Regulation of the respiratory central pattern generator by chloride-dependent inhibition during development in the bullfrog (Rana catesbeiana)

Abstract

SUMMARYIsolated brainstem preparations from larval (tadpole) and adult Rana catesbeiana were used to examine inhibitory mechanisms for developmental regulation of the respiratory central pattern generator (CPG). Preparations were superfused at 20-22 °C with Cl--free artificial cerebrospinal fluid (aCSF) or with aCSF containing agonists/antagonists ofγ-aminobutyric acid (GABA) or glycine receptors. Respiratory motor output from the CPG, measured as neural activity from cranial nerve roots, was associated with fictive gill ventilation and lung ventilation in tadpoles and with fictive lung ventilation in adults. In tadpoles, fictive lung burst frequency was 0.8±0.2 min-1 and did not change significantly with Cl--free aCSF superfusion; however, lung burst amplitude increased by nearly 400 % (P<0.01). Fictive gill ventilation averaged 41.6±3.3 min-1 and was reversibly abolished by Cl--free aCSF. Superfusion with Cl--free aCSF abolished lung bursts in two of seven adult preparations, and overall lung burst frequency decreased from 3.1±0.7 to 0.4±0.03 min-1(P<0.01), but burst amplitude was unchanged. Low concentrations of GABA (0.5 mmol l-1) produced a significant increase in lung burst frequency followed by almost complete inhibition at 5.0 mmol l-1,accompanied by the abolition of gill ventilation at 2.5-5.0 mmol l-1. By contrast, fictive lung ventilation in adults was inhibited in a dose-dependent manner by glycine and GABA, and inhibition occurred at approximately 10-fold lower concentrations compared with tadpoles. The glycine receptor antagonist strychnine (2.5-25.0 μmol l-1) and the GABAA receptor antagonist bicuculline (1-10 μmol l-1)inhibited fictive gill ventilation and increased fictive lung ventilation in tadpoles. However, bicuculline and strychnine inhibited fictive lung ventilation in adults. These results suggest that lung ventilation in the tadpole brainstem may be driven by a pacemaker-like mechanism since Cl--free aCSF failed to abolish lung ventilation. Lung ventilation in adults and gill ventilation in tadpoles, however, appear to be dependent upon conventional Cl--mediated synaptic inhibition. Thus, there may be a developmental change in the fundamental process driving lung ventilation in amphibians. We hypothesize that maturation of the bullfrog respiratory CPG reflects developmental changes in glycinergic and/or GABAergic synaptic inhibitory mechanisms.