Physiologisches Institut, Christian-Albrechts-Universität, 24098 Kiel, Germany
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ABSTRACT |
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Häbler, H.-J., T. Bartsch, and W. Jänig. Rhythmicity in single fiber postganglionic activity supplying the rat tail. The temporal pattern of ongoing sympathetic vasoconstrictor activity may play an important role for neurovascular transmission. Here we analyzed the activity of postganglionic fibers projecting into the ventral collector nerve of anesthetized and artificially ventilated vagotomized Wistar rats with respect to the presence of rhythmic firing under normocapnic conditions. Most of the fibers studied were likely vasoconstrictor and involved in thermoregulation. Accumulated histograms of sympathetic activity were produced synchronized with the electrocardiogram to detect cardiac rhythmicity, with phrenic nerve activity to detect modulation with the central respiratory cycle, and with tracheal pressure to uncover a reflex modulation associated with artificial ventilation. Sympathetic activity, phrenic activity, and tracheal pressure also were examined by spectral analysis and autocorrelation to detect rhythmicities distinct from respiration. Twenty-seven filaments containing two to seven fibers with spontaneous activity and 51 single fibers were analyzed. Ongoing activity was 1.12 ± 0.65 imp/s (mean ± SD, n = 51); conduction velocity was 0.62 ± 0.06 m/s (n = 30). Cardiac rhythmicity in sympathetic activity was weak (46.2 ± 16.4%). The dominant rhythm in the activity of 19/27 few-fiber preparations and 37/51 single fibers corresponded to the central respiratory cycle. The pattern consisted of an inhibition during inspiration and an activation in expiration. In 10/19 few-fiber preparations and 21/37 single fibers of this group, there was also a concomitant, less prominent rhythm related to artificial ventilation. By contrast, 8/27 few-fiber preparations and 11/51 single fibers exhibited a dominant pump-related modulation, whereas phrenic-related rhythmicity was subordinate. The dominant rhythm in the activity of two single fibers was related to neither central respiration nor artificial ventilation. We conclude that the ongoing activity of most postganglionic neurons supplying the rat tail is modulated by the central respiratory rhythm generator, suggesting that changes in respiratory drive may alter perfusion of the tail and therefore heat dissipation. Reflex modulation in parallel with artificial ventilation, independent of vagal afferents and possibly due to ventilatory changes of baroreceptor activity, is also an important source of rhythmicity in these neurons.
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INTRODUCTION |
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Sympathetic activity often exhibits a phasic
modulation with the same frequency as respiration (see
Häbler et al. 1994b). The function of this
modulation may be to convert primarily unpatterned activity into
bursts, which may facilitate neurovascular transmission (Nilsson
et al. 1985
; Pernow et al. 1989
; see also
Häbler et al. 1994b
). There is evidence that one
mechanism that is responsible for the respiratory modulation in
sympathetic activity is a direct interaction between neurons of the
respiratory network and neurons of the cardiovascular system at the
level of the medulla oblongata (Haselton and Guyenet
1989
; McAllen 1987
). This has led to the suggestion that a common cardio-respiratory neural network exists (Richter and Spyer 1990
) that controls both respiration
and the cardiovascular system in parallel. However, it can be shown
that there is some functional separation between these systems because they can be influenced independently by stimulation of arterial baroreceptors (Boczek-Funcke et al. 1991
), arterial
chemoreceptors (Koshiya and Guyenet 1996
), and laryngeal
afferents (Häbler et al. 1997
). An alternative
interpretation for the emergence of respiratory modulation in
sympathetic activity is that a central oscillator, which is under some
conditions entrained to respiration, generates sympathetic activity
(Barman and Gebber 1976
, 1981
).
The potential of afferent feedback to be involved in
respiration-related modulation of sympathetic activity is a matter of disagreement. There is evidence that the arterial baroreceptors that
encode the blood pressure waves associated with ventilation have a
powerful phasic influence on the ongoing discharge of neurons in the
lumbar and thoracic sympathetic outflow of the cat (see Häbler et al. 1994b). In the rat, these blood
pressure waves, although smaller, also translate into phasic
modulations of baroreceptor activity (Häbler et al.
1993
), and these probably elicit a ventilation-related modulation in some presympathetic barosensitive neurons of the rostral
ventrolateral medulla (Miyawaki et al. 1995
) and in
preganglionic neurons projecting to the superior cervical ganglion
(Häbler et al. 1996
).
