Fig 1.
OX-201 selectively activated OX2R and produced wake-promoting effects via OX2R activation in mice and rats.
(A) Chemical structure of OX-201. (B) Effects of OX-201 on calcium mobilization in hOX2R/CHO-K1 cells and hOX1R/CHO-K1 cells. The responses to 0.5% DMSO in the absence and presence of 100 nM OX-A (positive control) were used to represent the 0% and 100% responses, respectively. n = 4. (C) Effects of oral (p.o.) administration of OX-201 on wakefulness time in C57BL/6J mice. n = 10, **P = 0.003, ***P < 0.001 versus vehicle-treated mice. (D) Effects of OX-201 (p.o.) on wakefulness time in OX2R KO mice. n = 8. (E) Effects of OX-201 (p.o.) on wakefulness time in rats. n = 8, ***P < 0.001 versus vehicle-treated rats. Data are presented as mean ± standard error of the mean.
Fig 2.
Danavorexton, OX-201, and OX-A activated inspiratory neurons in the pre-Bötzinger complex in rat medullary slices.
(A) Representative traces by whole-cell patch clamp recording of inspiratory neurons in the pre-Bötzinger complex before and after perfusion with danavorexton at 3 μM. (B) The method to calculate peak amplitude, duration, and area of inspiratory synaptic currents from recorded signals. (C) Effects of danavorexton on the frequency, peak amplitude, duration, and area of inspiratory synaptic currents. Numbers of neurons recorded in each concentration (n) are 4 at 30 nM, 4 at 100 nM, 5 at 300 nM, 5 at 1 μM, 4 at 3 μM, and 4 at 10 μM (frequency); 4 at 30 nM, 3 at 100 nM, 5 at 300 nM, 3 at 1 μM, 4 at 3 μM, and 3 at 10 μM (peak amplitude and area); 4 at 30 nM, 3 at 100 nM, 5 at 300 nM, 4 at 1 μM, 4 at 3 μM, and 3 at 10 μM (duration). (D) Effects of OX-201 on the frequency, peak amplitude, duration, and area of inspiratory synaptic currents. Numbers of neurons recorded in each concentration (n) are 3 at 100 nM, 10 at 300 nM, 12 at 1 μM, 10 at 3 μM, and 6 at 10 μM. (E) Effects of OX-A on the frequency, peak amplitude, duration, and area of inspiratory synaptic currents. Numbers of neurons recorded in each concentration (n) are 4 at 1 nM, 3 at 3 nM, 6 at 10 nM, 1 at 30 nM, 7 at 100 nM, and 3 at 300 nM. Neuronal activity was recorded in the presence of vehicle followed by the application of drugs. Mean values in the frequency, peak amplitude, duration, and area of inspiratory synaptic currents during the last 1–2 min in the presence of vehicle were used as control values, and those during the last 1–2 min with stimulation by each concentration of drug were used to calculate percent changes from control values. *P < 0.05, ***P < 0.001 versus control. Data are presented as mean ± standard error of the mean.
Fig 3.
Danavorexton, OX-201, and OX-A increased burst activity in rat isolated brainstem−spinal cord preparations.
(A) Representative bandpass-filtered (0.01–3000 Hz) electrophysiological traces of burst activity recorded from the cervical (C3–C5) ventral root before and after perfusion with danavorexton at 10 μM. (B) The method to calculate burst amplitude, burst duration, and burst area from integrated signals. (C) Effects of danavorexton on burst frequency, burst amplitude, burst duration, and burst area. Numbers of tissues recorded in each concentration (n) are 5 at 1 μM, 5 at 3 μM, and 6 at 10 μM. (D) Effects of OX-201 on burst frequency, burst amplitude, burst duration, and burst area. Curve fitting was not implemented because only two concentrations of OX-201 were tested. Numbers of tissues recorded in each concentration (n) are 5 at 3 μM and 4 at 10 μM. (E) Effects of OX-A on burst frequency, burst amplitude, burst duration, and burst area. Numbers of tissues recorded in each concentration (n) are 6 at 3 nM, 6 at 10 nM, 8 at 30 nM, 14 at 100 nM, and 6 at 300 nM. Burst activity was recorded in the presence of vehicle followed by the application of drugs. Mean values in burst frequency, burst amplitude, burst duration, and burst area during the last 2 min in the presence of vehicle were used as control values, and those during the last 2 min of drug perfusion were used to calculate percent changes from control values. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control. Data are presented as mean ± standard error of the mean.
