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1 Department of Physiology, Dartmouth Medical School, 2 Department of Pediatrics, Dartmouth-Hitchcock Medical Center, and 3 Department of Neurology, Dartmouth- Hitchcock Medical Center, Lebanon, New Hampshire 03756
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ABSTRACT |
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 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Some victims of sudden infant death syndrome have arcuate nucleus abnormalities. The arcuate nucleus may be homologous with ventral medullary structures in the cat known to be involved in the control of breathing and the response to systemic hypercapnia. We refer to putative arcuate homologues in the piglet collectively as the rostral ventral medulla (RVM). We inhibited the RVM in awake and sleeping, chronically instrumented piglets by microdialysis of the GABAA receptor agonist muscimol. Muscimol dialysis (10 and 40 mM) had no effect on eupnea but caused a significant reduction in the response to hypercapnia during both wakefulness (34.8 ± 8.7 and 30.7 ± 10.1%, respectively) and sleep (36.7 ± 6.7 and 49.5 ± 8.9%, respectively). The effect of muscimol on the CO2 response was entirely via a reduction in tidal volume and appeared to be greater during non-rapid-eye-movement sleep. We conclude that the piglet RVM contains neurons of importance in the response to systemic CO2 during both wakefulness and non-rapid-eye-movement sleep. We hypothesize that dysfunction of homologous regions in the human infant could lead to impaired ability to respond to hypercapnia, particularly during sleep, which could potentially be involved in the pathogenesis of sudden infant death syndrome.
central chemoreceptors; sudden infant death; parapyramidal; raphé; retrotrapezoid nucleus; nucleus paragigantocellularis lateralis; arcuate nucleus
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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APPROXIMATELY ONE OF EVERY 1,000 infants born in the United States each year succumbs to sudden infant death syndrome (SIDS). Despite more than 30 years of research there is, as yet, no known cause or cure. In recent years, however, two major advances have taken place in SIDS research. The first was the "Back to Sleep" campaign, which encouraged parents to place sleeping infants supine rather than prone. This has resulted in a >40% reduction in the incidence of SIDS (16). Many theories have been proposed for the deleterious effects of prone sleeping, the most widely accepted being rebreathing of expired gases and asphyxia by an infant with his or her face down or covered by bedding (43). The second advance has been the discovery of abnormalities in the brain stems of SIDS victims. Some have severe developmental hypoplasia of the arcuate nucleus (10), whereas in many more there is reduced muscarinic (17) and kainate (32) receptor binding in the arcuate nucleus. More recently, a widespread reduction in serotonergic binding in the caudal raphé system, including the arcuate nucleus [with scattered serotonergic neurons (18)], has been reported (33).
In human infants, the arcuate nucleus is a thin layer of cells on the ventral surface of the medulla (9). The arcuate region has been proposed to be homologous with ventral medullary structures in animals, which are known to be involved in chemoreception and the control of breathing (9). No arcuate homologue has, as yet, been cytologically identified in the piglet. However, we have defined a region of the piglet brain stem as the rostral ventral medulla (RVM), the location of which corresponds anatomically to the rostral aspect of the human lateral arcuate nucleus (6). This area lies within the classic rostral (23) and intermediate (37) chemosensitive areas. The RVM contains respiratory related nuclei such as the retrotrapezoid nucleus (RTN), the juxtafacial aspect of the nucleus paragigantocellularis lateralis (PGCL), and the parapyramidal region.
In adult animals, the RVM region has anatomic connections to the dorsal and ventral respiratory groups (39). Some RVM cells show a respiratory rhythm and increase their firing rate in response to CO2 (5, 25). RVM lesions in anesthetized or decerebrate cats and rats inhibit eupneic breathing and reduce the response to systemic hypercapnia (24, 26, 27, 29, 30). In anesthetized piglets, cooling of the ventral surface of the rostral medulla reduced the response to systemic hypercapnia (22). More recently, our laboratory has demonstrated that RVM lesions in decerebrate piglets reduce eupneic phrenic activity and the response to systemic hypercapnia (6). Systemic hypercapnia produces c-Fos expression in the RVM in anesthetized cats (41) and conscious rats (13, 35, 42) indicating that this region may be either directly chemosensitive or involved in the integration of the response to hypercapnia. Decreasing local pH via acetazolamide injection (3, 28) or focal application of CO2 (19) stimulates breathing in anesthetized rats and cats, demonstrating that this area is chemosensitive.
