| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of 1 Physiology and 2 Medicine, Dartmouth Medical School, Lebanon, New Hampshire 03756
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The rostral ventral medulla (RVM) may be important in the control of cardiorespiratory interactions. We hypothesized that inhibition of the RVM would enhance inhibition of breathing associated with transient blood pressure elevations. In 25 piglets 3-16 days of age, we studied the effect of acutely increasing blood pressure, by systemic infusion of phenylephrine, on respiratory activity before and after inhibition of neural activity in the RVM by dialysis of 10 mM muscimol, a GABAA-receptor agonist. Muscimol dialysis through probes that were placed along the ventral medullary surface from ~1 mm rostral to the facial nucleus to ~0.5 mm caudal to the facial nucleus augmented the respiratory inhibition associated with acute increases in blood pressure. No similar enhancement of respiratory inhibition after phenylephrine treatment was seen in six control animals that did not receive muscimol dialysis. We conclude that the piglet RVM participates in cardiorespiratory interactions and that dysfunction of homologous regions in the human infant could lead to cardiorespiratory instability and may be involved in the pathogenesis of sudden infant death syndrome.
phrenic nerve; sudden infant death syndrome; parapyramidal nucleus; caudal raphé; retrotrapezoid nucleus; nucleus paragigantocellularis lateralis
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
THE SUDDEN INFANT DEATH SYNDROME (SIDS) is the most common cause of death in infants <1 yr of age in the developed world. In the United States, the present rate of SIDS is ~0.8 deaths per 1,000 births. The most widely accepted putative cause of SIDS is failure of respiratory or cardiovascular control during sleep. However, the mechanisms or triggers for such catastrophic failure are unclear. Recently, specific developmental abnormalities have been described in the ventral medulla of SIDS victims (12, 19, 22, 27, 28). These ventral medullary structures in the infant may be homologous with chemosensitive areas of the medulla in animals responsible for respiratory and cardiovascular control (11).
Control of the cardiovascular and respiratory systems is closely linked, both anatomically and physiologically. Acute increases in blood pressure inhibit breathing (2, 9, 29, 33), probably via carotid and aortic baroreceptor stimulation (9). However, the regions of the central nervous system (CNS) mediating cardiorespiratory interactions are not fully established. The ventral medulla contains areas involved in the control of cardiovascular and respiratory output. Some studies suggest that the rostroventrolateral medulla (RVLM), which contains the C1 region, may be the site of cardiorespiratory interactions (4, 8, 15). The pattern of activation of the RVLM may depend in turn on activity of the caudal ventrolateral medulla (CVLM) (31). We have been studying the effect of inhibition of a more rostral region of the ventral medulla, the rostroventral medulla (RVM), on ventilation and sleep (5-7). Inhibition of this region reduces the ventilatory response to CO2 (5, 6, 25) and hypoxia (25) and may reduce the ventilatory output during wakefulness (24) and sleep (26). We defined the RVM as a region within 1.2 mm of the ventral surface that extends rostocaudally the length of the facial nucleus and mediolaterally ~1.5-4.0 mm from the midline. This area encompasses the retrotrapezoid nucleus, the parapyramidal region, and juxtafacial parts of the nucleus paragigantocellularis lateralis (6). The RVM is, therefore, distinct from and rostral to the RVLM (the rostral extent of the RVLM is usually the caudal end of the facial nucleus). However, the caudal RVM does abut, and may overlap slightly, the rostral RVLM.
In the present study, we examined the effect of inhibition of the RVM on interactions between the respiratory and cardiovascular systems. We were particularly interested in the possibility that defects within the RVM may promote cardiorespiratory instability and contribute to the pathogenesis of SIDS. Therefore, we conducted our analysis of cardiorespiratory interactions in neonatal piglets. We subjected these animals to elevations in systemic blood pressure, which inhibits respiratory activity, before and after muscimol dialysis in the RVM. It was our hypothesis that elevating systemic blood pressure would more effectively inhibit respiration after dialysis of muscimol in the RVM.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Successful experiments were performed on a total of 31 piglets (3-16 days of age, 2.1-5.8 kg body wt). Piglets were housed with the sow in our on-site animal care facility. The Institutional Animal Care and Use Committee of Dartmouth College approved all protocols.
Surgery.
Animals were anesthetized with 2% halothane
(2-bromo-2-chloro-1,1,1-trifluoroethane; Halocarbon Laboratories) in
O2. Body temperature was monitored with a rectal probe and
maintained at 38-39°C using a heating pad and radiant heat
source. Femoral arterial and venous catheters were inserted for
measurement of blood pressure and administration of drugs,
respectively. Piglets were tracheostomized and artificially ventilated
(Dual-Phase Respirator, Harvard Apparatus, S. Natick, MA). The carotid
sinus regions were exposed bilaterally, and the internal and external
carotid arteries were ligated to facilitate decerebration. Care was
taken to avoid damaging the carotid sinus nerves. The vagus nerves were
sectioned bilaterally in the neck to prevent entrainment of the
respiratory activity of each animal to the ventilator. Each piglet was
placed prone with its head positioned in a stereotaxic apparatus (Kopf
Instruments, Tujunga, CA). After the skull was opened, each animal was
decerebrated at the level of the superior colliculus. Anesthesia was
discontinued after decerebration, and animals were paralyzed with
pancuronium bromide (1 mg/kg iv; Elkins-Sinn, Cherry Hill, NJ).
