Coupling changes in cortical and pontine sigma and theta frequency oscillations following monoaminergic lesions in rat.

PURPOSE: Sigma and theta frequency electroencephalogram (EEG) oscillations exhibit substantial and well-recognized shifts with transitions across sleep and wake states. We aimed in this study to test the changes in coupling between these characteristic oscillations of non-rapid-eye-movement (NREM)/rapid-eye-movement (REM) sleep within and between cortical and pontine EEGs following monoaminergic lesion, by using the Pearson's product-moment correlation coefficients. METHODS: Experiments were performed in 14 adult, male Sprague Dawley rats chronically instrumented for sleep recording. We lesioned the dorsal raphe nucleus axon terminals in four rats using PCA neurotoxin (p-chloroamphetamine; Sigma-Aldrich, MO) administered as two intraperitoneal (IP) injections (6 mg/kg) 24 h apart. Lesioning of locus coeruleus axon terminals was performed in five rats using DSP-4 neurotoxin (N-2-chloroethyl-N-ethyl-2-bromobenzilamine; Sigma-Aldrich, MO) in a single IP dose of 50 mg/kg. RESULTS & CONCLUSIONS: Our previous study [Saponjic et al., Physiol Behav 90:1-10, 2007] demonstrated that these systemically induced monoaminergic lesions failed to produce significant changes in sleep/wake distribution from control conditions. The present study, by using spectral analysis and by examining the Pearson's correlation coefficients and their approximate probability density (APD) distribution profiles in control and lesion condition, demonstrates significant augmentation of the sigma/theta coupling strength, an inversion of cortical sigma/theta coupling direction and emergence of an additional sigma/theta coupling "mode" specific to the post-lesion state only within the cortex. By using the Pearson's correlation coefficients and their APD profiles, instead of classical sleep/wake distribution analysis, as a measure of direction and strength of sigma/theta coupling within and between cortex and pons, we were able to uncover the impact of a tonically decreased level of brain monoamines as altered strength and mode of coupling between sigma and theta oscillations. Specifically, a new mode of sigma/theta coupling emerged following lesion, which was specific to NREM sleep, suggests that loss of monoaminergic signaling interferes with NREM sleep consolidation. Our results also indicate an importance of monoamines in control of the sleep spindle and theta rhythm generators.

Prospective trial of efficacy and safety of ondansetron and fluoxetine in patients with obstructive sleep apnea syndrome.

STUDY OBJECTIVE: Incremental withdrawal of serotonin during wake to sleep transition is postulated as a key mechanism that renders the pharyngeal airway collapsible. While serotonin promotion with reuptake inhibitors have demonstrated modest beneficial effects during NREM sleep on obstructive sleep apnea (OSA), animal studies suggest a potential therapeutic role for selective serotonin receptor antagonists (5-HT3) in REM sleep. We aimed to test the hypothesis that a combination of ondansetron (Ond) and fluoxetine (Fl) may effectively reduce expression of disordered breathing during REM and NREM sleep in patients with OSA. DESIGN AND SETTING: A prospective, parallel-groups, single-center trial in patients with OSA. PARTICIPANTS: 35 adults with apnea hypopnea index (AHI) > 10; range 10-98. INTERVENTION: Subjects were randomized to placebo, n = 7; Ond (24 mg QD), n = 9; Fl (5 mg QD) + Ond (12 mg QD), n = 9; and Fl (10 mg QD) + Ond (24 mg QD), n = 10. MEASUREMENTS AND RESULTS: AHI was measured by in-lab polysomnography after a 7-day no-treatment period (Baseline) and on days 14 and 28 of treatment. The primary endpoint was AHI reduction at days 14 and 28. OND+FL resulted in approximately 40% reduction of baseline AHI at days 14 and 28 (unadjusted P < 0.03 for each) and improved oximetry trends. This treatment-associated relative reduction in AHI was also observed in REM and supine sleep. CONCLUSIONS: Combined treatment with OND+FL is well-tolerated and reduces AHI, yielding a potentially therapeutic response in some subjects with OSA.

Local antagonism of intertrigeminal region metabotropic glutamate receptors exacerbates apneic responses to intravenous serotonin.

