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J Appl Physiol 98: 918-929, 2005. First published October 8, 2004; doi:10.1152/japplphysiol.00900.2004
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C57BL/6J and B6.V-LEPOB mice differ in the cholinergic modulation of sleep and breathing

Christopher L. Douglas, Grant N. Bowman, Helen A. Baghdoyan, and Ralph Lydic

Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan

Submitted 19 August 2004 ; accepted in final form 4 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Respiratory and arousal state control are heritable traits in mice. B6.V-Lepob (ob) mice are leptin deficient and differ from C57BL/6J (B6) mice by a variation in the gene coding for leptin. The ob mouse has morbid obesity and disordered breathing that is homologous to breathing of obese humans. This study tested the hypothesis that microinjecting neostigmine into the pontine reticular nucleus, oral part (PnO), of B6 and ob mice alters sleep and breathing. In B6 and ob mice, neostigmine caused a concentration-dependent increase (P < 0.0001) in percentage of time spent in a rapid eye movement (REM) sleeplike state (REM-Neo). Relative to saline (control), higher concentrations of neostigmine increased REM-Neo duration and the number of REM-Neo episodes in B6 and ob mice and decreased percent wake, percent non-REM, and latency to onset of REM-Neo (P < 0.001). In B6 and ob mice, REM sleep enhancement by neostigmine was blocked by atropine. Differences in control amounts of sleep and wakefulness between B6 and the congenic ob mice also were identified. After PnO injection of saline, ob mice spent significantly (P < 0.05) more time awake and less time in non-REM sleep. B6 mice displayed more (P < 0.01) baseline locomotor activity than ob mice, and PnO neostigmine decreased locomotion (P < 0.0001) in B6 and ob mice. Whole body plethysmography showed that PnO neostigmine depressed breathing (P < 0.001) in B6 and ob mice and caused greater respiratory depression in B6 than ob mice (P < 0.05). Western blot analysis identified greater (P < 0.05) expression of M2 muscarinic receptor protein in ob than B6 mice for cortex, midbrain, cerebellum, and pons, but not medulla. Considered together, these data provide the first evidence that pontine cholinergic control of sleep and breathing varies between mice known to differ by a spontaneous mutation in the gene coding for leptin.

acetylcholine; pontine reticular formation; respiratory control; obesity

RESPIRATORY (33, 73, 83, 84) and arousal state (30, 31, 57, 88, 90) control are heritable traits in mice that may be regulated by a limited number of genes. The C57BL/6J (B6) mouse strain has been validated as a model of sleep-disordered breathing (80). The B6.V-Lepob (ob) mouse originated from the spontaneous mutation of the leptin gene in the B6 line (45). The ob mouse is leptin deficient and morbidly obese. Changes in breathing associated with the ob phenotype are homologous to the aberrant breathing of obese humans (63, 64, 67, 85, 86). Human morbid obesity can include resistance to the circulating satiety hormone leptin (reviewed in Ref. 32), and the severity of obstructive sleep apnea (OSA) has been associated with increased body mass index in adults (98) and children (95).

Breathing is significantly modulated by pontine acetylcholine (ACh) (reviewed in Refs. 47, 50, 54). Pontine ACh also is critical for the generation of the rapid eye movement (REM) phase of sleep (reviewed in Refs. 3, 54). During REM sleep, when muscle tone is actively inhibited (reviewed in Ref. 49), breathing is severely disrupted (68, 87). Human OSA also has been correlated with a loss of pontine cholinergic projections to thalamus and striatum (34). The cholinesterase inhibitor physostigmine decreases REM sleep latency (reviewed in Ref. 54) and, in some OSA patients, reduces the frequency of apneic episodes during REM sleep (39).

Microinjecting the acetylcholinesterase inhibitor neostigmine into the pontine reticular formation of B6 mice causes a REM sleeplike state (REM-Neo) characterized by an activated electroencephalogram (EEG), motor hypotonia, and respiratory depression (18, 56). In B6 mice, the neostigmine-induced REM sleeplike state is blocked by pretreatment of the pontine reticular formation with the muscarinic antagonist methoctramine, consistent with mediation by muscarinic cholinergic receptors (18). Microdialysis data indicate that ACh release in the pontine reticular nucleus, oral part (PnO), of B6 mice also is modulated by muscarinic receptors (17). Parallel data are not available for ob mice, but leptin has been shown to inhibit choline acetyltransferase (ChAT), the synthetic enzyme that produces ACh (23). This finding suggests that cholinergic modulation of sleep and breathing may be altered in leptin-deficient ob mice. Therefore, the present experiments were designed to test the hypothesis that the cholinergic regulation of sleep and breathing differs between B6 and ob mice. Portions of these data have been presented as abstracts (11, 27, 69).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In Vivo Studies

