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Departments of Medicine, Pharmacology, and Physiology, University of Toronto
Division of Respirology, Toronto General Hospital Research Institute of the University Health Network, Toronto General Hospital, Toronto, Canada
ABSTRACT
Rationale: Previous studies modulating pharyngeal muscle activity with pharmacologic approaches have targeted membrane receptors on pharyngeal motoneurons. Whether modulation of intracellular pathways can increase pharyngeal muscle activity, however, has not been investigated but is relevant to pharmacologic treatments of obstructive sleep apnea.
Objectives: To determine if modulating the second messenger cyclic adenosine-3'-5'-monophosphate (cAMP) at the hypoglossal motor nucleus (HMN) will increase genioglossus activity across sleep– wake states.
Methods: Forty-eight 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 HMN to perfuse artificial cerebrospinal fluid and (1) forskolin (500 μM, adenylyl cyclase activator to increase cAMP), (2) a cAMP analog (500 μM), (3) iso-butyl-methylxanthine (IBMX; 300 μM, phosphodiesterase inhibitor), or (4) a cyclic guanosine-3'-5'-monophosphate (cGMP) analog (500 μM, 8-Br-cGMP).
Measurements and Main Results: Forskolin and the cAMP analog at the HMN increased respiratory-related and tonic genioglossus activities in wakefulness and non–REM sleep but not REM sleep. IBMX did not affect genioglossus activity in awake or sleeping rats. However, IBMX abolished the robust excitatory responses to serotonin and phenylephrine at the HMN, but responses to non-N-methyl-D-aspartate receptor activation remained. These effects of IBMX were mimicked by 8-Br-cGMP.
Conclusions: Genioglossus responses to manipulation of cAMP at the HMN are differentially modulated by sleep–wake state. Selective abolition of serotonin and phenylephrine responses after IBMX suggests that under conditions of nonspecific phosphodiesterase inhibition the HMN is unresponsive to certain, otherwise potent, excitatory inputs. Similar responses with 8-Br-cGMP suggest this effect is likely mediated by cGMP pathways.
Key Words: cAMP hypoglossal motor nucleus obstructive sleep apnea sleep
Second-messenger pathways play a critical role in regulating neuronal activity (1). Cyclic adenosine-3'-5'-monophosphate (cAMP) is an important intracellular second messenger associated with G protein–coupled receptors (GPCRs) (2, 3). Stimulatory GPCRs activate adenylyl cyclase, which increases intracellular levels of cAMP and protein kinase A (PKA) activity, whereas inhibitory GPCRs suppress adenylyl cyclase activity. Raising levels of intracellular cAMP increases the likelihood of neuronal excitation in a variety of model systems (4–8) but few studies have been performed at respiratory neurons and none at respiratory motoneurons in vivo. Studies using slices of neonatal brainstem showed that activation of the cAMP–PKA pathway increased hypoglossal motoneuron and respiratory neuron activities in vitro (9–11). In anesthetized animals in vivo, increasing cAMP–PKA activity increased activity of medullary respiratory neurons (12, 13). However, the effects of focally manipulating cAMP at a defined respiratory motoneuron pool on motor outflow to respiratory muscle have not been determined in freely behaving animals in vivo. Moreover, whether such motor responses to manipulation of intracellular signal-transduction mechanisms, normally studied in reduced preparations in vitro, are modulated by the prevailing sleep–wake states in vivo is unknown.
This study tests the hypothesis that activation of cAMP at the hypoglossal motor nucleus (HMN), the source of motor outflow to the genioglossus (GG) muscle of the tongue, will increase GG activity in wakefulness, non-REM sleep, and REM sleep. The GG muscle was studied because, in conjunction with other pharyngeal muscles (14), it helps maintain an open airway for effective breathing and prevent obstructive sleep apnea (OSA) (15), a common and serious sleep-related respiratory disorder affecting approximately 4% of adults (16, 17). Previous studies modulating pharyngeal muscle activity across natural sleep–wake states have targeted membrane receptors on pharyngeal motoneurons via local application of neurotransmitters (18–20) or systemic drug administration—for example, selective serotonin reuptake inhibitors in humans (21–30). Many of these interventions manipulate extracellular levels of neurotransmitters that act via GPCRs on pharyngeal motoneurons. However, none of these interventions have shown clinically significant beneficial effects, especially in REM sleep where OSA typically persists. Whether modulation of intracellular signaling molecules can increase pharyngeal muscle activity, however, has not been investigated but is potentially relevant to pharmacologic treatments of OSA as it may allow for sustained pharyngeal motoneuron activation despite changes in extracellular neurotransmitters across sleep–wake states.
METHODS
The methods are described in more detail in the online supplement. Experiments were performed on 48 male Wistar rats (mean body weight = 271 g, range = 251–315 g). Procedures conformed to the recommendations of the Canadian Council on Animal Care, and the University of Toronto Animal Care Committee approved the experimental protocols.
Anesthesia and Surgical Procedures
Sterile surgery was performed under general anesthesia for the chronic implantation of EEG and neck EMG electrodes for determination of sleep–wake states, and GG and diaphragm electrodes for respiratory-muscle recordings (18–20, 31, 32). Tests for the accurate placement of the GG electrodes and their function throughout the experiments are described in the online supplement. During surgery, microdialysis guides were targeted 3 mm above the HMN (18–20, 31). The rats recovered for an average of 6.8 d (range = 5–8 d) before the studies.
Protocol
On the day of the studies, the microdialysis probes were inserted into the HMN and flushed with artificial cerebrospinal fluid (ACSF) at a flow rate of 2.1 μl/min. In the first set of experiments in nine rats, a water- soluble derivative of forskolin (7-deacetylforskolin-7-hemisuccinate, 500 μM) was also applied to the HMN. Forskolin is an adenylyl cyclase activator that stimulates production of intracellular cAMP (11, 13, 33–35). In a second set of experiments in nine rats, increases in cAMP were produced by addition of a cAMP analog to the HMN (cAMP sodium salt, 500 μM). In a third set of experiments in nine rats, the nonspecific cyclic nucleotide phosphodiesterase (PDE) inhibitor iso-butyl methylxanthine (IBMX; 300 μM) was applied to the HMN to prevent the breakdown of endogenous cAMP.
