Designer Receptors Exclusively Activated by Designer Drugs Approach to Treatment of Sleep-disordered Breathing

Abstract

Rationale: Obstructive sleep apnea is recurrent upper airway obstruction caused by a loss of upper airway muscle tone during sleep. The main goal of our study was to determine if designer receptors exclusively activated by designer drugs (DREADD) could be used to activate the genioglossus muscle as a potential novel treatment strategy for sleep apnea. We have previously shown that the prototypical DREADD ligand clozapine-N-oxide increased pharyngeal diameter in mice expressing DREADD in the hypoglossal nucleus. However, the need for direct brainstem viral injections and clozapine-N-oxide toxicity diminished translational potential of this approach, and breathing during sleep was not examined.Objectives: Here, we took advantage of our model of sleep-disordered breathing in diet-induced obese mice, retrograde properties of the adeno-associated virus serotype 9 (AAV9) viral vector, and the novel DREADD ligand J60.Methods: We administered AAV9-hSyn-hM3(Gq)-mCherry or control AAV9 into the genioglossus muscle of diet-induced obese mice and examined the effect of J60 on genioglossus activity, pharyngeal patency, and breathing during sleep.Measurements and Main Results: Compared with control, J60 increased genioglossus tonic activity by greater than sixfold and tongue uptake of 2-deoxy-2-[¹⁸F]fluoro-d-glucose by 1.5-fold. J60 increased pharyngeal patency and relieved upper airway obstruction during non-REM sleep.Conclusions: We conclude that following intralingual administration of AAV9-DREADD, J60 can activate the genioglossus muscle and improve pharyngeal patency and breathing during sleep.

Keywords: chemogenetics; hypoglossal motoneurons; obstructive sleep apnea; pharmacotherapy.

Figure 1.

Histological images in AAV9-hSyn-GFP and AAV-hSyn-DIO-hM3D(Gq)-mCherry designer receptors exclusively activated by designer drugs (DREADD)–treated mice. Fluorescent microscopy images (×20) of the brain medulla show (A) GFP (green fluorescent protein) and (B) DREADD mCherry expression in the hypoglossal nucleus (12 N). Fluorescent images (×20) of the tongue also show (C) GFP and (D) DREADD expression. Cell nuclei were stained with DAPI (blue). Representative hematoxylin and eosin images of the tongue (×20) in (E) GFP- and (F) DREADD-treated mice showed no evidence of inflammation or tissue injury. Scale bars, 50 μm. Arrows represent the localization of DREADD or GFP (n = 27). The mixed effect multivariable linear regression was used for statistical comparisons. 4V = fourth ventricle; Ep = epithelium; M = muscle.

Figure 2.

Genioglossus EMG recordings. (A) Representative genioglossal electromyography (EMG_GG), moving average (∫EMG_GG), and respiratory effort recorded at baseline (left) and after J60 administration (right) in a mouse expressing designer receptors exclusively activated by designer drugs (DREADD) in the hypoglossal motoneurons. Note the robust increase in both phasic and tonic EMG activity after J60. (B) EMG response to J60 or saline in DREADD-treated animals (n = 9; baseline and 15 min after injections) and EMG response to J60 and mice treated with control GFP virus (n = 5, baseline and 15 min after injections). Tonic and phasic EMG values were normalized to peak phasic EMG at baseline and plotted separately. *P < 0.001. The mixed effect multivariable linear regression was used for statistical comparisons. a.u. = arbitrary units; Exp. = expiration; GFP = green fluorescent protein; Insp. = inspiration.

Figure 3.

Positron emission tomography data. Representative positron emission tomography scan images (A) in AAV9-hSyn-GFP–treated mice (n = 5) and (B) in AAV-hSyn-DIO-hM3D(Gq)-mCherry–treated mice (n = 7). Note the robust increase in tongue metabolic activity (white arrow) in mice treated with designer receptors exclusively activated by designer drugs (DREADD). 2-Deoxy-2-[¹⁸F]fluoro-d-glucose uptake in standardized uptake values in individual mice infected with (C) GFP or (D) DREADD. Bars reflect median values. *P < 0.001. The mixed effect multivariable linear regression was used for statistical comparisons. GFP = green fluorescent protein; SUV = standardized uptake value.

Figure 4.

Upper airway magnetic resonance imaging was performed after saline and J60 treatments in AAV9-hSyn-hM3D(Gq)-mCherry designer receptors exclusively activated by designer drugs (DREADD)-infected mice (n = 5), whereas only J60 treatment was performed in AAV9-hSyn-GFP–infected mice (n = 4). Representative axial magnetic resonance imaging of the pharynx of the mouse expressing DREADD is shown. The axial sections 5 mm caudal to the hard–soft palate junction after the injection of (A) saline or (B) J60 are shown. Note the dilatation of the upper airway following J60 infusion in comparison to saline (white arrows). (C) Increases in pharynx cross-sectional areas caudal to the hard–soft palate junction on inspiration (Inspiration – Expiration; I-E) before and after treatment were significant only at the 5 mm distance in DREADD-infected mice after J60. *P < 0.05 from baseline, compared with saline in the same mice and compared with J60 in GFP-infected mice. The mixed effect multivariable linear regression was used for statistical comparisons. Scale bar, 1 cm. GFP = green fluorescent protein.

Figure 5.

Sleep recordings. (A and F) Representative NREM sleep recordings (A) and REM (F) in the same mouse expressing AAV9-hSyn-hM3D(Gq)-mCherry designer receptors exclusively activated by designer drugs (DREADD) in the hypoglossal motoneurons after treatment with saline (left) versus J60 (right). + denotes breaths with inspiratory flow limitation. For NREM, (B) maximal inspiratory flow (Vi_max), (C) $\dot{\text{V}}$e in mice expressing DREADD, and (D) Vi_max and (E) $\dot{\text{V}}$e in mice expressing GFP in the hypoglossal motoneurons are shown. For REM, (G) Vi_max, (H) $\dot{\text{V}}$e in mice expressing DREADD, and (I) Vi_max and (J) $\dot{\text{V}}$e in mice expressing GFP in the hypoglossal motoneurons are shown. Every line in B–J represents an individual mouse treated with saline or J60. Means ± SEM are shown. *P < 0.05 by Wilcoxon rank-sum test. a.u. = arbitrary units; EXPIR = expiration; GFP = green fluorescent protein; INSP = inspiration; NREM = non-REM.