Reassessment of chemical control of breathing in undisturbed bullfrogs, Lithobates catesbeianus, using measurements of pulmonary ventilation

Abstract

Despite the importance of bullfrogs (Lithobates catesbeianus) as models in respiratory control, chemical control of breathing in conscious bullfrogs has never been assessed with methods that measure the tidal volume (VT). This has precluded the calculation of important respiratory variables like minute ventilation (V.E) and air convection requirement. To address this, we adapted airflow pneumotachography for use in bullfrogs and reassessed chemical control of breathing. We show that V.E measured with pneumotachography produces breathing pattern and metabolism values consistent with anurans. Second, we confirm that bullfrogs have small ventilatory responses to hypercarbia that include increases in tidal volume and a post-hypercarbic hyperpnea. We observed that the magnitude of the post-hypercarbic hyperpnea does not depend on ventilatory responses during hypercarbia. Finally, we showed that increases in breathing frequency and VT during hypoxia are differentially regulated with time. These findings comprise the first complete assessment of hypercarbic and hypoxic V.E responses in intact bullfrogs and emphasize the importance of measuring VT.

Control of lung ventilation following overwintering conditions in bullfrogs,Lithobates catesbeianus

“…During hypercarbia and hypoxia, ventilation was measured for 5 min, every 10 min, to elucidate the time course of the ventilatory response. Additionally, ventilation in the 1 min period after hypercarbia ( post-hypercarbia) was analyzed as this ventilatory response represents the 'stimulatory' ventilatory response to CO 2 independent of olfactory inhibition (Kinkead and Milsom, 1996) and is less variable between animals (Santin and Hartzler, 2016) presumably because of variable activation of inhibitory olfactory responses. VȮ 2 and breathing pattern were measured in the last 5 min of hypoxia and hypercarbia.…”

“…Bullfrogs fitted with the mask and pneumotach were then placed in a dark 875 ml experimental chamber overnight at 24°C and exposed to airflow of 1.1 l min −1 . The only methodological difference between this study and our previous study (Santin and Hartzler, 2016) is that we omitted the use of sutures to secure the mask before applying super glue. This step was deemed unnecessary as the super glue alone held the mask in place and remained sealed in most animals.…”

“…The only time point at which ventilation measurements were made immediately after altering the flow was the post-hypercarbic period. It is unlikely that changes in flow influence the increase in breathing observed in the post-hypercarbic period (1-2 min after hypercarbia) because breathing frequency in the 'post-hypoxic' period does not increase (Santin and Hartzler, 2016) (J.M.S., personal observation; data not shown). At the end of each experiment, the facemask was removed and 0.1-1.2 ml air injections were pushed through the facemask to calibrate the pneumotach.…”

“…We measured ventilation in bullfrogs acclimated to room temperature (22°C; n=8), 2°C with air access (n=7) and 2°C submerged (n=5) groups at 24°C (a temperature requiring lung ventilation as would occur in spring) using air-flow pneumotachography (Glass et al, 1978) closely following methods that we recently adapted for bullfrogs (Santin and Hartzler, 2016). Briefly, the evening prior to measurements, bullfrogs were lightly anesthetized with ∼0.5% (v/v) isoflurane until they lost toe pinch and eye reflexes.…”

“…The rates of O 2 consumption (VȮ 2 ) and CO 2 production (VĊ O2 ) were determined in concert with ventilation as described in Santin and Hartzler (2016). Open-flow respirometry was performed by pushing experimental gas mixtures at 1.1 ml min −1 through a flask half-filled with water to fully humidify the gas.…”