At the end of each individual experiment, 10 mM 5-hydroxytryptamine (serotonin, or 5-HT) was applied to the HMN as a positive control to confirm that it was still functional and able to respond to manipulation of neurotransmission (18–20). For reasons described in RESULTS, additional experiments were conducted after IBMX perfusion using the glutamate receptor agonist (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl) propionic acid (AMPA; 100 μM, n = 11 rats) and the 1-adrenergic receptor agonist phenylephrine (100 μM, n = 8 rats). Furthermore, because IBMX is a nonspecific PDE inhibitor and prevents the breakdown of both endogenous cAMP and cyclic guanosine-3',5'-cyclic monophosphate (cGMP), a final set of experiments was conducted to test the influence of the cGMP analog, 8-bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP; 500 μM, n = 4 rats), on GG activity and the responses to 5-HT, phenylephrine, and AMPA at the HMN. After each experiment the rats were reanesthetized and tests for GG electrode function were performed before the microdialysis sites were confirmed by histology.
Data Analysis
Sleep–wake states and respiratory-muscle activities were analyzed as previously described (19, 20, 31, 32) and detailed in the online supplement. GG activity was quantified as mean tonic activity, respiratory-related activity, and the overall geometric mean (i.e., the mean of all sampled data points constituting the GG signal including the tonic and respiratory-related components). Statistics were performed using analysis of variance with repeated measures (ANOVA-RM) and post hoc t tests were performed using Bonferroni corrected p values. All data are expressed as mean ± SEM.
RESULTS
Location of Microdialysis Probes
Figure 1A shows a lesion site made by a microdialysis probe in the HMN in one rat. Figure 1 also shows the distribution of individual microdialysis sites from all rats that were administered forskolin (Figure 1B; n = 9 rats), the cAMP analog (Figure 1C; n = 9 rats), and IBMX (Figure 1D; n = 9 rats). Microdialysis probes were successfully implanted into, or immediately adjacent to, the HMN in all animals. In each rat tongue, movement was observed in response to electrical stimulation of the GG electrodes both at the time of surgery and after the experiments, showing that these electrodes were in place throughout the study.
Responses to Forskolin at the HMN
Periods of wakefulness, non-REM sleep, and REM sleep were analyzed in all nine animals with the exception of REM sleep in one rat that did not have REM sleep in the presence of forskolin. Figure 2 shows an example of GG responses to microdialysis perfusion of forskolin into the HMN. Compared to ACSF controls, forskolin produced robust increases in respiratory-related and tonic GG activities in wakefulness and non-REM sleep. However, forskolin was unable to overcome the strong suppression of hypoglossal motor output to GG muscle in REM sleep and GG activity was similar to that during ACSF. Brief GG twitches in REM sleep occurred with both ACSF and forskolin. Such twitches, typical of REM sleep, have been observed previously in rats (18–20) as well as in other species (36) and are thought to be responsible for the periodic restorations of airflow during obstructive apneas in humans (37).
The grouped data in Figure 3A show that forskolin at the HMN increased respiratory-related GG activity compared with that of ACSF controls. Statistical analysis established that there was a significant effect of forskolin on GG activity (F1,8 = 6.95, p = 0.030, ANOVA-RM) with increased activity compared with ACSF (t8 = 2.62, p = 0.031, post hoc t test). Nevertheless, the effect of forskolin on GG activity was dependent on the prevailing sleep–wake states (F2,15 = 4.051, p = 0.039, ANOVA-RM). Post hoc analyses confirmed that forskolin increased respiratory-related GG activity during wakefulness (t8 = 3.43, p = 0.004, post hoc t test) and non-REM sleep (t8 = 2.58, p = 0.022) but not during REM sleep (t7 = 0.73, p = 0.478).
Figure 3B illustrates that forskolin at the HMN also increased tonic GG activity (F1,8 = 9.46, p = 0.015, ANOVA-RM) compared with ACSF (t8 = 3.05, p = 0.016). The effect of forskolin on tonic GG activity depended on sleep–wake state (F2,15 = 8.12, p = 0.004, ANOVA-RM) with increased tonic activity in wakefulness (t8 = 4.11, p = 0.002) and non-REM sleep (t8 = 3.17, p = 0.009) but not during REM sleep (t7 = 1.07, p = 0.305).
The latency to an increase in GG activity after a switch to forskolin was 16.5 ± 2.1 min. Once activated, GG activity remained elevated for as long as forskolin was applied (range = 110 to 350 min in the different experiments, mean = 245 ± 27 min) and returned to baseline levels approximately 1 h after a switch back to ACSF (range = 55 to 85 min, mean = 73 ± 3 min). At this time, responses to 5-HT, a known excitatory neurotransmitter at the HMN (18, 20, 38), were tested (see STIMULATION OF THE HMN WITH SEROTONIN).
Responses to forskolin at the HMN were specific to the GG muscle as there were no statistically significant effects on respiratory rate (F1,8 = 2.42, p = 0.158, ANOVA-RM), diaphragm amplitude (F1,8 = 0.281, p = 0.610), or neck EMG activity (F1,8 = 5.21, p = 0.051).
Responses to the cAMP Analog at the HMN
Periods of wakefulness, non-REM sleep, and REM sleep were analyzed in all nine animals with the exception of REM sleep for one rat that did not have REM sleep in the presence of the cAMP analog. The grouped data in Figure 3C shows that administration of the cAMP analog to the HMN increased respiratory-related GG activity compared with that of ACSF controls. There was a significant effect of the cAMP analog on GG activity (F1,8 = 9.90, p = 0.013, ANOVA-RM), with increased activity compared with ACSF (t8 = 3.13, p = 0.014, post hoc t test). The effects of the cAMP analog on GG activity, however, were dependent on the prevailing sleep–wake state (F2,15 = 4.06, p = 0.039, ANOVA-RM), with increased respiratory-related activity during wakefulness (t8 = 2.66, p = 0.015) and non-REM sleep (t8 = 3.85, p < 0.001) but not REM sleep (t7 = 1.92, P = 0.850). Figure 3D also illustrates that the effect of the cAMP analog on tonic GG activity was dependent on sleep–wake state (F2,15 = 3.87, p = 0.044, ANOVA-RM) with increased tonic GG activity during non–REM sleep (t8 = 3.43, p = 0.003) but not during wakefulness (t8 = 1.62, p = 0.121) or REM sleep (t7 = 0.164, p = 0.871).
