Vol. 284, Issue 2, R455-R466, February 2003
Influence of respiratory network drive on phrenic
motor output evoked by activation of cat pre-Bötzinger
complex
Irene C.
Solomon
Department of Physiology and Biophysics, State
University of New York at Stony Brook, Stony Brook, New York
11794-8661
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ABSTRACT |
10.1152/ajpregu.00395.2002. We have previously demonstrated
that microinjection of DL-homocysteic acid (DLH), a
glutamate analog, into the pre-Bötzinger complex (pre-BötC)
can produce either phasic or tonic excitation of phrenic nerve
discharge during hyperoxic normocapnia. Breathing, however, is
influenced by input from both central and peripheral chemoreceptor
activation. This influence of increased respiratory network drive on
pre-BötC-induced modulation of phrenic motor output is unclear.
Therefore, these experiments were designed to examine the effects of
chemical stimulation of neurons (DLH; 10 mM; 10-20 nl) in the
pre-BötC during hyperoxic modulation of CO2 (i.e.,
hypercapnia and hypocapnia) and during normocapnic hypoxia in
chloralose-anesthetized, vagotomized, mechanically ventilated cats. For
these experiments, sites were selected in which unilateral
microinjection of DLH into the pre-BötC during baseline
conditions of hyperoxic normocapnia [arterial
PCO2 (PaCO2) = 37-43
mmHg; n = 22] produced a tonic (nonphasic) excitation of phrenic nerve discharge. During hypercapnia
(PaCO2 = 59.7 ± 2.8 mmHg; n = 17), similar microinjection produced excitation in which phasic
respiratory bursts were superimposed on varying levels of tonic
discharge. These DLH-induced phasic respiratory bursts had an increased
frequency compared with the preinjection baseline frequency
(P < 0.01). In contrast, during hypocapnia (PaCO2 = 29.4 ± 1.5 mmHg; n = 11), microinjection of DLH produced nonphasic tonic excitation of
phrenic nerve discharge that was less robust than the initial
(normocapnic) response (i.e., decreased amplitude). During normocapnic
hypoxia (PaCO2 = 38.5 ± 3.7; arterial PO2 = 38.4 ± 4.4; n = 8) microinjection of DLH produced phrenic excitation similar to
that seen during hypercapnia (i.e., increased frequency of phasic
respiratory bursts superimposed on tonic discharge). These findings
demonstrate that phrenic motor activity evoked by chemical stimulation
of the pre-BötC is influenced by and integrates with modulation
of respiratory network drive mediated by input from central and
peripheral chemoreceptors.
respiratory rhythm generation; neural control of breathing; hypercapnia; hypocapnia; hypoxia
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INTRODUCTION |
THE
PRE-BÖTZINGER COMPLEX (pre-BötC) is
hypothesized to be the primary locus of respiratory rhythm generation
in mammals (20, 24). Activation of this region in vivo has
been demonstrated to increase the frequency of inspiratory bursts
(5, 14, 28, 34) and produce tonic excitation of
inspiratory motor activity (5, 28), while selective
neuronal destruction of this region in vivo abolishes normal breathing
(9) and the tachypnea response elicited by focal
activation (34). Although anatomic specificity within the
pre-BötC appears to explain some patterns of phasic modulation of inspiratory motor activity (i.e., rapid series of high-amplitude, rapid rate of rise, short-duration bursts) in vivo
(16, 28), other mechanisms by which focal activation of
the pre-BötC produces phasic vs. tonic excitation of respiratory motor output have not been examined.
One possible mechanism for different response types elicited by
activation of this region might be the level of intrinsic excitability
of pre-BötC rhythm-generating neurons. It has been proposed that
the rhythm-generating neurons located in the pre-BötC receive
synaptic inputs that synchronize and modify the basic rhythm generator
by affecting intrinsic membrane conductances of presumptive
rhythmogenic neurons (8, 19, 20, 24, 25), which in turn
provide rhythmic drive to the respiratory network during the
inspiratory phase of network activity (4, 8, 12, 20,
23-25). Thus the level of membrane depolarization of pre-BötC rhythm-generating neurons would vary with the level of
overall respiratory network drive, which in vivo is influenced by input
from both central and peripheral chemoreceptor activation.
In our previous experiments in the anesthetized cat, unilateral
microinjection of the glutamate analog DL-homocysteic acid (DLH) into the pre-BötC produced excitation of phrenic motor output that exhibited either increased phasic burst frequency or tonic
(i.e., nonphasic) discharge (28). Similar modulation of
expiratory motor output in response to unilateral microinjection of DLH
into the pre-BötC has also been observed in this animal model
(26), confirming that activation of the pre-BötC is
capable of eliciting both phasic and tonic (i.e., nonphasic) excitation of respiratory motor activity. Our previous studies, however, were
conducted under hyperoxic, normocapnic conditions; thus the influence
of modified (i.e., increased or decreased) respiratory network drive on
this DLH-induced pre-BötC-mediated excitation of respiratory
motor output was not examined. Therefore, the purpose of this study was
to specifically examine the effects of microinjection of DLH into the
same site in the pre-BötC on phrenic motor output during
modulation of CO2 (i.e., hypercapnia and hypocapnia) and O2 (i.e., hypoxia).
It has previously been demonstrated that respiratory network drive can
influence the pattern of expression (i.e., phasic vs. tonic) of
inspiratory and expiratory motoneuron discharges (22). In
the absence of respiratory rhythm (produced by hypocapnia), for
example, tonic excitation of respiratory motoneuron discharge is
observed. As chemical drive (i.e., increasing CO2 or
hypoxia) is increased, however, phasic activity replaces tonic firing. Thus, if the level of intrinsic excitability of pre-BötC
rhythm-generating neurons plays a role in determining the response type
(i.e., phasic or tonic) elicited by DLH-induced activation of this
region, then alterations in respiratory network drive should produce a
predictable modulation of phrenic motor output elicited by chemical
activation of this region. I hypothesized that repeated chemical
activation of the same site in the pre-BötC will evoke different
responses in phrenic motor output, which are dependent on the level of
respiratory network drive. I further hypothesized that during increased
respiratory network drive, activation of the pre-BötC would
elicit an increase in the frequency of phasic phrenic bursts, while
during reduced respiratory network drive, activation of the
pre-BötC would elicit solely nonphasic tonic excitation of
phrenic motor output.
