Young and middle-aged mouse breathing behavior during the light and dark cycles

Unrestrained barometric plethysmography is a common method used for characterizing breathing patterns in small animals. One source of variation between unrestrained barometric plethysmography studies is the segment of baseline. Baseline may be analyzed as a predetermined time-point, or using tailored segments when each animal is visually calm. We compared a quiet, minimally active (no sniffing/grooming) breathing segment to a predetermined time-point at 1 h for baseline measurements in young and middle-aged mice during the dark and light cycles. Additionally, we evaluated the magnitude of change for gas challenges based on these two baseline segments. C57BL/6JEiJ x C3Sn.BliA-Pde6b⁺ /DnJ male mice underwent unrestrained barometric plethysmography with the following baselines used to determine breathing frequency, tidal volume (VT) and minute ventilation (VE): (1) 30-sec of quiet breathing and (2) a 10-min period from 50 to 60 min. Animals were also exposed to 10 min of hypoxic (10% O₂ , balanced N₂ ), hypercapnic (5% CO₂ , balanced air) and hypoxic hypercapnic (10% O₂ , 5% CO₂ , balanced N₂ ) gas. Both frequency and VE were higher during the predetermined 10-min baseline versus the 30-sec baseline, while VT was lower (P < 0.05). However, VE/V_O2 was similar between the baseline time segments (P > 0.05) in an analysis of one cohort. During baseline, dark cycle testing had increased VT values versus those in the light (P < 0.05). For gas challenges, both frequency and VE showed higher percent change from the 30-sec baseline compared to the predetermined 10-min baseline (P < 0.05), while VT showed a greater change from the 10-min baseline (P < 0.05). Dark cycle hypoxic exposure resulted in larger percent change in breathing frequency versus the light cycle (P < 0.05). Overall, light and dark cycle pattern of breathing differences emerged along with differences between the 30-sec behavior observational method versus a predetermined time segment for baseline.