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Vol. 286, Issue 3, 1446-1452, September 1998
Department of Anesthesia, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Muscarinic autoreceptors regulate acetylcholine (ACh) release in several brain regions, including the medial pontine reticular formation (mPRF). This study tested the hypothesis that the muscarinic cholinergic receptor mediating mPRF ACh release is the pharmacologically defined M2 subtype. In vivo microdialysis was used to deliver muscarinic cholinergic receptor (MAChR) antagonists to the feline mPRF while simultaneously measuring endogenously released ACh. The lowest concentration of each antagonist that caused a significant increase in mPRF ACh release was determined and defined as the minimum ACh-releasing concentration. Data obtained from 41 mPRF dialysis sites in 10 animals showed that the order of potency (followed by the minimum ACh-releasing concentration) was scopolamine (1 nM) > AF-DX 116 (3 nM) > pirenzepine (300 nM). Comparison of these minimum ACh-releasing concentrations to the known affinities of the antagonists for the five mAChR subtypes is consistent with the conclusion that the autoreceptor regulating mPRF ACh release is the M2 subtype. Considerable evidence supports a role for cholinergic neurotransmission and postsynaptic M2 receptors in the mPRF in regulating levels of arousal. The present data suggest that presynaptic M2 receptors contribute to the regulation of arousal states by modulating mPRF ACh release.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Autoreceptors
are defined as presynaptically localized receptors that respond to a
neuron's own transmitter and function to regulate transmitter release
by feedback control (Kalsner, 1990
). Functional, anatomic and ligand
binding data support the existence of muscarinic, cholinergic
autoreceptors in many brain regions. Functionally, in vivo
microdialysis studies have shown that mAChRs regulate ACh release in
the striatum (Billard et al., 1995), cerebral cortex
(Quirion et al., 1994), medial septal area (Moor et
al., 1995) and hippocampus (Moor et al., 1995;
Nordström and Bartfai, 1980). Anatomically, the presence of
mAChRs on presynaptic terminals of basal forebrain cholinergic neurons
has been visualized through the use of electron microscopy and antibody
staining (Levey et al., 1995). Ligand binding studies have
demonstrated that lesions of cholinergic neurons cause changes in
terminal field mAChR density, providing evidence for the presence of
muscarinic autoreceptors in cerebral cortex (Mash et al.,
1985).
Muscarinic autoreceptors regulate ACh release in the feline mPRF (Roth
et al., 1996). The brain stem reticular formation modulates level of behavioral arousal, cardiopulmonary control, somatic motor
tone and pain sensation (Role and Kelly, 1991). Cholinergic and
noncholinergic, cholinoceptive pontine neurons play an important role
in the control of arousal states, particularly in the generation of
rapid eye movement (REM) sleep (Baghdoyan, 1997a; Steriade and
McCarley, 1990). Neurons of the mPRF, which are not cholinergic (Jones
and Beaudet, 1987), receive their cholinergic input from neurons of the
LDT and PPT nuclei (Mitani et al., 1988; Shiromani et
al., 1988). ACh is released in the mPRF from LDT/PPT neurons (Lydic and Baghdoyan, 1993), and the muscarinic autoreceptors present
in the mPRF are presumed to reside on LDT/PPT terminals (Roth et
al., 1996).
