Pontine Nitric Oxide Modulates Acetylcholine Release, Rapid Eye
Movement Sleep Generation, and Respiratory Rate
Timothy O. Leonard and Ralph Lydic
Department of Anesthesia and the Program in Neuroscience, The Pennsylvania State University College of Medicine,
Hershey, Pennsylvania 17033
Pontine cholinergic neurotransmission is known to play a key
role in the regulation of rapid eye movement (REM) sleep and to
contribute to state-dependent respiratory depression. Nitric
oxide (NO) has been shown to alter the release of acetylcholine
(ACh) in a number of brain regions, and previous studies indi-
cate that NO may participate in the modulation of sleep/wake
states. The present investigation tested the hypothesis that
inhibition of NO synthase (NOS) within the medial pontine
reticular formation (mPRF) of the unanesthetized cat would
decrease ACh release, inhibit REM sleep, and prevent cholin-
ergically mediated respiratory depression. Local NOS inhibition
by microdialysis delivery of NG-nitro-L-arginine (NLA) signifi-
cantly reduced ACh release in the cholinergic cell body region
of the pedunculopontine tegmental nucleus and in the cholino-
ceptive mPRF. A second series of experiments demonstrated
that mPRF microinjection of NLA significantly reduced the
amount of REM sleep and the REM sleep-like state caused by
mPRF injection of the acetylcholinesterase inhibitor neostig-
mine. Duration but not frequency of REM sleep epochs was
significantly decreased by mPRF NLA administration. Injection
of NLA into the mPRF before neostigmine injection also
blocked the ability of neostigmine to decrease respiratory rate
during the REM sleep-like state. Taken together, these findings
suggest that mPRF NO contributes to the modulation of ACh
release, REM sleep, and breathing.
Key words: acetylcholine; nitric oxide; pons; reticular forma-
tion; respiratory control; halothane anesthesia; REM sleep
Pontine cholinergic neurotransmission is involved in regulating
the rapid eye movement (REM) phase of sleep (Steriade and
McCarley, 1990; Jones, 1993; Lydic and Baghdoyan, 1994; Mc-
Carley et al., 1995). Both anatomical (Mitani et al., 1988; Shiro-
mani et al., 1988) and functional (Lydic and Baghdoyan, 1993)
studies have shown that cholinergic neurons of the laterodorsal
and pedunculopontine tegmental (LDT/PPT) nuclei project axon
terminals to the medial pontine reticular formation (mPRF),
where acetylcholine (ACh) is released. Electrical stimulation of
the LDT/PPT causes a monotonic increase in mPRF ACh release
(Lydic and Baghdoyan, 1993). Lesions of the LDT/PPT disrupt
REM sleep, and the amount of REM sleep reduction is correlated
positively with LDT/PPT cell destruction (Webster and Jones,
1988; Shouse and Siegel, 1992). Microinjection of cholinergic
agonists into the mPRF elicits a state with the behavioral and
electrophysiological traits of REM sleep (Baghdoyan et al., 1984;
Baghdoyan et al., 1989; Vanni-Mercier et al., 1989; Yamamoto et
al., 1990; Baghdoyan et al., 1993). This cholinergically evoked
REM sleep-like state also is characterized by upper airway muscle
hypotonia and depressed rate of breathing (Lydic and Baghdoyan,
1989; Lydic et al., 1989). ACh release in the mPRF increases
during the cholinergically evoked REM sleep-like state (Lydic et
al., 1991) and during natural REM sleep (Leonard and Lydic,
1995). Therefore, multiple lines of evidence have established the
involvement of cholinergic LDT/PPT cells and noncholinergic,
cholinoceptive mPRF neurons in the generation of REM sleep
and state-dependent respiratory depression. An important ques-
tion for understanding the cellular and molecular regulation of
REM sleep concerns the mechanisms by which pontine cholin-
ergic neurotransmission is controlled.
Nitric oxide (NO) is a modulator of neuronal function (Garth-
waite and Boulton, 1995; Zhang et al., 1995) and has been shown
to alter ACh release (Prast and Philippu, 1992; Guevara-Guzman
et al., 1994; Ohkuma and Kuriyama, 1994; Leonard and Lydic,
1995; Ohkuma et al., 1995; Prast et al., 1995). LDT/PPT cholin-
ergic neurons in cat stain positively for NADPH diaphorase
(Vincent et al., 1983; Mizukawa et al., 1989), which has been
identified as a neuronal nitric oxide synthase (NOS) (Dawson et
al., 1991; Hope et al., 1991). In rat (Kapas et al., 1994a) and rabbit
(Kapas et al., 1994b), systemic inhibition of NOS alters sleep. The
presence of NOS protein and mRNA in LDT/PPT neurons has
been confirmed (Bredt et al., 1991), and mPRF administration of
a NOS inhibitor reduces mPRF ACh release (Leonard and Lydic,
1995). These findings suggested that mPRF levels of NO might
modulate pontine cholinergic neurotransmission and possibly par-
ticipate in the regulation of arousal states. Therefore, the present
study has expanded these earlier findings by testing the hypothesis
that stereoselective inhibition of NOS in the mPRF would de-
crease local pontine ACh release, inhibit REM sleep, and prevent
cholinergically evoked respiratory rate depression.
MATERIALS AND METHODS
Electrodes for polygraphic monitoring of sleep and wakefulness were
implanted during halothane anesthesia (1–2% in O2) in 10 adult male
cats. Each cat was used for either microinjection or microdialysis exper-
iments. For microinjection studies (n ? 5 cats), 24 gauge stainless steel
guide tubes were implanted 5 mm above the mPRF using the stereotaxic
Received July 15, 1996; revised Oct. 30, 1996; accepted Oct. 31, 1996.
This work was supported by National Heart, Lung, and Blood Institute Grant
HL-40881 (R.L.) and the Departments of Neuroscience and Anatomy, and Anes-
thesia. We thank M. A. Fleegal and P. P. Myers for excellent technical and secretarial
Correspondence should be addressed to Prof. Ralph Lydic, Department of Anes-
thesia, The Pennsylvania State University, College of Medicine, Hershey, PA 17033.