Surgical preparation.   Adult male B6 (n = 12) and ob (n = 5) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences Press, 1996). Mice were anesthetized with 2–3% isoflurane (Abbott Laboratories, North Chicago, IL) in 100% O2 and placed in a David Kopf (Tujunga, CA) model 962 stereotaxic frame with a model 921 mouse adapter and model 907 mouse anesthesia mask. Delivered isoflurane concentration was reduced to 1.5%, and core body temperature was maintained at 36–37°C by a TP400 heat pump therapy system (Gaymar, Orchard Park, NY). Mice were implanted with 0.13-mm diameter stainless steel wire electrodes (California Fine Wire, Grover City, CA) for recording the cortical EEG and neck electromyogram (EMG). EEG electrodes were placed above the motor cortex, somatosensory cortex, and hippocampus. A stainless steel 26-gauge guide cannula occluded with a stylet (Plastics One, Roanoke, VA) was aimed stereotaxically for the PnO and implanted to permit subsequent drug administration by microinjection. Stereotaxic coordinates for the PnO microinjection site were 4.84 mm caudal to bregma, 0.65 mm lateral to the midline, and 5.0 mm ventral to the surface of the skull at bregma (66). All electrode leads were gathered into a plastic connector (Plastics One) and encased in dental cement with the guide tube and two stainless steel anchor screws. Animals were housed individually in constant illumination with free access to food, water, and nesting material. Mice were allowed to recover for at least 1 wk before the microinjection experiments were initiated. Mice were handled daily during the recovery period.

Microinjection procedure.   Microinjections (50 nl) were performed between 11:00 AM and 1:00 PM. Drug solutions were prepared by dissolving neostigmine bromide or atropine sulfate (Sigma-Aldrich, St. Louis, MO) in sterile isotonic saline immediately before use. A manual microdrive connected to a 31-gauge microinjector was used to deliver saline, neostigmine (1.33, 13.3, 133, and 1,330 ng, equivalent to 0.088, 0.88, 8.8, and 88 mM, respectively), and atropine (10.0 ng, equivalent to 0.3 mM) plus neostigmine (13.3 ng for B6 mice; 133 ng for ob mice). Microinjection duration was 1 min. The microinjector remained in the brain for an additional 1 min postinjection before being replaced by a stainless steel stylet.

Electrographic criteria of sleep and wakefulness.   Mice were adapted to a Raturn system (Bioanalytical Systems, West Lafayette, IN), with free access to food, water, and nesting material. Movement, EEG, and EMG were recorded via a counterweighted cable connected to the mouse headcap. A photoelectric device monitored turning of the cable generated by mouse movement. The monitoring device generated countermovement of the Plexiglas enclosure to ensure that the cable did not become twisted. Mice exhibited normal locomotor behavior. EEG and EMG signals were amplified using a Grass Instruments (West Warwick, RI) model 15RXi amplifier. Analog signals were digitized at 128 Hz and recorded using Grass Polyview software. EEG, EMG, and behavior were used to objectively determine states of sleep and wakefulness in 20-s epochs for 4 h postinjection. Fast-Fourier transformation analyses identified the dominant frequencies in 2-s bins from 0.5 to 25 Hz.

Figure 1 shows representative digital EEG and EMG recordings from the same B6 mouse made during wakefulness, non-REM (NREM) sleep, REM sleep, and REM-Neo. Wakefulness was indicated by an activated EEG with low-amplitude, mixed-frequency waves and the presence of movement artifacts in the EMG. While awake during the testing periods, mice engaged in normal behaviors (eating, drinking, exploring, nesting). The EEG of NREM sleep was characterized by low-frequency, high-voltage delta waves (1–4 Hz), and sleep spindles (7–14 Hz). EMG showed no movement artifacts during NREM sleep, and mice assumed normal sleep postures. REM sleep and REM-Neo were scored based on muscle hypotonia, as indicated by reduced EMG amplitude relative to wakefulness and NREM sleep and an activated EEG characterized by theta waves. Theta rhythms were between 4 and 8 Hz for spontaneous REM sleep and between 3 and 7 Hz for REM-Neo. The use of these physiological traits as criteria for behavioral-state classifications is consistent with mouse sleep scoring procedures in many laboratories (15, 31, 43, 56, 63, 72, 78, 81, 90, 93, 94). Dependent measures of arousal state included percentage of the 4-h recording period spent in wakefulness, NREM sleep, REM sleep, and REM-Neo; latency to onset (minutes) of the first postinjection REM sleep or REM-Neo episode (REM latency); the number of REM sleep or REM-Neo episodes per 4-h recording period; and the duration (minutes) of REM sleep or REM-Neo episodes.



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  		Fig. 1. Ten-second EEG and electromyogram (EMG) recordings from 1 C57BL/6J (B6) mouse show electrographic features representative of B6.V-Lepob (ob) and B6 mice during states of wakefulness, rapid eye movement (REM) sleep, non-REM (NREM) sleep, and the neostigmine-induced REM sleep-like state (REM-Neo). Bottom row of EEG and EMG recordings were obtained during REM-Neo caused by delivering 133 ng of neostigmine into the pontine reticular nucleus, oral part (PnO). There was good concordance between the electrographic traits of EEG and EMG and the behavioral manifestations of wakefulness and sleep.

 
Whole body plethysmography.   The effects of PnO microinjections on breathing were quantified using a Buxco (Sharon, CT) plethysmograph for unrestrained mouse (PLY3211) and BioSystem XA software. Animals were placed in calibrated whole body plethysmograph chambers after PnO microinjections. Breathing then was measured continuously for 20 min. Plethysmographic data were used to quantify respiratory rate (breaths/min), tidal volume (ml/breath), minute ventilation (ml/min), inspiratory time (in seconds), expiratory time (in seconds), total respiratory cycle time (inspiratory time + expiratory time; in seconds), inspiratory flow (in ml/s), and an index of inspiratory effort (in seconds), also referred to as duty cycle (73).

