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. C57 BL /6 JE iJ 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% O 2 , balanced N 2 ), hypercapnic (5% CO 2 , balanced air) and hypoxic hypercapnic (10% O 2 , 5% CO 2 , balanced N 2 ) 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 O 2 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.

Down syndrome (Ds) results from trisomy 21 and is the most common cause of mental retardation in the US. People with Ds display muscle weakness and cardiorespiratory complications that may contribute to their altered breathing. Ts65Dn mice, a model of Ds, have been shown to exhibit altered breathing at 12 months of age. It is unknown when breathing changes occur in these mice but it is hypothesized to be later in development based on muscle alterations reported in the literature. An additional study found hypoxemia to be more common in the dark vs. light cycle in Ts65Dn mice. We tested the hypothesis that 3‐month Ts65Dn mice would display an altered pattern of breathing compared to wild‐type (WT) mice, and that responses would differ between dark and light cycles. In order to test these hypotheses, unrestrained barometric plethysmography was used to quantify breathing frequency (breaths/min; BPM), tidal volume (TV; mL/breath) and minute ventilation (MV; mL/min) in 3‐month old Ts65Dn (n=3) and WT (n=4) mice. Mice were tested between hours 8–10 of the dark cycle and between hours 7–9 of the light cycle. Implantable LifeChips (Destron Fearing, Airport, TX) were used to monitor body temperature; changes of more than 1 degree were accounted for in the analyses. Ponemah software (Data Sciences International, St. Paul, MN) was used to analyze flow tracings during exposure to air (20.93% O2, balanced N2), hypoxia (10% O2, balanced N2), hypercapnia (5% CO2, balanced air) and hypoxic hypercapnia (10% O2, 5% CO2, balanced N2). Data are expressed as MEAN±SD; p<0.05; WT vs. Ts65Dn. Body weight was not different between WT and Ts65Dn mice (37.5±4.2 vs. 37.7±5.8g). The delta for dark minus light cycle were analyzed with repeated measures two‐way ANOVA, with strain and gas exposure as factors. An interaction between strain and gas exposures was found for MV and breathing frequency. No interaction was detected for TV. The delta for frequency are described for quiet breathing (12±7 vs. −3±36 BPM), hypoxia (6±31 vs. 8±15 BPM), hypercapnia (61±32 vs. 48±25 BPM), hypoxic hypercapnia (48±37 vs. 35±38 BPM) and recovery (−14±64 vs. 156±49 BPM). MV deltas are listed for quiet breathing (−0.9±21.9 vs. 20.8±33.8 mL/min), hypoxia (−0.9±38.8 vs. 24.9±21.9 mL/min), hypercapnia (32.5±72.3 vs. 71.1±68.1 mL/min), hypoxic hypercapnia (25.8±78.2 vs. 66.9±81.8 mL/min) and recovery (10.8±46.5 vs. 146.7±107.1 mL/min). Once more data are collected, the repeated measures two‐way ANOVA will be re‐run, and post‐hoc analysis will be performed for each gas exposure. Apnea count (no flow ≥0.5 sec) showed cycle (light/dark) and strain differences, along with a cycle by strain interaction. Light cycle apneas were more common (16±17/hr vs. 81±40/hr; WT vs. Ts65Dn) compared to the dark cycle (7±12/hr vs. 51±35/hr; WT vs. Ts65Dn); apneas were more prevalent in Ts65Dn mice overall. This preliminary study indicates the Ts65Dn mouse model may have a greater delta magnitude (dark minus light) when comparing the pattern of breathing to WT mice in dark and light cycles. The apnea count in Ts65Dn is suggestive of a lower neural drive to breath in these mice vs. WT. Additional data will be collected to identify if these initial findings continue in Ts65Dn and WT mice.Support or Funding InformationFunded by 1 R15 HD076379‐01A1, CNR supported by 1 R15 HD076379‐01A1S1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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Unrestrained barometric plethysmography (UBP) is a common technique used to quantify respiratory responses in mice; often air breathing values are compared to responses following respiratory stressors. Previous studies have reported a familiarization period (30–60 min), after which baseline measurements are quantified in mice, but these studies do not supply behavioral standards for what features determine a baseline. We had the overarching goal of quantifying a stable breathing pattern with air that could be compared to consistent breathing that is observed when mice are exposed to hypoxia and hypercapnia. The term calm breathing is used to define a segment of air breathing without sniffing and grooming, where >90% of breaths are accepted by the software. In order to compare a traditional method of baseline, we analyzed air breathing after habituation of 1 hour using a ten min average and compared it to a 30 s calm breathing segment collected during hours 1–3. Young (~4 months; n=7) and middle‐aged (~13 months; n=11) mice were tested during the light and dark cycle for breathing frequency (F; breaths/min), tidal volume (VT; ml/breath) and minute ventilation (VE; ml/min). Data were analyzed during exposure to air (20.93% O2, balanced N2), hypoxia (10 min; 10% O2, balanced N2), hypercapnia (10 min; 5% CO2, balanced air) and hypoxic hypercapnia (10 min; 10% O2, 5% CO2, balanced N2). Data are presented as mean±SD; 10 min air breathing vs. 30 s air breathing. The air breathing ten min segment was higher for F (light: 329±59, dark: 333±68 vs. light: 156±20, dark: 163±51; p=0.000) and VE (light: 114.0±37.1, dark: 138.5±50.4 vs. light: 65.7±16.4, dark: 89.7±44.2; p=0.004), but lower for VT (light: 0.38±0.14, dark: 0.44±0.13 vs. light: 0.41±0.08, dark: 0.57±0.31; p=0.004) vs. 30 s calm breathing. There was a main effect of circadian cycle for VE (p=0.020) and VT (p=0.021) as values during the dark cycle were higher than the light cycle. Percent change from the ten min air segment and the 30 s of calm breathing to the gas exposures were different for F (light: −17±21%, dark: −11±22% vs. light: 67±25%, dark: 84±37%; p=0.000), VT (light: 25±15%, dark: 38±39% vs. light: 10±13%, dark: 16±38%; p=0.006) and VE (light: 6±21%, dark: 24±34% vs. light: 90±37%, dark: 103±73%; p=0.000). Similar results were observed during hypercapnia and hypoxic hypercapnia for F, VT, and VE, although there was a significant main effect of circadian cycle only on frequency during hypoxia (p=0.048). Overall, the % change response to gases for the ten min air segment is lower for F and VE vs. calm breathing, but higher for VT. This response is due to the higher F and VE at baseline with the 10 min air segment. When air VE is normalized to VO2 (mL/min; 10 min: 171.69±42.0 ml/min, 30 s: 183.68±82.31 ml/min), no significant differences are observed (p=0.713) which suggests overall ventilation is not changing between the air segments, but the pattern of breathing is different. Therefore, documenting mouse behavior throughout UBP is essential. We recommend a shorter, calmer segment of air breathing for baseline.Support or Funding Information1 R15 HD076379‐01A1, 1 R15 HD076379‐01A1S1, American Physiological Society UGSRF (BE) and the Le Moyne College McDevitt Natural Science Fellowship (BE).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.