The use of power spectral analysis to determine cardiorespiratory control in the short-horned sculpin Myoxocephalus scorpius
Department of Physiology and School of Biological Sciences, University of Birmingham, B15 2TT, UK
* Author for correspondence (e-mail: s.egginton{at}bham.ac.uk)
Accepted 4 November 2003
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Summary |
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Key words: oxygen consumption, teleost fish, Myoxocephalus scorpius, electrocardiogram, heart rate variability, vagal control, power spectral analysis
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Introduction |
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Direct measurement of cardiac output in active teleost fish species has
shown that the increase during swimming in sub-carangiform mode is chiefly
dependent on increases in stroke volume (Vs) rather than
heart rate (fH)
(Stevens and Randall, 1967;
Farrell, 1981
). The resultant
increase in pulsatility may increase lamellar recruitment, thereby sustaining
oxygen uptake (Farrell et al.,
1980
; Altimiras et al.,
1995
). However, Lucas
(1994
) found
fH to be a useful indicator of changes in metabolic rate
in Atlantic salmon Salmo salar at low swimming speeds, and Armstrong
(1986
) found that
fH provided a precise estimate of apparent specific
dynamic action (SDA) and meal size in pike Esox lucius when rate of
oxygen consumption
(
) was
standardised between fish. The importance of fH modulation
as a response to changes in oxygen supply or demand is also emphasised by a
characteristic bradycardic response when fish are exposed to hypoxic
conditions (Axelsson et al.,
1990
) or startled (Priede,
1974
). It may therefore be concluded that in resting fish or at
low swimming speeds, reflex changes in heart rate may be a rapid and sensitive
method of regulating cardiac output. Although the hypoxic bradycardia may be
compensated by increased stroke volume to preserve cardiac output, reflex
slowing of the heart is initiated by oxygen-sensitive chemoreceptors and is
largely under vagal control (Taylor,
1992
).
Histological and pharmacological studies have shown that the teleost heart
receives inhibitory innervation via the vagus (Xth cranial) nerve
that stimulates muscarinic, cholinoceptors on the cardiac ganglion and
myocardium (Laurent et al.,
1983). This regulates heart rate by setting a resting vagal tonus
(Axelsson et al., 1987
). There
is less clear evidence for adrenergic excitatory control via
sympathetic innervation of the teleost heart, although adrenergic nerve fibres
have been found in a few species and endogenous or circulating catecholamines
may exert a stimulatory affect on cardiac function. However, the relative
importance of these neural and humoral influences remains unclear
(Taylor et al., 1999
). Most
studies of this dual mechanism affecting heart rate have been conducted by
pharmacological blockade (Axelsson et al.,
1987
; Axelsson,
1988
; Altimiras et al.,
1997
). Atropine, a muscarinic receptor antagonist introduced
via cannulae, blocks cholinergic post-synaptic receptors, thus
reducing inhibitory parasympathetic drive resulting in a tachycardia.
Alternatively, ß-adrenoceptor antagonists such as propranolol or sotalol
competitively antagonise endogenous catecholamines and result in a
bradycardia. A few studies have observed the degree of vagal tone by
sectioning the nerve, and observed that the apparent beat-to-beat variation
present in intact fish was greatly reduced in vagotomised animals
(Priede, 1974
;
Altimiras et al., 1995
).
A fluctuation in the instantaneous heart rate on a beat-to-beat basis is
termed the heart rate variability signal (HRVS). Simple measurement of the
variation in normal beat interval taken from electrocardiogram (e.c.g.)
records (RR interval) in the time domain (standard deviation of
RR interval, SDRR) provides an index of overall heart rate variability
(HRV), but as a measure of changes in sympatho-vagal balance it is limited.
