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First published online December 14, 2007
Journal of Experimental Biology 211, 66-78 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.011908
Singing with reduced air sac volume causes uniform decrease in airflow and sound amplitude in the zebra finch
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA
* Author for correspondence (e-mail: goller{at}biology.utah.edu)
Accepted 30 October 2007
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: birdsong, respiration, sensory feedback, motor control, Taeniopygia guttata
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Songs of many bird species are complex temporal sequences of alternating
sounds and silent periods, which are generated by elaborate respiratory
patterns (Suthers et al.,
1999
). Quiet respiration is characterized by its rhythmic
alternation between expiration and inspiration, each driven by similar
absolute changes in air sac pressure. Sound is normally produced during
expiration and, during song, the expiratory pressure increases at least
10–20-fold over that of quiet respiration. In addition, the duration of
respiratory phases becomes more variable (e.g.
Suthers and Goller, 1997;
Suthers et al., 1999;
Goller and Cooper, 2004;
Goller and Daley, 2001).
Silent periods during song are used to take short inspirations (minibreaths),
which are drawn with increased inspiratory pressure relative to that of quiet
breathing. In the zebra finch (Taeniopygia guttata), song consists of
a series of 4–8 different expiratory pulses, corresponding to the song
syllables, which alternate with minibreaths
(Fig. 1)
(Franz and Goller, 2002;
Goller and Cooper, 2004). This
sequence of stereotyped syllables (motif) is repeated a variable number of
times and forms a song bout.
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; Goller and
Daley, 2001). However, it is unknown how close to the limits of
air supply birds operate during song
(Goller and Cooper, 2004).
Second, it is largely unknown whether gas exchange is maintained during the
drastically altered ventilation patterns of song. Indirect evidence from
measurements of oxygen consumption indicates that gas exchange is not
compromised because birds do not incur an oxygen debt during song
(Oberweger and Goller, 2001;
Franz and Goller, 2003).
Decreased respiration after song in zebra finches and canaries suggests that
some individuals even hyperventilate during song, although hyperventilation in
the zebra finch is uncommon (F.G., unpublished observation). The ventilatory
pattern of song may therefore increase excretion of carbon dioxide
(Hartley and Suthers, 1989;
Franz and Goller, 2003).
Together, these findings suggest that during song, airflow through the lungs
is sufficient for gas exchange and may even be increased compared with
ventilation during quiet respiration. However, this may not be true for all
syllable types, because the volume exchanged during a
phonation–minibreath cycle may vary substantially. In canaries, airflow
during minibreath-syllables varies at least 6-fold, and during some syllables
the exchanged air volume is less than the tracheal deadspace
(Hartley and Suthers,
1989).
The avian respiratory system is highly efficient in exchanging oxygen and
carbon dioxide (e.g. Maina,
2000; Powell and Scheid,
1989), and this efficiency is in part attributed to the
unidirectional flow of air through the lung during both phases of the
respiratory cycle. This flow pattern is possible through the intricate
morphological design (Fig. 2),
where air sacs function as bellows and air reservoirs (e.g.
Scheid and Piiper, 1989) and
flow through the rigid lungs is primarily directed by aerodynamic valves (e.g.
Jones et al., 1981;
Banzett et al., 1987;
Brown et al., 1995). The
anterior and posterior thoracic sets of air sacs both contribute air to
expiratory flow. Air from the posterior air sacs perfuses the lungs and then
enters the primary bronchi, whereas air from the anterior air sacs is directly
routed into the primary bronchi. The vocal organ, the syrinx, is situated
where the primary bronchi merge into the trachea. Thus, the air stream for
phonation presumably originates in roughly equal parts from both the anterior
and posterior reservoirs. This model assumes that airflow patterns during song
are similar to those established in anesthetized, quietly breathing birds
(Bretz and Schmidt-Nielsen,
1972). Airflow patterns within the respiratory system have not
been directly studied during dynamic behaviors such as singing.
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). Only slight modification of song tempo occurs in different
social contexts (e.g. Cooper and Goller,
2006). The temporal pattern of song varies between males, possibly
causing variation in lung ventilation
(Franz and Goller, 2002). In
addition, the duration of different syllables within a song differs, leading
to a variation in the volume of air that is needed to complete each syllable.
One experiment showed that individual expiratory syllables require up to 0.3
ml of air (Goller and Daley,
2001).
Manipulations to the respiratory system could give insight into how dynamic
behaviors integrate with respiratory needs. Total occlusion of the thoracic
air sacs of chickens (Gallus domesticus) elicited no changes in quiet
respiration during exercise (Brackenbury et
al., 1989). Few studies have attempted to experimentally
manipulate respiratory dynamics during song. Small injections of air into an
anterior thoracic air sac during song in the northern cardinal (Cardinalis
cardinalis) elicited a compensatory decrease in expiratory effort in the
abdominal muscles. This indicates that cardinals use an on-line feedback
mechanism to monitor air sac pressure and airflow characteristics during song
(Suthers et al., 2002). The
small injections were unlikely to affect gas exchange or flow patterns, and
these experiments therefore do not address questions about respiratory
functions during song.
