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Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Central chemoreceptors are widespread within the brain stem. We hypothesize that function at different sites varies with arousal state. In unanesthetized rats, we produced focal acidification at single sites by means of microdialysis using artificial cerebrospinal fluid equilibrated with 25% CO2. Tissue acidosis, measured under anesthesia, is equivalent to that observed with 63 Torr end-tidal PCO2 and is limited to 600 µm. Focal acidification of the retrotrapezoid nucleus increased ventilation by 24% only in wakefulness via an increase in tidal volume (Li A, Randall M, and Nattie E. J Appl Physiol 87: 910-919, 1999). In this study of the medullary raphe, the effect of such focal acidification was in sleep (defined by electroencephalographic and electromyographic criteria): ventilation and frequency increased by 15-20% in non-rapid eye movement sleep, and frequency increased by 15% in rapid eye movement sleep. There was no effect in wakefulness. Chemoreception in the medullary raphe appears to be responsive in sleep. Central chemoreceptors at two different locations appear to vary in effectiveness with arousal state.
central chemoreception; arousal; carbon dioxide response; medulla; control of breathing
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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CENTRAL CHEMORECEPTORS MEDIATE changes in breathing and blood pressure in response to changes in CO2 and H+ within the brain (16, 20-22). Early work located central chemoreceptors at sites just beneath the ventral medullary surface (16); recent experiments in vitro and in vivo indicate a widespread distribution within the brain stem (3, 5, 14, 15, 20-22, 31). These locations include a superficial region just beneath the surface of the ventral lateral medulla, the region of the nucleus tractus solitarius, the region of the locus ceruleus, the midline raphe, and the region of the ventral respiratory group. Why are central chemoreceptors located at so many sites? As one possible explanation, we hypothesize that differences in response and physiological role of some sites depend on the state of arousal.
We focus on the chemosensitive region lying deep to the ventral medullary surface at the midline, the medullary raphe. Raphe neurons in vitro exhibit CO2-dependent changes in membrane potential and firing rate (31). In vivo, the firing rate of some medullary raphe neurons is increased by systemic hypercapnia in wakefulness but not in sleep (34). The medullary raphe has known anatomic connections with the respiratory control network (8). Destruction of the medullary raphe in anesthetized and decerebrate piglets reduces substantially the response of the phrenic and hypoglossal nerves to systemic CO2 stimulation (6). Focal acidification of the raphe by microinjection of acetazolamide in anesthetized, vagotomized animals increases the amplitude of the integrated phrenic nerve signal (3). Stimulation of the raphe electrically or by chemical injection can increase or decrease respiratory output (2, 7, 12). The medullary raphe also is involved in blood pressure regulation (30), control of brown fat metabolism (19), thermoregulation (4), modulation of sensory input and motor output (13), and autonomic integration (17), and it plays a role in the sleep-wake cycle (28, 29). Its overall role in normal autonomic physiology is not fully understood.
We use a microdialysis probe (15) to produce a focal
acidosis in the medullary raphe of unanesthetized, unrestrained rats. The probe tip with semipermeable membrane (pores <6,000 Da) is 1 mm
long and 240 µm diameter, with a volume of 45 nl. The guide tube and
dialysis probe are made of a rigid, sturdy material, allowing their use
in a chronic animal. The goal is to evaluate the effect of focal
acidification of the medullary raphe on ventilation during sleep and
wakefulness. In a prior, similar study (15), we dialyzed
CO2 into the retrotrapezoid nucleus (RTN) of unanesthetized rats, with sleep determined by behavioral criteria: the rat was considered to be asleep when curled up, motionless, with its eyes closed. We observed that focal acidification of the RTN increased ventilation (
E), solely by an effect on tidal volume
(VT), in wakefulness only. In this study, we use
electroencephalogram (EEG) and electromyogram (EMG) measures to
determine sleep and wakefulness objectively and continuously.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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General Preparation
Surgery. Eight male Sprague-Dawley rats (300-450 g) were anesthetized with ketamine (100 mg/kg im) and xylazine (20 mg/kg ip). The crown of the skull was shaved, and the skin was sterilized with povidone-iodine (Betadine) and alcohol. The head was placed into a Kopf stereotaxic holder, and a dialysis guide cannula (0.38 mm OD) with a dummy was implanted into the medulla. The coordinates for probe placement were 11.2 mm caudal and 0 mm lateral from bregma and 10.4-10.6 mm below the dorsal surface. The guide cannula was secured with cranioplastic cement. Three EEG electrodes were screwed into the right side of the skull: the frontal electrode 2 mm anterior to bregma and 2 mm lateral to the midline, the parietal electrode 2 mm anterior to lambda and 2 mm lateral to the midline, and the ground electrode between the frontal and parietal electrodes. For the EMG, a pair of wire electrodes was inserted deep into the neck muscle. The skull wound was sutured. The abdominal surface was shaved, the skin was sterilized, an incision was made through the linea alba, and a sterile telemetry temperature probe (TA-F20, Data Sciences, St. Paul, MN) was placed in the abdominal cavity. The incision was closed, and the animal was allowed to recover for 3-4 days.
CO2 dialysis solution. The artificial cerebrospinal fluid (aCSF) was equilibrated with 5 or 25% CO2. The composition of the aCSF was (in mM) 152 sodium, 3.0 potassium, 2.1 magnesium, 2.2 calcium, 131 chloride, and 26 bicarbonate. The calcium was added after the aCSF was warmed to 37°C and equilibrated with CO2. The pH of each solution was monitored to ensure that the equilibration was reliable. The dialysis pump was run at a speed of 45 µl/min.
