Maturation of cardiovascular control mechanisms in the embryonic emu (Dromiceius novaehollandiae)
1 Department of Ecology and Evolutionary Biology, University of California,
Irvine, CA 92697, USA
2 Department of Biology, University of Akron, OH 44325, USA
3 Department of Biological Sciences, University of North Texas, Denton, TX
76203, USA
* Author for correspondence (e-mail: dcrossle{at}uci.edu)
Accepted 1 May 2003
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Summary |
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Key words: cardiovascular regulation, embryogenesis, physiology, incubation period, cholinergic tone, adrenergic tone, emu, Dromiceius novaehollandiae
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Introduction |
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Studies have established that chickens exhibit several components of the
mature cardiovascular regulatory system early in ontogeny
(Ignarro and Shideman, 1968;
Pappano, 1977
). These include
responsive adrenergic and cholinergic receptor populations in the developing
cardiovascular system as well as anabolic and catabolic enzymes for
catecholamines and acetylcholine (Berry,
1950
; Zacks, 1954
;
Ignarro and Shideman, 1968
).
Despite the presence of regulatory components from both arms of the autonomic
nervous system, at day 12 [60% of the incubation period (60%I)] embryonic
white leghorn chickens (Gallus gallus) possess only a tonic
adrenergic stimulation of the cardiovascular system
(Crossley and Altimiras, 2000
).
Several factors may dictate the timing of onset and subsequent effectiveness
of cardiovascular regulatory mechanisms during chicken development. However,
due to the lack of data from any avian species other than chickens,
comparative hypotheses focusing on cardiovascular control during development
cannot be evaluated.
Ratite birds are a useful group for addressing evolutionary hypotheses
focusing on developmental cardiovascular physiology in birds. Ratites
represent a distinct lineage of avian vertebrates, separated from other
groups. Thus, the study of ratites could further the understanding of
cardiovascular control patterns that are conserved during development in avian
lineages (Burggren and Crossley,
2002).
The goal of this study was to characterize the development of cardiovascular control systems in the emu. We hypothesized that, given the taxonomic differences between emus and chickens, emus would possess different cardiovascular regulation mechanisms. Cardiovascular responses (change in heart rate and arterial pressure) to graded levels of hypoxia were used as indicators of the embryonic emus' ability to respond to the environment as well as to determine the timing of onset of a functional chemoreflex. Furthermore, cholinergic and adrenergic receptor antagonists were applied during the interval from 70%I to 90%I to assess the function of these receptors during this 30% developmental window of emu incubation.
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Materials and methods |
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Surgical procedure
Eggs were placed in a water-jacketed temperature control dish for surgery.
A 3 cm2 window was cut in the shell to expose the vessels of the
underlying chorioallantoic membrane (CAM). Both a CAM artery and vein were
occlusively catheterized using heat-pulled polyethylene tubing (PE-90) filled
with heparinized saline (0.9%) (Crossley
and Altimiras, 2000).
Following catheterization, eggs were placed in a 1200 ml temperature-controlled chamber fitted with a lid containing multiple ports for externalization of catheters and inflow tubes for changing gas mixtures within the chamber. The arterial catheter was then passed through a port and connected to a pressure transducer (WPI) attached to a bridge amplifier (CB Sciences 400). Humidified 36°C air was continuously circulated into each experimental chamber during the period of study at a rate of 1.5 litres min-1. This rate of gas flow was also used during the acute hypoxic response experiments. A data acquisitions system (Powerlab; AD Instruments, Colorado Springs, CO, USA) was used to collect the output signal at a sampling frequency of 100 Hz.
Experimental protocol
Eggs were equilibrated in their experimental chamber for 1 h to allow
cardiovascular values to stabilize. Prior to experimental manipulations,
baseline data were collected from 20 instrumented embryos for 30 min. Arterial
pressure was recorded directly and heart rate determined from the blood
pressure trace using a software tachograph. Two experimental series were
conducted on each instrumented embryo.
