Gasotransmitter modulation of hypoglossal motoneuron activity
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eLife assessment
The purpose of this study was to determine whether heme oxygenase -2 deficiency translates to deficiencies in motor neuron function. This paper plays a plausible mechanism by which heme oxygenase-2 deficiency can lead to obstructive apneas. Indeed, this is among the first papers to comprehensively describe a signaling pathway in motor neurons and the consequences of its deficiency. Furthermore, the work completed here may be relevant to other diseases in which motor neuron signal transmission is a key contributor.
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Abstract
Obstructive sleep apnea (OSA) is characterized by sporadic collapse of the upper airway leading to periodic disruptions in breathing. Upper airway patency is governed by genioglossal nerve activity that originates from the hypoglossal motor nucleus. Mice with targeted deletion of the gene Hmox2, encoding the carbon monoxide (CO) producing enzyme, heme oxygenase-2 (HO-2), exhibit OSA, yet the contribution of central HO-2 dysregulation to the phenomenon is unknown. Using the rhythmic brainstem slice preparation that contains the preBötzinger complex (preBötC) and the hypoglossal nucleus, we tested the hypothesis that central HO-2 dysregulation weakens hypoglossal motoneuron output. Disrupting HO-2 activity increased the occurrence of subnetwork activity from the preBötC, which was associated with an increased irregularity of rhythmogenesis. These phenomena were also associated with the intermittent inability of the preBötC rhythm to drive output from the hypoglossal nucleus (i.e. transmission failures), and a reduction in the input-output relationship between the preBötC and the motor nucleus. HO-2 dysregulation reduced excitatory synaptic currents and intrinsic excitability in inspiratory hypoglossal neurons. Inhibiting activity of the CO-regulated H 2 S producing enzyme, cystathionine-γ-lyase (CSE), reduced transmission failures in HO-2 null brainstem slices, which also normalized excitatory synaptic currents and intrinsic excitability of hypoglossal motoneurons. These findings demonstrate a hitherto uncharacterized modulation of hypoglossal activity through mutual interaction of HO-2/CO and CSE/H 2 S, and support the potential importance of centrally derived gasotransmitter activity in regulating upper airway control.
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Author Response
eLife assessment
The purpose of this study was to determine whether heme oxygenase -2 deficiency translates to deficiencies in motor neuron function. This paper plays a plausible mechanism by which heme oxygenase-2 deficiency can lead to obstructive apneas. Indeed, this is among the first papers to comprehensively describe a signaling pathway in motor neurons and the consequences of its deficiency. Furthermore, the work completed here may be relevant to other diseases in which motor neuron signal transmission is a key contributor.
We thank for their assessment and constructive comments. Based on their input below we performed additional analyses focused on the impact of HO-2 dysregulation on the rhythmogenesis from the preBötC.
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eLife assessment
The purpose of this study was to determine whether heme oxygenase -2 deficiency translates to deficiencies in motor neuron function. This paper plays a plausible mechanism by which heme oxygenase-2 deficiency can lead to obstructive apneas. Indeed, this is among the first papers to comprehensively describe a signaling pathway in motor neurons and the consequences of its deficiency. Furthermore, the work completed here may be relevant to other diseases in which motor neuron signal transmission is a key contributor.
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Reviewer #1 (Public Review):
The purpose of this study was to determine whether heme oxygenase -2 deficiency translates to deficiencies in motor neuron function. This paper plays a plausible mechanism by which heme oxygenase-2 deficiency can lead to obstructive apneas. Indeed, this is among the first papers to comprehensively describe a signaling pathway in motor neurons and the consequences of its deficiency.
The major strengths of this paper include comprehensive pharmacological and genetic methods, and the combination of histology and functional electrophysiological measures of neuronal function. While it is not clear the mechanism by which heme oxygenase-2 deficiency might occur in motor neuron pools, or the relevance to human disease, the authors identify several targetable molecules in the hypoglossal motor neurons that are …
Reviewer #1 (Public Review):
The purpose of this study was to determine whether heme oxygenase -2 deficiency translates to deficiencies in motor neuron function. This paper plays a plausible mechanism by which heme oxygenase-2 deficiency can lead to obstructive apneas. Indeed, this is among the first papers to comprehensively describe a signaling pathway in motor neurons and the consequences of its deficiency.
The major strengths of this paper include comprehensive pharmacological and genetic methods, and the combination of histology and functional electrophysiological measures of neuronal function. While it is not clear the mechanism by which heme oxygenase-2 deficiency might occur in motor neuron pools, or the relevance to human disease, the authors identify several targetable molecules in the hypoglossal motor neurons that are candidates for future study.
Furthermore, the work completed here may be relevant to other diseases in which motor neuron signal transmission is a key contributor.