E measurement.
The plethysmograph chamber used in these experiments is similar to the
setup described by Jacky (10) and Pappenheimer
(24). The analog output of the pressure transducer was
converted to a digital signal and directly sampled at 150 Hz by
computer using the DataPac 2000 system. The data were also recorded on
tape using a digital system (model 3000A, Vetter). The animal chamber
operates at atmospheric pressure, with the inflow and outflow of
inspired gases balanced to prevent hyper- or hypobaric conditions in
the box. The inflow gas was humidified, and the flow rate was
controlled by a flowmeter (model 7491T, Matheson). The outflow port was
connected to the in-house vacuum system via a flowmeter. A
high-resistance "bleed" of the outflow line provided ~100 ml/min
of outflow gas to the O2 and CO2 analyzers
(Applied Electrochemistry). The flow rate through the plethysmograph
was maintained at 
1.4 l/min to prevent CO2 rebreathing.
The plethysmograph was calibrated with 0.3-ml injections.
O2 consumption, CO2 production, and
temperature measurement.
O2 consumption (O2) and
CO2 production (CO2) were
measured using the Fick principle by calculating the difference in
O2 or CO2 content between inspired and expired
gas [O2 = (in × FIO2) 
(out × FOO2), where
in and out represent inflow and
outflow flow rate, FIO2 is fraction of
inflow O2, and FOO2 is
fraction of outflow O2] and normalized to milliliters per
gram of body weight per hour. The inflow O2 content was
measured at the beginning of each experiment, and the outflow content
of O2 and CO2 was read from the O2
and CO2 sensors constantly during the experiment. A
thermometer inside the chamber measured the chamber temperature. Rat
body temperature (Tb) was measured using the analog output
via telemetry from the temperature probe in the peritoneal cavity.
EEG and EMG signals. The signals from the EEG and EMG electrodes were sampled at 150 Hz, filtered at 0.3-50 and 0.1-100 Hz, respectively, and recorded directly on the computer with backup on tape.
Anatomic analysis. At the end of the experiment, the rats were killed and the medulla was quickly removed and frozen and then cut into sections (50 µm thick) with a Reichert-Jung cryostat. The sections were counterstained with cresyl violet. We identified anatomic landmarks and the site of dialysis probe placement using a rat brain atlas (25) for reference. The guide tubes were removed postmortem but before brain stem removal and sectioning, which required manipulation and produced tissue disruption. This procedure facilitated the anatomic verification of guide tube and probe tip location but also increased the volume of tissue disruption compared with that produced by simple insertion.
Data analysis.
For sleep analysis we used the raw EEG and EMG signals, the fast
Fourier transform of the EEG signal analyzed in 3.6-s epochs using
delta (0.3-5 Hz), theta (6-9 Hz), and sigma (10-17 Hz)
frequency bands, and behavioral observations. The rats were housed in a room with midnight to noon being the light or rest period and noon to
midnight being the dark or active period. All the experiments were performed between 9 AM and noon, during the end of their rest
period, or between noon and 4 PM, during the beginning of their active
period. The state of arousal was defined using criteria modified from
those of Bennington et al. (1) and Trachsel et al.
(33). The rat was judged to be awake when the EEG showed a
low-amplitude signal, delta power was low, the ratio of theta to delta
power was low, EMG activity was present, and the product of theta and
sigma power was low. The rat was judged to be in non-rapid eye movement
(NREM) sleep when the EEG showed a high-amplitude signal, delta power
was high, the ratio of theta to delta power was low, the EMG activity
was absent or low, and the product of theta and sigma power was
moderate to high. The rat was judged to be in rapid eye movement (REM)
sleep when the EEG signal showed low amplitude, delta power was low,
the ratio of theta to delta power was high, the EMG activity was absent
or low, and the product of sigma and theta power was moderate or high.
When the rat was resting quietly on all four paws with eyes open or
closed, it was sometimes difficult to distinguish light sleep from
quiet wakefulness. On occasion, we had to judge the state as
indeterminate. Data from indeterminate states are not included. We
applied this analysis to each experiment, as shown in Fig.
1. We determined NREM sleep, REM sleep,
and wakefulness visually from such records.
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100, and
up to 300, breaths at defined sleep and wakefulness periods during the
experiment. Sighs, sniffing, and recording artifacts were edited from
analysis. These data were exported to Sigmaplot 4.0 (Jandel
Scientific), and VT per 100 g body wt, frequency (f),
and E per 100 g body wt were calculated for
each breath using plethysmograph temperature and Tb for
that time period. In our initial analysis, we grouped the experiments
on the basis of the amount of time during the experiment the rat was
asleep: >50% defining "sleep" and <50% defining "awake."
For NREM sleep and wakefulness, we were able to obtain three to four
defined periods before CO2 exposure and two periods after
exposure. During the 30 min of test dialysis, we obtained NREM sleep
and wakefulness data representative of the first, second, and third
10-min periods. For our second analysis, we examined ventilatory data
in each state in all experiments. Here we calculated the maximum
percent change in each state comparing the maximum value during the
30-min test dialysis period with a baseline control value in that
state. Our REM sleep data are more fragmentary, in that REM sleep
periods in the rat are brief.
