Pharmacological Characterization of Serotonergic Receptor Activity in the Hypoglossal Nucleus

Center for Sleep and Respiratory Neurobiology and Division of Sleep Medicine, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

	ABSTRACT

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State-dependent reductions in serotonin delivery to upper airway dilator motoneuron activity may contribute to sleep apnea. The functional significance of serotonin receptor subtypes implicated in excitation of dilator motor neurons was evaluated in anesthetized, paralyzed, mechanically ventilated adult rats (n = 108). The effects of antagonists selective for serotonin receptor subtypes 2A, 2C, or 7 on intrinsic hypoglossal activity and on serotonin agonist (serotonin, 5-carboxamidotryptamine maleate, and RO-600,175) dose responses were characterized. All drugs were injected unilaterally into the hypoglossal nucleus. The 2A antagonist, MDL-100,907, dropped intrinsic hypoglossal nerve respiratory activity by 61 ± 6% (p < 0.001) and suppressed serotonin excitation of hypoglossal nerve activity (p < 0.05). The 2C antagonist, SB-242,084, dropped hypoglossal nerve activity 17 ± 6% (p < 0.05) and suppressed the dose–response curve for the 2C agonist. Rapid desensitization occurred with the 2C agonist only (p < 0.05). The 7 antagonist, SB-269,970, had no effect on either intrinsic activity or agonist responses. We conclude that serotonin 2A is the predominant excitatory serotonin receptor subtype at hypoglossal motor neurons. The serotonin 2C excitatory effects are of lower magnitude and are associated with rapid desensitization. There is no evidence for serotonin 7 activity in the hypoglossal nucleus. This characterization of serotonin receptor subtypes in the hypoglossal nucleus provides a focus for the development of pharmacotherapies for sleep apnea.

　

Key Words: motor neurons • respiratory • serotonin • microinjection • obstructive sleep apnea

Obstructive sleep apnea/hypopnea syndrome (OSAHS) is present in more than 2% of adults (1) and has been identified as an independent risk factor for several cardiovascular disease processes (2–5). Safe and widely effective pharmacotherapies are needed for OSAHS.

The pathophysiology of this disease, with compromised respiration only in sleep (6–8), suggests that this disorder should be amenable to drug therapies that would prevent sleep-related reductions in upper airway dilator motoneuronal activity. State-dependent changes in neurotransmitter delivery are complex and involve alterations in the delivery of many different neurochemicals in multiple regions involved in the control of respiratory drive to upper airway motor neurons (9, 10). An understanding of the neurochemical changes at all of these regions will be necessary to develop the most effective pharmacotherapies for OSAHS. We have chosen to focus first on changes occurring directly at dilator motor neurons. Here, we hypothesize that serotonin (5-HT) contributes, at least in part, to the state-dependent reductions in dilator motoneuronal activity.

The rationale to focus on 5-HT at upper airway motor neurons is based on the following observations. 5-HT excites adult upper airway dilator motor neurons (11–17) and provides intrinsic excitation at brainstem motor neurons in unanesthetized animals (13). The activity of neurons supplying 5-HT to motor neurons declines in sleep (18, 19). Furthermore, pretreatment of upper airway dilator motor neurons with 5-HT reduces sleep state-dependent suppression in upper airway dilator muscle activity (20).

The pharmacology of 5-HT modulation of upper airway dilator motor neurons is complex and appears to involve multiple 5-HT receptor subtypes (21–29). An understanding of receptor subtypes is essential for targeting drug therapies. Previous studies with partially selective 5-HT agonists and antagonists have suggested an excitatory effect through one or more of the 5-HT₂ receptor subtypes (11, 15, 21, 23, 27–29). 5-Carboxyamido-tryptamine (5-CT; a 5-HT agonist with greater affinity at 5-HT₁ and 5-HT₇), however, also appears effective in exciting hypoglossal neurons (15). We recently measured mRNA copy numbers for eight postsynaptic 5-HT receptor subtypes in individual XII motor neurons and found that 5-HT_2A was the predominant receptor subtype in motor neurons, and the mRNA for this receptor subtype was present in all XII motor neurons (30). Smaller amounts of 5-HT_2C were identified in half of the motor neurons, dispersed throughout the hypoglossal nucleus. The functional significance of mRNA for each of these receptor subtypes has not been characterized.