The peak incidence of SIDS occurs in the early morning hours. This suggests a role for sleep state in the occurrence of SIDS. In unanesthetized rats, RVM lesions reduced the ventilatory response to systemic hypercapnia by 39% (1). Focal acidification of the RVM in rats by using microdialysis stimulated ventilation during wakefulness but not during sleep (20), indicating that neurons in the region appear to be chemosensitive only during wakefulness in the rat. The role of this region in the control of breathing and the response to systemic hypercapnia, however, has not been examined in awake and sleeping neonates
The purpose of the present study was to examine the effect of RVM inhibition, by application of the GABAA receptor agonist muscimol via microdialysis, on ventilation and the response to systemic CO2 in awake and sleeping piglets. We hypothesized that RVM inhibition would reduce respiratory drive and the response to systemic hypercapnia. We further hypothesized that these effects would be magnified during periods of reduced respiratory drive as occur during non-rapid-eye-movement (NREM) and rapid-eye-movement (REM) sleep.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In total, 17 piglets were used in this study, ranging in age from 2 to 18 days old (mean ± SE = 10.4 ± 1.0 days) with an average weight of 2.7 ± 0.2 (SE) kg. Each animal was studied on the first day postsurgery and subsequently for up to 3 days after surgery. Some animals were studied only once, whereas four animals were also studied using a different protocol for a subsequent study (Darnall RA, Curran AK, Filiano JJ, Li A, and Nattie EE, unpublished results). No animal was studied more than twice for this study. Data from animals that were studied twice were averaged to yield one value for their response to CO2 and the effect of muscimol. Piglets were housed with the sow, pre- and postoperatively, in a farrowing crate in our on-site animal care facility. The Institutional Animal Care and Use Committee of Dartmouth College approved all surgery and protocols.
Chronic Instrumentation
All surgery was performed under general anesthesia (~2% isoflurane in O2) using sterile technique. Throughout the surgical procedure, the animals' heart rate (HR), end-tidal CO2, body temperature, and isoflurane levels were constantly monitored. Animals were pretreated with an intravenous antibiotic (20 mg/kg iv, cefazolin, Apothecon, Princeton, NJ) just before surgery and given daily antibiotic doses (120 mg, trimethaprim sulfa oral solution, Alpharma, Baltimore, MD) postoperatively until euthanasia. Analgesia was administered immediately postoperatively (0.1 mg/kg im, Buprenex, Reckitt & Coleman, Richmond, VA) and as required up to 24 h after surgery. All incisions were cleaned daily, and topical antibiotic ointment was applied.We implanted a dual-lumen catheter, for recording of blood pressure and sampling of arterial blood, in the abdominal aorta via the femoral artery. A thermistor probe was sewn, subcutaneously, in the abdominal area. Both the blood pressure catheter and the thermistor were tunneled subcutaneously and exited in the back, just caudal to the scapulae. The catheter lumens were filled with heparin (1,000 U/ml, Elkins-Sinn, Cherry Hill, NJ).
The animal was then placed prone in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA). A midline incision was made in the scalp, and stereotaxic coordinates were taken for lambda, bregma, and a fiducial mark on the right ear bar. These coordinates were used in a regression formula to predict the location of the RVM for microdialysis probe placement (40). An electroencephalogram (EEG)-electrooculogram (EOG) montage, consisting of two EEGs, two EOGs, and a common, a ground, and a bipolar neck electromyogram (EMG) were implanted. All wires were attached to brass connectors, which were, in turn, attached to a plastic pedestal. A small burr hole was drilled into the skull, through which a microdialysis guide tube and stylette (BAS, West Lafayette, IN) were inserted, to lie with its tip in the region of the RVM. The dialysis probe tip measured 1 mm long and 250 µm in diameter. The EEG pedestal and the microdialysis guide tube were affixed to the skull with cranioplastic cement. At least 24 h were allowed after the end of surgery before any studies were performed.