Injections of a broad spectrum glutamate receptor antagonist into the pontine intertrigeminal region (ITR) exacerbate vagal reflex apnea produced by intravenous serotonin infusion. This effect is not reproduced by ITR injections with either NMDA or AMPA receptor antagonists. Here, we tested the hypothesis that ITR injection with a metabotropic glutamate antagonist would alter respiratory responses to serotonin (5-HT) intravenous infusions. In anesthetized adult male rats (N=20; Sprague-Dawley) AIDA (1-aminoindan-1,5-dicarboxylic acid), a specific antagonist of the type 1 metabotropic glutamate receptor (mGlu1R), was microinjected unilaterally into the ITR to block 5-HT evoked apnea. Respiratory pattern changes evoked by ITR-glutamate injection and by intravenous serotonin (5-HT) infusion (0.5 microl, 0.05 M; or 2.5x10(-8) mol) were characterized according to apnea expression and duration, as well as coefficients of variation for breath duration (CVTT) and amplitude (CVVT) before and after ITR AIDA injection. Unilateral AIDA blockade of the ITR significantly increased the duration of apnea evoked by 5-HT infusion (p<0.03 for each dose tested) during the 30s following infusion in a dose-dependent fashion, with the two highest doses resulting in intermittent apneas for at least 10 min following a bolus 5-HT infusion. Similar prolonged increases in CVTT and CVVT with respect to control were associated with ITR AIDA injections. These findings suggest that brief perturbations of vagal afferent pathways can produce ongoing respiratory dysrhythmia, including spontaneous apnea, and that glutamatergic neurotransmission within ITR may be important for damping such disturbances. The present observations also suggest that such respiratory damping may be mediated by mGlu1 receptors. These findings extend our understanding of the role of the intertrigeminal region in modulating respiratory reflexes.

Pharmacology of vagal afferent influences on disordered breathing during sleep.

Sleep-related breathing disorders (SRBD) are a significant public health concern, with a prevalence in the US general population of approximately 2% of women and approximately 4% of men. Although significant strides have been made in our understanding of these disorders with respect to epidemiology, risk factors, pathogenesis and consequences, work to understand these factors in terms of the underlying cellular, molecular and neuromodulatory processes remains in its infancy. Current primary treatments are surgical or mechanical, with no drug treatments available. Basic investigations into the neurochemistry and neuropharmacology of sleep-related changes in respiratory pattern generation and modulation will be essential to clarify the pathogenic processes underlying SRBD and to identify rational and specific pharmacotherapeutic opportunities. Here we summarize emerging work suggesting the importance of vagal afferent feedback systems in sleep-related respiratory pattern disturbances and pointing toward a rich but complex array of neurochemical and neuromodulatory processes that may be involved.

Monotone Signal Segments Analysis as a novel method of breath detection and breath-to-breath interval analysis in rat.

We applied a novel approach to respiratory waveform analysis--Monotone Signal Segments Analysis (MSSA) on 6-h recordings of respiratory signals in rats. To validate MSSA as a respiratory signal analysis tool we tested it by detecting: breaths and breath-to-breath intervals; respiratory timing and volume modes; and changes in respiratory pattern caused by lesions of monoaminergic systems in rats. MSSA differentiated three respiratory timing (tachypneic, eupneic, bradypneic-apneic), and three volume (artifacts, normovolemic, hypervolemic-sighs) modes. Lesion-induced respiratory pattern modulation was visible as shifts in the distributions of monotone signal segment amplitudes, and of breath-to-breath intervals. Specifically, noradrenergic lesion induced an increase in mean volume (por=0.06). MSSA of timing modes detected noradrenergic lesion-induced interdependent changes in the balance of eupneic (decrease; p

Efficacy of mirtazapine in obstructive sleep apnea syndrome.

