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Pedunculopontine stimulation alters respi ration and
increases ACh release in the pontine retie ular fo rma
RALPH LYDIC AND HELEN A. BAGHDOYAN
Department
of
Anesthesia, College
of
Medicine, Pennsylvania State University,
tion
Hershey, Pennsylvania 17033
Lydic, Ralph, and Helen A. Baghdoyan. Pedunculopon-
tine stimulation alters respiration and increases ACh release in
the pontine reticular formation. Am. J. Physiol. 264 (Regulatory
Integrative Comp. Physiol. 33): R544-R554,1993.-The present
study examined the hypothesis that cholinergic neurons in the
pedunculopontine tegmental nucleus (PPT) can cause the re-
lease of acetylcholine (ACh) in the pontine reticular formation
and contribute to respiratory depression. In vivo microdialysis
of the gigantocellular tegmental field (FTG) was performed in
10 adult male cats while respiration was being measured. In
four intact, unanesthetized cats these measurements were ob-
tained during states of quiet wakefulness and during the rapid
eye movement (REM) sleeplike state caused by FTG microin-
jections of carbachol. The results demonstrate a simultaneous
time course of enhanced ACh release in the FTG and respira-
tory rate depression. In six barbiturate-anesthetized cats simi-
lar measurements were obtained while PPT regions containing
NADPH-positive neurons were electrically stimulated. PPT
stimulation caused increased ACh release in the FTG and
caused respiratory rate depression. Together, these findings
are consistent with the hypothesis of a causal relationship be-
tween ACh release in the FTG and respiratory depression.
microdialysis; respiration; control of breathing; sleep; cholin-
ergic; gigantocellular tegmental field; NADPH-diaphorase
ACETYLCHOLINE
(ACh) has been known for more than
50 years to be involved in the central nervous system
(CNS) regulation of respiration (8,
13).
Subsequent lo-
calization studies demonstrated that the major drive to
breathe originates from CO2 receptors near the ventro-
lateral surface of the medulla (reviewed in Ref.
11)
and
that CNS chemosensitivity is, at least in part, cholin-
ergically mediated (9,12,38,39). More than 20 years ago
ACh was also suggested to play a critically important
role in the generation of rapid eye movement (REM)
sleep (reviewed in Ref.
47).
Since the
197Os,
multiple
lines of evidence have localized cholinergic REM sleep-
generating mechanisms to the pontine brain stem (re-
viewed in Refs.
19, 28, 47).
In view of the foregoing observations, we wondered
whether alterations in CNS cholinergic neurotransmis-
sion contribute to the clinically significant problem of
sleep-dependent respiratory depression
(10,41).
A series
of investigations during the past 5 years has shown that
many of the respiratory changes occurring during REM
sleep are mimicked during the REM sleeplike state
caused by microinjecting cholinergic agonists into the
medial pontine reticular formation of intact, unanesthe-
tized cats. These studies discovered that, like natural
REM sleep, the cholinergically induced REM sleeplike
state is accompanied by upper airway muscle hypotonia
(33)) significant reductions in minute ventilation (27))
decreased discharge of parabrachial respiratory neurons
(14, 15),
a diminished ventilatory response to hyperoxic
hypercapnia
(24, 32),
and increased ACh release in the
gigantocellular tegmental field (FTG) of the medial pon-
tine reticular formation
(31).
These findings raise the
question: By what mechanisms do FTG regions known
to be involved in REM sleep generation also cause state-
dependent changes in respiration?
Neurons in the FTG are not cholinergic because they
contain neither ACh nor the ACh synthetic enzyme cho-
line acetyltransferase (2
1,48).
Because the FTG contains
receptors for ACh, this region of the pons is referred to
as cholinoceptive. In 1988, it was discovered that ACh
input to the FTG arises from the more rostrally and dor-
sally located laterodorsal tegmental (LDT) and pedun-
culopontine tegmental (PPT) nuclei (37,45). The potent
respiratory effects caused by injecting cholinergic ago-
nists into the FTG and the fact that cholinergic input to
the pontine FTG arises from the LDT/PPT led us to
hypothesize that the cholinergic LDT/PPT nuclei con-
tribute to state-dependent respiratory depression (3 1,
33).
Here we describe two studies designed to test the
foregoing hypothesis. First, measurements of ACh and
respiration obtained during the cholinergically induced
REM sleeplike state quantify the time course of en-
hanced ACh release in the FTG and the parallel time
course of declining respiratory rate. Second, experiments
performed on barbiturate-anesthetized cats demonstrate
that electrical stimulation of the PPT causes enhanced
ACh release in the FTG while simultaneously depressing
respiration. Portions of these results have been presented
in preliminary form (29, 30, 31).
METHODS
Chronic studies. As described in detail elsewhere (3l), four
adult male cats were anesthetized with halothane (2-3% in 0,)
and implanted with electrodes to permit the polygraphic record-
ing of sleep and wakefulness. Stainless steel guide tubes were
positioned 5 mm dorsal to the FTG, using the atlas of Berman
(6). These guide tubes made it possible to repeatedly position a
microinjection cannula into the left FTG and a microdialysis
probe into the right FTG at stereotaxic coordinates P3, Ll.5,
and H-5 to H-7. This region corresponds to plate 37 of Ber-
man’s stereotaxic atlas and is synonymously referred to as the
medial pontine reticular formation or the FTG. Throughout
this paper we retain Berman’s (6) FTG nomenclature and use it
to describe the pontine region specified by the foregoing ster-
eotaxic coordinates.
After surgical recovery, the cats were trained to sleep in a
head-stable position in the laboratory. After a 2- to 3-wk train-
ing period it was possible in these intact, unanesthetized cats to
microinject carbachol (0.4 or 4.0 pg/O.25 ~1) into the left FTG
while simultaneously microdialyzing the right FTG (see Fig. 1,
A and B). The two carbachol doses correspond to concentra-
tions of 8.8 and 88 mM, respectively. Rate of respiration was
measured using a thermistor placed at the nares.