The results for E, VT, f,
O2, CO2,
and Tb during 25 or 5% CO2 tests were compared
by two-way repeated-measures paired ANOVA with treatment and time as
factors (Sigmastat, Jandel Scientific) or by paired t-test.
Post hoc tests were performed when significant differences were found.
Experimental Protocol
We dialyzed each of eight rats with 5 and 25% CO2 during morning and afternoon measurement periods when the rats were expected to be asleep or awake, respectively. Ultimately, the rat was judged to be awake or asleep by the criteria outlined above. Rats were not always predominantly awake or sleep during the time period in the circadian cycle of greater likelihood for each state, so our initial paired experimental design did not work out in practice. As a result, we analyze our results using two approaches. In the first approach, we grouped responses to 5 and 25% CO2 during wakefulness or sleep as defined by our EEG and EMG criteria. If the rat was asleep for most (>50%) of the entire experiment and for most of the 30-min test dialysis period, the experiment was called a sleep experiment, and ventilatory data were analyzed only in the NREM sleep periods. If the rat was awake for most of the entire experiment and for the 30-min test dialysis period, the experiment was called an awake experiment, and ventilatory data were analyzed only in the periods of wakefulness. Some rats were dialyzed with 5 or 25% CO2 more than once. For data analysis and statistics, multiple runs were averaged, so that each rat contributed one set of data in any condition. Data analysis was paired; e.g., responses to 25 and 5% CO2 dialysis in rats of the sleep group were compared using data obtained from each of six rats. Insofar as the rats that slept for most of the experimental period tended to have NREM sleep periods with more consistently increased delta power and those that were awake for most of the experimental period had the opposite, this approach allowed us to clearly separate the effects in sleep from the effects in wakefulness.In the second approach, we analyzed all wakefulness, NREM sleep, and
REM sleep data in each experiment regardless of the amount of time the
rat was awake or asleep. This allowed a paired comparison of effects
but assumed that any sleep/wakefulness effect will be present
regardless of the amount of time spent in sleep or the depth of sleep.
In some experiments, there may be no periods of sleep or wakefulness.
The change in f, VT, and E was
calculated using a single maximum value obtained in any state during
the 30-min dialysis period and a value obtained in that same state during the preceding control period.
The rats would initially be weighed and then gently held while the dummy cannula was removed and the dialysis probe was inserted into the guide tube. The animals were placed into a plethysmograph chamber and allowed 30-40 min to acclimate. Dialysis with 5% CO2-equilibrated aCSF began when the rat was placed into the chamber and continued through the entire experimental period. All experiments were performed in the room air-breathing condition. Baseline measurements were made over the next 40 min. The dialysis solution was then changed to 25% CO2 or maintained at 5% CO2, and measurements were taken over 30 min. The dialysis tube and cannula dead space is taken into account along with the dialysis fluid flow rate, so that time 0 is the estimated time at which the test solution reaches the exchange membrane. After 30 min, the dialysis solution was changed to 5% CO2 or maintained at 5% CO2, and measurements were again made over 40 min.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Typical Experiment
Figure 1 shows, for a typical experiment, the raw EEG and EMG signals, the fast Fourier transform-derived parameters, and calculated f andE for the state-specific periods chosen
before, during, and after dialysis with aCSF equilibrated with 25%
CO2. Dialysis with aCSF equilibrated with 5%
CO2 occurs from beginning to end, except for the 30-min
test period. This experiment was judged to be in the sleep group,
inasmuch as in 50% of the total experiment and in 77% of the 30-min
25% CO2 dialysis period, the rat was in NREM sleep. The
rat was in REM sleep for 7.4% of the total period and for 6.5% of the
test dialysis period. The rat was awake for 43% of the total period,
much of this in the initial portion of the experiment, and for 16.7%
of the test dialysis period.
The power spectrum records show nicely the typical sleep cycling of the rat with periods of high delta power (NREM sleep) interspersed with periods of REM sleep and wakefulness. In the sleep group with 25% CO2 dialysis, there were 7.4 ± 0.5 (SE) NREM sleep periods per total experiment with a mean duration of 7.7 ± 0.4 min. During the 30-min test dialysis period, there were 3.2 ± 0.2 NREM sleep periods with a 6.9 ± 0.4 min average duration. In the sleep group with 5% CO2 dialysis, there were 7.0 ± 0.5 NREM sleep periods per total experiment with an average duration of 7.2 ± 0.6 min. During the 30-min test dialysis period, there were 3.4 ± 0.2 NREM sleep periods with a 5.9 ± 0.6 min average duration. Dialysis with 25% CO2 did not affect sleep cycling.
E, VT, and f were calculated for
100-300 breaths in periods of NREM sleep and wakefulness. For
grouped data, each experiment was placed into the sleep or the awake
group on the basis of the percentage of time in the whole experiment
and of the 30-min dialysis period in NREM sleep or wakefulness, and we
report ventilatory data only in that state. For this typical example,
we have calculated ventilatory results in all three states.
In wakefulness, f and E were unaffected by 25%
CO2 dialysis in the medullary raphe. In NREM sleep,
baseline E was similar to that in wakefulness and f
was lower. With 25% CO2 dialysis, E and
f increased during the first half of the dialysis period and then
returned to baseline levels toward the end of the 25% CO2
dialysis period. This recovery of E and f during
25% CO2 dialysis was not the usual response, as can be
seen in the grouped data discussed below. In REM sleep, baseline
E was lower and f was higher than in NREM sleep or wakefulness. With 25% CO2 dialysis, E
and f increased.