The purpose of this work was (1) to determine whether 5-HT_2A, 5-HT_2C, and/or 5-HT₇ receptors contribute to 5-HT excitation within a representative upper airway motor nucleus, the XII, and (2) to determine whether the 5-CT effect is mediated via activation of 5-HT₇ receptors.

	METHODS

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Surgery
Adult male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 350–390 g were preanesthetized with halothane (2.0–2.5%) and then anesthetized with urethane 1.2 g/kg intraperitoneally (Sigma-Aldrich, St. Louis, MO) (31). A femoral arterial line, femoral venous line, and tracheotomy tube were placed; the vagi were transected distal to the nodose ganglion (32). Cuff electrodes (self-fashioned) were placed on both hypoglossal nerves for recording. Blood pressure, end tidal CO₂, and both hypoglossal nerves were recorded after paralysis with pancuronium and mechanical ventilation as previously described (32). The dorsal surface of the medulla was exposed for microinjections. Procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

Drugs
5-HT; RO-60,0175, a selective 5-HT_2C agonist (RO); 5-carboxamidotryptamine maleate, a 5-HT agonist with µM affinities at the 5-HT_1A/B/D, 5-HT_2C, 5-HT_5A/B, 5-HT₆, and 5-HT₇ receptor subtypes (5-CT); SB-206,553 hydrochloride, a selective 5-HT_2C antagonist (SB-206); SB-242,084 dihydrochloride, a selective 5-HT_2C antagonist (SB-242); SB-269,970 hydrochloride, a selective 5-HT₇ antagonist (SB 269); and methiotepin mesylate, a broad-spectrum 5-HT antagonist, were obtained from Sigma-Aldrich. MDL-100,907 (MDL, a selective 5-HT_2A antagonist) was a gift of Guy Kennett (Cerebrus Pharmaceuticals, Winnersh, Berkshire, UK). All drugs were solubilized in normal saline.

Drugs were administered as microinjections targeting one hypoglossal nucleus using coordinates relative to the calamus scriptorius (0.2 mm lateral, 1.05 ventral, and 0.2–0.6 mm rostral). The main effect measured was ipsilateral hypoglossal nerve activity using methods as previously detailed (15, 31, 33) and illustrated in  .

fig.ommitted Figure 1. Digitized electrographic recording of measured physiological parameters during microinjection studies. Whole nerve recordings were performed on both the side of injection (in this case, the right hypoglossal, or XII) and the contralateral XII nerve (left XII, control). Calibrated blood pressure and end tidal CO2 (ETCO2) were also recorded, and events were tagged with an event marker. The first arrow marks the insertion of a pipette into the right XII nucleus, and the second arrow marks an injection of 20-nl 10-µM 5-HT. This is a typical response in magnitude and pattern, showing predominantly an increase in tonic XII activity.

 
5-HT and 5-CT (Nonselective Agonist) Effects on XII Nerve Activity Protocol
Effects of 5-HT and 5-CT on XII nerve activity were assessed in 35 rats, each receiving five to six microinjections (20 nl of each) targeted at the ventral region of the XII nucleus (34), as shown in  .  summarizes the concentrations tested and the number of animals per condition. The order of drug and concentration (including saline control) was randomized for each rat, with the exception that if randomized to a 5-mM dose, this would be administered last. The effect was measured 30 seconds after the injection. The interval between injections was 10 minutes (32).

fig.ommitted Figure 2. Coronal section within the medulla (30 µm), localizing the microinjection site. Large motor neuronal cell bodies, counterstained with neutral red, are evident within the ventral half of the hypoglossal nucleus. The darkly stained cell bodies (arrow) indicate the site of injection. Notice that the dye is localized to the ventral portion of the ipsilateral hypoglossal nucleus and that the architecture of the nucleus appears similar on both sides, despite seven microinjections into the nucleus.

 

fig.ommitted TABLE 1. Dose-dependent effects of serotonin and serotonin agonists on baseline hypoglossal nerve activity.