Acute hypoxia response
20 embryos were used during this series. Each embryo was exposed to 15%
O2 for 5 min while changes in heart rate and arterial pressure were
recorded. After a 30 min recovery period (a length of time that was sufficient
for cardiovascular variables to return to control levels), the hypoxic
procedure was then repeated with 10% O2. This hypoxic protocol was
completed in all of the embryos at each of the incubation intervals
tested.
Cholinergic and adrenergic tone
Following a 1.5 h recovery period, five embryos from three stages (70%I,
80%I and 90%I; total N=15) from the hypoxic series study were used to
assess the autonomic tone on the emu cardiovascular system. Three autonomic
receptor antagonists were serially injected to determine the endogenous
autonomic receptor (cholinergic and adrenergic) tone on heart rate and
arterial pressure.
Following the hypoxic recovery period, a single dose (1 mg kg-1)
of the muscarinic antagonist atropine was injected to determine the resting
cholinergic receptor stimulation (tone) on heart rate and arterial pressure.
Once a new baseline for heart rate and pressure was established, a second
drug, the ß-adrenergic antagonist propranolol (3 mg kg-1), was
injected into each embryo. Heart rate and blood pressure changes were recorded
and allowed to stabilize at a new resting level prior to the injection of the
-adrenergic antagonist phentolamine (3 mg kg-1).
After the injection of phentolamine, blood samples were taken to determine
catecholamine concentration in a subset of embryos. Blood samples
(approximately 100 µl) were collected by allowing blood to flow freely from
the arterial catheter into a 500 µl Eppendorf collection tube.
Catecholamine blood samples were mixed with 5 µl of an EGTA/glutathione
solution (0.2 mol l-1/0.2 mol l-1) to prevent
catecholamine oxidation and immediately centrifuged at 13 700
g for 5 min. High-performance liquid chromatography (HPLC)
analysis of plasma catecholamines was carried out as previously described by
Fritsche and Nilsson (1990).
Samples were maintained at -70°C until analysis was carried out (within
one month). These embryos were then euthanized with an intravenous injection
of sodium pentobarbital (50 mg kg-1). All embryos were then weighed
to the nearest 1.0 g todetermine wet mass of the animals
(Table 1).
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Calculations and statistics
Non-parametric tests were conducted on the data due to small sample sizes
and the comparison of percentage changes. A Wilcoxon non-parametric test was
used to assess statistical significance of the responses to treatments
(hypoxia and drug administration; atropine, propranolol and phentolamine) at
each stage of incubation studied in the emu. A MannWhitney U
test on the percentage change in heart rate (fH) and mean
arterial pressure () following each
treatment was used to determine differences in responses between developmental
intervals. Since repeated tests were carried out, thereby using the same data
more than once, the fiduciary limit (P<0.05) was
Bonferroni-corrected according to the number of times (2-3 times) each data
set was used (thereby comparing 60%I to 70%I, 70%I to 80%I, 70%I to 90%I and
80%I to 90%I). This procedure was conducted to reduce the possibility of
incorrectly finding a difference between incubation groups, i.e. a type 1
error. A MannWhitney U test was also conducted on the
catecholamine levels using the same correction described above. The limited
sample size prevented comparisons of the data acquired for the 60%I embryos.
All data are shown as means ± 1 S.E.M.
A fiduciary level of P<0.05 was used. Statistical analysis was
conducted using Statistica (Statsoft, version 5.0).
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Results |
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Hypoxic responses
Hypoxic exposure (both 10% and 15% O2) significantly decreased
fH at 60%I but had no effect from 70%I to 80%I. At 90%I,
10% O2 caused a significant increase in fH
(Fig. 2A,C). The fall in
fH at 60%I was accompanied by a reduction in
a during exposure to 15% and 10%
O2 (Fig. 2B,D). A
significant decrease in
a was also
recorded during 10% O2 in 70%I embryos and during 15% O2
in 80%I embryos (Fig. 2B,D).
