THE effects of tonic lung inflation on ventilation in the American bullfrog Rana catesbeiana Shaw
Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, V6T 1Z4, Canada
*Author for correspondence (e-mail: milsom{at}zoology.ubc.ca)
Accepted May 14, 2001
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Summary |
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Key words: frog, Rana catesbeiana, ventilation, breathing pattern, lung inflation, pulmonary stretch receptor, HeringBreuer reflex.
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Introduction |
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It is currently believed that, in mammals, phasic stimulation of slowly adapting pulmonary stretch receptors located throughout the tracheobronchial tree and lungs, as would occur with each breath, modulates the frequency of breathing by inhibiting further inhalation during the inspiratory phase or causing prolongation of exhalation during the expiratory phase (for reviews see Kubin and Davies, 1995; Tenney and Leiter, 1995). This is the HeringBreuer reflex. Tonic stimulation of these same receptors, as would occur with changes in resting lung volume (or functional residual capacity), alters the timing of inspiration relative to expiration in a manner that acts to stabilize the resting lung volume and resist change (DAngelo and Agostini, 1975; Muza and Frazier, 1983; Finkler and Iscoe, 1984; for review see Milsom, 1990). Vagotomy, the severing of the vagus nerve, eliminates these reflexes.
While it is generally believed that amphibians also possess a classic HeringBreuer reflex as seen in other vertebrates (for reviews see Tenney, 1979; Tenney and Leiter, 1995), the manner in which it is manifested must be significantly different. Since amphibians achieve pulmonary ventilation through a forcepump mechanism in which the lungs remain inflated during the resting phase of the ventilation cycle, amphibians must not experience the same impulse for exhalation in response to phasic lung expansion as do mammals. The consequences of tonic lung inflation in amphibians, and particularly their reflexes, are also poorly understood and controversial.
Kogo et al. (Kogo et al., 1994) showed that in decerebrate, chemically paralyzed and unidirectionally ventilated (UDV) bullfrogs (Rana catesbeiana), breathing frequency (as determined by the frequency of bursts of motor activity in the trigeminal nerve; fictive breathing) was decreased when all proprioceptive feedback from the lungs was removed through bilateral vagotomy. Kinkead and Milsom (Kinkead and Milsom, 1997), using a similar in situ preparation, also found that breathing frequency was decreased, both when proprioceptive feedback was abolished through bilateral vagotomy, and when the lungs were deflated. They also demonstrated that breathing frequency was increased when the lungs were inflated, but that the amplitude of the trigeminal motor output associated with fictive breaths in this preparation was inversely proportional to pulmonary stretch receptor feedback. When the pulmonary branch of the vagus nerve was artificially stimulated in in vitro brainstem preparations (Kinkead et al., 1994) to simulate pulmonary stretch receptor feedback for each fictive breath, fictive breath frequency also increased dramatically, suggesting that lung inflation should stimulate breathing (Kinkead and Milsom, 1997). These results, suggesting that lung inflation stimulates breathing and that lung deflation inhibits breathing in reduced preparations, are supported by several other studies (McLean et al., 1995a; McLean et al., 1995b; Kinkead and Milsom, 1996; Reid and Milsom, 1998).
On the other hand Reid et al. (Reid et al., 2000), using in situ preparations of Bufo marinus, found that while lung deflation promoted episodic breathing, lung inflation promoted not only small, continuous lung ventilations at higher levels of respiratory drive, but also periods of apnea at low levels of respiratory drive. Then, in a recent study using the same preparation, reducing lung volume increased the frequency of trigeminal nerve discharge and lung inflation reduced fictive breathing frequency (Wang et al., 1999). With higher levels of tonic inflation, the amplitude of neural discharge associated with lung inflation recorded from the mandibular branch of the trigeminal nerve was reduced and lung ventilations appeared to be replaced with buccal oscillations (Wang et al., 1999). Kogo et al. also showed that decerebrate, chemically paralyzed and unidirectionally ventilated (UDV) bullfrogs (Rana catesbeiana) increased their breathing frequency when their lungs were deflated (Kogo et al., 1994). These results, suggesting that lung inflation inhibits breathing and that lung deflation stimulates breathing, support earlier findings in Rana sp. and Xenopus laevis (De Marneffe-Foulon, 1962; Shelton and Boutilier, 1982).