Sleep
Analysis of the percentage of total time in NREM sleep and wakefulness for our rats, with the animals grouped by whether the measurements were obtained in the late sleep period (9 AM to noon) or the early active period (noon to 4 PM), shows that, for 22 runs in 8 rats obtained before noon, they are, on average, awake for 52 ± 7% (SE) of the time and in NREM sleep for 42 ± 6% of the time. For the experiments after noon (18 runs in 8 rats), the rats were awake for 47 ± 5% of the time and in NREM sleep for 45 ± 4% of the time. Thus these rats showed, as a group, approximately equal distributions of NREM sleep and wakefulness whether they were measured before or after noon.Initially, we grouped our data on the criterion of the state in which
the rat spent the most time during the entire experiment and
the 30-min test dialysis period. Rats that were mostly in NREM sleep
during the experiment, whether before or after noon, were grouped as
sleep. Rats that were mostly awake during the experimental period,
whether before or after noon, were grouped as awake. Table
1 shows the percentage of time in each
state during the entire experiment and during the 30-min test dialysis period for animals paired according to predominant sleep or
wakefulness. Six rats contributed paired data in sleep; six (not the
same rats) contributed paired data in wakefulness. The sleep group, on
average, spent 55-57% of the total experiment time and 65% of
the 30-min dialysis test period in NREM sleep. The awake group spent
67-73% of the total experiment time and 65% of the 30-min
dialysis test period awake.
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Ventilatory Responses to 25% CO2 Dialysis in the Medullary Raphe: Initial Approach
Figure 2 shows theE, VT, and f responses of the sleep
group to test period dialysis with 5 or 25% CO2. The
results are paired and plotted in 10-min bins. For example, ventilatory
data obtained in NREM sleep during the 10-min period before the onset of dialysis are grouped as the 5-min time point. E
is significantly greater with 25% CO2 dialysis. Two-way
repeated-measures paired ANOVA using treatment (25 vs. 5%
CO2 dialysis) and time showed a significant interactive
effect (P < 0.03), with a Bonferroni post hoc
comparison showing differences between treatments at 10 min
(P < 0.005), 20 min (P < 0.01), and
30 min (P < 0.01). Frequency is significantly greater
with 25% CO2 dialysis. Two-way repeated-measures paired
ANOVA using treatment (25 vs. 5% CO2 dialysis) and time
showed a significant interactive effect (P < 0.001),
with Bonferroni post hoc comparison showing differences between
treatments at 10, 20, and 30 min (P < 0.005).
VT was not significantly affected by 25 or 5%
CO2 dialysis in sleep. For these analyses, the four initial
baseline values were combined into a single value for each animal.
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Figure 3 shows responses of
E, VT, and f responses of the awake
group to test period dialysis with 5 or 25% CO2. Two-way repeated-measures paired ANOVA using treatment (25 vs. 5%
CO2 dialysis) and time showed no significant effects.
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To emphasize the most robust finding of the study, we have plotted in
Fig. 4 the percent change in f and
E, compared with the average baseline, for the sleep
group results. E and f are significantly greater
with 25% CO2 dialysis (P < 0.01 and
P < 0.004, respectively, by 2-way repeated-measures
paired ANOVA).
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O2,
CO2, and Tb
O2, CO2,
and Tb results for the sleep group during dialysis with 25 or 5% CO2. Two-way repeated-measures paired ANOVA using
treatment (25 vs. 5% CO2 dialysis) and time showed no
significant effects. Similar analysis for data during wakefulness also
showed no significant effects of treatment or time. Comparison of sleep
group data with awake group data in either treatment showed no
significant differences in O2 or CO2, but Tb was
significantly lower in sleep (P < 0.01, by 2-way ANOVA). The difference in mean values was between 0.3 and 0.4°C.
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Ventilatory Responses to 25% CO2 Dialysis in the Medullary Raphe: Second Approach
In the second approach, we did not group the experiments by amount of sleep or wakefulness but utilized, as much as possible, data in wakefulness, NREM sleep, and REM sleep in each experiment. We expressed the maximum increase in f andE in each
state during the 30-min test dialysis period as a percentage of the
closest pretest dialysis baseline period in that same state. The NREM sleep and wakefulness f data are shown in Fig.
6 as a function of the percentage of
total experimental time spent in sleep (NREM and REM). When the
observations during the 30-min test dialysis period are made in
wakefulness, there is no effect of 25% CO2 dialysis
regardless of the amount of time during the experiment the rat was
asleep. In marked contrast, when the observations of f are made during
NREM sleep, there is an effect of 25% CO2 dialysis, and
the degree of this effect is greater the more time the rat was asleep
during the experiment. Linear regression analysis shows
y = (0.45 ± 0.17)x 2.9 (r = 0.65) for 25% CO2 dialysis and y = (0.08 ± 0.08)x 4.9 (r = 0.33) for 5% CO2 dialysis. The slope for the 5% CO2 dialysis response lies
outside the 95% confidence interval for the 25% CO2
dialysis results. Paired t-test comparison of the
maximum percent increase in f during sleep in the 25%
CO2 dialysis data vs. that observed during wakefulness
shows P < 0.01. A similar analysis of the ventilation
data shows y = (0.41 ± 0.21)x 4.1 (r = 0.53) for 25% CO2 dialysis
and y = (0.04 ± 0.08)x + 3.9 (r = 0.2) for 5% CO2 dialysis. Here
again, the slope for the 5% CO2 dialysis data lies outside
the 95% confidence interval for the slope of the 25% CO2
dialysis data. In this analysis as well, focal acidosis of the
medullary raphe increases f and E in sleep, and the
effect is greater if more of the experimental time is spent in sleep.
