Effects of Cannabinoid Agonists and Antagonists on Sleep in Laboratory Animals.

The cannabinoids are a family of chemical compounds that can be either synthesized or naturally derived. These compounds have been shown to modulate a wide variety of biological processes. In this chapter, the studies detailing the effects of cannabinoids on sleep in laboratory animals are reviewed. Both exogenous and endogenous cannabinoids generally appear to decrease wakefulness and alter rapid eye movement (REM) and non-REM sleep in animal models. In addition, cannabinoids potentiate the effects of sedative-hypnotic drugs. However, the individual contributions of each cannabinoid on sleep processes is more nuanced and may depend on the site of action in the central nervous system. Many studies investigating the mechanism of cannabinoid effects on sleep suggest that the effects of cannabinoids on sleep are mediated via cannabinoid receptors; however, some evidence suggests that some sleep effects may be elicited via non-cannabinoid receptor-dependent mechanisms. More research is necessary to fully elucidate the role of each compound in modulating sleep processes.

Sleep fragmentation increases blood pressure and is associated with alterations in the gut microbiome and fecal metabolome in rats.

The gut microbiota, via the production of metabolites entering the circulation, plays a role in blood pressure regulation. Blood pressure is also affected by the characteristics of sleep. To date, no studies have examined relationships among the gut microbiota/metabolites, blood pressure, and sleep. We hypothesized that fragmented sleep is associated with elevated mean arterial pressure, an altered and dysbiotic gut microbial community, and changes in fecal metabolites. In our model system, rats were randomized to 8 h of sleep fragmentation during the rest phase (light phase) or were undisturbed (controls) for 28 consecutive days. Rats underwent sleep and blood pressure recordings, and fecal samples were analyzed during: baseline (days -4 to -1), early sleep fragmentation (days 0-3), midsleep fragmentation (days 6-13), late sleep fragmentation (days 20-27), and recovery/rest (days 28-34). Less sleep per hour during the sleep fragmentation period was associated with increased mean arterial pressure. Analyses of gut microbial communities and metabolites revealed that putative short chain fatty acid-producing bacteria were differentially abundant between control and intervention animals during mid-/late sleep fragmentation and recovery. Midsleep fragmentation was also characterized by lower alpha diversity, lower Firmicutes:Bacteroidetes ratio, and higher Proteobacteria in intervention rats. Elevated putative succinate-producing bacteria and acetate-producing bacteria were associated with lower and higher mean arterial pressure, respectively, and untargeted metabolomics analysis demonstrates that certain fecal metabolites are significantly correlated with blood pressure. These data reveal associations between sleep fragmentation, mean arterial pressure, and the gut microbiome/fecal metabolome and provide insight to links between disrupted sleep and cardiovascular pathology.

Autonomic regulation during sleep and wakefulness: a review with implications for defining the pathophysiology of neurological disorders.

Cardiovascular and respiratory parameters change during sleep and wakefulness. This observation underscores an important, albeit incompletely understood, role for the central nervous system in the differential regulation of autonomic functions. Understanding sleep/wake-dependent sympathetic modulations provides insights into diseases involving autonomic dysfunction. The purpose of this review was to define the central nervous system nuclei regulating sleep and cardiovascular function and to identify reciprocal networks that may underlie autonomic symptoms of disorders such as insomnia, sleep apnea, restless leg syndrome, rapid eye movement sleep behavior disorder, and narcolepsy/cataplexy. In this review, we examine the functional and anatomical significance of hypothalamic, pontine, and medullary networks on sleep, cardiovascular function, and breathing.

Intracerebroventricular injections of dronabinol, a cannabinoid receptor agonist, does not attenuate serotonin-induced apnea in Sprague-Dawley rats.