Recently, making use of a focal recording technique, Johnson and
Gilbey (1996, 1998a
) reported that the dominant rhythm in the
activity of most postganglionic sympathetic neurons supplying the rat
tail artery and vein is not related to respiration but has a frequency
similar to but distinct from the latter. Furthermore none of the
postganglionic fibers studied exhibited rhythmicity associated with the
ventilation pump. The authors concluded that the dominant rhythm in
postganglionic neurons supplying the tail was not generated by the
respiratory network. However, in that study, only autocorrelation
analysis was performed in most cases, and the relationship of
sympathetic activity to the respiratory cycle was not considered in any
detail. Therefore it remained unclear whether, in addition to the
dominant rhythm, there was also respiratory modulation in the activity
of postganglionic neurons projecting to the tail.
The question whether or not the sympathetic innervation of a
thermoregulatory organ like the rat tail (Dawson and Keber
1979) is linked to respiration may be of specific functional
relevance because heat stress often is accompanied by increases of
respiratory drive (Cooper and Veale 1986
). Therefore the
aim of the present study was to analyze rhythmicity in sympathetic
fiber activity projecting in the ventral collector nerves. We used
several methods of analysis, namely, ventilation-pump- and
phrenic-triggered summation, spectral analysis (see Gootman and
Sica 1994
), and autocorrelation to detect rhythms related to
central respiration or related to the ventilation pump or rhythms
unrelated to respiration.
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METHODS |
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Animal maintenance
The experiments were conducted on 16 female Wistar rats (200-280 g), which were anesthetized with pentobarbital sodium (Nembutal, 60 mg/kg ip initially, maintenance by 10 mg/kg, dissolved in Tyrode's solution 1:3, intravenously every hour). A sufficient depth of anesthesia was judged from the absence of withdrawal reflexes in the unparalyzed state, from the absence of gross fluctuations of blood pressure and heart rate during muscular paralysis, and from a largely regular phrenic nerve discharge. Arterial blood pressure was monitored continuously through the cannulated femoral or ventral caudal artery. For administration of drugs and fluid, a catheter was placed in the jugular vein. The trachea also was cannulated. During surgery the animals breathed spontaneously. They were paralyzed (Pancuronium, Organon, 1 mg/kg iv initially, maintenance with 0.4 mg/kg when necessary) and artificially ventilated (for parameters see following text) before the recordings. At intervals we let muscular paralysis wear off and confirmed that withdrawal reflexes were absent. Blood gases were measured at intervals of 2-3 h (ABL 30, Radiometer, Copenhagen) and maintained within the following range: pH, 7.30-7.40; PCO2 35-50 mmHg; PO2 110-180 mmHg; base excess 0 ± 3 mM/l. The electrocardiogram (ECG) was monitored continuously in most experiments. Rectal temperature was kept close to 37°C by means of a servo-controlled heating blanket.
At the end of the experiments, the animals were killed under deep anesthesia by intravenous injections of a saturated solution of potassium chloride. All experiments had been approved by the local animal care committee of the state administration and were conducted in accordance with German Federal Law and with the National Institutes of Health Guide.
Nerve preparation and recording
In all experiments, the vagus nerves, the aortic depressor nerves and the superior laryngeal nerves were exposed bilaterally and cut. Central respiratory activity was monitored continuously by recording, with a pair of platinum hook electrodes, the activity of the left phrenic nerve (PHR), which was prepared in the neck and desheathed.
The ventral collector nerves were exposed about 10 cm distal to the
base of the tail, cut and desheathed. Filaments containing one to seven
spontaneously active postganglionic units were split from the nerves
using fine forceps. Sympathetic fiber activity was recorded with a
platinum electrode the indifferent electrode being connected to nearby
tissue. In five experiments, the left lumbar sympathetic trunk (LST)
was exposed using a retroperitoneal approach and placed on a pair of
platinum hook electrodes for electrical stimulation (single pulses,
supramaximal for C fibers, 0.5-ms duration, 0.2 Hz) between ganglia L2
and L3 (nomenclature after Baron et al. 1988) to
identify postganglionic neurons and to determine conduction velocities.
The nerves and exposed tissue were covered with warm paraffin oil in
pools made from skin flaps.
