DREADD activation increases diaphragm EMG output in wild-type mice

A representative example of diaphragm EMG activity before and after application of the J60 DREADD ligand is shown in the top panel. Examples of the individual inspiratory EMG bursts at baseline (a), after vehicle (b), and after J60 (c) are shown. The J60 ligand increased diaphragm output but did not impact respiratory rate. The mean responses (n = 11; n = 7 females) for EMG AUC, peak-to-peak amplitude, tonic activity, and respiratory rate are shown in panels d-g. For diaphragm EMG data (panels d-f) left hemidiaphragm EMG is represented in orange, while right hemidiaphragm EMG is blue. Error bars depict ±D1 SEM. Statistical reports for all panels are provided in Supplemental Table 1. * and ‡ symbols indicate significant main effects (p < 0.05) on One-Way RM ANOVA for the left and right hemidiaphragm, respectively. Dia = diaphragm, AUC = area under the curve, amp = peak amplitude, BL = baseline, SL = saline (sham injection).

DREADD activation increases diaphragm EMG output in ChAT-Cre mice

A representative example of diaphragm EMG activity before and after application of the J60 DREADD ligand is shown in the top panel. Examples of the individual inspiratory EMG bursts at baseline (a), after vehicle (b), and after J60 (c) are shown. Mean responses (n = 9; n = 6 females) for EMG AUC, peak-to-peak amplitude, tonic activity, and respiratory rate are shown in panels d-g. The DREADD ligand caused a bilateral increase in diaphragm EMG AUC, peak-to-peak amplitude, and tonic activity. For all EMG parameters, the responses were greater on the right vs. left hemidiaphragm. Respiratory rate decreased over time. For panels d-f, the left hemidiaphragm EMG is represented in orange, while right hemidiaphragm EMG is blue. Error bars depict ±D1 SEM. Statistical reports for all panels are provided in Supplemental Table 2. * and ‡ symbols indicate significant main effects (p < 0.05) on One-Way RM ANOVA for the left and right hemidiaphragm, respectively. # indicates a significant main effect (p < 0.05) on One-Way RM ANOVA for respiratory rate data. Dia = diaphragm, AUC = area under the curve, amp = peak amplitude, BL = baseline, SL = saline (sham injection).

Wild type vs. ChAT-Cre mouse responses to DREADD activation

Direct comparisons of diaphragm EMG response parameters (a-f) and respiratory rate (g) at 30-minute post-J60 application (Wild type, n = 11; n = 7 females; ChAT-Cre, n = 9; n = 6 females). Left hemidiaphragm EMG AUC (a), peak-to-peak amplitude (b), and tonic activity (c) were similar between groups. However, the same parameters on the right hemidiaphragm (d-f) were greater in ChAT-Cre mice. Respiratory rate was similar between groups. Error bars depict ±ℒ1 SEM. Statistical reports for all panels are provided in Supplemental Table 3. * p < 0.05. AUC = area under the curve, amp = peak EMG amplitude, Dia = diaphragm, BL = baseline, resp rate = respiratory rate.

DREADD activation increases ventilation in unanesthetized ChAT-Cre rats

Summary plots (n = 9; n = 3 females) showing the impact of the J60 DREADD ligand on tidal volume, respiratory rate, and minute ventilation are shown in panels a-c. The normalized values (% of baseline) are shown in panels d-f. The DREADD ligand increased tidal volume compared to sham infusion (saline). Error bars depict ±D1 SEM. Statistical reports for all panels are provided in Supplemental Table 4. BL = baseline, IV = intravenous infusion period.

DREADD activation increases phrenic nerve output in ChAT-Cre rats

Representative data showing that the J60 DREADD ligand causes a rapid increase in phrenic nerve output (a). Mean data (n = 9; n = 3 females) showing the impact of J60 application on phrenic nerve raw (b) and normalized (c) peak-to-peak amplitude, raw (d) and normalized (e) tonic activity, systolic blood pressure (f), diastolic blood pressure (g), heart rate (h), mean arterial blood pressure (i), and respiratory rate (j). The J60 ligand caused an increase in phrenic peak-to-peak amplitude and tonic activity. Systolic, diastolic, and mean arterial blood pressure all decreased after J60 application. Heart rate and respiratory rate were not statistically different after J60. In panels b-e, the left phrenic is represented in orange, while right phrenic is blue. Error bars depict ±D1 SEM. Statistical reports for all panels are provided in Supplemental Table 6. * and ‡ symbols indicate significant main effects (p < 0.05) on One-Way RM ANOVA for the left and right hemidiaphragm, respectively. # indicates a significant (p < 0.05) effect on One-Way RM ANOVA for respiratory rate data. Phr = phrenic, amp = amplitude, BL = baseline, SP = systolic pressure, DP = diastolic pressure, HR = heart rate, MAP = mean arterial pressure.

Histological assessment of mCherry expression in the C4/C5 spinal segments

Representative photomicrographs of mid-cervical spinal sections from a wild-type mouse (a-ai), a ChAT-Cre mouse (b-bi), and a ChAT-Cre rat (c-ci). Wild-type mice (a-ai) showed a nonspecific pattern of expression throughout the mid-cervical grey matter. ChAT-Cre mice and rats (b-ci) showed expression limited to neurons in the ventral horns. Red color indicates positive and mCherry fluorescence. Dashed white line indicates the approximate white-gray matter demarcation.

Qualitative assessment of mCherry expression in the mid-cervical spinal cord

Spinal segments C3-C6 were assessed in quadrants broken into dorsal, ventral, left, and right. Spinal segments were counted as “positive” if they showed any evidence of mCherry expression in neuronal soma or fibers. The counts therefore indicate the number of animals of a given cohort that were mCherry positive for a given spinal segment quadrant. All animals showed a slight trend for more mCherry expression moving rostral to caudal and for more expression in the ventral vs the dorsal lamina. This trend was more prominent in the ChAT-Cre animals. At the bottom of the table, a heatmap is provided for easier assessment of the distribution of positive mCherry counts across quadrants and spinal segments.