STUDY OBJECTIVES: Decreased serotonergic facilitation of upper-airway motor neurons during sleep has been postulated as an important mechanism rendering the upper airway vulnerable to obstruction in patients with obstructive sleep apnea syndrome (OSA). Although serotonin reuptake inhibitors have been shown to produce modest reductions in the apnea-hypopnea index (AHI) during non-rapid eye movement (NREM) sleep, they have not been proven to be generally effective as treatments for OSA. Conversely, antagonists of type 3 (5-HT3) serotonin receptors effectively have been shown to reduce the frequency of central apneas during rapid eye movement (REM) sleep in a rodent model of sleep-related breathing disorder. We sought to determine whether mirtazapine, a mixed 5-HT2/5-HT3 antagonist that also promotes serotonin release in the brain would effectively reduce AHI during both NREM and REM sleep in patients with OSA. DESIGN: A randomized, double-blind, placebo-controlled, 3-way crossover study of mirtazapine in patients with OSA. SETTING: Laboratory studies were conducted in the Center for Sleep and Ventilatory Disorders at the University of Illinois Medical Center. PATIENTS: Seven adult men and 5 adult women with newly diagnosed (treatment-naïve) and medically uncomplicated OSA were randomized into the study. INTERVENTIONS: Each subject self-administered oral medications 30 minutes before bedtime each night for 3 consecutive 7-day treatment periods. These treatments comprised (1) placebo, (2) 4.5 mg per day of mirtazapine, and (3) 15 mg per day of mirtazapine. The order of treatments was randomized for each subject, and orders were counterbalanced for the overall study. MEASUREMENTS AND RESULTS: Each subject charted his or her sleep-wake schedule throughout the study and completed the Stanford Sleepiness Scale every 2 hours during the seventh day of each treatment period. Subjects were studied by laboratory polysomnography on the seventh night of each treatment period. With respect to placebo treatment, 4.5 mg of mirtazapine significantly reduced the AHI in all sleep stages to 52%, with 11 of 12 subjects showing improvement over placebo; 15 mg of mirtazapine reduced the AHI to 46%, with 12 of 12 subjects showing improvement over placebo. Sleep fragmentation was reduced only by the higher dose of mirtazapine. Gross changes in sleep architecture were unremarkable. CONCLUSIONS: Daily administration of 4.5 to 15 mg of mirtazapine for 1 week reduces AHI by half in adult patients with OSA. This represents the largest and most consistent drug-treatment effect demonstrated to date in a controlled trial. These findings suggest the therapeutic potential of mixed-profile serotonergic drugs in OSA and provide support for future studies with related formulations. Mirtazapine also is associated with sedation and weight gain-2 negative side effects in patients with OSA. In view of the above, we do not recommend use of mirtazapine as a treatment for OSA.

Effects of intertrigeminal region NMDA and non-NMDA receptors on respiratory responses in rats.

Respiratory disturbance, including apnea, can be induced by microinjection of glutamate into the intertrigeminal region (ITR) of the lateral pons, a region that is anatomically coupled to both the dorsal and ventral respiratory groups of the medulla. We showed that the ITR plays a functional role in regulating both vagal reflex apnea and spontaneous sleep-related apnea in rats, but the mechanisms have not been determined. This study shows that functional NMDA receptors are expressed in the ITR since the blockade of these receptors by AP5, a specific NMDA receptor antagonist, was fully effective in blocking apnea induced by glutamate injection within this region. Selective blockade of ITR NMDA receptors had no effect on the immediate apnea evoked by an intravenous 5-HT bolus, whereas the nonspecific glutamate receptor antagonist kynurenic acid significantly increased the duration of this vagal reflex apnea. These findings are of interest because pontine NMDA receptors participate in inspiratory off-switch mechanisms and have been implicated in various short- and long-term potentiation and depression phenomena. These data support the involvement of ITR non-NMDA receptors in modulation of reflex apnea per se, whereas NMDA receptors play a role in damping respiratory responses to transient disturbances.

Impact of intertrigeminal region AMPA receptor blockade on respiratory responses in rats.

We hypothesized that functional glutamatergic AMPA receptors are expressed in the intertrigeminal region (ITR) of the lateral pons, and that blockade of these receptors by a specific AMPA receptor antagonist (NBQX) would alter respiratory responses both to ITR glutamate injections as well as to vagally mediated reflex apnea induced by intravenous infusion of serotonin. Non-selective blockade of ITR glutamate receptors using kynurenic acid previously was shown to alter both of these respiratory responses. Unilateral ITR injections in 19 anaesthetized spontaneously breathing adult Sprague-Dawley rats with the vagi intact demonstrated that NBQX (10mM): (1) shortened glutamate-induced apnea duration (p=0.006), (2) reduced glutamate-induced apnea frequency (p=0.034) and (3) decreased glutamate-induced apnea density (p=0.006). The same dose of NBQX did not affect vagally mediated reflex apnea induced by intravenous infusion of serotonin. These results show that functional glutamate AMPA receptors are expressed in the ITR but fail to directly demonstrate any specific physiologic role for these receptors in respiratory control. In contrast to antagonism of NMDA receptors which completely blocked the central apnea evoked by glutamate, antagonism of AMPA receptors only partially blocked the effects of glutamate. In contrast to kynurenic acid, antagonists to NMDA and AMPA receptors given separately did not potentiate the duration of reflex apnea.