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METHODS |
General.
All experiments were performed under protocols approved by the
Institutional Animal Care and Use Committee at the State University of
New York at Stony Brook in compliance with the Animal Welfare Act and
in accordance with the American Physiological Society's "Guiding
Principles for Research Involving Animals and Human Beings " (1). A detailed description of the general methods has
been published previously (28).
In brief, anesthesia was induced in adult cats (3.3-4.7 kg;
n = 22) with halothane (5%) in oxygen and maintained
with intravenous 
-chloralose (initial 35-50 mg/kg; supplemental
3-5 mg/kg). The adequacy of anesthesia was regularly verified by
absence of a withdrawal reflex (in the unparalyzed state) or blood
pressure response (during muscular paralysis) to a noxious paw pinch.
If the cat withdrew its limb during the absence of paralysis or if an
increase in blood pressure was evoked, additional anesthesia was given.
The right brachial vein and both brachial arteries were cannulated for
administration of drugs, measurement of arterial blood pressure
(Statham transducer, P23XL), and sampling of arterial blood. The
trachea was cannulated, the cat was vagotomized bilaterally (to
eliminate the influence of cardiopulmonary afferent input to the
central respiratory network), and the lungs were mechanically ventilated with 40% O2 in a balance of N2.
Bilateral pneumothoraxes were established, and the expiratory
outlet of the ventilator was placed under 2-3 cmH2O to
prevent collapse of the lungs during expiration. The cat was then
paralyzed with vecuronium bromide (0.2-0.4 mg/kg iv), supplemented
as needed. The dorsal surface of the brain stem was exposed, and the
C5 rootlet of one or both phrenic nerves was isolated for
recording. Raw phrenic nerve discharge was amplified (10,000 times) and band-pass filtered from 100 Hz to 10 kHz; the
filtered signal was rectified, and a moving average was obtained using
a third-order Paynter filter with a 100-ms time constant.
Experimental protocol.
I examined the effects of DLH-induced activation of neurons located in
the pre-BötC on phrenic nerve discharge during hyperoxic (PO2 
175 mmHg) modulation of
CO2 and during normocapnic hypoxia. Sites in the
pre-BötC were initially localized using predetermined stereotaxic
coordinates relative to the calamus scriptorius, functionally identified using DLH (28), and histologically confirmed
after completion of the experiments. In all experiments, spontaneous phasic phrenic nerve activity was observed under hyperoxic, normocapnic conditions before functional identification. For these experiments, only functionally identified pre-BötC sites in which unilateral microinjection of DLH (10 mM; 
20 nl; Sigma-Aldrich Chemical, St.
Louis, MO) produced a tonic (nonphasic) excitation of phrenic nerve
discharge under hyperoxic, normocapnic conditions were selected. To
assess repeatability of this response, control experiments (n = 5) were conducted in which microinjection of DLH
was repeated in the same site at least three times under hyperoxic,
normocapnic conditions. In the remaining experiments, after functional
identification of pre-BötC sites, one of the following changes in
the ventilation parameters was made: 1) the fraction of
CO2 in the inspired gas mixture was increased to 5 or 7%
(hypercapnia), 2) the frequency of the ventilator was
increased (hyperventilation/hypocapnia), or 3) the fraction
of O2 in the inspired gas mixture was decreased to
12-14% O2 in a balance of N2 (i.e.,
peripheral chemoreflex). The DLH microinjection was then repeated using
the same volume used for functional identification. In all experiments,
the volume of injectate was measured by observing the displacement of
the fluid meniscus using a microscope equipped with an eyepiece
reticule. Typically, the ventilation parameters were changed two or
three times at each site before returning back to control levels to demonstrate recovery of the initial response. Changes in ventilation parameters were made in a randomized order, and at least 15 min of
recovery were allowed before moving to the next trial. Under each
condition, arterial PO2
(PaO2), PCO2
(PaCO2), and pH were measured (Radiometer ABL-500)
immediately preceding microinjection of DLH. To control for nonspecific
effects, equivalent volumes of saline or larger volumes (120 nl) of
2% Fast green dye, which was used to mark injection sites, were
microinjected into all sites. At the end of each experiment, the brain
stem was removed for subsequent histological analysis to confirm that
the injection site was in the pre-BötC (see Location of
injection sites).
Data acquisition and analysis.
Both raw and averaged phrenic nerve discharge were recorded on tape
(Vetter, model 4000A) and on a chart recorder (Astro-Med, model MT95K2)
throughout the experimental protocol. Appropriate segments of data were
then transferred to a Macintosh PowerBook 3400c computer for offline
analyses (PowerLab, Chart 3.6.1, AD Instruments).
Peak amplitude of integrated phrenic nerve discharge, inspiratory
duration (TI), expiratory duration (TE), and
frequency of phasic phrenic bursts were determined in response to
unilateral microinjection of DLH into the pre-BötC under each of
the ventilation conditions described above. Preinjection baseline
values were determined by averaging the values obtained for the 60-s
period preceding DLH microinjection. Response values were determined as
the peak change from preinjection baseline values for a tonic nonphasic
excitation of phrenic nerve discharge or by averaging the values
obtained for five consecutive breathing cycles displaying the greatest
change from preinjection baseline values for phasic phrenic nerve
discharge responses. Further, for tonic nonphasic excitation of phrenic
nerve discharge, TI represents the duration of tonic
firing, and TE was not determined. Amplitude of integrated phrenic nerve discharge and frequency of phasic phrenic bursts are
reported as a percent change from preinjection baseline levels of
discharge, which were set at 100% in each cat. The onset latency for
DLH-induced responses was measured from the beginning of microinjection.
All values are reported as means ± SE. Responses to DLH
microinjection are presented as paired data. Student's paired
t-tests, the paired nonparametric Wilcoxon signed-rank test,
or two-way repeated measures ANOVA, followed by Scheffé's
post hoc test, as appropriate, were used to determine statistical
significance, for which the criterion level was set at
P < 0.05.
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RESULTS |
General effects of DLH microinjection.