Our study was designed to address this controversy. Does lung inflation stimulate or inhibit breathing in frogs? Based on the evidence to date, we hypothesized that lung inflation should stimulate a transition to the production of deflation breaths involving reduced motor activity rather than the conversion of lung breaths to buccal oscillations. Using simultaneous recordings of electroneurograms (ENGs) from the mandibular branch of the trigeminal nerve (Vm, as an indication of the frequency and force of contraction of the buccal pump), and the laryngeal branch of the vagus nerve (Xl, as an indication of glottal opening, and hence fictive lung ventilation), in conjunction with changes in lung pressure, we monitored the responses to changes in tonic lung volume and pressure under conditions of high and low CO2-related respiratory drive, to determine whether this was so.
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Materials and methods |
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Decerebration
A reduced frog preparation comparable to that developed by Kogo et al. (Kogo et al., 1994) was used in this study. Before surgery, the frogs were quarantined for at least 48h following their last feeding to prevent complications. They were anaesthetized by submersion in a 1.5gl-1 aqueous solution of MS-222 (3-aminobenzoic acid ethyl ester), pH 7.0 (balanced with sodium bicarbonate to neutrality), until there was no response to toe pinching and no eye-blink reflex (approx. 30min). An incision was made along the sagittal plane on top of the head, between the eyes, towards the back of the skull. Using a Dremmel® moto-tool, a hole was then drilled into the skull, centered between the frontoparietal plates at a point slightly posterior to where they fuse rostrally to the exoccipital bones, to allow access to the brain. An incision was made transversely through the thalamus, between the cerebral hemispheres and the optic lobes, and the telencephalon was subsequently removed by suction, thereby eliminating cognitive function, pain perception and all higher cortical influences on breathing. The vacant cranium was then packed with cotton pellets and small (23mm3) pieces of Gelfoam® sterile sponge to promote clotting and maintain cranial pressure. The cranial opening was sealed with Vaseline, and a small piece of dental dam was affixed to the skull with Krazy Glue® to prevent water from entering the cranium. Following this, the skin was closed with sutures. The frogs were then allowed to recover from the surgery for at least 48h before further experimentation ensued.
Experimental set-up
On the day of an experiment, a small incision (1cm) was made through the body wall on either side of the abdomen just anterior to the rear limbs. Through these openings, both lungs were cannulated with polyethylene tubing (PE 240) at their apical ends to allow unidirectional ventilation of the respiratory system (air flow into one lung and out of the other). Another incision was made through the body wall more anteriorly into one lung to accommodate a third catheter of polyethylene tubing (PE 80) to allow measurement of the internal lung pressure with a strain-gauge pressure transducer. Following insertion into the pulmonary lumen, the catheters were secured in place with 5-0 silk sutures. The skin and the hypaxial muscles of the body wall were then sutured closed separately around the catheters.
The frog was then placed on its back and an incision was made starting behind the margin of the mouth on one side and carried anteriorly along the mandible to the chin. The skin was retracted so that the mandibular branch of one trigeminal nerve (Vm) was located and isolated from the surrounding fascia. A piece of 5-0 silk was tied around the nerve before it was cut distally. Placing the frog on its abdomen, a dorsal approach was used to locate the vagus nerve on one side. The skin was cut transversely just anterior to the scapula and longitudinally along the spinal column. The fascia and muscles attached to the suprascapula were severed and the suprascapula retracted. The laryngeal branch of the vagus nerve (Xl) was located, isolated from surrounding fascia, and severed as far distally as possible.
To minimize movement during the experiment, the sciatic nerves innervating the rear limbs were severed in the region of the pelvic joint, and the brachial nerve on the same side as the exposed trigeminal nerve was also severed. This procedure was chosen over paralysis since our protocol (see below) required that animals be able to produce spontaneous inflations/deflations. The tympanic membranes were slit and the frog was then further immobilized by being placed in a stereotaxic head restraint.