However, hypoxia (10% and 15%) had no effect on
a in embryonic emus that had reached
90%I (Fig. 2B,D).
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Autonomic blockade response
Fig. 3 represents the
response of fH and
a of an embryonic emu at 90%I to
injections of cholinergic and adrenergic receptor antagonists. Following
cholinergic blockade, fH rose while
a was unchanged. ß-adrenergic
receptor blockade then caused a decrease in fH and an
increase in
a.
-adrenergic
receptor blockade caused both fH and
to fall
(Fig 3A,B).
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At all incubation ages tested, fH significantly
increased following cholinergic blockade: 70%I (increase of 12±3 beats
min-1), 80%I (increase of 20±3 beats min-1) and
with the greatest increase at 90%I (increase of 50±3 beats
min-1) (Fig. 4A). Cholinergic blockade had no effect on
a during this period
(Fig. 4B).
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ß-adrenergic blockade with propranolol, following cholinergic
blockade, caused changes in fH and
a at each measured age of emu
development (Fig. 5A,B).
Propranolol elicited a bradycardia that was significantly weaker at 70%I (-33
beats min-1) compared with at 80%I (-43 beats min-1) and
90%I (-46 beats min-1) (Fig.
5A). Propranolol had the opposite effect on arterial pressure,
which increased at 70%I (0.15 kPa), 80%I (0.3 kPa) and 90%I (0.9 kPa)
(Fig. 5B). But although the
absolute change in pressure caused by propranolol appeared greatest in the
90%I age group, the change in pressure relative to the resting arterial
pressure for each age group was constant.
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Injection of the non-specific -adrenergic blocker phentolamine,
after muscarinic and ß-adrenergic blockade, produced a significant
decrease in fH and
a
(Fig. 3A,B). Phentolamine
decreased fH by a similar amount in embryos at 70%I (-19
beats min-1), 80%I (-28 beats min-1) and 90%I (-25 beats
min-1) (Fig. 5C).
Phentolamine injection also caused a significant decrease in
a at 70%I (-0.35 kPa), 80%I (-0.60
kPa) and 90%I (-1.50 kPa) (Fig.
5D). Again, while the absolute change in pressure caused by
phentolamine appeared greatest in the 90%I age group, the change in pressure
relative to the resting arterial pressure for each age group was constant.
Changes in circulating catecholamines
Plasma concentrations of norepinephrine increased progressively with
embryonic development to a maximum of 80.0±21.0 ng ml-1 at
90%I (Table 2). The maximum
epinephrine level was 53±24 ng ml-1 at 90%I, but the levels
did not differ statistically during incubation
(Table 2).
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Discussion |
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Critique of the experimental protocol
The heart rate and arterial pressure responses to hypoxia and reaction to
autonomic antagonists were conducted in all embryos tested at 70%I, 80%I and
90%I. A recovery period between the hypoxic exposures and autonomic blockade
allowed blood chemistry [lactate, pH, oxygen partial pressure
(PO) and carbon dioxide partial pressure
(PCO)] changes associated with hypoxia to return to
control values. The recovery period (1.5 h) was based on blood chemistry
recovery profiles following 5 min hypoxic exposures (15% and 10%
O2) in embryonic white leghorn chickens. Following each hypoxic
exposure in embryonic white leghorn chickens, blood parameters returned to
near control values within 1 h (D. A. Crossley, unpublished data). Therefore,
the time period allowed prior to the initiation of the autonomic blockade
series (1.5 h) in embryonic emus was probably sufficient to allow blood
chemistry to return to control levels.