Measurements
Studies were performed using barometric plethysmography. To prevent piglets from becoming entangled in wires and tubing in the box, they were placed in a custom-made sling that was suspended from a metal frame inside the box. The plethysmograph was connected to a reference chamber via a slow leak to minimize large pressure fluctuations. A constant vacuum from the box exactly matched a constant flow of warmed, humidified air into the box, both via high resistance ports. This allowed continuous measurement of breathing.Pressure fluctuations in the box due to warming of the inspired air were measured by use of a pressure transducer (Validyne, Northridge, CA) attached in series between the plethysmograph and the reference chamber. A slow leak between the plethysmograph and reference chamber reduced artifact related to pressure fluctuations due to animal movement or changes in room pressure due to opening or closing of doors, and so on. Blood pressure and HR were recorded from one lumen of the arterial catheter. By using the second lumen of the arterial catheter, blood-gas samples could be withdrawn without interruption of the blood pressure record. EEG and EOG signals were amplified and band-pass filtered at 0.3-30 Hz, and neck EMG was amplified and band-pass filtered at 10-100 Hz. A small amount of gas was withdrawn from the plethysmograph outlet and analyzed for O2 (Applied Electrochemistry, model S-3A/II, Pittsburgh, PA) and CO2 (CWI, Capstar-100, Ardmore, PA). Inlet O2 was also measured in a similar manner. Box and animal temperatures were also continuously measured (YSI, Yellow Springs, OH). All signals were recorded on a computerized data acquisition system (PowerLab, ADInstruments, Castle Hill, Australia) at 1,000 Hz and stored for later analysis. A backup of all signals was recorded on digital tape (Vetter, model 4000A, Redersburg, PA). In addition, the animals were continuously videotaped with a time stamp synchronized with the computer record. This tape was used for behavioral analysis of sleep.
Protocol
At least 1 h before study, the piglet was brought into the laboratory to interact with researchers. This served the dual purposes of acclimatizing the piglet to human handling and making the piglet tired, thereby increasing the likelihood of achieving sleep in the plethysmograph. During this period the plethysmograph was sealed and temperature and humidity were allowed to stabilize before the box was calibrated. Calibration consisted of application of known gas mixtures to the gas analyzers and set pressures to the box and blood pressure transducers. In addition, the plethysmograph was calibrated by sequential triplicate injections of 1, 2, 3, and 5 ml of air and measuring the resultant pressure deflections for insertion into our tidal volume (VT) calculation formula. Calibration injections were made at a rate similar to the rate of pressure change produced by the piglets' breathing.Once placed into the plethysmograph, each piglet's RVM was continuously dialyzed with artificial cerebrospinal fluid (aCSF) at a flow rate of 8.5 µl/min. The piglet was allowed a rest period of at least 45 min to become accustomed to the box and for the box to reach the required temperature and 100% humidity before any data were recorded.
When box temperature, humidity, CO2, and O2 were constant, the first baseline was recorded. This consisted of a 40-min period of air breathing during which the animals cycled through wakefulness, NREM, and REM. After at least 40 min of baseline, CO2 was added to the box inflow to bring box CO2 level to 5%. This was achieved in <2 min. Box CO2 was maintained at 5% for 20 min, at which time recording was paused and the box was opened to flush out the CO2. When the box CO2 had fallen back to control, the box was resealed and another rest period was allowed for the box to return to recording conditions. At this point, a second baseline was recorded as before. At the end of the second baseline, the aCSF in the dialysis system was replaced with aCSF containing either 10 or 40 mM muscimol. The muscimol was dialyzed for a total of 40 min before washout was begun. After 20 min of muscimol dialysis, we again performed a 5% CO2 challenge for the final 20 min of muscimol.
