-influencing neural components would have to be located very rostrally within the spinal cord, slightly caudal to the 12th cranial nerve, since in vitro preparations exhibit episodic breathing patterns. The second hypothesis involvess the accuracy of variables measured following a transection at the optic tectum-medulla border. More specifically, it is possible that a statistically-significant change in the overall breathing frequency may be evident with an increase in subject numbers. If an overall increase in breathing frequency was observed, this would suggest that the transection at the rostral spinal cord removed an excitatory input, stimulating episodes to reappear as the respiratory drive was decreased. If, conversely, a significant decrease in the overall breathing frequency was recorded, the spinal cord 82 would appear to provide an inhibitory input which when removed, re-elevated the drive to breathe and subsequently re-instated the episodic breathing pattern. The latter hypothesis follows the logic describing the results subsequent to the previous two transections performed within the optic tectum. More specifically, transections in the rostral optic tectum removed an inhibitory input thereby mimicking an increase in the respiratory drive, whereas the transection in the mid-optic tectum abolished an excitatory input akin to decreasing the drive to breathe. In order, then, to reclaim the episodic breathing pattern following the former transection, a decrease in the respiratory drive was required (removing the C 0 2 from the ventilating air), while an increase in drive stimulated episode production in the latter case (inflation of the lungs). Is the transection in the caudal optic tectum at the site of the nucleus isthmi? The present study is unique in its examination of bullfrog respiration via the use of transections through the optic tectum of an in situ preparation. Similar results were observed, however, in a previous study (also employing an in situ model), involving chemical lesions to a region within the optic tectum between the roof of the midbrain and the cerebellum, known as the nucleus isthmi (Kinkead et al., 1997). Following bilateral microinjections of kainic acid into this region, the breathing pattern was converted from an episodic one to one of evenly-spaced single breaths (periodic, non-episodic), akin to the results of the present transection experiment. Furthermore, in both cases, lung inflation no longer influenced breathing following the ablations. This poses the question of whether these are the same sites. Upon reviewing the results from both studies, it does not appear that the sites are identical. More specifically, following the chemical lesioning of the nucleus isthmi, a C 0 2 level of 3.5% was required to evoke a neural response; that 83 is, the preparation became less C02-sensitive with the nucleus isthmi ablated. This was not the case following the present transections, as a C 0 2 level of 2.5% was maintained throughout the entire experiment. Furthermore, following histological analyses of the transection locations in the present study, it appears that the nucleus isthmi lies slightly rostral to the transection performed the caudal optic tectum. Indeed, ablating the nucleus isthmi and transecting at the mid-optic tectum have a similar effect on the product breathing pattern, however the region excised by the transection in the present study comprises more than the excitatory input associated with the nucleus isthmi. The effects of transection between the Vth and Xth cranial nerves This set of experiments was designed to investigate the potential presence of multiple central rhythm generators for breathing in the bullfrog. The responses following the said transections were unwavering. In 7 of 7 animals, the episodic breathing pattern was eliminated following the transection, as was all neural activity from both nerves (Figs. 22 and 23). These results are supported by previous studies examining respiratory rhythmogenesis in bullfrogs. Using a "brain-in-a-dish" preparation, fictive respiratory activity from the Vth, Xth, and Xllth cranial nerves was monitored prior to and following microinjections of lidocaine and GAB A into the region between the Vth and Xth cranial nerves (McLean et al., 1995). Following the injection of both lidocaine and GABA (the former is a neurotransmitter blocker which targets both cell membranes and axons while the latter is specific to cell bodies), the frequency of the respiratory activity decreased and eventually became completely arrested before reappearing concurrent with the dissipation of the neurotransmitter substances. A third in vitro preparation reported arrest of activity 84 from the Xth cranial nerve following transections caudal to the trigeminal nerve (Perry et al., 1995). The results of these two in vitro studies, in accordance with the present in situ work suggests the presence of a region between the Vth and Xth cranial nerves, which is essential to the expression of neural activity from the trigeminal, the vagus, and possibly the hypoglossal nerves. Whether it is the lone central rhythm generator, or an integral part of a more complex network remains speculative. 