Copyright ? 1997 Society for Neuroscience 0270-6474/97/170774-12$05.00/0
The Journal of Neuroscience, January 15, 1997, 17(2):774–785
coordinates [2.0 mm posterior (P); 1.5 mm lateral (L); ?5.0 mm hori-
zontal (H)] of Berman (1968). For experiments involving microdialysis
(n ? 5 cats), the cranial acrylic encasement surrounding the sleep scoring
electrode array was equipped with a plastic well that permitted subse-
quent placement of microdialysis probes into the mPRF. After recovery
from surgery and before beginning microdialysis or microinjection exper-
iments, all cats were trained for 1–2 months to sleep in the laboratory in
a head-stable position. Animals were studied in this head-restrained
position and all experiments strictly adhered to the National Institutes of
Health guidelines for the care and use of laboratory animals (National
Institutes of Health Publication No. 85-23, 1985).
mPRF ACh measurement
Microdialysis. Before in vivo mPRF dialysis, a microdialysis probe (CMA/
10, Acton, MA) with a polycarbonate membrane of 20 kDa pore size was
placed in a vial containing a known concentration of ACh and was
perfused (CMA/100 microinjection pump) with a modified Ringer’s so-
lution, pH 6.0, 147 mM NaCl; 4.0 mM KCl; 2.4 mM CaCl2; 10 ?M
neostigmine bromide (Sigma, St. Louis, MO). As demonstrated previ-
ously, (Lydic et al., 1991) 10 ?M neostigmine does not alter arousal state.
This procedure was used to determine the preexperiment recovery of
ACh by the microdialysis probe. At the conclusion of each mPRF dialysis
experiment, probe recovery of ACh from a standard solution verified that
in vivo measurement of changes in mPRF ACh release was not attribut-
able to mechanical alteration of the probe membrane. Only data from
experiments in which preexperiment in vitro probe recovery did not differ
from postexperiment ACh recovery are included in this report.
For each experiment, a microdialysis probe was placed in the mPRF
using stereotaxic coordinates: P ? 1.5–3.0 mm; L ? 0.8–1.5 mm; H ?
?5.0 to ?6.5 mm; probe angle ? 30? P. The dialysis probe was perfused
continuously with Ringer’s solution (control) at 3 ?l/min, and endoge-
nous ACh was recovered in 30 ?l dialysate samples. Each 10 min mPRF
dialysate sample was collected during unambiguously scored states of
wakefulness, non-REM (NREM) sleep, or REM sleep. After 2–3 hr of
sample collection during Ringer’s perfusion, the probe was perfused with
10 mM NG-nitro-L-arginine (NLA; RBI, Natick, MA) dissolved in Ring-
er’s. Dialysate samples again were collected during wakefulness, NREM
sleep, and REM sleep for determination of mPRF ACh release in the
presence of the NOS inhibitor NLA. After collection of samples during
Ringer’s dialysis, the mPRF was dialyzed with Ringer’s containing 10 mM
NG-nitro-D-arginine (NDA; Bachem, Torrance, CA) instead of NLA.
NDA is a stereoisomer of NLA that has been shown to be less potent in
its ability to inhibit NOS (Wang et al., 1993). Because NDA is a relatively
inactive enantiomer of NLA, it has been suggested that NDA can serve as
an effective pharmacological control for NLA administration (Griffith
and Stuehr, 1995). Multiple experiments using different mPRF sites in the
same animal were separated by at least 5 d.
High performance liquid chromatography (HPLC). Each 30 ?l mPRF
dialysate sample was injected into an HPLC system (Bioanalytical Sys-
tems, West Lafayette, IN) and carried in a 50 mM Na2HPO4mobile
phase, pH 8.5, at 1.0 ml/min (pressure ? 13–15 MPa). Samples passed
through an analytical separation column before entering an immobilized
enzyme reactor column, where H2O2was produced from ACh in stoichi-
ometric amounts. H2O2was detected at a platinum electrode with an
applied potential of 500 mV relative to an Ag?/AgCl reference electrode.
The generated current created a chromatogram peak that was recorded
on a flat-bed recorder and processed by a computer software program
(Inject). Mean retention time for the ACh chromatogram was 5.30 min.
The chromatogram peak areas are proportional to the ACh content in
each dialysis sample. Chromatogram areas were compared with a series
of ACh standards (0.1–3.0 pmol) to express ACh values as pmol/10 min
for each brain sample.
PPT ACh measurement
Additional microdialysis experiments in two cats examined the effect of
PPT NLA delivery on ACh release within the PPT. The animals were
anesthetized with halothane (1–2% in O2) delivered through a mask.
Once anesthetized, cats were intubated with a #4 cuffed endotracheal
tube and placed in stereotaxic head restraint. A microdialysis probe was
placed in the PPT according to the coordinates of Berman (1968): P ? 0.8
mm; L ? 3.0 mm; H ? ?2.5 mm; angle ? 30? P. The probe was constantly
perfused at 3 ?l/min with Ringer’s solution. A Raman spectrophotometer
sampled expired gas from the endotracheal tube and measured end tidal
CO2and halothane concentration. Halothane anesthesia was maintained
at 1.2% (in O2). End tidal CO2was maintained at 20–25 mmHg by
adjusting minute ventilation. During 1.2% halothane anesthesia, 30 ?l
dialysate samples were collected and ACh content was measured as
pmol/10 min. After termination of halothane anesthesia, ACh released
into the PPT was measured during wakefulness. Wakefulness was deter-
mined by (1) measurement of end tidal halothane; (2) polygraphic re-
cordings (EEG desynchrony, return of muscle tone, conjugate eye move-
ments); and (3) behavioral observation (limb movements, tracking eye
movements). Finally, the microdialysis probe was perfused with 10 mM
NLA and samples were analyzed for ACh content as a result of delivery
of a NOS inhibitor during a state of quiet wakefulness. From these
experiments, it was possible to quantify the effects of both 1.2% halo-
thane anesthesia and PPT NOS inhibition on ACh release within
Drug administration. Because the brain parenchyma is devoid of nocicep-
tors, it was possible to make repeated microinjections into the mPRF of
unanesthetized cats while they were in a state of quiet wakefulness.