In an attempt to study the integration between respiratory needs and song, we here describe a chronic manipulation of respiratory functions. We manipulated respiration during song in zebra finches by eliminating part or all of the air volume contained in the posterior thoracic air sacs. We then recorded changes in respiratory and airflow patterns as well as acoustic output during singing.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Birds were
euthanized with an overdose of isoflurane. Flexible tubing (Silastic
laboratory tubing with inner diameter of 0.76 mm and outer diameter of 1.65
mm; Dow Corning, Midland, MI, USA) was inserted into the glottis of the bird
and secured with tissue adhesive, so that there was no leaking around the
insertion. The tubing was attached to a reservoir, which was later filled with
MICROFIL CP-101 (Flowtech Inc., Carver, MA, USA), a liquid silicone compound
for microvascular injection, which is cured by stannous octoate and a
crosslink agent, ethyl silicate. The bird was placed in an airtight glass
chamber and the chamber was attached through another tube to a vacuum source.
The chamber and the zebra finch were evacuated to 1.2–5.0 psi
(8.3–34.5 kPa) below the outside air pressure. When the pressure is at
its lowest, the CP-101 is released through the glottal tube into the
respiratory system of the bird, driven by the higher pressure outside the
chamber. The rate of repressurization was kept low via a regulatory
valve. To avoid rupture of membranes inside the bird, the chamber was also
vented slowly as the bird was filling with casting material. After all
pressures were equalized, the casting material was left to cure for several
minutes. The bird was then placed into a refrigerator to allow full curing of
the CP-101 overnight. The body of the bird was then placed into a container
with KOH for 3–4 days to expose the cast of the respiratory system. To
estimate volumes of different air sacs, we first determined the density of the
casting material. We inserted samples into a 1 ml syringe and determined the
volume of displaced fluid. These samples were then weighed and the
relationship between mass and volume calculated. The volume of air sacs was
estimated by weighing the casts.
Injection experiment
Male zebra finches (Taeniopygia guttata Gould) were isolated in a
small cage, which was placed into a wooden box lined with 5 cm-thick acoustic
foam. After the bird resumed singing in the new environment, it was fitted
with an elastic belt around its thorax with a VelcroTM tab situated on
the back. Birds were tethered with a wire leash attached to the VelcroTM
tab. The other end of the leash was led through the top of the wire cage and
was attached to a tether arm, which allowed free movement of the bird and was
counterbalanced to any additional weight attached to the backpack. After the
bird sang again, surgical implantation of a pressure cannula and airflow probe
followed.
Timeline
For 1–3 days, the bird was placed in a cage alone and allowed to
acclimate to his environment. Once he began to sing, he was belted and leashed
and allowed to acclimate again. Once song began again (1–3 more days),
surgery was performed, during which a cannula and/or flow probes or wire
electrodes for recording of electromyograms (EMG), where applicable, were
implanted. This was day 1 of the experiment. Birds usually took 24 h until
they resumed singing. Once sufficient data were collected (at least 20 song
bouts), which was typically finished on day 2, the left posterior thoracic air
sac of the bird was injected late on day 2. On day 3, 20 more song bouts were
collected. The bird then received a second injection. Day 4 and beyond, we
collected as much song as we could until signal quality deteriorated. The bird
was then euthanized and placement and size of the injections were
determined.
Measurements of air sac pressure
After food and water deprivation for one hour, birds were anesthetized with
isoflurane. A cannula (silastic tubing with 0.76 mm inner and 1.65 mm outer
diameter) was inserted below the last rib into the left anterior thoracic air
sac and sutured to the rib cage. Tissue adhesive was applied to seal the
insertion site. The free end of the cannula was connected to a piezoresistive
pressure transducer (FPM-02PG; Fujikura, Tokyo, Japan), which was mounted on
the VelcroTM tab on the backpack. A more detailed explanation can be
found in Franz and Goller (Franz and
Goller, 2002).
Airflow measurements
Tracheal airflow was measured in four birds. Flow probes were custom-built
by attaching microbead thermistors (0.13 mm; BB05JA202; Thermometrics, Edison,
NJ, USA) to small wires with conductive epoxy and then insulating the contacts
with non-conductive epoxy. The skin was opened above the furcula at the
midline, exposing the trachea. Just above the membrane of the interclavicular
air sac, a small hole was made into the connective tissue between two
cartilages of the trachea. The tip of the flow probe was inserted into this
hole and then secured in place by a suture around the cartilage just cranial
to the flow probe. The wires were routed subcutaneously to microconnectors on
the backpack. Airflow was determined by a feedback circuit (Hector
Engineering, Ellettsville, IN, USA) and is proportional to the current
required to maintain the thermistor at a constant temperature. A more detailed
description can be found in Goller and Daley
(Goller and Daley, 2001).