Artificial ventilation
While recording the animals were ventilated artificially with positive pressure (respirator RUS-1301, FMI, Egelsbach, Germany) using O2-enriched room air. The ventilation was adjusted to 75 strokes per minute with a minute volume of 120-180 ml. Tracheal pressure (TPR) was monitored continuously and served as an indicator for the pump cycle. Because of the bilateral vagotomy, the cycle of central respiration and the cycle of artificial ventilation were mostly desynchronized but sometimes a loose entrainment remained (see RESULTS).
Data processing
All nerve signals and the ECG were amplified (input resistance
10 M), band-pass filtered (120-1200 Hz) and fed into window discriminators. Discrimination of single units from filaments containing more than one active fiber (few-fiber preparations) was done
mostly by window discrimination, making use of different units
differing in the size of their action potentials. The accuracy of spike
discrimination was controlled by an electronic delay unit. For this
purpose, the discriminated action potentials were displayed on a
storage oscilloscope, delayed by 5 ms, using the output of the window
discriminator as a trigger (see Figs. 3F, 4F, 6F, and 8F). In a few cases action
potentials were discriminated according to their shape using a special
computer program ("spike" by C. Forster). Signals were fed into
an IBM compatible computer at 50- to 100-Hz sample frequency (software
CARDS by S. Tiedemann). For off-line analysis, all original signals
were stored on a digital tape recorder (DTR-2602, Bio-Logic, Claix, France).
The rhythmicity in sympathetic activity was analyzed off-line in several ways from recording periods of 250-300 s (binwidth 10-20 ms): 1) phrenic (PHR)-triggered histograms were constructed by summing up all recorded parameters during 80-300 respiratory cycles. Summation was synchronized by the onset of PHR nerve activity. 2) Pump-triggered histograms were constructed by summation of all recorded parameters during 150-375 ventilation cycles. Summation was synchronized with the onset of inflation obtained from the continuous TPR reading. However, the synchronization of pump-triggered histograms with TPR does not imply a causative role of afferents from the respiratory tract in the pump-related modulation of sympathetic activity. The accumulated sympathetic impulses obtained in PHR- and pump-triggered histograms always were recalculated to 100 sweeps to facilitate comparison of sympathetic discharge rates between different recordings. The pulsatile blood pressure waves were dampened with a low-pass filter (cutoff-frequency 1.5 Hz). At the ventilation rate set to 75 strokes per minute, filtering produced a phase lag of ~120 ms and a reduction in amplitude of 30%, which were not compensated in the figures. The difference in central and peripheral conduction time between PHR and sympathetic pathways introduced a lag of sympathetic activity behind PHR activity by ~350 ms, which is also not compensated in the figures (see Fig. 4A, stippled lines). 3) Fast Fourier transformations (FFT, computer program by U. Wittmann) were performed from data blocks of consecutive 212-214 bins. The TPR signal and the discriminated PHR and sympathetic activities (sample frequency 50-100 Hz) were used to construct power spectra. The sample frequencies and block lengths used allowed a valid data analysis in a frequency range 0.012-25 Hz. However, because the main interest lay in the frequency range around respiration, power spectra were truncated >2.5 Hz. 4) Autocorrelograms (computer programs by V. Banarer and C. Forster) were computed from the original TPR signal and the discriminated PHR and sympathetic activities. 5) Interspike interval histograms (ISIH) were constructed from the activity of single sympathetic fibers (computer programs by V. Banarer and C. Forster). And 6) the modulation by the pressure pulse wave of postganglionic activity ("cardiac rhythmicity," CR) was determined by constructing post-R-wave histograms over two cardiac cycles (binwidth 4 ms).
The degree of respiratory-related modulation (RM) of sympathetic
activity was calculated from both the PHR-triggered and from the
pump-triggered histograms by means of a computer program using the
formula: RM = 100 [1 (min/max)], with minimum (min) and maximum (max) activity being determined by summing the activity of
every eight consecutive 20-ms bins (or 16 bins of 10 ms), i.e., 160 ms,
in a given histogram (see Boczek-Funcke et al. 1991
;
Häbler et al. 1993
). Cardiac rhythmicity was
quantified in the same way using eight consecutive 4-ms bins to
calculate minimum and maximum (Häbler et al.
1994a
). These quantifications were made only for the activity
in filaments containing more than one unit because the low ongoing
discharge in single fibers tends to result in quantitative values which
overestimate the rhythmicity. Results are expressed as means ± SD
or means ± SE as indicated. Statistical analysis was carried out
using Student's t-test.