R544 0363-6119/93 $2.00 Copyright 0 1993 the American Physiological Society
PONTINE ACETYLCHOLINE RELEASE AND RESPIRATORY DEPRESSION
R545
B
I
8
l
L
2 min
Ach
L,
A
U
C
STlMULATlON n
P
D
ACh
Fig.
1.
Schematic drawings of coronal sections
through the cat pons illustrating placement of
microinjection cannula and microdialysis probe
in gigantocellular tegmental field (FTG) of the
pontine reticular formation and placement of
stimulating electrode in pedunculopontine teg-
mental nucleus (PPT). A illustrates methods
used in conscious cat for microinjecting
1
FTG
while microdialyzing the contralateral FTG.
Horizontal arrow on dialysis probe indicates
output tube from which dialysis sample was col-
- lected in 30-4 volumes. B: high performance liq-
DCARB - uid chromatography (HPLC) chromatograms
measuring ACh during wakefulness and during
the carbachol-induced REM sleeplike state
(DCarb). Amount of ACh present was quantified
from area under ACh peaks. C and D schematize
experimental methods used for barbiturate-
anesthetized cats in which a stimulating elec-
trode was placed in the pedunculopontine teg-
mental (PPT) nucleus (C) and a microdialysis
probe was placed in the ipsilateral FTG (D).
Stereotaxic coordinates of posterior
1.0 (Pl)
and
3.0 (P3) are from the stereotaxic atlas of Berman
(6). 5N, trigeminal nerve; BC, brachium con-
junctivum; BP, brachium pontis; CS, superior
central nucleus; FTP, paralemniscal tegmental
field; LDT, laterodorsal tegmental nucleus; LL,
lateral lemniscus; IC, inferior colliculus; KF,
Kolliker-Fuse nucleus; P, pyramidal tract; PB,
parabrachial nucleus; V4, fourth ventricle.
Acute
studies. For the second set of experiments, six cats were
anesthetized with an intravenous injection of pentobarbital so-
dium (35 mg/kg) and placed in a stereotaxic frame. The cats
were allowed to breathe spontaneously, and core body temper-
ature was maintained at 37°C. A small hole was made in the
skull, permitting the insertion of a bipolar stimulating electrode
in the left PPT. The stereotaxic coordinates for the PPT-stim-
ulating electrode were PO.& L3.0, H-2.5, 30” posterior. A mi-
crodialysis probe was placed in the left FTG at P3, Ll.5, H-5
(see Fig. 1, C and 0). Respiratory measures in the anesthetized
cats were obtained with a thermistor at the nares
(n = 3)
or via
an endotracheal tube
(n =
3). The endotracheal tube was at-
tached to a pneumotachograph for measuring airflow, and tidal
volumes were obtained from a Grass 7PlO integrated flow sig-
nal.
Microdialysis and high-performance liquid chromatography
(HPLC). The microdialysis probe (CMA/lO, Carnegie Medicin,
Stockholm) had a 2-mm polycarbonate membrane with a mo-
lecular mass cutoff of ~20 kDa. The microdialysis probe was
continuously perfused (3 pl/min) with Ringer solution contain-
ing 10 PM neostigmine bromide. Neostigmine was necessary to
prevent the enzymatic degradation of ACh, and we have previ-
ously shown that 10 PM neostigmine does not alter the sleep-
wake cycle during microdialysis (31) or when microinjected into
the FTG (2). Microdialysis samples were collected on ice every
10 min and these 30-~1 samples were injected into an ampero-
metric detector (BAS LC-4B) with an applied potential of 500
mV on the platinum working electrode vs. Ag/AgCl. The sample
was carried to the analytic column in a 50 mM disodium hy-
drogen phosphate mobile phase (pH 8.5) at a flow rate of 1
ml/min and a pressure of 11.9- 12 MPa. The sample first passed
through a polymeric analytic column that separated ACh and
choline (Ch), and these two analytes then passed through an
immobilized enzyme reactor (IMER) column. Because ACh and
Ch are not normally electroactive, the IMER column containing
covalently bonded acetylcholinesterase and choline oxidase per-
formed enzyme catalysis, giving off H202 as an electroactive
product in amounts proportional to the ACh and Ch in the
microdialysis sample (42, 44). Electrochemical detection pro-
duced chromatograms (Fig. 1B) measured at 1 nA full scale.
For each experiment using chronically implanted cats, at
least four consecutive lo-min microdialysis samples were col-
lected from the FTG before microinjection of carbachol into the
contralateral FTG. During this preinjection period, animals
were in a state of quiet wakefulness. Carbachol microinjection
caused the onset of a REM sleeplike state. This desynchronized
(D) sleeplike state, caused by pontine administration of carba-
chol (Carb), is abbreviated using the term DCarb. After the
onset of the REM sleeplike state, the FTG was dialyzed for 40
min or more.
In each of the acute experiments, baseline microdialysis sam-
ples were collected from the FTG for 60 min before electrical
R546
PONTINE ACETYLCHOLINE RELEASE AND RESPIRATORY DEPRESSION
stimulation of the PPT nucleus was initiated. After the collec-
tion of these control data, the PPT was stimulated electrically
while the FTG was microdialyzed. The stimulus parameters
were identical to those previously shown to cause increased ACh
release in the cortex when applied to the nucleus basalis of
Meynert (22). PPT stimulation consisted of 0.5-ms pulses with
a frequency of 50 Hz, delivered in a lo-min train throughout the
course of each 10 min of microdialysis. Current amplitude was
systematically varied from 0.1 to 0.9 mA. For each PPT stim-
ulus level a corresponding lo-min microdialysis sample was ob-
tained from the FTG.
Quantitative chromatography and statistical analyses.
The
area under each chromatographic ACh peak (Fig. 1B) was in-
tegrated with a 386-based computer and the Inject software
package (Interactive Microware, State College, PA). A standard
curve was created before each experiment by injecting five
known concentrations of ACh (0.1-3 pmol) into the HPLC
machine. The amount of ACh in each pontine microdialysis
sample was calculated from
1)
the area under the chromato-
graphic peak and 2) flow rate of the microdialysis pump. The
percent of ACh recovered by the probe was calculated by placing
it in a known ACh standard. For all of these experiments a 10%
recovery rate was typical for the dialysis probes. One-way anal-
ysis of variance (ANOVA) was used to compare ACh release and
respiratory frequency in wakefulness to similar measures ob-
tained during the carbachol-induced REM sleeplike state.
