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TRANSLATIONAL PHYSIOLOGY
Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan
Submitted 6 August 2005 ; accepted in final form 17 January 2006
ABSTRACT
ACh regulates arousal, and the present study was designed to provide insight into the neurochemical mechanisms modulating ACh release in the pontine reticular formation. Nitric oxide (NO)-releasing beads microinjected into the pontine reticular formation of C57BL/6J (B6) mice significantly (P < 0.0001) increased ACh release. Microdialysis delivery of the NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12) to the mouse pontine reticular formation also caused a concentration-dependent increase in ACh release (P < 0.001). These are the first neurochemical data showing that ACh release in the pontine reticular formation of the B6 mouse is modulated by NO. The signal transduction cascade through which NO modulates ACh release in the pontine reticular formation has not previously been characterized. Therefore, an additional series of studies quantified the effects of a soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), on ACh release in the cat medial pontine reticular formation. During naturally occurring states of sleep and wakefulness, but not anesthesia, ODQ caused a significant (P < 0.001) decrease in ACh release. These results show for the first time that NO modulates ACh in the medial pontine reticular formation of the cat via an NO-sensitive sGC signal transduction cascade. Isoflurane and halothane anesthesia have been shown to decrease ACh release in the medial pontine reticular formation. The finding that ODQ did not alter ACh release during isoflurane or halothane anesthesia demonstrates that these anesthetics disrupt the NO-sensitive sGC-cGMP pathway. Considered together, results from the mouse and cat indicate that NO modulates ACh release in arousal-promoting regions of the pontine reticular formation via an NO-sensitive sGC-cGMP pathway.
arousal state control; sleep; mechanisms of anesthetic action
The mouse is a particularly valuable species for neurochemical studies of arousal state control, because anesthetic susceptibility (46) and sleep propensity (47) are heritable phenotypes. The Mouse Phenome Project specified the C57BL/6J (B6) mouse as a high-priority strain for phenotyping (40). ACh in the pontine reticular formation of the B6 mouse is a lower-level phenotype regulating the cortical EEG (10, 15) and REM sleep (7, 16, 33). The actions of NO on cholinergic neurotransmission within the pontine reticular formation of the B6 mouse have not been characterized. Therefore, the present study examined the hypothesis that NO in the pontine reticular formation of the B6 mouse modulates ACh release.
The short duration of the mouse sleep cycle and limitations of neurochemical detection preclude measurement of ACh from the pontine reticular formation of the B6 mouse during continuous stages of any sleep state. In contrast, the long duration of the cat sleep cycle makes it possible to deliver drugs via dialysis while ACh is measured during electrographically determined states of sleep and wakefulness (29, 52). Volatile anesthetics decrease ACh release in the medial pontine reticular formation of the intact cat (26), and in vitro studies of the rat demonstrate that volatile anesthetics disrupt NO-sensitive soluble guanylate cyclase (sGC) (36). No previous studies have determined whether the NO-sensitive sGC pathway is a mechanism modulating ACh release in regions of the medial pontine reticular formation that regulate arousal. Therefore, in a second series of experiments, sleeping and anesthetized cats were used to test the hypothesis that dialysis delivery of an sGC inhibitor to the medial pontine reticular formation modulates ACh release in the medial pontine reticular formation.
MATERIALS AND METHODS
Animals
All experiments were approved by the University of Michigan Committee on the Use and Care of Animals and were conducted in accordance with the US Public Health Service policy on humane care and use of laboratory animals (NIH Publ. No. 80-23). Adult male B6 mice (n = 18; Jackson Laboratory, Bar Harbor, ME) were used to measure ACh release in the pontine reticular formation. Additional microdialysis experiments were performed in adult male cats (n = 13) to measure ACh release in response to dialysis delivery of an sGC inhibitor during states of sleep and wakefulness and during anesthesia.