The latency to an increase in GG activity with the cAMP analog was 12.2 ± 1.0 min. However, unlike responses to forskolin in which GG activity remained elevated for as long as forskolin was applied (mean = 245 ± 27 min), GG activity with the cAMP analog remained elevated for approximately 2 h (mean = 142 ± 12 min, range = 102 to 155 min) after which time GG activity declined, but still remained elevated compared with ACSF. This difference in duration of response with forskolin and the cAMP analog was statistically significant (t16 = 3.44, p = 0.003, unpaired t test). After a switch back to ACSF, GG muscle activity returned to baseline levels after approximately 1 h (range = 45 to 85 min, mean = 56 ± 4 min) at which time responses to application of 5-HT were tested (see below).
Responses to application of the cAMP analog to the HMN were specific to the GG muscle as there were no effects on respiratory rate (F1,8 = 0.589, p = 0.464, ANOVA-RM), diaphragm amplitude (F1,8 = 0.819, p = 0.391), or neck EMG activity (F1,8 = 0.841, p = 0.385).
Responses to IBMX at the HMN
Periods of wakefulness, non-REM sleep, and REM sleep were analyzed in all nine animals with the exception of REM sleep for one rat that did not have REM sleep in the presence of IBMX. Figures 3E and 3F show that compared with ACSF, IBMX at the HMN did not affect respiratory-related GG activity (F1,8 = 0.118, p = 0.740, ANOVA-RM) or tonic GG activity (F1,8 = 1.17, p = 0.311, ANOVA-RM). After administration of IBMX, the perfusion medium was switched back to ACSF for approximately 1 h (range = 40 to 120 min, mean = 67 ± 9 min) before testing of the ability of the HMN to respond to manipulation of neurotransmission (see below).
IBMX at the HMN had no significant effects on respiratory rate (F1,8 = 0.197, p = 0.668, ANOVA-RM) or diaphragm amplitude (F1,8 = 0.716, p = 0.422). However, neck EMG activity with IBMX was different from that with ACSF (F1,8 = 12.47, p = 0.008), an effect that was dependent on sleep–wake state (F1,8 = 17.40, p < 0.001). A significant decrease in neck EMG was observed in wakefulness (t8 = 6.64, p < 0.001, post hoc paired t test) although no differences were observed in non-REM (t8 = 1.08, p = 0.293) or REM sleep (t7 = 0.289, p = 0.776).
Stimulation of the HMN with Serotonin
For each rat in each experiment (i.e., forskolin, cAMP analog, and IBMX treatments) 10 mM serotonin was applied to the HMN after a switch back to ACSF to confirm an intact HMN that was able to respond to manipulation of neurotransmission (18–20). Mean GG activity, rather than tonic or respiratory-related activity, was used to quantify these effects because the aim was simply to determine if GG activity increased with 5-HT at the HMN. We have previously shown that 5-HT at the HMN mainly increases tonic GG activity but respiratory-related activity can also increase (18, 39); analysis of the mean for these experiments aiming to confirm an intact HMN takes into account both of these possibilities.
Figure 4 shows examples of GG responses in non-REM sleep to 5-HT at the HMN. Non-REM sleep was chosen for these comparisons as it is the most stable physiologic state and reliably occurred in all rats at the end of the experiments. In the presence of ACSF after washout of forskolin, 5-HT produced robust increases in GG activity in all rats (Figure 4A) with a latency of 6.7 ± 1.0 min, a lag time consistent with previous studies (18, 20). Similar excitatory responses to 5-HT occurred in each rat after the cAMP analog (Figure 4B).
However, in contrast to the forskolin and cAMP experiments in which all animals responded positively to 5-HT, none of the nine animals treated with IBMX showed increased GG activity with 5-HT at the HMN across any sleep–wake state. Figure 4C shows such a response. Group GG responses to 5-HT in the different experiments are shown in Figure 5A; 5-HT at the HMN significantly increased GG activity after forskolin (t8 = 9.37, p < 0.001, paired t test) and the cAMP analog (t8 = 3.42, p = 0.009) but not IMBX (t8 = 1.15, p = 0.285).
Although GG activity in the presence of IBMX was unchanged compared with that of ACSF controls across sleep–wake states (Figures 3E–3F), further interventions were performed to confirm that the HMN was intact and still able to respond to neuromodulation. Given that 5-HT excites hypoglossal motoneurons via metabotropic 5-HT2 receptors (40, 41), further studies were performed in an additional three rats after IBMX in which 5-HT responses were compared with responses after application of AMPA to the HMN (Figure 5B); AMPA is an agonist for ionotropic non-N-methyl-D-aspartate (non-NMDA) glutamate receptors that excite hypoglossal motoneurons (34, 42). Figure 5B shows that although these animals were unresponsive to 5-HT following IBMX (t2 = 0.310, p = 0.786, paired t test), AMPA produced robust increases in GG activity (t2 = 4.64, p = 0.043; Figure 5B) with a latency of 12.6 ± 2.1 min. These data suggested that IBMX prevented excitation of hypoglossal motoneurons via effects on metabotropic 5-HT–mediated responses but not those associated with ionotropic mechanisms mediated by AMPA.
To determine whether the IBMX effects were limited to pathways associated with metabotropic 5-HT receptors, additional studies were performed in five rats in which responses to phenylephrine were also compared with AMPA after IBMX (Figure 5C). Phenylephrine is an excitatory neurotransmitter at the HMN (43), and in these experiments phenylephrine increased GG activity by 82 ± 10% (t2 = 7.85, p = 0.016, paired t test, data not shown). After IBMX, however, each animal was also unresponsive to phenylephrine at the HMN (t4 = 1.62, p = 0.181, paired t test; Figure 5C), similar to the lack of response to 5-HT after IBMX, whereas AMPA again produced robust activation (t4 = 2.89, p = 0.045; Figure 5C). Phenylephrine is an agonist for metabotropic 1-adrenergic receptors that converge onto the same intracellular pathways as 5-HT2 receptors (34, 44). Accordingly, these data suggested that IBMX, which prevents the breakdown of endogenous cAMP by PDE, abolished excitatory responses associated with these metabotropic receptors while preserving excitatory responses to ionotropic receptor activation.