Electromyograms from abdominal expiratory muscles
In six birds, EMG were recorded from the abdominal muscle sheet. No effort
was made to record specifically from only one of the muscles. Electrodes were
placed into the muscle sheet so that they most likely recorded from all three
main muscles: m. obliquus abdominis externus, m. obliquus abdominis internus
and m. transversus abdominis. Bipolar EMG electrodes were implanted after a
small area of the muscle sheet was exposed by opening the skin and connective
tissue. Electrode tips were pushed into the muscle sheet, the wires were
looped to provide slack and then routed subcutaneously to the back. The
incision in the skin was then closed with suture and tissue adhesive. EMG
signals were amplified (1 K gain) and band-pass filtered (100–3000 Hz)
with a DAGAN EX4-400 amplifier (Minneapolis, MN, USA).
Injecting
In order to reduce the volume of available air within air sacs, we injected
dental impression medium (Reprosil Type 1 Hydrophilic Vinyl Polysiloxane
Impression Material; Milford, DE, USA). After food deprivation, the bird was
again anesthetized with isoflurane. Impression material was filled into a
syringe and injected through a hypodermic needle (size 18 G) into a posterior
thoracic air sac. Within minutes, the injected material cures into a block but
retains some flexibility. Although every effort was made to inject the same
amount of material each time, the volume injected into the air sac ranged from
0.08 to 0.23 ml of the impression material. This variation was caused by
differing amounts of leakage from the injection site. The puncture in the body
wall was closed with surgical suture and tissue adhesive as needed. Typically,
we injected first the left air sac and, in a subsequent injection, the right
air sac. Once enough song was collected following the second injection, the
bird was euthanized. We first determined the placement of the injection in
situ and then extracted the impression material for determining its
volume.
Recording physiological measurements and song
During each stage of the experiment (preinjection, after one injection and
after two injections), song was recorded together with physiological data. A
female zebra finch was placed in front of the cage at a constant distance to
the perch to induce the male to sing. All song used for analysis was directed
song to assure that the singing bird faced forward towards the microphone.
Song was recorded with an Audiotechnica AT8356 microphone (Stow, OH, USA)
and amplified with a Brownlee amplifier (Model 410; San Jose, CA, USA). The
voltage output of the pressure transducer was recorded simultaneously with the
sound either on a TEAC 135T multi-channel digital recorder at a sample rate of
24 kHz or directly onto computer (Avisoft recorder, 26–32 kHz sample
rate; National Instruments PCI-6220 M, Austin, TX, USA) using Avisoft Recorder
software (Avisoft Bioacoustics, Berlin, Germany). Flow and EMGs were recorded
simultaneously on separate channels [see also Franz and Goller
(Franz and Goller, 2002) for
more detailed description].
Analysis
We digitized the TEAC recordings (Data translation 2821G AD converter at a
40 kHz sample rate; Marlboro, MA, USA). We then analyzed the data using SIGNAL
3.1 software (Engineering Design, Berkeley, CA, USA). We looked for temporal
and amplitude differences in pressure patterns between the pre-injection and
post-injection song bouts.
The respiratory pattern was analyzed during quiet respiration and during song for the various stages of the experiment. For quiet respiration and for each expiratory pulse of the song motif, we measured the duration of the expiratory phase as well as respiratory rate. In addition, we used integrated voltage of each expiration as a measure of total expiratory effort, assuming that syringeal resistance did not change between treatments. Airflow was quantified in a similar fashion, using uncalibrated voltage output. Calibration was not possible for the various stages of the experiment. In order to assess whether the voltage response of the flow probe deteriorated over the course of the experiment, we compared changes in quiet respiration to changes in song syllables.
In order to compare EMG recordings across days, we plotted EMG activity for individual expiratory pulses against air sac pressure during quiet respiration and song. We assumed that changes in quiet respiration reflect a deterioration in the EMG signal over the course of the experiment and used this information to interpret changes during song.
In addition, we quantified various temporal parameters of song. The duration of song bouts, the duration of syllables, and other temporal changes were measured using the air sac pressure pattern. Onset and offset of respiratory pulses can be determined more accurately using air sac pressure than is possible from sound recordings.
Sound amplitude was determined for each syllable by rectifying the voltage signal (i.e. absolute values calculated with 0.1 ms window) and integrating voltage (time window 2 ms) for each syllable. Spectrograms were calculated at each stage of the experiment, and visual comparison of acoustic features was used to identify potential changes in acoustic structure.