ANOVA and paired t tests were used to determine whether ACh
A
release in the FTG was significantly increased by electrical
stimulation in the PPT nucleus.
Histological analyses.
The 10 brain stems were each cut in
40-pm-thick sagittal sections. The sections were collected seri-
ally and stained with cresyl violet to visualize the three-dimen-
sional stereotaxic placement of the dialysis probes, the micro-
injection cannulas, and the stimulating electrodes. With the use
of a charge-coupled device camera, a computer-based imaging
system, and National Institutes of Health public domain soft-
ware (Image 1.43), the injection, stimulation, and dialysis sites
were localized with reference to the stereotaxic coordinates of
Berman (6). To localize the position of the stimulating electrode
relative to choline@ neurons in the PPT, selected alternate
sections were stained using the NADPH-diaphorase protocol
(49). In the cat PPT, all cholinergic neurons have been shown to
stain positively for NADPH-diaphorase (48, 49).
RESULTS
Chronic
studies. The histological localization of typical
microinjection sites in one FTG and a dialysis site in the
contralateral FTG is illustrated in Fig. 2. For each of the
15 experiments performed using chronically implanted
cats, the microinjection sites were localized to the left
FTG and the dialysis probes were found to have been
positioned in the right FTG.
C
Fig. 2. HistologIcal localization of microinjection and microdialysis sites.
Top:
sagittal sections of cat brain stem at
lateral (L) = 1.9
(A)
and L = 1.2 (C), with rostra1 to the right. Bottom: enlarged photomicrographs of boxed areas in
A
and C. B: arrows show 2 glial scars used to identify 2 microinjection sites within the FTG.
D:
arrow shows lesion in
contralateral FTG made by microdialysis probe. Calibration bar = 1 mm. 6, abducens nucleus; 6N, abducens nerve; 7G,
genu of facial nerve; TB, trapezoid body; TV, ventral tegmental nucleus.
PONTINE ACETYLCHOLINE RELEASE AND RESPIRATORY DEPRESSION
R547
Figure 3 illustrates the results obtained from one ex- lease returned to control levels when the animal awoke
periment designed to measure ACh release and respira- from the carbachol-induced REM sleeplike state (SPl-
tion in a chronically implanted cat during electrographi- SP5).
tally defined states of wakefulness and sleep. ACh release The time course of ACh release and respiration mea-
was stable at -4 pmol/lO min during 50 consecutive min sured across 880 min is illustrated in Fig. 4. Respiratory
of wakefulness
(Wl-W5)
and the following 50 consecu- rate was significantly depressed during the carbachol-
tive min of non-REM sleep (Sl-S5). Microinjection of induced REM sleeplike state compared with waking (F =
carbachol (4 pg/O.25 ~1) into the FTG caused the REM 6.46; df = 1,54;
P
= 0.014). The number of breaths per
sleeplike state (Fig. 3, DCarb) and an increase in ACh minute (mean t SD) was greater during wakefulness
release in the contralateral FTG (DCl-DC5). ACh re- (38.21 t 12.99) than during the carbachol-induced REM
Wakefulness I
Respiration
EMG ]
50
” ’ ‘I’ “1. r
LGB 150
EOG 1 soo
Left
EOG 1 500
Right
EEG 3 50
16 -
E
E: 10 -
0
c 08 -
’ 2 06-
DCarb
A A *
.- 1
ilu O4
c)
a
02
00
Fig. 3. Simultaneous recordings of respiration, states
of consciousness, and ACh release. Top
left:
typical
segment of wakefulness. Top right: desynchronized
(D) or REM sleeplike state (DCarb) caused by mi-
croinjection of carbachol. EEG, electroencephalo-
gram; EMG, electromyogram; EOG, electroocculo-
gram; I, inspiration; LGB, lateral geniculate body
recordings. Vertical calibration bars show pV.
Bot-
tom:
histograms showing state-dependent changes in
ACh release during wakefulness (W), non-REM
sleep (S), REM sleeplike state (DC), and during a
dissociated state characterized by the presence of
occasional EEG spindles (S) and PGO waves (P) in
an awake animal. Since each histogram represents a
IO-min dialysis sample, these data summarize a 200-
min experiment.
0 ~1 ~2 ~3 w4 wfj s1 s2 ~3 S4 S5 DC1 DC2 DC3 DC4 DC5 SPl SP2 SP3 SP4 SP5
R548
2.0
z
E 1.6
0
-7
z
E 1.2
a
PONTINE ACETYLCHOLINE RELEASE AND RESPIRATORY DEPRESSION
, T 1 1 RESPIRATORY RATE ,
T
ACEZ'YILHOLINE RELEASE
lb 2'0 3'0 40 50 60 70 80
TIME
(min)
Fig. 4. Time course of respiratory rate and ACh release in the FTG.
Circles, respiratory rate in breaths/min; ACh release in pmol/lO min
was measured simultaneously and is plotted here as a function of time
and state of consciousness (histograms). Each lo-min time bin repre-
sents a lo-min microdialysis interval. Open circles and cross-hatched
bars, measurements obtained during quiet wakefulness. After the 40th
min, carbachol was microinjected into the FTG contralateral to the
microdialysis probe. Closed circles and closed bars, measurements taken
during carbachol-induced REM sleeplike state. Respiratory rate was
obtained by counting no. of breaths/min for 3 min of recording during
the midportion of the dialysis samples. Each histogram represents av-
erage of
11
dialysis samples for waking and
11
samples during REM
sleeplike state. Because dialysis sample bins were each 10 min, histo-
grams compare ACh release for identical times during wakefulness (440
min) and during REM sleeplike state (440 min). All values plot means
t SD.
