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 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.

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 hemi-diaphragm EMG is represented in orange, while right hemi-diaphragm EMG is blue. Error bars depict ± 1 SEM. Statistical reports for all panels are provided in Supplemental Table 1. 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 hemi-diaphragm. Respiratory rate decreased over time. For panels d-f, the left hemi-diaphragm EMG is represented in orange, while right hemi-diaphragm EMG is blue. Error bars depict ± 1 SEM. Statistical reports for all panels are provided in Supplemental Table 2. 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 hemi-diaphragm EMG AUC (a), peak-to-peak amplitude (b) and tonic activity (c) were similar between groups. However, the same parameters on the right hemi-diaphragm (d-f) were greater in ChAT-Cre mice. Respiratory rate was similar between groups. Error bars depict ± 1 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 ± 1 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 ± 1 SEM. Statistical reports for all panels are provided in Supplemental Table 6. 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 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 approximate white-gray matter demarcation.

Impact of DREADD activation on diaphragm EMG in ChAT-Cre rats.

Example trace of rectified and integrated diaphragm EMG activity from a ChAT-Cre rat that had previously undergone injections of AAV9-hSyn-DIO-hM3D(Gq)-mCherry into the C4 ventral horns before and after application of the DREADD ligand, J60. Callout panels below show example activity at baseline (a), after injection of vehicle (b), and after application of the DREADD ligand, J60 (c).

Example quantification of mCherry expression in the C3-C6 spinal cord.

Mid-cervical spinal cord images from ChAT-Cre animal (left) with positive mCherry expression. A grid has been placed on each image to highlight the quadrant system (i.e., left dorsal, right dorsal, left ventral, right ventral) used to score the tissue. The right column shows an outline of each section on the left with a score for each quadrant based on mCherry expression. A given quadrant was scored with a “+” if it contained any mCherry positive neurons or fibers else it was given a “-“ signifying no mCherry expression.

mCherry expression in the mid-cervical ventral horn corresponds with the laterality of the diaphragm EMG DREADD response.

Example spinal histology (left) from two ChAT-Cre mice showing unilateral (a) and bilateral (b) expression of mCherry (red) in the ventral horn(s) and corresponding diaphragm EMG traces. Unilateral ventral horn mCherry expression (a) results in DREADD responses that were limited to the ipsilateral hemi-diaphragm. Animals with bilateral ventral horn DREADD expression responded to DREADD activation with a bilateral increase in diaphragm output.

Statistical summary for impact of DREADD activation on diaphragm EMG in wild type mice.

Time points are in reference to minutes passed since J60 infusion. Summary data presented in Figure 1. EMG = electromyography, AUC = area under the curve, RM = repeated measures, df = degrees of freedom. Bolded p-values indicates p<0.05.

Statistical summary for impact of DREADD activation on diaphragm EMG in ChAT-Cre mice.

Time points are in reference to minutes passed since J60 infusion. Summary data presented in Figure 2. EMG = electromyography, AUC = area under the curve, RM = repeated measures, df = degrees of freedom. Bolded p-values indicates p<0.05.

Statistical summary for impact of DREADD activation on diaphragm EMG in in wild type mice vs. ChAT-Cre mice at the 30-min post J60 infusion time point.

Summary data presented in Figure 3. EMG = electromyography, AUC = area under the curve, df = degrees of freedom. Bolded p-values indicates p<0.05.

Statistical summary for impact of DREADD activation on plethysmography outcomes in unanesthetized ChAT-Cre rats using two-way repeated measures ANOVAs.

Each outcome measure is presented normalized to body weight (with the exception of respiratory rate) and normalized to values at baseline. Summary data presented in Figure 4. RM = repeated measures, ml/kg = milliliters of air per kilogram of animal’s body weight, df = degrees of freedom. Bolded p-values indicates p<0.05.

Statistical summary for impact of DREADD activation on phrenic nerve activity in ChAT-Cre mice.

Time points are in reference to minutes passed since J60 infusion. Summary data presented in Figure 5. RM = repeated measures, df = degrees of freedom. Bolded p-values indicates p<0.05.