Abstract
A low arousal threshold (LAT) is a pathophysiological trait of obstructive sleep apnea (OSA) that may be associated with brainstem ascending reticular activating system-cortical functional connectivity changes. We evaluated resting-state connectivity between the brainstem nuclei and 105 cortical/subcortical regions in OSA patients with or without a LAT and healthy controls. Twenty-five patients with moderate to severe OSA with an apnea–hypopnea index between 20 and 40/hr (15 with and 10 without a LAT) and 15 age- and sex-matched controls were evaluated. Participants underwent functional magnetic resonance imaging after overnight polysomnography. Three brainstem nuclei—the locus coeruleus (LC), laterodorsal tegmental nucleus (LDTg), and ventral tegmental area (VTA)—associated with OSA in our previous study were used as seeds. Functional connectivity values of the two brainstem nuclei (LC and LDTg) significantly differed among the three groups. The connectivity of the LC with the precuneus was stronger in OSA patients than in controls regardless of the concomitant LAT. The connectivity between the LDTg and the posterior cingulate cortex was also stronger in OSA patients regardless of the LAT. Moreover, OSA patients without a LAT showed stronger LDTg-posterior cingulate cortex connectivity than those with a LAT (post hoc p = 0.013), and this connectivity strength was negatively correlated with the minimum oxygen saturation in OSA patients (r = − 0.463, p = 0.023). The LAT in OSA patients was associated with altered LDTg-posterior cingulate cortex connectivity. This result may suggested that cholinergic activity may play a role in the LAT in OSA patients.

Introduction
Obstructive sleep apnea (OSA) is a condition where the upper airway narrows or collapses during sleep. This can cause frequent arousals, sleep fragmentation, intermittent desaturation, and sympathetic activation1,2. Anatomically, most OSA patients have a narrowed upper airway that results in increased negative pressure during inspiration3. Moreover, nonanatomical factors, such as low arousal threshold (LAT), high loop gain, and poor upper airway muscle responsiveness, also contribute to the pathophysiology of OSA3,4. Arousal is associated with apnea termination and can play a protective role in OSA4. However, frequent arousal from sleep is also linked to sympathetic activation and leads to cardiometabolic complications or memory disturbance observed in OSA patients5,6.

The arousal threshold is a measure of the respiratory effort level that triggers arousal during sleep. Arousal responses differ between OSA patients, and 30–50% of them have a LAT7. Those with a LAT are prone to be easily aroused from sleep in response to relatively mild respiratory stimuli. The LAT can be identified noninvasively according to the following polysomnography results: (apnea–hypopnea index (AHI) < 30/hr) + (minimum oxygen saturation > 82.5%) + (fraction of hypopnea > 58.3%). Each criterion is scored as 1, and a score of more than 2 indicates a LAT in OSA patients8.

OSA patients with a LAT (OSA + LAT) are less obese, older, more likely to be female, and more likely to have rapid eye movement sleep-predominant OSA than those without a LAT (OSA-LAT)7,9,10. A LAT is associated with sleep discontinuity and poor compliance with PAP therapy9,11. Moreover, a LAT may be a therapeutic target in selected patients with OSA12. However, no studies have evaluated neural substrates or possible pathomechanisms associated with a LAT in OSA patients.

The ascending reticular activating system is a network of brainstem nuclei that is connected with cortical and subcortical regions and is involved in arousal and vigilance13. Patients with OSA may have a neural arousal-associated pattern generator that responds to an obstructive respiratory event14. We previously reported altered resting-state functional connectivity among three brainstem nuclei [the locus coeruleus (LC), laterodorsal tegmental nucleus (LDTg), and ventral tegmental area (VTA)] and cortical/subcortical regions in patients with moderate to severe OSA compared to that in controls15.

We hypothesized that the LAT in OSA patients may be related to the altered brainstem nuclei-cortical/subcortical functional connectivity. Our aim was to assess resting-state functional connectivity between the three preidentified brainstem nuclei and 105 cortical/subcortical regions in patients with moderate to severe OSA with or without a LAT compared to that in healthy controls.

Results
Patient characteristics
Twenty-five patients with moderate to severe OSA [apnea–hypopnea index (AHI) between 20/hr and 40/hr] and fifteen age- and sex-matched controls without OSA were evaluated. The mean participant age was 48 years old, and 34 (85.0%) were male, which was similar among the three groups. The Pittsburg Sleep Quality Index score was higher than that of the controls only in the OSA+LAT group (post hoc p = 0.005). The mean apnea, hypopnea, AHI, and minimum oxygen saturation in OSA patients were 8.6 ± 6.0/hr, 20.6 ± 5.9/hr, 29.2 ± 5.4/hr, and 81.0%, respectively. OSA + LAT patients showed a lower apnea index (post hoc p = 0.006) and higher minimum oxygen saturation than OSA-LAT patients (post hoc p = 0.001). No significant difference in sleep quality and daytime sleepiness was observed between the two groups.