WAKE DCARB DCARB
04% wg
Fig. 5. Dose-dependent ACh release measured in the FTG of 1 cat
during 100 min of wakefulness (wake) and during
100
min of carbachol-
induced REM sleeplike state (DCarb) caused by 0.4 and
4.0
pg carba-
chol. Compared with preinjection waking levels of ACh, the 0.4~pg dose
of carbachol caused a
100%
increase in ACh release and the 4.0~pg dose
caused a
141%
increase in ACh release. All values reflect mean + SD.
sleeplike state (30.45 t 9.57). As previously reported (31),
ACh release (mean t SD) in pmol/lO min was less during
waking (0.398 t 0.111) than during the REM sleeplike
state (0.695 t 0.284). Thus during the carbachol-induced
REM sleeplike state there was a 20% depression in res-
piratory rate and a 74.5% enhancement in ACh release
within the FTG.
Previous studies have shown that the carbachol-in-
A
I
I
L
i
09
l
Fig. 6. Chromatographic peaks representing ACh measured in FTG of
barbiturate-anesthetized cat. As described in
METHODS,
area under
curve of each chromatogram is proportional to amount of ACh mea-
sured during
10
min of microdialysis. A: ACh peaks of 5 consecutive
microdialysis samples obtained from FTG before electrical stimulation
of PPT (control). B: chromatograms from the same experiment during
electrical stimulation of PPT. Numbers below ACh peaks indicate cur-
rent amplitude (mA) used to stimulate the PPT.
.
PPT STIMULUS AMPLITUDE(mA)
Fig. 7. ACh release caused by PPT stimulation in 6 barbiturate-anes-
thetized cats. All levels of PPT stimulation except 0.1 mA caused ACh
release significantly greater than control (no PPT stimulation). Values
are means t SD; symbols indicate statistically significant differences.
duced REM sleeplike state is dose dependent (1). In the
present study, increased levels of ACh release were de-
tected in response to increased doses of carbachol (Fig. 5).
Acute studies.
The acute studies were performed to test
the hypothesis that ACh release and respiration would be
altered by stimulation of the PPT. In every one of the six
PONTINE ACETYLCHOLINE RELEASE AND RESPIRATORY DEPRESSION
R549
0.9
B
110
2 100
0
g
90
g 80
u
6\" 70
V
E 60
2 50
z
40
g ii? 30
k 20
z 10
0
CONTROL (0.0) 0.1 0.3 0.5
PPT STIMULATION (mA)
barbiturate-anesthetized cats, electrical stimulation of
the PPT increased ACh release in the ipsilateral FTG.
The chromatograms in Fig.
6
show ACh release within
the FTG before (Fig.
6A)
and during (Fig. 6B) PPT stim-
ulation in one experiment. The progressive increase in
peak height shows that more ACh was released in re-
sponse to increasing PPT stimulus amplitude.
Figure
7
presents average histograms reflecting ACh
release measured in the FTG of six cats, each of which
was exposed to five different levels of electrical stimula-
tion delivered to the PPT. There was a linear increase
(r = 0.992) in ACh release within the FTG caused by
increasing PPT stimulus amplitude (Fig.
7).
ANOVA re-
vealed a statistically significant main effect due to PPT
stimulus amplitude on levels of ACh release (F =
12.29; df
=
5,63;
P <
0.0001).
Levels of ACh release during differ-
ent amplitudes of PPT stimulation were compared by t
test to ACh release with no PPT stimulation (control).
There were statistically significant increases in ACh re-
lease within the FTG during PPT stimulation at
0.3
mA
(t = 2.46;
df =
40; P =
0.018), 0.5
mA
(t
= 5.01;
df = 40;
P
< O.OOOl), 0.7 mA
(t
= 6.36;
df
= 40;
P
< 0.0001) and
0.9 mA
(t
= 7.09;
df = 40; P <
0.0001).
The acute experiments were also designed to test the
hypothesis that state-dependent changes in respiratory
frequency could be caused, at least in part, by electrical
stimulation of neurons within the PPT. Figure 8 illus-
trates the slowing of respiratory frequency and the pro-
longed apneas that were caused by PPT stimulation. For
the histograms shown at the bottom of Fig. 8, ANOVA
revealed that respiratory rate was significantly depressed
by PPT stimulation
(F = 70.45;
df =
3,76; P c
0.0001).
Histological evaluation demonstrated that for each of
the six acute experiments the dialysis probe was in the
FTG. Similar histological analyses showed that the stim-
ulating electrode was implanted in the PPT region of the
mesopontine tegmentum (20). The NADPH-diaphorase
stain was used to confirm that the stimulating electrode
was placed in the cholinergic portion of the PPT. This
stain colocalizes with choline acetyltransferase immu-
noreactivity in the PPT nucleus of both cat (48, 49) and
rat (25). Figure 9 shows that the PPT-stimulating elec-
trode was placed within a field of NADPH-positive neu-
rons.
DISCUSSION
Respiratory depression has been observed to accom-
pany the loss of waking consciousness in all mammals so
far studied. The biological ubiquity of this respiratory
depression suggests the existence of a potent functional
overlap between neuronal mechanisms regulating mam-
malian sleep and respiration. Although there are no major
Fig. 8. Polygraphic recordings and histograms illustrating effects of
PPT stimulation on respiration. A:
top
trace in each of the 4 respiratory
recordings indicates tidal volume (Vr) calibration bar = 20 ml;
bottom
trace in each shows airflow with inspiration indicated by an upward
deflection. Time moves
left
to
right
in all 4 recordings. Number below
each airflow tracing is stimulus amplitude (mA). * Onset of PPT stim-
ulation. B: respiratory rate as a function of PPT stimulus amplitude.
These histograms summarize 799 respiratory cycles and illustrate that
PPT stimulation significantly decreased respiratory frequency.
R550
PONTINE ACETYLCHOLINE RELEASE AND RESPIRATORY DEPRESSION
Fig. 9. Localization of stimulating electrode
placement in PPT of brain stem relative to
NADPH diaphorase-positive neurons. A: out-
line of a sagittal brain stem section at L3.5 (ros-
tral to right). Small box marked by arrow is
enlarged in the color plate shown below. B: X10
enlargement showing lesion caused by PPT-
stimulating electrode as a brown stain at top
margin. C: x40 enlargement of NADPH-posi-
tive neurons (dark blue to black), indicating
that they are clearly differentiated from sur-
rounding neurons, which did not stain posi-
tively for NADPH diaphorase. 5, trigeminal
motor nucleus; 7N, facial nerve; IO, inferior
olive.