Fig 4.
Danavorexton, OX-201, and OX-A activated hypoglossal motoneurons in rat medullary slices.
(A) Representative traces by whole-cell patch clamp recording of hypoglossal motoneurons before and after perfusion with danavorexton at 10 μM. (B) The method to calculate peak amplitude, duration, and area of inspiratory synaptic currents from recorded signals. (C) Effects of danavorexton on the frequency, peak amplitude, duration, and area of inspiratory synaptic currents. Numbers of neurons recorded in each concentration (n) are 5 at 30 nM, 7 at 100 nM, 7 at 300 nM, 8 at 1 μM, 5 at 3 μM, and 5 at 10 μM. (D) Effects of OX-201 on the frequency, peak amplitude, duration, and area of inspiratory synaptic currents. Numbers of neurons recorded in each concentration (n) are 10 at 100 nM, 11 at 300 nM, and 10 at 1 μM. (E) Effects of OX-A on the frequency, peak amplitude, duration, and area of inspiratory synaptic currents. Numbers of neurons recorded in each concentration (n) are 10 at 3 nM, 10 at 10 nM, and 8 at 30 nM. Neuronal activity was recorded in the presence of vehicle followed by the application of drugs. Mean values in the frequency, peak amplitude, duration, and area of inspiratory synaptic currents during the last 1–2 min in the presence of vehicle were used as control values, and those during the last 1–2 min with stimulation by each concentration of drugs were used to calculate percent changes from control values. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. Data are presented as mean ± standard error of the mean.
Fig 5.
Intravenous (i.v.) OX-201 administration significantly increased burst frequency of the diaphragm in anesthetized rats.
(A) Representative traces by EMG recording of the diaphragm before and after administration of vehicle or OX-201 at 3 mg/kg. (B) The method to calculate burst amplitude and tonic activity from integrated EMG signals. (C) Effects of OX-201 on burst frequency, burst amplitude, and tonic activity of the diaphragm. Tonic activity was defined as the mean value of integrated EMG signals between bursts. Mean values in burst frequency, burst amplitude, and tonic activity during 2 min before vehicle or OX-201 administration were used as control values, and those during 10 min after vehicle or OX-201 administration were used to calculate percent changes from control values. n = 8, *P = 0.040, ***P < 0.001 versus vehicle-treated rats. Data are presented as mean ± standard error of the mean.
Fig 6.
Intravenous (i.v.) OX-201 administration significantly increased burst amplitude of the genioglossus muscle in anesthetized rats.
(A) Representative traces by EMG recordings of the genioglossus muscle before and after administration of vehicle or OX-201 at 1 mg/kg. (B) The method to calculate burst amplitude and tonic activity from integrated EMG signals. Tonic activity was defined as the mean value of integrated EMG signals between bursts. (C) Effects of OX-201 on burst frequency, burst amplitude, and tonic activity of the genioglossus muscle. Mean values in burst frequency, burst amplitude, and tonic activity during 2 min before vehicle or OX-201 administration were used as control values, and those during 10 min after vehicle or OX-201 administration were used to calculate percent changes from control values. n = 8, *P = 0.011, ***P < 0.001 versus vehicle-treated rats. Data are presented as mean ± standard error of the mean.
Fig 7.
Oral (p.o.) administration of OX-201 significantly increased respiratory activity in free-moving mice.
Effects of OX-201 on the (A) respiratory rate, (B) tidal volume, (C) minute volume, (D) peak inspiratory flow, (E) peak expiratory flow, (F) inspiratory time, and (G) expiratory time were measured. The mean values in each parameter during 3 h after vehicle or OX-201 administration were calculated. n = 16, *P = 0.035, **P = 0.0017, ***P < 0.001 versus vehicle-treated mice. Data are presented as mean ± standard error of the mean.