Molecular cloning studies have identified five subtypes of mAChRs
(m1-m5) linked to different G proteins and intracellular signal
transduction pathways (Brown et al., 1997; Felder, 1995; Hosey, 1992). Muscarinic receptors also are classified
pharmacologically according to affinity binding profiles of muscarinic
antagonists (Caulfield, 1993). The pharmacologically defined M1-M3
mAChR subtypes correspond fairly well to the molecularly identified
m1-m3 subtypes, respectively, but it is important to note that there
are no mAChR antagonists with exclusive selectivity for any one of the
molecularly identified subtypes (Buckley, 1990; Dörje et
al., 1991). Although the functional roles of mAChR subtypes are
beginning to be elucidated, lack of subtype-specific ligands makes such
studies difficult (Felder, 1995). The pharmacologically defined M2
subtype has been demonstrated to regulate ACh release in several brain
areas (Billard et al., 1995; Moor et al., 1995),
satisfying the operational definition of an autoreceptor. Given the
importance of the pontine brain stem for regulating behavioral arousal
(Steriade and McCarley, 1990), and given that the predominant mAChR
subtype in the mPRF is M2 (Baghdoyan, 1997b; Baghdoyan et
al., 1994), the present study used in vivo
microdialysis to test the hypothesis that the mAChR mediating ACh
release in the mPRF is the pharmacologically defined M2 subtype.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animal and drug preparation.
All experiments were conducted
in accordance with the Guide for the Care and Use of Laboratory
Animals (7th ed., National Academy of Sciences Press, Washington,
DC, 1996). Adult male cats (N = 10) were anesthetized
with halothane or isoflurane (1-2% in O2) and implanted
with standard electrodes for recording the cortical
electroencephalogram and nuchal electromyogram. As described previously
(Leonard and Lydic, 1997), a craniotomy was performed over the
cerebellum to allow access of the microdialysis probe to the mPRF.
Experiments were started after a 3-wk, postsurgical recovery period.
; Leonard and Lydic, 1997; Moor et
al., 1995; Quirion et al., 1994). AF-DX 116 (Boehringer
Ingleheim, Ridgefield, CT) was dissolved in 0.05 N HCl and p-FHHSiD
(Research Biochemicals International, Natick, MA) was dissolved in
100% EtOH. It was possible to use these solvents because they were
shown to have no effect on ACh release. All drugs were diluted serially
to their final concentrations using Ringer's solution.
In vivo microdialysis and HPLC/EC.
Microdialysis
probes (CMA/10; CMA Microdialysis, Acton, MA) had a 2-mm long
polycarbonate membrane, a 0.5-mm diameter and a 20-kDa cut-off.
Quantification of pontine ACh release using HPLC/EC (Bioanalytical
Systems Inc., West Lafayette, IN) has been described in detail (Keifer
et al., 1996; Leonard and Lydic, 1997; Lydic and Baghdoyan,
1993). Briefly, a CMA/100 pump ensured continuous flow of Ringer's
solution through the dialysis probe at a rate of 3 µl/min. Dialysis
samples were collected every 10 min, and the dialysate was injected
immediately into the HPLC/EC system to determine the amount of ACh.
Upon injection, a 50 mM Na2HPO4 mobile phase
(pH = 8.5) carried the dialysate through a column that separated
ACh and choline. An immobilized enzyme reactor column produced hydrogen
peroxide in concentrations proportional to the amount of ACh present in
the sample. Hydrogen peroxide then was quantified using a platinum
electrode with an applied potential of 500 mV in reference to a
silver/silver chloride electrode. The resulting signal was recorded on
a flatbed recorder and simultaneously digitized for off-line analysis.
The areas under the chromatogram peaks were integrated using the Inject
program (Bioanalytical Systems Inc.).
Experimental design. Every experiment began by creating a standard curve based on seven known concentrations of ACh. In addition, before positioning the microdialysis probe in the brain, the probe was placed in an ACh solution of known concentration and dialyzed to determine the percent ACh recovery. This procedure was repeated after collection of the last brain dialysis sample. Pre- and postexperimental probe recoveries were compared by t test to ensure that any changes in ACh observed during the experiment were due to pharmacological manipulations rather than to alteration of the dialysis probe membrane. Data reported below were obtained from experiments showing no significant difference between pre- and postexperimental probe recovery.