Anatomy
The anatomical analysis used in this study has been previously described (6). Briefly, microinjections of 20-50 µl of 1% potassium permanganate were used to mark the site of dialysis (40). Potassium permanganate was injected rather than dialyzed because of its tendency to precipitate on the dialysis membrane, thereby blocking the pores. Brain stems were placed in a cryoembedding medium (Tissue-Tek OCT, Sakura Finetek, Torrance, CA), along with two pieces of pasta to act as fiducial marks for later reconstruction, and frozen in isopentane at
70°C. Brain stems were
cut at 50 µm in a cryostat at 18°C, and sections were
mounted on gelatinized glass slides. Sections were fixed in formal
alcohol overnight and stained with cresyl violet (2, 21).
The locations of the dialysis probe tips were plotted on a standardized grid relative to the caudal end of the facial nucleus. The caudal end of the facial nucleus was chosen as the reference point because of the unreliability of traditional references such as lambda and bregma in a developing piglet (6). The grid represents the average length and width of the facial nucleus, derived previously from a group of 12 piglets aged 4-12 days. The position of the probe tip was considered to affect the RVM, on the basis of the spread of a fluorescent marker used in a previous study (6), if it was within 2 mm of the ventral surface, at least 0.5 mm from the midline, and within 2 mm rostral and caudal and 1 mm lateral to the facial nucleus. Accordingly, some probes may have been placed outside the RVM but were still considered to have affected the RVM on the basis of our previous measure of the spread of a fluorescent marker, which predicts that the muscimol would spread into the RVM (6).
Data Analysis
Beat-to-beat mean arterial pressure (MAP) and HR were derived from the continuous recordings of arterial blood pressure. Similarly, breath-to-breath VT, respiratory frequency (fR), and minute ventilation (
E) were
calculated from plethysmograph respiratory pressure fluctuations. EEG
signals were resampled at 100 Hz and filtered with a bandwidth of
0.1-30 Hz. The EEG record was then divided into 5-s epochs, and
absolute spectral power density was computed over a frequency range of
0-50 Hz. The average power for each epoch was assigned to delta
(0.1-4.0 Hz), theta (5.0-9.0 Hz) or sigma (10.0-14.0 Hz)
frequency bands. The videotape of the animal was also reviewed, and
each 5-s epoch was scored for eye openings and body movement. Using
visual inspection of continuous plots of VT, MAP, HR, EEG
power density, EOG movements, and eye openings, we designated periods
of wakefulness, NREM, and REM. Characteristically, animals cycled
through NREM followed by a period of REM, which terminated with a short
arousal and a period of wakefulness before returning to NREM. NREM was
defined as a period of high delta power and EEG amplitude, minimal body
movement, and closed eyes. REM periods were characterized by low delta
power, the presence of rapid-eye-movements with eyes closed, nose and
ear twitching, and decreased VT and MAP. Wakefulness was
characterized by the presence of intermittent gross body movements,
with the eyes open and low delta power. The effects of muscimol,
CO2, and their interactions on ventilation and blood
pressure were analyzed separately for each sleep state using two-way
analysis of variance, with the Tukey post hoc test. The effect of sleep
on eupneic ventilation was analyzed using one-way ANOVA with the
Tukey's post hoc test. For all statistical tests, differences were
considered significant if P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Anatomy
The positions of the dialysis probe tips (n = 17) are shown schematically in Fig. 1. A total of 14 animals had probes with their tips either within the region of the RVM or in positions where the spread of the muscimol would affect portions of the RVM (6). Three probes were placed outside the RVM, either too rostral or caudal or too deep relative to the ventral surface. The position of one dialysis probe is shown on a cross section through the medulla of one piglet in Fig. 2.
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Effects of Sleep State
The effect of sleep state on ventilation and the response to CO2 in nine animals, with dialysis of aCSF only, is summarized in Fig. 3. There was no significant difference between wakefulness and NREM sleep for any variable during eupnea or 5% CO2. REM sleep caused a significant fall in VT andE during
eupnea relative to both wakefulness (19.1 ± 3.9% and 32.4 ± 4.1% reductions, respectively) and sleep (27.2 ± 2.0% and
31.6 ± 3.4% reductions, respectively) and a significant
reduction in fR (14.0 ± 6.3% reduction) relative to
wakefulness. The VT, fR, and
E at 5% CO2 were also significantly reduced relative to wakefulness (27.3 ± 2.7, 24.5 ± 2.9, and 44.8 ± 4.2% reductions, respectively) and NREM sleep
(29.7 ± 2.1%, 26.7 ± 3.8%, and 47.2 ± 4.6%
reductions, respectively). The slope of the CO2 response
was also significantly reduced for VT, fR, and
E.
