Keywords: Pulmonary stretch receptors, respiratory control, Bufo marinus, toad, ventilatory variability
ABSTRACT
Previous studies reveal that chemoreceptors and pulmonary stretch receptor
(PSR) afferent input have distinct effects on respiratory patterns, and
mechanics in toads. Removal of pulmonary feedback by vagotomy modified
behavior of the glottal and narial valves, but had no effects on respiratory
patterns. Since vagotomy may have a transformational effect on ventilation,
in this paper I decided to further investigate the relationships between
pulmonary feedback and ventilatory mechanics, pattern, and variability
by unidirectionally ventilating (UDV) toads' lungs to set lung pressure
and volume. This approach allows for the dampening of oscillations in both
slowly and rapidly adapting PSRs. Step changes in lung pressure were found
not to affect narial and glottal timing during inflation breaths in UDV
toads. This result indicates that the coordination of the narial and glottal
valves do not depend directly on PSR output and that studies reporting
alteration of valve timing in vagotomized toads may be looking at a transformational
effect caused by the denervation. The buccal pump stroke, for both respiratory
breaths and non-respiratory buccal oscillations (NRBO), became significantly
slower with increasing static lung volumes. Ventilatory entropy, an index
of breathing variability (or regularity) indicates a more regular system
at higher lung pressures. I conclude that levels of PSR output is relevant
to the control of buccal pumping frequency but not valve timings and that
qualitative aspects of PSR activity (presence of oscillations) are important
for generation of adaptive variation in the system's output.
INTRODUCTION
Breathing in amphibians is a remarkably complex motor synergy that produces
much wider array of respiratory behaviors and patterns than typically seen
in mammals or other continuous breathers. Their ventilatory behavior can
be described as a series of breathing episodes (consisting of one or more
breaths taken in sequence) interspersed by apneic periods of varying lengths.
This mode of ventilation is characteristic of other lower vertebrates as
well and is called episodic breathing. A breath in Bufo marinus starts
with expiration and ends with lung filling and is controlled by the coordinated
action of two valves (nares and glottis) and a buccal force pump. An analogy
with a piston pump illustrates well the mechanism (figure
1). The activity of the buccal pump can lead to ventilation of the
lungs (respiratory breath) as well as to non-respiratory buccal oscillations
(cycles of the pump in which the glottis valve remains closed and no exchange
of gas takes place between the buccal cavity and the lungs). A typical
example of the changes in pressure and airflow accompanying the mechanical
events of a respiratory breath are shown in figure 2.
Respiratory breaths in Bufo marinus, as well as in most anurans, can be
subdivided in three basic types according to the net transfer of gas between
the lungs and the buccal cavity (2). If lung pressure
is higher after a breath than it was immediately before, the breath is
called an "inflation breath", if it becomes smaller, than the breath is
a "deflation breath", and if lung pressure remains the same as it was before
the breath, the breath is called a "balanced breath".
Among the afferent signals that contribute to ventilatory control in
anurans, pulmonary stretch receptors (PSR) have been show to contribute
to the modulation of Bufo marinus' respiratory pattern and variability
(4, 2). These receptors (lung stretch
receptors) can be divided in two types: slowly adapting (tonic) receptors
and rapidly adapting (phasic) receptors. Slowly adapting receptors (SAR)
convey information about the level of inflation of the lung while rapidly
adapting receptors (RAR) fire in response to transient changes in lung
volume.
The present approach of controlling pulmonary mechanoreceptor output
has advantages over pulmonary denervation (used in other studies). Removal
of pulmonary mechanoreceptor feedback by sectioning the pulmonary branches
of the vagus appears to be "interpreted" as an empty lung, and results
in delayed glottal opening and advanced narial closure, thus producing
lung over-inflation (3). Consequently, vagotomy may produce
significantly different effects than controlled stimulation of pulmonary
mechanoreceptors.