Anesthesia began by mask induction with halothane (3-3.5% in O2). After the loss of wakefulness, laryngoscopy was performed, lidocaine (1%) was sprayed on the vocal cords and a cuffed endotracheal tube was inserted in the trachea. The animal was placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) equipped with a Kopf 880 semi-chronic head holder. Electroencephalogram and electromyogram were recorded continuously on a Grass model 7 Polygraph (Astro-Med Inc., West Warwick, RI). End-tidal carbon dioxide and halothane concentrations were measured using a Raman spectrometer (Ohmeda Rascal II, Louisville, CO) and maintained at 28 to 30 mm Hg and 1.4%, respectively. Core body temperature was held at 37°C using a T/Pump Heat Therapy System (Gaymar, TP 400 Series, Orchard Park, NY) and a rectal thermometer. Oxygen saturation and heart rate were monitored continuously using an Ohmeda Biox 3700 Pulse Oximeter (Boulder, CO). Blood pressure readings were taken noninvasively every 10 min using a Dinamap (Critikon, Tampa, FL), and heart and breath sounds were monitored regularly using an esophageal stethoscope. The microdialysis probe was placed in the mPRF through the craniotomy according to the stereotaxic coordinates of Berman (1968) and perfused
continuously with Ringer's solution.
Once mPRF ACh release was determined to be stable, five dialysis
samples (50 min) were collected to determine baseline (control) levels
of ACh release. Using a CMA/110 liquid switch, the mPRF then was
dialyzed with Ringer's solution containing either scopolamine, AF-DX
116, pirenzepine, p-FHHSiD or carbachol. Nine sequential dialysis
samples were obtained during drug administration, and only one dose of
one drug was dialyzed per experiment. On completion of each experiment,
the dialysis probe was removed from the brain and the craniotomy was
closed. After extubation, animals were kept under continuous
observation until recovery from anesthesia. A minimum of 7 days
separated experiments in the same animal. Each animal was used for six
to eight experiments. The dialysis probe was placed in a different site
within the mPRF for each experiment.
One goal of the present study was to determine the lowest concentration
of each antagonist that caused a significant increase in mPRF ACh
release (defined as the minimum ACh-releasing concentration) (Billard
et al., 1995). This required a series of experiments conducted according to the following procedure (Billard et
al., 1995). An initial concentration of each antagonist was
dialyzed in one experiment, and the effects on ACh release were
quantified. If that initial concentration caused no change in ACh
release, then the antagonist concentration was increased by one-half
log unit and the experiment was repeated until a concentration was found that caused a statistically significant increase in ACh release.
Similarly, if the initial concentration did cause increased ACh
release, then the antagonist concentration was reduced sequentially by
one-half log unit until a concentration was found that caused no change
in ACh release. As described above, only one dose of one drug was
delivered during each experiment. The criteria for establishing the
minimum ACh-releasing concentration of an antagonist required three
experiments showing a significant increase in ACh release at a given
concentration, and each antagonist was tested in at least three
different animals. Similar requirements were applied for establishing
the highest antagonist concentration that had no effect on ACh release
(defined as the highest no-effect concentration) (Billard et
al., 1995). Descriptive statistics and one-way ANOVA with
Dunnett's multiple comparison test were used to determine the effect
of dialyzed drugs on ACh release. The significance level was P < .05.
Histology.
After the last dialysis experiment, halothane
administration was discontinued and pentobarbital was injected i.v.
(35-40 mg/kg). The brain was perfused in situ with 10%
formalin, removed and fixed in formalin for at least 2 wk. The brain
stem then was placed in 30% sucrose + 10% formalin for 7 days
and serial sections were cut on a freezing microtome at 40 µ in
thickness. All sections were slide-mounted and stained with Cresyl
violet. The laterality of every section that included the mPRF was
identified by comparing each section with the sagittal plates of
Berman's atlas (Berman, 1968). The stereotaxic coordinates of all
dialysis sites were determined from the probe-induced lesions appearing
in these sections, as described previously (Lydic and Baghdoyan, 1993).
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Figure 1 shows a digitized image of
a Cresyl violet-stained, sagittal section through the cat brain stem.
