Richard L. Horner

Obstructive sleep apnea as a cause of systemic hypertension. Evidence from a canine model.

Several epidemiological studies have identified obstructive sleep apnea (OSA) as a risk factor for systemic hypertension, but a direct etiologic link between the two disorders has not been established definitively. Furthermore, the specific physiological mechanisms underlying the association between OSA and systemic hypertension have not been identified. The purpose of this study was to systematically examine the effects of OSA on daytime and nighttime blood pressure (BP). We induced OSA in four dogs by intermittent airway occlusion during nocturnal sleep. Daytime and nighttime BP were measured before, during, and after a 1-3-mo long period of OSA. OSA resulted in acute transient increases in nighttime BP to a maximum of 13.0+/-2.0 mmHg (mean+/-SEM), and eventually produced sustained daytime hypertension to a maximum of 15.7+/-4.3 mmHg. In a subsequent protocol, recurrent arousal from sleep without airway occlusion did not result in daytime hypertension. The demonstration that OSA can lead to the development of sustained hypertension has considerable importance, given the high prevalence of both disorders in the population.

Afferent pathway(s) for pharyngeal dilator reflex to negative pressure in man: a study using upper airway anaesthesia.

1. To determine the afferent pathways mediating pharyngeal dilator muscle activation in response to negative airway pressure in man, we recorded genioglossus electromyogram (EMG) activity (via intra‐oral bipolar surface electrodes) in response to 500 ms duration pressure stimuli of ‐15 and ‐25 cm H2O in normal, conscious, supine subjects relaxed at end‐expiration; responses were compared before and after upper airway anaesthesia. 2. Six rectified and integrated EMG responses were bin averaged for pressure stimuli applied with the glottis open (GO) and closed (GC) and to the outside of the face only (controls). Response magnitude was quantified as the ratio of the EMG activity for an 80 ms post‐stimulus period (before the subjects reaction time for tongue protrusion) to an 80 ms pre‐stimulus period. 3. In eight subjects, upper airway anaesthesia reduced the EMG responses with GC to a level indistinguishable from controls. After anaesthesia, responses with GO remained higher than those with GC. 4. With GC, the mean EMG responses decreased by 43% after selective anaesthesia of the nasal mucosa (trigeminal nerves) in two subjects, 32% after selective anaesthesia of the laryngeal mucosa (superior laryngeal nerves) in six subjects and by 21% after selective anaesthesia of the oropharyngeal mucosa (glossopharyngeal and lingual nerves) in four subjects. 5. We conclude that upper airway afferents mediate pharyngeal dilator muscle activation in response to negative pressure with GC and that subglottal receptors caused the increased activation with GO. With GC, the trigeminal and superior laryngeal nerves mediate an important component of the responses with the glossopharyngeal nerves playing a less important role.

The effect of sleep on reflex genioglossus muscle activation by stimuli of negative airway pressure in humans.

The present study was designed to determine the effect of sleep on reflex pharyngeal dilator muscle activation by stimuli of negative airway pressure in human subjects. Intra‐oral bipolar surface electrodes were used to record genioglossus electromyogram (EMG) responses to 500 ms duration pressure stimuli of 0 and ‐25 cmH2O applied, via a face‐mask, in four normal subjects. Stimuli were applied during early inspiration in wakefulness and in periods of non‐rapid‐eye‐movement (non‐REM) sleep, defined by electroencephalographic (EEG) criteria. The rectified and integrated EMG responses to repeated interventions were bin averaged for the 0 and ‐25 cmH2O stimuli applied in wakefulness and sleep. Response latency was defined as the time when the EMG activity significantly increased above prestimulus levels. Response magnitude was quantified as the in ratio of the EMG activity for an 80 ms post‐stimulus period to an 80 ms prestimulus period; data from after the subjects voluntary reaction time for tongue protrusion (range, 150‐230 ms) were not analysed. Application of the ‐25 cmH2O stimuli caused genioglossus muscle activation in wakefulness and sleep, but in all subjects response magnitude was reduced in sleep (mean decrease, 61%; range, 52‐82%; P = 0.011, Students paired t test). In addition, response latency was increased in sleep in each subject (mean latency awake, 38 ms; range, 30‐50 ms; mean latency asleep, 75 ms; range, 40‐110 ms; P = 0.072, Students paired t test). Application of the ‐25 cmH2O stimuli caused arousal from sleep on 90% occasions, but in all cases the reflex genioglossus muscle responses (maximum latency, 110 ms) always proceeded any sign of EEG arousal (mean time to arousal, 643 ms; range, 424‐760 ms). These results show that non‐REM sleep attenuates reflex genioglossus muscle activation by stimuli of negative airway pressure. Attenuation of this reflex by sleep may impair the ability of the upper airway to defend itself from suction collapse by negative pressures generated during inspiration; this may have implications for the pathogenesis of obstructive sleep apnoea.