Activity recordings.   Locomotor activity in freely moving mice was quantified using real-time video recordings. Immediately after PnO microinjection of saline or neostigmine (133 ng), mice were placed in an open field and allowed to freely explore. The field was digitally rendered as video pixels, and each pixel crossing was summed. Locomotor activity was quantified for 1 h postinjection.

Histological localization of microinjection sites.   On completion of all experiments, mice were deeply anesthetized and decapitated. Brains were removed, frozen, and sectioned coronally at 40 µm. Brain stem sections were mounted serially on glass slides, fixed in hot (80°C) paraformaldehyde vapor, and stained with cresyl violet. Microinjection sites were histologically localized using a mouse stereotaxic atlas (66). Each microinjection site was assigned millimeter coordinates in the rostrocaudal, dorsoventral, and mediolateral planes.

Statistical analyses.   Descriptive statistics summarized time spent in states of sleep and wakefulness as means + SD. Differences in dependent measures of sleep and wakefulness between five microinjection treatment conditions (1 saline and 4 neostigmine concentrations) and between mouse lines (ob and B6) were analyzed by two-way ANOVA with Satterthwaite treatment for unequal variances and post hoc Tukey's statistic with Bonferroni correction. One assumption of ANOVA is equal variance. The Satterthwaite treatment is a highly conservative approach to ANOVA that penalizes the number of degrees of freedom (df) when variance in the data is unequal (71). The atropine-blocking studies were analyzed by one-way ANOVA with Dunnett's post hoc test. Locomotor activity data are presented as means + SD pixel crossings x 10,000. Comparisons between microinjection treatment condition (saline and neostigmine) and between congenic mouse lines (B6 and ob) were made using one-tailed t-test and completely randomized two-way ANOVA followed by the nonparametric Wilcoxon two-sample signed-rank test. Differences in the dependent measures of breathing between saline and neostigmine treatment conditions and between B6 and ob mice were evaluated by t-test. Differences in anatomic location of microinjection sites between B6 and ob mice were evaluated by t-test. A P value of <0.05 indicated a statistically significant difference.

In Vitro Studies

Brain tissue preparation.   The in vitro studies quantified the brain region-specific expression of proteins coding for the M2 subtype of muscarinic cholinergic receptor. B6 and ob mice (15 per group, mean age 4 mo) were decapitated, and brains were rapidly removed. Brains were dissected into five regions: cerebral cortex, cerebellum, midbrain, pons, and medulla. Each brain region was homogenized in ice-cold homogenization buffer (50 mM Tris·HCl, pH 8.0, 10 mM EGTA, and protease inhibitors, 10 mg/ml PMSF, 25 µg/ml aprotinin, and 100 mM sodium orthovanadate). The homogenates were centrifuged for 10 min at 3,500 rpm and 4°C to remove cell debris. The resulting supernatants were ultracentrifuged at 38,000 rpm and 4°C for 1 h, and the pellet (membrane fraction) was resuspended in the smallest possible volume of homogenization buffer. Protein content was measured in an aliquot of each sample by the Bradford method (Bio-Rad protein assay kit, Bio-Rad, Hercules, CA), using bovine serum albumin as the standard. Suspended membrane fractions were frozen at –80°C until analysis by Western blot.

Western blotting.   Tissue samples (40 µg) from each of the five brain regions were thawed, fractionated by SDS-polyacrylamide gel electrophoresis (12% Tris-glycine polyacrylamide gels, Jule Biotechnologies, Milford, CT), and transferred to a polyvinylidene diflouride membrane (Hybond-P, pore size 0.45 µm, Amersham Pharmacia Biotech, Piscataway, NJ). After transfer, the membrane was incubated in a blocking buffer (10 mM Tris·HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20, 5% nonfat dry milk) at room temperature for 1 h, followed by overnight incubation at 4°C with the primary antibody solution (polyclonal rabbit anti-M2, 1:800 in 3% blocking buffer). The next day, the membrane was washed three times in 0.1% Tris-buffered saline-Tween 20 (15 min/wash) and incubated for 45 min with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG, 1:20,000 in 5% blocking buffer). The membrane was washed again, and the bound antibodies were detected using the enhanced chemiluminescent method (ECL detection kit, Amersham Pharmacia Biotech).

For quantification purposes, the membrane was stripped and reprobed for actin and detected using a polyclonal rabbit anti-actin antibody (Sigma-Aldrich) followed by a secondary peroxidase-conjugated goat anti-rabbit antibody and enhanced chemiluminescence detection. The bands were analyzed by scanning densitometry (at 600 dots/in.) using a Microtek ScanMaker 5 scanner and Kodak one-dimensional image analysis software. Optical density measures of M2 protein were normalized using actin immunoreactivity for each lane. The primary anti-M2 antibody was purchased from Calbiochem (San Diego, CA), and the secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA). The M2-GST fusion protein (the immunogen for the M2 antibody) was obtained from Calbiochem and was used as a positive control. Negative controls were performed either by omission of the primary antibody incubation or by preincubating the primary antibody with the immunizing M2-GST fusion protein. The presence of M2 muscarinic-receptor protein was verified by detection as a prominent band with a molecular weight in the range of previous reports (50 kDa) (16, 74) and by absence of the band in the negative controls.