This section contains a typical lesion made in the mPRF by a dialysis
probe (arrow). The lesion was used to confirm probe placement in the
mPRF. The ACh measurements reported below were obtained from 41 mPRF
dialysis sites in 10 animals. The mPRF dialysis sites ranged from P 1.0 to P 4.0, L 0.3 to L 2.3 and H 
4.0 to H 6.5 (stereotaxic
coordinates according to the sagittal plates in Berman, 1968).
Mean ± S.E.M. stereotaxic coordinates for all dialysis sites were
P 2.6 ± 0.1, L 1.2 ± 0.1 and H 5.3 ± 0.1. The H
coordinate indicates the midpoint of the 2-mm long dialysis membrane.
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Results illustrating how each dialysis experiment was performed are
shown in figure 2. The Ringer's
(control) and scopolamine ACh chromatograms (fig. 2A) correspond to 0.7 and 2.2 pmol/10 min, respectively. During the first 10 min of dialysis
with scopolamine, mPRF ACh was increased 90% over the mean control
level (fig. 2B, first scopolamine sample vs. the mean of the
five Ringer's samples). By 20 min of mPRF dialysis with scopolamine,
ACh was increased 221% over the average control ACh level (fig. 2B,
second scopolamine sample). This scopolamine-induced increase in ACh
release (fig. 2C) is typical of recent findings demonstrating the
presence of muscarinic autoreceptors in the mPRF (Roth et
al., 1996).
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Further evidence supporting the regulation of mPRF ACh release by muscarinic autoreceptors is provided by the finding that mPRF dialysis with the agonist carbachol (10 mM) significantly decreased ACh release below control levels (t = 1.8, d.f. = 28). Thus, although the Ringer's solution contained 10 µM neostigmine, the autoreceptors were not maximally inhibited during control dialysis and could respond to an agonist by decreasing ACh release.
This study next sought to identify the subtype of mAChR regulating ACh release by comparing the relative potencies of different mAChR antagonists in their ability to increase mPRF ACh release. The data are reported in figures 3, 4 and 5. Figure 3 illustrates the time course of one experiment using AF-DX 116 (squares), another experiment with pirenzepine (circles) and a third experiment with p-FHHSiD (triangles). Mean ± S.E.M. basal levels of mPRF ACh were 0.59 ± 0.03, 0.52 ± 0.01 and 0.32 ± 0.14 pmol/10 min preceding dialysis with AF-DX 116, pirenzepine and p-FHHSiD, respectively. Dialysis of the mPRF with AF-DX 116 and with pirenzepine significantly increased mPRF ACh release to 1.48 ± 0.05 and 0.70 ± 0.23 pmol/10 min, respectively. In contrast, administration of p-FHHSiD did not alter mPRF ACh release (0.36 ± 0.01 pmol/10 min).
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Figure 4 summarizes results based on 27 experiments in 10 cats. Cross-hatched histograms indicate average ACh
level (pmol/10 min) in the mPRF during control (Ringer's) dialysis and
solid histograms indicate mPRF ACh levels during dialysis with the
minimum concentration of antagonist needed to produce a significant
increase in ACh release over control levels. This concentration of
antagonist is referred to as the minimum ACh-releasing concentration
(Billard et al., 1995), and was determined to be 1 nM for
scopolamine (fig. 4A), 3 nM for AF-DX 116 (fig. 4B) and 300 nM for
pirenzepine (fig. 4C). p-FHHSiD was tested at concentrations ranging
from 10 to 1000 nM, but did not cause a significant increase in mPRF
ACh release. Figure 4 also shows the highest concentration of each antagonist that had no effect on ACh release (diagonally hatched histograms). This highest no-effect concentration (Billard et al., 1995) was one half log unit below the minimum ACh-releasing concentration (fig. 4, A-C).