Frequency domain analysis is preferable, whereby mathematical dissection of
the signal reveals the amplitude of the oscillatory components hidden in the
variability of the e.c.g. signal as distinct peaks in spectral amplitude
(Malik, 1996). In mammalian
systems, Akselrod (1981) demonstrated that random process analysis of the HRVS
provided a sensitive, quantitative measure of rapidly reacting cardiovascular
control mechanisms, revealing three distinct components. These were the high
frequency component (0.30.4 Hz) associated with central respiratory
drive and solely vagally mediated, a mid (0.10.3 Hz) and low
(0.070.1 Hz) frequency component associated with blood pressure control
systems, and thermal vasomotor activity, respectively. Both of these latter
components had a mixture of sympathetic and vagal contributions
(Bootsma et al., 1994
). In the
reptile (Gallotia gallot) two separate peaks were revealed (Gonzalaz
and Porcell, 1988). Although these did not show the beat-to-beat
cardiorespiratory synchrony present in mammals, they were thought to
correspond to cutaneous vasomotor thermoregulation (0.032 Hz at 20°C to
0.07 Hz at 35°C) and endogenous pressure vasomotor activities (0.039 Hz at
20°C to 0.1 Hz at 35°C).
The few power spectral studies undertaken in fish have found interesting
interspecific differences. A dual spectral peak was observed for rainbow trout
(DeVera and Priede, 1991) and
sea bream Sparus aurata (Altimiras
et al., 1995
), whilst only a single main component was found in
pike, brown trout (Armstrong et al.,
1988
), ballen wrasse (Altimiras
et al., 1995
) and Atlantic salmon
(Altimiras et al., 1996
). This
disparity between species in HRV may reflect differences in the degree of
cholinergic inhibition and/or adrenergic excitation, with a predominance of
vagal control generating high frequency components in the HRVS
(Taylor et al., 1999
). It has
previously been shown, through pharmacological blockade and nerve transection,
that the degree of cardiac vagal or adrenergic tone on the fish heart varies
greatly between species, and with activity levels, temperature or oxygen level
within species (Taylor, 1992
).
Consequently, a systematic study of variations in tonic control of the heart
in fishes, at a range of temperatures and activity levels, may serve to
clarify the mechanisms of beat-to-beat control of the fish heart.
In this study we explored the use of power spectral analysis further in
examining the neural influences on the teleost heart, adapting the general
methodology and application of spectral techniques for teleost e.c.g. as
described previously (Altimiras et al.,
1994,
1995
;
Altimiras, 1999
). The
short-horned sculpin Myoxocephalus scorpius was chosen as an example
of a labriform swimmer of limited aerobic scope associated with sit-and-wait
predation. It has not previously been examined by spectral analysis
techniques, although pharmacological estimates of cholinergic and adrenergic
control (Axelsson et al.,
1987
) facilitated this comparative study.
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Materials and methods |
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Instantaneous heart rate
Two 30 cm lengths of 0.02 mm diameter, multi-strand Teflon coated stainless
steel wire (A-M Systems, USA) were utilised as e.c.g. recording electrodes. A
2 mm section of insulation was removed from the end of the wire, and this was
implanted into anaesthetised (MS222 0.5 g l-1) fish (N=7)
by inserting a 0.5 mmx25 mm hypodermic needle, carrying the wire, just
behind each pectoral fin and advancing it a few millimetres underneath the
pectoral girdle. The needle was then extracted and each wire was anchored into
the pharyngeal skeleton. The electrodes were inserted at different
rostrocaudal positions either side of the heart to maximise the gain
from the vectored electrical signal. A loop was put into each wire close to
the site of body entry, and secured to the fish underside by a single suture
(7/0 braided silk); both wires were brought around the left flank of the fish,
twisted together and sutured in front of the dorsal fin. The whole surgical
procedure took <8 min, after which the fish recovered in the holding
aquarium and was then placed into a respirometry chamber (see below). The fish
was unrestrained within the respirometry chamber. The electrode wires were
passed up through an extended chimney in the lid of the chamber, such that
recordings of e.c.g. (PowerLab 4e with animal bio-amp) could be taken with
minimal disturbance to the fish (Fig.