Serotonin and noradrenaline modulate respiratory pattern disturbances evoked by glutamate injection into the pedunculopontine tegmentum of anesthetized rats.

STUDY OBJECTIVES: We hypothesized that 2 important neurotransmitters related to behavioral state control, serotonin and noradrenaline, could also be modulators of pedunculopontine tegmental nucleus (PPT)-induced respiratory dysrhythmia. DESIGN: We examined the impact of serotonin and noradrenaline at respiratory control sites in the PPT functionally identified by immediate apnea of 2.5- to 10-second duration, followed by increased variability of breath time (CVT(T)) (P < .04) after locally injecting glutamate in anesthetized rats. SETTING: Basic sleep and respiratory neurobiology laboratory. PARTICIPANTS: Sixteen adult, male Sprague-Dawley rats. MEASUREMENTS AND RESULTS: Glutamate-induced respiratory responses, including increases of total apnea duration and CVT(T), were not different between groups of rats in which we further tested monoaminergic modulatory effects (for CVT(T) P = .98, and for total apnea duration, P = .80). Serotonin or noradrenaline injected at the same sites as glutamate had equal impact on CVT(T) (P = .34) and on mean total apnea duration (P = .80), but pretreatment of PPT sites with serotonin blocked (remained equal to preinjection; P = .11), whereas pretreatment with noradrenaline potentiated (P = .04) the increment of respiratory-timing variability induced by glutamate. The serotonergic-blocking effect on glutamate-induced respiratory dysrhythmia was specific to the PPT: the respiratory responses induced by glutamate injection outside the PPT were not modulated by serotonin (for CVT(T), P = .46, and for mean apnea duration, P = .99). CONCLUSIONS: The opposed impact of serotonin and noradrenaline on PPT-induced respiratory dysrhythmia, in contrast to their convergent regulatory role in behavioral state control, suggests a functionally distinct role for the PPT in respiratory-pattern control independent of rapid eye movement sleep control.

An automated pontine-wave detection system.

STUDY OBJECTIVES: Pontine-waves (P-waves), the pontine component of ponto-geniculo-occipital waves, represent a close marker of brainstem phasic events and are associated with cardiorespiratory changes in sleep. Because visual scoring is subjective and cumbersome, we developed an automated P-wave analysis system, which could detect and classify P-waves as clusters or as isolated events in bipolar recordings of the pontine electroencephalogram (EEG) of conscious rats. DESIGN: A computer algorithm was developed to extract and normalize each half-wave of the pontine EEG according to the background noise level. Candidate events for different P-wave patterns (uniphasic, biphasic, and triphasic) were compared to a corresponding set of amplitude and duration thresholds to identify P-waves and to reject artifacts. PARTICIPANTS: Ten adult male Sprague-Dawley rats were instrumented for chronic polysomnography. MEASUREMENTS AND RESULTS: Two human experts manually scored each recording, and their consensus score was used as the "gold standard" for algorithm optimization and validation. The algorithm's scoring thresholds were optimized on a training set of 5 six-hour polysomnographic records, yielding 96.8% accuracy and 97.7% sensitivity versus human consensus scoring. Validation of the algorithm, using the optimized threshold values, was conducted using a set of 5 independent recordings, resulting in 94.8% accuracy and 94.7% sensitivity versus human consensus scoring. CONCLUSIONS: We have developed and validated an automated system for detection and classification of P-waves in conscious rats with advantages over human scoring, including increased speed and perfect reliability.

Pontine intertrigeminal region attenuates sleep apneas in rats.