Statistical analyses were performed using SPSS and SigmaPlot 8.0 software (SYSTAT, San Jose, CA, USA). Because syllables and corresponding air sac pressure patterns are individually characteristic, we initially tested all syllables for changes with injection treatments using two-tailed Student's t-tests on the original measurements. Subsequently, to test for overall effects, we compared percentage change values between treatment groups with two-tailed unpaired or, where appropriate, paired t-tests. If multiple tests were performed using the same data set, degrees of freedom were adjusted. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Utah.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Injection into the posterior thoracic air sacs
In 11 vigorously singing birds, we injected dental impression medium
initially into the left posterior thoracic air sac. Birds sang readily after
the injection, and song and air sac pressure were recorded again. We then
injected dental impression medium into the right posterior thoracic air sac
and recorded again. Injections ranged from 0.102 to 0.23 ml, thus taking up
between 46 and 100% of one posterior thoracic air sac
(Table 1). Smaller injections
sometimes resulted in the apparent occlusion of the connection from the air
sac to the lung. In eight birds, it appeared that one or both injections had
protruded into the ostium (Table
1), presumably removing the entire volume of the occluded air sac
from the air reservoir. However, it cannot be assessed whether or not the
ostium was completely occluded under the pressurized conditions of song. The
four birds in which the first injection missed the air sac serve as a control
(Table 1) (see below).
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Injections affect quiet respiration
Amplitude of air sac pressure pulses and respiratory rate are highly
variable during quiet respiration, making comparisons between treatments
difficult. We selected 3 s-long segments of quiet respiration
(N=35–55; at least 10 s before or after song and not including
any calls) for each treatment and calculated respiratory rate and mean
expiratory air sac pressure amplitude. Despite substantial variability,
respiratory rate showed a highly significant increase after two injections in
all birds and a significant increase after one injection into an air sac in
all but one bird (Table 2).
Missed injections did not have the same effect. Changes to the amplitude of
expiratory air sac pressure pulses were much less consistent. One bird showed
a drastic increase in air sac pressure, while others ranged from a small
increase to a decrease (Table
2). Whereas birds responded to the injections with timing changes
during quiet respiration, the respiratory pattern of song remained remarkably
unchanged (see below).
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One bird, B4, was apneic following almost all preoperational song bouts,
indicating hyperventilation during song
(Franz and Goller, 2003). The
duration of apnea was positively correlated with song bout duration before
injections of dental impression medium. With each injection, however, apnea
length decreased for bouts of similar length. After the second injection, most
song bouts were no longer followed by apnea, although air sac pressure
amplitude was still reduced for the first few breaths
(Fig. 3).
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Tracheal airflow was monitored in three birds with injections into both posterior thoracic air sacs and in one bird with one missed injection (Table 1). Expiratory airflow decreased with the first injection, and decreased even further with the second (Fig. 5). In two birds, airflow decreased proportionally to the decrease in air sac pressure. In the other two birds, airflow decreased substantially more than would be expected from reduced air sac pressure. Degradation of the voltage response of flow probes over the course of the experiment may have contributed to this disproportionate decline in airflow. Overall, airflow decreased by 8.3% to 56.4%, with an average of 35.1% after the second injection.
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The pressure amplitude of individual pulses, corresponding to syllables,
typically was decreased uniformly throughout the duration of the syllable
(Fig. 6). However, two birds
showed a differential effect over the duration of syllables. For example, in
one syllable of W42, air sac pressure after two injections was 70% of
pre-injection values at the onset of the syllable but declined to 40% at the
end (Fig. 7). All four
syllables in this bird's motif showed a similar decrease over the course of
the pressure pulse, regardless of their varying durations ranging from 103 to
144 ms. In the other bird (R9) the air sac pressure declined more towards the
end of one long syllable (245 ms), descending from 80% of pre-injection value
at the onset to 30% at the end. Changes to shorter syllables (
100 ms)
were not as pronounced.
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In one bird, whose air sac pressure decreased uniformly over the course of syllables, airflow also decreased uniformly. The slopes of cumulative airflow plots run parallel to each other for the different treatments (Fig. 8). However, in another bird with uniform decrease in air sac pressure, airflow did not decrease uniformly. After injections, the slope of cumulative airflow plots changed from that observed during pre-injection song. For example, the slope decreased near the end of each syllable, indicating a greater reduction in flow than was observed during the early portions of the pulses (Fig. 6, indicated by arrows).
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The temporal structure of song bouts changed in five individuals. Mean motif length decreased in four birds due to variation in how much of the full motif was sung. Pre-injection song in these birds occasionally included incomplete motifs, but their occurrence increased after injections. One bird sang 97% full motifs prior to injection and only 29.2% after two injections. One individual stayed within 5% of pre-injection motif length. The change could have been based on small sample size. Three other birds always sang their full pre-injection motif and continued to do so throughout the experiment.
Mean bout duration decreased significantly after the first injection. Duration was normalized to the maximum recorded bout duration for each individual to allow pooling of data between birds. Mean bout duration prior to injection was 0.45±0.03 of maximal bout duration and dropped to 0.28±0.02 after the first injection and 0.24±0.03 after the second (Student's t-test; pre- vs first injection, t=4.45; P<0.00001, d.f.=166; first vs second, t=0.99, P=0.32, d.f.=160). However, birds were still able to sing long bouts, but did so less frequently (Fig. 9). Because bout duration also depends on motivation, it is possible that this decrease reflects reduced motivation and not a direct respiratory effect of the injections.