Unilateral microinjection of DLH into 22 sites in the
pre-BötC produced a nonphasic tonic excitation of phrenic nerve
discharge under hyperoxic, normocapnic conditions
(PaO2 = 189.6 ± 5.6 mmHg; PaCO2 = 39.4 ± 2.3 mmHg). This response was
characterized by an abrupt rise in phrenic nerve discharge to a plateau
level, which in some cases gradually decayed, and had durations ranging
from 20 to 165 s. In general, the peak amplitude of integrated
phrenic nerve discharge at the onset of the response was higher or the same as that seen during preinjection baseline phrenic bursts, and
recovery consisted of either a gradual return of phasic phrenic bursts
or a transient postexcitatory depression of phrenic nerve discharge. In
some cases (n = 7), phasic phrenic bursts returned as
tonic activity began to wane (i.e., before complete cessation of tonic activity).
Repeatability of the DLH-induced response.
Repeated microinjection of DLH into the same site in the pre-BötC
without modulation of the ventilation parameters (i.e., maintained
under hyperoxic, normocapnic conditions; n = 5)
demonstrated that the DLH-induced response was reproducible within the
same site (Fig. 1). In general, at least
15 min was allowed for recovery before attempting to demonstrate
repeatability, and at least three microinjections were made into the
same site. An example of the results obtained from one of these
experiments is provided in Fig. 1A, and summary data
describing the changes in peak amplitude of integrated phrenic nerve
discharge and TI for each microinjection trial from all
five experiments are illustrated in Fig. 1B. As shown,
repeated microinjection of DLH into the same site in the pre-BötC
elicited a reproducible increase in phrenic nerve discharge (Fig.
1A), with no differences observed in either the DLH-induced increase in peak amplitude of integrated phrenic nerve discharge or the
DLH-induced increase in TI (Fig. 1B).
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Fig. 1.
Example demonstrating the effects of repeated
DL-homocysteic acid (DLH)-induced activation of the
pre-Bötzinger complex (pre-BötC) during hyperoxic
normocapnia. A: repeated microinjection of DLH into the same
site in the pre-BötC elicited a reproducible increase (i.e.,
tonic nonphasic excitation) in phrenic nerve discharge. Contra,
contralateral; PaCO2, arterial
PCO2. B: summary data illustrating
the effects of repeated DLH-induced activation of the pre-BötC on
peak amplitude of integrated phrenic nerve discharge ( Phrenic) and
inspiratory duration (TI). Although DLH-induced activation
of the pre-BötC significantly increased peak amplitude of
integrated phrenic nerve discharge and TI, no differences
were noted between trials. * Significant difference
(P < 0.05) from preinjection baseline values; ns, no
significant difference.
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Effects of modulation of CO2 on the DLH-induced
response.
I examined the effects of altering inspired CO2 on phrenic
nerve activity in response to DLH-induced activation of the
pre-BötC. Responses from 17 sites were examined during
hypercapnia, and responses from 11 sites were examined during
hypocapnia. Arterial blood gases and pH, which were measured
immediately preceding unilateral microinjection of DLH into the
pre-BötC during normocapnia (baseline), hypercapnia, and
hypocapnia, are provided in Table 1, and
an example of the results obtained from one experiment examining the
effects of both hypercapnia and hypocapnia on DLH-induced activation of
the pre-BötC is provided in Fig. 2.
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Fig. 2.
Example demonstrating the effects of DLH-induced
activation of the pre-BötC during modulation of CO2.
A: during hyperoxic normocapnia (i.e., baseline),
microinjection of DLH into the pre-BötC evoked a nonphasic tonic
excitation of phrenic nerve discharge. ipsi, Ipsilateral. B:
increasing the fraction of inspired CO2 (5%
CO2) increased the amplitude of phrenic nerve discharge,
and subsequent microinjection of DLH produced a tonic excitation of
phrenic nerve discharge in which phasic respiratory bursts were
superimposed. These phasic bursts had an increased frequency compared
with the preinjection baseline frequency. C and
D: increasing the rate of the ventilator decreased phrenic
neurogram amplitude (C), with further increases in rate
producing phrenic apnea (D). During hypocapnia,
microinjection of DLH produced a tonic excitation of phrenic nerve
discharge, which was associated with no phasic respiratory bursts.
E: after a return to normocapnic levels (i.e., recovery),
microinjection of DLH evoked a nonphasic tonic excitation of phrenic
nerve discharge. Note that in this experiment, phasic phrenic bursts
returned as tonic activity began to wane.
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In these experiments, during normocapnia, unilateral microinjection of
DLH into the pre-BötC produced a nonphasic tonic excitation of
phrenic nerve discharge in each of the sites examined (as described above). Hypercapnia elicited an increase in the amplitude of phrenic bursts with little or no effect on burst frequency (i.e., preinjection baseline). During hypercapnia, similar microinjection of DLH into the
same sites in the pre-BötC produced excitation of phrenic nerve
discharge in which phasic bursts were superimposed on varying levels of
tonic activity. The onset latency for DLH-induced excitation of phrenic
nerve discharge was similar during normocapnia and hypercapnia
(P > 0.05), with responses being observed within
1-3 s from the beginning of microinjection.
Data were obtained during either moderate or severe hypercapnia, which
was produced by adding 5 or 7% CO2 to the inspired gas,
respectively. Examples demonstrating the effects of moderate and/or
severe hypercapnia on the DLH-induced response are provided in Figs. 2
and 3. Regardless of the severity of the
hypercapnic challenge, microinjection of DLH into the pre-BötC
during hypercapnia modified the DLH-induced response, such that it
included phasic phrenic bursts. In contrast, the level of DLH-induced
tonic activity appeared to be dependent on the severity of the
hypercapnic challenge, such that higher levels of CO2 were
accompanied by lower levels of underlying tonic discharge. Examples
demonstrating the effect of the severity of hypercapnia on the
magnitude of the DLH-induced underlying tonic activity during both
moderate and severe hypercapnia can be seen in Fig. 3 (also see Fig.
2B for effects of moderate hypercapnia). In addition, the
duration of the DLH-induced tonic excitation also appeared to be
dependent on the severity of the hypercapnic challenge, such that
shorter durations of underlying tonic discharge were observed at higher
levels of CO2 (Fig.