Most studies on the control of breathing in amphibians focus on the
effects of different levels of afferent input on the animal's breathing
pattern (i.e., the temporal arrangement of breaths) as well as average
breathing amplitude and frequency. In the present paper, I propose to further
characterize the roles of pulmonary mechanoreceptors in both mechanical
and dynamical aspects of the toad's ventilation by looking at breathing
mechanics (valve timing) and pattern as represented by pumping frequency
and ventilatory variability in unidirectionally ventilated (UDV) toads
held at several static levels of lung inflation. By unidirectionally ventilating
toads, the activity of pulmonary RARs is greatly reduced (since lung volume
oscillations from ventilatory efforts are dampened by UDV) while tonic
SAR activity is still present. With larger set lung volumes, RAR activity
is further reduced while SAR activity is enhanced as it reflects lung stretch.
This experimental setup allowed the assessment of the importance of pulmonary
mechanoreceptor information in the modulation of key aspects of the toad's
ventilatory behavior without the transformational effects that a denervation
might cause.
MATERIALS AND METHODS
Experimental: Marine toads (n=10) were anesthetized in a 0.3% solution of MS-222 and had each lung implanted with polyethylene catheters (PE-205). A buccal catheter (PE-160) was introduced through the tympanum to monitor buccal pressure variations. The lungs were unidirectionally ventilated with the help of a gas mixer (Cameron GF-3). A known gas mixture (2% CO2, 15% O2, 83% N2, humidified) was set to flow through the toad's lungs (entering through one catheter and exiting through the other) at a controlled rate by means of a mass-flowmeter (Aalborg GFM-1700) connected in series between the gas mixer and the toad. A pressure transducer (Validyne DP-45) monitored lung pressure. Another DP-45 pressure transducer was used to monitor buccal pressure. Airflow through the nares was measured by means of a pneumotachograph outfitted to a plastic facemask glued to the toad's snout (figure 4) . Different lung volumes/pressures (0,1, 2, and 4 cm H2O) were obtained by changing the gas flow rate with the mass flowmeter, since the lungs resistance to the flow remained the same, the pressure (thus volume) of the lung increased as airflow increased. The airflow was adjusted until the desired pressure was obtained.
Analytical: Respiratory mechanics was analyzed through measurements of 50 steady state respiratory inflation breaths in each animal at each lung pressure level. The onset of glottal opening (GO, marked 1 on figure 2), and narial closure (NC, 3 on figure 2) were measured in milliseconds from the beginning of the buccal pulse and milliseconds after GO, respectively. Averages were taken and a one-way ANOVAs were done for each variable to check for differences in valve timings in response to lung pressure changes. Buccal pumping frequency was determined by means of FFTs calculated from 15-minute segments (including both respiratory and non-respiratory movements) of buccal pressure traces. In the spectra, the operating frequency of the buccal pump stands out as large peak in the vicinity of 1 Hz. A one way ANOVA was used to assess the effects of lung pressure in buccal pumping frequency.
The regularity of the breathing pattern or lack thereof was assessed by a measure of signal entropy calculated from the FFT spectra of the aforementioned 15-minute segments of breathing traces. The estimation of the respiratory signal entropy is as described by Shannon (1949) (8):
Considering the spectrum (resulting from the Fourier transform) of the
signal as a vector of n frequency components j, 
is the total energy of the signal and
is
the energy at a given frequency component j such that
.
Regression analysis was used to test the effects of lung pressure in
the regularity of the breathing patterns as quantified by shannon's entropy.
RESULTS
Changes in lung volume had evident effects in respiratory pattern (figure 4). Both the basal speed of the buccal pump and the regularity in the time-distribution of respiratory events were affected strongly by the qualitative and quantitative changes in lung mechanoreceptor output.
The timing of narial and glottal vales was not affected in any statistically significant way by the level of activity of lung mechanoreceptors. Averages and standard errors for glottal opening and narial closure timing variables (GO and NC) at each level of inflation of the lungs are shown on table 1.