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Reviewer #2 (Public Review):
The tongue muscles play a major role in many important behaviors including suckling, swallowing, and ensuring that the upper airway remains open during breathing. Hypoglossal motoneurons innervate tongue muscles and as such play a key role in homeostasis. Previous work has shown that heme-oxygenase-2 (HO-2) null mice have severe airway obstruction. HO-2 forms carbon monoxide, suggesting that CO contributes to hypoglossal motoneuron excitability. In addition to HO-2, the present study also shows that hypoglossal motoneurons express cystathionine ϒ-lyase (CSE), which produces H2S and is regulated by CO. Interestingly, the authors show that H2S reduced transmission of the breathing-related drive to hypoglossal motoneurons. Together these observations suggest that CO and H2S interact to maintain the excitability …
Reviewer #2 (Public Review):
The tongue muscles play a major role in many important behaviors including suckling, swallowing, and ensuring that the upper airway remains open during breathing. Hypoglossal motoneurons innervate tongue muscles and as such play a key role in homeostasis. Previous work has shown that heme-oxygenase-2 (HO-2) null mice have severe airway obstruction. HO-2 forms carbon monoxide, suggesting that CO contributes to hypoglossal motoneuron excitability. In addition to HO-2, the present study also shows that hypoglossal motoneurons express cystathionine ϒ-lyase (CSE), which produces H2S and is regulated by CO. Interestingly, the authors show that H2S reduced transmission of the breathing-related drive to hypoglossal motoneurons. Together these observations suggest that CO and H2S interact to maintain the excitability of hypoglossal motoneurons. An imbalance of these competing influences may underlie tongue muscle dysfunction in conditions such as obstructive sleep apnea and dysphagia. Thus, the novel observations reported here open new avenues that will lead to a better understanding of the highly complex tongue muscle motor system.
The team used brain stem slices containing hypoglossal motoneurons that were receiving rhythmic, breathing-related depolarizing drive from the pre-Botzinger complex, the putative respiratory central pattern generator. Extracellular population activity was recorded from the pre-Botzinger complex and the ipsilateral hypoglossal motor nucleus-and in some experiments-the hypoglossal premotor population located in the ipsilateral reticular formation. In these rhythmic slices, the fidelity between hypoglossal and pre-Botzinger bursts approached 100% and blocking HO-2 with drugs reduced fidelity by about 25%. In addition, HO-2 null mice showed an even larger reduction in pre-Botzinger-hypoglossal bursting fidelity of 40-45%. Since these interventions did not significantly impact the pre-Botzinger population activity, the data show convincingly that CO contributes to hypoglossal motoneuron excitability. While whole cell intracellular recordings of hypoglossal motoneurons showed that blocking HO-2 reduced the magnitude of inspiratory drive currents and the number of action potentials generated in response to the depolarizing drive, these effects were relatively modest, suggesting that the mechanisms that underlie these important observations are unsettled.
The other key part of these experiments was demonstrating that the H2S forming enzyme CSE is expressed in the hypoglossal nucleus, and that exogenous application of NaHS, an H2S donor, reduces the fidelity of pre-Botzinger-hypoglossal coupling. Interestingly, H2S activity was lower in HO-2 null mice, and HO-2-dependent uncoupling could be rescued by bath application of the CO donor CORM-3. Moreover, failed transmission between pre-Botzinger and hypoglossal population bursts was not observed in preparations from mice null for both HO-2 and CSE. These data support excitatory-inhibitory interactions that are triggered by the gases CO and H2S.
Mechanistic experiments showed that blockade of CSE with L552 propargylglycine could restore transmission fidelity, and this was accompanied by a substantial increase in the magnitude of inspiratory-related drive currents in hypoglossal motoneurons. The team also showed that blocking small-conductance potassium channels with apamin in slices where HO-2 was first blocked pharmacologically enhanced inspiratory drive currents, though this observation needs re-examination given that the effect was large in two neurons, but very small in the remaining four (Fig. 8 A3). Finally, similar experiments focused on the ATP-sensitive potassium channel (KATP), which was previously shown to be enhanced by H2S. Blockade of KATP with tolbutamide had no impact on hypoglossal motoneuron output. In conclusion, the experiments reveal a very important contribution of gas-mediated transmitters in setting the balance of excitation and inhibition of hypoglossal motoneuron excitability. More work will be needed to unravel the mechanisms behind these exciting observations.
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Reviewer #3 (Public Review):
Previous work on HO-2 null mice suggest that the increased occurrence of central and obstructive sleep apneas observed in these animals is linked to hyperactivation of carotid bodies through a CO-dependent H2S increase mechanism within the carotid bodies. Hyperoxia, genetic ablation or pharmacological bloc of CSE (a CO-dependent H2S producing enzyme) reduced the occurrence of both central and obstructive apneas.
Here, the authors propose an alternate, or complementary view to address occurrence of OSA in HO-2 null mice. In in vitro medullary slice preparations they used the same pharmacological and genetic approaches to manipulate levels of CO and H2S and observe that inhibition or elimination of HO-2 induces a transmission failure between the preBotC and hypoglossal motoneurons that is potentially linked to …
Reviewer #3 (Public Review):
Previous work on HO-2 null mice suggest that the increased occurrence of central and obstructive sleep apneas observed in these animals is linked to hyperactivation of carotid bodies through a CO-dependent H2S increase mechanism within the carotid bodies. Hyperoxia, genetic ablation or pharmacological bloc of CSE (a CO-dependent H2S producing enzyme) reduced the occurrence of both central and obstructive apneas.
Here, the authors propose an alternate, or complementary view to address occurrence of OSA in HO-2 null mice. In in vitro medullary slice preparations they used the same pharmacological and genetic approaches to manipulate levels of CO and H2S and observe that inhibition or elimination of HO-2 induces a transmission failure between the preBotC and hypoglossal motoneurons that is potentially linked to a post-synaptic effect on hypoglossal motoneurons, in particular at the level of apamin sensitive small conductance potassium channels.
Although drugs were bath applied rather than local applied to XII motoneurons, the authors provide evidence that HO-2 and CSE modulation affects the Input/Output relationship between the preBötC and the hypoglossal nucleus.
Given the occurrence on central apneas in these mice in vivo, the potential effects of H2S on preBotC neurons, the use of bath application in these experiments and the apparent effects on rhythmogenesis, additional assessment of preBötC function in these mice would benefit the study.
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