Microinjections were given through 31 gauge stainless steel tubing placed
in the implanted guide tube. A 250 nl volume of saline (vehicle control)
or drug was injected into the mPRF over a 30 sec period using a 1 ?l
Hamilton syringe (Thomas Scientific, Swedesboro, NJ) and a manual
microdrive assembly. For 2 hr after the microinjection, states of sleep and
wakefulness were recorded on a Grass polygraph. A thermistor placed at
the nares also permitted polygraphic quantification of respiratory rate. In
this way, effects of mPRF drug administration on sleep/wake states were
determined for the following six microinjection conditions: (1) saline; (2)
NLA (22.8 mM: 1.25 ?g/0.25 ?l); (3) NDA (22.8 mM: 1.25 ?g/0.25 ?l); (4)
neostigmine bromide (40.0 mM: 3.0 ?g/0.25 ?l saline); (5) 22.8 mM NLA
microinjected 15 min before a 40.0 mM neostigmine injection; and (6)
22.8 mM NDA microinjected 15 min before a 40.0 mM neostigmine
injection. In addition, respiratory rate was quantified after mPRF injec-
tion of saline; 40.0 mM neostigmine alone; 22.8 mM NLA 15 min before
neostigmine; and 22.8 mM NDA before neostigmine. All experiments in
which a drug (NLA, NDA, or neostigmine) were microinjected into the
mPRF of the same animal were separated by at least 3 d.
State and breathing quantification. For each experiment, 2 hr poly-
graphic recordings were divided into 120 bins, and each min was scored
as wakefulness, NREM sleep, or REM sleep. Polygraphic variables re-
corded from the implanted electrodes were used to objectively score
states of wakefulness, NREM sleep, and REM sleep according to stan-
dard criteria (Ursin and Sterman, 1981). For each recording, 10 min of
each of the three states was randomly selected and respiratory rate
(breaths/min) was tabulated.
For microdialysis and microinjection experiments, descriptive statistics
and ANOVA were used to quantify drug effects on the following depen-
dent measures: mPRF and PPT ACh release (pmol/10 min); percent
wakefulness, NREM sleep, and REM sleep; REM sleep latency; REM
sleep epoch frequency and duration; and rate of breathing. Post hoc
multiple pairwise comparisons were performed using Tukey’s tests for
analysis of state effect on mPRF and PPT ACh release. For mPRF
microdialysis experiments, a priori independent t tests were used to test
for statistical significance in the difference between ACh release during
control (Ringer’s) or drug (10 mM NLA or 10 mM NDA) dialysis within
states of wakefulness, NREM sleep, and REM sleep. To test the effects
of mPRF microinjection on the percent time spent in REM sleep, NREM
sleep, and wakefulness; REM sleep epoch duration, frequency, and
latency; as well as injection effect on respiratory rate within each state,
multiple pair-wise comparisons were made using independent t tests with
Bonferroni correction factors ( pactual? 0.05/number of comparisons).
From four of the five animals, it was possible to obtain three measures of
breathing and arousal state in each of the six microinjection conditions.
From the fifth animal, three measures of breathing and arousal state were
obtained for four of the six microinjection conditions. For all statistical
comparisons, a significance level of p ? 0.05 was chosen.
At the completion of mPRF microdialysis or microinjection experiments,
cats were deeply anesthetized with sodium pentobarbital and transcardi-
ally perfused with isotonic saline followed by 10% phosphate buffered
formalin, pH 7.0, (Fisher Scientific, Houston, TX). Brains were removed
and soak-fixed first in the buffered formalin and then in 30% sucrose-
formalin for 1–2 weeks. Brainstems were sectioned (40 ?m thick) on a
Leonard and Lydic • NO Alters Pontine ACh Release and REM SleepJ. Neurosci., January 15, 1997, 17(2):774–785 775
freezing microtome, mounted on chrome-alum-coated slides, stained with
cresyl violet, and coverslipped.
The results were obtained from a total of 1870 min of mPRF
microdialysis, 330 min of PPT microdialysis, and 82 mPRF micro-
injection experiments. These data are the first to show the ability
of NOS inhibition within specific brain regions to alter simulta-
neously (1) neurotransmitter release, (2) states of sleep and
wakefulness, and (3) state-dependent respiratory depression. Por-
tions of these mPRF microdialysis data were published previously
in a brief report (Leonard and Lydic, 1995). The present results
describe for the first time an increased number mPRF dialysis
samples, ACh release from PPT brain regions, the stereoselective
effects of NLA, and the ability of mPRF NOS inhibition to alter
REM sleep and breathing during REM sleep.
Identification of microdialysis and microinjection sites
For all microdialysis experiments, confirmation of dialysis probe
placement in the mPRF and the PPT was achieved by successful
in situ recovery of ACh and histological visualization of probe-
induced lesions. Figure 1, A and B, demonstrates visualization and
localization of probe-induced lesions within the mPRF and the
PPT, respectively. Likewise, stereotaxic placement of microinjec-
tors in the mPRF was verified by histological analyses. For all
microinjection studies, a 3 ?g injection of neostigmine was able to
cause a REM sleep-like state (Baghdoyan et al., 1984) during at
least 50% of a 2 hr recording period. A typical mPRF microin-
jection lesion is illustrated in Figure 1C.
mPRF ACh release varied across states
mPRF ACh release during states of sleep and wakefulness
To test the hypothesis that ACh release in the mPRF would
increase during REM sleep relative to NREM sleep and wakeful-
ness ACh levels, 10 min (30 ?l) mPRF dialysate samples (n) were
collected during objectively defined states of wakefulness (n ?
41), NREM sleep (n ? 33), and REM sleep (n ? 18) from five
different cats. Quantification of these samples revealed state-
dependent differences in mPRF ACh release (F(2,92)? 42.10; p ?