Statistical analyses.   Differences between mouse lines and between brain regions in M2 muscarinic receptor/actin optical density ratios were evaluated using two-way ANOVA for repeated measures and the Newman-Keuls post hoc test. A P value of <0.05 was required to designate statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Microinjection Sites Were Localized to the PnO

Figure 2 demonstrates that all microinjection sites were localized within the PnO. There were no significance differences between microinjection sites in B6 and ob mice. Mean ± SD stereotaxic coordinates for the microinjection sites were 4.67 ± 0.16 posterior to bregma, 0.81 ± 0.16 lateral to the midline, and –4.73 ± 0.35 ventral to bregma for B6 mice, and 4.81 ± 0.15 posterior to bregma, 0.91 ± 0.42 lateral to the midline, and –4.56 ± 0.41 ventral to bregma for ob mice.



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  		Fig. 2. Histology illustrates microinjection sites in mouse pontine reticular formation. A: representative, cresyl violet-stained, coronal section at the level of the PnO. The arrow marks 1 microinjection site. B: microinjection sites from 12 B6 ({bullet}) and 5 ob (*) mice are indicated on coronal brain stem schematics from the mouse brain atlas (66). Numbers at top right of both A and B indicate mm posterior to bregma.

 
Pontine Neostigmine Caused Concentration-dependent REM Sleep Enhancement in B6 and ob Mice

Figure 3 shows one representative time course plot of arousal states for each microinjection condition. PnO administration of neostigmine caused a concentration-dependent increase in REM-Neo for B6 and ob mice. Figure 3 illustrates typical increases in the duration and number and decreases in latency to onset of REM-Neo. Figure 3 shows that PnO neostigmine caused the REM sleeplike state to be evoked directly from wakefulness. The normal sequential occurrence of wakefulness, followed by NREM sleep and then REM sleep, was restored by coadministration of atropine.



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  		Fig. 3. Time course of sleep and wakefulness recorded from 1 B6 (left) and 1 ob (right) mouse for 4 h after PnO microinjection. These time course plots demonstrate the REM sleep enhancement caused by increasing concentrations of pontine neostigmine. The bottom 2 plots illustrate atropine block of REM-Neo. Note the similarity in the distribution of sleep and wakefulness after microinjection of atropine (bottom 2 plots) and microinjection of saline (top 2 plots).

 
Figure 4 summarizes group data showing the effects of neostigmine on six dependent measures of sleep and wakefulness. The effects of PnO neostigmine on percent time spent in wakefulness were concentration dependent and varied between B6 and ob mice (Fig. 4A). Two-way ANOVA revealed a main effect of mouse line (F = 22.58; df = 1, 6; P = 0.003), a neostigmine concentration-main effect (F = 25.51; df = 4, 24; P < 0.0001), and an interaction between mouse line and neostigmine concentration (F = 4.16; df = 4, 24; P = 0.0107) on percent wakefulness. Post hoc tests showed that ob mice spent a greater percentage of time awake than B6 mice after PnO microinjections of saline (control, 0 ng neostigmine), 1.33 ng of neostigmine, and 13.3 ng of neostigmine (P < 0.05). Asterisks in Fig. 4 indicate that 133 ng of neostigmine decreased time spent in wakefulness below control levels (P < 0.05) in ob mice and that 1,330 ng of neostigmine decreased wakefulness in B6 mice.



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  		Fig. 4. Microinjection of neostigmine into the PnO of B6 and ob mice caused concentration-dependent changes in sleep and wakefulness. A: %wake; B: %NREM; C: %REM; D: REM latency; E: no. of REM episodes; F: REM duration. *Significant (P < 0.05) changes from the saline (control) microinjection condition. {dagger}Significant differences between B6 and ob mice (P < 0.05).

 
The percent time spent in NREM sleep after PnO microinjections also varied between B6 and ob mice and was concentration dependent (Fig. 4B). ANOVA showed a significant difference between B6 and ob mice (F = 37.56; df = 1, 6; P = 0.0009) and a neostigmine concentration main effect (F = 33.95; df = 4,24; P < 0.0001) on percent NREM sleep. There was no interaction between B6 and ob congenic lines and neostigmine concentration on percent NREM sleep time. B6 mice spent a greater percentage of time in NREM sleep than ob mice after PnO microinjections of saline (0 ng of neostigmine), 1.33 ng of neostigmine, and 133 ng of neostigmine (P < 0.05). As described for percent wakefulness, the amount of neostigmine that decreased percent NREM sleep was lower (P < 0.05) for ob (133 ng) than for B6 (1,330 ng) mice.

For percent REM sleep after PnO microinjections (Fig. 4C), two-way ANOVA revealed a neostigmine concentration main effect (F = 99.38; df = 4, 8.65; P < 0.0001) and a mouse line by neostigmine concentration interaction (F = 11.6; df = 4, 8.65; P = 0.0016). There was no main effect of mouse line on percent REM sleep. Post hoc Tukey's statistic showed that 13.3 ng of neostigmine increased percent REM sleep in B6 but not ob mice (P < 0.05). Figure 4C shows that percent REM sleep of ob mice was significantly (P < 0.05) increased over control levels by administering 133 ng of neostigmine. Microinjecting 133 ng of neostigmine also caused a greater increase in percent REM sleep in ob than in B6 mice (P < 0.05).