Buccal pumping frequency showed significant decrease with higher levels of inflation of the lungs (F=23.7, p < 0.001) (figure 5). The increase in the duration of an inflation cycle can be appreciated even visually when we look at the respiratory traces for the two extreme lung pressure levels used (figure 4).
The toads breathing pattern also became more regular (figure
6) with the increase in lung pressure as indicated by significant reduction
in ventilatory entropy (R2 = 0.38; p = 0.03).
DISCUSSION AND CONCLUSIONS
Larger lung volumes had two main effects in the breathing pattern of Bufo marinus. 1- Ventilation became more regular at larger lung volumes. This could be the due to the increased SAR activity or to the removal of variability in overall lung mechanoreceptor information (due to the attenuation of RAR activity). 2- Buccal pumping frequency decreased with more inflated lungs. The physiological relevance of such response is not clear but it is, nevertheless, an interesting result that indicates that lung stretch information modulates the most basic rhythm of breathing in these animals.
Reduction in lung volume has been associated in past studies (6) with an increased breathing frequency. In this study I found that the basic pumping frequency does increase but that does not imply a larger frequency of respiratory breaths (figure 4). Moreover, the relationship of breathing rate to lung volume seems to be species-specific among amphibians (1;5).
Step changes in lung pressure were found not to affect narial and glottal timing during inflation breaths in UDV toads. This result indicates that the coordination of the narial and glottal valves do not depend directly on PSR output and that studies (3) reporting alteration of valve timing in vagotomized toads may be looking at a transformational effect caused by the denervation.
The fact that in this study, breathing pattern (distribution of breaths over time and buccal pumping frequency) was influenced by the change in mechanoreceptor output independently of breathing mechanics (valve timings) seem to reinforce the hypothesis of the existence of separate areas in the central nervous system to control these parameters. These areas have been termed "central rhythm generator (CRG)", generating basic pumping frequency and its regularity, and "central pattern generator (CPG)", generating the complex patterns of motor activation associated with breathing (i.e., breathing mechanics). The present data also supports the idea that both respiratory breaths and non-respiratory buccal oscillations share a common origin since they are modulated as a single frequency. The differentiation between respiratory and non-respiratory breaths may be determined by the output of the CPG as it controls the dynamics of the glottis and narial valves.
It has been argued that variability in ventilatory output is related
to variability in the afferent signals that modulate it (4,
7). The data presented here supports this idea since
UDV and high lung volumes dampen fluctuations in lung volume. However,
it is not possible to rule out the possible effects of increased SAR activity
on ventilatory variability.
ACKNOWLEDGEMENTS
This research was supported by the Brazilian research Council - CNPq
REFERENCES
2. Baker, T. L. Effects of pulmonary denervation on ventilatory mechanics of the toad, Bufo Marinus. 1-94. 1997. Thesis. The University of Texas at Arlington.
3. Baker, T.L. and N.J. Smatresk. Effects of pulmonary denervation on ventilatory mechanics of the toad, Bufo marinus. The FASEB Journal 11: A29, 1997.(Abstract)
4. Coelho, Flávio C. Afferent signal oscillations and the control of breathing in Bufo marinus. 1-100. 1999. Dissertation. The University of Texas at Arlington.
5. Kinkead, R., M.B. Harris, and W.K. Milsom. The role of the nucleus isthmi in respiratory pattern formation in bullfrogs. The Journal of Experimental Biology 200: 1781-1793, 1997. MEDLINE
6. Milsom, W.K. Mechanoreceptor modulation of endogenous respiratory rhythms in vertebrates. Am.J.Physiol. 259: R898-R910, 1990. MEDLINE
7. Modarreszadeh, M. and E.N. Bruce. Ventilatory variability induced by espontaneous variations of PaCO2 in Humans. J.Appl.Physiol. 76: 2765-2775, 1994. MEDLINE
8. Shannon, C.E. and W. Weaver. The mathematical theory of communication. Chicago: The University of Illinois Press, 1949, p. 1-125.