0.0001) (Fig. 2A, hatched bars). During REM sleep, mPRF ACh
release increased significantly, rising 100% over waking levels and
124% above ACh release during NREM sleep. In every experi-
ment in each animal, mPRF ACh release measured during REM
sleep was at least 80% greater than ACh release during either
wakefulness or NREM sleep. There was no significant difference
in ACh levels recovered from the mPRF comparing wakefulness
and NREM sleep ( post hoc Tukey’s test).
NLA decreased mPRF ACh release
To test the hypothesis that NO regulates pontine ACh release, the
mPRF of intact, unanesthetized cats was dialyzed with the NOS
inhibitor NLA while simultaneously measuring ACh release dur-
ing wakefulness, NREM sleep, and REM sleep. Figure 2A shows
that during every state, NLA caused a significant reduction in
mean mPRF ACh release. Compared with Ringer’s dialysis, NLA
caused decreases in average mPRF ACh release of 39% during
wakefulness (t ? 6.01; df ? 74; p ? 0.0001); 44% decrease during
NREM sleep (t ? 4.47; df ? 61; p ? 0.0001); and 45% during
REM sleep (t ? 3.52; df ? 27; p ? 0.0016).
During NLA dialysis, state-dependent changes in mPRF ACh
release also were observed (F(2,76)? 12.82; p ? 0.0001) (Fig. 2A,
solid bars). REM sleep ACh release (n ? 11) was significantly
greater than wakefulness ACh release (82% increase) and NREM
showing histological localization of representative microdialysis and mi-
croinjection sites. Rostral is to the right. A, The black arrow marks the tip
of the lesion in the mPRF (also referred to as the gigantocellular tegmen-
tal field, or FTG, by Berman, 1968) caused by the microdialysis probe. The
tip of the lesion was localized to the coordinates P ? 3.0 mm; L ? 1.5 mm;
H ? ?5.5 mm. B, Cat brainstem cut through the PPT nuclei at 2.9 mm
lateral to midline. The tip of the microdialysis probe lesion is located at
P ? 0.5 mm and H ? ?2.5 mm and is indicated by the black arrow. C,
Brainstem section illustrating a microinjection site in the cat mPRF (black
arrow) localized to P ? 2.0 mm; L ? 1.6 mm; H ? ?6.0 mm. Scale bars
(lower right corners), A–C, 2 mm. 5MET, Mesencephalic trigeminal tract; 6,
abducens nucleus; 6N, abducens nerve; 7, facial nucleus; 7G, genu of facial
nerve; 7N, facial nerve; BC, brachium conjunctivum; CB, cerebellar cortex;
CBM, medial nucleus of the cerebellum; CBM/IN, medial and interpositus
nuclei of the cerebellum; IC, inferior colliculus; IO, inferior olive; mPRF,
medial pontine reticular formation (or FTG); P, pyramidal tract; PAG,
periaqueductal gray; SC, superior colliculus; SO, superior olive; TB, trap-
Sagittal sections of cat brainstem stained with cresyl violet and
776 J. Neurosci., January 15, 1997, 17(2):774–785Leonard and Lydic • NO Alters Pontine ACh Release and REM Sleep
sleep ACh values (121% increase). Average ACh release during
wakefulness (n ? 35 samples) was not significantly different from
NREM sleep ACh release (n ? 30).
mPRF ACh release was not altered by dialysis with NDA
Additional microdialysis experiments were designed to confirm
that mPRF administration of NLA decreased mPRF ACh release
because of specific enzymatic inhibition of NOS. These experi-
ments involved dialyzing the mPRF with 10 mM NDA, the less
active stereoisomer of NLA. Figure 2B shows that compared with
control, NDA did not significantly alter mPRF ACh release dur-
ing wakefulness, NREM sleep, or REM sleep. Dialysis with NDA
also did not produce any observable behavioral or electrographic
effects on states of arousal.
PPT ACh release was decreased by halothane
anesthesia and PPT NOS inhibition
Halothane anesthesia has been shown to decrease ACh release
from cholinergic terminals in the mPRF (Keifer et al., 1994).
Additionally, stereoselective NOS inhibition now has been shown
to decrease ACh release within the mPRF (Fig. 2). Because the
mPRF is known to contain PPT axon terminals, these results
encouraged experiments designed to test the hypothesis that halo-
thane and NLA would decrease ACh release in the cholinergic
cell body region of the PPT. Figure 3A illustrates chromatogram
peaks representative of PPT ACh release during 1.2% halothane
anesthesia (left), during quiet wakefulness with Ringer’s dialysis
(middle), and during quiet wakefulness while dialyzing the PPT
with 10 mM NLA (right). Figure 3B shows that both 1.2% halo-
thane and delivery of the NOS inhibitor NLA during wakefulness
caused a significant decrease in PPT ACh release compared with
ACh levels of release during quiet wakefulness with Ringer’s
dialysis. Mean (? SD) ACh release (pmol/10 min) in the PPT was
reduced 15% ( p ? 0.05) by 1.2% halothane anesthesia (n ? 11)
compared with wakefulness (n ? 10). PPT ACh release was
decreased by 36% ( p ? 0.01) with NLA dialysis (n ? 11) during
quiet wakefulness compared with ACh levels of release during
Ringer’s dialysis. The reduction in PPT ACh release caused by
NLA was greater than the reduction caused by 1.2% halothane
anesthesia ( p ? 0.01, post hoc Tukey’s test).
mPRF microinjection of NOS inhibitor decreased
Having demonstrated that NLA significantly decreased mPRF
ACh release (Fig. 2), and knowing that REM sleep is generated,
in part, by cholinergic stimulation of the mPRF, this study also
tested the hypothesis that mPRF NLA microinjection would de-
Ringer’s (control, hatched bars) or 10 mM NLA (solid bars) during wakefulness, NREM sleep, and REM sleep. Values on the ordinate are expressed as mean
? SD pmol of ACh recovered from the mPRF per 10 min of dialysis. Asterisks designate a significant difference ( p ? 0.05; independent t tests) in mean mPRF
ACh release between NLA dialysis and Ringer’s dialysis within each state. B, Mean ? SD mPRF ACh release from separate experiments dialyzing the mPRF
with either Ringer’s solution (control, hatched bars) or 10 mM NDA (stippled bars) during states of wakefulness, NREM sleep, or REM sleep. Note that NDA,
the less active stereoisomer of NLA, had no statistically significant effect on mPRF ACh release compared with Ringer’s control.