4A). Summary data illustrating
these effects of moderate (n = 10) and severe
(n = 7) hypercapnia on the DLH-induced changes in peak amplitude and TI of the tonic component of integrated
phrenic nerve discharge are provided in Fig. 4A. For these
experiments, comparisons were made between the DLH-induced responses
during normocapnia and hypercapnia as well as between the two different levels of hypercapnia.
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Fig. 3.
Examples demonstrating the effects of DLH-induced
activation of the pre-BötC during moderate (A) and
severe (B) hypercapnia. A1 and B1:
during hyperoxic normocapnia (i.e., baseline), unilateral
microinjection of DLH into the pre-BötC produced a nonphasic
tonic excitation of phrenic nerve discharge. A2 and
B2: increasing the fraction of inspired CO2 (5%
CO2 in A2; 7% CO2 in B2)
increased the amplitude of phrenic nerve discharge, and subsequent
microinjection of DLH produced a lower level of tonic excitation of
phrenic nerve discharge in which phasic respiratory bursts were
superimposed. These phasic bursts had an increased frequency compared
with the preinjection baseline frequency. Dashed line in traces of
integrated phrenic nerve discharge identifies the nadir of phrenic
nerve activity. Note that the magnitude of underlying tonic activity in
B2 was reduced to a greater extent than that observed in
A2.
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Fig. 4.
Summary data illustrating the effects of DLH-induced
activation of the pre-BötC during modulation of CO2
on tonic excitation of phrenic nerve discharge. A: during
both moderate and severe hypercapnia, microinjection of DLH
significantly reduced the amplitude of the tonic component of the
integrated phrenic nerve discharge. The reduction in the DLH-induced
tonic excitation was more pronounced during severe hypercapnia. In
addition, during severe hypercapnia, the duration of the DLH-induced
tonic excitation was significantly shorter than during normocapnia.
* Significant difference (P < 0.05) from the
response observed during normocapnia and between moderate and severe
hypercapnic trials. B: during hypocapnia, microinjection of
DLH significantly reduced the amplitude of integrated phrenic nerve
discharge and produced a slight, but not statistically significant,
increase in TI. * Significant difference
(P < 0.05) from the DLH-induced response during
normocapnia.
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Although microinjection of DLH into the pre-BötC during
hypercapnia elicited phasic phrenic bursts, both the amplitude and the
timing of these phasic bursts were different from those recorded during
the preinjection baseline. This DLH-induced modulation appeared to be
independent of the severity of the hypercapnic challenge, and therefore
these data have been combined for statistical analyses. Summary data
illustrating the DLH-induced changes in peak amplitude of integrated
phasic phrenic nerve discharge, frequency of phasic phrenic bursts,
TI, and TE for all 17 of the hypercapnia experiments are provided in Fig. 5. In
general, microinjection of DLH into the pre-BötC elicited a small
increase in the peak amplitude of integrated phasic phrenic nerve
activity (Fig. 5A; P < 0.05), although a
decrease in the peak amplitude of integrated phasic phrenic nerve
activity was observed in 5 of the 17 sites examined (including the
responses shown in Figs. 2B and 3A2). In
addition, the DLH-induced phasic bursts had an increased frequency compared with the preinjection baseline frequency (Fig. 5B;
P < 0.01). This increased frequency resulted
predominantly from a reduction in TE (Fig. 5D;
P < 0.001) although a decrease in TI (Fig.
5C; P < 0.05) was also observed in 14 of
the 17 experiments.
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Fig. 5.
Summary data illustrating the effects of DLH-induced
activation of the pre-BötC during hypercapnia on the timing and
patterning characteristics of phrenic nerve discharge. During
hypercapnia (PaCO2 = 59.7 ± 2.8 mmHg),
microinjection of DLH significantly increased the amplitude of
integrated phrenic nerve discharge (A) and the frequency of
phasic phrenic bursts (B) and reduced both TI
(C) and expiratory duration (TE; D).
* Significant difference (P < 0.05) from
preinjection baseline values.
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I also examined the effects of hypocapnia on the DLH-induced response
in 11 sites in the pre-BötC. For these experiments, inspired
CO2 was reduced by increasing the rate of the ventilator. In two experiments, the level of CO2 was reduced to produce
depression of phrenic nerve activity of 40-50% and then the level
of CO2 was further reduced to produce phrenic apnea (i.e.,
apneic threshold). An example of the results obtained from one of these
experiments is provided in Fig. 2. In the remaining experiments, the
level of CO2 was reduced to produce phrenic apnea. During
phrenic apnea, spurious action potential discharges were observed in
some cases.
In these experiments, during normocapnia, unilateral microinjection of
DLH into the pre-BötC produced a nonphasic tonic excitation of
phrenic nerve discharge in each of the sites examined (as described above). During hypocapnia, similar microinjection of DLH into the same
sites in the pre-BötC also produced a nonphasic tonic excitation
of phrenic nerve activity; however, this response was less robust than
the initial (normocapnic) DLH-induced response (Fig. 2). In all
experiments, there was an attenuation of the DLH-induced increase in
peak amplitude of integrated phrenic nerve discharge compared with the
DLH-induced response during normocapnia (P < 0.05). In
addition, two patterns (or types) of nonphasic tonic excitation of
phrenic nerve discharge were observed during hypocapnia-induced apnea:
1) an abrupt rise in phrenic nerve discharge to a plateau
level (similar to that seen during normocapnia; depicted in Fig.
2D) and 2) a gradual increase in phrenic nerve
discharge, which peaked at ~15-20 s after the beginning of
microinjection (not shown). It should be noted that although
variability was observed in the latency to peak amplitude under these
conditions, the onset latency for DLH-induced excitation of phrenic
nerve discharge was similar during normocapnia and hypocapnia
(P > 0.05), with responses being observed within
1-3 s from the beginning of microinjection. In contrast, the
duration of the DLH-induced tonic excitation during hypocapnia was
quite variable. Overall, during hypocapnia there was a small increase
in the DLH-induced prolongation of TI compared with the
DLH-induced response during normocapnia; however, this modulation of
TI during hypocapnia was not statistically significant
(P = 0.053; n = 11). In seven of these
experiments, there was an increase in the DLH-induced prolongation of
TI (mean ± SE, 164 ± 87%; range, 16-760%
above normocapnic duration), while in two experiments, there was a
decrease in the DLH-induced prolongation of TI (23 and 63%
below normocapnic duration). In the remaining two experiments, there
was no detectable difference in the DLH-induced prolongation of
TI between the hypocapnic and normocapnic trials. Summary
data describing the DLH-induced changes in peak amplitude and
TI of integrated tonic phrenic nerve discharge for all 11 of the hypocapnia experiments are illustrated in Fig. 4B.