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In a comparison of REM sleep periods during the 25% CO2
exposure with REM sleep periods in the prior baseline period with 5%
CO2 dialysis (n = 5 rats), f increased by
15 ± 4.5%, VT decreased by 3 ± 5%, and
E increased by 5.1 ± 5.9%. In rats with
continuous 5% CO2 dialysis, comparing REM sleep data in
the 30-min test period with prior baseline REM sleep periods
(n = 6 rats), we observed a 2.5 ± 2.7% increase
in f, a 1 ± 3.5% change in VT, and a 3 ± 3%
change in E. The increase in f during 25%
CO2 dialysis compared with 5% CO2 dialysis was
significant (P < 0.04, by t-test).
The changes in VT and E were not.
Anatomy
In Fig. 7, we show for each of the eight rats the cross section of the medulla that contains the greatest area of tissue disruption caused by the guide tube and dialysis probe. The top two cross sections represent actual stained sections of typical results for more rostral and caudal probe placements. The schematic sections below each stained section show the location of the probes in the other six rats grouped into the rostral and caudal sections. This rostral and caudal grouping is for ease of presentation; all probe sites were within the rostral-to-caudal confines of the facial nucleus.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Methods
Using microdialysis of aCSF equilibrated with 25% CO2, we produce focal acidification within a single chemoreceptor site in an unanesthetized rat. The microdialysis probe tip has dimensions of 1 mm × 0.24 µm; volume is 45 nl. Tissue damage and gliosis are present in the region surrounding the probe tip location (Fig. 7). We allow 3-4 days of recovery from surgery before conducting an experiment. In our experience, the rat is eating and gaining weight by this time, and we diminish the likelihood of probe or EEG/EMG connector damage and, perhaps, of greater tissue reaction to the presence of the probe by conducting experiments at this time. We dialyze at a high flow rate, 45 µl/min, which we have found necessary to deliver sufficient amounts of highly diffusible CO2 to the tissue to produce the focal acidification. This flow rate does not, by itself, produce changes in breathing or metabolism or in the sleep-wakefulness cycle, as is evident in the responses to control experiments with 3 h of dialysis with 5% CO2 in the dialysate. The tissue reaction does not seem to interfere with the ability of this dialysis approach to acidify the tissue. We have evaluated in anesthetized rats the intensity and spread of the focal pH change induced by this method (15). At the probe, the pH change is similar to that observed with an increase in end-tidal PCO2 to 63 Torr; with increasing distance from the probe, the pH change lessens, such that there is no detectable change at 600 µm. We are, at present, measuring arterial and medullary tissue pH in the unanesthetized rat during exposure to microdialysis-induced focal CO2 and to systemic increases in CO2. Preliminary data suggest that in the unanesthetized rat the degree of pH change is less.In our initial study of focal CO2 application in the RTN region, we relied on behavioral criteria to distinguish sleep from wakefulness. In the present study, we use EEG and EMG data to determine continuously the sleep/wakefulness state of the rat. Our observations indicate that when the rat is curled up, motionless with its eyes closed and its head tucked between the forepaws, it is usually in NREM sleep, as judged in our prior study (15). However, our present observations, similar to those of others (33), indicate that the rat arousal state cycles continuously, with wakefulness, NREM sleep, and REM sleep having different frequencies and durations depending on the time in the light/dark circadian period. Constant recordings of EEG and EMG information give access to greater amounts of usable data during any experiment and allow reliable determination of periods of quiet wakefulness (see below). Other improvements in experimental design include continuous dialysis with aCSF through the entire experiment. This adds an important control for any effects of dialysis per se. We also analyze 100-300 breaths for each defined sleep/wakefulness state. This large sample of breathing in any state minimizes breath selection bias and gives a better overall estimate of breathing.
We report only data obtained from rats with the dialysis guide tubes placed within the rostral aspect of the medullary raphe, as proven by postmortem anatomic analysis. Our probe locations are constrained to within the rostral-to-caudal dimension of the facial nucleus. We are affecting only a portion of the medullary raphe, but it is a portion that is known to have connections to other brain stem sites involved with the control of breathing (2, 3, 7, 12), that contains chemosensitive neurons (31), and that expresses chemosensitivity (2).