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In zebra finch Y34, short segments of song were omitted (arrows in Fig. 5). Sound production during these segments was prevented by closing the syringeal valves to airflow, as indicated by zero tracheal flow. One of these segments was an entire, short syllable. The acoustic structure of these segments does not suggest an obvious reason for why closure of the labial valves occurred.
Injection does not increase EMG activity in expiratory muscles
In six birds, we measured EMG activity of the abdominal expiratory muscles
to assess whether injection into posterior thoracic air sacs caused changes in
activity. Because the experiments lasted 3–4 days, changes in EMG
activity have to be interpreted carefully. Impedance at the electrode tips can
change over the course of a few days and might therefore result in differences
in the recorded EMG amplitude.
In five birds, the amplitude of EMG bursts associated with specific expiratory pulses of the song did not increase after injections (Fig. 11). Because air sac pressure was lower after injection, the points for each expiratory pulse are shifted to the left, indicating lower EMG activity for the specific syllable but higher EMG activity for the achieved air sac pressure than during pre-injection song (Fig. 11B–D). These results indicate that a similar expiratory effort results in generation of lower expiratory pressure and that no compensation for the reduced air sac pressure is evident from the EMG recordings. One bird showed an increase in EMG activity following the first injection. However, EMGs during quiet respiration also increase substantially after the first injection, suggesting a change in the impedance and not a compensatory adjustment.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Mechanics of breathing and injections of dental impression medium
Injections of dental impression medium replace part or all of the volume of
an air sac and therefore reduce the air reservoir available for singing. The
reduction of the total volume of air available for expiration ranged from
approximately 8 to 19%, but injections into the posterior thoracic air sacs
reduced the air volume for perfusion of the lung during expiration by as much
as 25.6%. In chickens, filling of all thoracic air sacs with cotton wool had a
minimal effect on breathing during rest and running
(Brackenbury et al., 1989).
Because the cotton wool became suffused with fluid within the air sacs, the
whole air sac volume was unusable to the bird. Singing requires much less
increase in oxidative metabolism than running
(Oberweger and Goller, 2001;
Franz and Goller, 2003),
suggesting that in our experiment gas exchange might not have been limited
either. A potentially important difference between the experiment on chickens
and this study is that we injected the posterior thoracic air sac without
similar injections into the anterior thoracic air sac. Such an asymmetric
injection might have altered flow patterns differently from those achieved
after asymmetrical filling of the anterior thoracic air sacs or the
symmetrical filling of all thoracic air sacs in the chicken
(Brackenbury et al., 1989).
The cured dental impression medium could also present a physical obstacle
to the thoracic movements associated with respiration. Dorso-ventral and
lateral motion of the rib cage during breathing
(McLelland, 1989;
Fedde, 1976) could be impeded
by the addition of a solid mass within the air sacs. Although the reduced
effect of injections that missed the air sac partially controls for mechanical
interference, a physical obstruction of respiratory movements cannot be ruled
out completely.
The missed injections provide a good control for the added mass resulting from replacing air with dental impression medium. Because this additional mass must be moved with every breath, it is possible that metabolism might have been increased as a consequence. However, birds with missed injections had to accelerate similar additional mass with every breath as did the birds with air sac injections but did not show increased ventilation, thus making it unlikely that the observed changes are caused by the addition of mass to the thorax.
Respiratory airflow and injections
In order to assess the potential effects of the injections on song, flow
patterns of air during both respiratory phases need to be considered. Because
no data are available for songbirds, we base this discussion on the avian
model obtained from anesthetized non-songbirds (e.g.
Bretz and Schmidt-Nielsen,
1972). The posterior set of air sacs (posterior thoracic and
abdominal air sacs) serves as an air reservoir
(McLelland, 1989), which fills
during inspiration and provides air that flows through the lung during
expiration (Fig. 2). The
routing of air is thought to be controlled by aerodynamic valving (e.g.
Jones et al., 1981;
Banzett et al., 1987;
Brown et al., 1995), which is
possibly enhanced by physical modifications
(Maina and Africa, 2000). The
efficiency of the valving is decreased at low airflow
(Banzett et al., 1987).
When air space was removed from the posterior thoracic air sacs, we
measured a slight increase or no significant change in air sac pressure during
quiet respiration. Air sac pressure amplitude can vary substantially with
fluctuating levels of activity, making these comparisons between treatments
difficult. However, injections into the posterior thoracic air sacs resulted
in a consistent increase in respiratory rate
(Table 2). These data therefore
suggest that the reduction in available air volume did affect gas exchange
during quiet breathing and birds compensated with increased ventilation.
Surprisingly, a much larger reduction in volume in chickens did not cause
significant changes in gas exchange during rest and running
(Brackenbury and Amaku, 1990;
Brackenbury et al., 1989).