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Effects of Muscimol
The baseline postmuscimol data shown in Figs. 4-9 are from the period just preceding the CO2 test. This was 20 min after the beginning of muscimol dialysis. There was no difference in any variable after 20 min of either 10 or 40 mM muscimol dialysis. The sleep architecture was disrupted, however, with less NREM and lower delta power during NREM periods. In a total of six animals, three who received 10 mM and three who received 40 mM muscimol, sleep cycling was completely abolished, leaving only wakefulness. The effects of muscimol on eupnea and sleep architecture were investigated in a separate study without CO2 testing (Darnall, Curran, Filiano, Li, and Nattie, unpublished results).
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Wakefulness CO2 Response
The effects of 10 and 40 mM muscimol dialysis in the RVM on eupnea and the CO2 response were studied in eight and six animals, respectively, during wakefulness. A further three animals with the microdialysis probe outside the RVM were dialyzed with 10 mM muscimol only. The baseline wakeful CO2 response in the 17 animals as a group consisted of a significant increase in VT (172.4 ± 4.8% of control), fR (144.7 ± 7.3% of control), andE (250.9 ± 13.3% of control)
(paired t-test, P < 0.05).
Dialysis with 10 mM muscimol.
In eight animals during wakefulness, dialysis of 10 mM muscimol had no
effect on eupneic ventilation or blood pressure. However, the
VT and E at 5% CO2 were
significantly reduced, by 10.9 ± 5.9 and 19.5 ± 5.1%,
respectively, with no effect on the fR response. In
addition, the slope of the CO2 response was significantly
reduced for both E (34.8 ± 8.7% reduction)
and VT (43.5 ± 14.0% reduction). Blood pressure was
not significantly affected by either CO2 or muscimol. These
data are summarized in Fig. 4. In the three animals with the probe
outside the RVM, there was no effect of muscimol on eupnea or the
CO2 response for any variable measured. These data are
summarized in Fig. 5.
Dialysis with 40 mM muscimol.
In six animals during wakefulness, dialysis of 40 mM muscimol for 40 min had effects similar to those for 10 mM. Eupneic ventilation and
blood pressure were unaffected by dialysis of 40 mM muscimol. However, the VT and E at 5%
CO2 were significantly reduced, by 15.7 ± 5.8 and
20.3 ± 6.2%, respectively, with no effect on the fR
response. In addition, the slope of the CO2 response was significantly reduced for both E (30.7 ± 10.1% reduction) and VT (42.0 ± 13.7% reduction).
Blood pressure was not significantly affected by either CO2
or muscimol. These data are summarized in Fig.
6.
NREM CO2 Response
The effects of 10 and 40 mM muscimol dialysis in the RVM on eupnea and the CO2 response were studied in five and three animals, respectively, during NREM sleep. The baseline CO2 response in all eight animals consisted of a significant increase in VT (164.4 ± 9.6% of control), fR (168.5 ± 10.3% of control), andE (284.9 ± 21.2% of control), with no significant effect on blood pressure (paired t-test, P < 0.05).
Dialysis with 10 mM muscimol.
In five animals during NREM sleep, dialysis of 10 mM muscimol had no
effect on eupneic ventilation or blood pressure during NREM sleep.
However, the E at 5% CO2 was
significantly reduced by 23.2 ± 5.7%, with no significant effect
on VT or fR. In addition, the slope of the
CO2 response was significantly reduced for
E (36.7 ± 6.7% reduction), VT
(41.0 ± 8.4% reduction), and fR (15.5 ± 7.9%
reduction). Blood pressure was not significantly affected by 10 mM
muscimol. These data are summarized in Fig.
7. An example of a typical record
obtained during NREM sleep is shown in Fig. 8.
Dialysis with 40 mM muscimol.
