Development of gill and lung breathing in amphibia

In the 25 morphological stages of larval bullfrog development there exists a gradual transition from gill to lung ventilation associated with a developmental decrease in the contribution of the skin in gas exchange. Bath application of GABA and/or glycine inhibited gill but not lung burst activities of cranial nerve (CN) VII in the premetamorphic (stages 16–19) in vitro tadpole brainstem preparation [1]. It was proposed that the neural basis of gill rhythmogenesis involved network inhibition, whereas lung rhythmicity was pacemaker driven [1]. Bath application of a bicuculline/strychnine solution abolished gill and enhanced lung bursting in stages 16–19 in vitro [1]. Bath application of the GABA_B receptor antagonist 2-hydroxy-saclofen disinhibited the lung central pattern generator (CPG) resulting in precocious lung bursting patterns as early as developmental stage 6 [2].

We recorded efferent activity from CNVII and spinal nerve (SN)II in the in vitro tadpole brainstem preparation in three successive developmental groups (3–9; 10–15; 16–19) before and after bath application of a 10 μM bicuculline and 5 μM strychnine solution. We also exposed the brainstem to bath pH7.4, 7.8, and 8.2 before and after bath application of bicuculline/strychnine. Bicuculline/strychnine produced lung ventilatory bursts in all developmental stages tested, indicating the presence of the lung CPG as well as excitatory synapses to respiratory motor neurons as early as stage 3.

We also designed an experiment to examine the importance of lung ventilation on the developmental shift from gill to lung bursting. Two groups of tadpoles were hatched from eggs. Control tadpoles had free access to the air-water interface throughout development, whereas "barrier" tadpoles were denied access to the air-water interface via the placement of Plexiglas 2.5 cm below the surface of the water. CNVII and SNII efferent activities were recorded in vitro at bath pH7.4, 7.8, and 8.2 before and after bath application of 10 μM bicuculline and 5 μM strychnine. Postmetamorphic barrier tadpoles exhibited different motor patterns than stage-matched controls. Tadpoles reared in the barrier condition to stages 20–25 possessed fictive gill and lung ventilatory activities of premetamorphic tadpoles. All barrier tadpole preparations exhibited robust, spontaneous lung burst activity following bath application of bicuculline/strychnine.

We propose that developmentally dependent GABA- and glycinergic mechanisms lead to disinhibition of the lung CPG such that early in development gill motor patterns are the dominant respiratory rhythm, whereas in late development the lung CPG is the dominant respiratory rhythm. Denying the tadpole the ability to "practice" lung breathing during metamorphosis produces a morphologically correct postmetamorphic tadpole with an immature, or premetamorphic respiratory rhythm. We propose that prevention of lung inflation in barrier tadpoles leads to premetamorphic levels of GABA- and glycinergic inhibition, and that this inhibition may be altered by pulmonary stretch receptor feedback in control tadpoles.