PONTINE ACETYLCHOLINE RELEASE AND RESPIRATORY DEPRESSION
R551
clusters of respiratory neurons within the FTG (II), the
present results show that acetylcholine release in this
reticular region can contribute to respiratory depression.
These new data demonstrate that 1) the time course of
ACh release in the FTG paralleled the time course of
respiratory depression during the carbachol-induced
REM sleeplike state, and 2) electrical stimulation of the
PPT caused both respiratory depression and enhanced
ACh release in the FTG. This second finding documents
for the first time that ACh release within the FTG is
regulated by mesopontine cholinergic neurons localized to
the PPT. These data are discussed with regard to the
ability of the cholinoceptive FTG to cause state-depen-
dent respiratory depression and with regard to the newly
suggested role of the PPT in respiratory regulation.
The time course
of
respiratory rate depression parallels
enhancement
of
ACh release in the FTG.
Cholinergic ma-
nipulation of the FTG (Fig. 1) caused the onset of a REM
sleeplike state (Fig. 3, DCarb) and increased ACh release
in the contralateral FTG (Fig. 3, histograms). As noted
previously, measurements of ACh release were site-spe-
cific within the pons (31). Site specificity (3) refers to the
fact that carbachol-induced increases in pontine ACh re-
lease only occurred when the microinjection and dialysis
probes were both in FTG regions shown to be effective for
causing the REM sleeplike state (Fig.
1A).
The site-spec-
ificity, chromatogram retention times (Fig. lB), and the
time course of ACh release (Figs. 3 and 4) ail indicate that
the FTG dialysis measurements reflect endogenous ACh
release and not carbachol or a carbachol reaction product
diffusing into the dialysis site.
The parallel time course for respiratory rate depression
and simultaneous enhancement of ACh release in the
FTG is summarized for the intact, unanesthetized cats in
Fig. 4. Previously published time course profiles have
demonstrated that the carbachol-induced REM sleeplike
state decreases as a function of time postinjection (see
Fig. 3B of Ref.
1).
In the present study, carbachol was
microinjected into the FTG between the
40-
and 50-min
time marks in Fig. 4. Note that the greatest depression in
respiratory rate occurred with the onset of the REM
sleeplike state, in association with the largest increase in
ACh release (50-min time bin). From 50 to 80 min, as
ACh release declined, rate of breathing returned toward
waking levels. Thus the time course data of Fig.
4
extend
our earlier report of an overall correlation
(r =
0.93) be-
tween respiratory frequency and FTG levels of ACh re-
lease (31) by showing that state-dependent respiratory
depression and FTG levels of ACh are also dynamically
related in the time domain. Because pontine microinjec-
tions of carbachol cause a REM sleeplike state and cause
respiratory depression, the time course relationships of
Fig.
4
are consistent with our hypothesis that FTG levels
of ACh causally alter sleep and respiration.
Functional evidence
for
reciprocal connections between
the cholinoceptive pontine reticular formation and cholin-
ergic neurons in the mesopontine tegmentum.
Immuno-
histochemical studies of cat brain have consistently
shown that neurons in the FTG region of the pontine
reticular formation do not produce ACh (21, 48). Retro-
grade tracing studies identified the PPT and LDT nuclei
within the mesopontine tegmentum as the source of cho-
linergic input to the pontine reticular formation
(37, 45).
Subsequent anterograde labeling studies have revealed
projections from the pontine FTG to the PPT (see Fig.
1A
of Ref. 18). Thus the finding that unilateral pontine
carbachol administration caused increased ACh release in
the contralateral pons provided the first functional evi-
dence suggesting a reciprocal projection from the FTG to
the LDT/PPT (31).
In vitro intracellular recordings have shown that most
neurons in the medial pontine reticular formation are
depolarized by carbachol
(16, 17).
Therefore, we hypoth-
esized that carbachol-induced enhancement of FTG neu-
ron discharge is relayed back to the LDT/PPT, which
then increases ACh release in the pontine reticular for-
mation
(31).
If this concept is correct, then lower doses of
carbachol should exert less of a depolarizing effect within
the pontine reticular formation. Such a diminished influ-
ence within FTG should be relayed to the LDT/PPT, and
there should be a diminished release of ACh measured in
the contralateral FTG.
Physiological data supporting the existence of a pro-
jection from FTG to LDT/PPT and the foregoing func-
tional concepts are presented in Fig. 5. Unilateral FTG
microinjection of carbachol at a dose of
0.4
pg caused a
smaller enhancement in ACh release in the contralateral
FTG than a 4.0-pg dose of carbachol (Fig. 5). This finding
is consistent with previous studies showing that the per-
cent of time spent in the REM sleeplike state after pon-
tine microinjections of carbachol is dose dependent (see
Fig. 4 of Ref. 1). When the dose of pontine carbachol was
increased from
0.4
pg to
4.0
pg there was a
125%
increase
in the REM sleeplike state
(1).
Considered together, these data provide further physi-
ological evidence for a projection from the pontine retic-
ular formation to the LDT/PPT. These data also suggest
that an exciting agenda for future studies will include
specifying the feedforward and feedback parameters be-
tween the cholinoceptive neurons in the pontine reticular
formation and the cholinergic neurons in the LDT/PPT.
Because the LDT/PPT nuclei comprise the only concen-
tration of cholinergic neurons in the pontine tegmentum
of cat
(21, 47, 48)
and because the LDT and PPT have
been shown to provide cholinergic terminals to the FTG
(3’7,
45),
it is probable that the stimulation-evoked in-
crease in ACh release in the present study arose from
neurons localized to the PPT/LDT. The next section
discusses results obtained from barbiturate-anesthetized
cats. These acute studies represent a crude, but essential,
first step aiming to specify the synaptic relationship be-
tween cholinergic neurons in the PPT and cholinoceptive
neurons in the pontine reticular formation.
The PPT regulates ACh release in the FTG and influ-
ences respiration.