In three animals during NREM sleep, dialysis of 40 mM muscimol for 40 min had effects similar to those for 10 mM. Eupneic ventilation and
blood pressure were unaffected by dialysis of 40 mM muscimol during
NREM sleep. However, the VT and E at 5% CO2 were significantly reduced, by 28.4 ± 1.7% and
33.9 ± 3.7%, respectively, with no effect on fR. In
addition, the slope of the CO2 response was significantly
reduced for E (49.5 ± 8.9% reduction) and
VT (79.1 ± 10.9% reduction). The reduction in the slope of the VT response to CO2 was such that
CO2 no longer produced a significant increase in
VT after 40 mM muscimol. Blood pressure was not
significantly affected by muscimol during NREM sleep. These data are
summarized in Fig. 9.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The main findings of this study are as follows. 1) Inhibition of the RVM in piglets has no effect on eupnea but reduces the response to systemic hypercapnia. 2) The reduction in the CO2 response is due to a reduced VT response. 3) The effect of RVM muscimol on the CO2 response appears to be more pronounced during NREM sleep.
Study Limitations
The microdialysis technique has many advantages and is a useful technique for application of neuroactive agents focally in the brain. We are unable to precisely determine the area affected by muscimol in this study, however. In a previous study, we used the spread of a fluorescent dye, fluorescein, which has a similar molecular weight to muscimol, to estimate the area affected by muscimol (6). In that study, the fluorescein spread formed an ellipsoid that covered an area that overlapped only 13.6% of one side of the RVM. This is a small portion of the RVM and may be insufficient for adequate assessment of the true contribution of the RVM to the CO2 response. The small size of the area affected by muscimol may explain the absence of an effect on eupnea. However, in light of the effect on the response to hypercapnia, we believe that this is a true indication of the role of the RVM in the control of breathing.The use of fluorescein in this study was deemed inappropriate because the molecule does not bind to any receptors in the brain stem but continues to spread after the cessation of dialysis until the point at which the tissue is frozen. The time taken to remove a brain from a decerebrate animal is substantially shorter than that for a conscious animal, which must be euthanized and have its skull opened before removal and freezing of the brain stem. Accordingly, the area with fluorescein would be substantially larger and would not accurately reflect the area affected by muscimol.
Histological examination of the RVM region in the piglet has not revealed a collection of cells that are distinctly like those labeled as arcuate in the human. Instead there are sparse collections of small cells along the ventral medullary surface. In addition, the dimensions of the human arcuate differ from the piglet RVM, inasmuch as the arcuate nucleus has a longer rostral-caudal extent, whereas the piglet RVM extends deeper from the surface and is comprised of several cell types. Accordingly, we cannot make a direct inference from the results reported here to the consequences of the developmental abnormalities reported by Kinney and colleagues in the human arcuate nucleus (10, 17, 32, 33). However, similarities between the results of our previous studies in decerebrate piglets and those in adult cats indicate that these areas may play a similar role in the control of breathing (29). This implies that the homology reported by Filiano et al. (9) between the human arcuate and the cat RVM could also be extended to the piglet RVM.
One of the most striking effects of RVM muscimol dialysis in this study was the disruption of sleep (Darnall, Curran, Filiano, Li, and Nattie, unpublished results). NREM sleep was abolished in four animals, and in those who continued to cycle there was a reduction in both the duration of NREM periods and the level of delta power achieved during NREM. This phenomenon was investigated fully in a subsequent study, which demonstrated that the percent time spent in NREM sleep was decreased from 48% premuscimol to 12% postmuscimol, whereas the average delta power achieved during NREM sleep was reduced to 60% of premuscimol control levels (Darnall, Curran, Filiano, Li, and Nattie, unpublished results). Comparison of the CO2 response during NREM sleep before and after muscimol is complicated by the different sleep architecture produced by muscimol. However, we believe that there was sufficient NREM sleep after muscimol to make our comparisons valid. In particular, the greater effect of muscimol on the CO2 response during NREM sleep compared with wakefulness, despite the lower level of delta power during NREM sleep, suggests that the level of delta power was sufficient to differentiate that state from wakefulness.