Microinjection of neostigmine into the PnO decreased the latency to REM sleep onset (Fig. 4D). This effect varied significantly as a function of mouse line (F = 16.04; df = 1, 8.35; P = 0.0036) and neostigmine concentration (F = 14.16; df = 4, 3.47; P < 0.0001). There was no interaction between concentration and mouse line. Graphic summary of REM sleep latency for the ob line (Fig. 4D) suggests a reduction in REM latency with increasing amounts of neostigmine. High variability in the data prevented post hoc analyses from supporting the conclusion that PnO neostigmine significantly decreased REM latency in ob mice at any concentration. Post hoc analysis did not identify significant REM latency differences between the congenic mouse lines. In B6 mice, neostigmine (Fig. 4D; 13.3, 133, and 1,330 ng) significantly reduced REM latency below control (0 ng) levels (P < 0.05).

Neostigmine-induced REM sleep enhancement also was manifested by a significant increase in the number of REM sleep episodes (Fig. 4E). ANOVA revealed a main effect of mouse line (F = 39.25; df = 1, 6; P = 0.0008), a neostigmine concentration main effect (F = 21.01; df = 4, 24; P < 0.0001), and no mouse line by concentration interaction. Post hoc Tukey's statistic revealed more REM sleep episodes in B6 than in ob mice after 1.33 and 13.3 ng of neostigmine (P < 0.05). Neostigmine increased the number of REM sleep episodes above control levels in B6 (13.3 ng) and in ob (133 ng) mice (Fig. 4E; P < 0.05).

The effects of PnO neostigmine on REM sleep duration were concentration dependent (Fig. 4F). Two-way ANOVA revealed a neostigmine concentration main effect (F = 15.26; df = 4, 7.86; P = 0.0009), no mouse line main effect, and no interaction between mouse line and neostigmine concentration. Similar to findings for the other arousal state parameters (Fig. 4, A–C, E), there was a differential effect of PnO neostigmine on REM sleep duration in B6 and ob mice. Post hoc tests revealed that 133 ng of neostigmine induced longer bouts of REM sleep in ob mice than in B6 mice (Fig. 4F; P < 0.05). The amount of neostigmine that caused a significant increase in REM sleep duration was 133 ng for ob mice and 1,330 ng for B6 mice (P < 0.05).

Atropine Blocked Neostigmine-Induced REM Sleep Enhancement

The significant increases in the REM sleeplike state caused by pontine neostigmine were blocked by coadministration of the muscarinic receptor antagonist atropine (Fig. 5). One-way ANOVA revealed a drug main effect on percent time spent in REM-Neo (Fig. 5A) for B6 mice (F = 35.89; df = 2, 8; P = 0.0005) and ob mice (F = 128.0; df = 2, 8; P < 0.0001). The neostigmine-induced increase in percent REM sleep (Fig. 5A; P < 0.01) was blocked by coadministration of atropine in B6 and ob mice. There was also a drug main effect on REM latency (Fig. 5B) for B6 mice (F = 24.8; df = 2, 8; P = 0.0013) and ob mice (F = 6.57; df = 2, 8; P = 0.0308). Atropine blocked the neostigmine-induced decrease in REM latency for B6 and ob mice (Fig. 5B; B6, P < 0.01; ob, P < 0.05), and in B6 mice atropine significantly increased REM latency over control levels (Fig. 5B; P < 0.05). There was a drug main effect on the number of REM sleep episodes (Fig. 5C) for B6 mice (F = 8.47; df = 2, 8; P = 0.0179) and ob mice (F = 35.07; df = 2, 8; P = 0.0005). The significant increase in the number of REM sleeplike episodes caused by neostigmine in B6 (P < 0.05) and ob (P < 0.01) mice was blocked by atropine. For REM duration (Fig. 5D), ANOVA revealed a drug main effect in ob mice (F = 26.75; df = 2, 8; P = 0.001) but not in B6 mice. The significant (P < 0.01) increase in duration of REM-Neo episodes for ob mice after PnO neostigmine was blocked by atropine.



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  		Fig. 5. Coadministration of atropine to the PnO blocked the neostigmine-induced REM sleep enhancement. Pontine neostigmine significantly increased the percentage of time spent in REM-Neo (A), decreased latency to onset of REM-Neo (B), and increase in the number of REM-Neo episodes (C) for B6 and ob mice (*P < 0.05). Neostigmine also increased the duration of REM-Neo episodes (D) in ob mice (*P < 0.05). For each of these dependent measures, atropine blocked the effects of neostigmine.