BACKGROUND: Evidence suggests that vagal nerve activity may play a role in sleep apnea induction. In anesthetized rats, dronabinol, a cannabinoid (CB) receptor agonist, injected into the nodose ganglia attenuates reflex apnea and increases genioglossus activity, and reflex apnea attenuation is blocked by systemic pre-treatment with cannabinoid type 1 and/or type 2 receptor antagonists. However, it is unclear whether dronabinol has similar effects in the central nervous system; CB receptors are widely distributed in the brain, especially on neuronal circuitry important for respiration and upper airway activation. Here, we examine the effects of intracerebroventricular (ICV) injection of dronabinol on serotonin (5-HT)-induced apnea. METHODS: Adult male Sprague-Dawley rats were anesthetized and instrumented with bilateral electrodes to monitor genioglossi EMG and with a piezoelectric strain gauge to monitor respiratory pattern. Serotonin was intravenously infused into a femoral vein to induce reflex apnea. After baseline recordings, rats were placed in a stereotaxic apparatus. A unilateral osteotomy was made to allow access for injection to the right lateral ventricle, and the dura were carefully removed. Dronabinol (100, 10, 1, or 0.1 µg/3 µl DMSO) or control (3 µl DMSO) was injected into the right lateral ventricle and 5-HT infusion was repeated. Data (mean ± SEM) were analyzed using a mixed model analysis with a repeated/fixed measure. RESULTS: There was no main effect in 5-HT-induced apnea or breath duration, or in breath instability, between ICV dronabinol injected and ICV vehicle control injected groups. Moreover, there was no main effect in phasic or tonic genioglossus activity between ICV dronabinol injected and ICV vehicle control injected groups. CONCLUSION: Our data show that ICV injection of dronabinol did not decrease 5-HT-induced apneas, and did not increase genioglossus activity. This in contrast to published results of dronabinol's effect on apnea via the vagus nerve. Our results suggest that the effects of dronabinol on reflex apneas are peripherally mediated via suppression of vagal nerve activity.

Treatments for Obstructive Sleep Apnea.

OBJECTIVE: To review the efficacy of current treatment options for adults with obstructive sleep apnea (OSA). METHODS: Review of the literature. RESULTS: OSA, characterized by repetitive ≥ 10-second interruptions (apnea) or reductions (hypopnea) in airflow, is initiated by partial or complete collapse in the upper airway despite respiratory effort. When left untreated, OSA is associated with comorbid conditions, such as cardiovascular and metabolic diseases. The current "gold standard" treatment for OSA is continuous positive air pressure (CPAP), which pneumatically stabilizes the upper airways. CPAP has proven efficacy and potential cost savings via decreases in health comorbidities and/or motor-vehicle crashes. However, CPAP treatment is not well-tolerated due to various side effects, and adherence among OSA subjects can be as low as 50% in certain populations. Other treatment options for OSA include improving CPAP tolerability, increasing CPAP adherence through patient interventions, weight loss/exercise, positional therapy, nasal expiratory positive airway pressure, oral pressure therapy, oral appliances, surgery, hypoglossal nerve stimulation, drug treatment, and combining 2 or more of the aforementioned treatments. Despite the many options available to treat OSA, none of them are as efficacious as CPAP. However, many of these treatments are tolerable, and adherence rates are higher than those of the CPAP, making them a more viable treatment option for long-term use. CONCLUSION: Patients need to weigh the benefits and risks of available treatments for OSA. More large randomized controlled studies on treatments or combination of treatments for OSA are needed that measure parameters such as treatment adherence, apnea-hypopnea index, oxygen desaturation, subjective sleepiness, quality of life, and adverse events.

DMSO potentiates the suppressive effect of dronabinol on sleep apnea and REM sleep in rats.

INTRODUCTION: Dimethyl sulfoxide (DMSO) is an amphipathic molecule with innate biological activity that also is used to dissolve both polar and nonpolar compounds in preclinical and clinical studies. Recent investigations of dronabinol, a cannabinoid, dissolved in DMSO demonstrated decreased sleep apnea frequency and time spent in REM sleep in rats. Here, we tested the effects of dronabinol dissolved in 25% DMSO diluted in phosphate-buffered saline (PBS) to rule out potentiating effects of DMSO. METHODS: Sprague-Dawley rats were anesthetized and implanted with bilateral stainless steel screws into the skull for electroencephalogram recording and bilateral wire electrodes into the nuchal muscles for electromyogram recording. Each animal was recorded by polysomnography. The study was a fully nested, repeated measures crossover design, such that each rat was recorded following each of 8 intraperitoneal injections separated by three days: vehicle (25% DMSO/PBS); vehicle and CB1 antagonist (AM 251); vehicle and CB2 antagonist (AM 630); vehicle and CB1/CB2 antagonist; dronabinol (CB1/CB2 agonist); dronabinol and CB1 antagonist; dronabinol and CB2 antagonist; and dronabinol and CB1/CB2 antagonists. Sleep was manually scored into NREM and REM stages, and sleep apneas were quantified. RESULTS: Dronabinol dissolved in 25% DMSO did not suppress sleep apneas or modify sleep efficiency compared to vehicle controls, in contrast to previously published results. However, dronabinol did suppress REM sleep, which is in line with previously published results. CONCLUSIONS: Dronabinol in 25% DMSO partially potentiated dronabinol's effects, suggesting a concomitant biological effect of DMSO on breathing during sleep.