Our dialysis flow rate of 8.5 µl/min is higher than most investigators use. We have used this previously with good success, however (6). We were careful to ensure that no ultrafiltration of dialysate occurred as a result of potentially high pressure in the dialysis line. In addition, because we dialyzed aCSF throughout the experiment, we minimized any potential problem with washout of CNS neurotransmitters by allowing at least 60-90 min of aCSF dialysis before recording any data.
Sleep and Breathing
In piglets premuscimol, sleep state was an important determinant of breathing and the CO2 response in the neonatal piglets studied. Transition into REM sleep produced profound reductions in both VT andE. In
addition, the ventilatory response to CO2 during REM sleep
was significantly less than during either wakefulness or NREM sleep.
There were no significant differences in ventilation or the response to
systemic hypercapnia between wakefulness and NREM sleep. The reduced
ventilation and response to CO2 during REM sleep relative
to NREM sleep and wakefulness is substantial. Similar results have been
reported in adult animals (31, 34) and humans
(36). However, there is some variability in the responses to hypercapnia reported in human infants. Haddad et al.
(12) reported no difference in the response to hypercapnia
between REM and NREM sleep, whereas Cohen et al. (4)
reported that the hypercapnic response was greatly reduced during REM
relative to NREM. It has been proposed that these differences may be
methodological, with no difference reported in steady-state hypercapnic
challenges (12) and reductions in hypercapnic
responsiveness during CO2 rebreathing tests. Our study
demonstrates a clear reduction in the response to steady state
hypercapnia during REM sleep in piglets. The mechanism by which this
reduced response occurs is unclear and cannot be demonstrated in this
study. A reduced response to hypercapnia during REM sleep may be due to
the effects of REM sleep atonia on rib cage and accessory respiratory
muscles reducing ventilation for a given central neural output
(15). By this mechanism, the CO2 response, in
terms of neural output, would be similar between sleep states, but
ventilatory mechanics would be altered. Alternatively, the reduced
ventilation and hypercapnic response may be due to a reduced central
drive and insensitivity to chemoreceptor input during REM sleep
(38). This is supported by evidence that dogs are slower
to arouse in response to hypercapnia during REM sleep than during NREM
sleep (34), indicating a reduced general sensitivity to
CO2.
Muscimol and Breathing.
In our study, 20 min of muscimol dialysis had no effect on eupneic ventilation during wakefulness or NREM sleep. Because of the short time period during which the muscimol was dialyzed before a CO2 challenge, we did not see any REM sleep. Accordingly, any effects of muscimol on eupnea during REM were missed. This question, as well as the longer term effects of RVM muscimol dialysis on sleep architecture and eupnea, was examined in another study (Darnall, Curran, Filiano, Li, and Nattie, unpublished results).The absence of an effect of RVM muscimol dialysis on eupneic
ventilation is in contrast to studies in decerebrate and anesthetized animals in which phrenic nerve activity was reduced by lesion of the
RVM (24, 26, 27, 29, 30). In a previous study in
decerebrate piglets, we demonstrated a substantial reduction in phrenic
nerve activity in response to 10 min of 10 mM muscimol dialysis
(6). These differences seem likely to be due to the use of
anesthesia or decerebration. Studies in conscious rats demonstrated
that lesion of the RVM with ibotenic acid injections did not alter
baseline ventilation but substantially reduced the response to
CO2 over the course of 3 wk of study (1), a
result similar to that reported here. In combination with our results, these data suggest that the RVM may be of importance in the ventilatory response to systemic hypercapnia with little role in eupneic
ventilatory control in the unanesthetized state. Furthermore, our data
suggest that the role of the RVM in the response to systemic
hypercapnia lies entirely in the VT response, because
muscimol dialysis had no effect on the fR response.
Experiments in conscious rats have demonstrated a
VT-dependent increase in E in response
to microdialysis of aCSF containing 25% CO2 into the RVM
(20). This supports our findings that the RVM plays a role
in the VT response to hypercapnia. In contrast to our
findings, however, Li et al. (20) found that the increase
in ventilation due to RVM CO2 dialysis only occurred during
wakefulness with no effect during behaviorally defined sleep. Our data
suggest that the effect of RVM inhibition is more pronounced during
NREM sleep. This apparent contradiction may be explained by either
species and/or age differences. Alternatively, one may conclude that
the RVM modulates the activity of other regions involved in the
response to systemic hypercapnia. The intrinsic chemosensitivity of the
RVM may diminish during NREM sleep, whereas its role in modulation of
other sites or in the integration of peripheral chemoreceptor afferent
activity may remain unchanged or be enhanced.