 
Pontine Neostigmine Decreased Locomotor Activity

Figure 6 summarizes the effects of saline and neostigmine on locomotor activity recorded from freely moving mice over a 1-h period after pontine microinjection. Two-way ANOVA indicated a significant effect of mouse line (F = 66.83; df = 1, 9; P < 0.0001), drug (F = 119.98; df = 1, 9; P < 0.0001), and a mouse line-by-drug interaction (F = 72.37; df = 1, 17; P < 0.0001). Compared with saline, neostigmine significantly decreased locomotor activity in B6 and ob mice (P < 0.05). Figure 6 also shows that control (saline) locomotor activity was significantly lower in ob than in B6 mice (P < 0.01). There was no difference between the locomotor activity of B6 and ob mice after pontine microinjection of neostigmine.



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  		Fig. 6. Locomotor activity of B6 and ob mice in an open field (67 x 56 cm) after PnO injection of saline or neostigmine (133 ng). There was significantly ({dagger}P < 0.01) less locomotor activity in ob mice compared with B6 mice after pontine microinjection of saline. PnO neostigmine decreased locomotor activity below respective control levels in B6 and ob mice (*P < 0.05).

 
Pontine Neostigmine Caused Respiratory Depression

Figure 7 shows eight dependent measures of breathing obtained from B6 and ob mice after PnO microinjection of saline and neostigmine (133 ng). There was no difference between B6 and ob mice for any respiratory measure after pontine microinjection of saline. In contrast, PnO neostigmine caused a larger magnitude effect in B6 than in ob mice for every respiratory parameter examined. Microinjection of neostigmine significantly decreased respiratory rate below control levels (Fig. 7A) in B6 mice (t = 8.16; df = 20; P < 0.0001) and ob mice (t = 4.95; df = 20; P < 0.0001). Neostigmine decreased respiratory rate to a greater extent in B6 than in ob mice (t = –6.63; df = 20; P < 0.0001). Neostigmine decreased tidal volume (Fig. 7B) below control levels in ob mice (t = 8.13; df = 20; P < 0.0001) but not in B6 mice. Tidal volume after neostigmine microinjection was significantly lower in B6 mice than in ob mice (t = 4.29; df = 20; P = 0.0004). Minute ventilation (Fig. 7C) was significantly decreased in B6 mice (t = 4.58; df = 20; P = 0.0002) and ob mice (t = 8.60; df = 20; P < 0.0001) by pontine neostigmine. Minute ventilation after pontine neostigmine was significantly lower in B6 mice than in ob mice (t = 6.92; df = 20; P < 0.0001). Microinjection of neostigmine significantly increased total respiratory cycle time (Fig. 7F) in B6 mice (t = 6.78; df = 20; P < 0.0001) and ob mice (t = 4.44; df = 20; P = 0.0003) by increasing both the duration of inspiration (Fig. 7D; B6, t = 11.73, df = 20, P < 0.0001; ob, t = 6.28, df = 20, P < 0.0001) and duration of expiration (Fig. 7E; B6, t = 5.52, df = 20, P < 0.0001; ob, t = 3.72, df = 20, P = 0.0013). After PnO neostigmine, duration of inspiration (Fig. 7D; t = 7.27; df = 20; P < 0.0001), duration of expiration (Fig. 7E; t = 5.60; df = 20; P < 0.0001), and total respiratory cycle time (Fig. 7F; t = 6.03; df = 20; P < 0.0001) were greater in B6 than in ob mice. Inspiratory flow (Fig. 7G) significantly decreased after pontine neostigmine injection in B6 mice (t = 5.75; df = 20; P < 0.0001) and ob mice (t = 12.19; df = 20; P < 0.0001). The decrease in inspiratory flow was greater in B6 than in ob mice (t = 6.63; df = 20; P < 0.0001). Inspiratory effort or duty cycle (Fig. 7H) was not significantly altered from control levels by pontine neostigmine. Inspiratory effort was greater in ob mice than in B6 mice after pontine microinjection of neostigmine (t = 3.18; df = 20; P = 0.0047).



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  		Fig. 7. Quantification of breathing after PnO microinjection of saline and neostigmine in B6 and ob mice. Graphs illustrate means + SD for respiratory rate (A), tidal volume (B), minute ventilation (C), duration of inspiration (TI; D), duration of expiration (TE; E), total respiratory cycle time (Ttot; F), inspiratory flow (G), and inspiratory effort or duty cycle (H). *Significant difference from saline (P < 0.05). {dagger}Significant (P < 0.05) differences between B6 and ob mice. Breathing data for B6 mice are from Ref. 56.

 
Expression of M2 Muscarinic Receptor Protein Differs in B6 and ob Mice

The five brain regions in which M2 muscarinic receptor protein was quantified is illustrated by Fig. 8A. Two-way ANOVA revealed a significant effect of mouse line (F = 34.60; df = 1, 50; P < 0.0001), a brain region main effect (F = 9.35; df = 4, 50; P < 0.0001), and a mouse line-by-brain region interaction (F = 3.77; df = 4, 50; P = 0.0099) for expression levels of M2 muscarinic receptor protein (Fig. 8B). Post hoc Newman-Keuls test showed that M2 receptor protein density was greater for ob than for B6 mice in cortex (P < 0.01), midbrain (P < 0.05), pons (P < 0.01), and cerebellum (P < 0.01). The medulla revealed no differences in M2 receptor protein between B6 and ob mice.