1). A 30 min e.c.g. recording was taken 10 min after surgery, and
thereafter every 24 h for 144 h.
|
Identification of the cardiac vagus
Two fish were anaesthetised in 0.5 g l-1 MS222, placed ventral
side up on a small operating table and the gills irrigated with aerated,
seawater at 8°C, pH 8.1, containing 0.1 g l-1 MS222. A
polyethylene cannula (PE50, Smiths Medical, Hythe, UK) was inserted into the
ventral aorta via a third gill arch using the method described by
Axelsson and Fritsche (1994).
Once a regular blood pressure trace was established a small incision was made
through the body wall along the line of the opercular flap to expose the
underlying vagal nerve trunk. Hook electrodes were placed around the putative
cardiac branch of the vagus nerve, identified by dissection, and the branch
was stimulated for a 3 s period using a Biostimulator 1000 (Searle,
Birmingham, UK) with pulses of 0.4 ms duration at an intensity of 3 V and a
frequency of 1.1 Hz. A PowerLab 4e, bridge amplifier (AD Instruments,
Chalgrove, UK) and a Capto SP 844 pressure transducer (AD Instruments) was
used for detection and amplification of the signal, and the resulting trace
was recorded with Chart 4.1.2 software (ADI) for the PC. Positive
identification of the branch was confirmed by cessation of the heartbeat,
accompanied by a drop in ventral aortic pressure
(Fig. 2).
|
Surgical intervention
After the final e.c.g recording five of the fish were anaesthetised (MS222,
0.5 g l-1) and underwent bilateral cardiac vagotomy. A small
incision was made behind the fourth gill arch and the cardiac branch of the
vagus (identified previously) was exposed on either side. A 1 mm section was
removed using bow scissors, and a single suture used to close each wound. The
animal was replaced into the holding chamber. Recordings of e.c.g. were made
as previously for a 144 h period. To validate the vagotomy procedure two fish
underwent sham operations where the vagal nerves were exposed but left
intact.
Pharmacological intervention
Relative parasympathetic (vagal), cholinergic (chol.) or sympathetic,
adrenergic (adr.) tonus were calculated as follows:
![]() | (1) |
![]() | (2) |
In this study fish were both rested and undisturbed using our method of
recording, such that the degree of adrenergic tonus is assumed to be minimal.
In addition, data from other species has shown very similar calculated values
for vagal tone on the heart determined after either vagotomy or
pharmacological blockade (Taylor et al.,
1977; Taylor,
1992
)
Respirometry
Rate of oxygen consumption
() was
measured to indicate aerobic metabolic rate in fish whilst undertaking
fH measurements. Six respirometry chambers were
constructed out of airtight PVC boxes (25 cmx25 cmx12 cm, volume
6.5 l). Each chamber was supplied with filtered aerated seawater, which was
circulated by a small submersible pump (Interpet, Lancaster, UK). Each chamber
had a 2.5 h closed period and a 25 min flush period at a flow rate of 100 l
h-1. The five fish with e.c.g. electrodes attached were monitored
simultaneously, leaving one chamber to determine background (bacterial)
. Water
was sampled from each chamber by a rotor valve (Omnifit, Cambridge, UK) every
2.5 min and the water passed through a flow cell containing a 1302
Strathkelvin (Glasgow, UK) oxygen probe, monitored by a 781 Strathkelvin meter
and logged via a channel interface (949 Strathkelvin) onto a PC
computer. Using this method water oxygen tension within each chamber could be
sampled every 15 min. Each fish was starved for 96 h before commencement of
the experiment, and once the fish was placed inside the chamber oxygen
measurement commenced immediately and ran continuously for 144 h.