Dialysis probes were placed in the RVM at all levels from just caudal to the facial nucleus to the pontomedullary junction. The effects of muscimol did not correlate with the rostral-caudal position of the probe. All animals with probes in the RVM showed a reduced CO2 response. Probes bordering the pontomedullary junction, although rostral to the facial nucleus, would still affect the RVM region as indicated by predictions of spread of the muscimol (6). These probes might also be expected to affect the A5 region of the pons. This area has been suggested to provide noradrenergic inhibitory input to the respiratory rhythm generator in the neonatal rat brain stem preparation (8, 14) and shows c-Fos expression in response to systemic CO2 in adult rats (13). Accordingly, one might expect these probes to stimulate breathing. Of four probes in this study, at or slightly rostral to the pontomedullary junction, three of which we determined to be close enough to affect the RVM, none produced any excitation of breathing. The precise position of the A5 region in the piglet is not clear. Accordingly, our probes may not have affected this region in our study. Alternatively, the role of the A5 region in a sleeping piglet may differ from that in an in vitro rat brain stem preparation.
Implications for SIDS
These data are consistent with the hypothesis that developmental abnormalities of the ventral medulla may lead to impairment of the ability to respond to a chemoreceptor challenge. The triple-risk model has been proposed to explain the incidence of SIDS (11). According to this model, SIDS requires the confluence of three factors: a vulnerable infant faced with an exogenous stressor at a vulnerable stage in development. In the context of the triple-risk model, the arcuate nucleus abnormalities reported by the Kinney group (10, 17, 32, 33), mimicked in our study by local inhibition of the RVM, lead to an impairment of the ability to respond to CO2 as an exogenous stressor. The larger effect of muscimol during NREM sleep also agrees with the model, which suggests a vulnerable period, related to either age or sleep state. The absence of any effect on eupnea may be important in that there is no obvious manifestation of the effects of RVM inhibition until the exogenous stressor, in the form of systemic hypercapnia, is applied. Infants who succumb to SIDS appear completely normal up until the time of death, with no outward manifestations of their condition.In conclusion, our findings demonstrate the importance of the RVM in the control of the response to hypercapnia, particularly during NREM sleep in the newborn piglet. Furthermore, these data support the hypothesis that developmental abnormalities of the ventral medulla in human infants may play a role in the pathogenesis of SIDS.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical assistance of Laurie Hildebrandt and Geoff Walford. We are grateful for surgical and postoperative care assistance from Karen Moodie and assistance with anatomical analysis from Man-Hua Sun and Dan Peraza. We acknowledge the advice and assistance of Hannah Kinney, the director of the program project grant from which this work is derived.
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FOOTNOTES |
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This work was supported by National Institute of Child Health and Human Development Grant HD-36379; The SIDS Alliance grant SP0018; the W. R. Hearst Foundation; and the SIDS Foundation of Washington. 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-Hitchcock Medical Center, 1 Medical Center Drive, 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.
Received 7 August 2000; accepted in final form 11 October 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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, Position on the
ventral surface of the medulla of the dialysis probes that were outside
the rostral ventral medulla (RVM). 
, Position on the
ventral surface of the medulla of the dialysis probes that were in the
RVM. Right: hemisections labeled
A-E are taken at the correspondingly
lettered lines on the brain stem picture. These show the position of
probe tips in the dorsoventral plane. Probe positions are shown on the
nearest representative cross section. Slice A includes all
probes placed at or above the pontomedullary junction, slice
B shows those probes at the rostral end of the facial nucleus,
slice C shows all probes in the facial nucleus, slice
D shows probes placed at the caudal end of the facial nucleus, and
slice E shows probes placed caudal to the facial nucleus.
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.