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  		Fig. 8. Western blot analysis revealed brain site-specific location of M2 muscarinic receptor protein. A: schematic sagittal view of the B6 mouse brain showing the microdissection limits (dashed lines) of the 5 brain regions studied. Schematic outline is from the mouse brain atlas (66). B: graph represents means + SD of M2 receptor protein/actin optical density values from 10 assays (15 B6 mice and 15 ob mice) for 5 brain regions. Amount of M2 muscarinic receptor protein varied significantly (P < 0.0001) as a function of mouse line and brain region. M2 protein levels measured in the cortex, midbrain, pons, and cerebellum were significantly greater in ob than in B6 mice (*P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Pontine Neostigmine Altered Arousal

Enhancing cholinergic neurotransmission in medial regions of the pontine reticular formation causes a REM sleeplike state in cat (5, 6), rat (9, 44, 58, 76), and B6 mouse (18, 21, 56). Consistent with previous studies in B6 mice (18, 21, 56), the present neostigmine injections into the pontine reticular formation of B6 and ob mice caused the behavioral and electrographic features characteristic of REM sleep (Fig. 1). The present report is the first to extend this finding to ob mice. As reviewed elsewhere (2, 3, 54), the medial pontine reticular formation contains muscarinic receptors but does not synthesize ACh. Cholinergic projections to the medial pontine reticular formation arise from the laterodorsal and pedunculopontine tegmental (LDT/PPT) nuclei (59, 75). LDT/PPT neuronal discharge is positively correlated with REM sleep (28) and provides ACh to the medial pontine reticular formation (55).

The mouse PnO (Fig. 2) is homologous to the feline medial pontine reticular formation. Functional muscarinic receptors have been localized to PnO of B6 mice (21), and ACh release within the PnO of B6 mouse is regulated by muscarinic receptors (17). Immunohistochemical data regarding cholinergic projections from LDT/PPT to PnO are not yet available for B6 or ob mice.

Microinjecting saline into the PnO did not disrupt the temporal organization of sleep and wakefulness (Fig. 3). After PnO saline administration, B6 and ob mice cycled normally from wakefulness to NREM sleep to REM sleep. In contrast, even the lowest concentration of neostigmine caused a REM sleeplike state to emerge directly out of wakefulness, with no intervening NREM sleep. Thus cholinergically induced changes in the temporal organization of sleep in B6 and ob mice are homologous to REM sleep enhancement in cats caused by neostigmine (5), carbachol (6), and bethanechol (40, 52).

Neostigmine Enhancement of REM Sleep Differed Between B6 and ob Mice

For B6 and ob mice, neostigmine caused a concentration-dependent increase in the REM sleeplike state (Fig. 4). Neostigmine also caused concentration-dependent decreases in the amount of time spent in wakefulness and NREM sleep (Fig. 4). The finding that the cholinergically evoked REM sleeplike state is concentration dependent in B6 and ob mice is consistent with data from cats (4, 5, 40) and rats (9, 35). B6 and ob mice showed differential sensitivity to the REM sleep-enhancing effects of neostigmine. These data encourage future studies designed to elucidate the cellular and molecular mechanisms mediating these differences between congenic mouse lines.

Atropine Blocked Neostigmine-Induced Enhancement of REM Sleep

Atropine blocked REM-Neo in B6 and ob mice (Figs. 3 and 5), a finding consistent with the interpretation that neostigmine caused an increase in endogenous ACh and that REM sleep enhancement was mediated by muscarinic cholinergic receptors localized to the PnO. Muscarinic receptors are coupled to guanine nucleotide binding (G) proteins. The data in Figs. 3 and 5 fit well with the finding that muscarinic receptor antagonists block cholinergically activated G proteins in PnO of B6 mice (21). A complete evaluation of the view that murine models can help elucidate the neurobiological regulation of sleep (57) will require data characterizing similarities and differences between mice and other species (8). The present atropine results agree with data from rats (1, 42, 58) and cats (4, 5, 13).

Pontine Neostigmine Inhibited Locomotor Activity

Although ob mice spent more time awake than B6 mice (Fig. 4A), the ob mice exhibited 80% less spontaneous locomotor activity (Fig. 6). Neostigmine eliminated locomotor activity for the first hour after PnO microinjection in B6 (99% decrease) and ob (81% decrease) mice. Microinjecting neostigmine caused no difference in locomotor activity between B6 and ob mice. One factor that likely impacts the locomotor activity of ob mice is a physical limitation related to morbid obesity. The low level of locomotor activity displayed by ob mice relative to B6 mice is homologous to the inactivity associated with the common form of human obesity (19). The Fig. 6 data thus provide another line of evidence in support of the view that mouse models can provide unique insights into the polygenic causes of human obesity (22).