 
RO (2C Agonist) Effects on XII Nerve Activity
Effects of RO (2C agonist) on XII nerve respiratory activity were evaluated in a separate 20 rats. Each rat received three injections of randomly selected doses, as described previously here for 5-HT and 5-CT. The effect was measured 30 seconds after the injection. The interval between injections was 1 hour.

Effects of 5-HT Antagonists on Baseline XII Nerve Activity
In a separate 25 rats, one of five antagonists was tested to characterize the effect of the drug on baseline XII nerve activity. For each of these trials, each rat received three 20-nl injections: one into the site used for agonist injections and then one 0.2 mm rostral and one 0.2 mm caudal to that site. Effect of antagonist was measured 15 minutes after the third injection. After MDL (2A antagonist) injections, methiotepin mesylate (using same dosing) was injected as mentioned previously here to assess for additional effect.

Effects of Selective Antagonists on 5-HT, 5-CT, and RO (2C Agonist) Dose–Response Curves
In 20 of the 25 rats used in the antagonist protocol described previously here, in all 35 rats used in the 5-HT, 5-CT dose response curves, and in an additional 18 rats without prior injections, the effects of selective antagonists on dose responses were tested. The effects of one antagonist on agonist dose–response curves were tested per animal, using the same randomized doses shown in . Five minutes after the three 20-nl antagonist injections, agonist dose responses were performed as in the previously mentioned agonist protocol.

Histologic Confirmation of Drug Placement into the XII Nucleus
After completion of the previously mentioned studies, a final injection (20 nl) of 5 mM 5-HT with Pontamine Blue dye was injected at the same coordinates that were used for agonist injection studies. The animal was then euthanized with 1 g of urethane IV, and the brainstem was prepared for histologic review after counterstaining with Neutral Red, as in .

Data Analysis
The main effect analyzed was the percentage of change in XII nerve peak respiratory activity, relative to baseline XII nerve respiratory amplitude (15, 32, 33). Data analyzed and reported were obtained from recording periods with stable blood pressure and end-tidal CO₂ signals. One-way analysis of variance was used to determine the overall effect of agonist on excitation of XII motor neurons. Where the analysis of variance F test was less than 0.05 and a Gaussian distribution was confirmed for all but normal saline condition, a Tukey-Kramer multiple comparison of means was performed for specific concentrations compared with saline. An antagonist effect on baseline was examined using the Student's paired t test with Bonferroni correction. An antagonist effect on agonist dose–response was performed with two-way analysis of variance.

	RESULTS

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Dose–Responses for 5-HT, 5-CT, and RO 60-0175
A summary of effects of drugs, sample sizes, range of effect, and statistical significance of effect relative to saline injection is provided in . Dose–response curves are illustrated in  . The response pattern of RO (2C agonist) differed from that of 5-HT in several ways. First, the maximum response (at 1,000 µM) was significantly less in the RO (2C agonist) trials (two-tailed unpaired t, t = 2.4, p < 0.05). In addition, the lowest dose for which a significant increase was observed was 0.1 µM for 5-HT (p < 0.05) and 10 µM for RO (2C agonist) (p < 0.01). Finally, RO (2C agonist) injections resulted in a rapid desensitization, as shown in  .

fig.ommitted Figure 3. Hypoglossal respiratory activity dose–response curves for 5-HT and 5-HT agonists in vivo. Data shown are average normalized values ± SE for effects on hypoglossal nerve respiratory activity at each concentration tested. The log dose–response for 5-HT microinjected into the hypoglossal nuclei in 28 adult rats is depicted with closed circles and a solid line. 5-CT injected into the XII nucleus results in a very similar dose–response curve (n = 23; open diamonds and dashed lines). RO-601075 (5-HT2C agonist) has a reduced maximum response (n = 17; open circles and solid line). Significant increases are seen for 5-HT and 5-CT for all doses of more than 0.1 µM (p < 0.05). For RO-600175, significant responses are present for all doses of more than 1 µM (p < 0.05).

 

fig.ommitted Figure 4. Repeated injections of a selective 5-HT 2C agonist into the hypoglossal nucleus. Without moving the pipette, three injections of 20 nl of RO-600175 are administered at intervals of 15 minutes into the right hypoglossal nucleus (right XII). In contrast to repeated injections of 5-HT, injections of the 2C agonist resulted in rapid return to baseline activity and diminished responses with repeated injections.