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The relative potencies of the mAChR antagonists for increasing ACh release are compared in figure 5. The solid histograms plot the mean percent increase in ACh release induced by the minimum ACh-releasing concentration for each antagonist. The order of potency was scopolamine > AF-DX 116 > pirenzepine, indicating that the mAChR regulating ACh release in the mPRF is the pharmacologically defined M2 subtype.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The present study establishes the minimum ACh-releasing concentration for scopolamine, AF-DX 116 and pirenzepine in the mPRF; demonstrates that mPRF carbachol administration decreases mPRF ACh release; and supports the view that the M2 subtype of mAChR functions as an autoreceptor in the mPRF. The ensuing discussion considers the use of muscarinic antagonists for identifying mAChR subtypes, and the potential role of muscarinic autoreceptors in modulating the REM phase of sleep.
Muscarinic receptor pharmacology and the use of in vivo
microdialysis to identify autoreceptor subtypes.
Evidence for the
existence of muscarinic autoreceptors that modulate ACh release in the
mPRF recently was provided by data showing that mPRF scopolamine
administration produced a dose-dependent increase in mPRF ACh release
(Roth et al., 1996). To identify the mAChR subtype
functioning as an autoreceptor, the present study used in
vivo microdialysis to deliver a range of concentrations of four
mAChR antagonists to the mPRF while simultaneously quantifying ACh
release. One important aspect of this approach is that the mAChR
antagonists used exhibit different affinities for the different mAChR
subtypes. By comparing potencies of the different antagonists in their
ability to increase mPRF ACh release with known affinities of these
antagonists for m1-m5 mAChRs, inferences can be made about the mAChR
subtype that functions as an autoreceptor in the mPRF. This approach
recently was used to identify the muscarinic autoreceptor subtype as M2
in rat striatum (Billard et al., 1995).
; Bolden et
al., 1992), and thus was predicted to be the most potent
antagonist (fig. 5). The minimum ACh-releasing concentrations for AF-DX
116 and pirenzepine were 3 nM (fig. 4B) and 300 nM (fig. 4C),
respectively. p-FHHSiD was ineffective in increasing ACh release at
concentrations up to 1000 nM (see below). Based on the binding
affinities of AF-DX 116 and pirenzepine for mAChR subtypes (Billard
et al., 1995; Bolden et al., 1992; Dörje
et al., 1991; Jones et al., 1992), only the m2
receptor would be expected to produce a functional response at
concentrations of less than 100 nM AF-DX 116 and more than 100 nM
pirenzepine. The conclusion that the m2 subtype modulates mPRF ACh
release is consistent with data from in vitro
autoradiographic studies demonstrating that the mPRF contains
predominantly M2 binding sites in cat (Baghdoyan et al.,
1994) and rat (Baghdoyan, 1997b), and with a new, preliminary report
demonstrating the presence of m2 receptors on the terminals of
cholinergic neurons in cat pontine reticular formation (Ray et
al., 1997).
One limitation of the present study is the impossibility, for all
practical purposes, of determining the antagonist concentration in the
mPRF region sampled by the dialysis probe. This issue has been
discussed in detail (Billard et al., 1995), and emphasizes the importance of testing a range of dosages for several different antagonists in their ability to increase mPRF ACh release. Only by
comparing relative (vs. absolute) potencies of these
antagonists is it possible to make conclusions about which mAChR
subtype modulates mPRF ACh release.
Another potential methodological limitation is the concentration of
neostigmine in the Ringer's solution with which the dialysis probe was
perfused. The rapid and powerful degrading actions of acetylcholinesterase require that neostigmine be used to prevent the
breakdown of ACh. Previous studies have shown that neostigmine in the
microdialysis buffer can stimulate the autoreceptor such that no
further reduction in ACh release can be evoked (Moor et al.,
1995). The finding that dialysis with carbachol significantly decreased
ACh release below control levels demonstrates that the autoreceptors
were not maximally inhibited during the present experiments.