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RESULTS |
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General
Here we describe the rhythmicity in postganglionic neurons
supplying the tail under baseline conditions, i.e., in vagotomized animals with intact carotid sinus nerves under artificial ventilation with blood gases maintained in the physiological range. The changes in
rhythmicity occurring during experimental interventions altering the
central respiratory rhythm generator, such as hypocapnic apnea, hypercapnia, and hyperthermia, and the rhythmicity remaining after abolishing the baro- and chemoreceptors by sino-aortic denervation will
be described in a companion paper (unpublished results). Adopting the
nomenclature of Johnson and Gilbey (1996), we will call
the rhythm that was most pronounced in the autocorrelogram and power
spectra the "dominant rhythm."
We analyzed spontaneous activity in 27 fiber preparations containing
two to seven active units and in 51 single fibers of which 42 were
extracted electronically from the preparations containing more than one
unit. The rate of spontaneous activity determined in the 51 single
fibers was 1.12 ± 0.65 imp/s (mean ± SD, median 0.89, range
0.23-2.6 imp/s; Fig. 1A). The
conduction velocity of 30 single fibers (Fig. 1B) identified
by electrical stimulation of the LST between ganglia L2 and L3
(Baron et al. 1988) was 0.62 ± 0.06 m/s (mean ± SD, range 0.46-0.83 m/s; Fig. 1C). The PHR bursts showed
a frequency of 0.69 ± 0.03 Hz (mean ± SE, n = 36), whereas that of artificial ventilation was set to 1.25 Hz.
Tracheal pressure (TPR) was 7.1 ± 0.3 mmHg (mean ± SE,
n = 36) and arterial blood pressure 109.5 ± 2.8 mmHg (n = 34).
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Because of the relatively low ongoing activity of single units, autocorrelogram analysis of 250-300 s recording periods showed respiratory and other rhythmicity less clearly than phrenic-triggered histograms or spectral analysis. Thus despite respiratory rhythmicity clearly being present in the PHR-triggered histogram and in power spectra, there was no rhythm detectable at all in the corresponding autocorrelogram in 16 cases. Generally PHR-triggered histograms proved to be the most sensitive means to detect respiratory modulation.
The activity of all postganglionic neurons exhibited short interspike
intervals 100 ms (Figs. 3E, 4E, 6E,
and 8E). In addition, when a postganglionic neuron showed a
sufficient level of ongoing activity, there was often a peak related to
the dominant rhythmicity of the unit's activity (Figs. 3E
and 8E). This peak had a latency slightly shorter than what
would have been expected from the frequency of the dominant rhythm.
However, in some ISIHs there was no peak related to the dominant rhythm
(Figs. 4E and 6E). In no case did the ISIH show a
single peak corresponding to a rhythm equal or similar to respiration
without also showing short intervals (type A in Johnson and
Gilbey 1996
).
Modulation by the pressure pulse wave (CR) was examined in the activity
of 23/27 multifiber preparations. Ten few-fiber preparations exhibited
no CR (CR 40%) (see Boczek-Funcke et al. 1991
;
Häbler et al. 1994a
), 9 showed weak (40% < CR
60%), and 4 exhibited strong CR (CR > 60%). On
average, CR was weak (46.2 ± 16.4%, mean ± SD).
Dominant sympathetic rhythm showing the same frequency as phrenic activity
The dominant rhythm in the activity of 19/27 few-fiber preparations and 37/51 single fibers, i.e., in the majority of postganglionic neurons, was correlated with PHR rhythmicity (Figs. 2-4). This central respiratory modulation was always observed in the PHR-triggered histogram (Figs. 2A, 3A, and 4A), in the power spectra (Figs. 2C, 3C, and 4C), and, in the presence of sufficient spontaneous activity, also in the autocorrelograms (Figs. 2D, 3D, and 4D). In particular the power spectra showed a perfect match between the frequency components of the PHR bursts and those of the dominant rhythm in the sympathetic neurons. Respiratory modulation generally showed the expiratory pattern, consisting of a depression during inspiration and either a circumscribed peak in early expiration (see Figs. 2A and 3A), a broad activity peak during expiration (see Fig. 5A) or two separate peaks, one each in early expiration and in late expiration (Fig. 4A).