Microdialysis perfusion of 5-HT into hypoglossal motor nucleus differentially modulates genioglossus activity across natural sleep-wake states in rats

1 Serotonin (5‐hydroxytryptamine, 5‐HT) excites hypoglossal (XII) motoneurons in reduced preparations, and it has been suggested that withdrawal of 5‐HT may underlie reduced genioglossus (GG) muscle activity in sleep. However, systemic administration of 5‐HT agents in humans has limited effects on GG activity. Whether 5‐HT applied directly to the XII motor nucleus increases GG activity in an intact preparation either awake or asleep has not been tested. 2 The aim of this study was to develop a novel freely behaving animal model for in vivo microdialysis of the XII motor nucleus across sleep‐wake states, and test the hypothesis that 5‐HT application will increase GG activity. 3 Eighteen rats were implanted with electroencephalogram and neck muscle electrodes to record sleep‐wake states, and GG and diaphragm electrodes for respiratory muscle recording. Microdialysis probes were implanted into the XII motor nucleus and perfused with artificial cerebrospinal fluid (ACSF) or 10 mm 5‐HT. 4 Normal decreases in GG activity occurred from wakefulness to non‐rapid eye movement (non‐REM) and REM sleep with ACSF (P < 0.01). Compared to ACSF, 5‐HT caused marked GG activation across all sleep‐wake states (increases of 91‐251 %, P < 0.015). Importantly, 5‐HT increased sleeping GG activity to normal waking levels for as long as 5‐HT was applied (3‐5 h). Despite tonic stimulation by 5‐HT, periods of phasic GG suppression and excitation occurred in REM sleep compared with non‐REM. 5 The results show that sleep‐wake states differentially modulate GG responses to 5‐HT at the XII motor nucleus. This animal model using in vivo microdialysis of the caudal medulla will enable the determination of neural mechanisms underlying pharyngeal motor control in natural sleep.

Serotonin at the Laterodorsal Tegmental Nucleus Suppresses Rapid-Eye-Movement Sleep in Freely Behaving Rats

Serotonin [5-hydroxytryptamine (5-HT)] is believed to play an important inhibitory role in the regulation of rapid-eye-movement (REM) sleep. 5-HT may exert this effect on neurons of the laterodorsal tegmental (LDT) nuclei that are implicated as important in the generation of REM sleep and phasic REM events such as ponto-geniculo-occipital (PGO) waves and respiratory variability. In rat brainstem in vitro, 5-HT hyperpolarizes and inhibits the bursting properties of LDT neurons assumed to be involved in generating REM sleep and PGO waves. This study tests the hypothesis that in vivo 5-HT at the LDT nuclei suppresses REM sleep and phasic REM events. Ten rats were implanted with bilateral cannulae aimed at the LDT and with electrodes for recording the electroencephalogram, neck electromyogram, PGO waves, and diaphragm electromyogram. During REM sleep, 5-HT (100 nl; 1–1.5 mm), saline, or sham microinjections were performed; repeated microinjections were separated by ∼1 hr. After the first microinjection, REM sleep as a percent of the total sleep time was reduced with 5-HT (mean percent REM, 19.9 ± 2.5% for 5-HT vs 26.8 ± 2.4% for saline; p = 0.02). REM duration was reduced by 37% with 5-HT (p = 0.01), but REM episode frequency was changed less consistently (p = 0.21), suggesting that 5-HT mainly disrupted REM sleep maintenance. Per unit time of REM sleep, 5-HT had no effect on the amount or variability of REM PGO activity (p > 0.740) or on the mean or coefficient of variation of REM respiratory rate (p > 0.11). With subsequent microinjections, the effects of 5-HT on REM sleep were similar. A dose-dependent REM sleep suppression with 5-HT was observed in five rats tested. These data suggest that in vivo 5-HT at the LDT nuclei suppresses REM sleep expression. Although 5-HT did not disproportionately reduce the occurrence of phasic events within REM, total REM phasic activity was reduced because of less REM sleep after 5-HT.