Supplemental doses of pancuronium bromide were given as required,
usually at a rate of 0.5 mg · kg
1 · h1. The phrenic
nerve was exposed in the neck, and a branch of the nerve was placed on
bipolar recording electrodes to monitor respiratory output. Peak
integrated phrenic nerve activity, body temperature, end-tidal
PCO2 and PO2, and blood
pressure were recorded on a computer-based data acquisition system
(PowerLab, ADI, Castle Hill, Australia) for later analysis.
Experimental protocol.
Throughout the experiment, the RVM was dialyzed at a rate of 8.5 µl/min with an artificial cerebrospinal fluid (aCSF) solution equilibrated with 5% CO2 (this dialysis flow rate caused
no volume transduction of fluid across the dialysis membrane). After
placement of the dialysis probe, 
60 min elapsed before any blood
pressure challenges were performed. When blood pressure and ventilatory variables were stable and unchanged for 10 min, a small dose of
sodium cyanide (0.1 mg/kg iv) was used to ensure that the carotid bodies were intact and innervated. A brief increase in peak integrated phrenic nerve amplitude and/or frequency >125% of control was taken
as evidence that the carotid bodies were intact. Absence of such a
response was taken as an indication of chemodenervation, and results
from two piglets were excluded from analysis on this basis.
-adrenergic-receptor agonist, to elevate
systemic blood pressure and elicit baroreflex inhibition of
respiration. We allowed a recovery period of 15 min after the sodium
cyanide test before testing the baroreflex, at which time each
animal's baseline responses to intravenous administration of
phenylephrine were measured. The response of individual animals to
phenylephrine differed; thus each animal was tested to determine the
dose required to produce an increase in mean arterial blood pressure
(MAP) of ~40-50 mmHg. MAP was calculated on-line using Chart
software (PowerLab). The responses after two or three infusions of
phenylephrine were recorded to determine the baseline response. Each
infusion was separated by 5 min, during which full recovery of blood
pressure and phrenic nerve activity occurred. After these control
studies, the aCSF in the dialysis syringe was replaced with 10 mM
muscimol in aCSF. Muscimol was dialyzed for 10 min before the muscimol
was replaced with aCSF alone, and dialysis was resumed to begin a
washout period. The phenylephrine challenges were repeated during the
washout phase of dialysis exactly as they had been during the initial
control period. The muscimol solution contained a small amount of fast
green, which allowed us to estimate the time at which the muscimol
solution reached the brain. Control animals were subjected to aCSF
dialysis throughout the study without muscimol, and the time course of
sham "before" and "after" trials remained the same as for test
animals. One control animal was not subjected to dialysis, but the time
course of phenylephrine challenges remained the same.
Neuroanatomy. On termination of the experiment, 20-50 µl of filtered 1% aqueous KMnO4 solution were microinjected through a dialysis probe with the tip broken back slightly to mark the site of dialysis (30). The brain stem was removed, and the ventral surface was photographed. The permanganate was often visible on the ventral surface of the brain and easily visible when the brain was sectioned. This facilitated identification of the position of the dialysis probe tip. A photograph of the KMnO4 on the ventral surface of the brain provided a permanent record of the site of muscimol microdialysis with respect to external brain stem landmarks (see Fig. 4). The external landmarks were correlated with the location in computer reconstructions and further analyses of cut sections.
Immediately after removal, the brain stem was placed in cryoembedding medium (Tissue-Tek OCT 458, Sakura Finetek, Torrance, CA) and frozen in isopentane at
70°C. The brain stem was cut into 20- to
50-µm sections in a cryostat at 18°C, and the sections were
mounted on gelatinized glass slides. Sections were fixed in formol
alcohol overnight and stained with cresyl violet (1, 21).
The rostrocaudal dimensions of the brain stem differed significantly
among piglets over the ages we studied, and coordinates expressed in
millimeters relative to the bregma or interaural line did not
accurately portray the location of probes with respect to ventral
medullary nuclei. Therefore, we decided to express the location of each
probe with respect to medullary structures, which we believe are the
anatomically relevant sites. The medullary nuclei move in a predictable
way relative to the external landmarks as animals age, and the dialysis
probes were placed according to a regression equation using animal age
and external landmarks (30). However, the position of the
dialysis probe within the brain stem was described with respect to the
caudal end of the facial nucleus and the midline and ventral surface of
the brain stem. The site of each dialysis probe was plotted on a grid,
which represented the average ventral surface area of the facial
nucleus (5), and the location of each dialysis probe was
expressed by three dimensions in millimeters: a normalized rostrocaudal dimension referenced to the caudal end of the facial nucleus, an
absolute mediolateral dimension referenced to the midline, and an
absolute dorsoventral dimension referenced to the ventral surface of
the brain stem (5, 6).
Data analysis and statistics. We measured the fractional end-tidal CO2 and integrated phrenic activity. The peak phrenic activity, instantaneous respiratory frequency, and minute phrenic activity (phrenic amplitude × respiratory frequency) of each breath were calculated from the integrated phrenic activity. Baseline respiratory activity was estimated from the 10 breaths preceding the phenylephrine infusion. The response to phenylephrine was determined from the relationship between peak MAP and ventilation. The peak MAP was related to the single breath that showed maximum inhibition after infusion of phenylephrine. The maximally inhibited breath was normally associated with the maximal level of blood pressure achieved and occurred within 5.8 ± 0.5 s after the onset of the blood pressure increase. If the maximal respiratory response occurred before the peak blood pressure after phenylephrine injection, the average blood pressure measured during a period equal to 150% of the time of the preceding breath was calculated as the blood pressure producing the response. The blood pressure response to phenylephrine declined more quickly than the respiratory response, and the average blood pressure over the entire respiratory response would have underestimated the value of the putative baroreceptor stimulus.