Volume of air needed for singing
The effects of the reduced air reservoir on song generation must depend on
the volume of air exchanged during different song syllables. The posterior
reservoir, composed of posterior thoracic and abdominal air sacs, holds about
0.9 ml of air. Two large injections of 0.22 ml each into the posterior
thoracic air sacs cause a reduction of at least 48%, leaving nearly 0.46 ml of
air in the posterior reservoir. Similarly, in cases where the ostium was
blocked by the injections, a reduction of the same amount was possible. Goller
and Daley used calibrated tracheal airflow measurements to estimate the volume
of air exhaled during individual expiratory pulses, corresponding to song
syllables (Goller and Daley,
2001). The largest expired volume for a syllable in songs of three
zebra finches approached 0.3 ml, which would use 50–75% of the available
posterior air volume after large bilateral injections. This estimate indicates
that long song syllables, which are generated with high airflow, might be
limited by the available air volume.
It is striking that air sac pressure and airflow were typically reduced for the entire song motif, independent of the variable volume of air exchanged during different syllables. This global reduction in airflow was unexpected. Instead, we expected to find that long syllables with large air requirements would be reduced in duration when air supply is exhausted, whereas short syllables should not be affected by the reduced air supply at all. Only one bird (B4) appeared to reach the limit of air supply and reduced the duration of an exceptionally long syllable from 612 ms to 554 ms (Fig. 8). However, even in this bird all syllables were produced with reduced air sac pressure amplitude after the injection.
Another possibility for how injections might affect song is a passive decline in air sac pressure as the available reservoir is depleted. In this case, a growing decrease in air sac pressure throughout long syllables would be predicted. This pattern was found only in two zebra finches (W42 and R9), and the progressively growing decline in air sac pressure amplitude during the course of a syllable was seen in short and long syllables alike (Fig. 7). Why these individuals showed such a different response to injection is unknown.
With the above-mentioned exception, the temporal pattern of the song motif did not change despite reduced amplitude of air sac pressure, airflow and sound amplitude. This suggests that the stereotyped respiratory motor program of song is generated unless physical limitations prevent its completion. The consequences of reduced airflow on gas exchange during song are not known, but quiet respiration after song did not indicate a limitation. Reduced bout duration after injection, however, could indicate a potential limitation caused by need for gas exchange, although a motivational explanation for this reduction cannot be ruled out.
Somatosensory feedback
Respiration is regulated by feedback from an array of different sensory
systems. Chemoreceptors in the avian lung, especially carbon dioxide
receptors, can affect respiratory rate and depth of breath on a
breath-by-breath time scale (Gleeson and
Molony, 1989). The decreased air volume in the posterior air
reservoir reduced flow through the lungs and altered gas exchange during quiet
respiration. This change was presumably effected by chemoreceptors. During
song, only one bird hyperventilated, as indicated by periods of apnea after
song bouts. Apnea was reduced after injections, indicating reduced gas
exchange during song. The other individuals were not apneic after song, and
injections did not change respiration after song noticeably (data not shown).
In general, these observations indicate that gas exchange during song was not
sufficiently compromised by the injections to cause altered respiratory
patterns after song. This strongly suggests that during song in zebra finches,
gas exchange is enhanced relative to quiet breathing. This confirms the
indirect evidence from oxygen consumption measurements, which do not show an
increase in oxygen consumption after song (oxygen debt) and also suggest
hyperventilation during song in some individuals
(Oberweger and Goller, 2001;
Franz and Goller, 2003).
Mechanoreceptors in the respiratory system are potentially important for
controlling the timing and duration of song bouts
(Wild, 2004). These
mechanoreceptors are most likely located in the air sac system
(Kubke et al., 2004) and
probably respond to volume changes, particularly during the inspiratory phase
(Ballam et al., 1982;
Molony, 1974). In cardinals,
an increase in air sac volume by injection of small air pulses into the
anterior thoracic air sac during song resulted in decreased EMG activity in
the abdominal expiratory muscles (Suthers
et al., 2002). This compensatory reduction in muscle effort
suggested regulation of air sac pressure and airflow. In contrast to these
results, an air sac permanently filled by an injection of dental impression
medium elicited no compensatory response in the abdominal muscles to maintain
air sac pressure and airflow for song. Comparisons of absolute amplitudes of
EMG activity across several days of recording can be problematic if the
impedance at the electrode tips changes over this time. Nevertheless, by
comparing EMG activity during quiet respiration before and after injection, we
can rule out large changes in impedance. Compensatory changes in EMG activity
during song after injections are not present in any of the birds. In one bird,
EMG activity during quiet respiration and song increased similarly after the
first injection (Fig. 11B),
which is consistent with an impedance change and probably does not indicate
compensation. This interpretation is confirmed by the data after the second
injection, where the pattern is the same as that found in all the other birds
(Fig. 11).
Mechanoreceptors fire preferentially during inspiration, when volume
increases and pressure decreases (Gleeson
and Molony, 1989). Perhaps the compensatory response in the
cardinal occurred because the injection of air simulated an inspiratory event.
In our experiment, the injected air sacs were permanently full and therefore
volume changes did not occur in these air sacs. Because volume change is the
most likely physical variable to alter the firing rate of these receptors
(Ballam et al., 1982;
Molony, 1974), feedback
information may not have been available to correct for our manipulation.