Experimental procedures
Frogs were unidirectionally ventilated (UDV) with mixed gases administered from a Cameron GF-3/MP gas-mixing flowmeter at a rate of up to 500mlmin-1. The gas was humidified by being bubbled through water in an Erlenmeyer flask before being conveyed to the inflow catheter of the unidirectional ventilation system. The degree of inflation of the lungs was regulated by controlling the resistance of the exhaust catheter by immersing its free end in a beaker of water. Unidirectional ventilation of the frogs commenced 1h before the experiment began.
The isolated branches of the vagus and trigeminal nerves (Xl and Vm, respectively) were each placed on bipolar platinum hook electrodes and covered with a 1:1 mixture of Vaseline and mineral oil to protect against desiccation. Electrical nerve activity recorded from the bipolar electrodes was amplified and filtered, full-wave rectified and integrated (Gould) in 375ms intervals. The ENGs were viewed on an oscilloscope and recorded on a polygraph and on computer (WinDaqTM version 1.32 data-acquisition system; DI-205, DATAQ Instruments) sampling at a frequency of 400Hz per channel.
The pressure catheter was connected to a strain-gauge pressure transducer and the signal from this transducer was amplified and also recorded on the polygraph and computer data-acquisition system.
Experimental protocol
This experiment investigated the effects of changes in tonic lung inflation under varying degrees of CO2-related respiratory drive. The frogs were placed on UDV, as described above, with gas mixtures containing one of six different levels of CO2 in air (air, 1%, 2%, 3%, 4% and 5% CO2). The gas mixtures were administered in a random order. Once respiratory drive was established at each level of inspired gas, lung volume was set at different levels by immersing the UDV outflow catheter in a beaker of water to manipulate the outflow resistance of the UDV exhaust and thereby control lung pressure (PL). Pulmonary pressure was initially set at 0cmH2O (1cmH2O=98.1Pa) and then subsequently increased in 1cmH2O increments at 5min intervals to a maximum resistance of 5cmH2O.
To acquire data representative of deflation breaths, frogs were inflated to 5cmH2O with air and then the lung cannulae were occluded and UDV terminated to allow the frog to breathe as naturally as this preparation would permit. Similarly, for inflation breaths, UDV was stopped, the cannulae were opened to allow deflation of the lungs, and the cannulae subsequently closed to allow the frog to breathe more naturally. Balanced-breath data were acquired by terminating UDV, closing the cannulae, and allowing the frog to breathe on its own for 5min in order to establish equilibrium.
Analytical procedures
Breathing frequency was quantified by analyzing the number of trigeminal (Vm) bursts that had corresponding vagal (Xl) bursts (a fictive breath) per unit time (termed absolute frequency; fL,abs) as well as by measuring the period between each two successive, uninterrupted, integrated Vm bursts of discharge within any lung ventilation episode. The period was measured from the beginning of the onset of the first integrated Vm ENG discharge to the beginning of the subsequent integrated Vm ENG discharge. The inverse of this period was then multiplied by 60 to obtain the instantaneous frequency of breathing within an episode, fL,inst.
The amplitude and area of Vm ENG discharge were recorded as a measure of respiratory effort and approximate tidal volume (Sakakibara, 1984b). Values for Vm amplitude were taken from the peak of the integrated ENG discharge (Vs). The area of Vm discharge was the area under the trace of the integrated signal, measured from the onset of the fictive breath to its termination (Vs2).
Apnea was subjectively defined as a period between two fictive breaths, with a duration of at least two fictive buccal oscillations. Apnea duration was measured from the end of the last integrated Vm ENG discharge of an episode to the onset of the first integrated Vm ENG burst of the subsequent episode. Breath episodes longer than 1min in duration with no apparent apneas were subjectively deemed to be continuous breathing.
For natural breaths (non-UDV), only the first fictive breath of an inflation or deflation breath series was analyzed since these were the breaths most strongly representative of an inflation or deflation pattern, respectively.
Data analysis
Values for fictive breathing variables were obtained by analyzing the events recorded during the last minute of exposure to a particular sequence (specific CO2 content and outflow resistance) before the setting was changed. All data are presented as means ± S.E.M. The results were analyzed statistically using one-way repeated-measures analysis of variance (RM ANOVA; P<0.05) followed by a StudentNewmanKeuls test (P<0.05) to discern the statistical relevance of responses to changes in pressure at each gas setting, and two-way RM ANOVA (P<0.05) to determine the overall statistical relevance of changing each variable, taking into account the effects of the other variable.