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Although the dominant rhythm corresponded to that of the PHR bursts, at the same time there was usually an additional modulation at the frequency of artificial ventilation (10/19 few-fiber and 21/37 single-fiber preparations), as seen in the pump-triggered histograms (Fig. 2B) and in the power spectra (Fig. 2C). PHR- and pump-related modulations interacted as there was a detectable deformation and sometimes an augmentation of the dominant PHR-related peak when inflation and a phrenic burst occurred approximately at the same time (Fig. 2D, see Fig. 5D, asterisks). However, often there was still some entrainment between the pump and central respiratory cycles (Fig. 2C) despite section of the vagus nerves. Therefore theoretically part of the modulation seen in the pump-triggered histograms might be related secondarily to the central respiratory rhythmicity. However, in the example illustrated (Fig. 2C), there is a separate narrow peak exactly at pump frequency (asterisk), which is superimposed on a broad peak at twice the PHR frequency (i.e., its 1st harmonic). This sympathetic peak at pump frequency is larger than would be expected if it was secondarily due to PHR activity. In accordance, a pump-related subordinate rhythmicity that is independent of the dominant PHR-related rhythm, is apparent in parts of the corresponding autocorrelogram (Fig. 2D). This allows the conclusion that a PHR- and a pump-related modulation were expressed concomitantly, independently of each other, in sympathetic activity.
In 9/19 few-fiber preparations and 16/37 single fibers, only central respiratory modulation was observed but no modulation by artificial ventilation (Fig. 3). In these cases, there was a clear PHR-related periodicity in the PHR-triggered histogram (Fig. 3A), the power spectrum (Fig. 3C), and the autocorrelogram (Fig. 3D), but no rhythmicity in the pump-triggered histogram despite some entrainment between central respiration and artificial ventilation (Fig. 3B). In the power spectrum, around the pump frequency only a sympathetic peak at the first harmonic of PHR frequency was seen but no pump-related peak independent of PHR activity (Fig. 3C). The autocorrelogram also showed no pump-related rhythm (Fig. 3D).
A relatively frequent observation (3 few-fiber preparations and 7 single fibers) was a rhythm in the sympathetic neurons with twice the frequency of central respiration (Fig. 4). In these cases, the frequency of the PHR bursts was significantly lower than in the other experiments (0.51 ± 0.03 Hz, n = 7, vs. 0.73 ± 0.03 Hz, n = 29, P < 0.001, t-test). The activity peaks came in early expiration and in late expiration, separated by two depressions, one during the PHR burst and one in midexpiration (Fig. 4A). In most cases, a concomitant pump-related weaker rhythm also was present (Fig. 4B), which was apparent in the power spectrum (Fig. 4C, asterisk) but only rudimentary in the autocorrelogram (Fig. 4D). However, the dominant PHR-related rhythm in sympathetic activity was unrelated to the pump cycle indicating that the two rhythms were independent of each other.
Dominant sympathetic rhythm showing the same frequency as artificial ventilation
The dominant rhythm in the activity of 8/27 few-fiber preparations and 11/51 single fibers was related to the frequency of artificial ventilation (Figs. 5 and 6). All units except one (Fig. 6) displayed an additional, less prominent, modulation in parallel with PHR activity. While it was in some cases hard to decide from the PHR- and pump-triggered histograms which of the two modulations was more pronounced, the peak in the power spectrum (Fig. 5C) at the pump frequency dominated over that at the PHR frequency. In the autocorrelogram (Fig. 5D), the dominant rhythm clearly was coupled to the pump. The influence of the concomitant PHR-related modulation was only seen by an enhancement of the pump-related peaks when they coincided with a PHR burst (Fig. 5D, asterisks). The even distribution of TPR in the PHR-triggered histograms (Fig. 5A) and of PHR activity in the pump-triggered histograms (Fig. 5B) indicated that both cycles were totally desynchronized. Hence the dominant pump-related modulation was independent of the PHR-related modulation.
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In two of the few-fibers preparations and three single fibers discriminated from them, the dominant modulation occurred in parallel with every second pump cycle in the autocorrelogram (Fig. 6). Although the pump-triggered histogram showed a small modulation with each cycle (Fig. 6B), the power spectrum revealed a relatively broad sympathetic peak at exactly half the pump frequency and a peak at the full pump frequency (Fig. 6C). In the autocorrelogram, a clear sympathetic rhythmicity with every second pump cycle is apparent (Fig. 6D). In this case, there was no clear modulation with the PHR cycle (Fig. 6, A and C).