Statistical analysis
Geometrical analysis of e.c.g. parameters was carried out on periods of
recording selected as having no ectopic beats or signal artefacts. Student's
two-tailed t-test for non-paired samples and the F-test were
utilised, and data subjected to log transformation when distributions were not
normally distributed. Suitability of line fits were tested by analysis of
variance (ANOVA) and comparison of fitted regression lines by analysis of
covariance (ANCOVA). Frequency domain analysis methods have been described
previously (Altimiras et al.,
1994; Altimiras,
1999
), with the fH data being converted to
RR interval (ms). A dataset of 256 consecutive RR intervals was
selected in an e.c.g. trace and tested for stationarity using the run test;
subtracting the mean heart rate normalised the data. A Fast Fourier
Transformation was then applied using a Hanning window to minimise spectral
leakage, and the resulting output plotted graphically.
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Results |
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|
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Power spectral analysis of the instantaneous heartbeat from individual sculpin, undisturbed for 120 h after surgery (Fig. 6), revealed a dual peaked spectrum. The frequency of the larger spectral amplitude peak was 0.018 Hz and the smaller spectral peak 0.048 Hz, with the power at both peaks increasing with time after surgery (Fig. 5). All fish measured showed an abolition of beat-to-beat variability immediately post-surgery, with an increase in spectral power at mean frequencies of 0.0180.2 Hz and 0.0460.5 Hz (Fig. 6), on recovery. This gives an oscillatory period for the low frequency of 55.550 s and the higher frequency peak of 21.720 s. Using mean resting RR interval (2.9 s), this corresponds to an oscillatory period in heart rate approximately every 7 and 17 beats. This corroborates the geometric statistics, showing that there is very little HRV after surgery, and implies that on recovery this variability has an oscillating waveform of fixed periodicity.
|
|
The effects of bilateral vagotomy on the instantaneous heart rate were dramatic, with an elevation in fH of approximately 50% after 144 h rest and abolition of the beat-to-beat variability evident in the RR interval (Fig. 7). Immediately after vagotomy both spectral peaks were abolished, and spectra showed a similar pattern to that previously obtained in a fish stressed due to anaesthesia and placement of electrodes. The beat-to-beat interval did increase with time post-surgery, increasing by 25% 144 h later. This was still 25% higher than the RR interval of the intact fish, and no increase in spectral amplitude developed with time after surgery. The two sham-operated animals showed an increase in RR interval and SDRR with time post surgery, and after 144 h a dual spectral peak was evident in the fH trace, similar to that seen for intact fish.
|
The relative cholinergic and adrenergic tonus on the heart was estimated
from recordings of heart rate taken from stressed, resting and vagotomised
fish. Cholinergic tonus was 41%, calculated using
Equation 1, and adrenergic tonus
was
20%, using Equation
2.
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Discussion |
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Some of the contrasts between the results of this study and earlier work
may have arisen as most studies have looked at variations in
fH and
whilst
exercising the fish in a flume. M. scorpius is a labriform swimmer
and would not readily swim in a steady flow. Consequently, the current
observations were made not on an exercising fish. Instead a range of
fH and
was
measured whilst metabolic rate recovered from its elevated rate following the
disturbance of surgical and handling stress. Few studies have taken this
approach so it is not clear whether the same would be true for other species,
although Armstrong (1986
) found
a good correlation between fH and
in
pike Esox lucius after feeding. Similarities between M.
scorpius and E. lucius may reflect the similar mode of life of
the two species. While one is marine and one freshwater, both species are not
subject to prolonged periods of activity, but are sit-and-wait predators.
Therefore, elevated oxygen consumption will tend to be associated with
digestion of food (specific dynamic action, SDA) or recovery from exhaustive
exercise. Heart rate changes that can be up- or down-regulated quickly may be
preferable for this mode of life rather than changes in stroke volume, which
may be little affected by cholinergic innervation but more by an increase in
venous return (Starling relationship) in the exercising animal due to muscle
pumping (Lillywhite et al.,
1999
).