For these experiments, comparisons were made between the DLH-induced
responses during normocapnia and hypocapnia.
Effects of modulation of O2 on the DLH-induced
response.
I examined the effects of altering inspired O2 on phrenic
nerve activity in response to DLH-induced activation of the
pre-BötC. Responses from eight sites were examined during
systemic hypoxia (i.e., peripheral chemoreflex). Arterial blood gases
and pH immediately preceding unilateral microinjection of DLH into the
pre-BötC during hyperoxia (baseline) and hypoxia are provided in
Table 2, and an example of the results
obtained from one of these experiments is provided in Fig.
6.
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Fig. 6.
Example demonstrating the effects of DLH-induced
activation of the pre-BötC during systemic hypoxia (i.e.,
peripheral chemoreflex). A1: during hyperoxic normocapnia
(i.e., baseline), microinjection of DLH into the pre-BötC evoked
a nonphasic tonic excitation of phrenic nerve discharge. A2:
decreasing the fraction of inspired O2 (12%
O2) increased the amplitude of phrenic nerve discharge, and
subsequent microinjection of DLH produced a tonic excitation of phrenic
nerve discharge in which phasic respiratory bursts were superimposed.
These phasic bursts had an increased frequency compared with the
preinjection baseline frequency, and some of these bursts exhibited a
high-amplitude, short-duration burst component. A3: after
reoxygenation (i.e., recovery), microinjection of DLH evoked a
nonphasic tonic excitation of phrenic nerve discharge. B:
expanded time scale demonstrating the augmented burst pattern often
elicited in response to microinjection of DLH into the pre-BötC
during hypoxia from 2 different experiments. Data shown in
B1 come from the response shown in A2.
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In these experiments, during hyperoxic normocapnia, unilateral
microinjection of DLH into the pre-BötC produced a nonphasic tonic excitation of phrenic nerve discharge in each of the sites examined (as described above). Systemic hypoxia (12-14%
O2) elicited an increase in the amplitude of phrenic bursts
with little or no effect on burst frequency (i.e., preinjection
baseline). During systemic hypoxia, similar microinjection of DLH into
the same sites in the pre-BötC produced excitation of phrenic
nerve discharge in which phasic bursts were superimposed on varying
levels of tonic activity. The onset latency for DLH-induced excitation
of phrenic nerve discharge was similar during hyperoxic normocapnia and
systemic hypoxia (P > 0.05), with responses being
observed within 1-3 s from the beginning of microinjection. In all
experiments, microinjection of DLH into the pre-BötC during
systemic hypoxia modified the DLH-induced response, such that it
included phasic phrenic bursts. Although microinjection of DLH into the
pre-BötC during systemic hypoxia elicited phasic phrenic bursts,
the patterning of these phasic phrenic bursts was quite variable and
typically included a high-amplitude, short-duration burst component,
such as those associated with the augmented burst (i.e., sigh) pattern. Examples of augmented bursts produced in response to microinjection of
DLH into the pre-BötC during hypoxia are presented in Fig. 6,
with Fig. 6B providing an expanded time scale. It should be noted that the augmented bursts shown in Fig. 6B1 are a
subset of bursts from the response illustrated in Fig. 6A2,
while the augmented bursts shown in Fig. 6B2 were obtained
from an experiment in a different animal (full response not shown).
In addition to the patterning changes noted, the timing of these bursts
was also different from those recorded during the preinjection
baseline. Summary data illustrating the DLH-induced changes in peak
amplitude of integrated phasic phrenic nerve discharge, frequency of
phasic phrenic bursts, TI, and TE for all eight
of the systemic hypoxia experiments are illustrated in Fig.
7. Overall, microinjection of DLH into
the pre-BötC during systemic hypoxia was ineffective in changing
the peak amplitude of integrated phasic phrenic nerve activity (Fig.
7A; P > 0.05). It should be noted that a
decrease in the peak amplitude of integrated phasic phrenic nerve
activity was observed in five of the eight sites examined (including
the response shown in Fig. 6A), while an increase in the
peak amplitude of integrated phasic phrenic nerve activity was observed
in the remaining three sites examined. It should also be noted that the
high-amplitude, short-duration burst components are not included in the
measurement of the peak amplitude of integrated phrenic nerve
discharge. The DLH-induced phasic bursts had an increased frequency
compared with the preinjection baseline frequency (Fig. 7B;
P < 0.01). This increased frequency resulted from a reduction in both TE (Fig. 7D; P < 0.01) and TI (Fig. 7C; P < 0.01).
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|
Fig. 7.
Summary data illustrating the effects of DLH-induced
activation of the pre-BötC during systemic hypoxia (i.e.,
peripheral chemoreflex) on the timing and patterning characteristics of
phrenic nerve discharge. Microinjection of DLH during systemic hypoxia
(PaO2 = 38.4 ± 4.4 mmHg) significantly
increased the phrenic burst frequency (B) and reduced both
TI (C) and TE (D). No
significant changes were observed in the amplitude of integrated
phrenic nerve discharge (A). * Significant difference
(P < 0.05) from preinjection baseline values.
|
|
Location of injection sites.
The distribution of sites in which DLH was microinjected into the
pre-BötC is shown in Fig. 8. As
landmarks for identifying the rostrocaudal level of the pre-BötC,
I identified the caudal pole of the retrofacial nucleus, nucleus
ambiguus, the rostral pole of the lateral reticular nucleus, and the
rostral pole of the hypoglossal nucleus. All sites in the
pre-BötC were identified with reference to the caudal pole of the
retrofacial nucleus.
View larger version (29K):
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Fig. 8.
