Control of lung ventilation after extended disuse caused by overwintering submergence in the bullfrog
Authors: Santin Joseph , Hartzler Lynn 
Published: November 2023

Many clinical scenarios result in reduced sensorimotor activity which leads to functional deficits. Although inactive motor systems of laboratory species and humans typically degrade, many animals escape prolonged neuromotor disuse unscathed. Semiaquatic frogs from northern latitudes survive the winter months aerobically at cold temperatures in water completely covered by ice because of sufficient skin gas exchange, leaving the neuromotor‐chemosensory system that regulates pulmonary ventilation entirely unused. Rapidly after spring emergence from underwater habitats, frogs perform costly behaviors at higher temperatures that urgently require breathing despite months of disuse. Determining whether bullfrogs preserve sensorimotor capacity of the respiratory system in the absence of function could provide a novel physiologically and ecologically relevant animal for model for countering neuromotor disuse and sensory deprivation. We tested the hypotheses that bullfrogs, Lithobates catesbeianus, could 1.) breathe to satisfy resting metabolism and 2.) combat challenges to blood gas homeostasis upon transition from a cold‐submerged (2°C; 6–9 weeks; no breathing) to a warm‐terrestrial (24°C; ~16 hours after emergence) environment as occurs following emergence in early spring. We found that recently emerged bullfrogs in vivo had resting ventilation comparable to warm‐acclimated (control) bullfrogs (p=0.9; one‐way ANOVA) and also ventilated similarly to match metabolism (ventilation:metabolism; p=0.80; one‐way ANOVA). Using an in vitro brainstem preparation to record discharge from cranial nerves innervating respiratory muscles, we found that the shape and frequency of the motor output was preserved and facilitated, respectively, in recently emerged frogs. When exposed to hypoxia (5% O2) in vivo, control and recently emerged bullfrogs both responded with increases in ventilation (p=0.48; two‐way ANOVA). However, the increase in breathing typically evoked by elevated CO2 (5% CO2) was reduced compared to control bullfrogs (p=0.004; two‐way ANOVA). To confirm that the reduction in ventilatory CO2 chemosensitivity resulted from diminished central chemosensitivity, we isolated the central respiratory control system in vitro. Frequency of respiratory nerve discharge in vitro increased during hypercapnia in preparations from control, but not in preparations from recently emerged bullfrogs (p<0.0001; two‐way ANOVA). Finally, we assessed CO2/pH sensitivity in locus coeruleus neurons, a chemosensory brain region involved breathing control, to confirm that overwintering decreased cellular chemosensitivity. Whole‐cell patch clamp recordings from individual chemosensitive neurons from brainstem slices revealed that cold‐acclimated frogs had reduced firing increases upon stimulation by hypercapnia (5% CO2) (p=0.01; two‐way ANOVA). These results demonstrate that bullfrogs maintain baseline respiratory‐motor function and the ability to compensate challenges to oxygen lack after ~2 months without lung ventilation. In contrast, ventilatory responses to acid‐base disturbances in early spring are blunted through reductions in central chemosensitivity. Thus breathing control in the bullfrog after overwintering provides a unique model to elucidate mechanisms underlying both preservation and loss of sensorimotor function in the same species, in the same individual, within the same neural system after natural, extended disuse.Support or Funding InformationNSF‐IOS 1257338 (LH), Wright State Biomedical Sciences PhD Program (JS)