 
Baseline XII Nerve Activity Response to Selective 5-HT Antagonists Injected into the XII Nucleus
The effects of five different antagonists were tested on baseline XII nerve activity and are summarized in  . The largest effect was seen with the MDL (2A antagonist) (62 ± 7% reduction in respiratory activity, n = 5, p < 0.01). Methiotepin mesylate (broad-spectrum antagonist) injected after MDL (2A antagonist) did not result in a further reduction in activity (65 ± 17%, n = 4, paired t, t = 0.9, p = NS).

fig.ommitted Figure 5. Effects of locally injected antagonists (2 µM, 60 l) on baseline hypoglossal nerve activity. The largest effect is observed with the selective 5-HT2A antagonist, MDL-100,907. Baseline hypoglossal nerve activity was reduced by 62 ± 7% (n = 5, p < 0.01). Methiotepin (MTP) suppressed activity by 20 ± 6% (n = 5, p < 0.05). The 5-HT7 antagonist, SB-269,970, (SB269, n = 4), had no effect on hypoglossal activity. The two 5-HT2C antagonists minimally suppressed hypoglossal activity. SB-206,553 (SB206) reduced activity to 95 ± 6% (n = 5, NS), and SB-242,084 (SB242) reduced activity to 83 ± 7% (n = 4, p < 0.05).

 
Effects of Selective 5-HT Antagonists on Dose Response Curves of 5-HT, 5-CT, and RO 60-0175
The effects of selective antagonists on the 5-HT and 5-CT (broad-spectrum agonist) dose–response curves are depicted in  . MDL (5-HT_2A antagonist) significantly altered the 5-HT dose effects (F = 68, p < 0.001). MDL significantly reduced the 5-HT responses for the following concentrations of 5-HT: 10 µM, p < 0.05, and for 100 µM, 1,000 v, and 5,000 µM, p < 0.001. In contrast, there was no effect of MDL on the 5-CT dose response curve (F = 2, p = NS). SB269 (5-HT₇ antagonist) had no effect on either the 5-HT (F = 0.4) or the 5-CT response (F = 0.3). SB242 (5-HT_2C agonist) had no effect on 5-HT (F = 0.2) but did reduce the 5-CT response (overall, F = 26, p < 0.001, with differences for the 10, 100, and 1,000 µM, p values all < 0.05). Sample sizes for each dose for RO (5-HT_2C agonist) experiments did not allow a statistical comparison. The effects are shown in  .

fig.ommitted Figure 6. Effects of selective 5-HT antagonists on serotonergic facilitation of hypoglossal respiratory activity. (A) and (B) show the effect of MDL-100907 on responses to 5-HT (A) and 5-CT (B). Data are expressed as mean percent change from baseline ± SE. Pretreatment of the hypoglossal nucleus with this 5-HT2A antagonist largely suppresses 5-HT excitation of hypoglossal (XII) nerve activity (A). MDL-100,907 has less effect on the 5-CT facilitation of hypoglossal nerve activity (B). The selective 5-HT7 antagonist, SB-269,970 (C, D) did not alter 5-HT or 5-CT responses. The lower panels show a minimal effect of the selective 5-HT2C antagonist, SB-242804 on the 5-HT (E) and 5-CT (F) effect. This 5-HT2C antagonist reduces the 5-CT response. Together, these response patterns suggest that in this animal preparation, the predominant 5-HT receptor subtype involved in 5-HT excitation of the hypoglossal nerve is the 5-HT2A receptor. The 5-CT response may be explained at least in part by 5-HT2A and 5-HT2C receptor activation. Finally, there is no evidence that either 5-HT or 5-CT excitation of hypoglossal nerve activity within the hypoglossal nucleus is mediated by 5-HT7 activation.