It is unclear why p-FHHSiD had no effect on ACh release at
concentrations (100-1000 nM) which would be expected to block m2 receptors (Dörje et al., 1991). Previous work also
found that 1000 nM p-FHHSiD failed to increase ACh release when
delivered directly to cortical and hippocampal slices, where muscarinic autoreceptors of the M1, M2 and M4 subtype were identified (Vannucchi and Pepeu, 1995). Further studies are necessary to reconcile the apparent lack of potency of p-FHHSiD in functional studies with its
high affinity in binding assays.
As has been emphasized elsewhere (Billard et al., 1995),
more than one subtype of mAChR may function as an autoreceptor, and the
generalization that muscarinic autoreceptors are always of the M2
subtype is premature. For example, a preliminary report shows that m3
receptors are present on some presynaptic, cholinergic terminals (Rye
et al., 1995). An M3-like autoreceptor has been suggested to
regulate ACh release in rat striatum (DeBoer et al., 1990),
and both M1 and M4 receptors have been suggested to regulate ACh
release in the hippocampus (Vannucchi and Pepeu, 1995). The agreement
between the present minimum ACh-releasing concentrations of mAChR
antagonists (fig. 4) and previously determined affinities of these
antagonists at cloned mAChR subtypes (Billard et al., 1995;
Bolden et al., 1992; Dörje et al., 1991;
Jones et al., 1992) supports the conclusion that the M2
receptor functions as an autoreceptor in the feline mPRF. Converging
lines of evidence obtained using a variety of methodologies will be
helpful in establishing the subtype identity of muscarinic
autoreceptors in additional brain regions.
mPRF ACh release and REM sleep generation.
ACh in the mPRF
contributes to the generation of REM sleep (reviewed in Baghdoyan,
1997a; McCarley et al., 1995; Steriade and McCarley, 1990).
During REM sleep, pontine ACh release is increased over both waking and
non-REM sleep levels (Kodama et al., 1990; Leonard and
Lydic, 1997). LTD/PPT neurons, which regulate ACh release in the mPRF
(Lydic and Baghdoyan, 1993), increase their discharge rates before and
during REM sleep (El Mansari et al., 1989; Kayama et
al., 1992), and neurotoxic lesions of the dorsolateral
pontomesencephalic tegmentum cause a long-term suppression of REM sleep
that is correlated with the degree of damage to cholinergic LDT/PPT
neurons (Webster and Jones, 1988). Most recently, electrical
stimulation of LDT/PPT has been shown to enhance REM sleep (Thakkar
et al., 1996).
; McCarley et al., 1995). For
example, postsynaptic M2 receptors have been visualized in the mPRF
(Ray et al., 1997; Rye et al., 1995; Vilaró
et al., 1992), and natural REM sleep can be blocked by
pontine microinjections of methoctramine but not pirenzepine or
p-FHHSiD (Imeri et al., 1994). In addition, the
cholinergically evoked REM sleep-like state can be inhibited by
blocking an m2/m4-linked signal transduction pathway involving a
pertussis toxin-sensitive G protein, adenylyl cyclase, cAMP and protein
kinase A (Capece and Lydic, 1997; Shuman et al., 1995), and
carbachol is now known to cause a dose-dependent, atropine-sensitive
activation of G proteins in the pontine reticular formation (Capece
et al., 1998). Intracellular recordings made in
vitro have shown that carbachol depolarizes mPRF neurons by activating a postsynaptic, pirenzepine insensitive (i.e.,
non-M1) mAChR (Greene et al., 1989). Taken together, these
multiple lines of evidence are consistent with the hypothesis that ACh
released from LDT/PPT terminals into the mPRF causes REM sleep, in
part, by activating postsynaptic M2 receptors localized to the same mPRF region explored in the present study. The data reported here suggest, for the first time, that presynaptic M2 receptors in the mPRF
also may contribute to REM sleep generation by modulating mPRF ACh
levels.