85 CONCLUSIONS Based on the results from the present study, a synoptic description may be presented, defining the roles of component regions of the bullfrog brain with respect to respiratory rhythm, burst pattern, and breathing pattern, and breathing frequency (Fig. 25). Respiratory Rhythm Transections between cranial nerves V and X affected the arrest of all neural activity from both nerves, in 7 of the 7 animals tested. These results imply the presence of a critical centre at this site (Fig. 25, transection #5), necessary for respiratory rhythmogenesis in the bullfrog, Rana catesbeiana. Burst Pattern The rostral optic tectum does not appear to play a significant role in the modulaiton of burst pattern, as neither burst duration nor amplitude were significantly altered following transections removing this region. By contrast, however, transections at the mid-optic tectum affected a significant increase in both components of burst pattern (amplitude and duration), suggesting the region between the 2 n d and 3 r d transections provides an inhibitory input with respect to burst pattern. Finally, the caudal optic tectum also appears to influence burst pattern, however only with respect to burst duration. Following transections to this region, burst duration increased significantly while burst amplitude remained unaltered, suggesting the presence of an inhibitory input between the 3 r d and 4 th transections, affecting only the duration of the bursts. The observation that the amplitude remained unaltered while the average duration of the bursts increased implies that the transection served to decrease the respiratory drive. 86 Breathing Pattern No transection in the optic tectum of the bullfrog brain served to eliminate the episodes unconditionally. Transections at the rostral optic tectum converted the episodic breathing pattern to a continuous one which could be reverted to episodes by decreasing the respiratory drive via removing the C 0 2 from the ventilated air. Transections at the mid-optic tectum transfromed the breathing pattern from periodic episodic to periodic non-episodic (evenly-spaced single breaths), however episodes reappeared consequent to increasing the respiratory drive via lung inflation. Finally, transections through the caudal optic tectum, akin to the former two transections, initially eliminated the episodic breathing pattern, however the episodes returned following a transection at the rostral spinal cord (slightly caudal to the 12th cranial nerve). The conclusion that emerges, then, is that the optic tectum serves as a respiratory pattern modulator rather than being necessary for the formation of the episodic breathing pattern exhibited by these animals. Respiratory Frequency Region a (Fig. 25) appears to provide an inhibitory input with respect to breathing frequency. When ablated, the result is akin to increasing the respiratory drive beyond the level at which the animal exhibits episodic breathing. An episodic pattern may therefore be reclaimed via decreasing the drive to breathe. Region b (Fig. 25), on the other hand, provides an excitatory input with respect to breathing. As a result, a transection at the mid-optic tectum parallels the effects of lowering the respiratory drive below that which produces episodic breathing. When the lungs are inflated, thereby increasing the drive to breathe, the episodes return. The importance of regions c and d (Fig. 25) with respect to breathing pattern remain largely speculative. While it appears that the both caudal optic 87 tectum and rostral spinal cord influence the respiratory pattern in the bullfrog, the nature of their affects (that is, whether excitatory, inhibitory, or neither with respect to breathing frequency) remains contentious. What is apparent, however, is that the influence of the vagus circumvents the caudal region of the optic tectum (Fig. 25c) and instead acts rostral to this region; therefore the effects of inflating and deflating the lungs are of no consequence following a transection at the optic tectum-medulla border. This implication is based on the observation that inflating the lungs following a transection in the mid-optic tectum restores the episodes, while no effect is seen with lung inflations following a transection in the caudal optic tectum. Finally, the paramount observation that episodes may occur following a transection at both the caudal optic tectum and rostral spinal cord, suggests a new theory with respect to respiratory pattern in the bullfrog. More specifically, the implication is that the medulla, not the optic tectum as initially hypothesised, possesses the neural components necessary for the production of episodes in the bullfrog, Rana catesbeiana. 88 REFERENCES Burggren, W.W. and N.H. West (1982). Changing respiratory importance of gills, lungs and skin during metamorphosis in the bullfrog Rana catesbeiana. Respir. Physiol. 47: 151-164. Cohen, A. and P. Wallen (1980). The neuronal correlate of locomotion in fish, 'fictive swimming' induced in an in vitro preparation of the lamprey spinal cord. Exp. Brain Res. 41: 11-18. deJongh, H.J. and C. Gans (1969). On the mechanism of respiration in the bullfrog, Rana catesbeiana: a reassessment. J. Morph. 127:259-290. Fortin, G., F. Kato, A. Lumsden and J. 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