Microdialysis of B6 Mouse Pontine Reticular Formation
Drug preparation. Drugs were dissolved in Ringer solution containing 147 mM NaCl, 2.4 mM CaCl2, 4.0 mM KCl, and 10 µM neostigmine bromide (Sigma Aldrich, St. Louis, MO). Two sets of experiments were performed to evaluate the effects of NO-altering compounds on ACh release in the pontine reticular formation of the B6 mouse. The first set of studies used silicone dioxide beads carrying a diazeniumdiolate donor loaded with NO (38). The NO donor beads were mixed in Ringer solution (1.62 mg/ml) and sonicated for 4–5 min. Sonication disrupted aggregation of the beads, making it possible for the beads to be microinjected into the pontine reticular formation. The half-life for NO release after the donor beads are placed in Ringer solution is 144 min. Silicone dioxide beads not loaded with NO (control) were prepared and delivered to the pontine reticular formation in the same manner. A second series of experiments measured ACh release in the mouse pontine reticular formation before and after dialysis delivery of the NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12; Dojindo Chemical, Gaithersburg, MD). In a 37°C solution, NOC-12 releases NO with a half-life of 100 min (22). ACh release was measured during dialysis with 5, 50, 158, and 500 µM NOC-12.
In vivo microdialysis and HPLC. B6 mice were anesthetized with isoflurane (Abbott Laboratories, North Chicago, IL) in 100% O2. After the onset of anesthesia, the mouse was transferred to a stereotaxic device (model 962, Kopf, Tujunga, CA) and held in place by a mouse bite-bar adaptor (model 921, Kopf) and rat ear bars (models 921 and 957, respectively, Kopf). While the animals were in the stereotaxic frame, isoflurane was delivered through an anesthesia mask (model 907, Kopf). Delivered isoflurane concentration was measured continuously using a spectrometer (Cardiocap/5, Datex-Ohmeda, Louisville, CO). Before microdialysis sample collection, stable anesthetic depth was maintained by monitoring reaction to a hindpaw pinch, breathing rate, and rectal temperature.
The procedures for in vivo microdialysis and analysis of ACh in the mouse brain have been described in detail elsewhere (7, 9, 14, 15). A microdialysis probe was aimed stereotaxically for the pontine reticular formation of each mouse at stereotaxic coordinates of 4.7 mm posterior, 0.9 mm lateral, and 5.2 mm depth relative to bregma, according to the atlas of Paxinos and Franklin (41). The microdialysis probe was perfused at a rate of 2.0 µl/min with Ringer solution (control) or NOC-12-containing Ringer solution. One dialysis sample was collected every 12.5 min (25 µl/sample).
For dialysis delivery of NOC-12, experiments were conducted using CMA/11 probes (CMA/Microdialysis, North Chelmsford, MA; 1 mm membrane length, 0.24 mm membrane diameter, 6 kDa cutoff). For all experiments involving administration of diazeniumdiolate-loaded silicone dioxide beads, IBR-2 combination probes (BAS, West Lafayette, IN; 1 mm membrane length, 0.32 mm membrane diameter, 5 kDa cutoff) were used. IBR-2 probes contain inlet and outlet ports for dialysis and a 40-µm-diameter injection cannula that allowed for microinjection delivery of the silicone dioxide bead mixture (Fig. 1A).
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Dialysis samples were injected into an HPLC system (BAS) coupled to an electrochemical detector (EC; model CC-5, BAS). Dialysis samples were carried through the HPLC-EC by a 50 mM Na2HPO4 mobile phase (pH 8.5) at a flow rate of 1.0 ml/min and a pressure of 3,000–3,500 psi. An analytic column (model MF-6150; 2.1 mm diameter, 100 mm long) separated choline and ACh, and an immobilized enzyme reactor column (model MF-6151) converted ACh to H2O2. The EC detector measured H2O2 at a 500-mV applied potential on a platinum electrode referenced to a Ag-AgCl electrode. The amount of H2O2 was proportional to the amount of ACh in the dialysis sample. Chromatograms were digitized using ChromGraph software (BAS). A standard curve was created before each experiment, and the area under the chromatographic peak of every brain dialysis sample was referenced to the standard curve to express ACh as picomoles per 12.5 min.