Data were analyzed using a two-way repeated-measures ANOVA in which muscimol (control vs. 10 mM dialysis) and phenylephrine (control vs. infusion) were within-subject factors (Systat 9.0, SPSS Science, Chicago, IL). When the ANOVA indicated that significant differences existed among groups (the F test was significant at P
0.05), we used Bonferroni's method to adjust
P values for multiple preplanned comparisons.
We performed cluster analysis to determine whether we could identify
neuroanatomic groupings of dialysis sites with similar respiratory
responses to phenylephrine after muscimol dialysis (Systat 9.0). In the
cluster analysis, we defined muscimol responsiveness as follows
|
![= <FENCE><FR><NU>[(f<SUB>cntrl</SUB> − f<SUB>PE</SUB>)<SUB>postmusc</SUB> − (f<SUB>cntrl</SUB> − f<SUB>PE</SUB>)<SUB>premusc</SUB>]</NU><DE>(f<SUB>cntrl</SUB> − f<SUB>PE</SUB>)<SUB>premusc</SUB></DE></FR></FENCE> × 100](/content/vol92/issue6/fulltext/2554/img002.gif)
|
0.05 for all possible
comparisons within a particular analysis. Scheffé's method is
the most stringent of tests used to make multiple comparisons and
protects against type I errors (incorrectly rejecting the null
hypothesis). Values are means ± SE.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Effect of phenylephrine on ventilation before and after muscimol.
The response of a single piglet to phenylephrine before and after
muscimol is shown in Fig. 1. In Fig.
1A, the integrated phrenic nerve activity, the end-tidal
CO2, and blood pressure are shown as a function of time.
Phenylephrine infusion increased the blood pressure, and associated
with the increase in blood pressure, the respiratory frequency slowed
and the amplitude of phrenic activity declined. Respiratory activity
recovered as the blood pressure returned to the control level. After
muscimol dialysis into the RVM at the caudal and medial aspect of the
facial nucleus, the respiratory frequency increased, and the inhibition
of respiration associated with phenylephrine was enhanced.
|
0.0001). The
level of MAP at baseline and after phenylephrine was ~5 mmHg less
after muscimol than during the control period (P = 0.003). However, the change in MAP after phenylephrine was similar
before and after muscimol dialysis. The average end-tidal
PCO2 was 38 Torr, and there was no change in
end-tidal PCO2 among the control and test
conditions (data not shown). We did occasionally observe a transient
fall in end-tidal PCO2 during the
phenylephrine-induced episodes of hypertension. We attributed this to
an acute decline in cardiac output and decreased delivery of
CO2 to the lungs. The fall in end-tidal
PCO2 was transient, not statistically
significant, and appeared to follow, rather than cause, the episodes of
respiratory inhibition.
Baroreceptor stimulation by phenylephrine infusion inhibited
respiratory activity in the control period (Fig.
2). Respiratory frequency fell
consistently after phenylephrine infusion (P < 0.001),
and phenylephrine treatment reduced the peak integrated phrenic
amplitude (P < 0.001). Finally, phenylephrine infusion decreased minute phrenic activity significantly (P < 0.001).
|
Time control for muscimol dialysis.
Muscimol has a long half-life binding to the GABAA
receptor. Therefore, muscimol dialysis always followed the control
study period, and muscimol treatment might have been confounded by a time effect or by the duration of dialysis with aCSF. To examine the
contribution of time alone to the "muscimol" effect, we performed control studies in six piglets. In five piglets, we continuously dialyzed the RVM with aCSF and studied the phenylephrine response during control and test periods that matched the times of the control
and muscimol tests described above. The dialysis probes in these
control animals were within the same ventral medullary region as the
actual muscimol dialysis experiments (see Fig. 5). In the remaining
piglet, we did not place a dialysis probe but followed the temporal
sequence of the muscimol experiments in all other respects. The average
responses of MAP, respiratory frequency, peak integrated phrenic
amplitude, and minute phrenic activity from these studies are shown in
Fig. 3. Baseline values of MAP,
respiratory frequency, phrenic amplitude, and minute phrenic activity
before phenylephrine administration were stable over the course of the
experiment. Phenylephrine increased MAP (P = 0.001) and
reduced respiratory frequency (P = 0.007) and minute phrenic activity (P = 0.007). The effects of
phenylephrine on these variables were similar in the first and second
time periods. The phrenic amplitude was similar among all conditions
tested. Thus neither time alone nor prolonged dialysis can account for the enhanced respiratory inhibition caused by phenylephrine infusion after muscimol dialysis.
|
Anatomic location of dialysis probes.
An example of the location of a dialysis probe is shown in a cross
section of the medulla in Fig. 4. The
locations of the dialysis probes in the muscimol and control
experiments are shown schematically in Fig.