The influence of induced hypocapnia and sleep on the endogenous respiratory rhythm in humans.

1. Ventilation has been studied during hypocapnia produced by passive mechanical ventilation in ten normal human subjects. 2. During wakefulness, disconnection of the ventilator led to inconsistent apnoea of only brief duration. During sleep, at a similar degree of hypocapnia, disconnection of the ventilator led more consistently to apnoea which was also of much longer duration; the deeper the sleep stage, the longer the apnoea. 3. The resumption of breathing during sleep could precede or follow arousal or be unaccompanied by arousal; in the absence of prior arousal, the evidence suggests that a starting end‐tidal CO2 pressure (PET, CO2) less than 41 mmHg could result in an apnoea during sleep stages I and II. 4. Subjects did not report any common sensation which led them to breathe following an apnoea whilst awake. 5. Prior hyperoxia in one subject prolonged the apnoea duration in both slow‐wave sleep and rapid eye movement sleep. 6. The results are interpreted as showing that even during light sleep, the maintenance of the respiratory rhythm is critically dependent on the arterial CO2 and O2 tensions. During wakefulness, other behavioural drives, which may not reach consciousness, supervene.

Role of inhibitory amino acids in control of hypoglossal motor outflow to genioglossus muscle in naturally sleeping rats

The hypoglossal motor nucleus innervates the genioglossus (GG) muscle of the tongue, a muscle that helps maintain an open airway for effective breathing. Rapid‐eye‐movement (REM) sleep, however, recruits powerful neural mechanisms that can abolish GG activity even during strong reflex stimulation such as by hypercapnia, effects that can predispose to sleep‐related breathing problems in humans. We have developed an animal model to chronically manipulate neurotransmission at the hypoglossal motor nucleus using in vivo microdialysis in freely behaving rats. This study tests the hypothesis that glycine receptor antagonism at the hypoglossal motor nucleus, either alone or in combination with GABAA receptor antagonism, will prevent suppression of GG activity in natural REM sleep during room air and CO2‐stimulated breathing. Rats were implanted with electroencephalogram and neck muscle electrodes to record sleep–wake states, and GG and diaphragm electrodes for respiratory muscle recording. Microdialysis probes were implanted into the hypoglossal motor nucleus for perfusion of artificial cerebrospinal fluid (ACSF) and strychnine (glycine receptor antagonist, 0.1 mm) either alone or combined with bicuculline (GABAA antagonist, 0.1 mm) during room air and CO2‐stimulated breathing. Compared to ACSF controls, glycine receptor antagonism at the hypoglossal motor nucleus increased respiratory‐related GG activity in room air (P= 0.010) but not hypercapnia (P= 0.221). This stimulating effect of strychnine in room air did not depend on the prevailing sleep–wake state (P= 0.625) indicating removal of a non‐specific background inhibitory glycinergic tone. Nevertheless, GG activity remained minimal in those REM sleep periods without phasic twitches in GG muscle, with GG suppression from non‐REM (NREM) sleep being > 85% whether ACSF or strychnine was at the hypoglossal motor nucleus or the inspired gas was room air or 7% CO2. While GG activity was minimal in these REM sleep periods, there was a small but measurable increase in GG activity after strychnine (P < 0.05). GG activity was also minimal, and effectively abolished, in the REM sleep periods without GG twitches with combined glycine and GABAA receptor antagonism at the hypoglossal motor nucleus. We conclude that these data in freely behaving rats confirm that inhibitory glycine and GABAA receptor mechanisms are present at the hypoglossal motor nucleus and are tonically active, but that such inhibitory mechanisms make only a small contribution to the marked suppression of GG activity and reflex responses observed in periods of natural REM sleep.