NLA dialysis significantly reduced mPRF ACh release compared with Ringer’s control. A, mPRF ACh release while dialyzing the mPRF with
Leonard and Lydic • NO Alters Pontine ACh Release and REM SleepJ. Neurosci., January 15, 1997, 17(2):774–785 777
crease natural REM sleep and would block the neostigmine-
induced REM sleep-like state (Baghdoyan et al., 1984). Figure 4
shows polygraphic recordings obtained from the present experi-
ments demonstrating the electrographic traits of wakefulness,
NREM sleep, REM sleep, and the REM sleep-like state induced
by 3 ?g neostigmine mPRF injection (REM-Neo). Figure 5 illus-
trates the typical patterns of waking, NREM sleep, and REM
sleep states during 120 min after mPRF microinjection for six
different microinjection conditions. The Figure 5 data also show
the ability of neostigmine and NLA to alter the temporal organi-
zation of REM sleep
mPRF microinjection of NLA, but not NDA, inhibited
Figure 6 illustrates the effect of mPRF microinjection of saline
(control), NLA, or NDA on the percent of time spent in states of
sleep and wakefulness during the first 2 hr after mPRF microin-
jection. The NOS inhibitor NLA significantly reduced the time
spent in REM sleep (Fig. 6A) compared with saline (t ? 4.22;
df ? 19; p ? 0.01; Bonferroni correction applied) and compared
with NDA (t ? 4.11; df ? 19; p ? 0.01). NDA microinjection,
however, had no effect on REM sleep percentage compared with
control. There was no significant effect of mPRF microinjection of
NLA or NDA on the percent time spent in NREM sleep (Fig. 6B)
or wakefulness (Fig. 6C).
mPRF microinjection of NLA, but not NDA, blocked the
Figure 7A shows the ability of mPRF NLA to block the REM-Neo
state. Compared with saline control, neostigmine injection caused
a 408% increase in the percent time occupied by the REM
sleep-like state (t ? 13.36; df ? 26; p ? 0.01). NLA injected 15
min before neostigmine significantly reduced the neostigmine-
induced REM sleep-like state by 64.2% (t ? 6.71; df ? 28; p ?
0.01 compared with Neo alone). NDA pretreatment, however,
had no effect on neostigmine’s ability to cause an increase in REM
sleep percentage. Figure 7, B and C, shows that the REM sleep-
enhancing effect of Neo occurred with a concomitant decrease in
NREM sleep, not wakefulness. Figure 7B shows that significantly
less time was spent in NREM sleep (?49.2%) after Neo com-
pared with saline (t ? 21.96; df ? 26; p ? 0.01). NLA injected
before neostigmine significantly blocked (47.2%; t ? 4.10; df ?
28; p ? 0.01) the neostigmine-induced decrease in NREM sleep.
NREM sleep time after NLA/Neo injections still was significantly
less (?26%) than after saline injection (t ? 4.71; df ? 26; p ?
0.01). NDA pretreatment did not alter the neostigmine-induced
decrease in NREM sleep. Figure 7C shows that there was no
effect of Neo, NLA/Neo, or NDA/Neo injections on time spent
mPRF microinjection of NLA, but not NDA, altered the
temporal organization of REM sleep
Figure 8 illustrates the effect of mPRF microinjection on both the
frequency and duration of REM sleep epochs. There was a
statistically significant main effect of mPRF microinjection on the
mean duration of REM sleep epochs (F(5,404)? 8.30; p ? 0.0001)
and the number of REM sleep epochs over a 2 hr period (F(5,73)
? 5.25; p ? 0.0004). Independent t test comparisons revealed that
mean REM sleep epoch duration after NLA injection was re-
duced significantly (?60%) compared with saline (NLA vs saline),
whereas NDA had no effect on REM sleep duration (NDA vs
saline). Neostigmine injection significantly increased REM sleep
epoch duration (?103%) compared with saline (Neo vs saline).
NLA administration before neostigmine completely blocked the
epoch duration enhancement induced by neostigmine (NLA/Neo
vs Neo) and returned the REM sleep epoch duration to control
levels (NLA/Neo vs saline). NDA pretreatment had no effect on
the REM sleep epoch duration enhancement caused by neostig-
mine (NDA/Neo vs Neo). Compared with saline, neostigmine
injection significantly increased (?155%) the number of REM
sleep epochs (Neo vs saline). Neither NLA nor NDA pretreat-
ment affected the ability of neostigmine to enhance the number of
REM sleep epochs (NLA/Neo or NDA/Neo vs Neo). Injection of
NLA or NDA alone did not alter the frequency of naturally
occurring REM sleep epochs (NLA or NDA vs saline). Taken
together, these data show that mPRF NLA injection had selective
effects on the temporal organization of REM sleep, causing a
significant decrease in the duration, but not the number, of REM
sleep and REM-Neo episodes.
release. A, Chromatograms show peak areas proportional to ACh content
present in 30 ?l (10 min) dialysate samples indicating PPT ACh release under
three different conditions. The chromatogram to the far left is representative
of PPT ACh release during 1.2% halothane anesthesia while dialyzing with
Ringer’s. The center peak shows ACh release during quiet wakefulness with
Ringer’s dialysis. The effect of NLA dialysis on ACh release during quiet
wakefulness is illustrated on the far right. The numbers below each chromato-
gram indicate the amount of ACh (pmol/10 min). B, Mean ? SD PPT ACh
release is shown on the ordinate during administration of 1.2% halothane
with Ringer’s dialysis (open bar) during wakefulness (0% halothane) with
Ringer’s dialysis (hatched bar) and during wakefulness in the presence of
NLA dialysis (solid bar). There was a significant main effect of dialysis and
anesthetic condition on PPT ACh release (F(2,32)? 23.59; p ? 0.0001).