Axial views of differences in voxelwise functional connectivity between the LC and the cortex or LDTg and the cortex when comparing patients with OSA-LAT vs. OSA + LAT vs. healthy controls. (A) Statistical maps of the regions where functional connectivity with the LC significantly differed among the three groups. (B) Statistical maps of the regions where connectivity with the LDTg significantly differed among the three groups (cluster-level p < 0.01, with false discovery rate correction p < 0.05). (C) The average functional connectivity value of the LC to the precuneus among controls, OSA-LAT, and OSA + LAT patients. (D) The average FC value of the LDTg to the posterior cingulate cortex among controls, OSA-LAT patients, and OSA + LAT patients. *p < 0.017, **p < 0.01, compared between two groups, post hoc Bonferroni correction. Abbreviations: OSA, obstructive sleep apnea; LAT, low arousal threshold; LC, locus coeruleus; LDTg, laterodorsal tegmental nucleus. Patients with OSA + LAT usually awake more frequently during sleep, which may lead to lower sleep quality16. Accordingly, we found that only OSA + LAT, not OSA-LAT showed higher PSQI score thant the controls. However, because no significant difference in sleep questionnaire score was found between the OSA-LAT and OSA + LAT, and our experimental correlation analysis showed no significant association, effect of sleep-related symptom on functional connectivity was considered to be limited. LDTg-posterior cingulate cortex connectivity differed according to the LAT in OSA patients. Pertinently, cholinergic neurons from the LDTg are part of the ascending reticular activating system, which is associated with arousal, and our results suggested that cholinergic activity may contribute to the concomitant LAT in OSA patients. The posterior cingulate cortex is also known as a crucial structure associated with arousal, and its functional connectivity changes according to arousal or awareness status17. Posterior cingulate cortex activation may be affected by cholinergic function because enhancing cholinergic activity through acetylcholine esterase inhibitors increases cerebral blood flow to the posterior cingulate cortex18. Among the factors determining LAT, minimum oxygen saturation showed the strongest association with the LDTg-posterior cingulate cortex connectivity strength. OSA-LAT patients are often exposed to a higher hypoxic burden than OSA + LAT patients because arousal may not occur even in situations of severe hypoxia. In healthy volunteers, sustained hypoxia delays the time to arousal and increases the respiratory arousal threshold19. Hypoxic burden may affect LDTg-cortical connectivity. Hypoxia decreases the number of cholinergic neurons20 and acetylcholine synthesis in rat brains21. Exposure to hypoxia also reduces cerebral blood flow to the posterior cingulate cortex22. Increased FC between LDTg and posterior cingulate cortex in OSA may be a compensatory mechanism for hypoxic burden in patients with OSA, which can be more severe in OSA-LAT patients than in those with OSA + LAT. Our results may imply that cholinergic activity is associated with OSA and the LAT. This suggested that modulating cholinergic function may be a therapeutic option that can improve OSA severity or the arousal threshold. Several studies have reported the effect of the acetylcholine esterase inhibitor donepezil on OSA severity. A placebo-controlled randomized trial showed that 10 mg donepezil in Alzheimer's disease patients with OSA improved AHI values and oxygen saturation23. Another study with nondemented participants reported improvement in AHI values, desaturation index values, and minimum oxygen saturation after one month of donepezil use24. However, the effect was not consistent. One randomized double-blind crossover study did not find a significant reduction in the AHI, minimum oxygen saturation or arousal threshold after donepezil administration for a single night25. Another randomized study also did not find changes in AHI values or minimum oxygen saturation after patients using donepezil for 1 month26. Several limitations should be considered when interpreting the results of our study. This was a single-center study with a limited number of participants. To minimize the effect of OSA severity on brainstem-cortical FC, we only evaluated patients with an AHI between 20 and 40/hr. Moreover, the demographics of our OSA + LAT patients differed from those in prior studies. Therefore, caution is warranted when generalizing our results to other populations. Age was included as a covariate, but OSA disease duration couldn’t be due to practical limitations. The LAT was not measured directly but was estimated using the polysomnography parameter, which is known to have high sensitivity and specificity8. Moreover, the association between the LAT and LDTg- posterior cingulate cortex functional connectivity does not imply a causal relationship between the two. Further pharmacological or neuromodulation studies will be needed to establish a causal relationship between the connectivity and LAT. Conclusions This study suggested that cholinergic LDTg– posterior cingulate cortex functional connectivity is associated with the LAT in patients with moderate to severe OSA. Among the factors determining LAT, minimum oxygen saturation exhibited the strongest correlation with LDTg- posterior cingulate cortex connectivity. Future studies with larger numbers of patients are needed to elucidate the clinical implications of LDTg- posterior cingulate cortex connectivity for determining individualized treatment options in OSA patients.