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Results |
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Discussion |
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Whereas all breaths in mammals consist of a single, tidal cycle (inspirationexpirationpause), there are four standard cycles that make up the anuran breathing repertoire, each having its own specific characteristics: buccal oscillations, balanced lung ventilations, lung inflation cycles, and lung deflation cycles. These are distinguished on the basis of mechanical events and the challenge has been to provide a basis for correlating fictive breathing with these behavioural events. Amalgamating data from the literature with the analysis of trigeminal (Vm) and laryngeal vagal (Xl) motor outputs obtained in the present study, however, the following picture emerges.
Buccal oscillations ventilate the oropharynx alone. The nares remain open while the floor of the buccal cavity moves up and down continuously, without ever forcing air into the lungs (De Jongh and Gans, 1969). With fictive breathing, such events can only be distinguished accurately from small lung ventilations in the presence of simultaneous evidence to indicate that the glottis remains closed (such as an absence of discharge in the laryngeal branch of the vagus nerve).
Balanced lung ventilations are thought to be the typical breath exhibited in resting anurans (De Jongh and Gans, 1969; West and Jones, 1975; Sakakibara, 1984a; Sakakibara, 1984b; Shelton and Vitalis, 1990; Kogo et al., 1994; Kinkead et al., 1994; Kinkead and Milsom, 1996). In these breaths, the glottis opens following buccal expansion and lung deflation is immediately followed by inflation with an equal volume of air. Our data suggest that buccal compression commences soon after glottal opening (0.05±0.02s) and thus there is little lag in the occurrence of motor output in Vm following that in Xl.
Either under conditions of high respiratory drive, or following lung deflation, multiple breaths may occur in rapid succession without allowing time for lung emptying. Such lung inflation cycles are characterized by an increase in the air pressure and volume in the lungs at the end of the cycle relative to that before the initiation of the cycle (West and Jones, 1975; Macintyre and Toews, 1976; Vitalis and Shelton, 1990; T. Baker and N. Smatresk, personal communication). Pulmonary pressure and lung volume generally increase in a ramp-like manner with each breath of the cycle (West and Jones, 1975). There is a reduction in the lung-emptying phase of each breath and thus contraction of the buccopharyngeal musculature occurs earlier in the cycle, pushing air into the lungs before the air already in the lungs is able to leave (West and Jones, 1975; Vitalis and Shelton, 1990). West and Jones (West and Jones, 1975) report that occasionally the buccal floor is elevated before the glottis opens, indicating a shift in timing of the various motor outputs involved in ventilation, and our data confirm that trigeminal discharge (which would initiate contraction of the buccal musculature) generally occurs before (0.33±0.18s) the motor output in the laryngeal branch of the vagus nerve (which would initiate contraction of the glottal dilator muscles) with inflation cycles.
Lung deflation cycles have not been studied extensively. With the lung deflation cycle, air pressure and volume in the lungs at the end of the cycle is lower than it was at the onset of the cycle (Vitalis and Shelton, 1990; T. Baker and N. Smatresk, personal communication). Vitalis and Shelton report a prolongation of phases 1 and 2 (inspiration and lung emptying) before the onset of phase 3 (lung filling) in Rana pipiens during deflation breaths and our data confirm that under these circumstances, trigeminal discharge precedes the motor output in the laryngeal branch of the vagus nerve by 0.323±0.855s.
From these data we can draw several conclusions. Fictive lung ventilations can be distinguished from fictive buccal oscillations by the presence of simultaneous discharge in the laryngeal branch of the vagus nerve (indicative of glottal opening) and the trigeminal nerve (indicative of buccal compression). By careful analysis of the timing of these two bursts of discharge relative to one another, these motor patterns can be further interpreted as events likely to produce deflation, balanced or inflation breaths. Given this, we can now more closely examine the effects of tonic lung inflation on fictive breathing patterns.