5. The probes were placed over the
superficial aspect of the RVM. The region affected by the probes
extended rostrally 5.22 mm from the caudal pole of the facial nucleus
and 0.57 mm caudally from the end of the facial nucleus. It extended 0.90-4.88 mm medially from the midline and was 0.00-2.47 mm
deep from the ventral surface. Within this volume, probes were placed homogenously with respect to the rostrocaudal and mediolateral dimensions. Among the probes placed for muscimol dialysis experiments, two probes were >1.2 mm deep to the ventral surface, but the remaining 23 probes were located superficially within 1.2 mm of the ventral surface.
|
|
Variable responses to phenylephrine among piglets. We noted considerable variability among responses in different piglets. We investigated this variability in two ways: we analyzed the relationship of the magnitude of the response to phenylephrine infusion as a function of the neuroanatomic location of the dialysis probe, and we examined the character of the responses of the subset of piglets with large responses to phenylephrine.
Response magnitude and dialysis location within the RVM.
The RVM was defined previously by the effect of muscimol dialysis on
ventilatory responses to CO2 (5, 6). Because
we did not analyze the ventilatory response to hypercapnia in this study, we used cluster analysis based on anatomy and responses to
phenylephrine and muscimol to determine whether small regions within
the area we tested were associated with greater inhibition of
respiratory frequency after phenylephrine infusions. Cluster analysis
is an empirical statistical approach, and quite different results may
be obtained with modest changes in model conditions. Therefore, we used
a hierarchical protocol with complete linkage and the
K-means routine to obtain clusters, and we used a variety of
metrics. We were looking for responders vs. nonresponders to muscimol,
so we arbitrarily examined only the two cluster solutions. These
multiple methods and conditions resulted in remarkably similar results.
The results of the K-means cluster protocol using complete linkage are shown in Table 1. The results
indicate that different clusters do exist on the basis of rostrocaudal
coordinates. However, these clusters do not differ with respect to the
other two dimensions or percent change in frequency. The clustering
simply identified a rostral and a caudal grouping of probe locations
without respect to muscimol responsiveness. We confirmed this by
performing an ANOVA on the RVM groups identified by cluster analysis,
and muscimol dialysis enhanced the inhibitory effect of phenylephrine
on respiratory frequency similarly in both clusters. Hence, there was
no evidence that specific regions within the RVM, defined anatomically
or empirically, differed with respect to the capacity of muscimol to
enhance respiratory inhibition after phenylephrine infusion.
|
Analysis of the subset of large responders.
Although we could not identify any anatomic criterion to select the
most responsive animals, inspection of the responses of individual
piglets suggested that the piglets fell into two groups: responders and
nonresponders. Therefore, we examined the average results of animals
with large responses to phenylephrine after muscimol dialysis. An
example of the profound enhancement of respiratory inhibition that we
observed in some piglets is shown in Fig.
6. We selected a group of piglets with
large responses by identifying animals in which muscimol dialysis
enhanced the response of respiratory frequency to phenylephrine by
>50% and in which the absolute change in respiratory frequency during
phenylephrine infusion after muscimol dialysis was >5 breaths/min. In
two piglets with large responses as defined above, respiratory
frequency actually rose when phenylephrine was given after muscimol
dialysis, but we also identified nine piglets in which enhancement of
the inhibitory effect of muscimol was large. The anatomic locations of
the dialysis probes in these groups were not remarkable; the probe
locations were distributed throughout the RVM with no evidence of
anatomic clustering. The average responses of these nine piglets with
large responses are presented in Fig. 7.
The responses of these animals contributed significantly to the results
we observed for the entire group, since there was no significant effect
of muscimol dialysis on the phenylephrine response if we analyzed the
responses of the remaining piglets (including the two piglets with
"paradoxical" phenylephrine responses after muscimol dialysis).
Although this is a post hoc analysis, closer inspection of results from
the large responders may provide some insight into the mechanism of the
responses to muscimol. The blood pressure stimulus was similar before
and after muscimol dialysis in these animals, and the phenylephrine treatment increased blood pressure significantly (P < 0.001; all P values were adjusted using Scheffé's
method for these post hoc comparisons). The respiratory frequency rose
significantly after muscimol and before phenylephrine treatment
(P < 0.05), and phenylephrine treatment slowed the
respiratory frequency (P < 0.05). The frequency drop
after phenylephrine was significantly enhanced by muscimol
(P < 0.001). Muscimol decreased phrenic amplitude (P = 0.02), but the decrease in phrenic amplitude after
phenylephrine was not significant (P = 0.25), and there
was no effect of the combination of phenylephrine and muscimol on
phrenic amplitude. Phrenic minute activity fell significantly after
muscimol dialysis before administration of phenylephrine
(P < 0.05). The inhibition of minute activity after
phenylephrine administration was greater after muscimol dialysis
(P = 0.05). The foregoing results differ from the
average responses of the entire group of 25 piglets: there was greater
inhibition of respiratory frequency and minute activity by muscimol
dialysis even before phenylephrine was infused, and phenylephrine had
no effect on phrenic amplitude. The effect of phenylephrine on
respiratory frequency was also larger before and after muscimol
dialysis in these animals (as it must have been on the basis of our
selection criteria).
|
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The main finding of this study is that muscimol dialysis in the RVM substantially accentuated the ventilatory depression associated with an acute elevation of blood pressure in the decerebrate piglet. The effect of muscimol dialysis on the response to phenylephrine was variable among piglets. The responses of nine piglets were large and accounted almost completely for the average responses of the entire group. The dialysis probe locations in the piglets with large responses were spread throughout the RVM and not clearly different from the nonresponders.