Schematic drawing of coronal sections of the medulla showing
location of pre-BötC sites that received microinjection of DLH
during trials demonstrating repeatability of the DLH-induced response
(A) and trials examining the effects of modulation of
CO2 and O2 on the DLH-induced response
(B). All sites in the pre-BötC are identified with
reference to the caudal pole of the retrofacial nucleus (0 mm). Each
section is meant to encompass level indicated ±0.2 mm (rostrally and
caudally). RFN, retrofacial nucleus; NA, nucleus ambiguus; LRN, lateral
reticular nucleus; 5SP, spinal nucleus of the trigeminal nerve; 5ST,
spinal tract of the trigeminal nerve; ION, inferior olivary
nuclei; P, pyramidal tract.
|
|
Histological analyses revealed that all microinjection sites
functionally identified as pre-BötC were located within the anatomic boundaries described for the pre-BötC in adult cat
(6, 18, 21, 28). The microinjection sites were located
within 320 µm (i.e., 0-320 µm caudal) of the caudal pole of
the retrofacial nucleus in the rostrocaudal plane, 3.72-4.04 mm
lateral to midline in the mediolateral plane, and 4.20-4.36 mm
ventral to the dorsal surface of the medulla in the dorsoventral plane.
No differences were detected in the location of sites used for
experiments examining repeatability of the DLH-induced response (Fig.
8A) vs. those examining the effects of modulation of
CO2 and hypoxia on the DLH-induced response (Fig.
8B).
| |
DISCUSSION |
In the present study, I have demonstrated that modulation of
respiratory network drive alters the phrenic motor output response elicited by DLH-induced activation of the pre-BötC in vivo. I have shown that although chemical stimulation of the pre-BötC under hyperoxic normocapnic conditions can produce a tonic (nonphasic) excitation of phrenic nerve discharge, during increased respiratory network drive, produced by either hypercapnia or hypoxia, chemical stimulation of this region elicits phasic phrenic bursts that are
superimposed on varying levels of tonic phrenic nerve discharge. Furthermore, these phasic phrenic bursts exhibit an increased burst
frequency compared with the preinjection baseline burst frequency.
Conversely, during decreased respiratory network drive produced by
hypocapnia, chemical stimulation of the pre-BötC evokes only a
low-amplitude nonphasic tonic increase in phrenic nerve discharge. To
my knowledge, this is the first demonstration that
pre-BötC-induced excitation of phrenic motor output can be
modulated by alterations in respiratory network drive. I interpret these findings to suggest that phrenic motor activity evoked by chemical stimulation of the pre-BötC is influenced by and
integrates with modulation of respiratory network drive mediated by
input from central and peripheral chemoreceptors.
Limitations of the current study.
There are four main limitations in the current investigation. First,
the effects of altered respiratory network drive on the phrenic nerve
discharge responses evoked by DLH-induced activation of the
pre-BötC were examined in chloralose-anesthetized adult cats.
Therefore, the patterns of evoked phrenic nerve activity observed in
the current investigation (as well as in our previous study; 28) may
have been influenced by the effects of anesthesia. It should be noted
that anesthesia has been previously demonstrated to depress respiratory
network activity due to enhanced inhibitory synaptic interactions; thus
it remains to be determined whether alterations in respiratory network
drive would similarly modify DLH-induced pre-BötC-mediated
phrenic nerve discharge responses in the unanesthetized (awake and/or
decerebrate) state.
Second, because respiratory network drive was altered by systemic
modulation of CO2 and O2 in the current
experiments, neuronal excitability within the pre-BötC may have
been modified by either direct or indirect mechanisms. Systemic
hypercapnia and systemic hypoxia are well known to activate central and
peripheral chemoreceptors, resulting in increased excitatory synaptic
input to the brain stem respiratory centers. In addition, in in vitro
transverse medullary slices obtained from neonatal mice (postnatal
days 0-22), an increase in the amplitude of excitatory
synaptic drive potentials has been demonstrated in ~50% of
pre-BötC inspiratory neurons in response to hypoxia
(17). Although increased excitatory synaptic input
mediated by enhanced release of excitatory neurotransmitters and
neuromodulators in the pre-BötC can explain modulation of neuronal excitability within the pre-BötC during systemic
hypercapnia and systemic hypoxia, recent studies have demonstrated
intrinsic CO2/H+ and hypoxic chemosensitivity
within the pre-BötC (11, 29, 31, 33), and
presumptive rhythmogenic pacemaker neurons appear to be the
CO2/H+- and hypoxia-chemosensitive neurons in
this region (11, 33). The direct effects of focal
modulation of CO2/H+ and hypoxia in the
pre-BötC on DLH-induced responses, however, were not assessed in
the current experiments; therefore, the contribution of intrinsic
pre-BötC chemosensitivity to modulation of the DLH-induced responses observed is unknown. It should be noted that during severe
hypoxia that is sufficient to produce gasping, microinjection of DLH
into functionally identified (during hyperoxic normocapnia) nonphasic
tonic sites in the pre-BötC produces frequency modulation of
gasplike phrenic bursts (27). The level of hypoxia used in the current experiments, however, was not severe (which appears to be a
prerequisite for intrinsic hypoxic chemosensitivity in the in vivo
pre-BötC; 29), and therefore, it is unlikely that intrinsic
hypoxic chemosensitivity played a significant role in modulation of the
DLH-induced responses observed (i.e., increased excitatory synaptic
inputs would be primarily responsible). From the current experiments,
however, I cannot exclude the possibility that intrinsic
CO2/H+ and/or hypoxic chemosensitivity within
the pre-BötC played a role, at least in part, in the modulation
of the DLH-induced responses observed.