A remaining concern, however, was that the abolition of responses to both 5-HT and phenylephrine were tested after washout and not in the actual presence of IBMX—that is, following the same protocol as the forskolin and cAMP analog experiments in which this effect on 5-HT responses was first revealed. Although our observations suggested that IBMX was exerting persistent effects even after washout, we could not definitively conclude that under conditions of IBMX-mediated inhibition of PDE the GG muscle was unresponsive to these excitatory neurotransmitters. Accordingly, from an additional series of experiments in three rats, results showed that even in the continued presence of IBMX (i.e., no attempted washout) the GG muscle was unresponsive to both 5-HT and phenylephrine at the HMN (t2 = 0.676, p = 1.00, post hoc paired t tests after ANOVA-RM) but responses to ionotropic glutamate receptor activation (t2 = 16.29, p < 0.001) remained. Grouped data for these experiments are shown in Figure 6A.
A possible mechanism for the selective abolition of responses to 5-HT and phenylephrine after IBMX was increased activation of PKA due to inhibition of PDE. As a result, it is possible that PKA may then have phosphorylated both metabotropic 5-HT2 and 1-adrenergic receptors and caused these receptors to become unresponsive to the applied 5-HT and phenylephrine. To test this possibility, additional experiments were performed in another three rats where 5-HT was applied to the HMN in the continued presence of forskolin (i.e., without washout). Because forskolin is an adenylyl cyclase activator that stimulates production of intracellular cAMP and activates PKA (11, 13, 33–35), if PKA activation were the predominant reason for the abolition of responses to 5-HT after IBMX, then similar abolition of responses would be expected in these additional forskolin experiments. However, Figure 7 shows that application of 5-HT in the continued presence of forskolin also led to significant increases in GG activity compared to forskolin alone (t2 = 11.245, p = 0.008, paired t test) and, as expected, responses to AMPA also persisted. These results, in addition to the observations that IBMX did not mimic the GG-activating effects of forskolin and the cAMP analog (Figure 3), supported the concept that the effects of IBMX were mediated by pathways independent of cAMP.
IBMX is a nonspecific inhibitor of PDE that prevents breakdown of both endogenous cAMP and cGMP. Consequently, cGMP may have played a significant role in abolishing the responses to applied 5-HT and phenylephrine at the HMN. To test this possibility, a final set of experiments was performed in four rats to determine whether cGMP at the HMN was involved in the selective abolition of responses to 5-HT and phenylephrine. These studies revealed that in the presence of the cGMP analog, responses to 5-HT and phenylephrine at the HMN were also abolished (t3 = 0.675, p = 1.00, post hoc paired t tests after ANOVA-RM) but responses to ionotropic glutamate receptor activation with AMPA remained (t3 = 5.08, p < 0.001; Figure 6B). These results are identical to those obtained from the IBMX experiments (Figures 6A vs. 6B) and suggested that some of the observed effects of IBMX may be mediated by cGMP pathways.
DISCUSSION
Pharyngeal muscle activity is critical to the maintenance of airway patency in OSA (15) and pharmacologic interventions may prove useful to increase pharyngeal muscle activity in sleep and prevent obstructive apneas (45–47). Previous studies in animals and humans aiming to modulate pharyngeal muscle activity in sleep have targeted receptors on pharyngeal motoneurons via manipulation of extracellular neurotransmitters (18–30, 45–47). However, recent in vitro experiments show that respiratory motoneuron excitability is dynamically modulated by a variety of intracellular signaling molecules that are amenable to manipulation (9–11, 34, 48). Modulation of intracellular targets downstream from surface receptors may offer new and alternative approaches to develop pharmacologic strategies for OSA (49). The present study is the first to examine the effects on respiratory- muscle activity of manipulating intracellular signaling molecules at respiratory motoneurons in freely behaving animals in vivo, the effects of prevailing sleep–wake states on these responses, and the important effects of these manipulations on subsequent responses to locally applied neurotransmitters.
Genioglossus Muscle Responses to Forskolin and the cAMP Analog at the HMN
Microdialysis perfusion of either the adenylyl cyclase activator forskolin or a cAMP analog into the HMN increased tonic and respiratory-related GG muscle activity. This selective GG motor activation, with no significant changes in other physiologic variables such as respiratory rate, diaphragm amplitude, or neck EMG, suggested that responses were localized to the HMN. Although pharyngeal muscle recordings were made only from the GG in this study, it is likely that the interventions also affected other hypoglossal motoneurons, that is, those innervating both extrinsic (protruder and retractor) and intrinsic tongue muscles (50). Both extrinsic and intrinsic muscles are involved in respiratory control of the upper airway and their coactivation is thought to be beneficial for the maintenance of airway patency (14, 51, 52). It is not known whether coactivation of these muscles could itself indirectly influence GG activity and contribute to the observed GG responses to the applied drugs. However, it is most likely that the major component of the observed GG responses to interventions at the HMN is due to direct effects because GG motoneurons contain the necessary receptors for the applied agonists (i.e., 5-HT, 1 and non-NMDA receptors) and responses recorded in vitro to intracellular perfusion of agents to modulate the cAMP–PKA pathway (34, 53) are consistent with those observed in vivo in the present experiments.
Indeed, the observed increase in respiratory-related GG activity is consistent with in vitro experiments showing augmentation of inspiratory drive currents in hypoglossal motoneurons following activation of the cAMP–PKA pathway (9). The present experiments in intact animals, however, also showed that forskolin and the cAMP analog increased tonic GG activity, a result not observed in previous experiments likely due to the extensive deafferentation accompanying in vitro procedures that removes a significant source of tonic excitatory drives. Any differences between responses observed in neonatal animals in vitro and adult animals in vivo may also be due to the age of the preparations. Increases in tonic GG activity in the intact animal are especially relevant because tonic pharyngeal motor activity results in increased upper airway size and stiffness and promotes airway patency (54, 55). Nevertheless, in addition to augmentation of inspiratory drive currents in hypoglossal motoneurons after activation of the cAMP–PKA pathway in vitro (9), it has also been observed that PKA can simultaneously potentiate inspiratory-related inhibition and even neutralize inspiratory activation in some hypoglossal motoneurons (10). It is unknown if similar augmentation of both inspiratory activation and inhibition occurs at hypoglossal motoneurons in vivo after activation of the cAMP–PKA pathway. However, if both excitatory and inhibitory effects are produced, the results show that the excitatory effects predominate as increased GG activity is observed (Figure 2), with the balance in favor of excitation also observed in vitro (34).