Experimental design. For experiments using silicone dioxide beads, initial microdialysis samples were collected to ensure the stereotaxic placement of the IBR-2 combination probe in a region of the pons from which ACh could be measured. Once ACh release and anesthetic state were stable, three samples were collected for determination of basal levels of ACh release. After the third sample was collected, 200 nl of the NO-releasing beads were injected through the injection port of the IBR-2 combination probe. Injection duration was 2 min. After pontine reticular formation injection of NO-releasing or control beads, ACh was measured for 75 min. The dialysis probe was perfused continuously with Ringer solution. For experiments using NOC-12, ACh release first was measured for 75 min (6 samples) during pontine reticular formation dialysis with Ringer solution. The dialysis solution then was changed from Ringer solution to NOC-12-containing Ringer solution by means of a CMA/110 liquid switch. Six additional dialysis samples were collected and analyzed to determine the effect of microdialysis delivery of NOC-12 on ACh release.
Data analysis. Descriptive statistics and one-way ANOVA were used to evaluate the effect of NO-releasing beads and NOC-12 on ACh release in the B6 mouse pontine reticular formation. Post hoc comparisons were performed using Dunnett's and Tukey-Kramer's multiple-comparison statistics. P < 0.05 was considered statistically significant.
Histology. At 3–5 days after each microdialysis experiment, mice were deeply anesthetized and decapitated. Brains were removed and immediately frozen for serial sectioning with a cryostat. Coronal sections were cut at 40 µm and stained with cresyl violet. Dialysis probe placement was localized according to the mouse brain atlas (41). All results are from studies in which dialysis probe placement was confirmed to be within the pontine reticular formation, which includes the oral and caudal parts of the pontine reticular nucleus (41).
Microdialysis of Cat Medial Pontine Reticular Formation
Cats were anesthetized with isoflurane and implanted with electrodes for recording cortical EEG, electrooculogram, and neck muscle electromyogram and with thalamic electrodes for recording the geniculate body component of pontogeniculooccipital waves. During the same surgery, a craniotomy was performed, and a polyacrylic well was fashioned to allow subsequent access to the medial pontine reticular formation. The animals were allowed to recover from surgery for 
4 wk and were trained to sleep in the laboratory.
During experiments, a dialysis probe was positioned stereotaxically in the medial pontine reticular formation (1.5–3.0 mm posterior, 1.0–2.0 mm lateral, –5.0 mm horizontal, 
= 30° posterior) according to the cat brain stem atlas of Berman (2). The dialysis probes (CMA/Microdialysis) had a polycarbonate membrane (2 mm long, 0.5 mm diameter) with a cutoff of 20 kDa. During experiments, the dialysis probe was perfused continuously at 3 µl/min with Ringer solution (control) followed by Ringer solution containing 10 µM 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; Sigma Aldrich), a selective inhibitor of sGC (19). Dialysis samples of 30 µl were collected every 10 min and injected into the HPLC-EC system. As described above for the mouse experiments, the performance of the microdialysis probe was determined in vitro by dialysis in a known concentration of ACh before and after each experiment. Only experiments in which there was no significant change in percentage of ACh recovered by the dialysis probe are reported here.
Microdialysis during sleep and wakefulness. Cats were trained to sleep in a stereotaxic frame (model 880, Kopf). The brain has no pain receptors, and after pontine reticular formation placement of the microdialysis probe, the animals continued to exhibit normal sleep. An electrically shielded cable was used to connect the implanted EEG, electrooculogram, pontogeniculooccipital, and electromyogram electrodes to a polygraph (model 7, Grass Instruments, West Warwick, RI). The animals spontaneously progressed through cycles of sleep and wakefulness. Microdialysis samples from the medial pontine reticular formation were obtained during polygraphically determined states according to objective criteria (51). ACh release was quantified during electrographically confirmed states of wakefulness, non-REM (NREM) sleep, and REM sleep. A liquid switch was used to change the dialysis solution from Ringer solution to Ringer solution containing 10 µM ODQ. At the end of dialysis sample collection, the microdialysis probe was withdrawn, and the cat was returned to its home cage.