Third, although the pre-BötC is a bilateral structure, activation
of this region was restricted to a unilateral microinjection; thus the
effects of bilateral activation of the pre-BötC on phrenic nerve
discharge (during hyperoxic normocapnia and altered respiratory network
drive) were not examined. I believe, however, that the modulation of
the DLH-induced responses observed in these experiments was specific to
chemical stimulation of the pre-BötC and did not result from
spread of DLH to the adjacent Bötzinger complex (BötC) or
rostral ventral respiratory group (rVRG). Although I cannot exclude the
possibility of spread, I suggest that this is unlikely. In our previous
experiments, we have demonstrated that nonphasic tonic excitation of
phrenic nerve discharge and modulation of phrenic burst frequency were
only observed in response to chemical stimulation of the pre-BötC
(28), while similar activation of the adjacent BötC
and rVRG regions has been shown to elicit changes in only amplitude of
phrenic nerve discharge (3, 5, 13, 14, 28). Furthermore,
preliminary data suggest that increased respiratory network drive,
produced by hypercapnia, attenuates the changes in amplitude of phrenic
nerve discharge elicited by DLH-induced activation of these adjacent regions (unpublished observations). I cannot exclude, however, the
possibility that microinjection of DLH had an effect on dendrites whose
cell bodies were distant from the site of injection.
Finally, for the current experiments, I only selected sites in the
pre-BötC in which microinjection of DLH produced a nonphasic tonic excitation of phrenic nerve discharge under hyperoxic,
normocapnic conditions. Although I often encountered sites in which
DLH-induced activation of the pre-BötC evoked other patterns of
excitation of phrenic nerve discharge (as previously reported; 28),
these sites were avoided because the DLH-induced response already
included modulation of phrenic burst frequency. The sites not included in the current investigation were predominantly those in which DLH-induced excitation of phrenic nerve activity included a
high-amplitude, short-duration burst component. When this type of
response was encountered, the microinjection pipette was moved
200-300 µm rostral, as this response type is generally obtained
from sites caudal to those in which tonic discharge is evoked
(16). Thus, whether respiratory network drive influences
these other pre-BötC-mediated DLH-induced patterns of excitation
of phrenic nerve discharge remains to be determined.
Chemical activation of the pre-BötC in vivo.
Previous in vivo studies, including work from our laboratory, have
demonstrated an increase in the frequency of inspiratory bursts
(5, 14, 26, 28, 34) as well as tonic (nonphasic) excitation of inspiratory motor activity (26, 28) in
response to chemical stimulation of this region; however, these studies did not assess the effects of pre-BötC activation during
alterations in respiratory network drive. In our previous experiments
in the anesthetized cat, we reported that microinjection of DLH into the pre-BötC under hyperoxic normocapnic conditions elicited multiple patterns of excitation of phrenic nerve discharge, including tonic excitation with phasic respiratory bursts superimposed
(28). Although in our previous experiments the cats were
maintained normocapnic during the experimental protocol, the apneic
threshold was not identified. Thus one possible explanation for our
previous finding is that in some of our experiments, the cats may have been farther away from their apneic threshold, and therefore, overall
respiratory network drive may have been relatively higher in those
animals. It should be noted, however, that in sites in which
DLH-induced activation of the pre-BötC produces tonic excitation with phasic respiratory bursts superimposed during hyperoxic
normocapnia, reducing CO2 in some of these animals shifts
the DLH-induced response to a nonphasic tonic excitation (unpublished
observations), consistent with the effects of reducing CO2
in the present investigation.
Although multiple patterns of excitation of phrenic nerve discharge
have been elicited by chemical stimulation of the pre-BötC in
vivo (5, 14, 28), the precise mechanism(s) by which focal
pre-BötC activation produces phasic vs. tonic excitation of
phrenic nerve discharge remain to be resolved. In our previous experiments, histological analyses revealed some degree of site specificity for some patterns of DLH-induced phasic activity (i.e., rapid series of high-amplitude, rapid rate of rise, short-duration bursts), but our histological analyses could not distinguish sites in
which DLH-induced activation produced nonphasic tonic discharge from
those that produced tonic discharge with phasic bursts superimposed (28). In the current experiments, DLH-induced activation
of a single site in the pre-BötC during increased respiratory
network drive (i.e., hypercapnia and hypoxia) elicited modulation of
phasic phrenic burst frequency, which was not observed in response to similar activation during either baseline conditions (i.e., hyperoxic normocapnia) or hypocapnia; thus modulation of respiratory network drive appears to be one mechanism capable of influencing the response type evoked by repeated activation of a single site in the
pre-BötC. The current experiments, however, did not investigate
the precise cellular mechanism(s) within the pre-BötC responsible
for this modulation of the DLH-induced response.
DLH-induced tonic (nonphasic) phrenic nerve discharge.
In our previous experiments, we suggested that nonphasic tonic
excitation of phrenic nerve discharge in response to activation of the
pre-BötC may result from a shift in the membrane potential of
presumptive rhythmogenic pre-BötC neurons from a level of quiescent or phasic (or oscillatory bursting) activity to a more depolarized level, leading to tonic (or beating) action potential generation, a response similar to that observed by depolarizing the
voltage-dependent pacemaker cells identified in the neonatal rodent
pre-BötC (8, 12, 20, 24, 25). The pre-BötC in
adult cat and rat, however, is characterized by a mixture of neurons
exhibiting inspiratory-modulated, expiratory-modulated, and
phase-spanning (including preinspiratory) discharge patterns (6,
10, 21, 32) and appears to contain neuronal elements that may be
essential for respiratory rhythm generation (9, 18).
Therefore, other possible explanations for the production of nonphasic
tonic excitation of phrenic nerve discharge in response to activation
of the pre-BötC must be considered. I suggest that nonphasic
tonic excitation may, alternatively, result from DLH-induced stimulation of multiple classes of respiratory-modulated neurons located in this region, which in turn could lead to a reduction or loss
of the synchronized phasic neuronal activity required for the
generation or expression of phasic phrenic bursts. This explanation
does not require that the phasic (or rhythmic) activity of the
presumptive rhythmogenic neurons be abolished but does suggest that the
phasic (or rhythmic) activity of presumptive rhythmogenic
pre-BötC neurons may not be sufficient (or adequately synchronized) to allow for expression of phasic phrenic motor activity.