The results also showed for the first time in vivo that the robust GG muscle activation produced in response to forskolin and the cAMP analog at the HMN was dependent on the prevailing sleep–wake state. Forskolin increased GG activity in wakefulness and non-REM sleep but not during REM sleep; similar responses were observed with the cAMP analog. This powerful influence of the prevailing sleep–wake state on hypoglossal motor responses to manipulation of cAMP could not have been predicted from previous in vitro experiments. These data highlight that the activation responses produced in respiratory motoneurons by manipulation of intracellular mechanisms under constant conditions in vitro are not intractable but are importantly modulated by ongoing behavioral states in the intact organism in vivo.
Forskolin increased GG activity for as long as it was applied (up to 6 h in the different experiments), whereas responses to the cAMP analog were not as large in magnitude (Figures 3A and 3B vs. Figures 3C and 3D) or as long lasting. Although the doses of each applied drug were the same, this difference may relate to the doses not being functionally equivalent—that is, forskolin may have amplified levels of endogenous cAMP (56) sufficient to sustain more of an increase in GG activity compared with the exogenous cAMP analog. Nevertheless, the results showed that forskolin and the cAMP analog both increased GG activity in non-REM sleep to levels exceeding those observed in normal wakefulness with control delivery of ACSF (Figure 3). These excitatory responses, however, were overcome by the neuronal processes associated with REM sleep, although the transient GG muscle twitches typical of REM (18, 20, 36) remained (Figure 2).
The neural mechanisms responsible for the periods of GG motor suppression in REM sleep remain to be determined but we have shown that postsynaptic inhibition mediated via glycine and -aminobutyric acid (GABA) and loss of facilitation via 5-HT play only a minor role in intact animals (19, 20, 31). Nevertheless, although glycine and GABA play only a small but measurable role in mediating GG suppression in REM sleep (19, 20), it is possible that this suppression effect may be augmented in the presence of PKA (34) due to the aforementioned augmentation of inspiratory inhibition at hypoglossal motoneurons (10). Accordingly, such a mechanism may explain a component of the suppression of GG activity in REM sleep in the presence of forskolin and the cAMP analog in this study (Figures 2 and 3). However, a recent study using the carbachol model of REM sleep in anesthetized rats implicates a more important role of loss of facilitation in the suppression of hypoglossal motor activity via withdrawal of noradrenaline (57), although this needs to be tested in natural sleep. The overall clinical relevance of these observations is that once the distinct mechanisms underlying the control of pharyngeal muscle activity in REM sleep have been identified, the results of the present study suggest that a combination of pharmacologic approaches may be the most effective approach to achieve sustained increases in GG activity across the sleeping period as different neural mechanisms are operative in non-REM and REM sleep.
Genioglossus Muscle Responses to IBMX and cGMP at the HMN
In contrast to the increased GG activity following forskolin and the cAMP analog at the HMN, the nonspecific PDE inhibitor IBMX produced no changes in either respiratory-related or tonic GG activities across sleep–wake states. Although it could be questioned whether an absent GG response to IBMX was due to a dose insufficient to mimic the effects of forskolin or the cAMP analog, preliminary studies showed that higher (500 μM and 1 mM) and lower (100 μM) doses of IBMX also did not alter GG activity. That reduced breakdown of endogenous cAMP did not produce the same activation response as forskolin or the cAMP analog suggests that endogenous levels of cAMP and adenylyl cyclase activity were likely relatively low in these behaving animals such that inhibiting breakdown of cAMP had little effect on GG activity. This result suggests that, unlike neonatal in vitro preparations (9, 34), the cAMP–PKA pathway is not constitutively active at the HMN in vivo. Further experiments with a variety of both PDE inhibitors and cAMP–PKA modifying drugs, including agents to block cAMP formation, are necessary to definitely prove this suggestion but these were beyond the scope of the present report. Although IBMX at the HMN did not alter respiratory rate or diaphragm activity, in addition to the absent effects on GG, it was noted that a decrease in neck EMG activity occurred after IBMX compared with ACSF. However, this effect was confined to wakefulness only and did not persist into non-REM or REM sleep, suggesting that it was most likely due to a nonspecific effect associated with less behavioral activity in those wakefulness periods rather than a specific effect of IBMX per se.
However, although not directly affecting GG muscle activity, IBMX did have robust physiological effects as judged by abolition of responses associated with excitatory metabotropic 5-HT and 1-adrenergic receptor activation at the HMN. This observation is significant because 5-HT and phenylephrine at the HMN otherwise cause robust excitation of hypoglossal motor activity in this preparation (see RESULTS) and as reported in other studies (18, 41, 43, 53, 58, 59). However, the abolition of these 5-HT and 1-mediated excitatory responses at the HMN were specific as responses coupled to ionotropic non-NMDA glutamate receptor activation remained. Because responses to IBMX did not mimic the effects of forskolin or the cAMP analog, the results suggested that these novel responses to IBMX were mediated by pathways independent of cAMP. Indeed, because IBMX is a nonspecific PDE inhibitor and prevents the breakdown of both endogenous cAMP and cGMP, the selective abolition of responses to 5-HT and phenylephrine after administration of IBMX may have been due to accumulation of cGMP. This suggestion was supported by the additional experiments showing that application of the cGMP analog to the HMN mimicked the effects of IBMX and abolished responses to subsequent application of 5-HT and phenylephrine (Figure 6). Further experiments with more specific PDE inhibitors and cGMP activators will be able to determine if accumulation of cGMP is solely responsible for the selective abolition of responses to 5-HT and 1-receptor activation at the HMN.
To our knowledge this is the first report of the interaction between interventions modulating PDE and cGMP and the subsequent abolition of otherwise potent excitatory motoneuron responses to 5-HT and 1-receptor stimulation while responses to ionotropic non-NMDA receptor activation remained. Although the cellular and molecular mechanisms underlying this observation remain to be determined and are outside the scope of the present report, additional experiments did suggest that the abolition of these responses to the applied monoamines was not likely due to cAMP–PKA mediated receptor desensitization (60, 61) because application of 5-HT in the presence of forskolin was still able to significantly increase GG activity compared with application of forskolin alone (Figure 7). Rather, the important implication of the selective abolition of responses to 5-HT and phenylephrine is that, under natural conditions and behaviors in which cGMP would be increased at the HMN, motor activity would be unresponsive to these important excitatory monoaminergic inputs.