Validation of PiezoSleep Scoring Against EEG/EMG Sleep Scoring in Rats.

Introduction: Current methods of sleep research in rodents involve invasive surgical procedures of EEG and EMG electrodes implantation. Recently, a new method of measuring sleep, PiezoSleep, has been validated against implanted electrodes in mice and rats. PiezoSleep uses a piezoelectric film transducer to detect the rodent's movements and respiration and employs an algorithm to automatically score sleep. Here, we validate PiezoSleep scoring versus EEG/EMG implanted electrodes sleep scoring in rats. Methods: Adult male Brown Norway and Wistar Kyoto rats were implanted with bilateral stainless-steel screws into the skull for EEG recording and bilateral wire electrodes into the nuchal muscles for EMG assessment. In Brown Norway rats, the EEG/EMG electrode leads were soldered to a miniature connector plug and fixed to the skull. In Wistar Kyoto rats, the EEG/EMG leads were tunneled subcutaneously to a telemetry transmitter implanted in the flank. Rats were allowed to recover from surgery for one week. Brown Norway rats were placed in PiezoSleep cages, and had their headsets connected to cable for recording EEG/EMG signals, which were then manually scored by a human scorer in 10-sec epochs. Wistar Kyoto rats were placed in PiezoSleep cages, and EEG/EMG signals were recorded using a telemetry system (DSI). Sleep was scored automatically in 4-sec epochs using NeuroScore software. PiezoSleep software recorded and scored sleep in the rats. Results: Rats implanted with corded EEG/EMG headsets had 85.6% concurrence of sleep-wake scoring with PiezoSleep. Rats implanted with EEG/EMG telemetry had 80.8% concurrence sleep-wake scoring with PiezoSleep. Sensitivity and specificity rates were similar between the EEG/EMG recording systems. Total sleep time and hourly sleep times did not differ in all three systems. However, automatic sleep detection by NeuroScore classified more sleep during the light period compared to the PiezoSleep. Conclusion: We showed that PiezoSleep system can be a reliable alternative to both automatic and visual EEG/EMG- based sleep-wake scoring in rat.

Update on the treatment of narcolepsy: clinical efficacy of pitolisant.

Narcolepsy is a neurological disease that affects 1 in 2,000 individuals and is characterized by excessive daytime sleepiness (EDS). In 60-70% of individuals with narcolepsy, it is also characterized by cataplexy or a sudden loss of muscle tone that is triggered by positive or negative emotions. Narcolepsy decreases the quality of life of the afflicted individuals. Currently used drugs treat EDS alone (modafinil/armodafinil, methylphenidate, and amphetamine), cataplexy alone ("off-label" use of antidepressants), or both EDS and cataplexy (sodium oxybate). These drugs have abuse, tolerability, and adherence issues. A greater diversity of drug options is needed to treat narcolepsy. The small molecule drug, pitolisant, acts as an inverse agonist/antagonist at the H3 receptor, thus increasing histaminergic tone in the wake promoting system of the brain. Pitolisant has been studied in animal models of narcolepsy and used in clinical trials as a treatment for narcolepsy. A comprehensive search of online databases (eg, Medline, PubMed, EMBASE, the Cochrane Library Database, Ovid MEDLINE, Europe PubMed Central, EBSCOhost CINAHL, ProQuest Research Library, Google Scholar, and ClinicalTrials.gov) was performed. Nonrandomized and randomized studies were included. This review focuses on the outcomes of four clinical trials of pitolisant to treat narcolepsy. These four trials show that pitolisant is an effective drug to treat EDS and cataplexy in narcolepsy.

Effects of Cannabinoid Agonists and Antagonists on Sleep and Breathing in Sprague-Dawley Rats.