Pontine Cholinergic Effects on Breathing were Different in B6 and ob Mice

Changes in arousal are accompanied by changes in breathing that are modulated by pontine cholinergic (47, 54) and monoaminergic (38) neurotransmission. Breathing was depressed by PnO neostigmine in B6 and ob mice but to a greater extent in B6 mice (Fig. 7). Thus blocking the degradation of endogenously released ACh in the PnO (17) likely alters respiratory control (37). The Fig. 7 data also are consistent with the finding that dialysis delivery of the cholinergic agonist carbachol to PnO of anesthetized B6 mice significantly decreases respiratory rate (20). The finding that neostigmine caused less of a respiratory effect in ob mice likely is due to numerous factors. Genotype is one factor known to alter control of breathing (33, 36, 77, 82, 83) and arousal (30, 57, 79, 88, 90). The ob mouse is morbidly obese, and the respiratory mechanics accompanying the obese phenotype significantly alter ventilation (64, 86). Pontine cholinergic neurotransmission in cats inhibits spinal motoneurons (60, 61) and contributes to the REM sleep atonia of antigravity (51) and upper airway muscles (53). Data from cats and rats show that pontine LDT/PPT neurons modulate motor control (41), respiratory rhythm generation (55), and REM sleep (48, 91). The mechanisms that account for differences in breathing between B6 and ob mice (Fig. 7) remain to be determined.

All of the foregoing in vivo data were derived from microinjecting drugs directly into the pontine reticular formation. Limitations of the microinjection technique are discussed in detail elsewhere (6, 14). The small size of the mouse brain makes drug diffusion after microinjection a potential limitation. Previous microinjection studies using radiolabeled drugs showed that, at a time point 30 min after injection, 72% of a 500-nl injection remained within a radius of 750 µm (96). The present experiments used 50-nl injections, and the mouse PnO has a volume of ~500 µm3. Cholinergic evocation of the REM sleep-like state has been shown to vary significantly with the site of drug microinjection in cats (6, 70, 92, 97) and rats (9, 35). Similar mapping studies using mice comprise an important future direction.

Pontine Muscarinic Receptor Protein Differed Between B6 and ob Mice

Western blot analysis revealed that ob mice had greater expression of M2 muscarinic receptor protein than B6 mice in the pons, cortex, midbrain, and cerebellum, but not in the medulla. Western blot analysis cannot be interpreted to indicate functional receptors, but quantitative autoradiography has demonstrated functional muscarinic receptors in PnO of B6 mice (21). Despite these limitations, the Fig. 8 data encourage future studies that test the hypothesis that B6 and ob mice differentially express muscarinic receptors and cholinergically activated G proteins in brain regions known to regulate sleep and breathing.

Conclusions

This study shows for the first time in mice that pontine cholinergic modulation of sleep and breathing is subject to genetic influence. The known difference between ob and B6 mice is the absence of circulating leptin caused by a mutation in the leptin gene. This singular difference and the results from this study warrant consideration of mouse chromosome 14 as a regulatory element for functional expression of both ACh and leptin systems.

Cholinergic neurons are phenotypically defined by the presence of ChAT, the enzyme responsible for ACh synthesis. A vesicular ACh transporter actively transports ACh into presynaptic vesicles. This process is directly relevant for arousal state control as demonstrated by the discovery that disrupting the ACh transporter blocks REM sleep enhancement by pontine microinjection of neostigmine (13). In mice, the vesicular ACh transporter gene and the gene that encodes ChAT are colocalized on chromosome 14 (62). Colocalization can promote coordinated gene expression. Chromosome 14 also contains genes that may contribute to the regulation of behavioral arousal (89). These genes regulate the expression of {alpha}1A- adrenergic receptor and the thyroid hormone receptor TR-{beta} (7, 29).

Leptin is a satiety factor that can alter cholinergic neurons by inhibiting ChAT (23). Mutations in the ChAT gene are associated with episodic apneas in humans (65). Quantitative trait loci analysis also has identified genes on mouse chromosome 14 that can alter serum levels of leptin (12). Quantitative trait loci analyses have identified additional genes on mouse chromosome 14 that can cause respiratory defects, including apneas and lung abnormalities, increased glucose levels, and increased adiposity (7, 29). The foregoing discussion emphasizes the need for data mapping the distribution of ChAT-positive neurons in the pons of B6 and ob mice. Leptin administration to ob mice alters the breathing phenotype (86), and the present results raise the question of how leptin administration to PnO of ob mice might alter sleep.

When mouse strains or congenic lines such as B6 and ob exhibit a significant difference in phenotype in the face of a singular difference in genotype, one parsimonious inference is that the missing or altered gene may account for differences in phenotype. Serum leptin levels are higher in patients with OSA (46), but common human obesity is associated with leptin resistance rather than lack of leptin (10). Despite this limitation, there is support for the view that natural allele effects in mice can provide insight into the genetic basis for common human obesity (22). Studies of B6 mice show that ACh in the pontine reticular formation (17) can be viewed as a lower level phenotype modulating the higher level phenotype of cortical excitability (24–26), sleep (18), and breathing (20, 56). The present results extend this hypothesis by demonstrating allele-associated differences in the cholinergic regulation of sleep and breathing. The proximity of sleep- and breathing-related genes suggests targets for future examination of genetic mechanisms related to sleep-disordered breathing.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by National Institutes of Health Grants HL-65272, HL-40881, HL-57120, and MH-45361, and the Department of Anesthesiology.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
N. Goldberg, F. Liu, M. A. Norat, and C. Puscau in the Department of Anesthesiology and K. Welch in the University of Michigan Center for Statistical Consultation and Research provided expert assistance. We thank Dr. G. Poe for use of the digital video monitoring system and Dr. E. Gozal for help with the Western blot assay.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Lydic, Dept. of Anesthesiology, Univ. of Michigan, 7433 Med. Sci. I, 1150 W. 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.


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 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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