STUDY OBJECTIVES: To determine whether the pontine intertrigeminal region (ITR), with recently described anatomic connections and an effect on vagally induced reflex apnea, has an impact on spontaneous sleep apneas in rats. DESIGN: Respiration, electroencephalogram (EEG), and electromyogram (EMG) were recorded in rats with lesions of the pontine ITR and in control animals. PARTICIPANTS: 9 adult male Sprague-Dawley rats. INTERVENTIONS: Rats were implanted with EEG and EMG electrodes and were polygraphically recorded for 6 hours, and their respiration was monitored by placing each animal inside a single-chamber plethysmograph. Subsequently, a respiratory-related intertrigeminal site was identified by probing on dorsoventral tracks with 2 to 5 nL glutamate (10 nL, 10 mmol) injections from a multibarrel glass pipette. This site was then lesioned by injecting ibotenic acid (10 nL, 50 mmol) from a second pipette barrel. Animals were again recorded for 6 hours on days 2, 7, and 14 after the lesion. MEASUREMENTS AND RESULTS: ITR lesions exerted no impact on mean respiratory pattern during any sleep-wake state, compared to baseline recordings. In contrast, apnea frequency during non-rapid eye movement sleep increased following ITR lesion, more than doubling by day 14. CONCLUSIONS: This study demonstrates that a small and well-localized unilateral lesion of the ITR region in the lateral pons can increase sleep apnea expression in freely moving rats over a 2-week period. The present findings are in agreement with the general modulatory role of pontine structures in activities including respiration, heart rate, and regulation of blood pressure.

Functional role for cannabinoids in respiratory stability during sleep.

STUDY OBJECTIVES: Serotonin, acting in the peripheral nervous system, can exacerbate sleep-related apnea, and systemically administered serotonin antagonists reduce sleep-disordered respiration in rats and bulldogs. Because cannabinoid receptor agonists are known to inhibit the excitatory effects of serotonin on nodose ganglion cells, we examined the effects of endogenous (oleamide) and exogenous (delta9-tetrahydrocannabinol; delta9THC) cannabimimetic agents on sleep-related apnea. DESIGN: Sleep architecture, respiratory pattern, and apnea expression in rats were assessed by polysomnography. A repeated measures, within-subjects, fully nested crossover design was used in which each animal was recorded on exactly 12 occasions. PARTICIPANTS: Eleven adult male Sprague-Dawley rats were instrumented for chronic polysomnography. INTERVENTIONS: Animals were recorded following intraperitoneal injection of various doses of delta9THC, oleamide, and serotonin, alone and in combination. MEASUREMENTS AND RESULTS: Our data show that delta9THC and oleamide each stabilized respiration during all sleep stages. With delta9THC, apnea index decreased by 42% (F=2.63; p=0.04) and 58% (F=2.68; p=0.04) in NREM and REM sleep, respectively. Oleamide produced equivalent apnea suppression. This observation suggests an important role for endocannabinoids in maintaining autonomic stability during sleep. Oleamide and delta9THC blocked serotonin-induced exacerbation of sleep apnea (p<0.05 for each), suggesting that inhibitory coupling between cannabinoids and serotonin receptors in the peripheral nervous system may act on apnea expression. CONCLUSIONS: This study demonstrates potent suppression of sleep-related apnea by both exogenous and endogenous cannabinoids. These findings are of relevance to the pathogenesis and pharmacological treatment of sleep-related breathing disorders.

Intertrigeminal region attenuates reflex apnea and stabilizes respiratory pattern in rats.

The goal of the present study was to determine whether the newly described anatomical pathway involving the pontine intertrigeminal region (ITR), (Chamberlin and Saper, 1998), has a physiological role in mediating or modulating vagally-induced reflex apnea. We explored the ITR impact on vagal reflex apnea elicited by intravenuously injected 5-HT in ten anesthetized rats. The animals had a catheter inserted into the femoral vein for administration of 5-HT (0.00375 mg) and respiration was recorded by piezo-electric crystal. Multibarrel pipettes were used to pressure inject glutamate (5-10 nl, 10 mM), kynurenic acid (10 nl, 50 mM, a glutamate receptor antagonist), and red dye into the ITR, unilaterally and bilaterally. Intravenous administration of 5-HT produced an immediate 3-s apnea. Microinjections of glutamate into the ITR produced apneas, while microinjections of kynurenic acid blocked the glutamate effect. Following glutamate antagonism, subsequent administration of 5-HT produced apneas of much longer duration (8 s). Acute ponto-medullary transection in two animals yielded even greater prolongation of 5-HT-induced apnea. We conclude that a physiological role for the ITR in respiration is to attenuate vagally-induced reflex apneas. This finding is in agreement with the general modulatory role of pontine structures in autonomic activities including respiration, heart rate and regulation of blood pressure. In addition, our data indicate that medullary circuits, independent of pontine structures, are sufficient to produce 5-HT induced reflex apnea.

Respiratory pattern modulation by the pedunculopontine tegmental nucleus.

