Hattie Liu

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.

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.

GABAA receptor antagonism at the hypoglossal motor nucleus increases genioglossus muscle activity in NREM but not REM sleep

The pharyngeal muscles, such as the genioglossus (GG) muscle of the tongue, are important for effective lung ventilation since they maintain an open airspace. Rapid‐eye‐movement (REM) sleep, however, recruits powerful neural mechanisms that can abolish GG activity, even during strong reflex respiratory stimulation by elevated CO2. In vitro studies have demonstrated the presence of GABAA receptors on hypoglossal motoneurons, and these and other data have led to the speculation that GABAA mechanisms may contribute to the suppression of hypoglossal motor outflow to the GG muscle in REM sleep. We have developed an animal model that allows us to chronically manipulate neurotransmission at the hypoglossal motor nucleus using microdialysis across natural sleep‐wake states in rats. The present study tests the hypothesis that microdialysis perfusion of the GABAA receptor antagonist bicuculline into the hypoglossal motor nucleus will prevent the suppression of GG muscle activity in REM sleep during both room‐air and CO2‐stimulated breathing. Ten 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) or 100 μm bicuculline during room‐air and CO2‐stimulated breathing (7 % inspired CO2). GABAA receptor antagonism at the hypoglossal motor nucleus increased respiratory‐related GG activity during both room‐air (P= 0.01) and CO2‐stimulated breathing (P= 0.007), indicating a background inhibitory GABA tone. However, the effects of bicuculline on GG activity depended on the prevailing sleep‐wake state (P < 0.005), with bicuculline increasing GG activity in non‐REM (NREM) sleep and wakefulness both in room air and hypercapnia (P < 0.01), but GG activity was effectively abolished in those REM periods without phasic twitches in the GG muscle. This abolition of GG activity in REM sleep occurred regardless of ACSF or bicuculline at the hypoglossal motor nucleus, or room‐air or CO2‐stimulated breathing (P > 0.63). We conclude that these data in freely behaving rats confirm previous in vitro studies that GABAA receptor mechanisms are present at the hypoglossal motor nucleus and are tonically active, but the data also show that GABAA receptor antagonism at the hypoglossal motor nucleus does not increase GG muscle activity in natural REM sleep.

Opposing muscarinic and nicotinic modulation of hypoglossal motor output to genioglossus muscle in rats in vivo

The genioglossus (GG) muscle of the tongue, innervated by the hypoglossal motor nucleus (HMN), helps maintain an open airway for effective breathing. In vitro studies in neonatal rodents have separately characterized muscarinic and nicotinic receptor influences at the HMN but the net effects of combined nicotinic and muscarinic receptor activation and increased endogenous acetylcholine have not been determined in adult animals in vivo. Urethane‐anaesthetized, tracheotomized and vagotomised rats were studied. Microdialysis perfusion of acetylcholine into the HMN significantly decreased respiratory‐related GG activity (28.5 ± 11.0% at a threshold dose of 0.1 mm). Application of the cholinergic agonists carbachol and muscarine have similar suppression effects (GG activity was decreased 11.8 ± 4.3 and 20.5 ± 5.8%, respectively, at 0.01 μm). Eserine, an acetylcholinesterase inhibitor, also decreased the amplitude of respiratory‐related GG activity (36.4 ± 11.3% at 1.0 μm) indicating that endogenous acetylcholine modulates GG activity. Although these results showed that suppression of GG activity predominates during cholinergic stimulation at the HMN, application of the nicotinic receptor agonist dimethyl‐4‐phenylpiperazinium iodide significantly increased tonic and respiratory‐related GG activity (156 ± 33% for respiratory activity at 1.0 mm) showing that excitatory responses are also present. Consistent with this, 100 μm carbachol decreased GG activity by 44.2 ± 7.5% of control, with atropine (10 μm) reducing this suppression to 13.8 ± 4.0% (P < 0.001). However, the nicotinic receptor antagonist dihydro‐β‐erythroidine (100 μm) increased the carbachol‐mediated suppression to 69.5 ± 5.9% (P= 0.011), consistent with a role for nicotinic receptors in limiting the overall suppression of GG activity during cholinergic stimulation. Application of eserine to increase endogenous acetylcholine also showed that inhibitory muscarinic and excitatory nicotinic receptors together determine the net level of GG activity during cholinergic stimulation at the HMN. The results suggest that acetylcholine has mixed effects at the HMN with muscarinic‐mediated GG suppression masking nicotinic excitation.

5-HT at hypoglossal motor nucleus and respiratory control of genioglossus muscle in anesthetized rats

Serotonin (5-HT) from medullary raphe neurons excites hypoglossal motoneurons innervating genioglossus (GG) muscle. Since some raphe neurons also show increased activity in hypercapnia, we tested the hypothesis that serotonergic mechanisms at the hypoglossal motor nucleus (HMN) modulate GG activity and responses to CO2. Seventeen urethane-anesthetized, tracheotomized and vagotomized rats were studied. Microdialysis probes were used to deliver mianserin (5-HT receptor antagonist, 0 and 0.1 mM) or 5-HT (eight doses, 0-50 mM) to the HMN during room air or CO2-stimulated breathing. Mianserin decreased respiratory-related GG activity during room air and CO2-stimulated breathing (P<0.001), and also suppressed GG responses to CO2 (P=0.05). In contrast, GG activity was increased by 5-HT at the HMN, and was further increased in hypercapnia (P<0.02). However, 5-HT increased respiratory-related GG activity at levels lower (1 mM) than those eliciting tonic GG activity (10-30 mM 5-HT). The results show that 5-HT at the HMN contributes to the respiratory control of GG muscle.

Endogenous glutamatergic control of rhythmically active mammalian respiratory motoneurons in vivo.