Muscimol effects on ventilation before phenylephrine infusion. The values of respiratory frequency and the pattern of peak phrenic amplitude and minute phrenic activity are similar to those previously reported in decerebrate piglets (5). Minute phrenic activity was stable in the control period preceding phenylephrine infusion before and after muscimol dialysis. However, the respiratory frequency rose significantly, and phrenic amplitude fell. The changes in respiratory frequency and minute activity after muscimol dialysis were even greater in the group of large responders (Fig. 7). The large responders were selected on the basis of the response to phenylephrine, but the group also had large responses to muscimol alone before phenylephrine was administered. Thus muscimol altered the respiratory pattern and reduced respiratory drive even before phenylephrine was given. The fall in amplitude and the rise in frequency probably reflect a single process, since there tends to be a reciprocal relationship between phrenic amplitude and frequency even in vagotomized decerebrate animals when respiratory drive changes (5, 10). Dreshaj et al. (10) noted a similar change in respiratory frequency after inhibition in the raphé, another CO2-sensitive region within the medulla, with lidocaine or ibotenic acid.
The dialysis probe not only added muscimol to the RVM (when muscimol was added to the dialysate), but dialysis also takes substances up from the CNS into the dialysate. Dialysis with aCSF alone might have had a modest inhibitory effect by reducing neurotransmitter concentrations in the region of the probe over time. Moreover, simply placing the probe may also disrupt the function of the region. However, the effects of muscimol dialysis are unlikely to be attributed to placement of the probe or prolonged dialysis, since there were no time-dependent changes in respiratory frequency, peak integrated phrenic activity, or minute phrenic activity in the control studies we conducted or in the control periods before and after muscimol dialysis in our previous study using a protocol with longer dialysis periods (5).Muscimol effects on phenylephrine-induced respiratory inhibition. Phenylephrine raises the blood pressure and stimulates baroreceptors, and baroreceptor stimulation inhibits respiratory activity (18). Muscimol dialysis enhanced the respiratory inhibition associated with phenylephrine infusion in the piglets we studied. Muscimol is a GABAA agonist with high affinity for the GABA receptor, and GABA receptors are ubiquitous. We believe that muscimol inhibits neurons in the RVM and reduces the excitatory drive to breathe. As a result, respiratory inhibition after phenylephrine was more profound after muscimol dialysis. Thus a facilitatory drive, originating in the RVM, stabilizes the respiratory output in the presence of inhibitory stimuli. However, we do not believe that the excitation arising within the RVM is specific for baroreceptor inhibition of respiration, since manifestations of the laryngeal chemoreflex, which is associated with respiratory inhibition, are also enhanced in intact sleeping piglets after muscimol dialysis in the RVM (32).
The enhanced inhibitory effect of phenylephrine infusion after muscimol was manifest only on frequency. Phrenic amplitude was reduced by phenylephrine infusion and by muscimol dialysis, but there was no interaction between the two treatments, and the reduction in phrenic amplitude was similar before and after muscimol dialysis. The effect on frequency also seems to be specific for the interaction between phenylephrine and muscimol, since the effect of muscimol dialysis on the CO2 response of decerebrate piglets was mediated exclusively through an effect on tidal volume (5).Muscimol responsiveness to phenylephrine: relationship to CO2 chemosensitivity. We did not test the ventilatory response to CO2 in this study, but in our previous studies, muscimol dialysis in the RVM reduced the ventilatory response to CO2 (5, 6). CO2 chemosensitivity seems to be present homogeneously within the RVM; we have not seen such clear distinctions between nonresponders and responders when examining the effect of muscimol dialysis on ventilatory responses to CO2 (5, 6). The heterogeneity of the phenylephrine responses to muscimol dialysis suggests that the RVM may contain small clusters of neurons involved in cardiorespiratory control. These neurons may be CO2 sensitive, but additional regions within the RVM are probably CO2 sensitive but not involved in cardiorespiratory interactions. On the other hand, muscimol may also act by CO2-independent mechanisms. In a series of studies of awake and anesthetized goats, Forster et al. (14) and Ohtake et al. (24) used cooling thermodes placed bilaterally on the ventrolateral surface of the medulla to inhibit neural activity. Brief cooling of the ventral surface inhibited respiration equally during hypoxia, hypercapnia, and exercise: there was no specific effect of cooling on the hypercapnic ventilatory response. When the duration of cooling was prolonged, which increased the depth of neural inhibition into the ventral medulla, the respiratory inhibition was greater during hypercapnia. Similar results were obtained in neonatal goats (26). These data are consistent with the hypothesis that a tonic, CO2-insensitive, facilitatory input to respiratory activity originates in the superficial RVM. Muscimol dialysis in the RVM may have reduced this tonic facilitatory input and enhanced the respiratory inhibition associated with phenylephrine infusion. One might have expected evidence of respiratory inhibition after muscimol dialysis alone if this were the case. For example, bilateral cooling of the ventral surface caused apnea in anesthetized goats (24). However, we saw no inhibition of ventilatory output after muscimol dialysis until phenylephrine was administered when the entire group of animals treated by muscimol dialysis was analyzed. It may be pertinent that our dialysis probes were placed unilaterally, and the region affected by the dialysate was only a small fraction of the total volume of the RVM (bilateral inhibition of the RVM with muscimol does cause apnea in decerebrate piglets; unpublished observations). Furthermore, there was inhibition of ventilatory activity after muscimol dialysis in the animals with larger responses to phenylephrine before phenylephrine was given (Fig. 7). The reduction in ventilatory activity after muscimol dialysis in this group of large responders is consistent with loss of a facilitatory input from the RVM, but our data do not directly address the contribution of CO2 chemosensory mechanisms to this facilitatory drive to ventilation.