Statistical and histological analyses. Because the bilateral medial pontine reticular formation is relatively large, it was possible to perform several experiments in each cat without reusing the same dialysis sites. At least 1 wk was allowed between experiments in the same cat. This permitted an intensive, within-animal experimental design, appropriate for state-dependent quantification of neurotransmitter release (29, 48, 52). Dialysis results were analyzed using descriptive statistics, repeated-measures ANOVA, and t-test. Histological analysis confirmed the placement of microdialysis probes in the medial pontine reticular formation, which is located within the gigantocellular tegmental field, as defined by Berman (2).
RESULTS
Microdialysis Sites Were Localized to the Pontine Reticular Formation
Figure 1 illustrates pontine placement of microdialysis probes. The IBR-2 probe shown in Fig. 1A allowed measurement of ACh release before and after microinjection of NO-releasing beads. The representative histological section shown in Fig. 1B confirmed probe placement in the mouse pontine reticular formation. The mouse pontine reticular formation (Fig. 1B) and the cat medial pontine reticular formation (Fig. 1D) are homologous regions of the pontine reticular formation.
NO Increased ACh Release in the Pontine Reticular Formation of the B6 Mouse
Figure 2 illustrates the effect of NO-releasing beads on ACh release. Figure 2A shows results from a representative experiment. Microinjection of control beads into the pontine reticular formation did not alter ACh release. Figure 2B shows that when NO-releasing beads were microinjected into the pontine reticular formation, ACh release was increased. Time-course data from all mice consistently revealed a progressive increase in ACh release caused by microinjection of NO-releasing beads. Figure 2C summarizes data from eight mice showing that microinjection of NO-releasing beads significantly increased ACh release (F = 34.0, df = 2,68, P < 0.0001) compared with levels before bead injection and compared with ACh levels after microinjection of silicone beads that did not release NO (control beads).
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Because of the long duration of the feline sleep cycle, the cat is a better choice than the mouse for experiments designed to quantify ACh release during microdialysis intervals comprised entirely of wakefulness, NREM sleep, or REM sleep (29, 52). Therefore, in a second series of experiments, the cat was used to test the hypothesis that ACh release is decreased by dialysis delivery of the sGC inhibitor ODQ (19).
Because the brain contains no pain receptors, it was possible to measure ACh release in the medial pontine reticular formation during electrographically identified states of sleep and wakefulness. Figure 4 illustrates a typical experiment in which a cat slept and awoke spontaneously during measurement of ACh at 10-min intervals. During the initial 70 min of the experiment in which Ringer solution was delivered by dialysis (control condition), the cat spent 30 min awake, 30 min in NREM sleep, and 10 min in REM sleep. At the end of this initial 70-min period of dialysis, a liquid switch was activated to begin dialysis delivery of Ringer solution containing 10 µM ODQ. The cat continued to awaken and sleep spontaneously, and Fig. 4 illustrates that ODQ decreased ACh release during states of wakefulness, NREM sleep, and REM sleep.
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= 0.05, 
= 0.2) were used to estimate the number of experiments required to demonstrate statistically significant differences in ACh release. Those calculations indicated that significant differences in ACh release during microdialysis with Ringer solution vs. ODQ would require 178 experiments using isoflurane anesthesia and 2,265 experiments using halothane anesthesia.
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Four new findings emerged from this study. 1) In the B6 mouse, NO modulates ACh release in pontine reticular formation regions known to regulate arousal. 2) Experiments using intact, unanesthetized cats demonstrated for the first time that ACh release in arousal-promoting regions of the medial pontine reticular formation is modulated by an NO-sensitive sGC transduction cascade. 3) The volatile anesthetics isoflurane and halothane blocked the ability of an sGC inhibitor to significantly decrease ACh release in the medial pontine reticular formation. 4) The present comparison across species revealed for the first time that similar mechanisms regulate ACh release in pontine reticular formation regions that are homologous in the mouse and cat. These neurochemical data are discussed relative to their functional implications for arousal state control.