Sleep/Wakefulness States
We altered the circadian light/dark period to facilitate these experiments. Our hope was to make measurements in sleep from 9 AM to noon, which would be the end of the rat's imposed circadian sleep (light) period, and in wakefulness from noon to 4 PM, which would be the beginning of the imposed circadian active (dark) period. We observed approximately equal amounts of NREM sleep and wakefulness during our before-noon and after-noon experiments. Although this obviated our ideal design, the result is not surprising, given the published data on rat diurnal sleep-wakefulness cycling; i.e., at the end of the sleep period and beginning of the active period, there is a merging of the frequency of the NREM sleep and wakefulness states (Fig. 2) (33). Measurements obtained continuously in rats over 24-h periods (33) indicate that, early in the 12-h sleep period, NREM episodes show greater amounts of delta power and are more regular than they are later in the period. As the switchover time (noon in our case) approaches, there are more periods of wakefulness and the high delta power episodes are less robust. At the beginning of the active period, some NREM sleep, high delta power episodes persist while the periods of wakefulness increase in frequency and duration. In this early period of wakefulness, the rats are not as fully active as they are later in the dark period. This facilitates our measurements of breathing, which entail a whole body plethysmograph technique. For successful data collection, rat movement within the plethysmograph needs to be at a minimum. Thus this technique is less well suited for measurements of fully active, awake rats engaged in activities such as exploring, feeding, and grooming. Our state of wakefulness represents quiet wakefulness, not this fully active state with enhanced motor activity. Nevertheless, using the criteria in this study, we can clearly distinguish such periods of quiet wakefulness from NREM and REM sleep. We observed a frequency and duration of NREM sleep periods similar to previous reports for the rat with 24-h monitoring (33). In addition, 25% CO2 dialysis did not alter the frequency or duration of the NREM sleep episodes; our treatment did not alter sleep cycling. Our approach gives us a firm basis to examine whether the response of the raphe to focal acidification in NREM sleep differs from that in wakefulness. Our design also minimizes circadian influences on CO2 sensitivity (24).Medullary Raphe and Breathing
Earlier studies involving electrical or glutamate stimulation of the medullary raphe reported excitation (2, 7, 12) and inhibition of breathing (12, 18). Thus the raphe is known to have the capability of alteringE.
Chemoreception in the raphe has been studied in vivo and in vitro. Veasey et al. (34) reported that six neurons in the medullary raphe of unanesthetized cats increased their firing rate with systemic hypercapnia. In four of these neurons, responses to hypercapnia in NREM sleep were absent. These workers hypothesized that these raphe cells function as "gain setters" for motor neurons, here involved with breathing. Dreshaj et al. (6) disrupted the function of rather large volumes of the medullary raphe in decerebrate or anesthetized piglets by making up to five 300-nl injections of the local anesthetic lidocaine or the excitatory amino acid neurotoxin ibotenic acid. These were presumably placed in the rostral-to-caudal axis; no detailed anatomy of the entire region affected is available. Nevertheless, these experiments resulted in profound reductions in the response of ventilatory output to increased systemic CO2. The implication is that the medullary raphe has an important role in central chemoreception. These studies do not distinguish between an effect directly on chemoreceptors themselves and an effect via a reduction in a required excitatory activity emanating from the raphe to the respiratory network, which is permissive for the full chemoreceptor response.
More direct studies of chemoreception in the medullary raphe involve focal acidification by the microinjection of acetazolamide, an inhibitor of carbonic anhydrase (3). That respiratory output is increased after many, but not all, such injections is interpreted to indicate the presence within the medullary raphe of chemoreceptors, in that the animals, on ventilators, have normal CO2 levels and pH at other brain sites. Studies of medullary slice preparations that include the raphe and of isolated neurons from the medullary raphe have described neurons that can be excited, or inhibited, by CO2, providing evidence of putative chemoreceptive neuronal functions in the raphe (31).
Raphe Acidification Increases Breathing Frequency in Sleep
The major finding of this study is that focal acidification of the medullary raphe via microdialysis of CO2-laden aCSF increasesE in the unanesthetized rat during sleep
but not during wakefulness. In NREM sleep, there is a robust increase
in E, which occurs via an increase in f
(15-20%). In REM sleep, f increases by 15%, but a small decrease
in VT obviates any increase in E.
We used two approaches for data analysis. In the first, experiments were grouped by the amount of time during the experiment and during the 30-min test dialysis period that the rat was asleep: >50% defining the sleep group and <50% defining the awake group. In each group, responses to 25 or 5% CO2 dialysis were paired. This approach maximizes the amount of data available in sleep and wakefulness, allowing a detailed time course analysis as shown in Figs. 2-4. In this approach, we did not examine wakefulness data in the sleep group and vice versa, thereby losing some information.
In our second approach, we analyzed data in wakefulness, NREM sleep,
and REM sleep in each experiment. We measured the peak f and
E response during the 30-min test dialysis compared
with the f and E in the same arousal state before
the test dialysis period. This allowed a paired analysis in each state
of the maximum response to focal acidosis in the raphe but did not
allow any analysis of the time course of the response, since the amount of NREM and REM sleep in the experiments with predominant wakefulness was small and, in some cases, absent, and vice versa. This analysis did
uncover the surprising relationship, shown on Fig. 6, between the
degree of the focal acidification effect on breathing and the total
amount of experimental time in sleep.
We explain this relationship by the depth of NREM sleep. By inspection, in rats with less total experimental time in sleep, the depth of NREM sleep, on average, is smaller. To estimate this, we averaged for each animal the percent increase in delta power in all NREM periods in each experiment compared with the delta power during wakefulness in each case. We then averaged these data for the two groups. In the sleep group, the average increase in delta power was 243 ± 22% (n = 6); in the awake group, it was 192 ± 6% (P < 0.05, by t-test). We suggest that the degree of the effect of focal acidification in the medullary raphe on breathing in sleep depends on the depth of sleep.