There is one hint in our data that the baroreflex-mediated inhibition of respiration does not involve a CO2 chemosensory mechanism. Muscimol dialysis in the RVM reduced the ventilatory response to CO2 by reducing the tidal volume (6). We observed a fall in phrenic amplitude after muscimol dialysis before phenylephrine was administered. However, among the large responders, the interactive effect of phenylephrine and muscimol was solely manifest as a drop in respiratory frequency (Fig. 7); there was no effect of phenylephrine on phrenic amplitude alone or in combination with muscimol. This suggests that the phenylephrine effect was independent of factors controlling phrenic amplitude and tidal volume and, therefore, independent of CO2 chemosensory mechanisms in the RVM, since these seem to operate in neonatal piglets by affecting phrenic amplitude and tidal volume.Site of cardiorespiratory interactions. The neural substrate of phenylephrine-induced respiratory inhibition within the CNS is incompletely characterized. With respect to cardiovascular control, baroreceptor afferents arrive in the nucleus tractus solitarius, and the inhibition of sympathetic vasomotor tone and activation of vagal efferents are mediated by the CVLM and RVLM and cardiovagal motoneurons in the nucleus ambiguus, respectively (16, 31). We believe that the RVLM identified by Guyenet and others (15, 16) is caudal and slightly dorsal to the RVM, as we have defined it. However, the rostral RVLM and the caudal RVM may well overlap each other. Kynurenic acid uncoupled sympathorespiratory interactions when injected into the RVM (15). Guyenet et al. actually called the region affected by these injections the RVLM, but the injections that modified cardiorespiratory interactions were rostral to the caudal pole of the facial nucleus in the region we defined as the RVM (see Fig. 6 in Ref. 15), and similar effects on cardiorespiratory interactions were not seen after injections made caudal to the facial nucleus. Thus we believe that the RVM may play an important role specifically in cardiorespiratory interactions.
A circuit for respiratory inhibition by baroreceptor activation mediated through the RVM has not been described, but two possibilities occur to us. First, efferent projections from the nucleus tractus solitarius that are activated by baroreceptor stimulation may inhibit the central pattern generator within the dorsal and ventral respiratory groups. In this scheme, the effect of muscimol dialysis in the RVM would be mediated by withdrawal of a facilitatory input to the central pattern generator. This places the site of integration of cardiorespiratory interactions in the respiratory central pattern generator. Alternatively, inhibitory activity derived from baroreceptor stimulation might directly inhibit the neurons within the RVM that provide a facilitatory input to respiration. Baroreceptor information could even arrive at the RVM through the closely adjacent RVLM (16). This places the site of integration of cardiorespiratory interactions in the RVM. In favor of the latter hypothesis, Harper et al. (17) found that phenylephrine-induced respiratory depression was associated with marked diminution of neural activity on the surface of the RVM. If the RVM has an integrative function, this activity may be shared with other sites, since activation of the CVLM, for example, also modifies cardiorespiratory interactions (31).Implications for SIDS. The triple-risk model for the pathogenesis of SIDS suggests that three factors contribute to the death of each infant: an underlying vulnerability, a critical period of homeostatic control, and an exogenous stressor (13). We studied an exogenous stressor that inhibits ventilation (phenylephrine) in a neonatal piglet with an experimentally induced vulnerability (inhibition of the RVM by muscimol dialysis). The results of the study are consistent with the triple-risk model of SIDS, in that inhibition of the RVM enhanced the inhibition of respiration associated with phenylephrine infusion and destabilized the respiratory output. One may reasonably ask whether either of our experimental interventions is relevant to SIDS. We believe that they are. Sleep in neonates is punctuated by multiple arousals, many of which have a stereotypical pattern of startle, hypertension and tachycardia, apnea, and arousal (20, 23). We found a similar pattern of hypertension and tachycardia followed by apnea in neonatal piglets (3). Hence, hypertension as part of an arousal may precipitate an apnea. It is our hypothesis that loss of input from the RVM might prolong the apnea and increase respiratory instability associated with the arousal. Regardless of the plausibility of hypertension and respiratory inhibition as triggers in the pathogenesis of SIDS, the results of the present study provide evidence that neurons within the RVM sustain and stabilize respiratory activity. The effects of muscimol are also consistent with current hypotheses about the underlying vulnerability associated with SIDS. GABA receptors are ubiquitous, and muscimol dialysis mimics to some extent the loss of neurons observed in the arcuate nucleus in some babies who died of SIDS (12, 22). On the other hand, infants who died of SIDS have reduced muscarinic, kainate, and serotonergic- receptor binding within multiple regions of the brain stem (19, 27, 28), and the effect of muscimol is not specific for any of the actual neurotransmitter defects identified thus far in SIDS victims.
In summary, we have demonstrated that muscimol dialysis in an extended region of the RVM enhances respiratory inhibition after phenylephrine infusion in decerebrate neonatal piglets. Thus the loss of a facilitatory input to respiratory drive, which originates in the RVM and may or may not be related to CO2 sensitivity within the RVM, promoted respiratory instability in these animals.| |
ACKNOWLEDGEMENTS |
|---|
We acknowledge the helpful comments of Drs. H. Kinney and R. A. Darnall and the technical assistance of N. Turner, L. Hildebrandt, and M.-H. Sun.
| |
FOOTNOTES |
|---|
This work was supported by National Institute of Child Health and Human Development Grant HD-36379, the American Heart Association, and the Charles H. Hood Foundation. A. K. Curran is a Parker B. Francis Fellow in Pulmonary Research.