NO Modulates ACh Release in the Pontine Reticular Formation of the Mouse
Microinjection of NO-releasing beads into the pontine reticular formation of the B6 mouse increased ACh release. This finding is consistent with the fact that inhibitors of NOS decrease ACh release in the pontine reticular formation of the cat (29) and with the finding that REM sleep in the mouse is decreased by targeted disruption of the gene coding for neuronal NOS (5). Time-course data revealed no change in ACh release after injection of control beads that did not release NO (Fig. 2A) and a significant increase in ACh release after microinjection of the NO-releasing beads (Fig. 2B). The group data (Fig. 2C) illustrate a 98% increase in ACh release in the mouse pontine reticular formation by the NO-releasing beads compared with the control beads. ACh levels averaging 
0.2 pmol/12.5 min of dialysis (Fig. 2) agree with previously reported levels of ACh in the pontine reticular formation of the B6 mouse (7).
Microdialysis delivery of the NO donor molecule NOC-12 to the pontine reticular formation of B6 mice increased ACh release in a concentration-dependent manner (Fig. 3). The results are consistent with in vitro evidence that an NO-cGMP pathway facilitates ACh release (21). Cholinergic neurotransmission in the pontine reticular formation contributes to REM sleep generation in the B6 mouse (6–8, 16, 33). Results from the NO-releasing bead studies (Fig. 2) and the NOC-12 concentration response data (Fig. 3) provide the first evidence that an NO-sensitive pathway modulates ACh release in the pontine reticular formation of the B6 mouse. One implication of these findings is that, in the pontine reticular formation of the B6 mouse, NO contributes to REM sleep generation.
NO-Sensitive sGC Modulates ACh Release in the Medial Pontine Reticular Formation of the Cat
As reviewed elsewhere (32), cat medial pontine reticular formation neurons express muscarinic receptors but do not produce ACh. Neurons in the laterodorsal and pedunculopontine tegmental nuclei (LDT/PPT) synthesize ACh, and these cholinergic LDT/PPT neurons project to the medial pontine reticular formation, where they release ACh. LDT/PPT neurons also express neuronal NOS (53) and are activated by NO (28). Cholinergic LDT/PPT neurons increase their firing rate just before and throughout REM sleep, consistent with the discharge of these cells serving a causal, rather than a merely correlative, role in REM sleep generation.
The present study was designed to focus on arousal state-specific measures of ACh release in the pontine reticular formation. ODQ did not appear to alter the electrographic features of sleep or sleep architecture. An earlier report that NOS inhibitors cause a stereospecific inhibition of ACh release (29) demonstrated that NO modulates cholinergic neurotransmission in the medial pontine reticular formation and LDT/PPT. The present finding that ODQ decreased ACh release during wakefulness, NREM sleep, and REM sleep (Figs. 4 and 5) identifies for the first time an NO-sensitive sGC pathway as one mechanism regulating ACh release in the cat medial pontine reticular formation.
Volatile Anesthetics Blocked the Decrease in ACh Release Caused by the sGC Inhibitor ODQ
Results from the cat (Figs. 4–6) are relevant to efforts to understand the brain regions and mechanisms through which volatile anesthetics alter arousal. The data show for the first time that the ODQ-induced decrease in ACh release (Figs. 4 and 5) was prevented by volatile anesthesia (Fig. 6). This finding is consistent with evidence that isoflurane and halothane compete with NO for a shared binding site on sGC (24, 36). The finding that the ODQ-induced decrease in ACh release was blocked by isoflurane anesthesia also supports the conclusion that ACh release in the medial pontine reticular formation is modulated by an NO-sensitive sGC-cGMP pathway (Fig. 7). Therefore, Figs. 4–6 provide novel evidence supporting the hypothesis that neurochemical mechanisms that evolved to regulate states of sleep and wakefulness are preferentially involved in generating states of anesthesia (for review see Ref. 32).