Study Objectives: There are no pharmacological treatments for obstructive sleep apnea syndrome, but dronabinol showed promise in a small pilot study. In anesthetized rats, dronabinol attenuates reflex apnea via activation of cannabinoid (CB) receptors located on vagal afferents; an effect blocked by cannabinoid type 1 (CB1) and/or type 2 (CB2) receptor antagonists. Here, using a natural model of central sleep apnea, we examine the effects of dronabinol, alone and in combination with selective antagonists in conscious rats chronically instrumented to stage sleep and measure cessation of breathing. Methods: Adult male Sprague-Dawley rats were anesthetized and implanted with bilateral stainless steel screws into the skull for electroencephalogram recording and bilateral wire electrodes into the nuchal muscles for electromyogram recording. Each animal was recorded by polysomnography on multiple occasions separated by at least 3 days. The study was a fully nested, repeated measures crossover design, such that each rat was recorded following each of 8 intraperitoneal injections: vehicle; vehicle and CB1 antagonist (AM 251); vehicle and CB2 antagonist (AM 630); vehicle and CB1/CB2 antagonist; dronabinol; dronabinol and CB1 antagonist; dronabinol and CB2 antagonist; and dronabinol and CB1/CB2 antagonist. Results: Dronabinol decreased the percent time spent in rapid eye movement (REM) sleep. CB receptor antagonists did not reverse this effect. Dronabinol also decreased apneas during sleep, and this apnea suppression was reversed by CB1 or CB1/CB2 receptor antagonism. Conclusions: Dronabinol's effects on apneas were dependent on CB1 receptor activation, while dronabinol's effects on REM sleep were CB receptor-independent.

Forced Exercise Preconditioning Attenuates Experimental Autoimmune Neuritis by Altering Th1 Lymphocyte Composition and Egress.

A short-term exposure to moderately intense physical exercise affords a novel measure of protection against autoimmune-mediated peripheral nerve injury. Here, we investigated the mechanism by which forced exercise attenuates the development and progression of experimental autoimmune neuritis (EAN), an established animal model of Guillain-Barré syndrome. Adult male Lewis rats remained sedentary (control) or were preconditioned with forced exercise (1.2 km/day × 3 weeks) prior to P2-antigen induction of EAN. Sedentary rats developed a monophasic course of EAN beginning on postimmunization day 12.3 ± 0.2 and reaching peak severity on day 17.0 ± 0.3 (N = 12). By comparison, forced-exercise preconditioned rats exhibited a similar monophasic course but with significant (p < .05) reduction of disease severity. Analysis of popliteal lymph nodes revealed a protective effect of exercise preconditioning on leukocyte composition and egress. Compared with sedentary controls, forced exercise preconditioning promoted a sustained twofold retention of P2-antigen responsive leukocytes. The percentage distribution of pro-inflammatory (Th1) lymphocytes retained in the nodes from sedentary EAN rats (5.1 ± 0.9%) was significantly greater than that present in nodes from forced-exercise preconditioned EAN rats (2.9 ± 0.6%) or from adjuvant controls (2.0 ± 0.3%). In contrast, the percentage of anti-inflammatory (Th2) lymphocytes (7-10%) and that of cytotoxic T lymphocytes (∼20%) remained unaltered by forced exercise preconditioning. These data do not support an exercise-inducible shift in Th1:Th2 cell bias. Rather, preconditioning with forced exercise elicits a sustained attenuation of EAN severity, in part, by altering the composition and egress of autoreactive proinflammatory (Th1) lymphocytes from draining lymph nodes.

Cannabinoid type 1 and type 2 receptor antagonists prevent attenuation of serotonin-induced reflex apneas by dronabinol in Sprague-Dawley rats.

The prevalence of obstructive sleep apnea (OSA) in Americans is 9% and increasing. Increased afferent vagal activation may predispose to OSA by reducing upper airway muscle activation/patency and disrupting respiratory rhythmogenesis. Vagal afferent neurons are inhibited by cannabinoid type 1 (CB1) or cannabinoid type 2 (CB2) receptors in animal models of vagally-mediated behaviors. Injections of dronabinol, a non-selective CB1/CB2 receptor agonist, into the nodose ganglia reduced serotonin (5-HT)-induced reflex apneas. It is unknown what role CB1 and/or CB2 receptors play in reflex apnea. Here, to determine the independent and combined effects of activating CB1 and/or CB2 receptors on dronabinol's attenuating effect, rats were pre-treated with CB1 (AM251) and/or CB2 (AM630) receptor antagonists. Adult male Sprague-Dawley rats were anesthetized, instrumented with bilateral electrodes to monitor genioglossus electromyogram (EMGgg) and a piezoelectric strain gauge to monitor respiratory pattern. Following intraperitoneal treatment with AM251 and/or AM630, or with vehicle, serotonin was intravenously infused into a femoral vein to induce reflex apnea. After baseline recordings, the nodose ganglia were exposed and 5-HT-induced reflex apneas were again recorded to confirm that the nerves remained functionally intact. Dronabinol was injected into each nodose ganglion and 5-HT infusion was repeated. Prior to dronabinol injection, there were no significant differences in 5-HT-induced reflex apneas or phasic and tonic EMGgg before or after surgery in the CB1, CB2, combined CB1/CB2 antagonist, and vehicle groups. In the vehicle group, dronabinol injections reduced 5-HT-induced reflex apnea duration. In contrast, dronabinol injections into nodose ganglia of the CB1, CB2, and combined CB1/CB2 groups did not attenuate 5-HT-induced reflex apnea duration. However, the CB1 and CB2 antagonists had no effect on dronabinol's ability to increase phasic EMGgg. These findings underscore the therapeutic potential of dronabinol in the treatment of OSA and implicate participation of both cannabinoid receptors in dronabinol's apnea suppression effect.