Electrophysiological studies have shown that activation of M2 receptors
hyperpolarizes neurons via an inwardly rectifying K+
current (reviewed in Jones et al., 1992). The membrane
potential effects of muscarinic agonists on cholinergic terminals with
the mPRF are unknown, but the present finding that mPRF carbachol decreased mPRF ACh release is consistent with the possibility of an
M2-mediated hyperpolarization of the presynaptic terminal. Activation
of M2 receptors also has been shown to inhibit a high-voltage-activated component of the Ca++ current in rat basal forebrain
cholinergic neurons, suggesting another possible mechanism by which M2
autoreceptors may modulate ACh release (Allen and Brown, 1993).
This study was conducted using halothane anesthesia, which eliminates
REM sleep. Thus, the putative role of M2 autoreceptors in REM sleep
regulation was not addressed. Because ACh release in many brain areas
changes with arousal state (Jasper and Tessier, 1971; Kodama et
al., 1990; Leonard and Lydic, 1997; Lydic et al., 1991;
Williams et al., 1994), the present rationale for using anesthetized animals was to hold arousal state constant. Administration of muscarinic agonists and antagonists into the pontine reticular formation of conscious cat by microdialysis recently has been shown to
cause significant changes in behavioral state (Sakai and Onoe, 1997),
and pontine ACh release is known to change with changes in behavioral
state (Kodama et al., 1990; Leonard and Lydic, 1997; Lydic
and Baghdoyan, 1993; Lydic et al., 1991). By holding arousal
state constant with inhalation anesthesia, it was possible in the
present study to make inferences regarding presynaptic receptor
modulation of ACh release.
The present data do not prove, unequivocally, that muscarinic
autoreceptors exist in the mPRF. In fact, the existence of any autoreceptors regulating transmitter release has been questioned (Kalsner, 1990). It is possible, for example, that a neuronal feedback
loop between the mPRF and LDT/PPT exists, and that postsynaptic M2
receptors on neurons in that feedback loop modulate ACh release. The
most direct explanation of the present finding that mPRF ACh release
was decreased in response to an agonist and increased in response to
antagonists is that mPRF ACh release is modulated by M2 autoreceptors,
putatively localized to LDT/PPT neuron terminals. This interpretation
is supported by a recent study using immunohistochemistry in
conjunction with electron microscopy to demonstrate that, in cat
pontine reticular formation, m2 receptors are localized to terminals of
cholinergic neurons (Ray et al., 1997).
In conclusion, this study showed for the first time that in the mPRF,
AF-DX 116 was more potent than pirenzepine or p-FHHSiD in causing
increased ACh release. These findings are most consistent with the
interpretation that mAChRs of the M2 subtype function as autoreceptors
to modulate pontine ACh release.
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Acknowledgments |
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The authors thank Boehringer Ingelheim for providing AF-DX 116. J. L. DiVittore and P. P. Myers provided expert technical and secretarial assistance. We thank Dr. J. Ellis for critical discussions about muscarinic receptor pharmacology.
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Footnotes |
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Accepted for publication May 4, 1998.
Received for publication December 12, 1997.
1 This work was supported by Grants MH-45361 (H.A.B.), HL-40881 (R.L.) and HL-47749 (R.L.) and by the Department of Anesthesia.
Send reprint requests to: Dr. Helen A. Baghdoyan, Department of Anesthesia (H187), The Pennsylvania State University, College of Medicine, 500 University Drive, Hershey, PA 17033.