Auditory feedback
During song, birds also receive auditory feedback information. However,
altered auditory feedback does not cause rapid changes to the temporal pattern
of song in the zebra finch. Changes occurred only after several days of
receiving altered acoustic feedback (e.g.
Leonardo and Konishi, 1999;
Cooper and Goller, 2004).
Because our experiments were completed within 3–4 days and acoustic
changes were generally small, we did not expect altered auditory feedback to
cause changes to the temporal pattern of song.
The lack of response to the reduced sound amplitude, which the bird must
have perceived through auditory feedback, is surprising. Zebra finches show an
increase in song intensity by 1–3 dB if the receiver distance is
increased (Brumm and Slater,
2006). In addition, a significant Lombard effect was present, with
increases in song amplitude by 8–10 dB, when birds sang in elevated
background noise (Cynx et al.,
1998). Theoretically, neither of these responses requires zebra
finches to use auditory feedback from song output to adjust song amplitude. In
the case of distance adjustments, visual estimates of distance may be the
stimulus for changing song amplitude. In the case of the Lombard effect, the
bird may be responding to the level of background noise and not the amplitude
of its song. However, budgerigars (Melopsittacus undulatus) and
nightingales (Luscinia megarhynchos) respond with a Lombard effect if
noise occupies the same frequency band as their vocalizations
(Manabe et al., 1998;
Brumm and Todt, 2002),
suggesting that birds monitor their vocal output or the signal-to-noise
ratio.
Data from this study suggest a possible reliance on background noise. Two
large injections reduced the volume of song by as much as 13 dB in some
syllables. If birds had used auditory feedback to evaluate their sound output,
we would expect an increase in abdominal muscle activity to compensate for
this amplitude decrease, but no compensation was found. This suggests that
increased background noise is the main stimulus for the Lombard effect in the
zebra finch. If this sensitivity to background noise is most effective in the
frequency band of the song, our interpretation is also consistent with the
results in budgerigars and nightingales
(Manabe et al., 1998;
Brumm and Todt, 2002). However,
it is possible that the lack of amplitude control in our study may be a
non-specific effect of the injection procedure.
Conclusions
The reduction of air volume by filling posterior thoracic air sacs with
dental impression medium has very little impact on the timing of song. Only
very long syllables are shortened, but otherwise the temporal pattern remains
intact. Although oxygen exchange must be reduced by the treatment, as
indicated by the change in quiet respiration and reduced apnea, this reduction
appears to be insufficient for causing temporal changes to the motif
structure. Air sac pressure amplitude for all syllables is increasingly
reduced by the consecutive injections of medium, resulting in reduced airflow
and reduced sound amplitude. EMG recordings do not indicate that birds attempt
to compensate for the reduced pressure and sound amplitude, suggesting that
this chronic alteration may not generate feedback information that leads to
corrective changes in the motor gestures. These two observations indicate that
the stereotyped temporal pattern of song arises from a motor program, which
remained surprisingly unmodified by the chronic reduction in air supply.
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Ballam, G. O., Clanton, T. L. and Kunz, A. L.
(1982). Ventilatory phase duration in the chicken: role of
mechanical and CO2 feedback. J. Appl.
Physiol. 53,1378
-1385.
Banzett, R. B., Butler, J. P., Nations, C. S., Barnas, G. M., Lehr, J. L. and Jones, J. H. (1987). Inspiratory aerodynamic valving on gas density and velocity. Respir. Physiol. 70,287 -300.[Medline]
Brackenbury, J. H. and Amaku, J. (1990).
Effects of combined abdominal and thoracic air-sac occlusion on respiration in
domestic fowl. J. Exp. Biol.
152,93
-100.
Brackenbury, J. H., Darby, C. and El-Sayed, M. S.
(1989). Respiratory function in exercising fowl following
occlusion of the thoracic air sacs. J. Exp. Biol.
145,227
-237.
Bretz, W. L. and Schmidt-Nielsen, K. (1972).
The movement of gas in the respiratory system of the duck. J. Exp.
Biol. 56,57
-65.
Brown, R. E., Kovacs, C. E., Butler, J. P., Wang, N., Lehr, J. and Banzett, R. B. (1995). The avian lung: is there an aerodynamic expiratory valve? J. Exp. Biol. 198,2349 -2357.[Medline]
Brumm, H. and Slater, P. J. B. (2006). Animals can vary signal amplitude with receiver distance: evidence from zebra finch song. Anim. Behav. 72,699 -705.[CrossRef]
Brumm, H. and Todt, D. (2002). Noise-dependent song amplitude regulation in a territorial songbird. Anim. Behav. 63,891 -897.[CrossRef]
Cooper, B. G. and Goller, F. (2004). Partial muting leads to age-dependent modification of motor patterns underlying crystallized zebra finch song. J. Neurobiol. 61,317 -332.[CrossRef][Medline]
Cooper, B. G. and Goller, F. (2006).