PreBötzinger Complex Neurokinin-1 Receptor-Expressing Neurons Mediate Opioid-Induced Respiratory Depression

The analgesic properties of the opium poppy Papever somniferum were first mentioned by Hippocrates around 400 BC, and opioid analgesics remain the mainstay of pain management today. These drugs can cause the serious side-effect of respiratory depression that can be lethal with overdose, however the critical brain sites and neurochemical identity of the neurons mediating this depression are unknown. By locally manipulating neurotransmission in the adult rat, we identify the critical site of the medulla, the preBötzinger complex, that mediates opioid-induced respiratory depression in vivo. Here we show that opioids at the preBötzinger complex cause respiratory depression or fatal apnea, with anesthesia and deep-sleep being particularly vulnerable states for opioid-induced respiratory depression. Importantly, we establish that the preBötzinger complex is fully responsible for respiratory rate suppression following systemic administration of opioid analgesics. The site in the medulla most sensitive to opioids corresponds to a region expressing neurokinin-1 receptors, and we show in rhythmically active brainstem section in vitro that neurokinin-1 receptor-expressing preBötzinger complex neurons are selectively inhibited by opioids. In summary, neurokinin-1 receptor-expressing preBötzinger complex neurons constitute the critical site mediating opioid-induced respiratory rate depression, and the key therapeutic target for its prevention or reversal.

Opioid receptor mechanisms at the hypoglossal motor pool and effects on tongue muscle activity in vivo

Opioids can modulate breathing and predispose to respiratory depression by actions at various central nervous system sites, but the mechanisms operating at respiratory motor nuclei have not been studied. This study tests the hypotheses that (i) local delivery of the μ‐opioid receptor agonist fentanyl into the hypoglossal motor nucleus (HMN) will suppress genioglossus activity in vivo, (ii) a component of this suppression is mediated by opioid‐induced acetylcholine release acting at muscarinic receptors, and (iii) δ‐ and κ‐opioid receptors also modulate genioglossus activity. Seventy‐two isoflurane‐anaesthetised, tracheotomised, spontaneously breathing rats were studied during microdialysis perfusion into the HMN of (i) fentanyl and naloxone (μ‐opioid receptor antagonist), (ii) fentanyl with and without co‐application of muscarinic receptor antagonists, and (iii) δ‐ and κ‐opioid receptor agonists and antagonists. The results showed (i) that fentanyl at the HMN caused a suppression of genioglossus activity (P < 0.001) that reversed with naloxone (P < 0.001), (ii) that neither atropine nor scopolamine affected the fentanyl‐induced suppression of genioglossus activity, and (iii) that δ‐, but not κ‐, opioid receptor stimulation also suppressed genioglossus activity (P= 0.036 and P= 0.402 respectively). We conclude that μ‐opioid receptor stimulation suppresses motor output from a central respiratory motoneuronal pool that activates genioglossus muscle, and this suppression does not involve muscarinic receptor‐mediated inhibition. This μ‐opioid receptor‐induced suppression of tongue muscle activity by effects at the hypoglossal motor pool may underlie the clinical concern regarding adverse upper airway function with μ‐opioid analgesics. The inhibitory effects of μ‐ and δ‐opioid receptors at the HMN also indicate an influence of endogenous enkephalins and endorphins in respiratory motor control.

The neuropharmacology of upper airway motor control in the awake and asleep states: implications for obstructive sleep apnoea

Obstructive sleep apnoea is a common and serious breathing problem that is caused by effects of sleep on pharyngeal muscle tone in individuals with narrow upper airways. There has been increasing focus on delineating the brain mechanisms that modulate pharyngeal muscle activity in the awake and asleep states in order to understand the pathogenesis of obstructive apnoeas and to develop novel neurochemical treatments. Although initial clinical studies have met with only limited success, it is proposed that more rational and realistic approaches may be devised for neurochemical modulation of pharyngeal muscle tone as the relevant neurotransmitters and receptors that are involved in sleep-dependent modulation are identified following basic experiments.