Asterisks indicate significantly decreased ( p ? 0.05; Tukey’s test) ACh release
during 1.2% halothane and NLA dialysis.
Both 1.2% halothane and PPT NLA dialysis decreased PPT ACh
778 J. Neurosci., January 15, 1997, 17(2):774–785Leonard and Lydic • NO Alters Pontine ACh Release and REM Sleep
mPRF microinjection of NLA, but not NDA, altered
The ability of mPRF NLA injection to block the cholinergically
induced decrease in respiratory rate during the REM sleep state
is illustrated in Figure 9. In agreement with previously described
effects of mPRF carbachol (Lydic and Baghdoyan, 1989) and
bethanechol (Lee et al., 1995) on respiratory rate, mPRF injection
of neostigmine caused a significant reduction in respiratory rate
by mPRF microinjection of neostigmine (REM-Neo). During each state, polygraphic tracings record respiration (arrow marks a point of peak inspiratory
airflow), eye movements (EOG), cortical electroencephalogram (EEG), field potentials from the lateral geniculate body of the thalamus (LGB), and neck
muscle electromyogram (EMG). Time scale (each tick equals 1 sec) is shown at the bottom of each 1 min polygraphic record. Calibration bars show
amplitude of pen deflection equal to 100 ?V. Note that during REM-Neo, the REMs, EEG activation, presence of ponto-geniculo-occipital waves in the
LGB recording, and muscle atonia interrupted by periodic bursts of muscle activity are similar to those seen during natural REM sleep.
One minute samples of polygraphic recordings during states of wakefulness, NREM sleep, REM sleep, and the REM sleep-like state induced
Leonard and Lydic • NO Alters Pontine ACh Release and REM SleepJ. Neurosci., January 15, 1997, 17(2):774–785 779
during REM sleep compared with saline control (t ? 6.50; df ?
262; p ? 0.01; Bonferroni correction). NLA injection before
neostigmine completely blocked the ability of neostigmine to
decrease respiratory rate. NDA injection before neostigmine did
not alter the neostigmine-induced decrease in respiratory rate.
The results demonstrate that mPRF administration of the NOS
inhibitor NLA stereoselectively (1) decreased ACh release within
the mPRF, (2) inhibited REM sleep, and (3) prevented the
cholinergically induced decrease in respiratory rate during the
REM sleep-like state. These are the first data to suggest that NO,
produced within a specific brain region, the mPRF, altered sleep/
wake states by modulating the release of a specific neurotrans-
Local inhibition of NOS reduced pontine ACh release
Delivery of NLA to the mPRF by microdialysis caused a stereo-
selective decrease in mPRF ACh release. Although stereoisomers
of NOS inhibitors have been shown to produce weakly some of
the effects of NOS inhibition (Wang et al., 1991, 1993, 1994a),
many investigators have used the D-enantiomer of NLA (referred
to here as NDA) to demonstrate that the biological activity of
NLA is attributable to specific interaction with NOS and inhibi-
tion of NO production (Liu et al., 1991; Iadecola, 1992; Khalil and
Helme, 1992; Khanna et al., 1993; Tanaka et al., 1994; Wang et al.,
1994b; Fukuto and Chaudhuri, 1995; Griffith and Stuehr, 1995).
Therefore, the ability of NLA, but not the enantiomer NDA, to
significantly decrease ACh release within the mPRF (Fig. 2)
suggests that NO produced in the mPRF plays a role in regulating
mPRF ACh release.
tion. For each minute of the 2 hr polygraphic recording (shown on the
abscissa), the behavioral state is indicated as wakefulness [W (lowest
level), NREM sleep (S ? EEG Synchronization, middle level), or REM
sleep (D ? EEG Desynchronization, highest level)] on the ordinate. These
plots illustrate typical sleep/wake patterns for 120 min after each of six
different mPRF microinjection conditions (A–F). Note the increase in
REM sleep time evoked by Neo injection (D vs A). Note also the ability of
the NOS inhibitor NLA to decrease both natural REM sleep (B vs A) and
the REM sleep-like state induced by neostigmine (E vs D). NDA had no
effect on natural (C vs A) or neostigmine-induced (F vs D) REM sleep.
These plots also illustrate how the temporal organization of REM sleep
was quantified for (1) REM sleep latency (the time from mPRF injection
at min 0 to the onset of the first REM sleep episode: 22 min in plot A); (2)
REM sleep epoch frequency (the number of REM sleep epochs that
occurred over 2 hr: 3 for plot A); and (3) duration of individual REM sleep
epochs (2, 4, and 3 min for plot A).
Time course of sleep and wakefulness after mPRF microinjec-
NDA on percent time spent in REM sleep (A), NREM sleep (B), or
wakefulness (C) over a 2 hr polygraphic recording period. Percent time
(mean ? SD) spent in each state after microinjection of saline (control,
hatched bars), NLA (solid bars), or NDA (stippled bars) is shown on the
ordinate. NLA microinjection caused a 71% decrease in REM sleep time
but did not significantly alter the percent time spent in either NREM sleep
or wakefulness. Microinjection of NDA had no effect on the amount of
time spent in REM sleep, NREM sleep, and wakefulness; *p ? 0.01
(independent t tests).