This may be similar to the ectopic bursts observed between phasic
hypoglossal nerve discharges in most of the presumptive rhythmogenic
pacemaker neurons tested in the pre-BötC of neonatal rats
(postnatal days 0-3) in vitro (7). Thus,
if this prediction is correct, it could explain why during decreased
respiratory network drive (i.e., hypocapnia), microinjection of DLH
into the pre-BötC evoked nonphasic tonic phrenic nerve activity
instead of phasic discharge, which might be expected (based on the
hybrid "pacemaker-network" model of respiratory rhythm
generation; 4, 7, 8, 12, 20, 23, 25). It is possible, however, that
nonphasic tonic excitation, which was observed during both normocapnia
and hypocapnia, may result from a combination of the above effects. From the current experiments, it is not clear which, if any, of these
potential mechanisms is responsible for the production of nonphasic
tonic excitation of phrenic nerve discharge in response to DLH-induced
activation of the pre-BötC.
It should also be noted that the above explanations for DLH-induced
generation of nonphasic tonic phrenic nerve discharge assume that the
tonic discharge is not masking or occluding an underlying phasic
phrenic nerve discharge. I do not believe that the DLH-induced
"nonphasic" tonic excitation of phrenic nerve activity occludes
phasic motoneuron output because in our previous experiments
1) DLH-induced nonphasic tonic excitation of phrenic nerve
discharge was associated with both increased and decreased peak
amplitude of tonic phrenic nerve discharge compared with peak amplitude
of preinjection baseline phasic phrenic nerve discharge (26, 28,
29); 2) DLH-induced nonphasic tonic excitation was
observed simultaneously in both inspiratory and expiratory motor
outputs (26); and 3) DLH-induced nonphasic
tonic excitation suppressed both the phasic (inspiratory modulated) and
tonic components of sympathetic nerve discharge recorded from the
preganglionic cervical sympathetic nerve (30).
Modulation of DLH-induced phrenic nerve discharge by increased
respiratory network drive.
During increased respiratory network drive produced by hypercapnia or
hypoxia, DLH-induced activation of the pre-BötC would presumably
affect the same populations of neurons described above. Under these
conditions, however, stimulation of the pre-BötC could elicit a
different effect on phrenic nerve discharge because changes in the
strength of synaptic interactions and/or changes in activity of a
population of respiratory neurons can modify respiratory rhythm and
pattern (2, 15). In the current experiments, microinjection of DLH into the pre-BötC during increased
respiratory network drive elicited predominantly frequency modulation
of phasic phrenic nerve discharge. At first glance, it appears that
these data are inconsistent with the hybrid pacemaker-network model of
respiratory rhythm generation (4, 7, 8, 12, 20, 23, 25).
Based on this model, it might be expected that during increased
respiratory network drive, microinjection of DLH into the
pre-BötC would evoke tonic, not phasic, phrenic nerve discharge (see explanation above). I do not believe that these data are inconsistent with the proposed hybrid pacemaker-network model of
respiratory rhythm generation but may reflect a wide dynamic range of
neuronal excitability of presumptive rhythmogenic pre-BötC neurons (7) available for modulation of phrenic burst
frequency. It should be noted, however, that in the adult anesthetized
cat, "pacemaker" cells have not been identified nor has the
behavior of individual presumptive rhythmogenic pre-BötC neurons
been examined in response to increased excitability within the
pre-BötC.
With respect to the current findings, I suggest that during increased
respiratory network drive, the presumptive rhythmogenic pre-BötC
neurons are closer to threshold for eliciting a rhythmogenic (i.e.,
frequency modulation) response to DLH-induced activation of the
pre-BötC. Further, under these conditions, DLH-induced activation
of this region elicits less of a contribution of the respiratory-modulated, nonrhythmogenic pre-BötC neurons because these neurons are in a more excited state due to increased synaptic drive (a similar mechanism of reduced contribution to neuronal excitation might also explain the effects observed in response to
DLH-induced activation on adjacent regions during hypercapnia; see
above). If this prediction is correct, I would expect to see both
frequency modulation of phasic phrenic bursts and a reduction in the
level of tonic phrenic nerve discharge in response to microinjection of
DLH into the pre-BötC during increased respiratory network drive.
In the current experiments, I found that during increased respiratory
network drive, microinjection of DLH into the pre-BötC consistently added a phasic component, which included frequency modulation, to varying levels of tonic phrenic nerve discharge. Furthermore, with higher levels of respiratory network drive, such as
that produced by severe hypercapnia, the level of DLH-induced underlying tonic phrenic nerve discharge was substantially reduced.
In conclusion, I interpret the current findings to suggest
that the basic rhythm-generating circuitry located in the
pre-BötC (20, 23, 24) is still responsive to
activation during increased respiratory network drive and that the
predominant response under these conditions is modulation of frequency
of phrenic bursts. Furthermore, I suggest that the response elicited by
activation of the pre-BötC results not only from the level of
intrinsic excitability of presumptive rhythmogenic pre-BötC
neurons but also the level of rhythmic synaptic drive to other classes
of inspiratory-modulated neurons (including nonrhythmogenic
pre-BötC neurons) and the interaction of presumptive rhythmogenic
pre-BötC neurons with these other respiratory-modulated neurons.
In summary, the findings of the current investigation demonstrate that
the phrenic motor output responses evoked by chemical stimulation of
the pre-BötC in vivo are strongly modulated by the excitatory
state of the respiratory network. I suggest that increased respiratory
network drive increases neuronal excitability of presumptive
rhythmogenic pre-BötC neurons and enhances rhythmic synaptic
drive to other classes of inspiratory-modulated neurons (including
nonrhythmogenic pre-BötC neurons), resulting in modulation of the
pre-BötC-mediated DLH-induced response. The findings further indicate that the pre-BötC has the potential to play a role in frequency modulation of phrenic motor output during increased respiratory network drive because chemical stimulation of this region
under these conditions elicits an increase in frequency of phrenic
bursts. Thus this study provides additional in vivo evidence for a role
of this region in modulation of phasic respiratory activity.
| |
ACKNOWLEDGEMENTS |
The author thanks T. J. Halat for excellent technical assistance.
| |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-63175.
Address for reprint requests and other correspondence:
I. C. Solomon, Dept. of Physiology and Biophysics,
Basic Science Tower T6 Rm. 140, State Univ. of New York at Stony Brook,
Stony Brook, NY 11794-8661 (E-mail:
ICSolomon{at}physiology.pnb.sunysb.edu).
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.
10.1152/ajpregu.00395.2002
Received 1 July 2002; accepted in final form 17 September 2002.
| |
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