Quantitatively, the PHR-related modulation in sympathetic activity
recorded from all filaments containing more than one active unit was
calculated to be 77 ± 18% (mean ± SD, n = 27),
whereas the pump-related modulation was weaker (48 ± 20%). As
the pump-related modulation is thought to be due to the baroreceptor
reflex (Häbler et al. 1996), it was tested whether
the magnitude of the pump-related modulation was related to the level
of mean arterial pressure (Fig. 7). A
moderate but significant correlation was found (correlation coefficient
0.45, P < 0.05).
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Other rhythms
Only two single fibers showed a dominant rhythm that was distinct from both the phrenic and the pump cycle (Fig. 8). The rhythmicity in the activity of these postganglionic fibers showed no modulation in the PHR-triggered histogram (Fig. 8A) and in the pump-triggered histogram (Fig. 8B). However, a rhythm was apparent in the power spectrum (Fig. 8C) and in the autocorrelogram (Fig. 8D), which was slower than the central respiratory rhythm (0.85 vs. 1 Hz of PHR activity). Thus this rhythm seemed to be totally independent of respiration.
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The appearance of this rhythm was, in one case, related to a spontaneous change in central respiration. Thus the second of these fibers started with a dominant modulation in parallel with a totally regular PHR nerve activity. Independently of any intentional stimulus, the PHR rhythm slowed from 0.77 to 0.45 Hz and became irregular and the unit's modulation changed to a frequency that was faster (0.85 Hz) and independent of the respiratory and pump cycles. After ~15 min, the PHR cycle, while remaining somewhat irregular, spontaneously accelerated to its original mean frequency (0.75 Hz) and the fiber regained partial synchronization with the PHR rhythm, although it kept a dominant rhythm that was slightly faster (0.8 Hz).
Finally one single fiber showed no rhythmicity in its activity at all, neither with a frequency related to central respiration nor related to the ventilation pump nor any rhythm at a similar frequency.
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DISCUSSION |
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The results of the present study indicate that the dominant
rhythmicity that is present in the ongoing activity of postganglionic sympathetic neurons projecting into the ventral collector nerve of
vagotomized Wistar rats is related to respiration in the vast majority
of cases under normocapnic conditions. In most recordings, a modulation
by the central respiratory rhythm generator dominated, resulting in a
pattern the same as is found in the activity of sympathetic neurons
supplying other targets, i.e., an inhibition during inspiration with
main activity occurring during expiration (Bartsch et al.
1996; Czyzyk-Krzeska and Trzebski 1990
;
Darnall and Guyenet 1990
; Gilbey et al.
1986
; Häbler et al. 1993
; Numao et
al. 1987
).
In a significant proportion of cases, the dominant rhythm was
related to the cycle of artificial ventilation as recently found in
preganglionic neurons projecting to the superior cervical ganglion in
rats (Häbler et al. 1996) and in many sympathetic
neurons in the cat (Häbler et al. 1994b
). Often
both respiration- and pump-related rhythms were present at the same
time, generating a complex but consistent modulation in the
autocorrelograms of sympathetic activity. Moreover, we now have data to
show that, in a given neuron, the dominance of one modulation over the
other depends on the strength of respiratory drive (unpublished
observations). Finally in only two postganglionic fibers, the dominant
rhythm was related neither to central respiration nor to the pump
rhythm. Such a rhythmicity would be in line with the so-called tail
(T)-rhythm described by Johnson and Gilbey (1996
,
1998a
).
The results show that the activity in vasoconstrictor neurons that are
involved in thermoregulation (Dawson and Keber 1979) is
linked intimately to the central regulation of respiration. This may
have functional implications. Whereas generally the consequence of any
rhythmic modulation in sympathetic discharge may be the emergence of
bursts that may result in temporal facilitation at the neurovascular
junction (see Häbler et al. 1994b
), in the tail
the observed respiratory modulation also may contribute directly to
vasodilatation during hyperthermia or exercise. Under these conditions,
the enhanced inspiratory drive, the increased respiration rate and the
decreased duration of the expiratory phase may lead to a preponderance
of the inspiratory inhibition in postganglionic activity thereby
reinforcing vasodilatation.