The data discussed in the previous sec-
tion encouraged us to test the hypothesis that electrical
stimulation of the PPT would cause increased ACh re-
lease in the FTG and cause respiratory depression. Figure
6 illustrates typical chromatograms obtained from FTG
microdialysis with no PPT stimulation (control) and
R552
PONTINE ACETYLCHOLINE RELEASE AND RESPIRATORY DEPRESSION
with increasing amplitudes of electrical stimulation in the
PPT. In every experiment, we observed increased ACh
release in the FTG caused by PPT stimulation (r =
0.992). ACh release as a function of PPT stimulus am-
plitude is shown for six anesthetized cats in Fig. 7. The
data summarized by Figs. 6 and 7 represent the first
evidence that the PPT to FTG projections identified by
anatomic studies (37, 45) can functionally alter levels of
ACh release within the FTG.
The potent respiratory effects caused by injecting cho-
linergic agonists into the FTG and the anatomic data
noted above led us to hypothesize that the cholinergic
LDT/PPT can influence respiratory regulation (27, 33,
31). Figure 8 presents evidence showing that, in addition
to causing increased ACh release in the FTG, electrical
stimulation of the PPT can cause respiratory depression.
Respiratory rate depression was observed in four of six
animals. In two cats PPT stimulation onset also caused
the respiratory cycle to abruptly switch from inspiration
to expiration or from expiration to inspiration.
The neuronal groups surrounding the pontine brachii
conjunctivi are structurally interwoven with borders that
are impossible to demarcate strictly. The ability to accu-
rately ascribe functional roles to these neuronal groups is
further hindered by an anatomic nomenclature that has
made it difficult to differentiate PPT neurons from the
parabrachial nuclei. It has been emphasized that PPT
neurons are peribrachial in location, and the PPT should
not be confused with nearby parabrachial nuclei (46,47).
A cellular substrate for Lumsden’s (26) notion of pontine
respiratory regulation was first localized to the parabra-
chial nuclei by Cohen and Wang (7), but PPT neurons
have not been previously suggested to play a role in res-
piratory control.
Although respiratory changes paralleled changes in
ACh release within the FTG, this does not unequivocally
confirm that these respiratory changes are limited only to
increased ACh release within the FTG. It is possible that
LDT/PPT neurons provide parallel cholinergic input to
both the FTG and to pontomedullary respiratory neu-
rons. We are presently investigating this possibility, and
preliminary results from fluorescent retrograde tracing
studies in our laboratory have identified reciprocal con-
nections between the parabrachial nuclei and cholinocep-
tive regions of the FTG (23).
PPT neurons are not cytoarchitecturally distinct in the
cat (47), but they are known to correspond to the cholin-
ergic cell group designated by Mesulam et al. (35) as Ch5
(20,48). Because Ch5 cholinergic neurons in the cat stain
positively for NADPH-diaphorase (49), we used this
stain to confirm that our stereotaxic coordinates for the
PPT corresponded to a neuronal group that was NADPH
positive and therefore likely to be cholinergic (Fig. 9). To
the best of our knowledge, these are the first data sug-
gesting that putatively cholinergic neurons localized to
the PPT can alter breathing.
Limitations and conclusions. The present results dem-
onstrating respiratory rate depression and increased pon-
tine ACh release caused by PPT stimulation confirm for
the first time through in vivo measurement a previously
suggested relationship between pontine ACh and respira-
tory control (36). The present microdialysis measure-
ments obtained from intact, unanesthetized cats further
specify that ACh release in the FTG, known to be in-
volved in REM sleep generation (47), can contribute to
state-dependent respiratory depression. Although the res-
piratory measures were limited to rate of breathing, the
results encourage future studies using intact, unanesthe-
tized animals to quantify respiratory cycle timing, tidal
volume, and minute ventilation in relationship to ACh
release in the FTG.
A long-appreciated limitation of all microinjection
studies concerns the relationship between diffusion of the
injected compound and the ability to spatially resolve the
central site of drug action. The diffusion issue has been
discussed in detail elsewhere (4)) and calculated diffusion
profiles are known to be a complex function of drug con-
centration, injection volume, and microinjection site.
Systematic evaluation of the cholinergic model of REM
sleep shows that the carbachol dose (0.4 or 4 pg), concen-
tration (8.8 or 88 mM), and injection volume (0.25 ~1)
used in the present study are comparable to these vari-
ables reported by five recent investigations (see Table 4 of
Ref. 5). Thus the technical limitations of the present
microinjections are not significantly different from the
generalized use of this technique.
Two additional limitations should be clear for the acute
studies. First is the issue of anesthesia. Pentobarbital has
long been known to significantly disrupt cardiopulmon-
ary regulation (43). The finding that PPT stimulation
caused respiratory depression in anesthetized animals
cannot be directly interpreted to represent sleep-depen-
dent respiratory depression. Because anesthetics hyper-
polarize neurons (40), however, we anticipate that future
PPT stimulation experiments can be performed with in-
tact, unanesthetized cats with the use of much lower cur-
rent amplitudes. In these future studies it will be impor-
tant to examine the hypothesis that PPT-stimulated
enhancement of ACh release in the FTG varies as a func-
tion of state-dependent changes in neuronal excitability
(34). If the data ultimately support this concept, state-
dependent changes in neuronal excitability may provide
an important interpretive framework concerning the
mechanisms that contribute to state-dependent respira-
tory depression.
A second but related limitation of the acute experi-
ments concerns the electrical stimulation in the PPT.
The goal of the acute studies was not to mimic physio-
logical conditions but to determine whether ACh release
in the FTG could be causally manipulated from the PPT.
As noted in
METHODS,
similar stimulation parameters
previously have been shown to be useful for functional
pathway mapping (22). One can clearly anticipate current
spread with higher levels of electrical stimulation. It has
been noted elsewhere, however, that most of the fibers
passing through the PPT originate from neurons in the
LDT nucleus that are also cholinergic
(47).
Even with
these acknowledged limitations, the stimulation data
PONTINE ACETYLCHOLINE RELEASE AND RESPIRATORY DEPRESSION
R553
demonstrate that peribrachial neurons regulate ACh re- 18.
lease in the pontine FTG and can contribute to cholin-
ergically mediated alterations in respiratory control.