This study demonstrates respiratory modulation caused by stimulation of the pedunculopontine tegmental nucleus (PPT), a structure not classically included in the pontine respiratory neuronal network. The long-lasting increase in variability of respiratory parameters following glutamate microinjection into PPT in anesthetized, spontaneously breathing Sprague Dawley rats was more pronounced under ketamine than nembutal anesthesia. The induced respiratory perturbations were characterized by intermittent apneas and increased variability of expiratory (TE) and total (TT) breath durations in all animals. Although the baseline spontaneous breathing patterns (mean values of all respiratory parameters and their variabilities) were equivalent under ketamine and nembutal anesthesia, different anesthetic agents did affect respiratory responses to PPT stimulation by glutamate in terms of latency, duration, and structure. We conclude that glutamatergic stimulation of PPT has a significant impact on the brainstem respiratory pattern generator.

Modulation of reflex and sleep related apnea by pedunculopontine tegmental and intertrigeminal neurons.

We describe and summarize here our recent findings about the role in respiration of two pontine structures that are not classically included in the pontine respiratory group: the pedunculopontine tegmental nucleus (PPT) and the intertrigeminal region (ITR). We also discuss significant contributions of other workers in the field, especially, S. Datta [Cell. Mol. Neurobiol. 17: 341-365, 1997], R. Lydic and H. Baghdoyan [Sleep, 25: 617-622, 2002], and N. Chamberlin and C. Saper [J. Neurosci. 18: 6048-6056, 1998], who postulated a role for the ITR in modulating reflex apnea. In anesthetized and freely moving rats we have consistently documented that PPT and ITR have a role in respiration. Neurochemical manipulations of each area affected the brainstem respiratory pattern generator and respiratory pattern variability,observed as spontaneous disturbances during sleep or as induced reflex apnea. Although the exact central mechanisms of apnea cannot be determined from our studies to date, we postulate that reflex and sleep-related apneas in rats share some common brainstem pathways, which may include PPT and ITR.

Intranodose ganglion injections of dronabinol attenuate serotonin-induced apnea in Sprague-Dawley rat.

Obstructive sleep apnea represents a significant public health concern. Afferent vagal activation is implicated in increased apnea susceptibility by reducing upper airway muscle tone via activation of serotonin receptors in the nodose ganglia. Previous investigations demonstrated that systemically administered cannabinoids can be used therapeutically to decrease the apnea/hypopnea index in rats and in humans. However, cannabinoids have effects on both the central and peripheral nervous systems, and the exact mechanism of decreased apnea/hypopnea index with cannabinoids is unknown. Here, we hypothesized that intranodose ganglion injections of a cannabinoid will attenuate 5-HT-induced reflex apnea and increase upper airway muscle tone. We show that dronabinol injected locally into the nodose ganglia suppresses 5-HT-induced reflex apnea, and increases phasic, but not tonic, activation of the genioglossus. These data support the view that dronabinol stabilizes respiratory pattern and augments upper airway muscles by acting at the nodose ganglia. These findings underscore a therapeutic potential of dronabinol for the treatment of obstructive sleep apnea.

A method of nodose ganglia injection in Sprague-Dawley rat.

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Acute exacerbation of sleep apnea by hyperoxia impairs cognitive flexibility in Brown-Norway rats.

STUDY OBJECTIVES: To determine whether learning deficits occur during acute exacerbation of spontaneous sleep related breathing disorder (SRBD) in rats with high (Brown Norway; BN) and low (Zucker Lean; ZL) apnea propensity. DESIGN: Spatial acquisition (3 days) and reversal learning (3 days) in the Morris water maze (MWM) with polysomnography (12:00-08:00): (1) with acute SRBD exacerbation (by 20-h hyperoxia immediately preceding reversal learning) or (2) without SRBD exacerbation (room air throughout). SETTING: Randomized, placebo-controlled, repeated-measures design. PARTICIPANTS: 14 BN rats; 16 ZL rats. INTERVENTIONS: 20-h hyperoxia. MEASUREMENTS AND RESULTS: Apneas were detected as cessation of respiration ≥ 2 sec. Swim latency in MWM, apnea indices (AI; apneas/hour of sleep) and percentages of recording time for nonrapid eye movement (NREM), rapid eye movement (REM), and total sleep were assessed. Baseline AI in BN rats was more than double that of ZL rats (22.46 ± 2.27 versus 10.7 ± 0.9, P = 0.005). Hyperoxia increased AI in both BN (34.3 ± 7.4 versus 22.46 ± 2.27) and ZL rats (15.4 ± 2.7 versus 10.7 ± 0.9) without changes in sleep stage percentages. Control (room air) BN and ZL rats exhibited equivalent acquisition and reversal learning. Acute exacerbation of AI by hyperoxia produced a reversal learning performance deficit in BN but not ZL rats. In addition, the percentage of REM sleep and REM apnea index in BN rats during hyperoxia negatively correlated with reversal learning performance. CONCLUSIONS: Acute exacerbation of sleep related breathing disorder by hyperoxia impairs reversal learning in a rat strain with high apnea propensity, but not a strain with a low apnea propensity. This suggests a non-linear threshold effect may contribute to the relationships between sleep apnea and cognitive dysfunctions, but strain-specific differences also may be important.