The present results confirm recent findings by Johnson and
Gilbey (1998b) in showing that CR in sympathetic activity
supplying the tail is relatively weak. The degree of CR was similar to
that found in the activity of postganglionic neurons supplying hindlimb skin (Häbler et al. 1994a
) that also are involved
mainly in thermoregulation rather than in the maintenance of systemic
blood pressure. Despite the relative lack of CR, there was a
pump-related rhythmicity in the activity of many fibers. This rhythm
was interpreted previously as being generated reflexly by the arterial
baroreceptors (Häbler et al. 1994b
, 1996
). Here we
found a significant correlation between the level of arterial blood
pressure and the magnitude of the pump-related modulation that supports
the involvement of the baroreceptors. In accordance, Johnson and
Gilbey (1998b)
found that aortic nerve stimulation inhibited
postganglionic fibers supplying tail artery and vein despite the
absence of CR in their activity. Therefore in accordance with previous
findings (Häbler et al. 1994a
), the lack of CR in
sympathetic neurons cannot be equated with the lack of baroreceptor control.
Although the bilateral vagotomy excluded a role for vagal afferents, an
involvement of arterial chemoreceptors in the pump-related rhythmicity
cannot be excluded. They show some remaining activity even under
hyperoxic conditions (see Marshall 1994) and are very sensitive to ventilatory phasic changes of arterial pH (Band et al. 1969
). A relatively frequent observation was a partial
coupling of the PHR discharge to the pump cycle despite bilateral
vagotomy. There is evidence that oscillations imposed on steady
chemoreceptor discharge can influence respiration (Cross et al.
1986
; Takahashi et al. 1990
). Thus it appears
that the carotid chemoreceptors might be responsible for the observed
partial entrainment of respiration and ventilation. Additionally, and
independently of their effects on respiration, they also may mediate
part of the pump-dependent rhythm in postganglionic activity.
Alternatively, in the absence of vagal afferent feedback, thoracic
afferents or afferents from the diaphragm (Balkowiec et al.
1995
) may entrain central respiration to the pump (Iscoe
and Duffin 1996
). However, whether here these afferents are
responsible for a pump-dependent rhythm in sympathetic activity as has
been shown in the spinal neonatal swine (Sica et al.
1997
) is unclear, because this rhythmicity largely was abolished after vagotomy and sino-aortic denervation in a previous study (Häbler et al. 1996
). A similar observation
was made on postganglionic activity supplying the tail (unpublished observations).
Another interesting finding was that the ISIHs of the activity of all
single postganglionic neurons displayed short intervals in addition to
a potential peak corresponding to the dominant rhythmicity. It has been
found previously that the discharge of postganglionic neurons is
determined by one to three preganglionic fibers, which generate
suprathreshold excitatory postsynaptic potentials (EPSPs; strong
inputs), rather than by summation of subthreshold (weak) inputs
(McLachlan et al. 1997). Most of the preganglionic
neurons in the cervical sympathetic trunk giving rise to strong inputs
showed respiratory modulation, but none was active at short intervals,
i.e., they did not fire in bursts (McLachlan et al.
1998
). This suggests that probably all postganglionic neurons
in the present study were driven by convergence of more than one
suprathreshold preganglionic input. This also would explain the
observation that the second peak in the ISIH at longer intervals came
slightly earlier than would be expected from the frequency of the
dominant rhythm (McLachlan et al. 1998
). In contrast,
Johnson and Gilbey (1996
, 1998a
) found some
postganglionic neurons supplying tail artery and vein that lacked small
intervals in their firing pattern and therefore probably were driven by
only one suprathreshold input.
Our results are at variance with those of Johnson and Gilbey
(1996, 1998a
), who found that 36/51 postganglionic fibers
supplying the tail artery and 9/14 supplying the tail vein displayed a
dominant rhythm in their ongoing activity that was distinct from both
the central respiratory rhythm and the frequency of artificial
ventilation. While in some of their fibers the dominant rhythm was
identical with respiration, it corresponded to artificial ventilation
in no case. In contrast, in the present study, the T rhythm was an extremely rare finding. The explanation for these discrepancies between
the studies is unclear. The experimental conditions were similar under
which the discrepant results were obtained. Thus the frequency of the
PHR bursts in the group of animals with vagotomy was almost identical
in those studies (see Fig. 9 in Johnson and Gilbey 1996
)
and in the present study. The frequency of artificial ventilation was
also similar (1.15-1.8 vs. 1.25 Hz).