We gratefully acknowledge the support of J. F. Biebuyck and the 19.
Department of Anesthesia. For expert technical assistance we thank
2. Lorinc, M. A. Royles, J. Spotts, R. Spayde, M. Bogdan, and Bio-
analytical Systems of West Lafayette, IN.
R. Lydic was supported by National Heart, Lung, and Blood Insti- 20.
tute Grants HL-47749 and HL-40881 and H. A. Baghdoyan by National
Institute of Mental Health Grant MH-45361. 21 .
Received 11 May 1992; accepted in final form 15 October 1992.
REFERENCES 22.
1.
2.
3.
4.
5.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Baghdoyan, H. A., R. Lydic, C. W. Callaway, and J. A.
Hobson. The carbachol-induced enhancement of desynchronized
sleep signs is dose-dependent and antagonized by centrally admin-
istered atropine. Neuropsychopharmacology 2: 67-79, 1989.
Baghdoyan, H. A., A. P. Monaco, M. L. Rodrigo-Angulo, F.
Assens, R. W. McCarley, and J. A. Hobson. Microinjection of
neostigmine into the pontine reticular formation of cats enhances
desynchronized sleep signs. J. Pharmacol. Exp. Ther. 231: 173-
180, 1984.
Baghdoyan, H. A., M. L. Rodrigo-Angulo, R. W. McCarley,
and J. A. Hobson. Site-specific enhancement and suppression of
desynchronized sleep signs following cholinergic stimulation of
three brainstem regions. Brain Res. 306: 39-52, 1984.
Baghdoyan, H. A., M. L. Rodrigo-Angulo, R. W. McCarley,
and J. A. Hobson. A neuroanatomical gradient in the pontine
tegmentum for the cholinoceptive induction of desynchronized
sleep signs. Brain Res. 414: 245-261, 1987.
Baghdoyan, H. A., J. L. Spotts, and S. G. Snyder. Simulta-
neous pontine and basal forebrain microinjections of carbachol
suppress REM sleep. J. Neurosci. 13: 229-242, 1993.
Berman, A. L. The Brain Stem of the Cat. Madison: Univ. of
Wisconsin Press, 1968.
Cohen, M. I., and S. C. Wang. Respiratory neuronal activity in
the pons of cat. J. Neurophysiol. 22: 33-50, 1959.
Comroe, J. H. The effects of direct chemical and electrical stim-
ulation of the respiratory center in the cat. Am. J. Physiol. 139:
490-498, 1943.
Dev, N. B., and Loeschcke, H. H. A cholinergic mechanism
involved in the respiratory chemosensitivity of the medulla ob-
longata in the cat. Pfluegers Arch. 379: 29-36, 1979.
Douglas, N. J. Control of ventilation during sleep. In: Principles
and Practice of Sleep Medicine, edited by M. H. Kryger, T. Roth,
and W. C. Dement. Philadelphia, PA: Saunders, 1989, p. 249-256.
Feldman, J. Neurophysiology of breathing in mammals. In:
Handbook of Physiology. The Nervous System. Intrinsic Regula-
tory Systems of the Brain. Bethesda, MD: Am. Physiol. Sot., 1986,
sect. 1, vol. IV, chapt. 9, p. 463-524.
Fukuda, Y., and H. H. Loeschcke. A cholinergic mechanism
involved in the neuronal excitation by H+ in the respiratory
chemosensitive structures of the ventral medulla oblongata of rat
in vitro. Pfluegers Arch. 379: 125-135, 1979.
Gesell, R., and E. T. Hansen. Eserine, acetylcholine, atropine,
and nervous integration. Am. J. Physiol. 139: 371-385, 1943.
Gilbert, K. A., and R. Lydic. Parabrachial neuron discharge is
altered during the carbachol induced REM sleep-like state. Neu-
rosci. Lett. 120: 241-244, 1990.
Gilbert, K. A., and R. Lydic. Cholinergic reticular mechanisms
produce state-dependent decreases in parabrachial respiratory
neuron discharge. Neurosci. Abstr. 17: 620, 1991.
Greene, R. W., U. Gerber, and R. W. McCarley. Cholinergic
activation of medial pontine reticular formation neurons in vitro.
Brain Res. 476: 154-159, 1989.
Greene, R. W., H. L. Haas, U. Gerber, and R. W. McCarley.
Cholinergic activation of medial pontine reticular formation neu-
rons in vitro. In: Central Cholinergic Synaptic Transmission, ed-
ited by M. Frotscher and U. Misgeld. Boston, MA: Birkhauser,
1989, p. 122-137.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
Higo, S., K. Ito, D. Fuchs, and R. W. McCarley. Anatomical
interconnections of the pedunculopontine tegmental nucleus and
the nucleus prepositus hypoglossi in the cat. Brain Res. 536: 79-
85, 1990.
Hobson, J. A., and M. Steriade. Neuronal basis of behavioral
state control. In: Handbook of Physiology. The Nervous System.
Intrinsic Regulatory Systems of the Brain. Bethesda, MD: Am.
Physiol. Sot., 1986, sect. 1, vol. IV, chapt. 14, p. 701-826.
Jones, B. E. Paradoxical sleep and its chemical/structural sub-
strates in the brain. Neuroscience 40: 637-656, 1991.
Jones, B. E., and A. Beaudet. Distribution of acetylcholine and
catecholamine neurons in the cat brainstem: a choline acetyltrans-
ferase and tyrosine hydroxylase immunohistochemical study. J.
Comp. Neurol. 261: 15-32, 1987.
Kurosawa, M., A. Sato, and Y. Sato. Stimulation of the nu-
cleus basalis of Meynert increases acetylcholine release in the
cerebral cortex in rats. Neurosci. Lett. 98: 45-50, 1989.
Lee, L. H., J. Veino, and R. Lydic. Fluorescent dye tracing
reveals reciprocal connections between respiratory nuclei and the
medial pontine reticular formation. Neurosci. Abstr. 18: 124, 1992.
Lorinc, Z., J. Derr, M. Snider, and R. Lydic. Defining origin
of positive slope in hypercapnic ventilatory response curve. Am. J.