Endogenous cGMP is produced via activation of soluble guanylate cyclase by natriuretic peptides and nitric oxide. Both soluble guanylate cyclases and protein kinase G are expressed at the HMN (62, 63), and preliminary experiments show that application of 8-Br-cGMP, the same cGMP analog as used in the present study, decreases excitability of hypoglossal motoneurons in vitro (64). There are identified nitrergic projections from the ventromedial reticular formation to hypoglossal motoneurons (65). Cholinergic neurons of the mesopontine tegmentum (laterodorsal and pedunculopontine tegmental neurons) also express nitric oxide synthase (66, 67) and these neurons are importantly involved in sleep–wake regulation, especially the generation of REM sleep. Pedunculopontine tegmental neurons project to the HMN (68–71) where muscarinic receptor stimulation suppresses GG activity (72). It remains to be determined if the abolition of GG responses to local application of 5-HT to the HMN in REM sleep (18), and phenylephrine (unpublished data), is due to recruitment of nitric oxide and cGMP, and if such effects can be blocked by cGMP-PKG inhibitors. Increased cGMP in REM sleep would also explain why muscle twitches are preserved in this state because these excitatory events are produced by glutamatergic non-NMDA receptor mechanisms (73), and responses to non-NMDA receptor stimulation with AMPA were unaffected by IBMX and cGMP (Figure 6). Such interactions between cGMP and responses to 5-HT and phenylephrine at the HMN are potentially of significant clinical relevance because although pharmacologic strategies aiming to increase 5-HT at pharyngeal motoneurons have shown some beneficial effects in patients with OSA, these effects are largely observed in non-REM sleep with minimal effects in REM sleep (22–24, 27, 30, 45–47).
Conclusions
The results of the present study show that modulation of cAMP at the HMN alters GG activity across sleep–wake states, a result relevant to basic mechanisms of pharyngeal motor control. Targeting intracellular mechanisms also opens up potential new pharmacologic strategies to increase pharyngeal muscle activity in sleep relevant to OSA. In the long-term, such strategies may also include the opportunity for more selective delivery of drugs to those respiratory motoneurons innervating pharyngeal muscles while avoiding systemic approaches that are nonselective and dispersive throughout the central nervous system. Such potential future strategies may include topical application of agents engineered for retrograde transport to pharyngeal motoneurons, with such technology already in development for other more severe, although less common, clinical disorders involving the somatic muscles (74–76), including respiratory muscle (77, 78). While production of long-term increases (i.e., several hours) in pharyngeal motor activity via modulation of intracellular signal transduction mechanisms are relevant to OSA, such strategies have to be tempered by the possible interactions between intracellular pathways—for example, documented influences on the responses to 5-HT and phenylephrine as observed in this study. Appropriate in vivo testing, therefore, of pharmacologic strategies aiming to modulate pharyngeal muscle activity needs to be performed with this caveat in mind.
Acknowledgments
The authors thank Erin Chan, Hendrik Steenland, and John Peever, Ph.D., at the University of Toronto for helpful discussion.
FOOTNOTES
Supported by Canadian Institutes of Health Research grant MT-15563 and a Natural Sciences and Engineering Research Council of Canada scholarship (C.R.A.A.).
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200509-1469OC on December 1, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
REFERENCES
Kaczmarek LK, Letivan IB. Neuromodulation, the biochemical control of neuronal excitability. New York: Oxford University Press; 1987.
Rall TW, Sutherland EW. The regulatory role of adenosine-3',5'-phosphate. Cold Spring Harb Symp Quant Biol 1961;26:347–354.
Robison GA, Butcher RW, Sutherland EW. Cyclic AMP. Annu Rev Biochem 1968;37:149–174.
Wenzel B, Elsner N, Heinrich R. mAChRs in the grasshopper brain mediate excitation by activation of the AC/PKA and the PLC second-messenger pathways. J Neurophysiol 2002;87:876–888.
Wright NJ, Zhong Y. Characterization of K+ currents and the cAMP-dependent modulation in cultured Drosophila mushroom body neurons identified by lacZ expression. J Neurosci 1995;15:1025–1034.
Lu Y, Li Y, Herin GA, Aizenman E, Epstein PM, Rosenberg PA. Elevation of intracellular cAMP evokes activity-dependent release of adenosine in cultured rat forebrain neurons. Eur J Neurosci 2004;19:2669–2681.
Kameyama K, Lee HK, Bear MF, Huganir RL. Involvement of a postsynaptic protein kinase A substrate in the expression of homosynaptic long-term depression. Neuron 1998;21:1163–1175.
Hensch TK, Gordon JA, Brandon EP, McKnight GS, Idzerda RL, Stryker MP. Comparison of plasticity in vivo and in vitro in the developing visual cortex of normal and protein kinase A RIbeta-deficient mice. J Neurosci 1998;18:2108–2117.
Bocchiaro CM, Saywell SA, Feldman JL. Dynamic modulation of inspiratory drive currents by protein kinase A and protein phosphatases in functionally active motoneurons. J Neurosci 2003;23:1099–1103.
Saywell SA, Feldman JL. Dynamic interactions of excitatory and inhibitory inputs in hypoglossal motoneurones: respiratory phasing and modulation by PKA. J Physiol 2004;554:879–889.
Shao XM, Ge Q, Feldman JL. Modulation of AMPA receptors by cAMP-dependent protein kinase in preBotzinger complex inspiratory neurons regulates respiratory rhythm in the rat. J Physiol 2003;547:543–553.
Richter DW, Lalley PM, Pierrefiche O, Haji A, Bischoff AM, Wilken B, Hanefeld F. Intracellular signal pathways controlling respiratory neurons. Respir Physiol 1997;110:113–123.
Lalley PM, Pierrefiche O, Bischoff AM, Richter DW. cAMP-dependent protein kinase modulates expiratory neurons in vivo. J Neurophysiol 1997;77:1119–1131.