Changes in the fictive breathing pattern of bullfrogs in response to different levels of inflation and respiratory drive
Increasing lung inflation increased absolute breathing frequency by reducing the duration of apnea between breaths and promoting a change in breathing pattern, from no breathing to single breaths, breathing episodes, and finally to continuous breathing. Associated with this was a decrease in the amplitude and area of the integrated trigeminal electroneurogram associated with the lung breaths, indicative of a reduction in the force of the buccal pump. There was also a shift in the timing of the trigeminal and vagal discharge. Based on the temporal coordination of discharge in the trigeminal and vagus nerves during naturally occurring breaths as just described, we characterized fictive breaths as inflation, deflation or balanced breaths. Based on these interpretations, lung inflation also led to a shift from inflation to deflation breaths. Taken together the data suggest that lung deflation produces infrequent, large-amplitude inflation breaths or cycles. They also suggest that progressive lung inflation changes the breathing pattern to one of high-frequency attempts to deflate the lungs, which are largely passive and accompanied by contractions of the buccal pump that are not larger than those associated with normal buccal oscillations.
There has been some controversy surrounding the consequences of tonic lung inflation on breathing pattern in frogs and toads, which can possibly now be resolved in light of these findings. While one group of studies suggested that lung inflation stimulated breathing and that lung deflation inhibited breathing (Kinkead et al., 1994; Kinkead and Milsom, 1996; Kinkead and Milsom, 1997; Reid and Milsom, 1998), another group of studies suggested the opposite (De Marneffe-Foulon, 1962; Shelton and Boutilier, 1982; Wang et al., 1999) and a few studies obtained both results (Kogo et al., 1994; Reid et al., 2000). Invariably, in all studies, the amplitude of the trigeminal motor output associated with fictive breaths was inversely proportional to the volume of the lungs. Much of the controversy arises from interpretation of the high-frequency, low-amplitude bursts of motor output obtained from the trigeminal nerve with lung inflation. In some studies they were interpreted as small breaths, and hence as an increase in breathing frequency, while in other studies they were interpreted as buccal oscillations, and hence a reduction in breathing frequency. Based on the present study we conclude that the low-amplitude bursts of motor output seen in these previous studies were most likely a mix of small breaths and buccal oscillations and that the effect of the lung inflation was both inhibitory and excitatory; i.e. lung inflation inhibits large-amplitude inflation breaths and stimulates small-amplitude attempts to deflate the lungs. The remainder of the controversy may stem from differences in the level of respiratory drive present during lung deflation. While the data from the present study clearly show a transition from continuous breathing to breathing in episodes, single breaths and then apnea, as respiratory drive and lung volume are reduced, they also show that at low levels of respiratory drive (i.e. unidirectional ventilation with air), lung deflation leads to the reappearance of single breaths and, on rare occasions, some breathing episodes (Fig.6). As a rule, these breaths were attempts to inflate the lungs (Fig.9).
Biological significance
The emphasis of this study has been on the consequences of tonic changes in lung volume on breathing pattern. In mammals, where exhalation is relatively passive and the respiratory pause occurs at end-expiration, tonic lung inflation leads to a shortening of inspiration and a lengthening of expiration, which act to reduce lung volume. The net effect on respiratory frequency is generally a slowing or no change. In anuran amphibians, the respiratory pause occurs at end-inflation when the glottis closes, maintaining the lungs tonically in the inflated state. Our manipulations mimic this situation, and in this light the data suggest that increased lung volume leads to increased attempts to deflate the lungs. In both cases inspiratory efforts are reduced, expiration is promoted and the only real difference is the net effect on breathing frequency. This may be a necessary consequence of the differences in the respiratory pumps (aspiration versus force pump) and the position of the respiratory pause (end-expiratory versus end-inspiratory) in the ventilation cycle in the two groups of animals. In mammals, the pause occurs at end-expiration with the glottis open, allowing extended time for lung deflation to occur. In the frog, the pause occurs at end-inflation with the glottis closed, and extending the time available for deflation can only occur by increasing breathing frequency. It is interesting to note that increasing levels of inspired CO2 tended to produce similar effects to lung inflation. This may indicate that high levels of pulmonary CO2, regardless of the degree of lung inflation, promote increased attempts to deflate the lungs, perhaps in an attempt to turn over and replenish lung contents.
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Acknowledgments |
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References |
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