 

fig.ommitted Figure 7. Effects of local selective 5-HT2A and 5-HT2C antagonists on concentration response curves for the 5-HT2C agonist, RO-600,175, microinjected into the hypoglossal nucleus. The without antagonist reference curve (average percent changes relative to baseline for 17 rats) for the RO-600,175 response is shown as the solid line and closed circle. The effect of pretreating the hypoglossal nucleus with a 5-HT2A antagonist is minimal (dashed line, open circles, n = 12). In contrast, the effect of the 5-HT2C antagonist, SB-242,084 (n = 11), is more apparent, appearing to shift the EC50 to the right by several log concentrations.

 

	DISCUSSION

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Recently, we identified 5-HT_2A as the predominant 5-HT receptor subtype mRNA in individual XII motor neurons in adult rats, whereas smaller quantities of 5-HT_2C were present in a subset of XII motor neurons (30). The present study demonstrates a functional role for both receptor subtypes in 5-HT excitatory activity within the XII nucleus. The predominant effect is 5-HT_2A. The 5-HT_2A receptor is involved in intrinsic XII nerve respiratory activity in this preparation, and a selective 5-HT_2A antagonist largely suppresses the excitatory response to microinjections of 5-HT into the XII nucleus, across a large range of concentrations. The 5-HT_2C effect is distinct from the 5-HT_2A effect, with the 2C selective agonist showing different response patterns with agonist/antagonist combinations and a rapid desensitization not seen with repeated 5-HT or 5-CT injections. The 5-CT effect is best explained by agonist properties at 5-HT_2A and 5-HT_2C, and there is no evidence that either 5-HT or 5-CT act at either the 5-HT₇ receptor or an additional 5-HT receptor subtype in the XII nucleus to increase XII nerve activity. This study provides a pharmacologic characterization of 5-HT excitatory effects within a representative population of upper airway dilator motor neurons, the XII nucleus.

Pharmacologic Characterization of Serotonergic XII Motoneuron Excitation
Results in this study provide several lines of evidence for a strong 5-HT_2A excitatory effect at XII motor neurons. Specifically, the 5-HT_2A antagonist is highly selective (by several log-fold differences) for the 5-HT_2A receptor (35). This drug at a small concentration almost completely blocks the 5-HT excitation of XII nerve activity, and methiotepin, a drug with greater affinity at 5-HT receptor subtypes other than 2A, does not add to this suppression (36). The specificity of the MDL at 5-HT_2A receptors is evidenced by the much smaller effect of MDL on the 5-HT_2C agonist, RO-60,0175. Together, these data suggest that the incomplete blockade of 5-HT at higher doses is more likely the result of insufficient diffusion of MDL to all hypoglossal 5-HT_2A motor neuron receptors, rather than involvement of other receptor subtypes.

Evidence for a 5-HT_2C effect stems from the dramatic and rapid desensitization of the RO-60,0175 (2C agonist) response, together with no apparent desensitization effect apparent with 5-HT injections. Reduced responsiveness has been observed for 5-HT_2C receptor subtype activation in the cortex (37, 38). The observation that repeated 5-HT injections do not reveal desensitization suggests that 5-HT_2C receptors contribute little to the overall 5-HT excitation in our animal preparation. Desensitization did occur with the highest doses of 5-CT, a drug with lower affinity at 5-HT_2C receptors than RO (2C agonist) (39). In addition, MDL altered the dose–response less for RO (2C agonist) than for 5-HT, whereas the 5-HT_2C antagonist (40, 41) largely suppressed the RO response.

The results of these studies do not support a 5-HT₇–mediated excitation of XII respiratory activity within the XII nucleus. The absence of effect for the 5-HT₇ antagonist (42) on intrinsic XII activity or either the 5-HT or 5-CT effect, coupled with a significant reduction in 5-CT effect from the 2A and 2C antagonists, strongly suggests that 5-CT excitatory activity in the hypoglossal nucleus occurs via the 2A and 2C receptor subtypes. This is consistent with our recent mRNA XII motoneuron findings (30). 5-HT₇ mRNA was not detected in XII motor neurons or elsewhere within the nucleus, whereas it was found in significant quantities lateral to the XII nucleus. This study extends these findings by showing no functional significance for the 5-HT₇ receptor within the XII nucleus. In agreement with our present findings, 8-hydroxy-2-(di-N-propylamino)-tetralin, a 5-HT₁ and 5-HT₇ agonist, has a predominantly inhibitory effect on XII respiratory activity, consistent with a 5-HT₁ effect, rather than 5-HT₇ (39).