Effect of mPRF microinjection of 22.8 mM NLA or 22.8 mM
780 J. Neurosci., January 15, 1997, 17(2):774–785 Leonard and Lydic • NO Alters Pontine ACh Release and REM Sleep
ACh is released in the mPRF from terminals of cholinergic
LDT/PPT neurons (Lydic and Baghdoyan, 1993), and the activity
of these neurons is known to be important in the generation of
cortical activation characterizing both REM sleep and waking
states (Webster and Jones, 1988; El Mansari et al., 1990; Steriade
et al., 1990; Kayama et al., 1992). Synaptically mediated, inhibi-
tory modulation of cholinergic LDT/PPT neurons is effected by
the neurotransmitters serotonin (Luebke et al., 1992; Leonard
and Llinas, 1994), norepinephrine (Williams and Reiner, 1993),
and ACh (Luebke et al., 1993; Leonard and Llinas, 1994). The
presence of reciprocal cholinergic innervation by cholinergic
LDT/PPT neurons (Semba and Fibiger, 1992; Steininger et al.,
1992) suggests that the release of ACh in the LDT/PPT cholin-
ergic cell body region might serve to modulate the activity of these
cholinergic neurons and hence participate in REM sleep regula-
tion. Anatomical evidence has shown that NOS is present in the
axon terminals and the cell bodies of LDT/PPT cholinergic neu-
rons (Vincent et al., 1983; Mizukawa et al., 1989; Bickford et al.,
1993). In addition to decreasing ACh release in the mPRF, NOS
inhibition decreased ACh release in the cholinergic PPT cell body
region (Fig. 3). The ability of NO production to modulate ACh
release in the PPT nuclei suggests that levels of NO, by altering
ACh release, may influence the activity of cholinergic neurons
known to be important in the generation of REM sleep.
NOS inhibition and anesthesia induced alterations
The present study also used halothane anesthesia as an additional
tool for examining the relationship between levels of arousal and
NOS modulation of ACh release in the mPRF. The results indi-
cate that halothane anesthesia, like PPT NOS inhibition, reduced
ACh release in the cholinergic PPT cell body region (Fig. 3). The
finding that both PPT NOS inhibition and halothane anesthesia
diminished ACh release in the PPT is consistent with results
showing that halothane anesthesia significantly reduces mPRF
ACh release (Keifer et al., 1994). Volatile anesthetics, including
halothane, have been shown to inhibit NOS in rat cerebellum
(Tobin et al., 1994). In addition, halothane has been shown to
interfere with the stability of NO (Rengsamy et al., 1995) and the
ability of NO to cause vasodilation (Blaise et al., 1994). The idea
that NO contributes to the regulation of arousal states also has
been supported by data showing that in mice and rats, inhibition
of NOS augmented anesthesia, analgesia, and sedation caused by
isoflurane and halothane anesthesia (Johns et al., 1992; Ichinose
mPRF neostigmine (Neo) microinjection to increase the amount of time
spent in a REM sleep-like state over a 2 hr period. The ordinate shows
percent of time (mean ? SD) spent in a polygraphically defined REM
sleep state (A), NREM sleep (B), or wakefulness (C) after microinjection
of saline, 40 mM Neo, 22.8 mM NLA pretreatment to Neo (NLA/Neo), or
22.8 mM NDA pretreatment to Neo (NDA/Neo). Neo microinjection
increased the amount of time spent in REM sleep (A), while decreasing
NREM sleep time (B). NLA pretreatment (NLA/Neo) significantly atten-
uated the ability of Neo to enhance REM sleep time (A) and decrease
NREM sleep percentage (B). NDA pretreatment did not alter the effect of
Neo on REM sleep and NREM sleep time. (C) None of the mPRF
microinjections produced a significant effect on the percent time spent in
wakefulness compared with saline control. Asterisks indicate significant
difference compared with saline ( p ? 0.01; independent t tests, Bonfer-
roni correction); †, significant difference from NLA/Neo injections ( p ?
Effect of NLA or NDA mPRF microinjections on the ability of
but not the initiation of REM sleep. This graph shows the mean ? SEM
duration of REM sleep epochs after mPRF microinjection ( y-axis) versus
the mean ? SEM number of REM sleep epochs which occurred in the 2
hr period after mPRF microinjection (x-axis). The mPRF microinjection
conditions are indicated by the following symbols: f, saline; F, 22.8 mM
NLA; å, 22.8 mM NDA; ?, 40 mM Neo; E, 22.8 mM NLA/40 mM Neo; Ç,
22.8 mM NDA/40 mM Neo. Notice that mPRF NLA administration
reduced the duration of REM sleep epochs both for naturally occurring
REM sleep (solid symbols) and for the REM sleep-like state (open sym-
bols). The number of REM sleep epochs that occurred both naturally and
after Neo injection, however, was not altered by NLA injection. These
data suggest that there was an NLA-specific effect on the ability to
maintain REM sleep episodes but no effect on the ability to generate
mPRF NLA injection disrupted the maintenance of REM sleep
Leonard and Lydic • NO Alters Pontine ACh Release and REM SleepJ. Neurosci., January 15, 1997, 17(2):774–785 781
et al., 1995). The finding that NOS inhibition decreased ACh
release in the mPRF (Fig. 2) and PPT (Fig. 3) is consistent with
reports that both pontine cholinergic stimulation (Keifer et al.,
1996) and brain NO (Nistico et al., 1994) contribute to generation
of electrocortical (EEG) arousal. The present results (Fig. 3)
demonstrating that both NOS inhibition and halothane anesthesia
decreased ACh release in the PPT, therefore, are consistent with
the notion that NO and volatile anesthetics may have antagonistic
effects on cholinergic modulation of EEG and behavioral arousal.
mPRF NOS inhibition interfered with the maintenance
of REM sleep
Injection of NLA into the mPRF caused a reduction in the time
spent in natural REM sleep and attenuated the ability of mPRF
neostigmine injection to produce the REM sleep-like state illus-
trated by Figure 4. More specifically, mPRF NOS inhibition
diminished the duration of individual REM sleep epochs but did
not alter the latency to REM sleep onset or the frequency of REM
sleep episodes (Figs. 5–8). These data suggest that NO production
in the mPRF is important for maintaining REM sleep once it has
been initiated. NLA in the mPRF inhibited both naturally occur-
ring REM sleep (Fig. 6A) and the cholinergically induced REM
sleep state (Fig. 7A). Furthermore, NLA administration de-
creased the epoch duration of both natural REM sleep and
neostigmine-induced REM sleep (Fig. 8).