Brown Norway and Zucker Lean rats demonstrate circadian variation in ventilation and sleep apnea.

STUDY OBJECTIVES: Circadian rhythms influence many biological systems, but there is limited information about circadian and diurnal variation in sleep related breathing disorder. We examined circadian and diurnal patterns in sleep apnea and ventilatory patterns in two rat strains, one with high sleep apnea propensity (Brown Norway [BN]) and the other with low sleep apnea propensity (Zucker Lean [ZL]). DESIGN/SETTING: Chronically instrumented rats were randomized to breathe room air (control) or 100% oxygen (hyperoxia), and we performed 20-h polysomnography beginning at Zeitgeber time 4 (ZT 4; ZT 0 = lights on, ZT12 = lights off). We examined the effect of strain and inspired gas (twoway analysis of variance) and analyzed circadian and diurnal variability. MEASUREMENTS AND RESULTS: Strain and inspired gas-dependent differences in apnea index (AI; apneas/h) were particularly prominent during the light phase. AI in BN rats (control, 16.9 ± 0.9; hyperoxia, 34.0 ± 5.8) was greater than in ZL rats (control, 8.5 ± 1.0; hyperoxia, 15.4 ± 1.1, [strain effect, P < 0.001; gas effect, P = 0.001]). Hyperoxia reduced respiratory frequency in both strains, and all respiratory pattern variables demonstrated circadian variability. BN rats exposed to hyperoxia demonstrated the largest circadian fluctuation in AI (amplitude = 17.9 ± 3.7 apneas/h [strain effect, P = 0.01; gas effect, P < 0.001; interaction, P = 0.02]; acrophase = 13.9 ± 0.7 h; r (2) = 0.8 ± 1.4). CONCLUSIONS: Inherited, environmental, and circadian factors all are important elements of underlying sleep related breathing disorder. Our method to examine sleep related breathing disorder phenotypes in rats may have implications for understanding vulnerability for sleep related breathing disorder in humans.

Proof of concept trial of dronabinol in obstructive sleep apnea.

STUDY OBJECTIVE: Animal data suggest that Δ(9)-TetraHydroCannabinol (Δ(9)THC) stabilizes autonomic output during sleep, reduces spontaneous sleep-disordered breathing, and blocks serotonin-induced exacerbation of sleep apnea. On this basis, we examined the safety, tolerability, and efficacy of dronabinol (Δ(9)THC), an exogenous Cannabinoid type 1 and type 2 (CB1 and CB2) receptor agonist in patients with Obstructive Sleep Apnea (OSA). DESIGN AND SETTING: Proof of concept; single-center dose-escalation study of dronabinol. PARTICIPANTS: Seventeen adults with a baseline Apnea Hypopnea Index (AHI) ≥15/h. Baseline polysomnography (PSG) was performed after a 7-day washout of Continuous Positive Airway Pressure treatment. INTERVENTION: Dronabinol was administered after baseline PSG, starting at 2.5 mg once daily. The dose was increased weekly, as tolerated, to 5 mg and finally to 10 mg once daily. MEASUREMENTS AND RESULTS: Repeat PSG assessments were performed on nights 7, 14, and 21 of dronabinol treatment. Change in AHI (ΔAHI, mean ± SD) was significant from baseline to night 21 (-14.1 ± 17.5; p = 0.007). No degradation of sleep architecture or serious adverse events was noted. CONCLUSION: Dronabinol treatment is safe and well-tolerated in OSA patients at doses of 2.5-10 mg daily and significantly reduces AHI in the short-term. These findings should be confirmed in a larger study in order to identify sub-populations with OSA that may benefit from cannabimimetic pharmacologic therapy.