Our analysis with three different methods (PHR- and pump-triggered histograms, spectral analysis, and autocorrelograms) produced consistent results. For the autocorrelograms, we used the original signals of tracheal pressure and PHR nerve activity rather than a single trigger derived from these signals. This greatly aided the detection of pump- and PHR-related rhythms. Therefore it appears unlikely that we missed a potential T rhythm. The validity of our methods was confirmed because in two cases, we found a dominant rhythm that differed from both PHR and pump frequency. This rhythm appeared in the power spectra and in the autocorrelograms, but the PHR- and the pump-triggered histograms did not show any periodicity. Because our extensive single fiber analysis revealed a T rhythm in only two cases and the proportion of single fibers and few-fiber preparations exhibiting PHR- and pump-related modulation, respectively, were essentially similar, we felt justified not to distinguish between single fiber and "population" rhythmicity. It appears unlikely that the modulation of few-fiber preparations, which generally was found to be the same as that of single fibers discriminated from them, in reality consisted of different T rhythms that combined to a PHR- or pump-related modulation.
One possibility to explain the discrepant results might be that Johnson
and Gilbey recorded from sympathetic fibers with identified target
organs, i.e., tail artery and vein, whereas we recorded from fibers
projecting to the tail but with unknown functions. However, the only
targets in the rat tail are vascular and, in the present study, almost
all postganglionic fibers tested were inhibited during whole body
heating (unpublished observations), suggesting that they indeed were
involved in thermoregulation. A difference between rat strains can be
ruled out because Johnson et al. (1997) found a similar
T rhythm in Sprague-Dawley and Wistar rats. Anesthesia might be the
crucial factor because the spontaneous activities found by
Johnson and Gilbey (1996
, 1998a
) were 1.5-2 times
higher than those reported here. We used pentobarbitone whereas Johnson
and Gilbey initially used pentobarbitone and maintained anesthesia with
chloralose. However, using the same type of anesthesia, Chang
and Gilbey (1998)
observed a respiratory rhythm in the activity of the whole ventral collector nerve in almost all animals under control conditions. Häbler et al. (1993)
observed
similar patterns of respiratory modulation in postganglionic fiber
activity supplying the hindlimb under three different anesthetics.
Therefore anesthesia is unlikely to account for the discrepant findings.
Gilbey and Johnson (1996, 1998a
) explained the
PHR-related rhythmicity, which they found in some of their sympathetic
neurons, by entrainment of oscillators generating the T rhythm(s) to
the central respiratory rhythm generator, a well-established concept (Holst 1939
) that also has been suggested by other
authors to explain respiratory modulation in sympathetic activity
(Barman and Gebber 1976
; Koepchen 1983
).
However, in the cat, Bachoo and Polosa (1987)
convincingly demonstrated that coupling of two independent oscillators
could not explain the respiratory modulation in sympathetic neurons
projecting in the cervical sympathetic trunk. Entrainment of
independent oscillators cannot totally be ruled out as an explanation for the results of the present study. However, then the postulated T-rhythm generator must simultaneously have been entrained to two
external rhythms in most neurons, i.e., to the respiratory rhythm
generator and to the pump. Furthermore the question remains why under
very similar experimental conditions sympathetic neurons show a
free-running T rhythm in one study but a PHR- and/or pump-related modulation, i.e., entrainment, in the other.
In conclusion, the present study shows that under control conditions,
the activity of most postganglionic fibers supplying the rat tail
exhibits a strong respiratory modulation as the dominant rhythm. In a
significant proportion, however, a rhythm related to the ventilation
pump is most prominent. Both rhythms can interact in a complex manner.
The fibers exhibit weak cardiac rhythmicity that is characteristic for
sympathetic neurons supplying skin. The discrepancies with the results
of Johnson and Gilbey (1996, 1998a
), who under similar
conditions observed a dominant rhythm with a frequency different from
both central respiration and the ventilation pump in most sympathetic
fibers supplying tail artery and vein, at present remain unresolved.
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ACKNOWLEDGMENTS |
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We thank E. Tallone for making the illustrations and S. Augustin for technical help in the experiments. We are grateful to A. Just and U. Wittmann for providing the computer program for the spectral analysis and to V. Banarer, A. Boczek-Funcke, C. Forster, and S. Tiedemann for the other software used in this study. We thank E. M. McLachlan for valuable comments on the manuscript.
This work was supported by the Deutsche Forschungsgemeinschaft.
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FOOTNOTES |
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Address for reprint requests: H.-J. Häbler, Physiologisches Institut, Christian-Albrechts-Universität, Olshausenstrasse 40, 24098 Kiel, Germany.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 August 1998; accepted in final form 1 February 1999.
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REFERENCES |
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