The Presence of Two Related 5-HT Receptor Subtypes within the XII Nucleus
It is of interest that XII motor neurons have two very similarly configured 5-HT excitatory receptors with seemingly similar signal transduction (2A and 2C). In this study, we have shown that activation of each of these receptor subtypes results in distinct XII nerve activity responses, distinguished by magnitude of effect and rapid desensitization. Until recently, cellular signal transduction mechanisms were believed to be similar for the 2A and 2C receptor subtypes (43). One exception recently identified is that the 2C receptor is more prone to desensitization than 2A (in vitro, on similar cells). Moreover, very high receptor occupancy (more than 99%) restores 2A responsiveness but not that of 2C (37). The differences in desensitization and resensitization relate to which protein kinases are activated, for example, protein kinase C or calmodulin-dependent protein kinase II (41). We hypothesize that these differences in protein kinase activation result in differences in glutamate receptor subtype activation, and this may explain differences in desensitization. Understanding how the sensitization of upper airway motor neurons changes by receptor subtype activation and how upper airway obstruction, intermittent hypoxia, and hypercarbia alter 5-HT excitation of upper airway motor neurons will be important in elucidating drugs for OSAHS.

5-HT depolarizes brainstem and spinal motor neurons through at least two mechanisms, a reduction in the resting potassium conductance and enhancement of the hyperpolarization-activated inward rectifier current, I_H (28, 29, 44). Potassium channel closure is likely mediated by 5-HT_2A or 5-HT_2C (17, 45). The 5-HT receptor subtypes, however, involved in modulation of excitability through I_H are not known. 5-HT_2A and 5-HT_2C receptor activation may explain the phosphorylation-dependent I_H-mediated 5-HT modulation but leaves unexplained a phosphorylation-independent mechanism of 5-HT facilitation. Specifically, the 5-HT–induced inward current can be reduced by adenylyl cyclase inhibitors (29). I_H may be activated by other monoaminergic neuromodulators, including noradrenaline and metatrobic glutamate receptors (45). We hypothesize that the phosphorylation-independent 5-HT excitation of brainstem motor neurons may be predominantly a facilitatory effect on noradrenergic or glutamatergic excitation of XII motor neurons in the XII nucleus. A similar mechanism explains an additive effect of 5-HT on glutamatergic transmission in the cortex (46).

Previous studies have shown that systemically administered 5-HT antagonists may augment hypoglossal nerve activity (47, 48), and clinical trials have been initiated to test two 5-HT antagonists as drugs for OSAHS. The mechanisms through which 5-HT antagonists may increase respiratory drive are unknown but may involve both central (49) and peripheral effects (50). Our work predicts that 5-HT antagonists active at the 5-HT_2A receptors should reduce hypoglossal activity.

Conclusion
We have shown that within the XII nucleus of the normal anesthetized adult rat, the predominant 5-HT excitatory effects on XII nerve respiratory activity are mediated through activation of 5-HT_2A and 5-HT_2C receptors. These two receptors explain most, if not all, of the local 5-HT excitatory response. Response patterns are quite unique for each receptor subtype, and this may provide clues to the significance of having two similarly configured 5-HT receptor subtypes present at XII motor neurons. The protocols used in this study are transferable for work involving other receptors involved in excitation of upper airway dilator motor neuronal excitation. Thus, it is now possible to characterize the functional significance of many receptor subtypes in upper airway motor neuron control. This study focused exclusively on the XII nerve responses. The hypoglossal nerve innervates the largest upper airway dilator muscles, and whether other brainstem motor neurons have a similar pharmacological profile remains to be determined. This work provides a focus for serotonergic pharmacologic treatment of obstructive sleep apnea and a model system with which to characterize in detail the function of neurotransmitters on upper airway dilator nerve activity.

	Acknowledgments

 
The authors thank Dr. Irwin Lucki and Dr. Leszek Kubin for their helpful comments during the course of these studies.

eived in original form February 12, 2002; accepted in final form August 16, 2002

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