The validity of utilizing a 5 min exposure to assess the cardiovascular response of embryonic emus also must be addressed from two views. The first is that the 5 min period would not allow the PO to reach the desired level within the experimental chamber. The second is that the exposure period was sufficient to elicit a transient cardiovascular response only. While both of these issues are acknowledged, the flow rate of air into the chambers was sufficient to turn over the volume of gas to the new PO level in approximately 30 s. This was independently verified prior to each study (D. A. Crossley et al., unpublished data). Therefore, the chamber that the embryo was in reached the desired PO within the first minute. A 5 min exposure could also be questioned as insufficient in terms of allowing an equilibrium cardiovascular state to be reached by the embryo. The goal of these experiments was to determine the acute cardiovascular response to hypoxia in embryonic emus in an effort to study the cardiovascular chemoreflex and compare it with the response of embryonic white leghorn chickens. The changes in the acute hypoxic response (Fig. 2) and differences from that of the white leghorn chicken embryos indicates that a 5 min period was sufficient to assess the developmental and species response differences to acute hypoxia.
Each of the autonomic blockade agents in the study was given to each embryo
in series (atropine, propranolol and then phentolamine). This method of study
has been used successfully in embryonic chickens
(Girard, 1973;
Tazawa et al., 1992
;
Crossley and Altimiras, 2000
),
embryonic alligators (D. A. Crossley et al., unpublished data) and larval
bullfrogs (Burggren and Doyle,
1986
) to determine the presence and strength of cardiovascular
autonomic tone during ontogeny. Therefore, this method was chosen to allow
comparison with existing literature for other species. However, there is a
potential for the cardiovascular responses to differ based on the order of
drug injection, and this should be considered in all studies.
Drug injection volumes might have induced a hypervolemia in the developing
emu. Blood volumes in embryonic emus (Table
1) were estimated using the known relationship between embryonic
mass and blood volume in developing chickens
(Crossley and Altimiras, 2000).
Each drug injection and flush volume totaled maximally less than 6% of the
estimated blood volume in the earliest embryos (60%) studied. Estimated blood
volume of the developing emu was calculated as 7% of the embryonic wet mass
(Table 1) based on previously
determined values for embryonic emus (D. A. Crossley, unpublished data).
Therefore, the maximal volume of fluid added to the embryo during the length
of the study was less than 17% of the estimated blood volume. Using 7% of
embryonic mass as an estimation of blood volume may underestimate the actual
volume by as much as 25% (based on chickens;
Romanoff, 1967
). Therefore,
the volume of each injection probably did not alter cardiovascular function
(Crossley and Altimiras,
2000
).
Developmental changes: pressure and heart rate
Over the interval of emu development tested (60%I to 90%I), resting
a increased progressively and was
significant only between 60%I and 70%I
(Fig. 1B). Routine
fH gradually decreased over this time frame, possibly due
to the onset and progressive expression of parasympathetic cardiac regulation
(Fig. 1A). This speculation is
based on the observation that atropine produced progressively larger increases
in fH after 60%I (Fig.
4A). If this explanation is correct, ongoing maturation of the
cholinergic inhibitory system seems likely to continue after hatching because
fH was elevated (3.5 times higher) compared with adult
emus (Grubb et al., 1983
).
While this finding could be attributed to mass changes alone, maturation of
cholinergic receptor-mediated regulation may also contribute.
Cardiovascular response to hypoxia
60%I embryonic emus responded to acute hypoxia (15% or 10% O2)
with a bradycardia, while 90%I embryos exhibited a tachycardia during 10%
O2 exposure (Fig.
2). This reversal suggests that at 90%I embryonic emus may possess
central or reflexive regulation of fH. The sensory
components that detect changes in PaO may be functional
and the effector systems altering fH are active. If the
components of the fH chemoreflex are intact at 90%I, as
the data indicate, then the hypoxic tachycardia was probably induced by a
reduction of cholinergic depression and/or an increase in adrenergic
stimulation of fH. The latter has been demonstrated in
other avian species and could be largely responsible for the response
documented in this study (Giussani et al.,
1994; Dragon et al.,
1996
; Mulder et al.,
2000
; Crossley et al.,
2003
).