Fuller DD, Williams JS, Janssen PL, Fregosi RF. Effect of co-activation of tongue protrudor and retractor muscles on tongue movements and pharyngeal airflow mechanics in the rat. J Physiol 1999;519:601–613.
Remmers JE, deGroot WJ, Sauerland EK, Anch AM. Pathogenesis of upper airway occlusion during sleep. J Appl Physiol 1978;44:931–938.
Young T, Palta M, Dempsey J, Skatrud J, Weber S, Badr S. The occurrence of sleep-disordered breathing among middle-aged adults. N Engl J Med 1993;328:1230–1235.
Hla KM, Young TB, Bidwell T, Palta M, Skatrud JB, Dempsey J. Sleep apnea and hypertension: a population-based study. Ann Intern Med 1994;120:382–388.
Jelev A, Sood S, Liu H, Nolan P, Horner RL. Microdialysis perfusion of 5-HT into hypoglossal motor nucleus differentially modulates genioglossus activity across natural sleep-wake states in rats. J Physiol 2001;532:467–481.
Morrison JL, Sood S, Liu H, Park E, Liu X, Nolan P, Horner RL. Role of inhibitory amino acids in control of hypoglossal motor outflow to genioglossus muscle in naturally sleeping rats. J Physiol 2003;552:975–991.
Morrison JL, Sood S, Liu H, Park E, Nolan P, Horner RL. GABAA receptor antagonism at the hypoglossal motor nucleus increases genioglossus muscle activity in NREM but not REM sleep. J Physiol 2003;548:569–583.
Hedner J, Kraiczi H, Peker Y, Murphy P. Reduction of sleep-disordered breathing after physostigmine. Am J Respir Crit Care Med 2003;168:1246–1251.
Kraiczi H, Hedner J, Dahlof P, Ejnell H, Carlson J. Effect of serotonin uptake inhibition on breathing during sleep and daytime symptoms in obstructive sleep apnea. Sleep 1999;22:61–67.
Sunderram J, Parisi RA, Strobel RJ. Serotonergic stimulation of the genioglossus and the response to nasal continuous positive airway pressure. Am J Respir Crit Care Med 2000;162:925–929.
Veasey SC, Fenik P, Panckeri K, Pack AI, Hendricks JC. The effects of trazodone with L-tryptophan on sleep-disordered breathing in the English bulldog. Am J Respir Crit Care Med 1999;160:1659–1667.
Veasey SC, Panckeri KA, Hoffman EA, Pack AI, Hendricks JC. The effects of serotonin antagonists in an animal model of sleep-disordered breathing. Am J Respir Crit Care Med 1996;153:776–786.
Veasey SC, Chachkes J, Fenik P, Hendricks JC. The effects of ondansetron on sleep-disordered breathing in the English bulldog. Sleep 2001;24:155–160.
Berry RB, Yamaura EM, Gill K, Reist C. Acute effects of paroxetine on genioglossus activity in obstructive sleep apnea. Sleep 1999;22:1087–1092.
Berry RB, Hayward LF. Selective augmentation of genioglossus electromyographic activity by L-5-hydroxytryptophan in the rat. Pharmacol Biochem Behav 2003;74:877–882.
Ogasa T, Ray AD, Michlin CP, Farkas GA, Grant BJ, Magalang UJ. Systemic administration of serotonin 2A/2C agonist improves upper airway stability in Zucker rats. Am J Respir Crit Care Med 2004;170:804–810.
Hanzel DA, Proia NG, Hudgel DW. Response of obstructive sleep apnea to fluoxetine and protriptyline. Chest 1991;100:416–421.
Sood S, Morrison JL, Liu H, Horner RL. Role of endogenous serotonin in modulating genioglossus muscle activity in awake and sleeping rats. Am J Respir Crit Care Med 2005;172:1338–1347.
Horner RL, Liu X, Gill H, Nolan P, Liu H, Sood S. Effects of sleep-wake state on the genioglossus vs. diaphragm muscle response to CO(2) in rats. J Appl Physiol 2002;92:878–887.
Insel PA, Ostrom RS. Forskolin as a tool for examining adenylyl cyclase expression, regulation, and G protein signaling. Cell Mol Neurobiol 2003;23:305–314.
Feldman JL, Neverova NV, Saywell SA. Modulation of hypoglossal motoneuron excitability by intracellular signal transduction cascades. Respir Physiol Neurobiol 2005;147:131–143.
Capece ML, Lydic R. cAMP and protein kinase A modulate cholinergic rapid eye movement sleep generation. Am J Physiol 1997;273:R1430–R1440.
Richard CA, Harper RM. Respiratory-related activity in hypoglossal neurons across sleep-waking states in cats. Brain Res 1991;542:167–170.
Younes M. Contributions of upper airway mechanics and control mechanisms to severity of obstructive apnea. Am J Respir Crit Care Med 2003;168:645–658.
Morrison JL, Sood S, Liu X, Liu H, Park E, Nolan P, Horner RL. Glycine at hypoglossal motor nucleus: genioglossus activity, CO(2) responses, and the additive effects of GABA. J Appl Physiol 2002;93:1786–1796.
Sood S, Liu X, Liu H, Nolan P, Horner RL. 5-HT at hypoglossal motor nucleus and respiratory control of genioglossus muscle in anesthetized rats. Respir Physiol Neurobiol 2003;138:205–221.
Fenik P, Veasey SC. Pharmacological characterization of serotonergic receptor activity in the hypoglossal nucleus. Am J Respir Crit Care Med 2003;167:563–569.
Kubin L, Tojima H, Davies RO, Pack AI. Serotonergic excitatory drive to hypoglossal motoneurons in the decerebrate cat. Neurosci Lett 1992;139:243–248.
Funk GD, Smith JC, Feldman JL. Generation and transmission of respiratory oscillations in medullary slices: role of excitatory amino acids. J Neurophysiol 1993;70:1497–1515.
Parkis MA, Bayliss DA, Berger AJ. Actions of norepinephrine on rat hypoglossal motoneurons. J Neurophysiol 1995;74:1911–1919.
Strader CD, Fong TM, Tota MR, Underwood D, Dixon RA. Structure and function of G protein-coupled receptors. Annu Rev Biochem 1994;63:101–132.
Horner RL. The neuropharmacology of upper airway motor control in the awake and asleep states: implications for obstructive sleep apnoea. Respir Res 2001;2:286–294.