In a prior experiment with focal acidification of the same region of the medullary raphe produced by microinjection of acetazolamide in anesthetized, vagotomized rats (3), an increase in the amplitude of the integrated phrenic nerve activity was observed without a change in f. It is difficult to compare this experiment with the present experiment in the unanesthetized rat, inasmuch as the vagus nerve was sectioned and systemic PCO2 was maintained at a constant normal value.
We conclude that the major effect of focal acidification of the
medullary raphe in the unanesthetized rat is an increase in E in sleep that occurs via an increase in f. In
contrast, Veasey et al. (34) reported that a small number
of neurons in the medullary raphe increased their firing rate with
systemic hypercapnia, but only in wakefulness. It may be that these
workers recorded from neurons more directly attuned to the motor act of
breathing, neurons that were not chemosensitive. It may also be that
our focal acidosis stimulates nonserotonergic raphe neurons, although
the in vitro work of Richerson (31) showing specific
pH-sensitive responses of raphe neurons argues against this.
In respect to central chemoreception, we suggest that each of many
locations for central chemoreceptors has a specific role that depends
on arousal state and stimulus intensity. The overall sensitivity of the
system to a small increase in CO2 systemically, such that
all sites are stimulated, is quite large. Inhalation of 7%
CO2 in the unanesthetized rat increases
E by 200% (15). Focal acidification
of the RTN or the medullary raphe increases E by
15-24% with a predominant effect in one arousal state for either
site. It seems reasonable to hypothesize (22) that the fine overall sensitivity of the system to increased systemic
CO2 requires stimulation of multiple chemoreceptor sites
and that some sites operate more importantly in wakefulness, others in sleep. Thus the contribution of the raphe would be to increase f in sleep.
Medullary Raphe and Sleep
The medullary raphe seems to be involved in the sleep-wakefulness cycle, but its precise role is uncertain (28, 29, 32). Raphe neurons exhibit their greatest firing rate during wakefulness, a lower firing rate in NREM sleep, and a very low or absent firing rate in REM sleep (11, 34). It is suggested that the sleep-related decrease in firing rate disinhibits mesopontine cholinergic neurons that are active in REM sleep, that the raphe neurons are part of the brain stem mechanisms of sleep regulation (32). The source of the decrease in raphe firing rates in sleep is unknown. The rat breathes at a slower frequency in NREM sleep than in wakefulness, and in NREM sleep, raphe neurons fire at slower rates. It has been suggested that the sleep-related changes in firing rate of raphe neurons affect upper airway caliber, inasmuch as there are projections from the raphe to hypoglossal motor neurons, and direct dialysis of serotonin (5-hydroxytryptamine, 5-HT) on hypoglossal motor neurons increases genioglossus muscle activity, which would increase upper airway caliber (9). Thus one may presume that decreased firing in the raphe could result in less 5-HT release and less excitation with reduced upper airway motor tone (35), which occurs in sleep.How do our findings fit into this picture? We know from the in vitro
experiments of Richerson (31) that medullary raphe neurons
can be excited, or inhibited, by CO2. Our data indicate that, in the intact, unanesthetized rat, focal medullary raphe acidification increases f predominantly and that this occurs in NREM
and REM sleep but not in wakefulness. We would like to know whether
raphe neurons increase their firing rate with focal CO2 stimulation in vivo in NREM and REM sleep and in wakefulness. It seems
possible that, in wakefulness, these neurons, which are already firing,
cannot be further stimulated. During NREM and REM sleep, when their in
vivo firing rates are lower, focal CO2 can have a greater
effect. We hypothesize that focal CO2 increases the raphe
neuron firing rate in NREM and REM sleep. The focal CO2
stimulation of the raphe converts its input to the respiratory premotor
and motor neurons to input similar to that in wakefulness; that is,
focal acidification changes raphe firing to be more similar to that in
wakefulness. The result is a stimulation of 5-HT release at hypoglossal
and other respiratory neurons, which increases f. The rat remains
asleep, but breathing becomes similar to that in wakefulness. An
analogous interpretation was made by Berner et al. (4) in
their studies of raphe inactivation and thermoregulation. With
lidocaine injection into the raphe, responses to cold stress originating in the periphery or centrally in the hypothalamus were
inhibited. They observed decreases in O2
and muscle EMG activity similar to those observed with spontaneous
entry into sleep from wakefulness. It was as if the raphe inactivation
by lidocaine initiated thermoregulatory responses associated with sleep. In our case, raphe activation by CO2 initiated
respiratory responses associated with wakefulness.
If medullary raphe stimulation by focal CO2 application in sleep increases f by the removal of a normal inhibition of raphe neurons in this state, we would expect the resultant f to be similar to that observed in wakefulness. Examination of our paired data provides some support for this conclusion, inasmuch as the f values with CO2 stimulation of the raphe in sleep are only slightly greater than those observed in wakefulness.