The similarity between the effects of NOS inhibition on natural
REM sleep and the cholinergically induced REM sleep state
supports two conclusions. First, these data suggest that mPRF NO
production participates in natural REM sleep regulation by mod-
ulating pontine cholinergic neurotransmission. Second, these data
lend additional support to the premise that endogenous cholin-
ergic neurotransmission plays a major role in natural REM sleep
generation. McCarley et al. (1995) noted that REM sleep gener-
ation requires the coordinated activation of pools of cholinergic
LDT/PPT and noncholinergic, cholinoceptive mPRF neurons.
The present data suggest the possibility that NO may contribute to
the recruitment of both cholinergic and cholinoceptive neurons
The absence of a significant difference in ACh levels recovered
from the mPRF during wakefulness compared with NREM sleep
is consistent with previous microdialysis studies of mPRF (Lydic
et al., 1991, 1993; Lydic and Baghdoyan, 1993). Wakefulness is the
most heterogeneous of behavioral states, and ACh release in the
mPRF also may vary during specific waking behaviors. To the best
of our knowledge, all currently available data on pontine ACh
release in cat has been obtained from head-restrained animals.
Measures of ACh release from the pons of freely moving dog,
however, note that motor activity did not significantly alter ACh
levels (Reid et al., 1994).
It is interesting to note that microdialysis of large areas of rat
thalamus revealed increased ACh release during wakefulness
and REM sleep, compared with NREM sleep (Williams et al.,
1994). This suggests the possibility that cholinergic LDT/PPT
neurons, which selectively increase their discharge rates during
REM sleep, project to the mPRF, whereas LDT/PPT neurons
with discharge rates that are highest in waking and REM sleep
project to various thalamic nuclei (Steriade et al., 1990). Cho-
linergic LDT/PPT neurons have been noted to be good candi-
dates for disrupting the synchronized spindle oscillations in
thalamocortical systems during both arousal and REM sleep
(Steriade et al., 1990). Recently, it has been shown that mPRF
microinjection of the cholinergic agonist carbachol significantly
decreased cortical EEG spindles that normally accompany
halothane anesthesia (Keifer et al., 1996).
mPRF NOS inhibition blocked cholinergically mediated
respiratory rate depression
Microinjection of neostigmine into the mPRF is known to pro-
duce a REM sleep-like state (Baghdoyan et al., 1984). Presum-
ably, the REM-Neo state results from the accumulation of endo-
genously released ACh. This assumption is supported by recent
evidence showing that mPRF microinjection of vesamicol-like
compounds that inhibit vesicular packaging of ACh inhibit REM-
Neo (Lydic et al., 1996). The present study is the first to show that
REM-Neo also is characterized by respiratory rate depression
(Fig. 9). The data also show that NLA administration into the
mPRF prevented the neostigmine-induced depression in respira-
tory rate. These data suggest that a reduction in NO production,
caused by NLA, resulted in diminished ACh levels within the
mPRF and eliminated the neostigmine-induced reduction in re-
spiratory rate. This conclusion is supported by previous studies
indicating that pontine cholinergic neurotransmission contributes
to respiratory rate depression during the REM sleep-like state
caused by mPRF administration of cholinomimetics (Lydic and
Baghdoyan, 1992). Both neuroanatomical (Lee et al., 1995) and
electrophysiological (Gilbert and Lydic, 1994) data demonstrate
pathways whereby the mPRF may influence respiratory rate. The
specific mechanisms through which NO, ACh, and mPRF neurons
alter breathing remain unknown, but state-dependent respiratory
modulation has been shown to involve pertussis toxin-sensitive
G-proteins and adenylate cyclase (Shuman et al., 1995) and cAMP
signal transduction systems (Capece et al., 1995, 1996).
Limitations and conclusions
In the present study, the inferences regarding the role of NO in
modulating ACh release, REM sleep, and respiratory rate are
based on the effects of NLA and NDA administration. NOS
cholinergically induced, state-dependent decrease in respiratory rate.
Mean ? SD respiratory rate (breaths/min) is shown on the ordinate for
each of four different mPRF microinjection conditions (abscissa): saline;
neostigmine (Neo); NLA/Neo; and NDA/Neo. Compared with saline
control, REM sleep respiratory rate was significantly reduced by mPRF
injection of neostigmine (*p ? 0.01). Injection of NLA before neostigmine
(NLA/Neo) prevented the neostigmine-induced decrease in respiratory
rate. NDA pretreatment had no significant effect on the cholinergically
induced decrease in respiratory rate.
Injection of the NOS inhibitor NLA into the mPRF blocked the
782 J. Neurosci., January 15, 1997, 17(2):774–785Leonard and Lydic • NO Alters Pontine ACh Release and REM Sleep
inhibitors currently represent one of the most widely used re-
search tools for investigating the role of NO in biological systems
(Griffith and Stuehr, 1995). It is acknowledged that the conclu-
sions drawn from the results of these experiments would be
strengthened by the use of NO-generating compounds and the in
situ electrochemical measurement of NO. The use of NO scaven-
gers such as hemoglobin recently have been shown to provide a
technically difficult but promising technique for measuring levels
of NO (Williams et al., 1995). Additional studies measuring ACh
release in the LDT/PPT during REM sleep also are needed. Such
studies are technically difficult, and stereotaxic access to the
LDT/PPT in the cat is limited by the presence of an ossified
tentorium. Nonetheless, the data shown in Figure 3 represent the
first measurements of ACh release in the pontine cholinergic cell
body region in the awake and anesthetized cat.
Data presented here provide evidence for the role of NO in
facilitating pontine ACh release, maintaining REM sleep once
it has been initiated, and participating in the cholinergic mod-
ulation of respiratory rate. It is likely that NO influences REM
sleep and breathing during REM sleep via modulation of ACh
release. NO also may serve to potentiate and prolong the
duration of ACh release from presynaptic axon terminals
within the mPRF. The production of NO within the mPRF may
be clinically relevant in the cholinergic modulation of state-
dependent respiratory depression (Pack, 1995), narcolepsy
(Reid et al., 1994; Nishino et al., 1995), and REM behavior
disorder (Mahowald and Schenck, 1992).
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