Address for reprint requests and other correspondence: A. K. Curran, Dept. of Physiology, Dartmouth Medical School, 1 Medical Center Dr., Lebanon, NH 03756 (E-mail: aidan.k.curran{at}dartmouth.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.
First published February 8, 2002;10.1152/japplphysiol.00895.2001
Received 29 August 2001; accepted in final form 23 January 2002.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Bandroft, JD,
and
Cook HC.
The central and peripheral nervous system.
In: Manual of Histological Techniques and Their Diagnostic Application, edited by Bandoft JD.. New York: Churchill Livingstone, 1994, p. 350-351.
2.
Brunner, MJ,
Sussman MS,
Greene AS,
Kallman CH,
and
Shoukas AA.
Carotid sinus baroreceptor reflex control of respiration.
Circ Res
51:
624-636,
1982
3.
BuSha, BF,
Leiter JC,
Curran A,
Li A,
Nattie EE,
and
Darnall RA.
Spontaneous arousals during quiet sleep in piglets: a visual and wavelet-based analysis.
Sleep
24:
499-513,
2001[Web of Science][Medline].
4.
Cox, BF,
and
Brody MJ.
Mechanisms of respiration-induced changes in vasomotor control exerted by rostral ventrolateral medulla.
Am J Physiol Regulatory Integrative Comp Physiol
257:
R626-R634,
1989
5.
Curran, AK,
Chen G,
Darnall RA,
Filiano JJ,
Li A,
and
Nattie EE.
Lesion or muscimol in the rostral ventral medulla reduces ventilatory output and the CO2 response in decerebrate piglets.
Respir Physiol
123:
23-37,
2000[Web of Science][Medline].
6.
Curran, AK,
Darnall RA,
Filiano JJ,
Li A,
and
Nattie EE.
Muscimol dialysis in the rostral ventral medulla reduces the ventilatory response to CO2 in awake and sleeping piglets.
J Appl Physiol
90:
971-980,
2001
7.
Darnall, RA,
Curran AK,
Filiano JJ,
Li A,
and
Nattie EE.
The effects of GABAA agonist in the rostral ventral medulla on sleep and breathing in newborn piglets.
Sleep
24:
514-527,
2001[Web of Science][Medline].
8.
Darnall, RA,
and
Guyenet P.
Respiratory modulation of pre- and postganglionic lumbar vasomotor sympathetic neurons in the rat.
Neurosci Lett
119:
148-152,
1990[Web of Science][Medline].
9.
Dove, EL,
and
Katona PG.
Respiratory effects of brief baroreceptor stimuli in the anesthetized dog.
J Appl Physiol
59:
1258-1265,
1985
10.
Dreshaj, IA,
Haxhiu MA,
and
Martin RJ.
Role of the medullary raphe nuclei in the respiratory response to CO2.
Respir Physiol
111:
15-23,
1998[Web of Science][Medline].
11.
Filiano, JJ,
Choi JC,
and
Kinney HD.
Candidate cell populations for respiratory chemosensitive fields in the human infant medulla.
J Comp Neurol
293:
448-465,
1990[Web of Science][Medline].
12.
Filiano, JJ,
and
Kinney HC.
Arcuate nucleus hypoplasia in the sudden infant death syndrome.
J Neuropathol Exp Neurol
51:
394-403,
1992[Web of Science][Medline].
13.
Filiano, JJ,
and
Kinney HC.
A perspective on neuropathological findings in victims of the sudden infant death syndrome: the triple-risk model.
Biol Neonate
65:
194-197,
1994[Web of Science][Medline].
14.
Forster, HV,
Ohtake PJ,
Pan LG,
Lowry TF,
Korducki MJ,
Aaron EA,
and
Forster AL.
Effects on breathing of ventrolateral medullary cooling in awake goats.
J Appl Physiol
78:
258-265,
1995
15.
Guyenet, PG,
Darnall RA,
and
Riley TA.
Rostral ventrolateral medulla and sympathorespiratory integration in rats.
Am J Physiol Regulatory Integrative Comp Physiol
259:
R1063-R1074,
1990
16.
Guyenet, PG,
Koshiya N,
Huangfu D,
Baraban SC,
Stornetta RL,
and
Li YW.
Role of medulla oblongata in generation of sympathetic and vagal outflows.
Prog Brain Res
107:
127-144,
1996[Web of Science][Medline].
17.
Harper, RM,
Rector DM,
Poe G,
Frysinger RC,
Kristensen M,
and
Gozal D.
Rostral brain regions contributing to respiratory control.
Prog Brain Res
107:
145-156,
1996[Web of Science][Medline].
18.
Joels, N.
Reflex respiratory effects of circulating catecholamines.
In: Handbook of Physiology. Endocrinology. Adrenal Glands. Washington, DC: Am. Physiol. Soc, 1975, vol. VI, p. 491-505.
19.
Kinney, HC,
Filiano JJ,
Sleeper LA,
Mandell F,
Valdes-Dapena M,
and
White WF.
Decreased muscarinic receptor binding in the arcuate nucleus in sudden infant death syndrome.
Science
269:
1446-1450,
1995
20.
