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

Abstract

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.

Keywords: Hypercapnia; hypoxia; hypoxic hypercapnia.

Figure 1

Representative breathing traces for a: (A) young mouse and (B) middle‐aged mouse during each segment of air breathing where 1 = 30‐sec light cycle, 2 = 30‐sec dark cycle, 3 = 10‐min light cycle, and 4 = 10‐min dark cycle. The x‐axis represents 0–5 sec and the y‐axis is −2 mL/min to 2 mL/min. Tracing in (C) depicts the type of data used for quiet breathing analysis, where segments of quiet breathing are flanked by increased activity. The x‐axis represents zero to 80 sec and y‐axis is −2 mL/min to 2 mL/min. (D) Representative tracings from a single mouse (middle‐aged, dark cycle) during hypoxia (H), hypercapnia (HC) and hypoxic hypercapnia (HH). The x‐axis represents 5 sec and the y‐axis is −2 mL/min to 2 mL/min.

Figure 2

Breathing frequency (freq; breaths/min), tidal volume (VT; mL/breath), and minute ventilation (VE; mL/min) during air exposure (20.93% O₂, balanced N₂) in young and middle‐aged mice during a 30‐sec quiet breathing segment versus a 10‐min average analyzed from 50 to 60 min of air breathing. Quiet breathing was averaged for a 30‐sec period when mice were sitting with no locomotor movement in the cage; this often took 1–3 h to observe. Both time points were acquired during light and dark circadian cycles. Light testing period: hours 4–7 of light cycle. Dark testing period: hours 4–7 of dark cycle. Air breathing is shown for: (A) young mice (~4 months; n  = 6) frequency, (B) middle‐aged mice (~13 months; n  = 10) frequency, (C) young mice VE, (D) middle‐aged mice VE, (E) young mice VT, and (F) middle‐aged mice VT. *Significant differences were observed between 30 sec and 10 min of air breathing (P < 0.05) using a one‐way ANOVA. Significant differences between light and dark cycle were also detected for VT (P = 0.0015). Body weight was a significant covariate for both VE (P = 0.038) and VT (P < 0.001). Mice were excluded if values were > 3SD from the mean (one mouse excluded in each age group). All data are presented as mean ± SD.

Figure 3

Percent change from air breathing frequency to various gas exposures is larger with use of a 30‐sec baseline. Air breathing was calculated as the 30‐sec of quiet breathing or the 10‐min average at the end of the first hour of air breathing. Each mouse's individual percent (%) change for a given gas exposure (10‐min average) was calculated. Both cohorts were tested in light and dark conditions. Light testing period: hours 4–7 of light cycle. Dark testing period: hours 4–7 of dark cycle. Percent change from air breathing is shown for: (A) hypoxia (10% O₂, balanced N₂) in young mice (~4 months; n  = 6), (B) hypoxia in middle‐aged mice (~13 months, n  = 10), (C) hypercapnia (20.93% O₂, 5% CO ₂, and balanced N₂) in young mice, (D) hypercapnia in middle‐aged mice, (E) hypoxic hypercapnia (10% O₂, 5% CO ₂, balanced N₂) in young mice, and (F) hypoxic hypercapnia in middle‐aged mice. Analysis was performed using a mixed model ANOVA. *Significant main effect of baseline segment for all exposures (P < 0.001). Significant main effect of circadian cycle during hypoxia (P = 0.019). Mice were excluded if baseline values were > 3SD from the mean (one mouse excluded in each age group). All data are presented as mean ± SD.

Figure 4

Percent change from air breathing tidal volume (VT) to VT during various gas exposures is larger with the use of a 10‐min baseline. Air breathing was calculated as the 30‐sec of quiet breathing or the 10‐min average at the end of the first hour of air breathing. Each mouse's individual percent (%) change for a given gas exposure (10‐min average) was calculated. Both cohorts were tested in light and dark conditions. Light testing period: hours 4–7 of light cycle. Dark testing period: hours 4–7 of dark cycle. Percent change from air breathing is shown for: (A) hypoxia (10% O₂, balanced N₂) in young mice (~4 months; n  = 7), (B) hypoxia in middle‐aged mice (~13 months, n  = 11), (C) hypercapnia (20.93% O₂, 5% CO ₂, and balanced N₂) in young mice, (D) hypercapnia in middle‐aged mice, (E) hypoxic hypercapnia (10% O₂, 5% CO ₂, balanced N₂): (E) in young mice, and (F) hypoxic hypercapnia in middle‐aged mice. Analysis was performed using a mixed model ANOVA. *Significant main effect of baseline segment for all exposures (P > 0.05). Mice were excluded if the values were > 3SD from the mean (one mouse excluded in each age group). All data are presented as mean ± SD.

Figure 5

Percent change from air breathing minute ventilation (VE) to VE during various gas exposures is larger with the use of a 30‐sec baseline. Air breathing was calculated as the 30‐sec of quiet breathing or the 10‐min average at the end of the first hour of air breathing. Each mouse's individual percent (%) change for a given gas exposure (10‐min average) was calculated. Both cohorts were tested in light and dark conditions. Light testing period: hours 4–7 of light cycle. Dark testing period: hours 4–7 of dark cycle. Percent change from air breathing is shown for: (A) hypoxia (10% O₂, balanced N₂) in young mice (~4 months; n  = 7), (B) hypoxia in middle‐aged mice (~13 months, n  = 11), (C) hypercapnia (20.93% O₂, 5% CO ₂, and balanced N₂) in young mice, (D) hypercapnia in middle‐aged mice, (E) hypoxic hypercapnia (10% O₂, 5% CO ₂, balanced N₂) in young mice, and (F) hypoxic hypercapnia in middle‐aged mice. Analysis was performed using a mixed model ANOVA. *Significant main effect of baseline segment for all exposures (P < 0.001). Mice were excluded if values were > 3SD from the mean (one mouse excluded in each age group). All data are presented as mean ± SD.