The Diencephalon Contributes to Hypoxia‐Induced Facilitation of Fictive Air Breathing in Pre‐Metamorphic Tadpoles

Abstract

Introduction and hypothesis The onset of air breathing is an important milestone in the physiological development of the American bullfrog (Lithobates catesbeianus). The expression of the motor command driving lung ventilation is inhibited during the early tadpole stages and is progressively disinhibited as the animal enters metamorphosis. Environmental and endocrine signals regulate the onset of metamorphosis and our previous work showed that exposing isolated brainstems from pre-metamorphic tadpoles to acute hypoxia (98% N2 + 2% CO2; 15 min) facilitates the expression of air breathing, as indicated by an increase in fictive air breaths that persists for at least 2h after the end of hypoxic stimulation. Based on the growing evidence indicating that structures located rostral to the brainstem contribute to respiratory control, we tested the hypothesis that the presence of rostral structures augments hypoxia-induced facilitation of fictive air breathing in pre-metamorphic tadpoles. Methods Experiments were performed on pre-metamorphic tadpoles (TK stages V to XI). The central nervous system as isolated and placed in a recording chamber where it was superfused with an oxygenated artificial cerebrospinal fluid (98% O2, 2% CO2; pH = 7.9). The preparation was randomly assigned to one of three groups corresponding to a distinct level of brain transection: 1) Medullary preparation, 2) Diencephalon and 3) Telencephalon. The neural correlates of gill and lung ventilation was assessed by placing suction electrodes onto cranial nerves V and X. Following 1h of recovery, fictive breathing was recorded under baseline condition (15 min) before being exposed to hypoxia (98% N2 + 2% CO2; 15 min). The preparation was returned to baseline condition and time-ependent changes were recorded 30 min, 1h and 2h post-hypoxia. Fictive gill and lung breathing frequency and amplitude was measured in each time point. In a distinct series of experiments, preparations from each groupwere maintained under basal conditions to assess stability of the preparations over the course of the protocol. Result Hypoxia induced time-dependent changes in lung burst frequency that were greater than those observed in “time control” experiments. The largest increase was observed in preparations with diencephale whereas medullary preparations did not respond. Gill burst frequency remained constant; changes in burst amplitude (gill or lung) were not significant. Conclusion and perspectives The lack of response of medullary preparations is consistent with data showing that the locus coeruleus is an important central O2 sensing structure. However, results obtained from diencephalon preparations indicate the presence of structures that potentiate the long-term stimulatory effects of hypoxia on fictive air breathing. The fact that the time-dependent rise in lung burst frequency was not as important in more intact preparations suggest that more rostral structures attenuate those effects.