The change in the a response to
hypoxia over development suggests that a hypoxic reflexive becomes operational
during emu incubation. This suggestion is based on the pressure response
change from hypotension during 10% O2 (60%I and 70%I) to no change
in pressure in embryos at 80%I and 90%I
(Fig. 2B,D). An alternative
explanation for no change in
a in the
80%I and 90%I groups may be a reduced sensitivity to hypoxia. However, since
10% O2 was sufficient to induce changes in fH
at 90%I, it is likely that the embryos were sensitive to hypoxia at this point
(Fig. 2B).
Regulation of heart rate and blood pressure
Adrenergic tone on heart rate
In addition to a cholinergic tone, embryonic emus also had an important
ß-adrenergic receptor-mediated tone on fH
(Fig. 5A). This
ß-adrenergic tone rose between 70%I and 80%I
(Fig. 5A). In chicken embryos,
there is an increase in ß-adrenergic tone on fH that
has been partially attributed to an increased stimulation by elevated levels
of circulating catecholamines (Dragon et
al., 1996; Crossley and
Altimiras, 2000
; Mulder et
al., 2000
). Circulating levels of norepinephrine also increased
from 70%I to 80%I (Table 2) and
may account for the increase in ß-adrenergic receptor-mediated tone on
fH during this period.
Unlike the fH responses to ß-blockade during emu
development, the reaction to -blockade with phentolamine was constant
during the period of incubation studied (70%I to 90%I;
Fig. 5C). The change in
fH after
-blockade may be derived from a secondary
response to the accompanied vasodilation, which would increase filling time
and reduce fH. However, given the excitatory action of
cardiac
-receptors in adult, as well as neonatal and fetal, mammals
(Cohen, 1986
), direct
-receptor excitatory tone on fH may be present in
embryonic emus.
Adrenergic tone on pressure
Embryonic emus possessed a constant ß-adrenergic tone that decreased
a during incubation when corrected
for the developmental changes in resting arterial pressure
(Fig. 5B). These embryos also
possessed a constant
-adrenergic tone that increased
a
(Fig. 5D). In combination,
these findings indicate that the embryonic emu relies on both
- and
ß-adrenergic receptor types to maintain
a during the window of development
studied.
Comparison with white leghorn chickens
The development of cardiovascular regulation in birds has been studied
primarily in the domestic chicken. The present study reveals some distinct
differences between emus and chickens.
In emus, fH initially falls (between 60%I and 70%I)
then remains relatively constant (Fig.
1A), while embryonic chickens maintain fH over
the same period of incubation (Girard,
1973; Tazawa,
1981
). This difference may be attributed to the presence of a
cholinergic tone on fH in emus
(Fig. 4A) and its absence in
chickens of the white leghorn strain
(Crossley and Altimiras, 2000
).
Furthermore, the hypoxic tachycardia of late embryonic emus (90%I;
Fig. 2C) differs from the
bradycardia in white leghorn embryos. Again, this may also be attributed to
the differences in cholinergic tone between the species.
Differences in the mechanisms of regulation were also evident between
embryonic emus and chickens of the white leghorn strain. The most prominent
difference between these avian embryos was the presence of cholinergic tone on
fH in embryonic emus (present study) and the absence in
white leghorn chickens (Crossley and
Altimiras, 2000). Given our understanding of these two models of
avian development, the reason for the appearance of cholinergic
fH tone in emus and its absence in white leghorn chickens
is unclear. This comparison illustrates that the cardiovascular regulatory
mechanisms delineated in white leghorn chickens during development may be
different in other bird species.
Conclusions
During embryonic development, emu cardiovascular regulation mechanisms are
more advanced that those of embryonic white leghorn chickens over a similar
window of development. At 70%I, a clear cholinergic receptor-mediated tone
(regulating fH) and adrenergic receptor-mediated tone
(regulating fH and
a) are functional in the emu. As the
emu matures, the embryo relies on an increasing cholinergic tone on
fH and a ß-adrenergic control of
fH. Furthermore, embryonic emus exhibit temporal changes
in the cardiovascular responses to 10% O2. Therefore, maturation of
cardiovascular regulatory mechanisms should be expected to differ between
avian species during development.
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Acknowledgments |
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References |
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