Veasey SC. Pharmacotherapies for obstructive sleep apnea: how close are we Curr Opin Pulm Med 2001;7:399–403.
Smith IE, Quinnell TG. Pharmacotherapies for obstructive sleep apnoea: where are we now Drugs 2004;64:1385–1399.
Bocchiaro CM, Feldman JL. Synaptic activity-independent persistent plasticity in endogenously active mammalian motoneurons. Proc Natl Acad Sci USA 2004;101:4292–4295.
Greer JJ. Are biogenic amines involved in controlling upper airway patency during REM sleep Am J Respir Crit Care Med 2005;172:1237–1238.
Dobbins EG, Feldman JL. Differential innervation of protruder and retractor muscles of the tongue in rat. J Comp Neurol 1995;357:376–394.
Bailey EF, Fregosi RF. Coordination of intrinsic and extrinsic tongue muscles during spontaneous breathing in the rat. J Appl Physiol 2004;96:440–449.
Bailey EF, Janssen PL, Fregosi RF. PO2-dependent changes in intrinsic and extrinsic tongue muscle activities in the rat. Am J Respir Crit Care Med 2005;171:1403–1407.
Rekling JC, Funk GD, Bayliss DA, Dong XW, Feldman JL. Synaptic control of motoneuronal excitability. Physiol Rev 2000;80:767–852.
Kuna S, Remmers JE. Anatomy and physiology of upper airway obstruction. In: Kryger MH, Roth T, Dement WC, editors. Principles and practice of sleep medicine, 3rd ed. Philadelphia: W.B. Saunders; 2000. pp. 840–858.
Horner RL. Motor control of the pharyngeal musculature and implications for the pathogenesis of obstructive sleep apnea. Sleep 1996;19:827–853.
Ross EM. Signal sorting and amplification through G protein-coupled receptors. Neuron 1989;3:141–152.
Fenik VB, Davies RO, Kubin L. REM sleep-like atonia of XII motoneurons is caused by loss of noradrenergic and serotonergic inputs. Am J Respir Crit Care Med 2005;172:1322–1330.
Berger AJ, Bayliss DA, Viana F. Modulation of neonatal rat hypoglossal motoneuron excitability by serotonin. Neurosci Lett 1992;143:164–168.
Al-Zubaidy ZA, Erickson RL, Greer JJ. Serotonergic and noradrenergic effects on respiratory neural discharge in the medullary slice preparation of neonatal rats. Pflugers Arch 1996;431:942–949.
Freedman NJ, Lefkowitz RJ. Desensitization of G protein-coupled receptors. Recent Prog Horm Res 1996;51:319–351 (discussion 352–353).
Chuang TT, Iacovelli L, Sallese M, De Blasi A. G protein-coupled receptors: heterologous regulation of homologous desensitization and its implications. Trends Pharmacol Sci 1996;17:416–421.
de Vente J, Asan E, Gambaryan S, Markerink-van Ittersum M, Axer H, Gallatz K, Lohmann SM, Palkovits M. Localization of cGMP- dependent protein kinase type II in rat brain. Neuroscience 2001;108:27–49.
Lin LH, Talman WT. Soluble guanylate cyclase and neuronal nitric oxide synthase colocalize in rat nucleus tractus solitarii. J Chem Neuroanat 2005;29:127–136.
Neverova NV, Saywell SA, Feldman JL. The role of cGMP-mediated signaling pathways in modulation of hypoglossal (XII) motoneuron excitability. Soc Neurosci Abstr 2002;5:865.
Pose I, Fung S, Sampogna S, Chase MH, Morales FR. Nitrergic innervation of trigeminal and hypoglossal motoneurons in the cat. Brain Res 2005;1041:29–37.
Gautier-Sauvigne S, Colas D, Parmantier P, Clement P, Gharib A, Sarda N, Cespuglio R. Nitric oxide and sleep. Sleep Med Rev 2005;9:101–113.
Vincent SR, Kimura H. Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 1992;46:755–784.
Connaughton M, Priestley JV, Sofroniew MV, Eckenstein F, Cuello AC. Inputs to motoneurones in the hypoglossal nucleus of the rat: light and electron microscopic immunocytochemistry for choline acetyltransferase, substance P and enkephalins using monoclonal antibodies. Neuroscience 1986;17:205–224.
Woolf NJ, Butcher LL. Cholinergic systems in the rat brain: IV. Descending projections of the pontomesencephalic tegmentum. Brain Res Bull 1989;23:519–540.
Dobbins EG, Feldman JL. Differential innervation of protruder and retractor muscles of the tongue in rat. J Comp Neurol 1995;357:376–394.
Woolf NJ. Cholinergic systems in mammalian brain and spinal cord. Prog Neurobiol 1991;37:475–524.
Liu X, Sood S, Liu H, Horner RL. Opposing muscarinic and nicotinic modulation of hypoglossal motor output to genioglossus muscle in rats in vivo. J Physiol 2005;565:965–980.
Soja PJ, Lopez-Rodriguez F, Morales FR, Chase MH. Effects of excitatory amino acid antagonists on the phasic depolarizing events that occur in lumbar motoneurons during REM periods of active sleep. J Neurosci 1995;15:4068–4076.
Alisky JM, Davidson BL. Gene therapy for amyotrophic lateral sclerosis and other motor neuron diseases. Hum Gene Ther 2000;11:2315–2329.
Boillee S, Cleveland DW. Gene therapy for ALS delivers. Trends Neurosci 2004;27:235–238.
Azzouz M, Mazarakis N. Non-primate EIAV-based lentiviral vectors as gene delivery system for motor neuron diseases. Curr Gene Ther 2004;4:277–286.
Matecki S, Dudley RW, Divangahi M, Gilbert R, Nalbantoglu J, Karpati G, Petrof BJ. Therapeutic gene transfer to dystrophic diaphragm by an adenoviral vector deleted of all viral genes. Am J Physiol Lung Cell Mol Physiol 2004;287:L569–L576.
Dudley RW, Lu Y, Gilbert R, Matecki S, Nalbantoglu J, Petrof BJ, Karpati G. Sustained improvement of muscle function one year after full-length dystrophin gene transfer into mdx mice by a gutted helper-dependent adenoviral vector. Hum Gene Ther 2004;15:145–156.