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Abbreviations |
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ACh, acetylcholine; AF-DX 116, 11-2[(diethylamino)methyl]-1-piperidinyl)-acetyl]-5, 11-dihydro-6H-pyrido(2, 3-b)(1, 4)-benzodiazepine-one ; ANOVA, analysis of variance; EtOH, ethanol; H, horizontal; HPLC/EC, high-performance liquid chromatography with electrochemical detection; L, lateral; LDT, laterodorsal tegmental nucleus; mAChR, muscarinic cholinergic receptor; mPRF, medial pontine reticular formation; P, posterior; p-FHHSiD, p-fluoro-hexahydro-sila-difenidol hydrochloride; PPT, pedunculopontine tegmental nucleus; REM, rapid eye movement; 6, abducens nucleus; 6N, abducens nerve; 7G, genu of the facial nerve.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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-thio)-triphosphate binding in rapid eye movement sleep-related brainstem nuclei of rat.
J Neurosci
18:
3779-3785
characterization, coupling and function.
Pharmacol Ther
58:
319-379[Medline].This article has been cited by other articles:
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  D. V. Volgin, I. Rukhadze, and L. Kubin Hypoglossal premotor neurons of the intermediate medullary reticular region express cholinergic markers J Appl Physiol, November 1, 2008; 105(5): 1576 - 1584. [Abstract] [Full Text] [PDF]
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 J. Vazquez and H. A. Baghdoyan GABAA Receptors Inhibit Acetylcholine Release in Cat Pontine Reticular Formation: Implications for REM Sleep Regulation J Neurophysiol, October 1, 2004; 92(4): 2198 - 2206. [Abstract] [Full Text] [PDF]
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D.-K. Kim, N. R. Prabhakar, and G. K. Kumar Acetylcholine release from the carotid body by hypoxia: evidence for the involvement of autoinhibitory receptors J Appl Physiol, January 1, 2004; 96(1): 376 - 383. [Abstract] [Full Text] [PDF]
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C. L. Douglas, H. A. Baghdoyan, and R. Lydic Postsynaptic Muscarinic M1 Receptors Activate Prefrontal Cortical EEG of C57BL/6J Mouse J Neurophysiol, December 1, 2002; 88(6): 3003 - 3009. [Abstract] [Full Text] [PDF]
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 J. Vazquez, R. Lydic, and H. A. Baghdoyan The Nitric Oxide Synthase Inhibitor NG-Nitro-L-Arginine Increases Basal Forebrain Acetylcholine Release during Sleep and Wakefulness J. Neurosci., July 1, 2002; 22(13): 5597 - 5605. [Abstract] [Full Text] [PDF]
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C. L. Douglas, H. A. Baghdoyan, and R. Lydic Prefrontal Cortex Acetylcholine Release, EEG Slow Waves, and Spindles Are Modulated by M2 Autoreceptors in C57BL/6J Mouse J Neurophysiol, June 1, 2002; 87(6): 2817 - 2822. [Abstract] [Full Text] [PDF]
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 K. Tottori, M. Nakai, Y. Uwahodo, T. Miwa, S. Yamada, Y. Oshiro, T. Kikuchi, and C. A. Altar Attenuation of Scopolamine-Induced and Age-Associated Memory Impairments by the Sigma and 5-Hydroxytryptamine1A Receptor Agonist OPC-14523 (1-{3-[4-(3-chlorophenyl)-1-piperazinyl]propyl}-5-methoxy-3,4-dihydro-2[1H]-quinolinone monomethanesulfonate) J. Pharmacol. Exp. Ther., April 1, 2002; 301(1): 249 - 257. [Abstract] [Full Text] [PDF]
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C. L. Douglas, H. A. Baghdoyan, and R. Lydic M2 Muscarinic Autoreceptors Modulate Acetylcholine Release in Prefrontal Cortex of C57BL/6J Mouse J. Pharmacol. Exp. Ther., December 1, 2001; 299(3): 960 - 966. [Abstract] [Full Text] [PDF]
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A. U. Höglund, C. Hamilton, and L. Lindblom Effects of Microdialyzed Oxotremorine, Carbachol, Epibatidine, and Scopolamine on Intraspinal Release of Acetylcholine in the Rat J. Pharmacol. Exp. Ther., October 1, 2000; 295(1): 100 - 104. [Abstract] [Full Text]
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