Physiol. 261 (Regulatory Integrative Comp. Physiol. 30): R747-
R751, 1991.
Luebke, J. J., R. W. Greene, K. Semba, R. W. McCarley,
and P. B. Reiner. Serotonin hyperpolarizes cholinergic low
threshold burst neurons in the rat laterodorsal tegmental nucleus
in vitro. Proc. Natl. Acad. Sci. 89: 743-747, 1992.
Lumsden, T. The regulation of respiration. J. Physiol. Lond. 58:
81-91, 1923.
Lydic, R., and H. A. Baghdoyan. Cholinoceptive pontine re-
ticular mechanisms cause state-dependent changes in respiration.
Neurosci. Lett. 102: 211-216, 1989.
Lydic, R., and H. A. Baghdoyan. The neurobiology of REM
sleep. In: Sleep and Breathing (2nd ed.), edited by N. A. Saunders
and C. E. Sullivan. New York: Marcel Dekker, 1993, p. 47-78.
(Lung Biol. Health Dis. ser.)
Lydic, R., and H. A. Baghdoyan. Cholinergic pontine mech-
anisms causing state-dependent respiratory depression. News
Physiol. Sci. 7: 220-224, 1992.
Lydic, R., H. A. Baghdoyan, and 2. Lorinc. Microdialysis of
the medial pontine reticular formation reveals increased acetyl-
choline release caused by electrical stimulation of the peduncu-
lopontine tegmental nucleus (Abstract). Sleep Res. 21: 10, 1992.
Lydic, R., H. A. Baghdoyan, and 2. Lorinc. Microdialysis of
cat pons reveals enhanced acetylcholine release during state-de-
pendent respiratory depression. Am. J. Physiol 261 (Regulatory
Integrative Comp. Physiol. 30): R766-R770, 1991.
Lydic, R., H. A. Baghdoyan, R. Wertz, and D. P. White.
Cholinergic reticular mechanisms influence the state-dependent
ventilatory response to hypercapnia. Am. J. Physiol. 261 (Regu-
latory Integrative Comp. Physiol. 30): R738-R746, 1991.
Lydic, R., H. A. Baghdoyan, and C. W. Zwillich. Choli-
noceptive reticular mechanisms cause state-dependent hypotonia
in posterior cricoarytenoid muscles of the larynx. FASEB J. 3:
1625-1631, 1989.
Lydic, R., R. W. McCarley, and J. A. Hobson. Enhancement
of dorsal raphe discharge by medial pontine reticular formation
stimulation depends on behavioral state. Neurosci. Lett. 38: 35-40,
1983.
Mesulam, M. M., E. J. Mufson, B. H. Wainer, and A. I.
Levey. Central cholinergic pathways in the rat: an overview
based on an alternative nomenclature (Chl-Ch6). Neuroscience
10: 1185-1201, 1983.
Metz, B. Correlation between respiratory reflex and acetylcholine
content of pons and medulla. Am. J. Physiol. 202: 80-82, 1962.
Mitani, A., K. Ito, A. H. Hallanger, B. H. Wainer, K.
Kataoka, and R. W. McCarley. Cholinergic projections from
the laterodorsal and pedunculopontine tegmental nuclei to the
pontine gigantocellular tegmental field in the cat. Brain Res. 451:
397-402, 1988.
Monteau, R., D. Morin, and G. Hilaire. Acetylcholine and
central chemosensitivity: in vitro study in the newborn rat. Respir.
Physiol. 81: 241-254, 1990.
R554
PONTINE ACETYLCHOLINE RELEASE AND RESPIRATORY DEPRESSION
39.
Nattie, E., J. Wood, A. Mega, and W. Goritski.
Rostra1 ven-
trolateral medulla muscarinic receptor involvement in central
chemosensitivity.
J. Appl. Physiol. 66: 1462-1470, 1989.
40. Nicoll, R. A., and D. V. Madison.
General anesthetics hyper-
polarize neurons in the vertebrate central nervous system.
Science
Wash. DC 217: 1055-1057, 1982.
41. Phillipson, E. A., and
G.
Bowes.
Control of breathing during
sleep. In:
Handbook of Physiology. The Respiratory System. Con-
trol of Breathing.
Bethesda, MD: Am. Physiol. Sot.,
1986,
sect. 3,
vol. II, chapt.
19,
p. 649-689.
42. Potter, P. E., J. L. Meek, and
N.
H. Neff.
Acetylcholine and
choline in neuronal tissue measured by HPLC with electrochem-
ical detection.
J. Neurochem. 41: 188-194, 1983.
43. Priano, L. L., D. L. Traber, and R. D. Wilson.
Barbiturate
anesthesia: an abnormal physiologic situation.
J. Pharmacol. Exp.
Ther. 165: 126-135, 1969.
44. Robinson, T. E., and J. B. Justice.
Microdialysis in the Neu-
rosciences. In:
Techniques in the Behavioral and Neural Sciences,
edited by J. P. Huston. New York: Elsevier,
1991, vol. 7,
p. l-450.
45. Shiromani, P. J., D. M. Armstrong, and J. C. Gillin.
Cho-
linergic neurons from the dorsolateral pons project to the medial
pons: A WGA-HRP and choline acetyltransferase immunohis-
tochemical study.
Neurosci. Lett. 95: 19-23, 1988.
46. Steriade, M., and G. Buzaki.
Parallel activation of thalamic
and cortical neurons by brainstem and basal forebrain cholinergic
systems. In:
Brain Cholinergic Systems,
edited by M. Steriade and
D. Biesold. New York: Oxford Univ. Press, 1990, p. 3-64.
47. Steriade, M., and R. W. McCarley. Brainstem Control of
Wakefulness and Sleep. New
York: Plenum, 1990.
48. Vincent, S. R., and P. B. Reiner.
The immunohistochemical
localization of choline acetyltransferase in the cat brain.
Brain
Res. Bull. 18: 371-414, 1987.
49. Vincent, S. R., K. Sato, D. M. Armstrong, and H. C.
Fibiger.
NADPH-diaphorase: a selective histochemical marker
for the cholinergic neurons of the pontine reticular formation.
Neurosci. Lett. 43: 31-36, 1983.