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.

Innovative treatments for adults with obstructive sleep apnea.

Obstructive sleep apnea (OSA) affects one in five adult males and is associated with significant comorbidity, cognitive impairment, excessive daytime sleepiness, and reduced quality of life. For over 25 years, the primary treatment has been continuous positive airway pressure, which introduces a column of air that serves as a pneumatic splint for the upper airway, preventing the airway collapse that is the physiologic definition of this syndrome. However, issues with patient tolerance and unacceptable levels of treatment adherence motivated the exploration of other potential treatments. With greater understanding of the physiologic mechanisms associated with OSA, novel interventions have emerged in the last 5 years. The purpose of this article is to describe new treatments for OSA and associated complex sleep apnea. New approaches to complex sleep apnea have included adaptive servoventilation. There is increased literature on the contribution of behavioral interventions to improve adherence with continuous positive airway pressure that have proven quite effective. New non-surgical treatments include oral pressure devices, improved mandibular advancement devices, nasal expiratory positive airway pressure, and newer approaches to positional therapy. Recent innovations in surgical interventions have included laser-assisted uvulopalatoplasty, radiofrequency ablation, palatal implants, and electrical stimulation of the upper airway muscles. No drugs have been approved to treat OSA, but potential drug therapies have centered on increasing ventilatory drive, altering the arousal threshold, modifying loop gain (a dimensionless value quantifying the stability of the ventilatory control system), or preventing airway collapse by affecting the surface tension. An emerging approach is the application of cannabinoids to increase upper airway tone.

Forced-exercise attenuates experimental autoimmune neuritis.

Physical inactivity in combination with a sedentary lifestyle is strongly associated with an increased risk of development of inflammatory-mediated diseases, including autoimmune disorders. Recent studies suggest that anti-inflammatory effects of physical exercise may be of therapeutic value in some affected individuals. In this study, we determined the effects of forced-exercise (treadmill running) on the development and progression of experimental autoimmune neuritis (EAN), an established animal model of Guillain-Barré syndrome. Adult male Lewis rats were subjected to sedentary (control) or forced-exercise (1.2 km per day, 5 days a week) for three weeks prior to induction of EAN. P2 (53-78)-immunized sedentary control rats developed a monophasic course of EAN beginning on post-injection day 12.33 ± 0.59 (n = 18) and reaching peak severity on day 15.83 ± 0.35 (n = 18). At near peak of disease, ankle- and sciatic notch-evoked compound muscle action potential (CMAP) amplitudes in sedentary control rats were reduced (~50%) while motor nerve conduction velocity (MNCV) was slowed (~30%) compared with pre-induction evoked responses. In marked contrast, rats undergoing forced-exercise exhibited a significantly less severe clinical course of EAN beginning on post-injection day 12.63 ± 0.53 (n = 16) and reaching peaking severity on day 14.69 ± 0.73 (n = 16). At near peak of disease, ankle- and sciatic-notch-evoked CMAP amplitudes in forced-exercised rats were preserved while EAN-associated slowing of MNCV was modestly attenuated by exercise. Three weeks of forced-exercise reduced by 46% total plasma corticosterone content while elevating the levels of corticosteroid binding globulin. We conclude from this study that forced-exercise administered prior to and during development of EAN affords a novel measure of protection against autoimmune-associated deficits in peripheral nerve evoked responses independent of steroid-induced immune suppression.