The transmission of rhythmic drive to respiratory motoneurons in vitro is critically dependent on glutamate acting primarily on non-NMDA receptors. We determined whether both non-NMDA and NMDA receptors contribute to respiratory drive transmission at respiratory motoneurons in the intact organism, both in the state of anesthesia and in the same animals during natural behaviors. Twenty-seven rats were implanted with electroencephalogram and neck electrodes to record sleep–wake states and genioglossus and diaphragm electrodes for respiratory muscle recordings. Microdialysis probes were inserted into the hypoglossal motor nucleus (HMN). Under anesthesia, non-NMDA or NMDA receptor antagonism significantly decreased respiratory-related genioglossus activity, indicating a contribution of each receptor to respiratory drive transmission at the HMN. However, despite the presence of respiratory-related genioglossus activity in the same rats across sleep–wake states, neither non-NMDA receptor antagonism at the HMN nor glutamate uptake inhibition had any effect on respiratory-related genioglossus activity. These results showed that, compared with anesthesia, respiratory drive transmission through the non-NMDA receptor is low in the behaving organism. In contrast, glutamate uptake inhibition increased tonic genioglossus activity in wakefulness and non-rapid-eye-movement sleep, indicating a functional endogenous glutamatergic modulation of tonic, but not respiratory, motor tone. Such an effect on tonic drive may contribute to the suppression of both tonic and respiratory-related genioglossus activity in wakefulness and sleep with NMDA receptor antagonism at the HMN. These data do not refute previous identification of a glutamatergic (mostly non-NMDA receptor activating) respiratory drive to hypoglossal motoneurons, but this mechanism is more prominent in anesthetized or in vitro preparations.

Respiratory activation of the genioglossus muscle involves both non-NMDA and NMDA glutamate receptors at the hypoglossal motor nucleus in vivo

Brainstem respiratory neurons innervate the hypoglossal motor nucleus which in turn transmits this respiratory drive signal to the genioglossus muscle of the tongue. The mechanism of this transmission is important to help maintain an open airspace for effective breathing, and is thought to rely almost exclusively on non-N-methyl-d-aspartate (non-NMDA) glutamate receptor activation during respiration. However those studies were performed in slices of medulla from neonatal animals in vitro which may have led to an underestimation of the contribution of NMDA glutamate receptors that may normally operate in intact preparations. The current study tests the hypothesis that both NMDA and non-NMDA receptors contribute to respiratory drive transmission at the hypoglossal motor nucleus in vivo. Experiments were performed in urethane-anesthetized and tracheotomized adult Wistar rats in which vagus nerves were either intact or sectioned. In the presence of augmented genioglossus activity produced by vagotomy, microdialysis perfusion of either an NMDA receptor antagonist (D-2-amino-5-phosphonovaleric acid, 0.001-10 mM) or a non-NMDA receptor antagonist (6-cyano-7-nitroquinoxaline-2, 3-dione disodium salt, 0.001-1 mM) to the hypoglossal motor nucleus reduced respiratory-related genioglossus activity in a dose-dependent manner (P < 0.001) indicating that both NMDA and non-NMDA glutamate receptors are necessary for transmission of the respiratory drive signal to genioglossus muscle in vivo. Similar effects were observed in the vagus nerve intact rats. Further experiments demonstrated that each delivered antagonist had effects that were specific to its respective receptor. Regression analysis also revealed that the activity of both NMDA and non-NMDA receptors at the hypoglossal motor nucleus is related to levels of the prevailing respiratory drive. These results show that both NMDA and non-NMDA glutamate receptors at the hypoglossal motor nucleus are involved in transmission of the respiratory drive signal to genioglossus muscle in vivo.

Suppression of genioglossus muscle tone and activity during reflex hypercapnic stimulation by GABAa mechanisms at the hypoglossal motor nucleus in vivo

The genioglossus muscle is involved in the maintenance of an open airway for effective breathing. Inhibitory neurotransmitters may be responsible for the major suppression of hypoglossal motor output to genioglossus muscle that occurs in certain behaviours such as rapid-eye-movement sleep. There is evidence for GABA(A) receptor-mediated inhibition of hypoglossal motoneurons in vitro. However, comparable studies have not been performed in vivo and the interactions of such mechanisms with integrative reflex respiratory control have also not been determined. Urethane-anaesthetised, tracheotomized and vagotomized rats were studied whilst diaphragm and genioglossus muscle activities, blood pressure and the electroencephalogram were recorded. Microdialysis probes were implanted into the hypoglossal motor nucleus, with sites verified by histology. Genioglossus responses to microdialysis perfusion of muscimol (GABA(A) agonist: 0, 0.1, 1 and 10 microM in artificial cerebrospinal fluid) were recorded at inspired CO(2)s of 0, 5 and 7.5% in six rats. Responses to bicuculline (GABA(A) antagonist, 0, 1, 10, 100 and 1000 microM) were also studied in six rats with and without CO(2) stimulation. Genioglossus activity decreased with muscimol (P<0.0001), with major suppression at 1 and 10 microM during air breathing (decreases=70.2% and 92.8%, P<0.005). Genioglossus activity increased with CO(2) (P=0.003), but genioglossus activation with 5 and 7.5% CO(2) were almost abolished with 10-microM muscimol. Responses were specific to genioglossus muscle as there were no changes in diaphragm, respiratory rate or blood pressure with muscimol (P>0.144). Antagonism of GABA(A) receptors increased genioglossus activity (P<0.001). These results show that GABA(A) receptor stimulation at the hypoglossal motor nucleus suppresses both genioglossus muscle tone and activity in the presence of reflex stimulation produced by hypercapnia. Recruitment of such mechanisms may contribute to the major suppression of genioglossus activity observed with and without CO(2) stimulation in behaviours such as rapid-eye-movement sleep.