Physiological insights into the social-context-dependent changes in the rhythm
of the song motor program. J. Neurophysiol.
95,3798
-3809.
Cynx, J., Lewis, R., Tavel, B. and Tse, H. (1998). Amplitude regulation of vocalizations in noise by a songbird, Taeniopygia guttata. Anim. Behav. 56,107 -113.[CrossRef][Medline]
Duncker, H. R. (1971). The lung air sac system of birds: a contribution to the functional anatomy of the respiratory apparatus. Embryol. Cell Biol. 45, 1-171.
Fedde, M. R. (1976). Respiration. InAvian Physiology. 3rd edn (ed. P. D. Sturkie), pp.127 -131. New York, Berlin, Heidelberg: Springer-Verlag.
Franz, M. and Goller, F. (2002). Respiratory units of motor production and song imitation in the zebra finch. J. Neurobiol. 51,129 -141.[CrossRef][Medline]
Franz, M. and Goller, F. (2003). Respiratory
patterns and oxygen consumption in singing zebra finches. J. Exp.
Biol. 206,967
-978.
Gleeson, M. and Molony, V. (1989). Control of breathing. In Form and Function in Birds. Vol.4 (ed. A. S. King and J. McLelland), pp.439 -484. London: Academic Press.
Goller, F. and Cooper, B. G. (2004). Peripheral
motor dynamics of song production in the zebra finch. Ann. N. Y.
Acad. Sci. 1016,130
-152.
Goller, F. and Daley, M. A. (2001). Novel motor gestures for phonation during inspiration enhance the acoustic complexity of birdsong. Proc. R Soc. Lond. B Biol. Sci. 268,2301 -2305.[Medline]
Hartley, R. S. and Suthers, R. A. (1989). Air flow and pressure during canary song: direct evidence for minibreaths. J. Comp. Physiol. 165,15 -26.[CrossRef]
Jones, J. H., Effman, E. L. and Schmidt-Nielsen, K. (1981). Control of air flow in bird lungs: radiographic studies. Respir. Physiol. 45,121 -131.[CrossRef][Medline]
Kubke, M. F., Ross, J. M. and Wild, J. M. (2004). Vagal innervation of the air sacs in a songbird, Taenopygia guttata. J. Anat. 204,283 -292.[CrossRef][Medline]
Leonardo, A. and Konishi, M. (1999). Decrystallization of adult birdsong by perturbation of auditory feedback. Nature 399,466 -470.[CrossRef][Medline]
Maina, J. N. (2000). What it takes to fly: the structural and functional respiratory refinements in birds and bats. J. Exp. Biol. 203,3045 -3064.[Abstract]
Maina, J. N. and Africa, M. (2000). Inspiratory aerodynamic valving in the avian lung: functional morphology of the extrapulmonary primary bronchus. J. Exp. Biol. 203,2865 -2876.[Abstract]
Manabe, K., Sadr, E. I. and Dooling, R. J. (1998). Control of vocal intensity in budgerigars (Melopsittacus undulates): differential reinforcement of vocal intensity and the Lombard effect. J. Acoust. Soc. Am. 103,1190 -1198.[CrossRef][Medline]
McLelland, J. (1989). Anatomy of the lungs and air sacs. In Form and Function in Birds. Vol4 (ed. A. S. King and J. McLelland), pp.221 -279. London: Academic Press.
Molony, V. (1974). Classification of vagal afferents firing in phase with breathing in Gallus domesticus.Respir. Physiol. 22,57 -76.[CrossRef][Medline]
Oberweger, K. and Goller, F. (2001). The
metabolic cost of birdsong production. J. Exp. Biol.
204,3379
-3388.
Powell, F. L. and Scheid, P. (1989). Physiology of gas exchange in the avian respiratory system. In Form and Function in Birds. Vol. 4 (ed. A. S. King and J. McLelland), pp. 393-437. London: Academic Press.
Scheid, P. and Piiper, J. (1989). Control of breathing. In Form and Function in Birds. Vol4 (ed. A. S. King and J. McLelland), pp.369 -391. London: Academic Press.
Suthers, R. A. and Goller, F. (1997). Motor correlates of vocal diversity in songbirds. In Current Ornithology. Vol. 14 (ed. V. Nolan, Jr, E. D. Ketterson and C. F. Thompson), pp. 235-288. New York: Plenum Press.
Suthers, R. A., Goller, F. and Pytte, C. (1999). The neuromuscular control of birdsong. Philos. Trans. R. Soc. Lond. B Biol. Sci. 354,927 -939.[CrossRef][Medline]
Suthers, R. A., Goller, F. and Wild, J. M.
(2002). Somatosensory feedback modulates the respiratory motor
program of crystallized birdsong. Proc. Natl. Acad. Sci.
USA 99,5680
-5685.
Wild, J. M. (2004). Functional neuroanatomy of
the sensorimotor control of singing. Ann. N. Y. Acad.
Sci. 1016,438
-462.
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