Steffensen et al. (1994) observed an
of
1.4±0.27 mmol O2 kg-1 h-1 at 4.5°C
in M. scorpius, which is similar to the value recorded from our
recently disturbed fish. Thus, these high values may have been the result of
handling stress, as their fish were measured immediately after placement in
the respirometry chamber. In contrast, Johnston and Battram
(1993
) left their fish for a
minimum of 72 h before recording O2 consumption and obtained a mean
of
0.82± 0.044 mmol O2 kg-1 h-1 at
5°C, which was almost identical to our
values
for resting M. scorpius of 0.78±0.07 mmol O2
kg-1 h-1 at 8°C. Results in the present study show
that M. scorpius reduced
by
approximately 60% after handling stress, and only stabilized 96 h
posthandling. Although
measurements in the present study at 72 h were similar to those of Johnston
and Battram (1993
) our fish
were measured at a higher temperature, and it would be expected that this
would increase oxygen demand. Clearly, we had achieved resting levels in our
fish, despite the fact that they had undergone anaesthesia and surgery to
insert e.c.g. electrodes prior to placement in respirometer chamber, and this
may have induced a greater stress response then placement in a chamber
alone.
Immediately after bilateral vagotomy the fH parameters (a range of 2932 beats min-1 among individual fish) were similar to those observed in post-surgery intact fish (2832 beats min-1). Over the next 144 h, consistent with withdrawal of sympathetic tone, fH did decrease slightly (2425 beats min-1) although not to resting levels observed previously in intact animals (1821 beats min-1). No distinguishable increase in HRV was detectable with time post-vagotomy, suggesting that cholinergic control was the major influence on the cardiac pacemaker generating HRV in intact M. scorpius. The permanent abolition of HRV after cardiac vagotomy indicates that cholinergic innervation of the cardiac pacemaker regulates fH on a beat-to-beat basis. Confirmation of this was obtained by power spectral analysis (PSA) of the instantaneous heart rate. When expressed graphically it is evident that immediately after surgery no peaks were present, implying that, in intact fish, extrinsic neural influences were having little effect on fH, which was determined either by intrinsic cardiac pacemaker activity, or an adrenergic tachycardia influenced by circulating catecholamines. As mean fH decreased with time after surgery, two separate peaks became apparent and increased in amplitude over time, reaching a maximum 120144 h after surgery. The complete absence of HRV post-surgery implies that there was no cardiac vagal tone on the heart and the progressive return of HRV, illustrated in Fig. 5, may reflect its re-establishment.
The two peaks observed for M. scorpius (0.02 Hz and 0.048 Hz) were
fourfold lower in frequency compared to those observed in rainbow trout at
10°C (0.084 Hz and 0.171 Hz) (DeVera
and Priede, 1991). Thus, rainbow trout at 10°C have an
oscillatory period in fH of 11.9 s and 5.8 s, which is
45 times shorter than M. scorpius (50 s and 21 s). However,
when oscillatory period was calculated per number of heart beats, the higher
fH observed in rainbow trout (52.4 beats min-1)
means an oscillatory period every 10.4 and 5.2 beats, which is comparable to
the oscillatory period of 17.2 and 7.1 beats, observed for M.
scorpius. A dual spectral peak (0.13 Hz and 0.25 Hz) has also been
observed in the sea bream (Altimiras et
al., 1995
), whilst only a single spectral peak was found in pike
(0.014 Hz) and brown trout (0.027 Hz)
(Armstrong et al., 1988
). In
these studies mean fH was not given, so that the
oscillations cannot be related to number of beats. Altimiras et al.
(1996
) found that Atlantic
salmon males (0.027 Hz) had a lower frequency spectral peak compared to
females (0.065 Hz), corresponding to an oscillatory period every 13.8 beats
for male and 5.8 beats for female salmon. All fish used in the present study
were confirmed by dissection to be female, and the high frequency peak
observed by PSA for M. scorpius of an oscillatory frequency every 7.1
heart beats was close to that of the female Atlantic salmon. Both peaks fall
in the range that it has been suggested is linked to blood pressure control
(Altimiras et al., 1996
).