Physiological Roles of the Medullary Raphe
Two general theories for raphe function, possibly relevant to our study, have been proposed. Jacobs and Fornal (11) observed that in a wide variety of behavioral circumstances the firing rate of raphe neurons is closely correlated with motor activity, especially repetitive motor activity, such as breathing. They suggest that the raphe serves to facilitate motor activity, with sensory processing being suppressed. Auxiliary to this modulatory function is a coordination of autonomic function. Lovick (17) proposed that the raphe serves to modulate existing activity in autonomic neural control systems and that this modulation is state dependent.If the raphe serves as a modulatory system for somatic motor and autonomic control, then why should it exhibit chemosensitivity? We note that another brain stem region that may serve mainly in a modulatory capacity, the locus ceruleus, also exhibits chemosensitivity in vitro and in vivo (5, 23, 27). These two monoamine systems may work in concert to set the gain of motor output/sensory processing and autonomic control in different states of alertness and arousal. CO2 may serve as a fundamental chemical signal to these two sets of neurons to determine their level of activity, to affect the proposed gain function, and to coordinate autonomic processes. The ventilatory chemoreceptor aspects of raphe and locus ceruleus neurons may be adjunct to this broader CO2-dependent function.
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ACKNOWLEDGEMENTS |
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Dr. Jing Shi performed many of the experiments reported here; Eva Liu and Joshua Osborne performed early versions of the experiment.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grant HL-28066.
Address for reprint requests and other correspondence: E. E. Nattie, Dept. of Physiology, Dartmouth Medical School, Borwell Bldg., Lebanon, NH 03756-0001 (E-mail: Eugene.Nattie{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 9 October 2000; accepted in final form 10 November 2000.
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REFERENCES |
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TOP
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METHODS
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M. R. Hodges, C. Opansky, B. Qian, S. Davis, J. Bonis, J. Bastasic, T. Leekley, L. G. Pan, and H. V. Forster Transient attenuation of CO2 sensitivity after neurotoxic lesions in the medullary raphe area of awake goats J Appl Physiol, December 1, 2004; 97(6): 2236 - 2247. [Abstract] [Full Text] [PDF]
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M. R. Hodges, P. Martino, S. Davis, C. Opansky, L. G. Pan, and H. V. Forster Effects on breathing of focal acidosis at multiple medullary raphe sites in awake goats J Appl Physiol, December 1, 2004; 97(6): 2303 - 2309. [Abstract] [Full Text] [PDF]
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 R. W. Putnam, J. A. Filosa, and N. A. Ritucci Cellular mechanisms involved in CO2 and acid signaling in chemosensitive neurons Am J Physiol Cell Physiol, December 1, 2004; 287(6): C1493 - C1526. [Abstract] [Full Text] [PDF]
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A. Hewitt, R. Barrie, M. Graham, K. Bogus, J. C. Leiter, and J. S. Erlichman Ventilatory effects of gap junction blockade in the RTN in awake rats Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2004; 287(6): R1407 - R1418. [Abstract] [Full Text] [PDF]
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N. C. Taylor, A. Li, A. Green, H. C. Kinney, and E. E. Nattie Chronic fluoxetine microdialysis into the medullary raphe nuclei of the rat, but not systemic administration, increases the ventilatory response to CO2 J Appl Physiol, November 1, 2004; 97(5): 1763 - 1773. [Abstract] [Full Text] [PDF]
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M. R. Hodges, L. Klum, T. Leekley, D. T. Brozoski, J. Bastasic, S. Davis, J. M. Wenninger, T. R. Feroah, L. G. Pan, and H. V. Forster Effects on breathing in awake and sleeping goats of focal acidosis in the medullary raphe J Appl Physiol, May 1, 2004; 96(5): 1815 - 1824. [Abstract] [Full Text] [PDF]
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M. L. Messier, A. Li, and E. E. Nattie Inhibition of medullary raphe serotonergic neurons has age-dependent effects on the CO2 response in newborn piglets J Appl Physiol, May 1, 2004; 96(5): 1909 - 1919. [Abstract] [Full Text] [PDF]
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E. E. Nattie, A. Li, G. Richerson, and D. A. Lappi Medullary serotonergic neurones and adjacent neurones that express neurokinin-1 receptors are both involved in chemoreception in vivo J. Physiol., April 1, 2004; 556(1): 235 - 253. [Abstract] [Full Text] [PDF]
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H. Nakayama, C. A. Smith, J. R. Rodman, J. B. Skatrud, and J. A. Dempsey Carotid body denervation eliminates apnea in response to transient hypocapnia J Appl Physiol, January 1, 2003; 94(1): 155 - 164. [Abstract] [Full Text] [PDF]
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E. E Nattie and A. Li Substance P-saporin lesion of neurons with NK1 receptors in one chemoreceptor site in rats decreases ventilation and chemosensitivity J. Physiol., October 15, 2002; 544(2): 603 - 616. [Abstract] [Full Text] [PDF]
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Y. Okada, Z. Chen, W. Jiang, S.-I. Kuwana, and F. L. Eldridge Anatomical arrangement of hypercapnia-activated cells in the superficial ventral medulla of rats J Appl Physiol, August 1, 2002; 93(2): 427 - 439. [Abstract] [Full Text] [PDF]
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E. E. Nattie and A. Li CO2 dialysis in nucleus tractus solitarius region of rat increases ventilation in sleep and wakefulness J Appl Physiol, May 1, 2002; 92(5): 2119 - 2130. [Abstract] [Full Text] [PDF]
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, Wakefulness; 
, non-rapid eye
movement (NREM) sleep; 
, rapid eye movement (REM)
sleep. Arousal state is first defined using electroencephalogram and
electromyogram criteria. Then, within a period of a single arousal
state (wakefulness, NREM sleep, or REM sleep), 100-300 breaths
were analyzed, and single-breath values of f, tidal volume
(VT), and 