Lijowska, AS,
Reed NW,
Chiodini BAM,
and
Thach BT.
Sequential arousal and airway-defensive behavior of infants in asphyxial sleep environments.
J Appl Physiol
83:
219-228,
1997
21.
Luna, LG.
Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts. Gaithersburg, MD: American Histolabs, 1992.
22.
Matturri, L,
Biondo B,
Mercurio P,
and
Russi L.
Severe hypoplasia of medullary arcuate nucleus: quantitative analysis in sudden infant death syndrome.
Acta Neuropathol (Berl)
99:
371-375,
2000[Medline].
23.
McNamara, F,
Wulbrand H,
and
Thach BT.
Characteristics of the infant arousal response.
J Appl Physiol
85:
2314-2321,
1998
24.
Ohtake, PJ,
Forster HV,
Pan LG,
Lowry TF,
Korducki MJ,
Aaron EA,
and
Weiss EM.
Ventilatory responses to cooling the ventrolateral medullary surface of awake and anesthetized goats.
J Appl Physiol
78:
247-257,
1995
25.
Ohtake, PJ,
Forster HV,
Pan LG,
Lowry TF,
Korducki MJ,
Smith K,
and
Forster AL.
Effect on breathing of neuronal dysfunction in the caudal ventral medulla of goats.
J Appl Physiol
79:
1586-1594,
1995
26.
Ohtake, PJ,
Forster HV,
Pan LG,
Lowry TF,
Korducki MJ,
and
Whaley AA.
Effects of cooling the ventrolateral medulla on diaphragm activity during NREM sleep.
Respir Physiol
104:
127-135,
1996[Web of Science][Medline].
27.
Panigrahy, A,
Filiano JJ,
Sleeper LA,
Mandell F,
Valdes-Dapena M,
Krous HF,
Rava LA,
Foley E,
White WF,
and
Kinney HC.
Decreased serotonergic receptor binding in rhombic lip-derived regions of the medulla oblongata in the sudden infant death syndrome.
J Neuropathol Exp Neurol
59:
377-384,
2000[Web of Science][Medline].
28.
Panigrahy, A,
Filiano JJ,
Sleeper LA,
Mandell F,
Valdes-Dapena M,
Krous HF,
Rava LA,
White WF,
and
Kinney HC.
Decreased kainate receptor binding in the arcuate nucleus of the sudden infant death syndrome.
J Neuropathol Exp Neurol
56:
1253-1261,
1997[Web of Science][Medline].
29.
Saupe, KW,
Smith CA,
Henderson KS,
and
Dempsey JA.
Respiratory and cardiovascular responses to increased and decreased carotid sinus pressure in awake and sleeping dogs.
J Appl Physiol
78:
1688-1698,
1995
30.
Sun, MH,
Hildebrandt L,
Curran AK,
Darnall RA,
Chen G,
and
Filiano JJ.
Potassium permanganate can mark the site of microdialysis in brain sections.
J Histotech
23:
151-154,
2000[Web of Science].
31.
Tolentino-Silva, FP,
Haxhiu MA,
Ernsberger P,
Waldbaum S,
and
Dreshaj IA.
Differential cardiorespiratory control elicited by activation of ventral medullary sites in mice.
J Appl Physiol
89:
437-444,
2000
32.
Van der Velde, L,
Walford GA,
Curran AK,
Filiano JJ,
Bartlett D, Jr,
Darnall RA,
and
Leiter JC.
Laryngeal chemoreflex and sudden infant death syndrome (SIDS): a study in piglets.
Neurosci Abstr
26:
1460,
2000.
33.
Wasicko, MJ,
Giering RW,
Knuth SL,
and
Leiter JC.
Hypoglossal and phrenic nerve responses to carotid baroreceptor stimulation.
J Appl Physiol
75:
1395-1403,
1993
34.
Wilkinson, L,
Blank G,
and
Gruber C.
Desktop Data Analysis With Systat. Upper Saddle River, NJ: Prentice-Hall, 1996.
This article has been cited by other articles:
|
|
 |
|
  A. K. Curran and J. C. Leiter Baroreceptor-mediated inhibition of respiration after peripheral and central administration of a 5-HT1A receptor agonist in neonatal piglets Exp Physiol, July 1, 2007; 92(4): 757 - 767. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Curran, L. Xia, J. C. Leiter, and D. Bartlett Jr. Elevated body temperature enhances the laryngeal chemoreflex in decerebrate piglets J Appl Physiol, March 1, 2005; 98(3): 780 - 786. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. van der Velde, A. K. Curran, J. J. Filiano, R. A. Darnall, D. Bartlett Jr., and J. C. Leiter Prolongation of the laryngeal chemoreflex after inhibition of the rostral ventral medulla in piglets: a role in SIDS? J Appl Physiol, May 1, 2003; 94(5): 1883 - 1895. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
,
Position of the tip of the dialysis probes within the RVM during
control dialysis (no muscimol); 
, position of the tip
of the dialysis probes within the RVM during muscimol dialysis.
A-E on right: hemisections from
correspondingly lettered lines on photograph at left;
symbols indicating probe locations were placed on the closest relevant
section. 7N, facial nerve; SO, superior olive; TB, trapezoid body; VII,
facial nucleus; RP, raphé pallidus; X vagal motor nucleus; IO,
inferior olive; XII, hypoglossal motor nucleus; VIII, auditory nucleus;
NTS, nucleus tractus solitarius.