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These findings are consistent with four additional lines of evidence showing that ACh release is modulated by the signal transduction cascade illustrated in Fig. 7. 1) NO enhances cGMP synthesis in cholinergic neurons (12). 2) The present results agree with the finding that ODQ decreases ACh release in other brain regions, such as the nucleus accumbens (44) and striatum (50). 3) Cyclic nucleotides participate in the cholinergic (3, 35) and nitrergic (11) modulation of arousal. 4) An interaction between ACh and NO in regulating sleep and wakefulness is suggested by the finding that NOS inhibitors in the basal forebrain block the cortical EEG arousal normally caused by electrical stimulation of the basal forebrain (34).
Previous studies quantifying the effect of the intravenous anesthetic ketamine on ACh release in the medial pontine reticular formation (31) also support the view that suppression of arousal can be caused by disruption of the NO-sGC-cGMP cascade. Ketamine acts as an N-methyl-D-aspartate (NMDA) channel blocker, and activation of NMDA receptors promotes arousal, increases NO synthesis, activates sGC, and increases cGMP (for review see Ref. 43). Consistent with data in Fig. 6, it was previously found that systemically administered ketamine and dialysis delivery of NMDA channel blockers to the medial pontine reticular formation decrease ACh release and eliminate EEG and behavioral arousal (31).
Limitations and Conclusions
The strengths and limitations of microdialysis have been extensively reviewed (1, 45, 54). The size of presently available microdialysis probes relative to the size of the mouse brain is a technical limitation that restricts brain sampling to sites that can accommodate a dialysis probe without overlap into other nuclei. As demonstrated elsewhere (6, 7), this size factor is not a limitation for microdialysis of the pontine reticular formation in the B6 mouse.
The present results should not be interpreted to imply an exclusive relation between NO, ACh, and arousal state control. NO contributes to the regulation of arousal by modulating multiple neurotransmitters. Monoaminergic neurotransmission promotes wakefulness, and serotonergic dorsal raphe neurons discharge maximally during wakefulness and stop firing during REM sleep, consistent with a permissive role in REM sleep generation (for review see Ref. 20). Direct measurements from dorsal raphe reveal that levels of NO are state dependent and maximal during REM sleep (4). Injection of NO donors into the dorsal raphe nucleus enhances REM sleep, and dorsal raphe injections of NOS inhibitors decrease sleep (for review see Ref. 20). Similarly, the finding that volatile anesthetics blocked the ODQ-induced decrease in ACh release may involve mechanisms in addition to volatile anesthetics competing with NO for an sGC binding site. Volatile anesthetics, for example, also decrease NMDA receptor activity (49).
Studies using more than one species can provide unique insights into structure-function relations ranging from organ systems (42) to the genetics underlying complex traits (13). The power of comparative studies is illustrated by evidence that even small species-specific differences can be functionally significant (39). For example, although subtype 6 serotonin (5-HT6) receptors in the mouse, rat, and human share an 84% homology, the 5-HT6 receptors in the mouse have binding properties that differ significantly from 5-HT6 receptors in the rat and human (30). Considered together, the present results from the B6 mouse and the cat support the conclusion that an NO-sensitive sGC-cGMP pathway modulates ACh release in arousal-promoting regions of the pontine reticular formation.
GRANTS
This study was supported by National Institutes of Health Grants HL-40881, MH-45361, HL-65272, and HL-57120 and by the Department of Anesthesiology.
ACKNOWLEDGMENTS
Dr. M. E. Meyerhoff kindly provided the NO donor beads. We thank N. Goldberg, K. Heindl, S. Jiang, F. Liu, and M. A. Norat (Dept. of Anesthesiology) and K. Welch (University of Michigan Center for Statistical Consultation and Research) for expert assistance.
FOOTNOTES
Address for reprint requests and other correspondence: R. Lydic, Dept. of Anesthesiology, Univ. of Michigan, 7433 Medical Sciences Bldg. I, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0615 (e-mail: rlydic{at}umich.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
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