However, visual observations of ventilation rates in sculpin gave mean values
of 2.1±0.08 s immediately after surgery, and 3.33±0.2 s from
resting fish, values that are 7 times less than the rate of the high frequency
peak in the HRV signal. This peak in the PSA therefore probably does not
represent centrally generated respiration related activity, analogous to the
respiratory sinus arrhythmia described in recordings of HRV in mammals
(Taylor et al., 1999
), and
sculpin are more like reptiles, which also do not show cardiorespiratory
synchrony (Gonzalez and Porcell,
1988
).
The low-frequency peak in the HRVS may correspond to the adrenergic effects
described by DeVera and Priede
(1991). In intact fish,
adrenaline increases systemic resistance and decreases gill resistance. The
heart may show reflex responses to these changes, or direct responses of the
myocardium to catecholamines, generating the low frequency peak in the HRVS.
In the sculpin both peaks are abolished by vagotomy so that control seems to
be largely cholinergic. However, the vagus may contain mixed cholinergic and
adrenergic fibres in sculpin, similar to the vagosympathetic trunk observed in
other species of teleost fishes (Gibbons,
1994
).
This study highlights the importance of cholinergic innervation of the
heart in M. scorpius. Stimulation of the vagus caused cardiac
inhibition similar to that observed in plaice
(Cobb and Santer, 1972) and
carp (Saito, 1973
), suggesting
that cholinergic innervation has a major influence on the cardiac pacemaker.
In addition, sectioning of the vagus nerve led to an increase in
fH in resting fish and was previously shown to abolish the
approach reflex in fish, i.e. the period of bradycardia observed in intact
fish when disturbed (Priede,
1974
). Furthermore, the beat-to-beat variability observable in the
instantaneous fH of intact fish was abolished after
vagotomy. Calculation of relative cholinergic and adrenergic tonus on the
heart suggested that cholinergic tonus exerts a greater influence on
fH than adrenergic control. This differs from the results
of Axelsson et al. (1987
),
which put a relative adrenergic and cholinergic tonus for M. scorpius
of 25.9 and 11.9%, respectively. However, those observations were made with an
intrinsic heart rate (
43 beats min-1) that was below the
resting HR (
48 beats min-1), in contrast to the present study
where a resting fH as low as 18 beats min-1 was
obtainable in M. scorpius if left for >72 h, while the degree of
cholinergic tonus as measured by PSA did not reach its maximum until
120
h post surgery. As M. scorpius was only left for 18 h in the
pharmacological study by Axelsson et al.
(1987
), they presumably would
not have measured resting fH.
The degree of cholinergic tonus calculated in the present study assumed that at rest adrenergic tonus was minimal and cholinergic control maximum, whilst immediately after handling cholinergic control was minimal and adrenergic tone maximum. Previous estimates of relative tonus using pharmacological blockade make assumptions that the fH of a resting fish also is exhibiting maximum cholinergic tonus and minimal adrenergic tonus. However, methods used in this study allow the measurement of fH from animals undisturbed for a much longer time span than pharmacological studies allow, while the use of PSA to estimate HRV is crucial in determining a true resting fH and consequently relative tonus. Although we cannot assess adrenergic tonus directly, the reduction in fH 144 h after vagotomy, whilst HRV (generated by parasympathetic, vagal input) did not recover, suggests this reduction in fH is solely due to withdrawal of adrenergic tonus. We accept that the level of adrenergic tonus after surgery may not be the maximum possible for the fish and therefore our estimates of relative cholinergic and adrenergic tone must be regarded as maximum and minimum values, respectively.
In summary, the stress of anaesthesia, handling, minor surgery and
placement of sculpin into respirometers, caused an elevation in both
fH and
values. The increase in fH was accompanied by a loss of
HRV and paralleled the effects of cardiac vagotomy, indicating that both are
the consequence of a reduction in an inhibitory cholinergic influence on the
cardiac pacemaker. Mean fH was correlated with mean
in
resting sculpin, and was fine-tuned on a beat-to-beat basis by vagal control,
although the low frequency rate of oscillations in M. scorpius
fH were not correlated with ventilation rate.
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Acknowledgments |
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References |
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